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Human Neonatal Peripheral Blood Leukocytes Demonstrate Pathogen-Specific Coordinate Expression of TLR2, TLR4/MD2, and MyD88 During Bacterial Infection In Vivo
Abstract
Toll-like receptors (TLRs) play important roles in infection. We have previously reported TLR2 is up-regulated in neonatal Gram-positive (G+) bacteremia, whereas TLR4 is up-regulated in neonatal Gram-negative (G-) bacteremia. For functional signaling, TLR4 requires myeloid differentiation (MD)-2, and both TLR2 and TLR4 signal need myeloid differentiation factor (MyD88). However, it is unknown whether newborns can enhance expression of MD-2 and MyD88 with bacterial infection in coordination with TLR expression. We characterized neonatal peripheral blood leukocyte expression of MD-2 and MyD88 in relation to TLR2/4 in newborns. TLR2 mRNA expression by PBMCs and TLR2 protein expression by monocytes and granulocytes were significantly increased in the G+ bacteremia group. TLR4 mRNA on PMBCs and protein expression on monocytes and granulocytes were significantly increased in the G- bacterial group. Remarkably, although, MyD88 mRNA was increased in all patients with documented bacterial infection and correlated with both TLR2 and TLR4, MD-2 mRNA was selectively increased in G- bacterial group, wherein it correlated with TLR4 but not with TLR2 mRNA. Our findings demonstrate that during bacterial infection in vivo, newborns selectively and coordinately amplify the TLR2-MyD88 pathway in G+ bacterial infection and the TLR4/MD2/MyD88 pathway in G- bacterial infection, suggesting key roles for innate immune pathway in neonatal responses to bacterial infection.
... Conversely, TLR9-mediated cytokine responses to CpG oligonucleotide stimulation of cord blood cells induces significantly greater IL-6, CXCL8 (formerly IL-8), IL-1β and the anti-inflammatory cytokine IL-10 compared to peripheral blood from 3 month-old infants [30]. Collection of relatively low (< 10 mL) volumes of human neonatal and infant peripheral blood allows in vitro immune evaluation, as demonstrated in clinical vaccination trials [31][32][33][34]. ...
... In addition to whole blood assays, mononuclear cell (MC) cultures are also a popular approach to in vitro immune evaluation [33,35]. Such assays have highlighted numerous ontological immune differences. ...
... • Peripheral leukocytes may have distinct responses to those in tissues/nodes that are the targets of adjuvanted vaccines. [25,27,28,30,33,35,39,73,121,168,169] ...
The human immune system comprises cellular and molecular components designed to coordinately prevent infection while avoiding potentially harmful inflammation and autoimmunity. Immunity varies with age, reflecting unique age-dependent challenges including fetal gestation, the neonatal phase, and infancy. Here, we review novel mechanistic insights into early life immunity, with an emphasis on emerging models of human immune ontogeny, which may inform age-specific translational development of novel anti-infectives, immunomodulators, and vaccines.
... It is believed that TLR2 responds mainly to gram-positive and TLR4 to Gram negative bacteria. The findings of Zang et al. [26] demonstrate that during bacterial infection in vivo, newborns selectively and coordinately amplify the TLR2 pathway in Gram-positive bacterial infection and the TLR4 pathway in Gram negative bacterial infection. These results indicate that various bacteria differ in their dose-dependent patterns of induction of TLRs [24,26]. ...
... The findings of Zang et al. [26] demonstrate that during bacterial infection in vivo, newborns selectively and coordinately amplify the TLR2 pathway in Gram-positive bacterial infection and the TLR4 pathway in Gram negative bacterial infection. These results indicate that various bacteria differ in their dose-dependent patterns of induction of TLRs [24,26]. However studies mostly performed in vitro, in laboratory models or in newborns, may not reflect the true situation during infectious diseases because pathogens are equipped with various ligands which may activate multiple signaling pathways. ...
Objective
From 10 to 15% of children suffer from recurrent acute otitis media (AOM). An association between polymorphism in TLRs and their co-receptor CD14 with otitis media proneness has been described in children. Moreover, the experiments on animal models have shown that TLRs and their signaling molecules are critical for timely resolution of bacterial otitis.
Aim
The aim of this study was to determine the expression of TLR1, TLR2 and TLR4 on lymphocytes, monocytes and granulocytes in peripheral blood in children with recurrent or persistent AOM.
Methods
The study was performed on a group of 25 children hospitalized for recurrent AOM, failures of previous treatments and/or acute mastoiditis. The results were compared to the control group of healthy children at the same age. The expression of TLRs on peripheral blood white cells was measured by flow cytometric analysis. The results were expressed as mean fluorescence intensity (MFI). The statistical analysis was performed using the Mann–Whitney U test.
Results
The highest expression of TLR was found on monocytes, the lowest on lymphocytes in both groups of children (AOM and the control one). The expression of TLR1 was the lowest and expression of TLR4 was the highest on all examined cells. The expression of all examined TLRs on monocytes was significantly higher in the AOM group.
Conclusions
Peripheral blood monocytes are characterized by increased expression of TLRs in the course of recurrent AOM.
... In contrast, TLR2 expression is constitutively expressed on monocytes across gestational age and is therefore at similar levels in monocytes of preterms, full-term neonates, and adult monocytes [64]. Interestingly, Gram-positive bacteremia apparently induces increases in neonatal peripheral blood monocyte and granulocyte TLR2 expression in infected human newborns [67,68]. ...
... In a novel model of intrajugular infection in mice less than 24 hours of life, newborn mice demonstrate impaired weight gain when injected intravenously with SE compared to saline-injected controls [78]. Similar to the pattern noted in the peripheral blood mononuclear cells of preterm human newborns during Gram-positive bacteremia [67,68], TLR2 and MyD88 mRNA levels in the liver were significantly increased by injection of SE that induced inocula-dependent serum IL-6 and TNF concentrations [78]. SE-induced cytokine production from human neonatal mononuclear cells (MCs) in vitro is dependent on gestational age [79][80][81][82]. ...
Preterm infants are especially susceptible to late-onset sepsis that is often due to Gram-positive bacterial infections resulting in substantial morbidity and mortality. Herein, we will describe neonatal innate immunity to
Staphylococcus
spp. comparing differences between preterm and full-term newborns with adults. Newborn innate immunity is distinct demonstrating diminished skin integrity, impaired Th1-polarizing responses, low complement levels, and diminished expression of plasma antimicrobial proteins and peptides, especially in preterm newborns. Characterization of distinct aspects of the neonatal immune response is defining novel approaches to enhance host defense to prevent and/or treat staphylococcal infection in this vulnerable population.
... In addition, while neonates are able to activate TLR2 signaling, it has been suggested that they are less well-equipped to terminate the TLR-mediated inflammatory response, potentially explaining their susceptibility to the adverse effects of gram positive infections like GBS [37]. Our findings of a robust TLR2 response in neonates following inflammatory stimuli are in keeping with findings that human neonates increase TLR2 mRNA in peripheral blood mononuclear cells and TLR2 protein in both monocytes and granulocytes in response to infection [38]. ...
Objectives
Scientific evidence provides a widened view of differences in immune response between male and female neonates. The X-chromosome codes for several genes important in the innate immune response and neonatal innate immune cells express receptors for, and are inhibited by, maternal sex hormones. We hypothesized that sex differences in innate immune responses may be present in the neonatal population which may contribute to the increased susceptibility of premature males to sepsis. We aimed to examine the in vitro effect of pro-inflammatory stimuli and hormones in neutrophils and monocytes of male and female neonates, to examine the expression of X-linked genes involved in innate immunity and the miRNA profiles in these populations.
Methods
Preterm infants (n = 21) and term control (n = 19) infants were recruited from the Coombe Women and Infants University Hospital Dublin with ethical approval and explicit consent. The preterm neonates (eight female, 13 male) were recruited with a mean gestation at birth (mean ± SD) of 28 ± 2 weeks and corrected gestation at the time of sampling was 30 + 2.6 weeks. The mean birth weight of preterm neonates was 1084 ± 246 g. Peripheral blood samples were used to analyze immune cell phenotypes, miRNA human panel, and RNA profiles for inflammasome and inflammatory genes.
Results
Dividing neutrophil results by sex showed no differences in baseline CD11b between sexes among either term or preterm neonates. Examining monocyte CD11b by sex shows, that at baseline, total and classical monocytes have higher CD11b in preterm females than preterm males. Neutrophil TLR2 did not differ between sexes at baseline or following lipopolysaccharide (LPS) exposure. CD11b expression was higher in preterm male non-classical monocytes following Pam3CSK treatment when compared to females, a finding which is unique to our study. Preterm neonates had higher TLR2 expression at baseline in total monocytes, classical monocytes and non-classical monocytes than term. A sex difference was evident between preterm females and term females in TLR2 expression only. Hormone treatment showed no sex differences and there was no detectable difference between males and females in X-linked gene expression. Two miRNAs, miR-212-3p and miR-218-2-3p had significantly higher expression in preterm female than preterm male neonates.
Conclusions
This study examined immune cell phenotypes and x-linked gene expression in preterm neonates and stratified according to gender. Our findings suggest that the responses of females mature with advancing gestation, whereas male term and preterm neonates have very similar responses. Female preterm neonates have improved monocyte activation than males, which likely reflects improved innate immune function as reflected clinically by their lower risk of sepsis. Dividing results by sex showed changes in preterm and term infants at baseline and following LPS stimulation, a difference which is reflected clinically by infection susceptibility. The sex difference noted is novel and may be limited to the preterm or early neonatal population as TLR2 expression on monocytes of older children does not differ between males and females. The differences shown in female and male innate immune cells likely reflect a superior innate immune defense system in females with sex differences in immune cell maturation. Existing human studies on sex differences in miRNA expression do not include preterm patients, and most frequently use either adult blood or cord blood. Our findings suggest that miRNA profiles are similar in neonates of opposite sexes at term but require further investigation in the preterm population. Our findings, while novel, provide only very limited insights into sex differences in infection susceptibility in the preterm population leaving many areas that require further study. These represent important areas for ongoing clinical and laboratory study and our findings represent an important contribution to exiting literature.
... TLRs are part of a complex system of receptors that play an important role in the identification of microorganisms, modulation of the immune response and initiation of the effector response (Zhang et al., 2010). Furthermore, these receptors are part of a group of transmembrane proteins, responsible for effecting and participating in the differentiation of effector cells (Fleer and Krediet, 2007). ...
Infections caused by Candida spp. are frequent in critically hospitalized patients, especially among premature neonates, representing one of the most common healthcare-related infections. Although there is considerable production of current knowledge about the mechanisms of immune response, aspects involved in the newborn's innate defense are not fully understood. The aim of this study was to describe the innate immune mechanisms involved in the defense of neonates against invasive candidiasis. This is an integrative literature review from the Scopus, Scifinder, Medline, Web of Science databases and the electronic libraries ScienceDirect and Scielo, in the period between 2002 and 2020, with rescue based on primary descriptor Immunity Innate plus secondary descriptors Candidiasis Invasive AND Infant Newborn. We have observed the involvement of various mechanisms in the neonatal response against invasive candidiasis, including the recognition, signaling, recruitment, and initiation of an effective immune response. These mechanisms encompass the presence of antimicrobial peptides, phagocytosis, synthesis of reactive oxygen species, inflammatory mediators, and complex cell signaling systems mediated by Pattern Recognition Receptors (PRRs). With this study, it is expected to contribute to the expansion of knowledge about the immunological mechanisms involved in the innate immune response of the newborn against disseminated infections caused by Candida species, and in the same sense, highlight the importance of this knowledge as a reflex in the decrease in mortality in the neonatal period.
... It is worth acknowledging that we found a differential modulation of TLR expression in stimulated CB monocytes compared to adults-in particular, concerning TLR1, TLR2, and TLR4 expression. These findings are in keeping with previous studies in neonatal peripheral blood mononuclear cells, monocytes, and granulocytes [9,59,60]. In summary, differences in the post-transcriptional regulation of IL-10 expression, either caused or accompanied by a stimulus-driven modulation of TLR expression in neonatal monocytes, might impact the pattern of immune response. ...
Developmentally regulated features of innate immunity are thought to place preterm and term infants at risk of infection and inflammation-related morbidity. Underlying mechanisms are incompletely understood. Differences in monocyte function including toll-like receptor (TLR) expression and signaling have been discussed. Some studies point to generally impaired TLR signaling, others to differences in individual pathways. In the present study, we assessed mRNA and protein expression of pro- and anti-inflammatory cytokines in preterm and term cord blood (CB) monocytes compared with adult controls stimulated ex vivo with Pam3CSK4, zymosan, polyinosinic:polycytidylic acid, lipopolysaccharide, flagellin, and CpG oligonucleotide, which activate the TLR1/2, TLR2/6, TLR3, TLR4, TLR5, and TLR9 pathways, respectively. In parallel, frequencies of monocyte subsets, stimulus-driven TLR expression, and phosphorylation of TLR-associated signaling molecules were analyzed. Independent of stimulus, pro-inflammatory responses of term CB monocytes equaled adult controls. The same held true for preterm CB monocytes—except for lower IL-1β levels. In contrast, CB monocytes released lower amounts of anti-inflammatory IL-10 and IL-1ra, resulting in higher ratios of pro-inflammatory to anti-inflammatory cytokines. Phosphorylation of p65, p38, and ERK1/2 correlated with adult controls. However, stimulated CB samples stood out with higher frequencies of intermediate monocytes (CD14+CD16+). Both pro-inflammatory net effect and expansion of the intermediate subset were most pronounced upon stimulation with Pam3CSK4 (TLR1/2), zymosan (TR2/6), and lipopolysaccharide (TLR4). Our data demonstrate robust pro-inflammatory and yet attenuated anti-inflammatory responses in preterm and term CB monocytes, along with imbalanced cytokine ratios. Intermediate monocytes, a subset ascribed pro-inflammatory features, might participate in this inflammatory state.
... In mice, pre-treatment with Toll-like receptor (TLR) agonists enhances subsequent responses to polymicrobial sepsis (7) and treatment with BCG results in enhanced emergency granulopoiesis (8). Evidence that such trained immunity occurs in human newborns includes: (a) critically ill preterm newborns demonstrate enhanced pathogen-specific mononuclear cell pattern recognition receptor (PRR) expression in the setting of Grampositive or Gram-negative bacteremia (9); and (b) histologic chorioamnionitis affecting preterm infants is associated with a significantly reduced risk of late onset bacterial sepsis (10). These observations suggest the existence of neonatal innate memory that alters responses to subsequent unrelated microbial challenges (11). ...
Background
Newborns exhibit distinct immune responses and are at high risk of infection. Neonatal immunization with BCG, the live attenuated vaccine against tuberculosis (TB), is associated with broad protection against a range of unrelated pathogens, possibly reflecting vaccine-induced training of innate immune cells (“innate memory”). However, little is known regarding the impact of age on BCG-induced innate responses.
Objective
Establish an age-specific human monocyte in vitro training platform to characterize and compare BCG-induced primary and memory cytokine responses and immunometabolic shifts.
Design/Methods
Human neonatal and adult CD33-selected monocytes were stimulated for 24h with RPMI (control) or BCG (Danish strain) in 10% autologous serum, washed and cultured for 5 additional days, prior to re-stimulation with the TLR4 agonist LPS for another 24h. Supernatants were collected at Day 1 (D1) to measure primary innate responses and at Day 7 (D7) to assess memory innate responses by ELISA and multiplex cytokine and chemokine assays. Lactate, a signature metabolite increased during trained immunity, was measured by colorimetric assay.
Results
Cytokine production by human monocytes differed significantly by age at D1 (primary, BCG 1:750 and 1:100 vol/vol, p<0.0001) and D7 (innate memory response, BCG 1:100 vol/vol, p<0.05). Compared to RPMI control, newborn monocytes demonstrated greater TNF (1:100, 1:10 vol/vol, p<0.01) and IL-12p40 (1:100 vol/vol, p<0.05) production than adult monocytes (1:100, p<0.05). At D7, while BCG-trained adult monocytes, as previously reported, demonstrated enhanced LPS-induced TNF production, BCG-trained newborn monocytes demonstrated tolerization, as evidenced by significantly diminished subsequent LPS-induced TNF (RPMI vs. BCG 1:10, p <0.01), IL-10 and CCL5 production (p<0.05). With the exception of IL-1RA production by newborn monocytes, BCG-induced monocyte production of D1 cytokines/chemokines was inversely correlated with D7 LPS-induced TNF in both age groups (p<0.0001). Compared to BCG-trained adult monocytes, newborn monocytes demonstrated markedly impaired BCG-induced production of lactate, a metabolite implicated in immune training in adults.
Conclusions
BCG-induced human monocyte primary- and memory-innate cytokine responses were age-dependent and accompanied by distinct immunometabolic shifts that impact both glycolysis and training. Our results suggest that immune ontogeny may shape innate responses to live attenuated vaccines, suggesting age-specific approaches to leverage innate training for broad protection against infection.
... As described above, MHC class I upregulation may take place in a MyD88 dependent way and, therefore, it could be postulated that TLR-mediated MHC I enrichment in newborns is reduced, possibly resulting in impaired cross-presentation. However, it has been shown that newborn cells can increase MyD88 mRNA expression after bacterial infection (240), and potent nuclear translocation of NF-kB can be achieved using TLR7/8 agonists rather than TLR3 or TLR4 agonists. Whether this would also happen upon viral infection is unknown. ...
Infections are most common and most severe at the extremes of age, the young and the elderly. Vaccination can be a key approach to enhance immunogenicity and protection against pathogens in these vulnerable populations, who have a functionally distinct immune system compared to other age groups. More than 50% of the vaccine market is for pediatric use, yet to date vaccine development is often empiric and not tailored to molecular distinctions in innate and adaptive immune activation in early life. With modern vaccine development shifting from whole-cell based vaccines to subunit vaccines also comes the need for formulations that can elicit a CD8⁺ T cell response when needed, for example, by promoting antigen cross-presentation. While our group and others have identified many cellular and molecular determinants of successful activation of antigen-presenting cells, B cells and CD4⁺ T cells in early life, much less is known about the ontogeny of CD8⁺ T cell induction. In this review, we summarize the literature pertaining to the frequency and phenotype of newborn and infant CD8⁺ T cells, and any evidence of induction of CD8⁺ T cells by currently licensed pediatric vaccine formulations. In addition, we review the molecular determinants of antigen cross-presentation on MHC I and successful CD8⁺ T cell induction and discuss potential distinctions that can be made in children. Finally, we discuss recent advances in development of novel adjuvants and provide future directions for basic and translational research in this area.
... Previous studies have shown reduced expression of MYD88 protein in human neonatal peripheral blood leukocytes, and we hypothesized that altered MYD88 expression in neonatal mice may drive reduced cell shedding. 27 However, there were no significant differences in the levels of MYD88 protein in Figure 2C). To confirm our findings indicating there are no neonatal defects in cell shedding signaling pathways we also challenged mice IP with TNF-ɑ. ...
The early life gut microbiota plays a crucial role in regulating and maintaining the intestinal barrier, with disturbances in these communities linked to dysregulated renewal and replenishment of intestinal epithelial cells. Here we sought to determine pathological cell shedding outcomes throughout the postnatal developmental period, and which host and microbial factors mediate these responses. Surprisingly, neonatal mice (Day 14 and 21) were highly refractory to induction of cell shedding after intraperitoneal administration of liposaccharide (LPS), with Day 29 mice showing strong pathological responses, more similar to those observed in adult mice. These differential responses were not linked to defects in the cellular mechanisms and pathways known to regulate cell shedding responses. When we profiled microbiota and metabolites, we observed significant alterations. Neonatal mice had high relative abundances of Streptococcus, Escherichia, and Enterococcus and increased primary bile acids. In contrast, older mice were dominated by Candidatus Arthromitus, Alistipes, and Lachnoclostridium, and had increased concentrations of SCFAs and methyamines. Antibiotic treatment of neonates restored LPS‐induced small intestinal cell shedding, whereas adult fecal microbiota transplant alone had no effect. Our findings further support the importance of the early life window for microbiota‐epithelial interactions in the presence of inflammatory stimuli and highlights areas for further investigation.
... Several observations in human epidemiologic studies support the notion that trained immunity occurs in the human neonate. One observation that supports a role for trained immunity in early life is the association of bloodstream infections in critically ill preterm newborns with enhanced pathogen-specific mononuclear cell PRR expression in the setting of subsequent Gram-positive or Gram-negative bacteremia (Zhang et al., 2010). This finding suggests that the neonatal innate immune system can remember previous activation such that responses to subsequent microbial challenges are altered. ...
Vaccines have been traditionally developed with the presumption that they exert identical immunogenicity regardless of target population and that they provide protection solely against their target pathogen. However, it is increasingly appreciated that vaccines can have off-target effects and that vaccine immunogenicity can vary substantially with demographic factors such as age and sex. Bacille Calmette-Guérin (BCG), the live attenuated Mycobacterium bovis vaccine against tuberculosis (TB), represents a key example of these concepts. BCG vaccines are manufactured under different conditions across the globe generating divergent formulations. Epidemiologic studies have linked early life immunization with certain BCG formulations to an unanticipated reduction (∼50%) in all-cause mortality, especially in low birthweight males, greatly exceeding that attributable to TB prevention. This mortality benefit has been related to prevention of sepsis and respiratory infections suggesting that BCG induces “heterologous” protection against unrelated pathogens. Proposed mechanisms for heterologous protection include vaccine-induced immunometabolic shifts, epigenetic reprogramming of innate cell populations, and modulation of hematopoietic stem cell progenitors resulting in altered responses to subsequent stimuli, a phenomenon termed “trained immunity.” In addition to genetic differences, licensed BCG formulations differ markedly in content of viable mycobacteria key for innate immune activation, potentially contributing to differences in the ability of these diverse formulations to induce TB-specific and heterologous protection. BCG immunomodulatory properties have also sparked interest in its potential use to prevent or alleviate autoimmune and inflammatory diseases, including type 1 diabetes mellitus and multiple sclerosis. BCG can also serve as a model: nanoparticle vaccine formulations incorporating Toll-like receptor 8 agonists can mimic some of BCG’s innate immune activation, suggesting that aspects of BCG’s effects can be induced with non-replicating stimuli. Overall, BCG represents a paradigm for precision vaccinology, lessons from which will help inform next generation vaccines.
... Previous studies have shown reduced expression of MYD88 protein in human neonatal peripheral blood leukocytes, and we hypothesised that altered MYD88 expression in neonatal mice may drive reduced cell shedding [20]. However, there were no significant differences in the levels of MYD88 protein in homogenised spleen samples taken from 14 day old and adult mice ( Figure 2C). ...
The gut microbiota plays a crucial role in regulating and maintaining the epithelial barrier, particularly during early life. Notably, patients with chronic intestinal inflammation have a dysregulated process of renewal and replenishment of the intestinal epithelial cell (IEC) barrier, which is linked to disturbances in the gut microbiota. To date, there are no studies focussed on understanding the impact of inflammatory cell shedding events during the early life developmental window, and which host and microbial factors mediate these responses. Here we sought to determine pathological cell shedding outcomes throughout the postnatal developmental period (day 14, 21, 29 and week 8). Surprisingly neonatal mice (day 14 and 21) were highly refractory to induction of cell shedding after intraperitoneal administration of LPS, with day 29 mice showing strong pathological responses, more similar to those observed in adult mice. These differential responses were not linked to defects in the cellular mechanisms and pathways known to regulate cell shedding responses, although we did observe that neonatal mice had elevated anti-inflammatory (IL-10) responses. Notably, when we profiled microbiota and metabolites from these mice, we observed significant alterations. Neonatal mice had high relative abundances of Streptococcus, Escherichia and Enterococcus and increased primary bile acids. In contrast, older mice were dominated by Candidatus Arthromitus, Alistipes and Lachnoclostridium, and had increased concentrations of SCFAs and methyamines. Faecal microbiota transplant (FMT) and antibiotic studies confirmed the importance of early life gut microbiota in cell shedding responses. In these studies, neonates All rights reserved. No reuse allowed without permission. (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint. http://dx.doi.org/10.1101/789362 doi: bioRxiv preprint first posted online Oct. 1, 2019; 2 treated with antibiotics restored LPS-induced small intestinal cell shedding, whereas adult FMT alone had no effect. Our findings further support the importance of the early life window for microbiota-epithelial interactions in the presence of inflammatory stimuli and highlight areas for further investigation to probe underlying mechanisms to drive therapeutic development within the context of chronic inflammatory intestinal diseases.
... Significant progress has been made in determining differences between the neonatal and adult response to LPS challenge and TLR4 stimulation. While important differences between adults and neonates have been identified, most of the studies comparing early life TLR4 responsiveness have not evaluated for sex differences [92][93][94][95][96][97][98][99][100] . Furthermore, in the scant data that are available, reports are not consistent. ...
Being of the male sex has been identified as a risk factor for multiple morbidities associated with preterm birth, including bronchopulmonary dysplasia (BPD). Exposure to inflammatory stress is a well-recognized risk factor for developing BPD. Whether there is a sex difference in pulmonary innate immune TLR4 signaling, lung injury and subsequent abnormal lung development is unknown. Neonatal (P0) male and female mice (ICR) were exposed to systemic LPS (5 mg/kg, IP) and innate immune signaling, and the transcriptional response were assessed (1 and 5 hours), along with lung development (P7). Male and female mice demonstrated a similar degree of impaired lung development with decreased radial alveolar counts, increased surface area, increased airspace area and increased mean linear intercept. We found no differences between male and female mice in the baseline pulmonary expression of key components of TLR4-NFκB signaling, or in the LPS-induced pulmonary expression of key mediators of neonatal lung injury. Finally, we found no difference in the kinetics of LPS-induced pulmonary NFκB activation between male and female mice. Together, these data support the conclusion that the innate immune response to early postnatal LPS exposure and resulting pulmonary sequelae is similar in male and female mice.
... 18 Moreover, the peripheral blood monocytes can enhance the expression of TLRs in response to newborn sepsis, indicating that they are adequately expressed during infection. 19 Despite the normal expression levels of TLRs on newborn leukocytes, the cytokines produced on their activation differ drastically from those observed in adults. Intriguingly, TLR-mediated cytokine production is also distinct between preterm and term infants. ...
Necrotizing enterocolitis (NEC) remains a leading cause of preterm infant mortality. NEC is multifactorial and believed a consequence of intestinal immaturity, microbial dysbiosis, and an exuberant inflammatory response. Over the past decade, exaggerated Toll-like receptor 4 (TLR4) activity in the immature intestine of preterm neonates emerged as an inciting event preceding NEC. Increased TLR4 signaling in epithelial cells results in the initiation of an uncontrolled immune response and destruction of the mucosal barrier. This article discusses the state of the science of the molecular mechanisms involved in TLR4-mediated inflammation during NEC and the development of new therapeutic strategies to prevent NEC.
... The neonatal immune response to MRSA has not been elucidated so far [53]. Some data suggest that neonates are prone to developing hyperinflammation during staphylococcal infections [54,55]. Such imbalanced inflammation has been considered to be a key underlying mechanism of long-term neonatal morbidities, such as bronchopulmonary dysplasia, necrotizing enterocolitis, retinopathy of prematurity, and cerebral palsy [56]. ...
Methicillin-resistant Staphylococcus aureus (MRSA) is a ubiquitous human inhabitant and one of the important pathogens of neonatal infections. MRSA is associated with significant mortality and morbidity, especially in very immature preterm neonates. Moreover, MRSA may be implicated in adverse long-term neonatal outcomes, posing a substantial disease burden. Recent advances in molecular microbiology have shed light on the evolution of MRSA population structure and virulence factors, which may contribute to MRSA epidemic waves worldwide. Equipped with remarkable genetic flexibility, MRSA has successfully developed resistance to an extensive range of antibiotics including vancomycin, as well as antiseptics. In the face of these new challenges from MRSA, our armamentarium of anti-infective strategies is very limited and largely dependent on prevention measures. Active surveillance cultures followed by decolonization may be a promising approach to control MRSA infections, with its efficacy and safety in the specific population of neonates yet to be addressed by large multicenter studies.
... MyD88 is essential for the downstream signal of various TLRs. Children with deficiency of this signal molecules result in the recurrence of [55]. IL-1Rassociated kinase (IRAK)-4, a serine kinase with a N-terminal death domain, interacts with MyD88 to activate other IRAK family members such as IRAK-1 and IRAK-2 [56]. ...
The development of inflammation is mutually affected with damaged DNA and the abnormal expression of protein modification. Ubiquitination, a way of protein modification, plays a key role in regulating various biological functions including inflammation responses. The ubiquitin enzymes and deubiquitinating enzymes (DUBs) jointly control the ubiquitination. The fact that various ubiquitin linkage chains control the fate of the substrate suggests that the regulatory mechanisms of ubiquitin enzymes are central for ubiquitination. In inflammation diseases, the pro-inflammatory transcription factor NF-κB regulates transcription of pro-labour mediators in response to inflammatory stimuli and expression of numerous genes that control inflammation which is associated with ubiquitination. The ubiquitination regulates NF-κB signaling pathway with many receptor families, including NOD-like receptors (NLR), Toll-like receptors (TLR) and RIG-I-like receptors (RLR), mainly by K63-linked polyubiquitin chains. In this review, we highlight the study of ubiquitination in the inflammatory signaling pathway including NF-κB signaling regulated by ubiquitin enzymes and DUBs. Furthermore, it is emphasized that the interaction of ubiquitin-mediated inflammatory signaling system accurately regulates the inflammatory responses.
... 99 Moreover, TLR2 expression may be up-regulated in neonates with sepsis, supporting the hypothesis of dysregulated neonatal inflammatory response in favor of hyper-inflammation. 100,101 As for FPR2, we found no data concerning its expression in neonates. A study using a mouse model of neonatal sepsis indicated that at high bacterial concentrations, FPR2 and NOD2 may be more involved in the recognition of S. epidermidis than TLR2. ...
Staphylococcus epidermidis accounts for the majority of cases of neonatal sepsis. Moreover, it has been demonstrated to be associated with neonatal morbidities, such as bronchopulmonary dysplasia (BPD), white matter injury (WMI), necrotizing enterocolitis (NEC) and retinopathy of prematurity (ROP), which affect short-term and long-term neonatal outcome. Imbalanced inflammation has been considered to be a major underlying mechanism of each entity. Conventionally regarded as a harmless commensal on human skin, S. epidermidis has received less attention than its more virulent relative Staphylococcus aureus. Particularities of neonatal innate immunity and nosocomial environmental factors, however, may contribute to the emergence of S. epidermidis as a significant nosocomial pathogen. Neonatal host response to S. epidermidis sepsis has not been fully elucidated. Evidence is emerging regarding the implication of S. epidermidis sepsis in the pathogenesis of neonatal inflammatory diseases. This review focuses on the interplay among S. epidermidis, neonatal innate immunity and inflammation-driven organ injury.
... TLR2 (ENSP00000260010) is a member of the Toll-like receptor family, which mainly contributes to pathogen recognition and innate immune activation [104,105]. Recruiting MYD88 (ENSP00000379625), another inferred gene, TLR2 is mainly involved in the innate immunity against Gram positive bacteria [106,107]. According to recent publications, the initiation and progression of MD have been widely confirmed to be associated with the innate immune system and bacterial infection [22, 76,108]. ...
As a chronic illness derived from hair cells of the inner ear, Menière’s disease (MD) negatively influences the quality of life of individuals and leads to a number of symptoms, such as dizziness, temporary hearing loss, and tinnitus. The complete identification of novel genes related to MD would help elucidate its underlying pathological mechanisms and improve its diagnosis and treatment. In this study, a network-based method was developed to identify novel MD-related genes based on known MD-related genes. A human protein-protein interaction (PPI) network was constructed using the PPI information reported in the STRING database. A classic ranking algorithm, the random walk with restart (RWR) algorithm, was employed to search for novel genes using known genes as seed nodes. To make the identified genes more reliable, a series of screening tests, including a permutation test, an interaction test and an enrichment test, were designed to select essential genes from those obtained by the RWR algorithm. As a result, several inferred genes, such as CD4, NOTCH2 and IL6, were discovered. Finally, a detailed biological analysis was performed on fifteen of the important inferred genes, which indicated their strong associations with MD.
... Such a phenomenon is thought to have existed for millions of years as acquired systemic resistance in plants and also in invertebrates, neither of which have adaptive immune systems (17). Several studies have revealed enhancements of the neonatal innate immune response to Gram-positive and Gramnegative infections following previous encounters, mediated by different toll-like receptor pathways (20) or initiated by in utero inflammatory conditions such as histologic chorioamnionitis (21). Studies in adults show that trained innate cells, including monocytes and natural killer (NK) cells, appear to be epigenetically and metabolically reprogrammed to produce increased amounts of pro-inflammatory cytokines and display higher levels of surface activation markers in response to restimulation with toll-like receptor ligands or different whole microorganisms (22)(23)(24)(25). ...
Vaccination of infants with bacillus Calmette–Guérin (BCG) activates both the innate and adaptive arms of the immune response. The antimycobacterial effects of these responses most likely account for the ability of BCG to protect against childhood forms of tuberculosis (TB). There is also evidence for a heterologous protective effect of BCG vaccination against TB-unrelated mortality in low birth weight infants. A possible mechanism of action of this effect, the induction of trained innate immunity, has been demonstrated when cells from BCG-vaccinated adults are restimulated in vitro with non-related microbial stimuli. Our aim was to examine an extensive panel of secreted immune biomarkers to characterize the profile of trained innate immunity in infants. Stimulation of whole blood for 48 h was performed 4 months after BCG vaccination, or in control unvaccinated infants. Stimulants were lipopolysaccharide; Pam3Cys (P3C); heat-killed Candida albicans, Staphylococcus aureus, Escherichia coli, and a lysate of Mycobacterium tuberculosis. Culture supernatants were tested for secreted cytokines and chemokines by 42-plex bead array and monocytes and natural killer (NK) cells assessed for expression of activation markers by flow cytometry. BCG-vaccinated infants displayed increases in 11 cytokines and chemokines in response to different non-specific innate immunity stimuli: epidermal growth factor (EGF); eotaxin; IL-6; IL-7; IL-8; IL-10; IL-12p40; monocyte chemotactic protein-3; macrophage inflammatory protein-1α; soluble CD40 ligand and platelet-derived growth factor (PDGF)-AB/BB. Although each stimulant induced a distinct response profile, three analytes, EGF, IL-6, and PDGF-AB/BB, were commonly higher after stimulation with Pam3Cys, C. albicans, and S. aureus. Conversely, certain cytokines such as interferon gamma-inducible protein-10, IL-2, IL-13, IL-17, GM-CSF, and GRO were suppressed in BCG-vaccinated infants, while no increases in TNFα or IL-1β production were detected. We did not observe a concomitant, BCG-associated change in monocyte surface activation markers in response to non-specific stimuli, but we detected a significant increase in CD69 expression on NK cells in response to Pam3Cys. Pam3Cys-induced NK cell activation correlated with the magnitude of IL-12p40 and IL-10 responses to the same stimulant. This study reveals a novel cytokine/chemokine biomarker signature of BCG-induced trained innate immunity in infants and the involvement of NK cells in these responses.
... Many investigators have shown equal amounts of TLR expression in children and adults as well as an ability in children to upregulate TLRs in response to infection. [36][37][38][39] However, the downstream effects of TLR activation appear to differ by age (Table). 40 Possibly important for IAV infection are the observations that young children may produce large amounts of IL-6 21 and IL-10 18,40 in response to TLR stimulation and decreased amounts of IL-12, 21 type I IFNs, 41 and IFN-γ. ...
Infection with influenza A virus is responsible for considerable morbidity and mortality in children worldwide. While it is apparent that adequate activation of the innate immune system is essential for pathogen clearance and host survival, an excessive inflammatory response to infection is detrimental to the young host. A review of the literature indicates that innate immune responses change throughout childhood. Whether these changes are genetically programmed or triggered by environmental cues is unknown. The objectives of this review are to summarize the role of innate immunity in influenza A virus infection in the young child and to highlight possible differences between children and adults that may make children more susceptible to severe influenza A infection. A better understanding of age-related differences in innate immune signaling will be essential to improve care for this high-risk population.
... 163 Interestingly, term newborns suffering from bacterial sepsis appropriately up-regulate both TLR surface expression levels and intracellular signalling pathways. 164 ...
Fetal hematopoiesis is a highly regulated process in terms of time and location. It is characterized by the emergence of specific cell populations at different extra- and intra-embryonic anatomical sites. Trafficking of hematopoietic stem cells between these supportive niches is regulated by a set of molecules, i.e. integrins and chemokine receptors, which are also described for the recruitment of differentiated innate immune cells. In this review an overview will be given on fetal hematopoiesis as well as trafficking of hematopoietic stem cells during fetal life. In addition, we will focus on the appearance of the first differentiated myeloid cells in the fetal circulation and describe how they acquire the ability to roll, adhere and transmigrate into inflamed fetal tissue. Furthermore, we will discuss other effector functions of innate immune cells evolving during fetal ontogeny.
Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.
... response mediators, such as TLR2, AIM2, ATG5, and IRF7 in samples from Petrozavodsk was evident. Interestingly, TLR2 has been reported to be upregulated in mononuclear cells at birth upon infection with Gram-positive bacteria [44], and conversely downregulated in cord blood CD34+ progenitor cells of infants at high-atopic risk [45]. IRF7 on the other hand plays a central role in the viral defense and in the induction of the interferon response [46], and shows genetic association with susceptibility to type 1 diabetes [47]. ...
The prevalence of immune-mediated diseases, such as allergies and type 1 diabetes, is on the rise in the developed world. In order to explore differences in the gene expression patterns induced in utero in infants born in contrasting standards of living and hygiene, we collected umbilical cord blood RNA samples from infants born in Finland (modern society), Estonia (rapidly developing society) and the Republic of Karelia, Russia (poor economic conditions). The whole blood transcriptome of Finnish and Estonian neonates differed from their Karelian counterparts, suggesting exposure to toll-like receptor (TLR) ligands and a more matured immune response in infants born in Karelia. These results further support the concept of a conspicuous plasticity in the developing immune system: the environmental factors that play a role in the susceptibility/protection towards immune-mediated diseases begin to shape the neonatal immunity already in utero and direct the maturation in accordance with the surrounding microbial milieu.
... In contrast to older children, neonates and infants are more likely to develop lower respiratory tract pathology when exposed to viruses and bacteria (2). This can occur even though neonates have been shown to have near-normal Toll-like receptor (TLR) responses to bacterial and viral antigens (3)(4)(5). The adult response to ALI is characterized by an early, robust proinflammatory T helper (Th) 1 immune response (6), whereas the neonate has been reported to have a delayed inflammatory response, which can be manifested by slow clearance of airway antigens and prolonged disease symptomatology (2,7,8). ...
Neonates and infants have a higher morbidity and mortality associated with lower respiratory tract illnesses compared to older children. To identify age-related and longitudinal differences in the cellular immune response to acute lung injury (ALI) neonatal and juvenile mice were given E. coli lipopolysaccharide (LPS) using a novel minimally invasive aspiration technique. Methods: Neonatal and juvenile mice received between 3.75 to 7.5mg/kg LPS by intra-pharyngeal aspiration. Airway and lung cells were isolated and characterized by flow cytometry. Cytokine/chemokine mRNA expression from lung homogenates was quantified. Lung morphometry and injury scores were performed. LPS treated neonatal mice underwent adoptive transfer (AT) with adult T regulatory cells (Tregs). Results: Following LPS aspiration lung monocytes isolated from neonatal mice had a predominant M2 phenotype whereas lung monocytes from juvenile mice displayed a mixed M1/M2 phenotype. At 72 hours post LPS, neonatal lungs were slower to resolve inflammation and expressed lower mRNA levels of CCL2, CCL5, CXCL10, and IL10. Juvenile but not neonatal mice demonstrated a significant increase in airway Tregs post LPS. Adoptive transfer of adult Tregs into LPS-challenged neonatal mice resulted in reduced lung inflammation and improved weight gain. Conclusion: These findings underscore several vulnerabilities in the neonatal immune response to LPS-induced acute lung injury. Most striking was the deficiency in airway Treg cells post LPS aspiration. Adoptive transfer of adult Tregs mitigated LPS-induced ALI in neonatal mice highlighting the importance of age-related differences in Tregs and their response to acute lung injury during early postnatal development.
... IL-8 priming of neutrophils that occurs during labor significantly improves neutrophil chemotaxis over that seen with Caesarian delivery and even adult controls [34] . Zhang et al. [35] showed newborns amplify the TLR2-MyD88 pathway in Grampositive bacterial infection and the TLR4/MD2/MyD88 pathway in Gram-negative bacterial infection, suggesting infection-specific changes in innate immune signaling. Rather than demonstrate a tolerance phenomenon, these examples suggest the potential for enhanced innate immune function in human neonates following a stimulus. ...
The newborn and infant periods of early life are associated with heightened vulnerability to infection. Limited antigen exposure and distinct adaptive immune function compared to the adult places a greater burden on innate immunity for host defense to microbial challenge during this time. Trained immunity describes the phenomenon of augmented innate immune function following a stimulus that is not specific to the original stimulus. We review the concept of trained immunity in the context of the newborn's unique innate immune system function, the preclinical and clinical evidence that supports the tenet of innate immune memory in early life, and potential consequences of altered innate immune host responses. © 2013 S. Karger AG, Basel.
... Intraperitoneal administration of TLR agonists (TLRAs) to newborn mice enhance subsequent cytokine-and phagocyte-based responses to bacterial infection with associated improvement in survival from polymicrobial sepsis induced by intraperitoneal injection of a cecal slurry (33). Of note, bloodstream infection in critically ill preterm human newborns is associated with enhancement of pathogen-speci c mononuclear cell PRR expression in the setting of Gram-positive bacteremia (TLR2, MyD88) and Gram-negative bacteremia (TLR4, MD-2, and MyD88) (34). A novel newborn murine model of intravenous infection with S. epidermidis in the rst 24 h of life con rms selective TLR2 upregulation in liver mRNA (35). ...
Unique features of immunity early in life include a distinct immune system particularly reliant on innate immunity, with weak T helper (Th)1-polarizing immune responses, and impaired responses to certain vaccines leading to a heightened susceptibility to infection. To these important aspects, we now add an increasingly appreciated concept that the innate immune system displays epigenetic memory of an earlier infection or vaccination, a phenomenon that has been named "trained immunity." Exposure of neonatal leukocytes in vitro or neonatal animals or humans in vivo to specific innate immune stimuli results in an altered innate immune set point. Given the particular importance of innate immunity early in life, trained immunity to early life infection and/or immunization may play an important role in modulating both acute and chronic diseases.Pediatric Research (2013); doi:10.1038/pr.2013.214.
... On the other hand, the present results indicate that LPS stimulation of the human amniotic epithelium up-regulated the expression of MyD88, which has been reported also in human neonatal peripheral blood leukocytes infected with gram-negative bacteria 38 and in a rat model of intra-uterine inflammation. 39 In this study, we also demonstrated that following stimulation of TLR-4 with LPS and cotreatment with P4 there was a significant reduction in the expression of MyD88 at the protein level. ...
Infection of human fetal membranes elicits secretion of pro-inflammatory modulators through its innate immune capacities. We investigated the effect of lipopolysacharide (LPS) and progesterone (P4) upon expression of TLR-4/MyD88, TNFα, IL-6, IL-8, IL-10, and HBD2 on the human amniotic epithelium.
Explants of the human amniotic epithelium were pre-treated with 0.01, 0.1, and 1.0 μm of P4; then cotreated with 1000 ng/mL LPS. TLR-4 was immuno-detected, and concentrations of MyD88, TNFα, IL-6, IL-8, IL-10, and HBD2 were quantified by ELISA.
P4 significantly reduced the expression of LPS-induced TLR-4/MyD88. LPS increased the concentrations of TNFα, IL-6, IL-8, IL-10, and HBD2 by factors of 30-, eight, three, three, and fivefold, respectively. P4 at 1.0 μm was the most effective dose to blunt the secretion of TNFα, IL-6, and HBD-2. RU-486 blocks the effect of P4.
P4 inhibited LPS-induced TLR-4/MyD88 and pro-inflammatory factors in the human amniotic epithelium. These results could explain partially how P4 can protect the amniotic region of fetal membranes and generate a compensatory mechanism that limits the secretion of pro-inflammatory modulators, which could jeopardize the immune privilege during pregnancy.
... The expression of TLRs, as well as downstream signaling molecules, in mononuclear cells of healthy infants over the first 5 years of life appears to be stable and to occur at adult-like levels (Danis et al., 2008; Dasari et al., 2011; Reece et al., 2011; Tulic et al., 2011). Furthermore, human newborns with bacterial sepsis are able to appropriately upregulate TLR expression on peripheral blood mononuclear cells (Zhang et al., 2010). Thus, differential TLR expression appears unlikely to be a main cause for altered susceptibility to infection, inflammation, or vaccine response in early versus adult life. ...
Given the "inborn" nature of the innate immune system, it is surprising to find that innate immune function does in fact change with age. Similar patterns of distinct Toll-like-receptor-mediated immune responses come to light when one contrasts innate immune development at the beginning of life with that toward the end of life. Importantly, these developmental patterns of innate cytokine responses correlate with clinical patterns of susceptibility to disease: A heightened risk of suffering from excessive inflammation is often detected in prematurely born infants, disappears over the first few months of life, and reappears toward the end of life. In addition, risk periods for particular infections in early life reemerge in older adults. The near-mirror-image patterns that emerge in contrasts of early versus late innate immune ontogeny emphasize changes in host-environment interactions as the underlying molecular and teleologic drivers.
... SE-injected mice also demonstrated selective increases in liver mRNA encoding TLR2, a key innate immune receptor that mediates recognition and clearance of multiple Gram-positive organisms including SE [20,48,49,50,51], and of the TLR adaptor molecule, MyD88 [52]. Interestingly, transcription of TLR2 and MyD88 are both coordinately up-regulated during human neonatal Grampositive infection in vivo [53,54], highlighting how our murine model recapitulates the human neonatal response to Neonatal Mouse Model of IV SE Infection positive bacterial infection. SE also induced inoculum-dependent impairment in weight gain, a sensitive marker of neonatal wellbeing known to be negatively affected by infection in both mice and human newborns [39,40]. ...
Staphylococcus epidermidis (SE) causes late onset sepsis and significant morbidity in catheterized preterm newborns. Animal models of SE infection are useful in characterizing disease mechanisms and are an important approach to developing improved diagnostics and therapeutics. Current murine models of neonatal bacterial infection employ intraperitoneal or subcutaneous routes at several days of age, and may, therefore, not accurately reflect distinct features of innate immune responses to bacteremia. In this study we developed, validated, and characterized a murine model of intravenous (IV) infection in neonatal mice <24 hours (h) old to describe the early innate immune response to SE. C57BL/6 mice <24 h old were injected IV with 10(6), 10(7), 10(8) colony-forming units (CFU) of SE 1457, a clinical isolate from a central catheter infection. A prospective injection scoring system was developed and validated, with only high quality injections analyzed. Newborn mice were euthanized between 2 and 48 h post-injection and spleen, liver, and blood collected to assess bacterial viability, gene expression, and cytokine production. High quality IV injections demonstrated inoculum-dependent infection of spleen, liver and blood. Within 2 h of injection, SE induced selective transcription of TLR2 and MyD88 in the liver, and increased systemic production of plasma IL-6 and TNF-α. Despite clearance of bacteremia and solid organ infection within 48 h, inoculum-dependent impairment in weight gain was noted. We conclude that a model of IV SE infection in neonatal mice <24 h old is feasible, demonstrating inoculum-dependent infection of solid organs and a pattern of bacteremia, rapid and selective innate immune activation, and impairment of weight gain typical of infected human neonates. This novel model can now be used to characterize immune ontogeny, evaluate infection biomarkers, and assess preventative and therapeutic modalities.
... TLR2 forms heterodimers with TLR1 and TLR6, and these receptor complexes recognize molecules expressed on Gram-positive bacteria, such as peptidoglycan, lipopeptides, and lipoproteins, and they also mediate recognition of whole bacteria such as Staphylococcus epidermidis [10,11,12]. Of note, TLR2 is selectively up-regulated in the peripheral blood mononuclear cells of human newborns infected with Gram-positive bacteria [13]. With respect to the CNS, a role for TLR2 signaling in adult mouse brain injury has been suggested, as summarized by a most recent review [14] but there are few reports that define the role of TLR2 signaling in neonatal brain injury. ...
Inflammation is associated with perinatal brain injury but the underlying mechanisms are not completely characterized. Stimulation of Toll-like receptors (TLRs) through specific agonists induces inflammatory responses that trigger both innate and adaptive immune responses. The impact of engagement of TLR2 signaling pathways on the neonatal brain is still unclear. The aim of this study was to investigate the potential effect of a TLR2 agonist on neonatal brain development.
Mice were injected intraperitoneally (i.p.) once a day from postnatal day (PND) 3 to PND11 with endotoxin-free saline, a TLR2 agonist Pam(3)CSK(4) (5 mg/kg) or Lipopolysaccharide (LPS, 0.3 mg/kg). Pups were sacrificed at PND12 or PND53 and brain, spleen and liver were collected and weighed. Brain sections were stained for brain injury markers. Long-term effects on memory function were assessed using the Trace Fear Conditioning test at PND50. After 9 days of Pam(3)CSK(4) administration, we found a decreased volume of cerebral gray matter, white matter in the forebrain and cerebellar molecular layer that was accompanied by an increase in spleen and liver weight at PND12. Such effects were not observed in Pam3CSK4-treated TLR 2-deficient mice. Pam3CSK4-treated mice also displayed decreased hippocampus neuronal density, and increased cerebral microglia density, while there was no effect on caspase-3 or general cell proliferation at PND12. Significantly elevated levels of IL-1β, IL-6, KC, and MCP-1 were detected after the first Pam3CSK4 injection in brain homogenates of PND3 mice. Pam(3)CSK(4) administration did not affect long-term memory function nor the volume of gray or white matter.
Repeated systemic exposure to the TLR2 agonist Pam(3)CSK(4) can have a short-term negative impact on the neonatal mouse brain.
Sepsis, a dysregulated host immune response to infection, is one of the leading causes of neonatal mortality worldwide. Improved understanding of the perinatal immune system is critical to improve therapies to both term and preterm neonates at increased risk of sepsis. Our narrative outlines the known and unknown aspects of the human immune system through both the immune tolerant in utero period and the rapidly changing antigen-rich period after birth. We will highlight the key differences in innate and adaptive immunity noted through these developmental stages, and how the unique immune phenotype in early life contributes to the elevated risk of overwhelming infection and dysregulated immune responses to infection upon exposure to external antigens shortly after birth. Given an initial dependence on neonatal innate immune host responses, we will discuss the concept of innate immune memory, or "trained immunity", and describe several potential immune modulators which show promise in altering the dysregulated immune response in newborns and improving resilience to sepsis.
Background:
Late-onset neonatal sepsis (LONS) detection is problematic as no single examinations (blood culture, c-reactive protein (CRP), procalcitonin (PCT)) are reliable. Toll-like receptors (TLRs), which detect the presence of pathogen-associated molecular patterns is a promising novel biomarker, but less studied in LONS. This study aimed to determine neutrophils and monocytes TLR2 and TLR4 expression in LONS and their diagnostic value.
Methods:
A cross-sectional study conducted in May and June 2017 involving 52 neonates with clinical late-onset (>72 hours of age) sepsis. We examine complete blood count, I/T ratio, CRP, PCT, as well as TLR2 and TLR4 expression to compared with blood culture as the gold standard. We classified cases into proven or unproven sepsis.
Result:
The incidence of LONS was 32.6% in the subjects. The expression of TLR2 was low in LONS, while TLR4 was high. TLR4 neutrophil expression has 88.2% sensitivity, 20% specificity, 34.9% positive predictive value (PPV), 77.8% negative predictive value (NPV), and an AUC of 0.541. TLR4 monocyte expression has 92.1% sensitivity, 11.4% specificity, 34% PPV, 80% NPV, and an AUC of 0.528. The AUC of CRP is increased from 0.608 to 0.843 after combination with TLR4, comparable with CRP + PCT (AUC 0.829).
Conclusion:
The increase in TLR4 expression has good sensitivity but low specificity. TLR4 expression, in combination with CRP, could become a reliable biomarker for the diagnosis of LONS.
Objectives
Conventionally regarded as a harmless skin commensal, Staphylococcus epidermidis accounts for the majority of neonatal late-onset sepsis and is shown to be associated with neonatal inflammatory morbidities, especially bronchopulmonary dysplasia. This study addressed the pro-inflammatory capacity of different S. epidermidis strains on human alveolar epithelial cells.
Methods
A549 cell monolayers were stimulated by live bacteria of S. epidermidis RP62A strain (biofilm-positive) and ATCC 12228 strain (biofilm-negative) at a multiplicity of infection ratio of 10 for 24 h. LPS (100 ng/ml) and Pam3CSK4 (1 µg/ml) were used for comparisons. Cell viability was measured by MTT method. The mRNA and protein expression of inflammatory mediators and toll-like receptor (TLR)-2 were assessed using RT-PCR, immunoassays and immunofluorescence.
Results
Both S. epidermidis strains induced expression of tumor necrosis factor (TNF)-α, IL-1β, interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP)-1, interferon γ-induced protein 10 (IP-10) and intercellular adhesion molecule (ICAM)-1, but not IL-10. The stimulatory effect of RP62A exceeded that of LPS (p < 0.05). RP62A strain showed a trend towards higher induction of pro-inflammatory mediators than ATCC 12228 strain. The co-stimulation with RP62A strain decreased cell viability compared to control and TLR agonists (p < 0.05). RP62A but not ATCC 12228 stimulated mRNA and protein expression of TLR2.
Conclusions
S. epidermidis drives pro-inflammatory responses in lung epithelial cells in vitro. The pro-inflammatory capacity of S. epidermidis may differ between strains. Biofilm-positive S. epidermidis strain seems to induce more potent pulmonary pro-inflammation than biofilm-negative S. epidermidis strain.
Epilepsy is a group of neurological disorders characterized by epileptic seizures. In this study, we aim to explore the role of microRNA-421 (miR-421) in hippocampal neurons of epilepsy mice via the TLR/MYD88 pathway. Forty mice were randomly served as the normal and model (established as epilepsy model) groups. Hippocampal neurons were assigned into seven groups with different transfections. The RT-qPCR and western blotting were conducted to examine the expression of miR-421 TLR2, TLR4, MYD88, Bax, Bcl-2, p53, Beclin-1 and LC3II/LC3I. Cell proliferation and apoptosis were detected by MTT and flow cytometry.MYD88 is a target gene of miR-421. Model mice showed elevated expression of TLR2, TLR4, MYD88, Bax, p53, Beclin-1 and LC3II/LC3Ibut reduced expression of miR-421 and Bcl-2. In vitro experiments reveals that overexpression of miR-421 inhibited the TLR/MYD88 pathway. Besides, overexpressed miR-421 increased cell apoptosis but declined cell proliferation. It reveals that miR-421 targeting MYD88 could inhibit the apoptosis and autophagy of hippocampal neurons in epilepsy mice by down-regulating the TLR/MYD88 pathway. This article is protected by copyright. All rights reserved
Immature immunity may predispose preterm neonates to infections and necrotizing enterocolitis (NEC). Intravenous antibiotics are frequently given to prevent and treat sepsis while oral antibiotics are seldom used. We hypothesized that oral antibiotics promote maturation of systemic immunity and delay gut bacterial colonization and thereby protect preterm neonates against both NEC and bacteremia in the immediate postnatal period. Preterm pigs were given formula and administered saline (CON) or broad-spectrum antibiotics orally (ORA) or systemically (SYS) for 5 days after birth. Temporal changes in blood parameters and bacterial composition in the intestine, blood and immune organs were analyzed. Newborn preterm pigs had few blood neutrophils and a high frequency of progenitor cells. Neutrophils gradually matured after preterm birth with increasing CD14 and decreasing CD172a expressions. Preterm neutrophil and monocyte TLR2 expression and TLR2-mediated blood cytokine responses were low, relative to adults. ORA pigs showed enhanced blood neutrophil maturation with reduced cell size and CD172a expression. Only ORA, but not SYS pigs, were protected from a high density of gut Gram-positive bacteria, high gut permeability, Gram-positive bacteremia, and NEC. Neonatal oral antibiotics may benefit mucosal and systemic immunity via delayed gut colonization and enhanced blood neutrophil maturation just after preterm birth.
Toll-like receptors are vital transmembrane receptors that initiate the innate immune response to many micro-organisms. The discovery of these receptors has improved our understanding of host–pathogen interactions, and these receptors play an important role in the pathogenesis of multiple neonatal conditions such as sepsis and brain injury. Toll-like receptors, especially TLRs 2 and 4, are associated with necrotizing enterocolitis, periventricular leukomalacia and sepsis. Conclusion
Toll-like receptor modulation may potentially be used as immunomodulators in the management of neonatal sepsis.
Altered neonatal Toll-like receptor (TLR) function is hypothesized to contribute to the heightened susceptibility to infection and perpetuated inflammation in term and preterm neonates, clinically evident in neonatal sepsis and increased rates of inflammatory disorders. Current data indicate that basal TLR expression in term neonates equals adult expression patterns, while expression in preterm infants seems to increase, depending on gestational age. Regarding TLR signaling, some studies suggest TLR incompetence in neonates associated with impaired pro-inflammatory responses, others describe neonatal TLR function well developed and allude to its hyper-inflammation tendency. We discuss the competing positions and considerable limitations of research approaches and conclude that neonatal innate immunity is not generally less able to respond to TLR stimulation. Moreover, we describe pre-conditioning factors other than immaturity having a comparable impact. In the long term, better understanding of the complex interplay of pre- and postnatal conditions and maturation-dependent neonatal TLR function may provide new therapeutic approaches.
Subunit vaccine formulations often include adjuvants that primarily stimulate innate immune cells. While young infants represent the major target population for vaccination, effective immunization in this age group remains a challenge. Many parameters of innate immune responses differ quantitatively and qualitatively from newborns to infants and adults, revealing a highly regulated developmental program. Herein, we discuss the potential implications of innate immune ontogeny for the activity of adjuvants contained in licensed infant vaccines, as well as future directions for rational design of adjuvanted vaccines for this age group.
Neonates, particularly those born prematurely, are among the most vulnerable age group for morbidity and mortality due to infections. Immaturity of the innate immune system and a high need for invasive medical procedures in the context of a preterm birth make these infants highly susceptible to common neonatal pathogens. Preterm infants who survive may also suffer permanent disabilities due to organ damage resulting from either the infection itself or from the inflammatory response generated under an oxidative stress. Infections in preterm infants continue to pose important healthcare challenges. Yet, developmental maturation events in the innate immune system that underlie their excessively high vulnerability to infection remain largely understudied. In this review article, we identify pertinent knowledge gaps that must be filled in order to orient future translational research.
The innate immune system plays a critical role in protecting neonates against infections early in life and Toll-like receptors (TLRs) are key components of innate immune recognition of pathogens. This study examined the effects of age and stimulation with a TLR 7/8 agonist (R848) on TLR8 mRNA expression by foal neutrophils during the first month of life. We also examined the effects of R848 stimulation on mRNA expression of interleukin (IL)-6 and IL-8 at 1 and 14 days of life. We observed that TLR8 mRNA was constitutively expressed (P<0.05) at all ages examined, and its expression did not change upon stimulation with R848. Stimulation with R848 resulted in significantly increased (P<0.05) expression of IL-6 and IL-8 mRNA at both 1 and 14 days of life. Our results demonstrate that foal neutrophils express constitutive levels of TLR8 mRNA. The increased expression of IL-6 and IL-8, both downstream products of the TLR 7/8 signaling pathway, following stimulation with R848 suggests that additional experiments to confirm the signaling pathway by which R848 stimulates foal neutrophils, and to investigate its ability to stimulate functional responses of foal neutrophils, are merited.
Prevention of neonatal infection-related mortality represents a significant global challenge particularly in the vulnerable premature population. The increased risk of death from sepsis is likely due to the specific immune deficits found in the neonate as compared to the adult. Stimulation of the neonatal immune system to prevent and/or treat infection has been attempted in the past largely without success. In this review, we identify some of the known deficits in the neonatal immune system and their clinical impact, summarize previous attempts at immunomodulation and the outcomes of these interventions, and discuss the potential of novel immunomodulatory therapies to improve neonatal sepsis outcome.
Hospital-acquired infections (HAIs) represent an important cause of morbidity and mortality in neonatal intensive care units (NICUs).
All neonates admitted for > 48 hours between January 2003 and December 2006 in the NICU of the teaching hospital Umberto I of Rome, Italy were considered.
Of the 575 neonates evaluated, 76 (13.2%) developed a total of 100 HAIs, including 36 bloodstream infections (BSIs), 33 pneumonias, 19 urinary tract infections, 8 conjunctivitis, and 4 onphalitis. There were 7.8 HAIs/1000 patient-days and 12.5 BSIs/1000 days of umbilical catheterization. Logistic analysis identified an association with mechanical ventilation (odds ratio [OR] = 3.05; 95% confidence interval [CI] = 1.75 to 5.31; P < .01) and birth weight <or= 1500 g (OR = 2.34; 95% CI = 1.36 to 4.03; P < .01). Thirty-five neonates (6.1%) died. Klebsiella pneumoniae (37.7%) and coagulase-negative staphylococci (28.6%) were the most frequently isolated microorganisms. Only 3 Candida spp determined BSIs (8.3%). BSI mortality was higher in infections with gram-negative pathogens (36.4%) than in infections with gram-positive pathogens (4.5%).
Although we found a low infection and mortality rate, attention should be directed toward antibiotic-resistant gram-negative pathogens.
MyD88 is a key downstream adapter for most Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). MyD88 deficiency
in mice leads to susceptibility to a broad range of pathogens in experimental settings of infection. We describe a distinct
situation in a natural setting of human infection. Nine children with autosomal recessive MyD88 deficiency suffered from life-threatening,
often recurrent pyogenic bacterial infections, including invasive pneumococcal disease. However, these patients were otherwise
healthy, with normal resistance to other microbes. Their clinical status improved with age, but not due to any cellular leakiness
in MyD88 deficiency. The MyD88-dependent TLRs and IL-1Rs are therefore essential for protective immunity to a small number
of pyogenic bacteria, but redundant for host defense to most natural infections.
Previous research in our laboratory has shown that polymorphonuclear leukocytes (PMN) from neonates are not primed effectively in vitro with lipopolysaccharide (LPS) (from Escherichia coli 0111:B4) compared with priming of adult PMN. This finding led us to speculate that differences between neonatal and adult LPS receptors may account for the lower response by neonatal PMN to LPS. In these experiments, we investigated if CD14 or other LPS receptors contribute to the priming activity of PMN by LPS. We found that unprimed neonatal and adult PMN expressed similar numbers of CD14 (11.6 +/- 9.2 versus 18.6 +/- 2.7 fluorescence units [FlU]; P > 0.05) and LPS-binding sites (2.94 +/- 1.4 versus 4.94 +/- 0.79 FlU; P > 0.05). Monoclonal antibody against CD14 (MY4) did not significantly change the binding of LPS to adult unprimed PMN, suggesting that LPS receptors other than CD14 receptors are predominant on PMN. However, when PMN were pretreated with LPS (10 ng/ml) for 45 min at 37 degrees C, expression of CD14 on adult PMN increased to 33.8 +/- 4.9 FlU (P < 0.05 versus unprimed adult PMN) while that on neonatal PMN showed little change, increasing to 17.2 +/- 10.3 FlU (P > 0.05 versus unprimed neonatal PMN; P < 0.05 versus primed adult PMN). Furthermore, MY4 specifically blocked oxidative-radical production from PMN primed with LPS (10 ng/ml) compared with that from control PMN (P < 0.01). These studies suggest that LPS primes PMN by activating CD14 expression. We conclude that lower expression of CD14 or failure to up-regulate CD14 after LPS pretreatment contributes to the inability of neonatal PMN to be primed by LPS.
Mutations of the gene Lps selectively impede lipopolysaccharide (LPS) signal transduction in C3H/HeJ and C57BL/10ScCr mice, rendering them resistant
to endotoxin yet highly susceptible to Gram-negative infection. The codominantLps
d allele of C3H/HeJ mice was shown to correspond to a missense mutation in the third exon of the Toll-like receptor-4 gene
(Tlr4), predicted to replace proline with histidine at position 712 of the polypeptide chain. C57BL/10ScCr mice are homozygous
for a null mutation of Tlr4. Thus, the mammalian Tlr4 protein has been adapted primarily to subserve the recognition of LPS and presumably transduces
the LPS signal across the plasma membrane. Destructive mutations of Tlr4 predispose to the development of Gram-negative sepsis, leaving most aspects of immune function intact.
The recognition of mycobacterial cell wall components causes macrophages to secrete tumor necrosis factor alpha (TNF-alpha) and other cytokines that are essential for the development of a protective inflammatory response. We show that toll-like receptors are required for the induction of TNF-alpha in macrophages by Mycobacterium tuberculosis. Expression of a dominant negative form of MyD88 (a signaling component required for toll-like receptor signaling) in a mouse macrophage cell line blocks TNF-alpha production induced by M. tuberculosis. We identify toll-like receptor-2 (TLR2) as the specific toll-like receptor required for this induction by showing that expression of an inhibitory TLR2 (TLR2-P681H) blocks TNF-alpha production induced by whole M. tuberculosis. Further, we show that TLR2-dependent signaling mediates responses to mycobacterial cell wall fractions enriched for lipoarrabinomannan, mycolylarabinogalactan-peptidoglycan complex, or M. tuberculosis total lipids. Thus, although many mycobacterial cell wall fractions are identified to be inflammatory, all require TLR2 for induction of TNF-alpha in macrophages. These data suggest that TLR2 is essential for the induction of a protective immune response to mycobacteria.
The human MD-2 molecule is associated with the extracellular domain of human Toll-like receptor 4 (TLR4) and greatly enhances its LPS signaling. The human TLR4-MD-2 complex thus signals the presence of LPS. Little is known, however, about cell surface expression and LPS signaling of the TLR4-MD-2 complex in vivo. We cloned mouse MD-2 molecularly and established a unique mAb MTS510, which reacted selectively with mouse TLR4-MD-2 but not with TLR4 alone in flow cytometry. Mouse MD-2 expression in TLR4-expressing cells enhanced LPS-induced NF-kappaB activation, which was clearly inhibited by MTS510. Thioglycolate-elicited peritoneal macrophages expressed TLR4-MD-2, which was rapidly down-regulated in the presence of LPS. Moreover, LPS-induced TNF-alpha production by peritoneal macrophages was inhibited by MTS510. Collectively, the TLR4-MD-2 complex is expressed on macrophages in vivo and senses and signals the presence of LPS.
Stimulation of Toll-like receptors (TLRs) triggers activation of a common MyD88-dependent signaling pathway as well as a MyD88-independent
pathway that is unique to TLR3 and TLR4 signaling pathways leading to interferon (IFN)-β production. Here we disrupted the
gene encoding a Toll/IL-1 receptor (TIR) domain-containing adaptor, TRIF. TRIF-deficient mice were defective in both TLR3-
and TLR4-mediated expression of IFN-β and activation of IRF-3. Furthermore, inflammatory cytokine production in response to
the TLR4 ligand, but not to other TLR ligands, was severely impaired in TRIF-deficient macrophages. Mice deficient in both
MyD88 and TRIF showed complete loss of nuclear factor kappa B activation in response to TLR4 stimulation. These findings demonstrate
that TRIF is essential for TLR3- and TLR4-mediated signaling pathways facilitating mammalian antiviral host defense.
Toll-like receptors (TLRs) are innate recognition molecules for microbial products, but their direct interactions with corresponding ligands remain unclarified. LPS, a membrane constituent of gram-negative bacteria, is the best-studied TLR ligand and is recognized by TLR4 and MD-2, a molecule associated with the extracellular domain of TLR4. Although TLR4-MD-2 recognizes LPS, little is known about the physical interaction between LPS and TLR4-MD-2. Here, we demonstrate cell surface LPS-TLR4-MD-2 complexes. CD14 greatly enhances the formation of LPS-TLR4-MD-2 complexes, but is not coprecipitated with LPS-TLR4-MD-2 complexes, suggesting a role for CD14 in LPS loading onto TLR4-MD-2 but not in the interaction itself between LPS and TLR4-MD-2. A tentative dissociation constant (Kd) for LPS-TLR4-MD-2 complexes was approximately 3 nM, which is approximately 10-20 times lower than the reported Kd for LPS-MD-2 or LPS-CD14. The presence of detergent disrupts LPS interaction with CD14 but not with TLR4-MD-2. E5531, a lipid A antagonist developed for therapeutic intervention of endotoxin shock, blocks LPS interaction with TLR4-MD-2 at a concentration 100 times lower than that required for blocking LPS interaction with CD14. These results reveal direct LPS interaction with cell surface TLR4-MD-2 that is distinct from that with MD-2 or CD14.
Newborns are at increased risk of overwhelming infection, yet the mechanisms underlying this susceptibility are incompletely defined. In this study we report a striking 1- to 3-log decrease in sensitivity of monocytes in human neonatal cord blood, compared with monocytes in adult peripheral blood, to the TNF-alpha-inducing effect of multiple TLR ligands, including bacterial lipopeptides (BLPs), LPS, and the imidazoquinoline compound, imiquimod. In marked contrast, TNF-alpha release in response to R-848, a TLR ligand that is a congener of imiquimod, was equivalent in newborn and adult blood. Differences in ligand-induced TNF-alpha release correlated with divergent ligand-induced changes in monocyte TNF-alpha mRNA levels. Newborn and adult monocytes did not differ in basal mRNA or protein expression of TLRs or mRNA expression of functionally related molecules. Newborn monocytes demonstrated diminished LPS-induced, but equivalent R-848-induced, phosphorylation of p38 mitogen-activated protein kinase and altered BLP- and LPS-induced acute modulation of cognate receptors, suggesting that the mechanism accounting for the observed differences may be localized proximal to ligand recognition by surface TLRs. Remarkably, newborn plasma conferred substantially reduced BLP-, LPS-, and imiquimod-induced TNF-alpha release on adult monocytes without any effect on R-848-induced TNF-alpha release, reflecting differences in a plasma factor(s) distinct from soluble CD14. Impaired response to multiple TLR ligands may significantly contribute to immature neonatal immunity. Conversely, relative preservation of responses to R-848 may present unique opportunities for augmenting innate and acquired immunity in the human newborn.
The impaired infection control related to the functional immaturity of the neonatal immune system is an important cause of infection in preterm newborns. We previously reported that constitutive Toll-like receptor (TLR) 4 expression and cytokine secretion on lipopolysaccharide (LPS) stimulation increases with gestational age. Here, we analyzed constitutive monocyte TLR2 expression and evaluated the expression profiles of the proximal downstream adapter molecule myeloid differentiation factor 88 (MyD88). We further investigated activation of protein kinases p38 and extracellular regulated kinsase (ERK) 1/2 in CD14 monocytes after ex vivo stimulation with bacterial TLR ligands (LPS and lipoteichoic acid [LTA]). The functional outcome of the stimulation was determined by cytokine secretion. Monocytes from 31 preterm newborns (<30 weeks of gestation, n=16; 30-37 weeks of gestation, n=15), 10 term newborns, and 12 adults were investigated. In contrast to TLR4 expression, TLR2 levels did not differ between age groups. However, MyD88 levels were significantly lower in preterm newborns. Activation of p38 and ERK1/2 was impaired in all newborn age groups after stimulation with TLR-specific ligands. Accordingly, after LTA stimulation, the levels of interleukin (IL)-1 beta , IL-6, and IL-8 cytokine production were substantially lower (P<.001) in preterm newborns than in adults. The reduced functional response to bacterial cell wall components appears to be part of the functional immaturity of the neonatal immune system and might predispose premature newborns to bacterial infection.
The fetus and newborn face a complex set of immunological demands, including protection against infection, avoidance of harmful inflammatory immune responses that can lead to pre-term delivery, and balancing the transition from a sterile intra-uterine environment to a world that is rich in foreign antigens. These demands shape a distinct neonatal innate immune system that is biased against the production of pro-inflammatory cytokines. This bias renders newborns at risk of infection and impairs responses to many vaccines. This Review describes innate immunity in newborns and discusses how this knowledge might be used to prevent and treat infection in this vulnerable population.
Human interleukin (IL) 1 receptor-associated kinase 4 (IRAK-4) deficiency is a recently discovered primary immunodeficiency that impairs Toll/IL-1R immunity, except for the Toll-like receptor (TLR) 3- and TLR4-interferon (IFN)-alpha/beta pathways. The clinical and immunological phenotype remains largely unknown. We diagnosed up to 28 patients with IRAK-4 deficiency, tested blood TLR responses for individual leukocyte subsets, and TLR responses for multiple cytokines. The patients' peripheral blood mononuclear cells (PBMCs) did not induce the 11 non-IFN cytokines tested upon activation with TLR agonists other than the nonspecific TLR3 agonist poly(I:C). The patients' individual cell subsets from both myeloid (granulocytes, monocytes, monocyte-derived dendritic cells [MDDCs], myeloid DCs [MDCs], and plasmacytoid DCs) and lymphoid (B, T, and NK cells) lineages did not respond to the TLR agonists that stimulated control cells, with the exception of residual responses to poly(I:C) and lipopolysaccharide in MDCs and MDDCs. Most patients (22 out of 28; 79%) suffered from invasive pneumococcal disease, which was often recurrent (13 out of 22; 59%). Other infections were rare, with the exception of severe staphylococcal disease (9 out of 28; 32%). Almost half of the patients died (12 out of 28; 43%). No death and no invasive infection occurred in patients older than 8 and 14 yr, respectively. The IRAK-4-dependent TLRs and IL-1Rs are therefore vital for childhood immunity to pyogenic bacteria, particularly Streptococcus pneumoniae. Conversely, IRAK-4-dependent human TLRs appear to play a redundant role in protective immunity to most infections, at most limited to childhood immunity to some pyogenic bacteria.
Human newborns are more susceptible than adults to infection by gram-negative bacteria. We hypothesized that this susceptibility may be associated with a decreased response by leukocytes to lipopolysaccharide (LPS). In this study, we compared LPS-induced secretion of tumor necrosis factor alpha (TNF-alpha) by mononuclear cells (MNC) from adult peripheral blood and newborn umbilical cord blood in vitro and attempted to determine the mechanisms involved in its regulation. At a high concentration of LPS (10 ng/ml) and in the presence of autologous plasma, MNC from adults and newborns secreted similar amounts of TNF-alpha. However, in the absence of plasma, MNC from newborns secreted significantly less TNF-alpha compared to MNC from adults. Moreover, at a low concentration of LPS (0.1 ng/ml) and in the presence of plasma, TNF-alpha secretion was significantly lower for newborn MNC compared to adult MNC. Adults and newborns had similar numbers of CD14 and Toll-like receptor 4 (TLR-4)-positive cells as measured by flow cytometry. However, the intensity of the CD14 marker was greater for adult than for newborn cells. Incubation of cells with LPS led to an increase in CD14 and TLR-4 intensity for adult cells but not for newborn cells. The effect of LPS stimulation of adult or newborn cells was similar for ERK, p38, and IkappaBalpha phosphorylation, as well as IkappaBalpha degradation. Finally, we assessed levels of the TLR-4 adapter protein, the myeloid differentiation antigen 88 (MyD88). We found a direct relation between adult and newborn TNF-alpha secretion and MyD88, which was significantly decreased in newborn monocytes. Since TLR-4 signals intracellularly through the adapter protein, MyD88, we hypothesize that MyD88-dependent factors are responsible for delayed and decreased TNF-alpha secretion in newborn monocytes.
Host proinflammatory responses to minute amounts of endotoxins derived from many Gram-negative bacteria require the interaction of lipopolysaccharide-binding protein (LBP), CD14, Toll-like receptor 4 (TLR4) and MD-2. Optimal sensitivity to endotoxin requires an ordered series of endotoxin-protein and protein-protein interactions. At substoichiometric concentrations, LBP facilitates delivery of endotoxin aggregates to soluble CD14 (sCD14) to form monomeric endotoxin-sCD14 complexes. Subsequent interactions of endotoxin-sCD14 with TLR4 and/or MD-2 have not been specifically defined. This study reports the purification of a stable, monomeric, bioactive endotoxin-MD-2 complex generated by treatment of endotoxin-sCD14 with recombinant MD-2. Efficient generation of this complex occurred at picomolar concentrations of endotoxin and nanogram per milliliter doses of MD-2 and required presentation of endotoxin to MD-2 as a monomeric endotoxin-CD14 complex. TLR4-dependent delivery of endotoxin to human embryonic kidney (HEK) cells and cell activation at picomolar concentrations of endotoxin occurred with the purified endotoxin-MD-2 complex, but not with purified endotoxin aggregates with or without LBP and/or sCD14. The presence of excess MD-2 inhibited delivery of endotoxin-MD-2 to HEK/TLR4 cells and cell activation. These findings demonstrate that TLR4-dependent activation of host cells by picomolar concentrations of endotoxin occurs by sequential interaction and transfer of endotoxin to LBP, CD14, and MD-2 and simultaneous engagement of endotoxin and TLR4 by MD-2.
Neonates exhibit an increased risk of sepsis mortality compared with adults. We show that in contrast to adults, survival from polymicrobial sepsis in murine neonates does not depend on an intact adaptive immune system and is not improved by T cell-directed adaptive immunotherapy. Furthermore, neonates manifest an attenuated inflammatory and innate response to sepsis, and have functional defects in their peritoneal CD11b(+) cells. Activation of innate immunity with either a Toll-like receptor 4 (TLR4) or TLR7/8 agonist, but not a TLR3 agonist, increased the magnitude, but abbreviated the early systemic inflammatory response, reduced bacteremia, and improved survival to polymicrobial sepsis. TLR4 agonist pretreatment enhanced peritoneal neutrophil recruitment with increased oxidative burst production, whereas the TLR7/8 agonist also enhanced peritoneal neutrophil recruitment with increased phagocytic ability. These benefits were independent of the adaptive immune system and type I interferon signaling. Improving innate immune function with select TLR agonists may be a useful strategy to prevent neonatal sepsis mortality.
During infections, pathogens bind to toll-like receptor (TLR)4 and CD14 receptors and induce cytokine release, leading to inflammation. Here, we investigated TLR4 and CD14 expression on peripheral blood leukocytes (PBLs) and their roles in lipopolysaccharide (LPS)-induced cytokine and chemokine release. Full-term and preterm neonates and adults were studied. PBLs were pretreated with anti-TLR4- and anti-CD14-blocking antibodies and stimulated with LPS. Cytokine and chemokine levels were measured in supernatants. TLR4, CD14 expression, and LPS-induced CXCL8 release were higher in neonates, possibly contributing to aberrant inflammation. TLR4 blockade resulted in approximately 3-fold greater suppression of LPS-induced CXCL8 release in preterm neonates (38%) than in adults (14%). CD14 blockade (approximately 80%) in neonates induced approximately 3-fold greater inhibition of CXCL8 release, compared with anti-TLR4 (approximately 30%). Anti-TLR4 partly (50-60%) inhibited IL-10 and TNF-alpha, whereas anti-CD14 completely suppressed their release. Our findings reveal that neonates depend more on TLR4 for CXCL8 release. Furthermore, neonatal LPS-induced CXCL8 release, apart from TLR4/CD14-mediated signaling, is regulated by LPS interactions with other TLRs and/or immune receptors. IL-10 and TNF-alpha release depends on LPS binding not only to CD14/TLR4 but also to CD14 associated with another TLR. Our findings reveal the contribution of TLR4 and CD14 in neonatal cytokine and chemokine release and could aid in design of antagonists to prevent harmful inflammation.
While the Toll-like receptors (TLRs) are responsible for the recognition and response to pathogen ligands, increasing evidence suggests that the family of five cytosolic Toll/IL-1 receptor (TIR) adaptor proteins also play a crucial role in the specificity of the response. Genetic studies in mice, and increasingly in human polymorphic populations, have given us a greater understanding the role these adaptors play in orchestrating and coordinating the multifaceted immune response to multiple exogenous threats. Importantly, with growing evidence of the critical role TLRs play in responses to host danger signals and autoimmune disease, a more comprehensive understanding and appreciation of the role these adaptors play in disease progression may provide future targets for therapeutic intervention in human disease. Importantly, growing evidence supports the concept of pathway specific and inflammatory control by a better understanding of how these adaptors interact with other signalling mediators, where they localise within the cell and the inflammatory programs they initiate as a way of manipulating immune responses. This review deals with our current understanding of these TIR-containing adaptor proteins and how mutagenesis of specific residues and domains has increased our knowledge of their function in TLR immune responses.
Up- and down-regulation of inflammatory response was described in blood cells from septic patients, according to the stage of sepsis and the cells evaluated. This study aimed to evaluate the Toll-like receptor (TLR) signaling pathway gene expression in peripheral blood mononuclear cells (PBMC) and neutrophils in patients throughout the different stages of sepsis.
Prospective, observational study.
Two emergency rooms and two intensive care units in one university and one teaching hospital.
A total of 15 septic patients, five with sepsis, five with severe sepsis, and five with septic shock, in addition to five healthy volunteers were enrolled.
None.
The Human-TLR Signaling Pathway, which comprises 84 genes related to TLR-mediated signal transduction, was evaluated by real time polymerase chain reaction in PBMC and neutrophils obtained from patients and controls. The fold change for each gene (2(-Delta DeltaCt)) was compared between the groups. Genes with fold changes greater than 2 and significant changes in DeltaCT are reported as differently expressed. The fold change ratios in PBMC gene expression between septic patients and healthy controls revealed a dynamic process according to the stage of sepsis, tending toward down-regulation of the TLR signaling pathway in PBMC in the more severe forms of the disease. However, the differential gene expression was restricted to five down-regulated genes in septic shock patients, which are found in the effector and downstream pathways. Neutrophils showed a different pattern of adaptation. Patients with sepsis, severe sepsis, and septic shock presented a broad gene up-regulation, which included all functional groups evaluated and persisted throughout the stages of the disease.
TLR-signaling pathway genes are differently regulated in PBMC and neutrophils of septic patients, and are dynamically modulated throughout the different stages of sepsis.
Induction of the adaptive immune response depends on the expression of co-stimulatory molecules and cytokines by antigen-presenting cells. The mechanisms that control the initial induction of these signals upon infection are poorly understood. It has been proposed that their expression is controlled by the non-clonal, or innate, component of immunity that preceded in evolution the development of an adaptive immune system in vertebrates. We report here the cloning and characterization of a human homologue of the Drosophila toll protein (Toll) which has been shown to induce the innate immune response in adult Drosophila. Like Drosophila Toll, human Toll is a type I transmembrane protein with an extracellular domain consisting of a leucine-rich repeat (LRR) domain, and a cytoplasmic domain homologous to the cytoplasmic domain of the human interleukin (IL)-1 receptor. Both Drosophila Toll and the IL-1 receptor are known to signal through the NF-kappaB pathway. We show that a constitutively active mutant of human Toll transfected into human cell lines can induce the activation of NF-kappaB and the expression of NF-kappaB-controlled genes for the inflammatory cytokines IL-1, IL-6 and IL-8, as well as the expression of the co-stimulatory molecule B7.1, which is required for the activation of naive T cells.
MyD88 is a general adaptor protein that plays an important role in the Toll/IL-1 receptor family signalings. Recently, Toll-like receptors 2 and 4 (TLR2 and TLR4) have been suggested to be the signaling receptors for lipopolysaccharide (LPS). In this study, we demonstrate that MyD88 knockout mice lack the ability to respond to LPS as measured by shock response, B cell proliferative response, and secretion of cytokines by macrophages and embryonic fibroblasts. However, activation of neither NF-kappaB nor the mitogen-activated protein (MAP) kinase family is abolished in MyD88 knockout mice. These findings demonstrate that signaling via MyD88 is essential for LPS response, but the inability of MyD88 knockout mice to induce LPS-dependent gene expression cannot simply be attributed to lack of the activation of MAP kinases and NF-kappaB.
Toll-like receptors (TLR:) have recently been linked to the immunostimulatory function of microbial toxins in human and mice. TLR: signals activation of nuclear factor kappaB that leads to the production of a number of proinflammatory mediators. TLR:4 mediates the endotoxin-induced inflammatory response, whereas TLR:2 may be involved in the response to yeast and Gram-positive bacterial products. To better understand age-related changes in acute inflammatory response, we studied the ontogeny of TLR:2 and TLR:4 mRNA in murine fetal lung, liver, and placenta by quantitative reverse transcriptase-PCR. Different expression patterns were seen between the tissues and between the TLR: This is in accordance with the evidence that there are differences in the receptors for different microbial toxins and that the response is organ specific. We additionally show that the expression of TLR: was dependent on the stage of differentiation. In the liver, the levels of Tlr2 and Tlr4 were high regardless of the age. In the lung, Tlr2 and Tlr4 expression levels were barely detectable in immature fetus (d 14-15). Tlr2 and Tlr4 were increased several-fold during prenatal development and further increased after birth. The present results support the finding of a deficient inflammatory response of the immature lung to microbial toxins.
Toll-like receptor 4 (TLR4) mediates lipopolysaccharide (LPS) signaling in a variety of cell types. MD-2 is associated with the extracellular domain of TLR4 and augments TLR4-dependent LPS responses in vitro. We show here that MD-2(-/-) mice do not respond to LPS, do survive endotoxic shock but are susceptible to Salmonella typhimurium infection. We found that in MD-2(-/-) embryonic fibroblasts, TLR4 was not able to reach the plasma membrane and predominantly resided in the Golgi apparatus, whereas TLR4 was distributed at the leading edge surface of cells in wild-type embryonic fibroblasts. Thus, MD-2 is essential for correct intracellular distribution and LPS-recognition of TLR4.
This review will focus on the molecular mechanisms of macrophage activation and desensitization by bacterial lipopolysaccharide (LPS). The most recent advances in the understanding of the function of the LPS receptor complex and its role in the development of the septic shock syndrome and endotoxin tolerance will be discussed.
Preterm newborn infants are especially susceptible to Gram-negative sepsis that is associated with a lethality of up to 40%.
We tested whether polymorphonuclear leukocytes (PMN) from preterm infants exhibit an impaired antibacterial response upon stimulation with lipopolysaccharide (LPS) from Escherichia coli when compared to full term newborns or adults.
We studied the effect of LPS on the expression of the surface proteins CD11b and CD14 and the secretion of elastase by PMN from preterm infants, term infants and adults ex vivo.
We found a significantly reduced antibacterial activity of PMN from preterm infants upon stimulation with LPS as indicated by low surface expression of the adhesion molecule CD11b and the reduced secretion of PMN elastase. LPS-induced CD11b expression was dependent on binding of LPS to the surface protein CD14 as CD14 antibodies inhibited LPS dependent CD11b upregulation. Furthermore CD14 expression was lower on PMN from preterm infants than from adults. In addition, CD14 independent upregulation of CD11b in response to tumor necrosis factor (TNF-alpha), N-formyl peptides (FMLP) and phorbol ester (PMA) was impaired.
PMN from preterm infants are distinctly hyporesponsive to LPS, which may explain the predisposition of these children to invasive disease due to gramnegative bacteria.
The modulation of Toll-like receptors (TLR) 1, 2 and 4 was studied during experimental human endotoxaemia. Healthy volunteers received 2 ng/kg of lipopolysaccharide (LPS) endotoxin (n = 10). TLR1, 2 and 4 expression occurred on monocytes and neutrophils, with monocytes expressing higher baseline levels of TLR2. LPS infusion downmodulated TLR4 expression on neutrophils, with maximal downregulation occurring at 24 h (-62% from baseline; P < 0.03 versus baseline). Monocyte TLRs were upregulated in vivo (TLR1 and 2), and in vitro (TLR1, 2 and 4) 8 h after LPS bolus (P < 0.05 versus baseline). Therefore, neutrophils and monocytes differentially express surface TLRs, and endotoxaemia differentially regulates TLR expression.
Despite extensive research, bacterial sepsis and its associated systemic inflammation remain a major cause of morbidity and mortality in the pediatric intensive care unit. Advances in molecular biology, however, have improved our understanding of this disease process and have opened up new avenues of potential therapeutic approaches. One such exciting area has been the substantial and still growing evidence that the mammalian immune system uses a family of Toll-like receptors (TLRs) to generate a response to molecular patterns present on invading microorganisms. In particular, TLR4 is part of a recognition complex for bacterial lipopolysaccharide (LPS), thus raising the likelihood of its involvement in the inflammatory response to bacterial sepsis. This review highlights our understanding of the molecular biology of these receptors, focusing on the LPS response, and concluding with a summary of ongoing evaluation and potential therapeutic strategies for treating sepsis through blockade of TLR signaling.
Newborns and especially preterm infants show a unique susceptibility to severe bacterial infections that cause significant morbidity and mortality. As very few data are available on innate immune functions in human fetuses, we conducted a comprehensive study to investigate the expression of several adhesion molecules essentially involved in migration (CD11a, CD11b, CD11c, CD18, and CD62L). Furthermore, phagocytic activity, generation of respiratory burst products, and production of several proinflammatory cytokines were assessed. Various functions of the fetal innate immune system were demonstrated to be essentially different from those observed in term neonates or adults. Expression of several surface markers was significantly diminished on fetal granulocytes. Furthermore, a significantly reduced phagocytic activity of fetal granulocytes and monocytes was found, contrasted by an enhanced generation of reactive oxygen products. In addition, we demonstrate that significant numbers of fetal monocytes are capable of the production of proinflammatory cytokines in response to stimulation. However, the pattern of cytokine production is different from the more mature individuals: the number of IL-6- and tumor necrosis factor-alpha-positive monocytes were significantly diminished, whereas more IL-8-producing monocytes were found compared with adults. The results of our study add significantly to our understanding of the maturation and impairment of the innate immune response.
One of the mechanisms by which the innate immune system senses the invasion of pathogenic microorganisms is through the Toll-like receptors (TLRs), which recognize specific molecular patterns that are present in microbial components. Stimulation of different TLRs induces distinct patterns of gene expression, which not only leads to the activation of innate immunity but also instructs the development of antigen-specific acquired immunity. Here, we review the rapid progress that has recently improved our understanding of the molecular mechanisms that mediate TLR signalling.
Toll-like receptor 4 (TLR4) is a signaling receptor for lipopolysaccharide (LPS) but requires MD-2, a molecule associated with the extracellular TLR4 domain, to respond efficiently to LPS. The purpose of this study was to determine the critical stretch of primary sequence in the TLR4 region involved in MD-2 recognition. TLR4 and TLR4/2a chimera consisting of the TLR4 region Met(1)-Phe(54) and the TLR2 region Ala(53)-Ser(784) were coprecipitated with MD-2, but the deletion mutant TLR4(Delta E24-P34) in which the TLR4 region Glu(24)-Pro(34) was deleted failed to coprecipitate. In agreement with the MD-2 binding, LPS-conjugated beads sedimented TLR4 and TLR4/2a chimera but not TLR2 with MD-2. TLR4(Delta E24-P34) barely coprecipitated with LPS-beads. The cells that had been cotransfected with TLR4(Delta E24-P34) and MD-2 did not induce NF-kappa B activation in response to LPS. These results clearly demonstrate that the amino-terminal TLR4 region of Glu(24)-Pro(34) is critical for MD-2 binding and LPS signaling.
Premature newborns are highly susceptible to severe bacterial infections. This is partially due to their immature innate immune system, characterized by decreased neutrophil and monocyte activity as well as by reduced concentrations of complement factors. However, additional mechanisms might be important for innate immunity and are still the subject of considerable debate. The importance of pattern recognition domains such as Toll-like receptors (TLR) has been fully acknowledged within the last few years. Therefore, we investigated age-related monocyte TLR4 expression and lipopolysaccharide-induced cytokine secretion from very low birth weight infants (VLBWI) and from newborns after wk 30 of gestation in comparison to healthy adults. In VLBWI, expression of TLR4 surface protein, detected by flow cytometry, and TLR4-specific mRNA, quantified by real time-PCR, were significantly reduced in comparison to mature infants and to adults. Reduced TLR4 expression was paralleled by significantly diminished ex vivo LPS stimulated IL-1beta, IL-6, and tumor necrosis factor-alpha secretion into whole blood. We conclude that, in VLBWI, the minimized expression of TLR4 contributes to the susceptibility of VLBWI to infections with Gram-negative bacteria due to the lack of cytokines to boost initial immune response.
Despite recent identification of specific pattern recognition receptors (PRR) for distinct microbial structures, data indicating their relevance in human infectious diseases are limited. We determined the expression levels of the Toll-like receptor (TLR)2 and TLR4 by flow cytometry on granulocytes and monocytes of healthy neonates compared with healthy adults. The basal expression of TLR2 was only slightly lower in neonatal phagocytes, whereas no differences could be detected for TLR4. Analyzing neonates with sepsis, we found an impressive up-regulation of TLR2 on blood phagocytes already at initial presentation of symptoms. Comparison with C-reactive protein, IL-8, and IL-6 suggested that TLR2 expression on monocytes is comparably valuable as an early sepsis marker. TLR2 was differentially regulated during neonatal sepsis, showing a constant up-regulation on monocytes but only a transient increase on granulocytes. Surprisingly, TLR4 showed no remarkable changes. Our results revealed a mild deficiency of TLR2 expression in newborns and demonstrated a differential expression of TLR2 but not TLR4 in the course of neonatal sepsis, which could reflect specific inflammatory responses to distinct pathogens. The definition of TLR expression patterns might open a new field of therapeutic targets for neonatal sepsis.
Signal transduction pathways activated by Toll-like receptors (TLRs) have continued to be a major focus of research for investigators interested in the initiation of innate immune responses and the induction of pro-inflammatory cytokines and type I interferons during infection. Biochemical details of the major signalling pathways have now been obtained, and the specific signalling pathways activated by different TLRs are being elucidated. New insights into the activation of IRF family members, notably IRF3, IRF5 and IRF7, have been obtained, and interesting spatiotemporal aspects of signalling by MyD88 leading to IRF7 activation revealed. Adapters in TLR signalling are targets for inhibition, both by endogenous regulators and by virally derived proteins. Selective targeting of pathways by anti-inflammatory glucocorticoids also indicates the potential for modulating TLR signalling therapeutically.
Neonates are vulnerable to various infections because of their immature immune responses. Toll-like receptors could induce immune responses, both the innate and the acquired immune responses. The aim of the present study was to investigate the changes of TLR2 and TLR4 in neonatal infections, and to determine their roles in anti-infection immune reaction.
A total of 200 infants were divided into six groups: sepsis group (n = 21), bacterial pneumonia group (n = 70), bacterial meningitis group (n = 17), urinary tract infection group (n = 38), congenital syphilis group (n = 11) and non-infection group (n = 48). The TLR mRNA was determined by RT-PCR. The protein expression of TLR and the percentage of TLR positive cells were evaluated through flow cytometric analysis.
1. The TLR2 mRNA expression increased significantly in the sepsis group (6.14 +/- 0.80), most significantly in the Gram positive sepsis group (6.43 +/- 0.74). TLR2 mRNA expression was also significantly higher in the bacterial pneumonia group (5.49 +/- 0.62), the bacterial meningitis group (5.61 +/- 0.60) and the congenital syphilis group (5.89 +/- 0.38). TLR2 protein expression was the highest in the sepsis group and significantly increased in the bacterial pneumonia group, bacterial meningitis group and the congenital syphilis groups as well, all were higher than the TLR2 protein expression of the non-infectious group (1.27 +/- 0.75). The TLR2 protein expression in the Gram positive bacterial sepsis group was 2.54 +/- 0.68, that of Gram negative bacterial sepsis group was 1.25 +/- 0.51 (P < 0.05). The percentage of TLR2 positive cells in the neonatal infection group was (70.95 +/- 20.15)%, which did not differ significantly from that of non-infection group. 2. The mRNA expression of TLR4 was the highest in the sepsis group (6.20 +/- 1.59), while that in the Gram negative bacterial sepsis group was 6.78 +/- 1.79, higher than that of the Gram positive bacterial sepsis group, 5.39 +/- 0.78, (t = 2.29, P = 0.037). TLR4 mRNA expression increased significantly in the bacterial pneumonia group (5.33 +/- 1.07), the bacterial meningitis group (5.87 +/- 0.70) and the urinary tract infection group (5.38 +/- 0.91). There were no significant differences in TLR4 protein expression among these groups. The percentage of TLR4 positive cells in the neonatal infection groups was (0.71 +/- 0.31)%, higher than that of non- infection group (0.29 +/- 0.36)%. 3. In the Gram positive bacterial sepsis group, the mRNA expression of TLR2 (6.43 +/- 0.74) was higher than the mRNA expression of TLR4 (5.39 +/- 0.78), (t = 1.56, P = 0.024). In the Gram negative bacterial sepsis group, the mRNA expression of TLR4 (6.78 +/- 0.79) was significantly higher than the mRNA expression of TLR2 (5.64 +/- 0.68) (t = 2.63, P = 0.011). In the sepsis group, the TLR2 protein expression was significantly higher than the expression of TLR4 (t = 1.06, P = 0.044). The percentage of TLR4 positive cells was lower than the percentage of TLR2 positive cells among all these groups, P < 0.01. 4. Correlation analysis on gestational age and the mRNA expression, the protein expression and the percentage of TLR2 and TLR4 positive cells among all these groups did not show any statistical significance.
The mRNA and the protein expression of TLR2 and the mRNA expression of TLR2 increased significantly in the studied neonatal infection groups, especially in the severe sepsis groups. The mRNA expression of TLR2 increased mainly in the Gram positive bacterial infection groups, and the mRNA expression of TLR4 increased in the Gram negative bacterial infection groups, suggesting that both the TLR2 and TLR4 signal pathway took part in the immune mechanism of neonatal infection, providing new idea and experimental basis for further understanding of immune mechanism of neonatal infection.
Early childhood is a period of heightened susceptibility to infection due to immaturity of the immune system, and the nature of these developmental deficiencies is only partially understood. In this study, we focused on the ontogeny of the innate immune system by investigating the capacity of mononuclear cells to secrete a wide spectrum of inflammatory cytokines in response to interferon (IFN)-gamma priming and lipopolysaccharide (LPS) stimulation, namely IL-6, IL-10, IL-12, IL-18, IL-23, tumor necrosis factor (TNF)-alpha, and myxovirus resistance protein A, induced by type-I IFN, at several time points between birth (cord blood) and adulthood. Competence to produce all these cytokines followed a similar developmental pattern, with slow postnatal up-regulation from the response observed in cord blood. Unexpectedly, IL-6, IL-10, TNF-alpha, and IFN-gamma showed slow postnatal up-regulation but also elevated cord blood responses equal to or greater than the adult level. This was transient and not observed at 2 mo of age, and was not related to predelivery stress of the newborns. Variations in Toll-like receptor (TLR)4 function may account for these age related differences in cytokine responses, as TLR4 expression on neonatal monocytes post LPS stimulation was elevated and sustained relative to infants and adults.
The mammalian immune system has innate and adaptive components, which cooperate to protect the host against microbial infections. The innate immune system consists of functionally distinct 'modules' that evolved to provide different forms of protection against pathogens. It senses pathogens through pattern-recognition receptors, which trigger the activation of antimicrobial defences and stimulate the adaptive immune response. The adaptive immune system, in turn, activates innate effector mechanisms in an antigen-specific manner. The connections between the various immune components are not fully understood, but recent progress brings us closer to an integrated view of the immune system and its function in host defence.
MD-2 is the crucial cofactor of TLR4 in the detection of LPS. Here, we show that soluble MD-2 (sMD-2) circulates in plasma of healthy individuals as a polymeric protein. The total amount of sMD-2 in septic plasma was strongly elevated and contained both sMD-2 polymers and monomers, the latter representing the putative biologically active form of MD-2. Moreover, during experimental human endotoxemia, the monomeric and total sMD-2 content in plasma increased with the kinetics of an acute phase protein. The increase in sMD-2 monomers was paralleled by enhanced TLR4 costimulatory activity. The presence of functional sMD-2 during endotoxemia and sepsis was confirmed by immunodepletion. Immunohistochemistry revealed that MD-2 expression in septic patients was strongly enhanced on endothelium and multiple inflammatory cells in lung and liver. In vitro studies showed that sMD-2 release appears to be restricted to endothelial cells and dendritic cells. Release of sMD-2 by endothelial cells was strongly enhanced by LPS and TNF-alpha stimulation. Taken together, this study demonstrates the increase of both circulating polymeric and functional monomeric sMD-2 during endotoxemia and sepsis, and evidence is provided that the endothelium is involved in this process.
Pyogenic bacterial infections in humans with MyD88 deficiency
- H Von Bernuth
- C Picard
- Jin Z Pankla
- R Xiao
- H Ku
- C L Chrabieh
- M Mustapha
- I B Ghandil
- P Camcioglu
- Y Vasconcelos
- J Sirvent
- N Guedes
- M Vitor
- A B Herrero-Mata
- M J Aróstegui
- J I Rodrigo
- C Alsina
- L Ruiz-Ortiz
- E Juan
- M Fortuny
- C Yagüe
- J Antón
- J Pascal
- M Chang
- H H Janniere
- L Rose
- Y Garty
- B Z Chapel
- H Issekutz
- A Maródi
- L Rodriguez-Gallego
- C Banchereau
- J Abel
- L Li
- X Chaussabel
- D Puel
- A Casanova
von Bernuth H, Picard C, Jin Z, Pankla R, Xiao H, Ku CL, Chrabieh M, Mustapha IB, Ghandil P,
Camcioglu Y, Vasconcelos J, Sirvent N, Guedes M, Vitor AB, Herrero-Mata MJ, Aróstegui JI,
Rodrigo C, Alsina L, Ruiz-Ortiz E, Juan M, Fortuny C, Yagüe J, Antón J, Pascal M, Chang HH,
Janniere L, Rose Y, Garty BZ, Chapel H, Issekutz A, Maródi L, Rodriguez-Gallego C, Banchereau
J, Abel L, Li X, Chaussabel D, Puel A, Casanova JL. Pyogenic bacterial infections in humans with
MyD88 deficiency. Science. 2008; 321:691-696. [PubMed: 18669862]
Essential role of MD-2 in LPS responsiveness and TLR4 distribution
- S Ishihara
- Ma Rumi
- Y Kadowaki
- T Kitamura
- A Kosugi
- M Kimoto
- K Miyake
Ishihara S, Rumi MA, Kadowaki Y, Kitamura T, Kosugi A, Kimoto M, Miyake K
2002 Essential role of MD-2 in LPS responsiveness and TLR4 distribution. Nat
Immunol 3:667– 672
- A Poltorak
- X He
- I Smirnova
- M-Y Liu
- Van Huffel
- C Du
- X Birdwell
- D Alejos
- E Silva
- M Galanos
Poltorak A, He X, Smirnova I, Liu M-Y, Van Huffel C, Du X, Birdwell D, Alejos
E, Silva M, Galanos C 1998 Defective LPS signaling in C3H/HeJ and C57BL/
10ScCr mice: mutations in Tlr4 gene. Science 282:2085–2088
How toll-like receptors signal: what we know and what we don't know
- O Neill
O'Neill LA 2006 How toll-like receptors signal: what we know and what we don't
know. Curr Opin Immunol 18:3–9
Hospital-acquired infection surveillance in a neonatal intensive care unit
- G B Orsi
- G Ettorre
- A Panero
- F Chiarini
- V Vullo
- M Venditti
Orsi GB, d'Ettorre G, Panero A, Chiarini F, Vullo V, Venditti M. Hospital-acquired infection
surveillance in a neonatal intensive care unit. Am J Infect Control. 2009; 37:201-203. [PubMed:
19059676]
- Wynn JL