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IL-10 production in human cells in vitro after stimulation with flagella and purified FliC from UPEC CFT073. (A) Human uroepithelial cell monocyte cocultures stimulated (5 h) with flagellum-enriched protein (1 g) from CFT073 and CFT073ΔfliC strains or MC4100 with or without pflhDC. Significance was determined by t test for CFT073 strains (*, P 0.05). (B) Monocytes stimulated (5 h) with purified FliC (1 g) from CFT073Δ4 strain or carrier control (generated from CFT073Δ4ΔfliC). Significance was determined by t tests (*, P 0.05).
Source publication
Urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) engages interleukin-10 (IL-10) as an early innate immune response to regulate inflammation and promote the control of bladder infection. However, the mechanism of engagement of innate immunity by UPEC that leads to elicitation of IL-10 in the bladder is unknown. Here, we...
Contexts in source publication
Context 1
... cytokines, including IL-1, -1, -2, -4, -6, and TNF-, and multiple chemokines (e.g., granulocyte colony-stimulating factor [G-CSF]) were detected (see Fig. S1 in the supplemental material). Hyperflagellation in flhDC-complemented UPEC strains and an absence of flagellar expression in fliC-deficient strains was confirmed using immunoblots for FliC (Fig. S2A), as previously described (57); in addition, motility assays showed phenotypes consistent with hyperflagellation in flhDC-complemented UPEC strains (Fig. S2B). Taken together, these data show that flagellar expression in CFT073 and other UPEC strains, including UTI89 and EC958, induces the production of IL-10 in uroepithelial cell ...
Context 2
... in the supplemental material). Hyperflagellation in flhDC-complemented UPEC strains and an absence of flagellar expression in fliC-deficient strains was confirmed using immunoblots for FliC (Fig. S2A), as previously described (57); in addition, motility assays showed phenotypes consistent with hyperflagellation in flhDC-complemented UPEC strains (Fig. S2B). Taken together, these data show that flagellar expression in CFT073 and other UPEC strains, including UTI89 and EC958, induces the production of IL-10 in uroepithelial cell monocyte cocultures as well as significant induction of several other functionally opposed and related cytokines. IL-10 responses of cell cultures to enriched ...
Context 3
... cultures to enriched flagella and purified FliC. Experiments examining the responses of uroepithelial cell monocyte cocultures to flagellumenriched protein from CFT073 (isolated by shearing and ultracentrifugation) showed significant IL-10 responses to flagella from CFT073 WT (versus the CFT073ΔfliC mutant) and MC4100/pflhDC (versus MC4100 WT) ( Fig. 2A). Subsequently, we measured IL-10 levels in response to FliC purified to homogeneity from CFT073Δ4/pflhDC using fast protein liquid chromatography (FPLC), because flagellum-enriched preparations contain trace amounts of other outer membrane proteins that could contribute to IL-10 induction (57). Pure FliC triggered significantly more ...
Context 4
... to homogeneity from CFT073Δ4/pflhDC using fast protein liquid chromatography (FPLC), because flagellum-enriched preparations contain trace amounts of other outer membrane proteins that could contribute to IL-10 induction (57). Pure FliC triggered significantly more IL-10 than the carrier control that was generated from the CFT073Δ4 ΔfliC strain (Fig. 2B). Similar responses were observed for mouse macrophages but did not reach statistical significance due to higher basal levels of IL-10 detected in these experiments (data not shown). Taken together, these findings show that pure FliC stimulates significant IL-10 synthesis in human uroepithelial cell monocyte ...
Citations
... We reasoned that an additional inoculation after 1 week would be akin to receiving a dose of monophosphoryl lipid A, a modified component of Gram-neg ative lipopolysaccharide (and thus present on EC958), which is a commercially available adjuvant and has been used previously in UTI vaccine studies (56). The modest GC B cell responses could theoretically also be the result of adaptive immune suppression via cytokines, which is a widely cited reason for a lack of a strong adaptive response in the bladder (12,(57)(58)(59)(60). Early during the innate immune response to UPEC, IL-10 is secreted by mast cells to dampen inflammation and prevent bladder damage (57,61). This in turn decreases DC activation and CD4 + T cell and GC B cell responses in draining lymph nodes. ...
Bacterial urinary tract infections (UTIs) are both common and exhibit high recurrence rates in women. UTI healthcare costs are increasing due to the rise of multidrug-resistant (MDR) bacteria, necessitating alternative approaches for infection control. Here, we directly observed host adaptive immune responses in acute UTI. We employed a mouse model in which wild-type C57BL/6J mice were transurethrally inoculated with a clinically relevant MDR UTI strain of uropathogenic Escherichia coli (UPEC). Firstly, we noted that rag1 −/− C57BL/6J mice harbored larger bacterial burdens than wild-type counterparts, consistent with a role for adaptive immunity in UTI control. Consistent with this, UTI triggered in the bladders of wild-type mice early increases of myeloid cells, including CD11c hi conventional dendritic cells, suggesting possible involvement of these professional antigen-presenting cells. Importantly, germinal center B cell responses developed by 4 weeks post-infection in bladder-draining lymph nodes of wild-type mice and, although modest in magnitude and transient in nature, could not be boosted with a second UTI. Thus, our data reveal for the first time in a mouse model that UPEC UTI induces local B cell immune responses in bladder-draining lymph nodes, which could potentially serve to control infection.
... Identifying the microbial molecules that trigger the synthesis of this critical regulator of innate immune responses is one aspect of understanding the role of IL-10 in infectious illness. Streptococcus M protein, Staphylococcus peptidoglycan-embedded lipopeptides, cell wall glycopolymers, Salmonella flagella and Yersinia flagella, among others, have all been demonstrated to cause the production of IL-10 in experimental illness models (Acharya et al., 2019;Frodermann et al., 2011;McNally et al., 2007;Price et al., 2005;Wyant et al., 1999). The UPEC flagella play several roles in the pathogenesis of UTIs, including motility that is linked to bacterial ascent from the bladder to the kidneys, causing pyelonephritis (Lane et al., 2007). ...
The two types of bladder cancer, muscle invasive and non-muscle invasive (NMIBC), are among the most prevalent cancers worldwide. Despite this, even though muscle-invasive bladder cancer is more deadly, NMIBC requires more therapy due to a greater recurrence rate and more extended and expensive care. Immunotherapy, intravesical chemotherapy, cystoscopy, and transurethral resection (TUR) are among the treatments available. Crude scorpion venomand purified proteins and peptides, can suppress cancer metastasis in an in vitro or in vivo context, suppress cancer growth, halt the cell cycle, and cause cell apoptosis, according to an increasing number of experimental and preclinical studies. In this research, three novels discovered peptides (P2, P3 and P4. ProteomeXchange: PXD036231) from Buthotus saulcyi and, Odontobuthus doriae scorpions were used along with a peptide called pantinin (as a control). The phylogenetic tree showed that the peptides belong to Chaperonin HSP60, Chrysophsin2 and Pheromone-binding protein2, respectively. These peptides were docked with four known antigens, BAGE, BLCAP, PRAME and ROR1 related to bladder cancer and three bacterial antigens FliC, FliD and FimH to investigate their antimicrobial and anticancer properties. The results showed that peptides 2 and 3 have the best binding rate. The MD simulation results also confirmed the binding of peptides 2 and 3 to antigens. The penetration power of peptides 2 and 3 in the membrane of cancer cells and bacterial cells was also simulated, and the results of RMSD and PD confirmed it. QSAR suggests that peptides 2 and 3 can act as anti-cancer and anti-microbial peptides.
Communicated by Ramaswamy H. Sarma
... These receptors are also present in the brain and were previously implicated in schizophrenia [251][252][253][254]. Despite the bladder's anti-bacterial defenses, including the urothelial barrier, AMP-containing mucus, local ILCs, and MCs, uropathogenic bacteria can overcome these barriers by upregulating IL-10 [255]. Recent studies have found that IL-10 could also account for the absence of long-lasting antibodies in UTIs, and the frequent recurrences found in patients [236,[256][257][258]. ...
Gut microbes are immunologically tolerated in the gastrointestinal tract but trigger aggressive immune responses upon translocation across the gut barrier. Although oral tolerance, a physiological process that dampens immune responses to food proteins and commensal microbiota, remains poorly defined, significant progress was made during and after the Human Immunodefi-ciency Virus epidemic in the 1980s and the discovery of regulatory T cells in 1995. Additional insight was gained after the discoveries of innate lymphoid cells in 2008 and the functional elucidation of mucosal mast cells. Prior to the historical discovery of human pathogens, the etiologies of most human diseases were considered unknown. The same was true about many genetic disorders prior to the Human Genome Project. Here, we hypothesize that many of the remaining idiopathic conditions , including autoimmune, fibroproliferative, and neuropsychiatric diseases as well as some cancers , can be considered microbial translocation disorders triggered by the host immune responses to extraintestinal gut microbes and/or their constituent parts. In addition to microbial translocation, we also discuss potential interventions for intestinal barrier rehabilitation, including antibodies against tumor necrosis factor-like ligand 1A and membrane lipid replacement supplements.
... Thus, IL-6 secretion activates immunity and inhibits intracellular bacterial community (IBC) formation [130]. On the other hand, upon urinary-tract colonization of UPEC, survival, invasion, and biofilm formation, flagellin is synthesized and recognized by TLR-5, which contributes to IL-10 production in the bladder during UTI [131]. Altogether, the contribution of IL-6 and IL-10 in controlling infection should be tightly regulated for the success of treatment outcomes. ...
Complex pathological diseases, such as cancer, infection, and Alzheimer’s, need to be targeted by multipronged curative. Various omics technologies, with a high rate of data generation, demand artificial intelligence to translate these data into druggable targets. In this study, 82 marine venomous animal species were retrieved, and 3505 cryptic cell-penetrating peptides (CPPs) were identified in their toxins. A total of 279 safe peptides were further analyzed for antimicrobial, anticancer, and immunomodulatory characteristics. Protease-resistant CPPs with endosomal-escape ability in Hydrophis hardwickii, nuclear-localizing peptides in Scorpaena plumieri, and mitochondrial-targeting peptides from Synanceia horrida were suitable for compartmental drug delivery. A broad-spectrum S. horrida-derived antimicrobial peptide with a high binding-affinity to bacterial membranes was an antigen-presenting cell (APC) stimulator that primes cytokine release and naïve T-cell maturation simultaneously. While antibiofilm and wound-healing peptides were detected in Synanceia verrucosa, APC epitopes as universal adjuvants for antiviral vaccination were in Pterois volitans and Conus monile. Conus pennaceus-derived anticancer peptides showed antiangiogenic and IL-2-inducing properties with moderate BBB-permeation and were defined to be a tumor-homing peptide (THP) with the ability to inhibit programmed death ligand-1 (PDL-1). Isoforms of RGD-containing peptides with innate antiangiogenic characteristics were in Conus tessulatus for tumor targeting. Inhibitors of neuropilin-1 in C. pennaceus are proposed for imaging probes or therapeutic delivery. A Conus betulinus cryptic peptide, with BBB-permeation, mitochondrial-targeting, and antioxidant capacity, was a stimulator of anti-inflammatory cytokines and non-inducer of proinflammation proposed for Alzheimer’s. Conclusively, we have considered the dynamic interaction of cells, their microenvironment, and proportional-orchestrating-host- immune pathways by multi-target-directed CPPs resembling single-molecule polypharmacology. This strategy might fill the therapeutic gap in complex resistant disorders and increase the candidates’ clinical-translation chance.
... Macrophages in the lamina propria of the bladder are particularly prone to cell death, as a significant reduction in the numbers of macrophages was observed during UTI in an experimental model 9 . Cells of the urothelium, including uroepithelial cells, can produce cytokines of the IL-6 family, IL-8 families, and IL-10 families, in response to UTIs [58][59][60][61] . Although IL-6 has been shown to promote macrophage survival and activity in other models of injury [62][63][64] , disruptions in the cytokine microenvironment caused by exfoliation may have an influence on macrophage survival. ...
The bladder supports a diversity of macrophage populations with functional roles related to homeostasis and host defense, including clearance of cell debris from tissue, immune surveillance, and inflammatory responses. This review examines these roles with particular attention given to macrophage origins, differentiation, recruitment, and engagement in host defense against urinary tract infections (UTIs), where these cells recognize uropathogens through a combination of receptor-mediated responses. Time is an important variable that is often overlooked in many clinical and biological studies, including in relation to macrophages and UTIs. Given that ageing is a significant factor in urinary tract infection pathogenesis and macrophages have been shown to harbor their own circadian system, this review also explores the influence of age on macrophage functions and the role of diurnal variations in macrophage functions in host defense and inflammation during UTIs. We provide a conceptual framework for future studies that address these key knowledge gaps.
... In addition, the presence of certain structures on bacterial cells, such as lipopolysaccharides, in general stimulates cytokine production in host cells [4]. Analysis of the presence and concentration of certain types of cytokines can provide additional information on how different E. coli strains affect certain immune processes, and thus indirectly reveal their (potential) pathogenicity [5]. ...
Escherichia coli is known to be an important uropathogenic agent. Several models were developed for investigating the uropathogensis of E. coli, including the recent biomimetic porcine urothelial in vitro model. The aim of this study was to assess the cytokine response of the cells of the biomimetic porcine urothelial model to different E. coli strains. The production of nine different cytokines in response to E. coli infection was evaluated using the commercial pre-configured immunoassay multiplex Cytokine & Chemokine 9-Plex Porcine ProcartaPlex™ Panel 1 kit. Our results showed that cells of the biomimetic porcine urothelial model reacted to the presence of all the employed different E. coli strains, albeit with some differences in levels and types of cytokines produced. Increased production of IL-10, IL-8, TNF-α, IL-1β, IL-4 and IL-12p40 was observed. Statistical analysis (Fisher’s exact test) revealed a correlation between the high fold change in the immune response and the presence of the cnf1 gene that encodes the cytotoxic necrotizing factor. Our results shed light on the cytokine response of normal urothelial cells to different E. coli strains and have the potential to fuel the search for understanding the mechanisms behind the different cytokine responses to different E. coli strains.
... U937 monocytes were differentiated into human monocyte-derived macrophages (MDMs) as follows. Briefly, monocytes were seeded (5x10 5 per well such that 1x10 5 adhere) into the wells of a 96-well tissue culture-treated plate (Falcon) essentially as described elsewhere [83,84], except that ...
Metals such as copper (Cu) and zinc (Zn) are important trace elements that can affect bacterial cell physiology but can also intoxicate bacteria at high concentrations. Discrete genetic systems for management of Cu and Zn efflux have been described in several bacteria pathogens, including streptococci. However, insight into molecular cross-talk between systems for Cu and Zn management in bacteria that drive metal detoxification, is limited. Here, we describe a biologically consequential cross-system effect of metal management in group B Streptococcus (GBS) governed by the Cu-responsive copY regulator in response to Zn. RNAseq analysis of wild-type (WT) and copY -deficient GBS subjected to metal stress revealed unique transcriptional links between the systems for Cu and Zn detoxification. We show that the Cu-sensing role of CopY extends beyond Cu and enables CopY to regulate Cu and Zn stress responses that effect changes in gene function for central cellular processes, including riboflavin synthesis. CopY also supported GBS intracellular survival in human macrophages and virulence during disseminated infection in mice. In addition, we show a novel role for CovR in modulating GBS resistance to Zn intoxication. Identification of the Zn resistome of GBS using TraDIS revealed a suite of genes essential for GBS growth in metal stress. Several of the genes identified are novel to systems that support bacterial survival in metal stress and represent a diverse set of mechanisms that underpin microbial metal homeostasis during cell stress. Overall, this study reveals a new and important mechanism of cross-system complexity driven by CopY in bacteria to regulate cellular management of metal stress and survival.
... In the case of females with diabetes mellitus without long-term complications with known acute uncomplicated cystitis, quantitative counts < 10 5 CFU/mL are isolated from 20 to 250 Ä of premenopausal women and l00 Ä from postmenopausal women (28). 8. Diagnostic imaging-The amplified frequency of life-threatening complications of UTIs for obtaining diagnostic imaging along with accuracy and pathogen specificity (28). ...
The pervasiveness of urinary tract infections (UTIs) with their clinical manifestations in patients with diabetes mellitus has escalated amidst the past decade or so, as myriad predisposing factors contribute to its occurrence. Although the causative agent of UTI is Escherichia coli, the etiopathogenesis can be traced back to glycosuria in the renal parenchymal region. This has precipitated pyelonephritis and renal complications, including cytopathic and altered metabolism. Furthermore, impaired immunity with scarce IL-6, 8 in urine, urinary retention, and dysfunctional voiding raise susceptibility towards uropathogens, mainly E. coli. Treatment for UTI with diabetes is based on symptoms and severity, urologic abnormalities, renal function, bladder infections, and metabolic alteration. The treatment process or regimens for patients with type 2 diabetes with asymptomatic bacteriuria are very low or negligible. Adequate management with antibiotic regimens in symptomatic patients after critical diagnosis is crucial for prophylaxis and effective treatment.
... A significant increase in the release of IL-6 and IL-8 was observed after 3 and 5 h of infection with the purified FliC protein, and a decrease in IL-6 and IL-8 release was observed after 3 h of infection with the strain containing a mutation in the fliC gene. Acharya et al. [30] demonstrated that the FliC protein induces the release of IL-10 and other cytokines, including IL-6, via TLR5 in vivo and in vitro. Our data, consistent with other studies, demonstrated that the flagellin FliC stimulates the release of IL-6 and IL-8 in HMC-1 cells. ...
Background:
Urinary tract infections (UTIs) are a public health problem in Mexico, and uropathogenic Escherichia coli (UPEC) is one of the main etiological agents. Flagella, type I fimbriae, and curli promote the ability of these bacteria to successfully colonize its host.
Aim:
This study aimed to determine whether flagella-, type I fimbriae-, and curli-expressing UPEC induces the release of proinflammatory cytokines in an established coculture system.
Methods:
The fliC, fimH, and csgA genes by UPEC strain were disrupted by allelic replacement. Flagella, type I fimbriae, and curli were visualized by transmission electron microscopy (TEM). HTB-5 (upper chamber) and HMC-1 (lower chamber) cells cocultured in Transwell® plates were infected with these UPEC strains and purified proteins. There was adherence to HTB-5 cells treated with different UPEC strains and they were quantified as colony-forming units (CFU)/mL.
Results:
High concentrations of IL-6 and IL-8 were induced by the FimH and FliC proteins; however, these cytokines were detected in low concentrations in presence of CsgA. Compared with UPEC CFT073, CFT073ΔfimH, CFT073ΔfimHΔfliC, and CFT073ΔcsgAΔfimH strains significantly reduced the adherence to HTB-5 cells.
Conclusion:
The FimH and FliC proteins are involved in IL-6 and IL-8 release in a coculture model of HTB-5 and HMC-1 cells.
... U937 monocytes were differentiated into human monocyte-derived macrophages (MDMs) as follows. Briefly, monocytes were seeded (5 Â 10 5 per well, such that 1 Â 10 5 adhered) into the wells of a 96-well tissue culture-treated plate (Falcon), essentially as described elsewhere (59,60), except that U937 monocytes were differentiated by exposure to 30 ng/ml phorbol 12myristate 13-acetate (PMA) for 48 h and cells subsequently rested in medium without PMA for 72 h to enhance the morphological and phenotypic markers of MDMs (61). A multiplicity of infection (MOI) of 100 bacteria:macrophages for 1 h was used in RPMI without antibiotics. ...
Bacteria can utilize Copper (Cu) as a trace element to support cellular processes; however, excess Cu can intoxicate bacteria. Here, we characterize the cop operon in group B streptococcus (GBS), and establish its role in evasion of Cu intoxication and the response to Cu stress on virulence. Growth of GBS mutants deficient in either the copA Cu exporter, or the copY repressor, were severely compromised in Cu-stress conditions. GBS survival of Cu stress reflected a mechanism of CopY de-repression of the CopA efflux system. However, neither mutant was attenuated for intracellular survival in macrophages. Analysis of global transcriptional responses to Cu by RNA-sequencing revealed a stress signature encompassing homeostasis of multiple metals. Genes induced by Cu stress included putative metal transporters for manganese import, whereas a system for iron export was repressed. In addition, copA promoted the ability of GBS to colonize the blood, liver and spleen of mice following disseminated infection. Together, these findings show that GBS copA mediates resistance to Cu intoxication, via regulation by the Cu-sensing transcriptional repressor, copY . Cu stress responses in GBS reflect a transcriptional signature that heightens virulence and represents an important part of the bacteria’s ability to survive in different environments.
Importance
Understanding how bacteria manage cellular levels of metal ions, such as copper, helps to explain how microbial cells can survive in different stressful environments. We show how the opportunistic pathogen group B Streptococcus (GBS) achieves homeostasis of intracellular copper through the activities of the genes that comprise the cop operon, and describe how this helps GBS survive in stressful environments, including in the mammalian host during systemic disseminated infection.
