Differential signaling pathways are initiated in macrophages during infection depending on the intracellular fate of Chlamydia spp.

Chlamydia muridarum and Chlamydia caviae have equivalent growth rates in mouse epithelial cells but only C. muridarum replicates inside mouse macrophages, while C. caviae does not. Macrophages infected with C. muridarum or C. caviae were used to address the hypothesis that the early signaling pathways initiated during infection depend on the fate of chlamydiae in the host cell. Transmission electron microscopy of C. muridarum-infected macrophages showed intact chlamydial elementary bodies and reticulate bodies 2 h postinfection in compact vacuoles. Conversely, in macrophages infected with C. caviae, chlamydiae were observed in large phagocytic vacuoles. Furthermore, C. caviae infections failed to develop into inclusions or produce viable bacteria. Expression of proinflammatory cytokines TNFα, IL-1ß and MMP13 was similar in C. caviae- or C. muridarum-infected macrophages at 3 h postinfection, indicating that chlamydial survival is not required for initiation of these responses. IL-1ß secretion, dependent on inflammasome activation, occurred in C. caviae-infected macrophages despite no chlamydial growth. Conversely, IFNß mRNA was observed only in C. muridarum- but not in C. caviae-infected macrophages. These data demonstrate that differential signaling events are initiated during a productive versus nonproductive chlamydial infection in a macrophage.

MicroRNAs Modulate Pathogenesis Resulting from Chlamydial Infection in Mice.

Not all women infected with chlamydiae develop upper genital tract disease, but the reason(s) for this remains undefined. Host genetics and hormonal changes associated with the menstrual cycle are possible explanations for variable infection outcomes. It is also possible that disease severity depends on the virulence of the chlamydial inoculum. It is likely that the inoculum contains multiple genetic variants, differing in virulence. If the virulent variants dominate, then the individual is more likely to develop severe disease. Based on our previous studies, we hypothesized that the relative degree of virulence of a chlamydial population dictates the microRNA (miRNA) expression profile of the host, which, in turn, through regulation of the host inflammatory response, determines disease severity. Thus, we infected C57BL/6 mice with two populations of Chlamydia muridarum, each comprised of multiple genetic variants and differing in virulence: an attenuated strain (NiggA) and a virulent strain (NiggV). NiggA and NiggV elicited upper tract pathology in 54% and 91% of mice, respectively. miRNA expression analysis in NiggV-infected mice showed significant downregulation of miRNAs involved in dampening fibrosis (miR-200b, miR-200b-5p, and 200b-3p miR-200a-3p) and in transcriptional regulation of cytokine responses (miR-148a-3p, miR-152-3p, miR-132, and miR-212) and upregulation of profibrotic miRNAs (miR-142, and miR-147). Downregulated miRNAs were associated with increased expression of interleukin 8 (IL-8), CXCL2, IL-1ß, tumor necrosis factor alpha (TNF-α), and IL-6. Infection with NiggV but not NiggA led to decreased expression of Dicer and Ago 2, suggesting that NiggV interaction with host cells inhibits expression of the miRNA biogenesis machinery, leading to increased cytokine expression and pathology.

Formula diet alters small intestine morphology, microbial abundance and reduces VE-cadherin and IL-10 expression in neonatal porcine model.

BACKGROUND: Breastfeeding is associated with a variety of positive health outcomes in children and is recommended exclusively for the first 6 months of life; however, 50-70 % of infants in the US are formula-fed. To test the hypothesis that immune system development and function in neonates and infants are significantly influenced by diet, 2-day old piglets were fed soy or milk formula (n = 6/group/gender) until day 21 and compared to a sow-fed group (n = 6/gender). METHODS: Histomorphometric analyses of ileum, jejunum and Peyer's patches were carried out, to determine the inflammation status, mRNA and protein expression of pro-inflammatory, anti-inflammatory and growth-related chemokines and cytokines. RESULTS: In formula-fed animals, increases in ileum and jejunum villus height and crypt depth were observed in comparison to sow-fed animals (jejunum, p < 0.01 villus height, p < 0.04 crypt depth; ileum p < 0.001 villus height, p < 0.002 crypt depth). In formula-fed the lymphoid follicle size (p < 0.01) and germinal centers (p < 0.01) with in the Peyer's patch were significantly decreased in comparison to sow-fed, indicating less immune education. In ileum, formula diet induced significant up-regulation of AMCFII, IL-8, IL-15, VEGFA, LIF, FASL, CXCL11, CCL4, CCL25 and down-regulation of IL-6, IL-9, IL-10, IL-27, IFNA4, CSF3, LOC100152038, and LOC100736831 at the transcript level. We have confirmed some of the mRNA data by measuring protein, and significant down-regulation of anti-inflammatory molecule IL-10 in comparison to sow-fed piglets was observed. To further determine the membrane protein expression in the ileum, VE-cadherin, occludin, and claudin-3, Western blot analyses were conducted. Sow fed piglets showed significantly more VE-Cadherin, which associated with levels of calcium, and putrescine measured. It is possible that differences in GI tract and immune development are related to shifts in the microbiome; notably, there were 5-fold higher amounts of Lactobacillaceae spp and 3 fold higher Clostridia spp in the sow fed group in comparison to milk formula-fed piglets, whereas in milk formula-fed pigs Enterobacteriaceae spp was 5-fold higher. CONCLUSION: In conclusion, formula diet alters GI morphology, microbial abundance, intestinal barrier protein VE-cadherin and anti-inflammatory molecule IL-10 expression. Further characterization of formula effects could lead to modification of infant formula to improve immune function, reduce inflammation and prevent conditions such as allergies and infections.

Early microRNA expression profile as a prognostic biomarker for the development of pelvic inflammatory disease in a mouse model of chlamydial genital infection.

UNLABELLED: It is not currently possible to predict the probability of whether a woman with a chlamydial genital infection will develop pelvic inflammatory disease (PID). To determine if specific biomarkers may be associated with distinct chlamydial pathotypes, we utilized two Chlamydia muridarum variants (C. muridarum Var001 [CmVar001] and CmVar004) that differ in their abilities to elicit upper genital tract pathology in a mouse model. CmVar004 has a lower growth rate in vitro and induces pathology in only 20% of C57BL/6 mouse oviducts versus 83.3% of oviducts in CmVar001-infected mice. To determine if chemokine and cytokine production within 24 h of infection is associated with the outcome of pathology, levels of 15 chemokines and cytokines were measured. CmVar004 infection induced significantly lower levels of CXCL1, CXCL2, tumor necrosis factor alpha (TNF-α), and CCL2 in comparison to CmVar001 infection with similar rRNA (rs16) levels for Chlamydiae. A combination of microRNA (miRNA) sequencing and quantitative real-time PCR (qRT-PCR) analysis of 134 inflammation-related miRNAs was performed 24 h postinfection to determine if the chemokine/cytokine responses would also be reflected in miRNA expression profiles. Interestingly, 12 miRNAs (miR-135a-5p, miR298-5p, miR142-3p, miR223-3p, miR299a-3p, miR147-3p, miR105, miR325-3p, miR132-3p, miR142-5p, miR155-5p, and miR-410-3p) were overexpressed during CmVar004 infection compared to CmVar001 infection, inversely correlating with the respective chemokine/cytokine responses. To our knowledge, this is the first report demonstrating that early biomarkers elicited in the host can differentiate between two pathological variants of chlamydiae and be predictive of upper tract disease. IMPORTANCE: It is apparent that an infecting chlamydial population consists of multiple genetic variants with differing capabilities of eliciting a pathological response; thus, it may be possible to identify biomarkers specific for a given virulence pathotype. miRNAs are known to regulate genes that in turn regulate signaling pathways involved in disease pathogenesis. Importantly, miRNAs are stable and can reflect a tissue response and therefore have the potential to be biomarkers of disease severity. Currently, with respect to chlamydial infections, there is no way to predict whether an infected patient is more or less likely to develop PID. However, data presented in this study indicate that the expression of a specific miRNA profile associated with a virulent variant early in the infection course may be predictive of an increased risk of pelvic inflammatory disease, allowing more aggressive treatment before significant pathology develops.

Effect of inflammatory response on in vivo competition between two chlamydial variants in the guinea pig model of inclusion conjunctivitis.

In order to study the interaction of variants in in vivo infection, we employed an azithromycin-resistant mutant (AZ(2)) and its wild-type parent (SP(6)) in the guinea pig model of Chlamydia caviae conjunctival infection. When each strain was inoculated individually into conjunctiva, both attained the same level of growth, but AZ(2) elicited less pathology. However, when equal numbers of the two strains were inoculated together into the guinea pig conjunctiva, SP(6) produced a significantly greater number of inclusion-forming units than AZ(2), and the pathology reflected that of a SP(6) monoinfection. The goal of this study was to further characterize the dynamics of concomitant infection of these two distinct variants, with particular emphasis on the impact of the host response on the in vivo growth of each organism and the development of pathology. Animals infected with AZ(2) had reduced conjunctival infiltration with CD45(+) cells and neutrophils as well as a reduced interleukin-8 (IL-8) response. Gene expression of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), CCL2, and CCL5 was also significantly lower in AZ(2)-infected animals. The lower inflammatory response induced by AZ(2) was associated with its decreased ability to activate NF-κB via Toll-like receptor 2 (TLR2). In general, the inflammatory response in animals infected with both variants was greater than in infection with AZ(2) alone, resulting in lower numbers of AZ(2) than those of SP(6) in the mixed infection. Our results suggest that the ability to elicit an inflammatory response is an important factor in the dynamics of mixed infection with strains that display different pathological phenotypes.

In vivo ultrastructural analysis of the intimate relationship between polymorphonuclear leukocytes and the chlamydial developmental cycle.

We utilized a recently developed model of intracervical infection with Chlamydia muridarum in the mouse to elicit a relatively synchronous infection during the initial developmental cycle in order to examine at the ultrastructural level the development of both the chlamydial inclusion and the onset of the inflammatory response. At 18 h after infection, only a few elementary bodies attached to cells were visible, as were an occasional intracellular intermediate body and reticulate body. By 24 h, inclusions had 2 to 5 reticulate bodies and were beginning to fuse. A few polymorphonuclear leukocytes (PMNs) were already present in the epithelium in the vicinity of and directly adjacent to infected cells. By 30 h, the inclusions were larger and consisted solely of reticulate bodies, but by 36 to 42 h, they contained intermediate bodies and elementary bodies as well. Many PMNs were adjacent to or actually inside infected cells. Chlamydiae appeared to exit the cell either (i) through disintegration of the inclusion membrane and rupture of the cell, (ii) by dislodgement of the cell from the epithelium by PMNs, or (iii) by direct invasion of the infected cell by the PMNs. When PMNs were depleted, the number of released elementary bodies was significantly greater as determined both visually and by culture. Interestingly, depletion of PMNs revealed the presence of inclusions containing aberrant reticulate bodies, reminiscent of effects seen in vitro when chlamydiae are incubated with gamma interferon. In vivo evidence for the contact-dependent development hypothesis, a potential mechanism for triggering the conversion of reticulate bodies to elementary bodies, and for translocation of lipid droplets into the inclusion is also presented.

Galectin-3 is a substrate for prostate specific antigen (PSA) in human seminal plasma.

BACKGROUND: Galectin-3 is a multivalent carbohydrate-binding protein involved in cell adhesion, cell cycle control, immunomodulation, and cancer progression, including prostate cancer. Galectin-3 function is regulated by proteolytic cleavage that destroys galectin-3 multivalency while preserving carbohydrate-binding activity. In human semen, galectin-3 is present in seminal plasma and is also associated with prostasomes, exosome-like vesicles secreted by the prostate. In the current study, we characterized the proteolytic activity that cleaves galectin-3 in human seminal plasma. METHODS: An in vitro assay was developed to investigate galectin-3 cleavage in seminal plasma. The effect of protease inhibitors, divalent ion chelators, and Zn(2+) on the cleavage activity was determined. Proteases enriched from seminal plasma were tested for their ability to cleave galectin-3. Affinity purification and microsequence analysis were used to identify the cleavage site in galectin-3. RESULTS: Galectin-3 was identified in human seminal plasma in an intact and truncated form. Gelatinases enriched from seminal plasma did not cleave galectin-3. Inhibitor studies indicated that the galectin-3 cleavage activity in seminal plasma is a Zn(2+) sensitive, serine protease. Prostate specific antigen (PSA) was demonstrated to cleave galectin-3 between tyrosine¹°7-glycine¹°8 and produce a functionally active, monovalent lectin. CONCLUSIONS: PSA is a chymotrypsin-like serine protease secreted by the prostatic epithelium and normally functions in liquefaction of semen following ejaculation. Furthermore, PSA is implicated in the promotion of localized prostate tumors and bone metastases by its roles in immunomodulation, invasion, and apoptosis. Our results indicate that PSA regulates galectin-3 in human semen and may regulate galectin-3 function during prostate cancer progression.

Local host response to chlamydial urethral infection in male guinea pigs.

Very little is known about the host response to chlamydial genital infection in the male, particularly about the nature of the local response in the urethra. In this study, the pathological and immunologic responses to urethral infection of the male guinea pig with Chlamydia caviae (Chlamydophila caviae) were characterized both during a primary infection and following a challenge infection. A dose-response experiment found that the 50% infectious dose for male urethral infection was 78 inclusion-forming units. The histopathologic response was similar to that of the female, with an initial acute inflammatory response followed by a chronic inflammatory response and plasma cell infiltration. Production of IgG and IgA antibodies in local urethral secretions developed following infection, and levels of both increased in a typical anamnestic response following a challenge infection. CD4 and CD8 T cells, as well as B cells, were observed in the local site by flow cytometry, with a slightly increased number of CD8 cells. Following challenge infection, the dominant anamnestic response was solely in the B-cell compartment, with only a minimal number of T cells. The T-cell response was clearly a Th1 response, as judged by increased levels of gamma interferon (IFN-gamma), interleukin-12 p40 (IL-12p40), and IL-2. The proinflammatory cytokines and chemokines IL-8, IL-1beta, tumor necrosis factor alpha (TNF-alpha), CCL2 (monocyte chemoattractant protein 1 [MCP-1]), and CCL5 (RANTES) were elicited in the urethra following primary infection, but only CCL5 showed increased levels upon challenge. This study represents the first comprehensive analysis of the local immune response in the male urethra to a chlamydial genital infection.

Critical role for interleukin-1beta (IL-1beta) during Chlamydia muridarum genital infection and bacterial replication-independent secretion of IL-1beta in mouse macrophages.

Recent findings have implicated interleukin-1beta (IL-1beta) as an important mediator of the inflammatory response in the female genital tract during chlamydial infection. But how IL-1beta is produced and its specific role in infection and pathology are unclear. Therefore, our goal was to determine the functional consequences and cellular sources of IL-1beta expression during a chlamydial genital infection. In the present study, IL-1beta(-/-) mice exhibited delayed chlamydial clearance and decreased frequency of hydrosalpinx compared to wild-type (WT) mice, implying an important role for IL-1beta both in the clearance of infection and in the mediation of oviduct pathology. At the peak of IL-1beta secretion in WT mice, the major producers of IL-1beta in vivo are F4/80(+) macrophages and GR-1(+) neutrophils, but not CD45(-) epithelial cells. Although elicited mouse macrophages infected with Chlamydia muridarum in vitro secrete minimal IL-1beta, in vitro prestimulation of macrophages by Toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS) purified from Escherichia coli or C. trachomatis L2 prior to infection greatly enhanced secretion of IL-1beta from these cells. By using LPS-primed macrophages as a model system, it was determined that IL-1beta secretion was dependent on caspase-1, potassium efflux, and the activity of serine proteases. Significantly, chlamydia-induced IL-1beta secretion in macrophages required bacterial viability but not growth. Our findings demonstrate that IL-1beta secreted by macrophages and neutrophils has important effects in vivo during chlamydial infection. Additionally, prestimulation of macrophages by chlamydial TLR ligands may account for the elevated levels of pro-IL-1beta mRNA observed in vivo in this cell type.

Strain and virulence diversity in the mouse pathogen Chlamydia muridarum.

The mouse chlamydial pathogen Chlamydia muridarum has been used as a model organism for the study of human Chlamydia trachomatis urogenital and respiratory tract infections. To date, two commonly used C. muridarum isolates have been used interchangeably and are essentially taken to be identical. Herein, we present data that indicate that this is not the case. The C. muridarum Weiss isolate and C. muridarum Nigg isolate varied significantly in their virulences in vivo and possessed different growth characteristics in vitro. Distinct differences were observed in intravaginal 50% infectious doses and in challenge infections, with the Weiss isolate displaying greater virulence. Respiratory infection by the intranasal route also indicated a greater virulence of the Weiss isolate. In vitro, morphometric analysis revealed that the Weiss isolate produced consistently smaller inclusions in human cervical adenocarcinoma cells (HeLa 229) and smaller plaques in monolayers of mouse fibroblasts (L929) than did the Nigg isolate. In addition, the Weiss isolate possessed significantly higher replicative yields in vitro than did the Nigg isolate. In plaque-purified isolates derived from our stocks of these two strains, total genomic sequencing identified several unique nonsynonymous single nucleotide polymorphisms and insertion/deletion mutations when our Weiss (n = 4) and Nigg (n = 5) isolates were compared with the published Nigg sequence. In addition, the two isolates shared 11 mutations compared to the published Nigg sequence. These results prove that there is genotypic and virulence diversity among C. muridarum isolates. These findings can be exploited to determine factors related to chlamydial virulence and immunity.

Chlamydiae and polymorphonuclear leukocytes: unlikely allies in the spread of chlamydial infection.

While much is known about the attachment of the chlamydiae to the host cell and intracellular events during the developmental cycle, little is known about the mechanism(s) by which elementary bodies exit the cell. In this report, we use the guinea-pig conjunctival model of Chlamydia caviae infection to present in vivo ultrastructural evidence supporting two mechanisms for release of chlamydiae from the mucosal epithelia. Four days after infection, histopathologic observation shows an intense infiltration of polymorphonuclear leukocytes (PMN) in the conjunctival epithelium. Using transmission electron microscopy, a gradient-directed PMN response to chlamydiae-infected epithelial cells was observed. As PMN infiltration intensifies, epithelial hemidesmosome/integrin/focal adhesion adherence with the basal lamina is disconnected and PMNs literally lift off and release infected superficial epithelia from the mucosa. Many of these infected cells appear to be healthy with intact microvilli, nuclei, and mitochondria. While lysis of some infected cells occurs with release of chlamydiae into the extracellular surface milieu, the majority of infected cells are pushed off the epithelium. We propose that PMNs play an active role in detaching infected cells from the epithelium and that these infected cells eventually die releasing organisms but, in the process, move to new tissue sites via fluid dynamics.

Chlamydia trachomatis induces expression of IFN-gamma-inducible protein 10 and IFN-beta independent of TLR2 and TLR4, but largely dependent on MyD88.

IFN-gamma-inducible protein 10 (IP-10) is a chemokine important in the attraction of T cells, which are essential for resolution of chlamydial genital tract infection. During infections with Gram-negative bacteria, the IP-10 response mediated through type I IFNs usually occurs as a result of TLR4 stimulation by bacterial LPS. However, we found that levels of IP-10 in genital tract secretions of Chlamydia trachomatis-infected female wild-type mice were similar to those of infected TLR2- and TLR4-deficient mice but significantly greater than those of infected MyD88-deficient mice. We investigated the mechanism of IP-10 and IFN-beta induction during chlamydial infection using mouse macrophages and fibroblasts infected ex vivo. The induction of IP-10 and IFN-beta was unchanged in Chlamydia-infected TLR2- and TLR4-deficient cells compared with wild-type cells. However, infection of MyD88-deficient cells resulted in significantly decreased responses. These results suggest a role for MyD88-dependent pathways in induction of IP-10 and IFN-beta during chlamydial infection. Furthermore, treatment of infected macrophages with an endosomal maturation inhibitor significantly reduced chlamydial-induced IFN-beta. Because endosomal maturation is required for MyD88-dependent intracellular pathogen recognition receptors to function, our data suggest a role for the intracellular pathogen recognition receptor(s) in induction of IFN-beta and IP-10 during chlamydial infection. Furthermore, the intracellular pathways that lead to chlamydial-induced IFN-beta function through TANK-binding kinase mediated phosphorylation and nuclear translocation of IFN regulatory factor-3.

Role of proapoptotic BAX in propagation of Chlamydia muridarum (the mouse pneumonitis strain of Chlamydia trachomatis) and the host inflammatory response.

The BCL-2 family member BAX plays a critical role in regulating apoptosis. Surprisingly, bax-deficient mice display limited phenotypic abnormalities. Here we investigate the effect of BAX on infection by the sexually transmitted pathogen, Chlamydia muridarum (the mouse pneumonitis strain of Chlamydia trachomatis). Bax(-/-) cells are relatively resistant to Chlamydia-induced apoptosis, and fewer bacteria are recovered after two infection cycles from Bax(-/-) cells than from wild-type cells. These results suggest that BAX-dependent apoptosis may be used to initiate a new round of infection, most likely by releasing Chlamydia-containing apoptotic bodies from infected cells that could be internalized by neighboring uninfected cells. Nonetheless, infected Bax(-/-) cells die through necrosis, which is normally associated with inflammation, more often than infected wild-type cells. These studies were confirmed in mice infected intravaginally with C. muridarum; since the infection disappears more quickly from Bax(-/-) mice than from wild-type mice, secretion of proinflammatory cytokines is increased in Bax(-/-) mice, and large granulomas are present in the genital tract of Bax(-/-) mice. Taken together, these data suggest that chlamydia-induced apoptosis via BAX contributes to bacterial propagation and decreases inflammation. Bax deficiency results in lower infection and an increased inflammatory cytokine response associated with more severe pathology.