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Role of iNOS in chlamydial genital tract infection in vivo. The course of infection (solid line) and urine nitrate levels (dashed line) in wild-type C57BL/6 mice receiving either 50 mM L-arginine (A) or 50 mM L-NMMA (B) in their drinking water and in iNOS KO mice (C) is shown. Each data point represents the mean IFU from cervical-vaginal swabs of culture-positive mice collected 4, 7, 10, 14, 21, 28, 35, and 42 days postinfection. Above each data point, the ratio of the number of culture-positive animals to the total number of animals in each experimental group is given. The asterisks in panels B and C designate significant differences in the quantitative recovery of viable MoPn at the indicated times postinfection compared to that in the control mice (panel A). Urine was collected daily for nitrate determination. The urinary nitrate response was not assessed in iNOS KO mice.
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Mice lacking inducible nitric oxide synthase (iNOS) or treated with iNOS inhibitors resolved chlamydial genital tract infections. Additionally, treatment of primary murine cell cultures with gamma interferon restricted chlamydial growth in the absence of nitric oxide. From these results, iNOS activity is unnecessary for the resolution of chlamydial...
Citations
... Studies on human tissue are limited by the access to tissue and cells; mice on the other hand are easily accessible but they have three major limitations: (i) based on their small size tissue samples are limited; (ii) infections with relevant Ct strains are easily cleared; and (iii) the murine immune mechanisms differ from humans, including the arguably most important cytokine in the anti-Ct response-interferon (IFN)γ [17][18][19][20][21]. In contrast to human cells, IFN-γ does not activate the expression of indoleamine 2,3-dioxygenase (IDO) in murine epithelial cells; instead, mice have redundant mechanisms including nitric oxide synthase and others to deplete the intracellular tryptophan pools [22]. This tryptophan depletion is an important mechanism to limit Chlamydia propagation through tryptophan starvation [4,[23][24][25]. ...
Chlamydia trachomatis (Ct) causes the most prevalent bacterial sexually transmitted disease leading to ectopic pregnancy and infertility. Swine not only have many similarities to humans, but they are also susceptible to Ct. Despite these benefits and the ease of access to primary tissue from this food animal, in vitro research in swine has been underutilized. This study will provide basic understanding of the Ct host–pathogen interactions in porcine oviduct epithelial cells (pOECs)—the counterparts of human Fallopian tube epithelial cells. Using NanoString technology, flow cytometry, and confocal and transmission-electron microscopy, we studied the Ct developmental cycle in pOECs, the cellular immune response, and the expression and location of the tight junction protein claudin-4. We show that Ct productively completes its developmental cycle in pOECs and induces an immune response to Ct similar to human cells: Ct mainly induced the upregulation of interferon regulated genes and T-cell attracting chemokines. Furthermore, Ct infection induced an accumulation of claudin-4 in the Ct inclusion with a coinciding reduction of membrane-bound claudin-4. Downstream effects of the reduced membrane-bound claudin-4 expression could potentially include a reduction in tight-junction expression, impaired epithelial barrier function as well as increased susceptibility to co-infections. Thereby, this study justifies the investigation of the effect of Ct on tight junctions and the mucosal epithelial barrier function. Taken together, this study demonstrates that primary pOECs represent an excellent in vitro model for research into Ct pathogenesis, cell biology and immunity.
... Nitric oxide by virtue of its unpaired electron is a highly reactive free radical which in excess can damage protein, carbohydrates, nucleotides, and lipids [24]. The production of reactive nitrogen species through the cytokine-iNOS likely plays a contributing role in the elimination of chlamydial infection in vitro, although it is not necessary for resolution of mouse urogenital infection [25]. In our study, we detected significant upregulation of mRNA expression of iNOS in ECT in CT-infected SSA as compared with controls (p ¼ .0004) ...
Purpose: The aim of study was to evaluate expression of inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) in Chlamydia trachomatis (CT)-infected spontaneous aborters (SA).
Materials and methods: Endometrial curettage tissue was collected from 140 SA (sporadic SA- 70; recurrent SA- 70) (Group I) and 140 age-matched controls (Group II) from Department of Obstetrics and Gynecology, Safdarjung Hospital, New Delhi, India. Polymerase chain reaction was performed for diagnosis of CT. The expression of iNOS/ eNOS/ IFN-γ/ TNF-α was assessed by real-time polymerase chain reaction (PCR).
Results: 15.7% SA were CT-positive (Group I); none in controls. Sporadic spontaneous aborters (SSA) (n = 8/70), recurrent spontaneous aborters (RSA) (n = 14/70) diagnosed as CT-positive (Group-I). Significant upregulation of iNOS/ eNOS was found in CT-positive SSA/RSA compared with CT-negative SSA/RSA and healthy controls. TNF-α and IFN-γ were expressed in CT-positive SSA/RSA compared with negative SSA/controls. iNOS showed a significant strong positive correlation with TNF-α and IFN-γ in CT-infected SA. eNOS showed a significant positive correlation with TNF-α and no correlation with IFN-γ in CT-infected SA. TNF-α was positively correlated with IFN-γ.
Conclusions: Significantly high expression of iNOS/ eNOS and proinflammatory cytokines affected pregnancy in CT-infected RSA, thereby implying that there occurs cytokine-induced expression of nitric oxide synthase (NOS).
... While C. trachomatis can avoid IFN-γ-induced tryptophan starvation by expressing a tryptophan synthase, C. muridarum is not able to produce this enzyme (Nelson et al., 2005), probably as a consequence of a different effect of IFN-γ on human and murine epithelial cells. Contrary to human epithelial cells, murine epithelial cells do not express indoleamine 2,3-dioxygenase following IFN-γ exposure but instead have redundant mechanisms using nitric oxide synthase and others (Ramsey et al., 1998). Mice can be infected with C. muridarum as well as C. trachomatis, although infections with the later are usually mild and mice lacking functional T-cells are able to clear C. trachomatis infections (Tuffrey et al., 1982). ...
The pig has garnered more and more interest as a model animal to study various conditions in humans. The growing success of the pig as an experimental animal model is explained by its similarities with humans in terms of anatomy, genetics, immunology, and physiology, by their manageable behavior and size, and by the general public acceptance of using pigs for experimental purposes. In addition, the immunological toolbox of pigs has grown substantially in the last decade. This development led to a boost in the use of pigs as a preclinical model for various human infections including sexually transmitted diseases (STIs) like Chlamydia trachomatis. In the current review, we discuss the use of animal models for biomedical research on the major human STIs. We summarize results obtained in the most common animal models and focus on the contributions of the pig model towards the understanding of pathogenesis and the host immune response. In addition, we present the main features of the porcine model that are particularly relevant for the study of pathogens affecting human female and male genital tracts. We also inform on the technological advancements in the porcine toolbox to facilitate new discoveries in this biologically important animal model. There is a continued need for improvements in animal modeling for biomedical research inclusive STI research. With all its advantages and the highly improved toolbox, the porcine model can play a crucial role in STI research and open the door to new exciting discoveries.
... As the cytokine polarization paradigm evolved, these results were extended to include detrimental effects of Th17-skewed responses (29). A major finding suggesting that Th1 responses may not be sufficient to understand Chlamydia pathogenesis was that iNOS gene knockout mice cleared C. muridarum genital tract infections (30,31). This result suggested that clearance of Chlamydia from the genital tract might not be adequately explained by IFN-␥ results and, by extension, the Th1/Th2 paradigm. ...
For almost two decades results from
Chlamydia
pathogenesis investigations have been conceptualized using a cytokine polarization narrative. Recent viral immunity studies identifying protective tissue resident memory T cells (Trm) suggest an alternative paradigm based on localized immune networks. As
Chlamydia
vaccines enter the preclinical pipeline, and in the case of an attenuated trachoma vaccine go into human subjects, it may be useful to ask whether cytokine polarization is the appropriate framework for understanding and evaluating vaccine efficacy. In this review we revisit
C. trachomatis
pathogenesis data from mice and humans using a Trm narrative and note a comfortable concordance with the
Chlamydia
pathogenesis literature.
... The protective effect of IFN-g in C. trachomatis-infected murine cells was also shown to be dependent on iNOS (21,22). In other experiments, mice lacking iNOS were found to restrict chlamydial growth as efficiently as wild type mice (23,24). There is evidence that clearance of Chlamydia from the genital tract depends on IFN-g-induced iNOS and NO production and on iNOS-independent mechanisms (25). ...
Chlamydia trachomatis is considered the most common agent of sexually transmitted disease worldwide. As an obligate intracellular bacterium, it relies on the host for survival. Production of NO is an effective antimicrobial defense mechanism of the innate immune system. However, whether NO is able to arrest chlamydial growth remains unclear. Similarly, little is known about the mechanisms underlying subversion of cellular innate immunity by C. trachomatis. By analyzing protein and mRNA expression in infected human mesenchymal stem cells, combined with RNA interference and biochemical assays, we observed that infection with C. trachomatis led to downregulated expression of inducible NO synthase (iNOS) in human mesenchymal stem cells in vitro. Furthermore, infection upregulated the expression of the rate-limiting enzyme in the polyamine biosynthetic pathway, ornithine decarboxylase, diverting the iNOS substrate l-arginine toward the synthesis of polyamines. Inhibition of ornithine decarboxylase activity using small interfering RNA or the competitive inhibitor difluoromethylornithine restored iNOS protein expression and activity in infected cells and inhibited chlamydial growth. This inhibition was mediated through tyrosine nitration of chlamydial protein by peroxynitrite, an NO metabolite. Thus, Chlamydia evades innate immunity by inhibiting NO production through induction of the alternative polyamine pathway.
... Primary lung fibroblasts were prepared from wildtype and TRAIL-R-deficient mice, following the procedures previously described [75,76]. Fourteen days post isolation, the primary lung fibroblasts were seeded into 12 well plates for infection assays, as we previously described [77]. ...
The immune system eliminates Chlamydia trachomatis infection through inflammation. However, uncontrolled inflammation can enhance pathology. In mice, TNF-related apoptosis-inducing ligand receptor (TRAIL-R), known for its effects on apoptosis, also regulates inflammation. In humans, the four homologues of TRAIL-R had never been investigated for effects on inflammation. Here, we examined whether TRAIL-R regulates inflammation during chlamydial infection. We examined TRAIL-R1 single nucleotide polymorphisms (SNPs) in an Ecuadorian cohort with and without C. trachomatis infections. There was a highly significant association for the TRAIL+626 homozygous mutant GG for infection vs no infection in this population. To confirm the results observed in the human population, primary lung fibroblasts and bone marrow-derived macrophages (BMDMs) were isolated from wildtype (WT) and TRAIL-R-deficient mice, and TRAIL-R1 levels in human cervical epithelial cells were depleted by RNA interference. Infection of BMDMs and primary lung fibroblasts with C. trachomatis strain L2, or the murine pathogen C. muridarum, led to higher levels of MIP2 mRNA expression or IL-1β secretion from TRAIL-R-deficient cells than WT cells. Similarly, depletion of TRAIL-R1 expression in human epithelial cells resulted in a higher level of IL-8 mRNA expression and protein secretion during C. trachomatis infection. We conclude that human TRAIL-R1 SNPs and murine TRAIL-R modulate the innate immune response against chlamydial infection. This is the first evidence that human TRAIL-R1 is a negative regulator of inflammation and plays a role in modulating Chlamydia pathogenesis.
... Infection of mice deficient in the signaling adapter MyD88 exhibit increased chlamydial burdens, increased times to resolution, and decreased recruitment of CD4 ϩ T cells (41). Mice deficient in effector mechanisms such as the production of nitric oxide clear genital infections normally (42)(43)(44) yet show altered susceptibility to infection-associated pathologies such as hydrosalpinx (45). Finally, monocytes and neutrophils infiltrate sites of chlamydial infection (9). ...
Chlamydia trachomatis is a Gram-negative obligate intracellular bacterium that preferentially infects epithelial cells. Professional phagocytes
provide C. trachomatis only a limited ability to survive and are proficient killers of chlamydiae. We present evidence herein that identifies a
novel host defense protein, perforin-2, that plays a significant role in the eradication of C. trachomatis during the infection of macrophages. Knockdown of perforin-2 in macrophages did not alter the invasion of host cells but
did result in chlamydial growth that closely mirrored that detected in HeLa cells. C trachomatis L2, serovar B, and serovar D and C. muridarum were all equally susceptible to perforin-2-mediated killing. Interestingly, induction of perforin-2 expression in epithelial
cells is blocked during productive chlamydial growth, thereby protecting chlamydiae from bactericidal attack. Ectopic expression
of perforin-2 in HeLa cells, however, does result in killing. Overall, our data implicate a new innate resistance protein
in the control of chlamydial infection and may help explain why the macrophage environment is hostile to chlamydial growth.
... Mice treated with an anti-IL-12p40 antibody but not those treated with an IL-4 neutralizing antibody exhibit prolonged infection, and neutralization of IL-12 was associated with significantly reduced production of IFN upon restimulation of splenocytes(75). Although, Ifn -/mice clear 99.9% of C. muridarum infection from the genital tract with near normal kinetics(74,75), and inos -/mice appear to clear infection normally(93), significant deficits in host defense were observed in this mice. Mice deficient in IFN developed ...
Sexually transmitted infections with Chlamydia trachomatis are highly prevalent and can lead to irreversible tissue damage if infection ascends from the cervix to the upper reproductive tract. The goal of these studies was to determine key immune responses involved in either the development of oviduct pathology or protection from infection. Using the mouse model of Chlamydia genital tract infection, we determined that increased neutrophil recruitment and delayed apoptosis were associated with enhanced oviduct damage. Despite the important role of neutrophils in Chlamydia-induced tissue damage, we were unable to detect a central role for IL-17, IL-22, or IL-23 in neutrophil recruitment or the development of pathology. We found that IL-17 did promote Th1 immunity to infection but was not required for normal resolution of infection. Finally, we determined that expression of the adaptor molecule MyD88 by CD4+ T cells was required for efficient resolution of Chlamydia from the genital tract due to its role in enhancing the survival of these cells. These studies revealed that increased influx, survival, or activation of innate immune cells in the genital tract was associated with enhanced disease while prolonged survival of CD4+ T cells was associated with more efficient clearance of bacteria from the genital tract.
... In vitro Chlamydia-specific Th1 cells terminated replication by inducing epithelial nitric oxide production to chlamydiacidal levels through a combination of IFN-c and T cell-epithelial cell contact [9,10]. However in vivo studies were discordant as mice deficient in inducible nitric oxide synthetase (iNOS) resolved C. muridarum genital tract infections [11,12]; suggesting that nitric oxide was either irrelevant to bacterial clearance or redundant in the setting of a second independent clearance mechanism. ...
CD4 T cells are critical for clearing experimental Chlamydia muridarum genital tract infections. Two independent in vitro CD4 T cell mechanisms have been identified for terminating Chlamydia replication in epithelial cells. One mechanism, requiring IFN-γ and T cell-epithelial cell contact, terminates infection by triggering epithelial production of nitric oxide to chlamydiacidal levels; the second is dependent on T cell degranulation. We recently demonstrated that there are two independent in vivo clearance mechanisms singly sufficient for clearing genital tract infections within six weeks; one dependent on iNOS, the other on Plac8. Redundant genital tract clearance mechanisms bring into question negative results in single-gene knockout mice. Two groups have shown that perforin-knockout mice were not compromised in their ability to clear C. muridarum genital tract infections. Because cell lysis would be detrimental to epithelial nitric oxide production we hypothesized that perforin was not critical for iNOS-dependent clearance, but posited that perforin could play a role in Plac8-dependent clearance. We tested whether the Plac8-dependent clearance was perforin-dependent by pharmacologically inhibiting iNOS in perforin-knockout mice. In vitro we found that perforin was detrimental to iNOS-dependent CD4 T cell termination of Chlamydia replication in epithelial cells. In vivo, unexpectedly, clearance in perforin knockout mice was delayed to the end of week 7 regardless of iNOS status. The discordant in vitro/in vivo results suggest that the perforin's contribution to bacterial clearance in vivo is not though enhancing CD4 T cell termination of Chlamydia replication in epithelial cells, but likely via a mechanism independent of T cell-epithelial cell interactions.
... In vitro studies using a Chlamydia-specific CD4 T cell clone capable of terminating C. muridarum replication in epithelial cells identified a probable mechanism for sterilizing CD4 T cell-mediated immunity that was dependent on inducible NO synthase (iNOS), IFN-g, and T cell/epithelial cell contact via LFA-1/ICAM-1 (11)(12)(13)(14). However, follow-up studies showed that iNOS knockout mice were not compromised in clearance of C. muridarum genital tract infections (15,16), and that IFN-g knockout mice cleared 99.9% of C. muridarum from the genital tract with near normal kinetics (17,18). Furthermore, mice deficient in the known T cell killing mechanisms perforin and Fas ligand/Fas were also able to clear C. muridarum genital tract infections with normal kinetics (19). ...
Chlamydia trachomatis urogenital serovars replicate predominantly in genital tract epithelium. This tissue tropism poses a unique challenge for host defense and vaccine development. Studies utilizing the Chlamydia muridarum mouse model have shown that CD4 T cells are critical for clearing genital tract infections. In vitro studies have shown that CD4 T cells terminate infection by upregulating epithelial inducible NO synthase (iNOS) transcription and NO production. However, this mechanism is not critical, as iNOS-deficient mice clear infections normally. We recently showed that a subset of Chlamydia-specific CD4 T cell clones could terminate replication in epithelial cells using an iNOS-independent mechanism requiring T cell degranulation. We advance that work using microarrays to compare iNOS-dependent and iNOS-independent CD4 T cell clones. Plac8 was differentially expressed by clones having the iNOS-independent mechanism. Plac8-deficient mice had delayed clearance of infection, and Plac8-deficient mice treated with the iNOS inhibitor N-monomethyl-l-arginine were largely unable to resolve genital tract infections over 8 wk. These results demonstrate that there are two independent and redundant T cell mechanisms for clearing C. muridarum genital tract infections: one dependent on iNOS, and the other dependent on Plac8. Although T cell subsets are routinely defined by cytokine profiles, there may be important subdivisions by effector function, in this case CD4(Plac8).
