Rishein Gupta

Chlamydia muridarum infection associated host microRNAs in the murine genital tract and contribution to generation of host immune response

Chlamydia trachomatis (CT) is the leading sexually transmitted bacterial infection in humans and is associated with reproductive tract damage. However, little is known about the involvement and regulation of microRNAs (miRs) in genital CT.

Vaginal chlamydial clearance following primary or secondary infection in mice occurs independently of TNF-α

The role of TNF-α in chlamydial clearance is uncertain. Antibody-mediated depletion of TNF-α in mice and guinea pigs has been shown not to significantly affect chlamydial clearance, whereas production of TNF-α in addition to IFN-γ from T cells has been shown to correlate with enhanced clearance. The aim of our study is to evaluate the mechanistic role of TNF-α in clearance of primary and secondary chlamydial infection from the genital tract (GT) using C57BL/6 TNF-α deficient (TNF-α−/−) and wild type (WT) mice. Chlamydial shedding from the lower GT was evaluated following primary and secondary intravaginal challenge. Also, antibody and antigen specific cytokine responses were analyzed from the infected GT and spleens, and oviduct pathology determined to analyze the role of TNF-α in upper GT pathological sequelae. MHC II−/− mice, known to display muted adaptive immune responses and failure to resolve genital chlamydial infections, were used as a negative control. Following both primary and secondary genital chlamydial infection, TNF-α−/− mice exhibited elevated granzyme B production, but similar IFN-γ and antibody responses. Importantly, absence of TNF-α did not significantly alter the resolution of infection. However, TNF-α−/− mice displayed significantly reduced upper genital tract (UGT) pathology compared to WT mice. This study demonstrates mechanistically that optimal chlamydial clearance following primary and secondary chlamydial genital infection can occur in the complete absence of TNF-α, and considered with the reduction of upper GT pathology in TNF-α−/− mice, suggests that targeted induction of anti-chlamydial TNF-α responses by vaccination may be unnecessary, and moreover could be potentially pathogenic.

Murine MicroRNA-214 regulates intracellular adhesion molecule (ICAM1) gene expression in genital Chlamydia muridarum infection

The hallmark of chlamydial infection is the development of upper genital pathology in the form of hydrosalpinx and oviduct and/or tubal dilatation. Although molecular events leading to genital tissue presentation and cellular architectural remodelling are unclear, early‐stage host immune responses are believed to contribute to these long‐term sequelae. Recently, we reported the contribution of selected infection‐associated microRNAs (miRs) in the generation of host immunity at early‐stage infection (day 6 after intravaginal Chlamydia muridarum challenge in C57BL/6 mice). In this report, we describe the contribution of an infection‐associated microRNA, i.e. miR‐214, to host immunity. Chlamydia muridarum infection in the C57BL/6 mouse genital tract significantly down‐regulated miR‐214 while up‐regulating intracellular adhesion molecule 1 (ICAM1) gene expression. These in vivo observations were confirmed by establishing direct regulation of ICAM‐1 by miR‐214 in ex vivo genital cell cultures in the presence of miR‐214 mimic and inhibitor. Because, ICAM‐1 contributes to recruitment of neutrophils following infection, we also demonstrated that alteration of ICAM1 by miR‐214 in interleukin‐17A‐deficient (IL‐17A−/−) mice correlated with reduction of neutrophils infiltrating genital tissue at day 6 after challenge. Additionally, these early‐stage events resulted in significantly decreased genital pathology in IL‐17A−/− mice compared with C57BL/6 mice. This report provides evidence for early‐stage regulation of ICAM1 by microRNAs, resulting in reduction of genital pathology associated with chlamydial infection.

Use of a guinea pig-specific transcriptome array for evaluation of protective immunity against genital chlamydial infection following intranasal vaccination in guinea pigs

Guinea pigs have been used as a second animal model to validate putative anti-chlamydial vaccine candidates tested in mice. However, the lack of guinea pig-specific reagents has limited the utility of this animal model in Chlamydia sp. vaccine studies. Using a novel guinea pig-specific transcriptome array, we determined correlates of protection in guinea pigs vaccinated with Chlamydia caviae (C. caviae) via the intranasal route, previously reported by us and others to provide robust antigen specific immunity against subsequent intravaginal challenge. C. caviae vaccinated guinea pigs resolved genital infection by day 3 post challenge. In contrast, mock vaccinated animals continued to shed viable Chlamydia up to day 18 post challenge. Importantly, at day 80 post challenge, vaccinated guinea pigs experienced significantly reduced genital pathology - a sequelae of genital chlamydial infections, in comparison to mock vaccinated guinea pigs. Sera from vaccinated guinea pigs displayed antigen specific IgG responses and increased IgG1 and IgG2 titers capable of neutralizing GPIC in vitro. Th1-cellular/inflammatory immune genes and Th2-humoral associated genes were also found to be elevated in vaccinated guinea pigs at day 3 post-challenge and correlated with early clearance of the bacterium. Overall, this study provides the first evidence of guinea pig-specific genes involved in anti-chlamydial vaccination and illustrates the enhancement of the utility of this animal model in chlamydial pathogenesis.

Antigen specific immune response in Chlamydia muridarum genital infection is dependent on murine microRNAs-155 and -182

Anti-chlamydial immunity involves efficient presentation of antigens (Ag) to effector cells resulting in Ag-specific immune responses. There is limited information on inherent underlying mechanisms regulating these events. Previous studies from our laboratory have established that select microRNAs (miRs) function as molecular regulators of immunity in Chlamydia muridarum (Cm) genital infection. In this report, we investigated immune cell type-specific miRs, i.e. miR-155 and -182, and the role in Ag-specific immunity. We observed significant up-regulation of miR-155 in C57BL/6 bone marrow derived dendritic cells (BMDC), and miR-182 in splenic Ag-specific CD4+ T-cells. Using mimics and inhibitors, we determined that miR-155 contributed to BMDC activation following Cm infection. Co-cultures of miR-155 over-expressed in BMDC and miR-182 over-expressed in Ag-specific CD4+ T-cells, or miR-155−/− BMDC with miR-182 inhibitor treated Ag-specific CD4+ T-cells, resulted in IFN-γ production comparable to Ag-specific CD4+ T-cells isolated from Cm infected mice. Additionally, miR-182 was significantly up-regulated in intranasally vaccinated mice protected against Cm infection. In vivo depletion of miR-182 resulted in reduction in Ag-specific IFN-γ and genital pathology in Cm infected mice. To the best of our knowledge, this is the first study to report an interaction of miR-155 (in Cm infected DC) and miR-182 (in CD4+ T-cell) resulting in Ag specific immune responses against genital Cm.

Chlamydial protease-like activity factor mediated protection against C. trachomatis in guinea pigs.

We have comprehensively demonstrated using the mouse model that intranasal immunization with recombinant chlamydial protease‐like activity factor (rCPAF) leads to a significant reduction in bacterial burden, genital tract pathology and preserves fertility following intravaginal genital chlamydial challenge. In the present report, we evaluated the protective efficacy of rCPAF immunization in guinea pigs, a second animal model for genital chlamydial infection. Using a vaccination strategy similar to the mouse model, we intranasally immunized female guinea pigs with rCPAF plus CpG deoxynucleotides (CpG; as an adjuvant), and challenged intravaginally with C. trachomatis serovar D (CT‐D). Immunization with rCPAF/CpG significantly reduced vaginal CT‐D shedding and induced resolution of infection by day 24, compared with day 33 in CpG alone treated and challenged animals. Immunization induced robust anti‐rCPAF serum IgG 2 weeks following the last immunization, and was sustained at a high‐level 4 weeks post challenge. Upregulation of antigen‐specific IFN‐γ gene expression was observed in rCPAF/CpG‐vaccinated splenocytes. Importantly, a significant reduction in inflammation in the genital tissue in rCPAF/CpG‐immunized guinea pigs compared with CpG‐immunized animals was observed. Taken together, this study provides evidence of the protective efficacy of rCPAF as a vaccine candidate in a second animal model of genital chlamydial infection.

MicroRNA mediated regulation of immunity against gram-negative bacteria

ABSTRACT Evidence over the last couple decades has comprehensively established that short, highly conserved, non-coding RNA species called microRNA (miRNA) exhibit the ability to regulate expression and function of host genes at the messenger RNA (mRNA) level. MicroRNAs play key regulatory roles in immune cell development, differentiation, and protective function. Intrinsic host immune response to invading pathogens rely on intricate orchestrated events in the development of innate and adaptive arms of immunity. We discuss the involvement of miRNAs in regulating these processes against gram negative pathogens in this review.

Temporal proteomic profiling of Chlamydia trachomatis–infected HeLa‐229 human cervical epithelial cells

Chlamydia trachomatis is the leading causative agent of bacterial sexually transmitted infections worldwide which can lead to female pelvic inflammatory disease and infertility. A greater understanding of host response during chlamydial infection is essential to design intervention technique to reduce the increasing incidence rate of genital chlamydial infection. In this study, we investigated proteome changes in epithelial cells during C. trachomatis infection by using an isobaric tags for relative and absolute quantitation (iTRAQ) labeling technique coupled with a liquid chromatography‐tandem mass spectrometry (LC‐MS3) analysis. C. trachomatis (serovar D, MOI 1)–infected HeLa‐229 human cervical carcinoma epithelial cells (at 2, 4 and 8 h) showed profound modifications of proteome profile which involved 606 host proteins. MGST1, SUGP2 and ATXN10 were among the top in the list of the differentially upregulated protein. Through pathway analysis, we suggested the involvement of eukaryotic initiation factor 2 (eIF2) and mammalian target of rapamycin (mTOR) in host cells upon C. trachomatis infection. Network analysis underscored the participation of DNA repair mechanism during C. trachomatis infection. In summary, intense modifications of proteome profile in C. trachomatis–infected HeLa‐229 cells indicate complex host‐pathogen interactions at early phase of chlamydial infection.

IgA modulates respiratory dysfunction as a sequela to pulmonary chlamydial infection as neonates

Neonatal Chlamydia lung infections are associated with serious sequelae such as asthma and airway hyper-reactivity in children and adults. Our previous studies demonstrated the importance of Th-1 type cytokines, IL-12 and IFN-γ in protection against neonatal pulmonary chlamydial challenge; however, the role of the humoral arm of defense has not been elucidated. We hypothesized that B-cells and IgA, the major mucosal antibody, play a protective role in newborns against development of later life respiratory sequelae to Chlamydia infection. Our studies using neonatal mice revealed that all WT and IgA-deficient (IgA(-/-)) animals survived a sublethal pulmonary Chlamydia muridarum challenge at one day after birth with similar reduction in bacterial burdens over time. In contrast, all B-cell-deficient (μMT) mice succumbed to infection at the same challenge dose correlating to failure to control bacterial burdens in the lungs. Although IgA may not be important for bacterial clearance, we observed IgA(-/-) mice displayed greater respiratory dysfunction 5 weeks post challenge. Specifically, comparative respiratory functional analyses revealed a significant shift upward in P-V loops, and higher dynamic resistance in IgA(-/-) animals. This study provides insight(s) into the protective role of IgA in neonates against pulmonary chlamydial infection induced respiratory pathological sequelae observed later in life.

Acinetobacter baumannii Gastrointestinal Colonization Is Facilitated by Secretory IgA Which Is Reductively Dissociated by Bacterial Thioredoxin A

ABSTRACT Multidrug-resistant Acinetobacter baumannii is among the most common causes of infectious complications associated with combat-related trauma in military personnel serving overseas. However, little is currently known about its pathogenesis. While the gastrointestinal (GI) tract has been found to be a major reservoir for A. baumannii, as well as to potentially contribute to development of multidrug resistance, no studies have addressed the mechanisms involved in gut colonization. In this study, we address this critical gap in knowledge by first assessing the interaction between secretory IgA (SIgA), the principal humoral immune defense on mucosal surfaces, and the A. baumannii clinical isolate Ci79. Surprisingly, SIgA appeared to enhance A. baumannii GI tract colonization, in a process mediated by bacterial thioredoxin A (TrxA), as evidenced by reduction of bacterial attachment in the presence of TrxA inhibitors. Additionally, a trxA targeted deletion mutant (ΔtrxA) showed reduced bacterial burdens within the GI tract 24 h after oral challenge by in vivo live imaging, along with loss of thiol-reductase activity. Surprisingly, not only was GI tract colonization greatly reduced but the associated 50% lethal dose (LD50) of the ΔtrxA mutant was increased nearly 100-fold in an intraperitoneal sepsis model. These data suggest that TrxA not only mediates A. baumannii GI tract colonization but also may contribute to pathogenesis in A. baumannii sepsis following escape from the GI tract under conditions when the intestinal barrier is compromised, as occurs with cases of severe shock and trauma. IMPORTANCE Acinetobacter baumannii is an emerging bacterial pathogen recently classified as a serious threat to U.S. and global health by both the Centers for Disease Control and Prevention and the World Health Organization. It also is one of the leading causes of combat-related infections associated with injured military personnel serving overseas. Little is known regarding mechanisms of gastrointestinal tract colonization despite this site being shown to serve as a reservoir for multidrug-resistant (MDR) A. baumannii isolates. Here, we establish that secretory IgA, the major immunoglobulin of mucosal surfaces, promotes A. baumannii GI tract colonization via bacterial thioredoxin A as evidenced through significant reduction in colonization in IgA-deficient animals. Additionally, bacterial colonization and mortality were significantly reduced in animals challenged with a thioredoxin A-deficient A. baumannii mutant. Combined, these data suggest that thioredoxin A is a novel virulence factor, for which antithioredoxin therapies could be developed, for this important multidrug-resistant pathogen. IMPORTANCE Acinetobacter baumannii is an emerging bacterial pathogen recently classified as a serious threat to U.S. and global health by both the Centers for Disease Control and Prevention and the World Health Organization. It also is one of the leading causes of combat-related infections associated with injured military personnel serving overseas. Little is known regarding mechanisms of gastrointestinal tract colonization despite this site being shown to serve as a reservoir for multidrug-resistant (MDR) A. baumannii isolates. Here, we establish that secretory IgA, the major immunoglobulin of mucosal surfaces, promotes A. baumannii GI tract colonization via bacterial thioredoxin A as evidenced through significant reduction in colonization in IgA-deficient animals. Additionally, bacterial colonization and mortality were significantly reduced in animals challenged with a thioredoxin A-deficient A. baumannii mutant. Combined, these data suggest that thioredoxin A is a novel virulence factor, for which antithioredoxin therapies could be developed, for this important multidrug-resistant pathogen.