Chlamydia Muridarum Research Articles

Multistage vaccines containing outer membrane, type III secretion system and inclusion membrane proteins protects against a Chlamydia genital tract infection and pathology

Pathogens with a complex lifecycles can effectively evade host immunity in part due to each developmental stage expressing unique sets of antigens. Multisubunit vaccines incorporating signature antigens reflecting distinct developmental stages (multistage vaccines) have proven effective against viral, bacterial and parasitic infection at preventing pathogen evasion of host immunity. Chlamydia trachomatis is characterized by a biphasic extra/intracellular developmental cycle and an acute/persistent (latent) metabolic state; hence a multistage vaccine may prevent immune evasion and enhance clearance. Here we tested the efficacy of a multistage vaccine containing outer membrane (MOMP and PmpG), type three secretion system (T3SS) (CdsF and TC0873) and inclusion membrane proteins (IncA and TC0500) in mice against an intravaginal challenge with Chlamydia muridarum. Comparison of single (eg. MOMP) and double antigen vaccines (eg. MOMP and PmpG), largely targeting the extracellular stage, elicited significant yet comparable protection against vaginal shedding when compared to unimmunized control mice. Utilization of different adjuvants (ISCOMATRIX – IMX, PCEP/polyI:C/IDR1002 – VIDO, CTA1-DD and ADVAX) and numerous immunization routes (subcutaneous – SQ and intranasal – IN) further enhanced protection against infection. However, a multistage vaccine elicited significantly greater protection against vaginal shedding and upper genital tract pathology than vaccines targeting only extra- or intracellular stages. This indicates that protection elicited by a vaccine targeting extracellular chlamydial antigens could be improved by including chlamydial antigen expressed during intracellular phase.

The cryptic plasmid is more important for Chlamydia muridarum to colonize the mouse gastrointestinal tract than to infect the genital tract.

Chlamydia has been detected in the gastrointestinal tracts of both animals and humans. However, the mechanism by which Chlamydia colonizes the gut remains unclear. Chlamydia muridarum is known to spread from the genital to the gastrointestinal tracts hematogenously. The C. muridarum plasmid is a key pathogenic determinant in the mouse upper genital tract although plasmid-deficient C. muridarum is still able to colonize the upper genital tract. We now report that plasmid-deficient C. muridarum exhibits significantly delayed/reduced spreading from the mouse genital to the gastrointestinal tracts. C. muridarum with or without plasmid maintained similar levels in the mouse circulatory system following intravenous inoculation but the hematogenous plasmid-deficient C. muridarum was significantly less efficient in colonizing the gastrointestinal tract. Consistently, plasmid-deficient C. muridarum failed to restore normal colonization in the gastrointestinal tract even after intragastric inoculation at a high dose. Thus, we have demonstrated a plasmid-dependent colonization of C. muridarum in the gastrointestinal tract, supporting the concept that C. muridarum may have acquired the plasmid for adaptation to the mouse gastrointestinal tract during oral-fecal transmission. Since the plasmid is more important for C. muridarum to colonize the gastrointestinal tract than to infect the genital tract, the current study has laid a foundation for further defining the host pathways targeted by the plasmid-encoded or -regulated chlamydial effectors.

Cold-induced stress modulates the maturation of immune cells of mice during Chlamydia muridarum genital infection

Abstract Chlamydia genital infection caused by Chlamydia trachomatis is the most common bacterial sexually transmitted disease (STD) in the United States. Results in a cold-induced stress mouse model established in our laboratory show that cold-induced stress increases the intensity of chlamydia genital infection. This study was to survey the production of T helper cell type 1 (Th1) and (Th2) cytokines in the stress mouse model. Cells harvested from bone marrow or T cells from spleens of stressed and non-stressed mice during Chlamydia muridarum genital infection were allowed to proliferate in vitro. Cytokine production in culture supernatants was tested by ELISA. Gene expression of GATA 3, T-bet and INF-γ in splenic T cells were determined by QPCR. Production of IL-12 by dendritic cells of stressed mice was 0μg compared to 41.06 μg in non stressed mice. However, C. muridarum infection induced increased IL-12 production (97.27 μg and 123 μg) in stressed and non-stressed mice, respectively. Stress induced a 2-fold decrease in T-bet gene expression in T-cells compared to non-stressed group. In contrast, gene expression of GATA 3 in T cells of stressed mice was significantly increased compared to all other groups tested. INF-γ gene expression in T cells of stressed mice showed a 4-fold decrease compared to T cells of non stressed mice. Stress resulted in decreased IL-12 production in dendritic cells and macrophages, INF-γ production and gene expression of T-bet by Th1 cells. Furthermore, the increased gene expression of GATA 3 only in stressed chlamydia infected T cell suggests that cold-induced stress leads to a skewed immune response toward marked increase in production of Th2 cytokines that favor immunosuppression during chlamydia genital infection.

Evaluation of the pathogenesis of Chlamydia induced reactive arthritis

Abstract We hypothesized that reactive arthritis can be induced in the knees and ankles of wild-type C57BL/6 mice following infection with Chlamydia muridarum through the involvement of inflammatory mediators. Reactive arthritis is a chronic form of arthritis, which can also result in conjunctivitis and inflammation of the genital, urinary, or gastrointestinal systems. C. muridarum is a gram-negative obligate intracellular pathogen which can infect the epithelium of the cervix, urethra, and rectum. The exact pathological mechanism of reactive arthritis is unknown. It is understood to result from CD4+ and CD8+ T cells, and factors including TNF-alpha and perforin. Five groups of C57BL/6J mice were used including a positive control group, CD4+ T cells knockout mice, TNF-alpha knockout mice, perforin knockout mice and TNFRSF knockout mice. Mice from each group were intra-vaginally infected with 5×104 inclusion forming units of C. muridarum, marked as day 0. The mice were swabbed a few days after infection to confirm C. muridarum. The mice from each group were euthanized on day 7 and day 14, and sera was collected and analyzed for cytokine expression using an antibody array. Ankles collected from C57BL/6J positive control mice were histologically processed and graded to determine pathology. C57BL/6J positive control mice showed increased synovial lining thickening 14 days following infection. Cytokine expression from TNF-alpha knockout mice, CD4+ T cells knockout mice, perforin knockout mice, and TNFRSF knockout mice was generally reduced. The results show that chlamydial induced reactive arthritis can be detected in the ankles of C57BL/6J mice and suggest that TNF-alpha and CD4+ T cells may be involved in this inflammatory process.

IFN-ɛ regulated innate immune responses in the female reproductive tract during Chlamydia infection

Abstract The immune processes involved in the clearance and immunopathology of Chlamydia infection in the female reproductive tract (FRT) are not well understood. In previous ground-breaking studies we showed that IFN-ɛ, a novel type I IFN that is exclusively and constitutively expressed in the FRT, plays an important role in protecting against Chlamydia infections. Here, we examined the effects of IFN-ɛ on innate immune processes in the FRT in order to elucidate the mechanisms by which IFN-ɛ protects against Chlamydia infections. Female WT and IFN-ɛ−/− C57BL/6 mice were pre-treated with progesterone and infected intra-vaginally with Chlamydia muridarum or sham-infected. Uterine horns were harvested and the effects of IFN-ɛ deficiency on immune factor expression and cellular infiltration were assessed using microarray/bioinformatics analyses, real-time qPCR and flow cytometry. IFN-ɛ uniquely regulated the expression of 744 genes at baseline and 802 genes during Chlamydia infection, and universally regulated the transcription of 61 genes, regardless of infection status. Of note, pathways associated with protective innate responses, such as IFN regulatory factor activation and pattern recognition receptor signalling were down-regulated in Chlamydia-infected IFN-ɛ−/− mice compared to WT controls. These changes correlate with a decrease in the expression of many important IFN-γ signalling molecules and number of IFN-γ-producing NK cells in the FRT during infection. These findings suggest that IFN-ɛ may protect against Chlamydia FRT infections by potentiating innate immune processes important for the clearance of infection, particularly pathways associated with IFN-γ signalling and the activation of NK cell responses.

Interferon epsilon protects primary macrophages against HIV infection

Abstract Interferon epsilon (IFNe) is a unique type I IFN that is not induced by pattern-recognition response elements. IFNɛ is constitutively expressed in mucosal tissues including the female genital mucosa. Although the direct anti-viral activity of IFNe was thought to be weak compared to IFNa, IFNe controls Chlamydia muridarum and herpes simplex virus 2 in mice, possibly through modulation of immune response. We show here that IFNe induces an antiviral state in human macrophages that blocks HIV-1 replication. IFNe had little or no protective effect in activated CD4+ T cells or transformed cell lines unless activated CD4+ T cells were infected with replication competent HIV-1 at a low MOI. The block to HIV infection of macrophages was maximal after 24 h of treatment and was reversible. IFNe acted on early stages of the HIV life cycle including viral entry, reverse transcription and nuclear import. The protection did not appear to operate through known type I IFN-induced HIV host restriction factors such as APOBEC3A and SAMHD1. IFNe-stimulated immune mediators and pathways had the signature of type I IFNs but were distinct from IFNα2. IFNe induced significant phagocytosis and reactive oxygen species, which contributed to the block to HIV replication in macrophages. These findings indicate that IFNe induces an antiviral state in macrophages that is mediated by different factors than those induced by IFNa2. Understanding the mechanism of IFNe-mediated HIV inhibition through immune modulation has implications for prevention.

Early-life respiratory bacterial infection-induced chronic lung disease is driven by a novel TLR2/IL-13/miR-21/PI3K-dependent, but MyD88-independent signalling pathway

Abstract There is a critical window in early-life where the lung is still maturing and susceptible to infection. Indeed, severe respiratory infections in early-life are a risk factor for the development of chronic lung diseases. The aim of this study was to identify the mechanisms involved. Neonatal wild-type (WT), TLR2 deficient (−/−), IL-13−/−, MyD88−/− and STAT6−/− mice were infected with the natural mouse bacterial pathogen Chlamydia muridarum, as a model of severe respiratory tract infection in early-life. In some experiments WT mice were treated with miR-21 antagomirs, PI3K inhibitors, or relevant controls during early-life infection. The impact of targeting these specific immune molecules during early-life on infection-induced impairment of lung function and structure were assessed. Neonatal Chlamydia respiratory infection increased TLR2, IL-13-receptor, miR-21 and PI3K expression and/or activity in the lung. TLR2 signalling induced IL-13-receptor expression, IL-13 signalling induced miR-21 expression and miR-21 increased PI3K activity. TLR2 signalling also increased IL-13+ ILC2s in the lung. TLR2−/− and IL-13−/− mice were protected against infection-induced airway hyperresponsiveness (AHR), but not emphysema-like alveolar enlargement. This TLR2/IL-13 mediated phenotype was independent of MyD88, but dependent on STAT6. Specific targeting of miR-21 prevented AHR but not emphysema. Pan-PI3K inhibition did not affect AHR, but protected against emphysema. Interestingly, early-life infection-induced AHR was steroid insensitive. This study identifies a novel signalling network that may be targeted for the prevention of the long-term deleterious effects of early-life infection on lung function and structure.

Chlamydia trachomatis neither exerts deleterious effects on spermatozoa nor impairs male fertility

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection. However, whether Chlamydia trachomatis has a negative impact on sperm quality and male fertility is still controversial. Herein, we report the effects on sperm quality of the in vitro exposure of spermatozoa to Chlamydia trachomatis, and also the effects of male genital infection on male fertility using an animal model. Human and mouse sperm were obtained from healthy donors and cauda epididimys from C57BL/6 mice, respectively. Highly motile human or mouse spermatozoa were in vitro exposed to C. trachomatis (serovar E or LGV) or C. muridarum, respectively. Then, sperm quality parameters were analyzed. Moreover, male fertility of Chlamydia muridarum infected male C57BL/6 mice was assessed. Human or murine sperm in vitro exposed to increasing bacterial concentrations or soluble factors from C. trachomatis or C. muridarum, respectively, did not show differences in sperm motility and viability, apoptosis, mitochondrial membrane potential, DNA fragmentation, ROS production and lipid peroxidation levels, when compared with control sperm (p > 0.05). Moreover, no differences in fertility parameters (potency, fecundity, fertility index, pre- and post-implantation loss) were observed between control and infected males. In conclusion, our results indicate that Chlamydia spp. neither directly exerts deleterious effects on spermatozoa nor impairs male fertility.

Effect of cold water-induced stress on immune response, pathology and fertility in mice during Chlamydia muridarum genital infection.

Genital infection by Chlamydia trachomatis is the most common bacterial sexually transmitted disease worldwide. It causes serious reproductive health complications, including pelvic inflammatory disease and infertility. Stress is implicated as a risk factor for various infections; however, its effect on chlamydia genital infection is unknown. We previously showed that repeated exposure of mice to cold water results in increased severity of chlamydia genital infection. In this study, cold water-induced stress resulted in (i) elevated levels of norepinephrine (NE) and epinephrine in the spleen and genital tract of stressed mice; (ii) elevated IL-1β, TNF-α, IL-6 and nitric oxide production in macrophage-rich peritoneal cells of mice; (iii) supplement of NE in vitro exerts an immunosuppressive effect on splenic T-cell production of cytokines; (iv) decreased C. muridarum shedding in the genital tract of β1Adr/β2Adr receptor KO mice; and (v) a higher rate of infertility in infected mice. These results suggest that cold water stress induces the production of catecholamines, which may play a critical role in the modulation of the immune system leading to increased intensity of C. muridarum genital infection.

Vaccination with the polymorphic membrane protein A reduces Chlamydia muridarum induced genital tract pathology

Chlamydia trachomatis serovars D-K are one of the most frequent causes of sexually transmitted infections of the female genital tract, with possible complications such as hydrosalpinx, pelvic inflammatory disease, extra-uterine gravidity or infertility. We used the murine genital tract infection model with C. muridarum for vaccination studies and found that more than 70% of the infected mice suffered from uterus dilatations and/or hydrosalpinx. Systemic consequences of the vaginal infection were apparent by splenomegaly ten to fifteen days post infection. While cultivable microorganisms were detectable for the first 23days post infection, the first lesions of the genital tract developed at day 15, however, many lesions occurred later in the absence of cultivable bacteria. Lesions were not accompanied by pro-inflammatory cytokines such as IFNɣ, TNF and IL-6, since these cytokines were almost undetectable in the genital tract 43days post infection. To prevent genital tract lesions, we vaccinated mice with the polymorphic membrane protein (Pmp) A in combination with CpG-ODN 1826 as adjuvant. The vaccine lowered the chlamydial burden and the differences were significant at day 10 post infection but not later. More importantly the vaccine decreased the rate and severity of genital tract lesions. Interestingly, control vaccination with the protein ovalbumin plus CpG-ODN 1826 enhanced significantly the severity but not the rate of pathologic lesions, which was presumably caused by the activation of innate immune responses by the adjuvant in the absence of a C. muridarum-specific adaptive immune response. In summary, vaccination with recombinant PmpA plus CpG-ODN 1826 significantly reduced C. muridarum-induced tissue damage, however, CpG-ODN 1826 may aggravate C. muridarum-induced tissue injuries in the absence of a protective antigen.

PI3K/AKT/Bcl-2/Bax pathway activation and CD4+ T cell apoptosis induced by Chlamydia muridarum respiratory tract infection

Objective To investigate PI3K/AKT/Bcl-2/Bax pathway activation and CD4+ T cell apoptosis in a mouse model of Chlamydia muridarum (Cm) respiratory tract infection. Methods C57BL/6 mice were infected with 1×103 inclusion forming units (IFUs) of Cm through nasal inhalation. Mononuclear cells were isolated from lung tissues of the infected mice at different points. Expression of PI3K p110δ at mRNA level was detected by RT-PCR. Western blot was used to detect the expression of p-AKT, Bcl-2 and Bax at protein level. Apoptosis of CD4+ T cells in lung tissues of mice was detected by flow cytometry. Results The expression of PI3K p110δ at mRNA level and p-AKT, a downstream protein of PI3K, at protein level in lung tissues of mice with Cm infection were significantly higher than those in non-infected group on day 3, reached to the highest level on day 7 and decreased on day 14. The expression of apoptosis-related proteins Bcl-2 and Bax significantly increased on day 3 and peaked on day 7, while the Bcl-2/BAX ratio decreased following Cm infection. These results suggested that the anti-apoptotic effect gradually decreased and the apoptosis of CD4+ T cells increased. The early apoptosis (Annexin+ PI+ ) and the total apoptosis (Annexin+ PI) of CD4+ T cells induced by Cm infection in lung tissues of mice began to increase on day 3 and peaked on day 7. Conclusion Cm respiratory tract infection induces the activation of PI3K/AKT/Bcl-2/Bax pathway, which results in the apoptosis of CD4+ T cells. Key words: Chlamydia muridarum (Cm); PI3K/AKT; CD4+ T cell; Apoptosis

The cationic liposomal adjuvants CAF01 and CAF09 formulated with the major outer membrane protein elicit robust protection in mice against a Chlamydia muridarum respiratory challenge

Two cationic liposomal adjuvants CAF01 and CAF09 were formulated with the native or the recombinant Chlamydia muridarum major outer membrane protein (nMOMP and rMOMP). BALB/c mice were immunized with the four vaccine formulations using the subcutaneous followed by the intranasal (i.n.) routes. As positive controls mice were inoculated i.n. with live C. muridarum and negative controls received i.n. minimal essential medium (MEM). Four weeks after the last immunization mice were challenged i.n. with 104 inclusion forming units (IFU) of C. muridarum. Following the challenge the mice were weighed daily. At 10days post-challenge the mice were euthanized, their lungs weighed and the number of C. muridarum IFU determined. Serum collected the day before the challenge showed that all four groups of mice immunized with CAF01, or CAF09 and MOMP had significant C. muridarum-specific antibody titers. As determined by a T-cell lymphoproliferative assay, these four groups of mice also mounted robust cell mediated immune responses with high production of IFN-γ and IL17 and low levels of IL-4. Following the challenge the four groups of mice lost significantly less body weight than the MEM-immunized group. Lungs of mice vaccinated with CAF01, or CAF09, and nMOMP were significantly lighter than those from mice immunized using rMOMP. The number of IFU recovered from the lungs of mice vaccinated with CAF01, or CAF09, and nMOMP was similar to the number of IFU recovered from mice immunized with live EB. Mice that received rMOMP had significantly higher numbers of IFU than other groups. In conclusion, CAF01 and CAF09 elicited very robust protective humoral and cellular immune responses and were equally effective at adjuntavizing the C. muridarum MOMP. Mice vaccinated with nMOMP were significantly better protected than those immunized with rMOMP, indicative of the importance of the structural conformation of this antigen in protection.

CD103+ lung dendritic cells (LDCs) induce stronger Th1/Th17 immunity to a bacterial lung infection than CD11bhi LDCs.

Recent studies suggest differential roles for CD103+ and CD11bhi lung dendritic cells (LDCs) in host defense against viral and bacterial infections. In this study, we examined the contribution of these LDC subsets in protective immunity to chlamydial lung infection using a Chlamydia muridarum mouse infection model. We found that CD103+ LDCs showed higher expression of costimulatory molecules (CD40, CD80 and CD86) and increased production of cytokines (IL-12p70, IL-10, IL-23 and IL-6) compared with CD11bhi LDCs, but the expression of programmed death-ligand 1 (PD-L1) was similar between the two subsets. More importantly, we found, in adoptive transfer experiments, that the mice receiving CD103+ LDCs from Chlamydia-infected mice exhibited better protection than the recipients of CD11bhi LDCs, which was associated with more robust Th1/Th17 cytokine responses. In addition, in vitro experiments showed that CD103+ LDCs induced stronger IFN-γ and IL-17 responses, when cocutured with chlamydial antigen-primed CD4+ T cells, than CD11bhi LDCs. Furthermore, the blockade of PD1 in the culture of CD4+ T cells with either CD103+ or CD11bhi LDCs enhanced production of IFN-γ and IL-17. In conclusion, our data provide direct evidence that CD103+ LDCs are more potent in promoting Th1/Th17 immunity to chlamydial lung infection than CD11bhi LDCs.

Comparison of Murine Cervicovaginal Infection by Chlamydial Strains: Identification of Extrusions Shed In vivo.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections (STIs) and preventable blindness. Untreated, asymptomatic infection as well as frequent re-infection are common and may drive pelvic inflammatory disease, ectopic pregnancy, and infertility. In vivo models of chlamydial infection continue to be instrumental in progress toward a vaccine and further elucidating the pathogenesis of this intracellular bacterium, however significant gaps in our understanding remain. Chlamydial host cell exit occurs via two mechanisms, lysis and extrusion, although the latter has yet to be reported in vivo and its biological role is unclear. The objective of this study was to investigate whether chlamydial extrusions are shed in vivo following infection with multiple strains of Chlamydia. We utilized an established C3H/HeJ murine cervicovaginal infection model with C. trachomatis serovars D and L2 and the Chlamydia muridarum strain MoPn to monitor the (i) time course of infection and mode of host cell exit, (ii) mucosal and systemic immune response to infection, and (iii) gross and histopathology following clearance of active infection. The key finding herein is the first identification of chlamydial extrusions shed from host cells in an in vivo model. Extrusions, a recently appreciated mode of host cell exit and potential means of dissemination, had been previously observed solely in vitro. The results of this study demonstrate that chlamydial extrusions exist in vivo and thus warrant further investigation to determine their role in chlamydial pathogenesis.

IL-17-producing γδT cells promote neutrophils accumulation at early stage of Chlamydial airway infection

Objective To investigate the effects of IL-17-producing γδT cells on accumulation and chemotaxis of neutrophils in lung following Chlamydia muridarum (Cm) airway infection and the possible mechanism. Methods Wild type (WT) (C57BL/6) and TCRδ-/- mice were inoculated intranasally with 3×103 inclusion-forming units (IFU) of Cm strains to induce Chlamydial pneumonitis in mice. Mononuclear cells were isolated from the lungs of mice at early stage of chlamydial infection. The percentages of Gr1+ CD11b+ neutrophils in lung mononuclear cells were detected by flow cytometry. The levels of IL-17 secreted by γδT cells were measured with intracellular cytokine staining. The expression of IL-17, KC and IL-6 at mRNA level in lungs were measured by RT-PCR. Results Our data showed that γδT cells in lung tissues were activated rapidly and secreted large quantities of IL-17 at early stage of Cm infection. Compared with uninfected mice, the production of IL-17 by CD3+ γδT+ cells in infected mice significantly increased and reached the peak on the third day (P<0.001), and the levels of IL-17 remained high at 7 days post-infection (p.i.) (P<0.05). Compared with WT(C57BL/6) mice, TCRδ-/- mice showed decreased expression of IL-17 at mRNA level and lower percentages of CD11b+ Gr-1+ neutrophils in lungs at early stage of Cm infection (3 days p. i.). Further analysis showed that the expression of KC and IL-6 at mRNA level significantly decreased at 3 and 7 days after Cm infection. Conclusion IL-17-producing γδT cells might promote infiltration of neutrophils to lung tissues by promoting the expression of KC and IL-6 during Chlamydial pneumonitis. Key words: Chlamydia muridarum; Neutrophils; γδT cells; IL-17

Chronic Infection of the Prostate by Chlamydia muridarum Is Accompanied by Local Inflammation and Pelvic Pain Development.

Chlamydia trachomatis urogenital infections are the leading cause of sexually transmitted bacterial infections. Although the prevalence of chlamydial infection is similar in men and women, current research is mainly focused on women, neglecting the study of male genital tract infections. We, therefore, investigated Chlamydia infection in the rodent male genital tract. Male NOD and C57BL/6 mice were inoculated in the meatus urethra with C. muridarum. Bacterial DNA, leukocyte infiltration of male genital tract tissues, pelvic pain, and Chlamydia-specific immune responses were analyzed at different time points. The inoculation of C. muridarum in the meatus urethra of male mice resulted in an ascending and widely disseminated infection of the male genital tract. C. muridarum remained longer and with the highest bacterial burdens in the prostate, thus showing a special tropism for this organ. Infection caused leukocyte infiltration, mainly composed by neutrophils, and also induced early pelvic pain development that rapidly dropped and resolved as the infection became chronic. Bacterial load and leukocyte infiltration was observed in all prostate lobes, although dorsolateral prostate was the most affected lobe. Interestingly, immune responses induced by both mice strains were characterized by the production of high levels of IL-10 during early stages of the infection, with highest and sustained levels observed in NOD mice, which showed to be less efficient in clearing the infection. Chronic infection of the prostate accompanied by local inflammation and pelvic pain development described herein have important implications for the improvement of the diagnosis and for the design of new efficient therapies. Prostate 77:517-529, 2017. © 2017 Wiley Periodicals, Inc.

IFN-ε protects primary macrophages against HIV infection.

IFN-ε is a unique type I IFN that is not induced by pattern recognition response elements. IFN-ε is constitutively expressed in mucosal tissues, including the female genital mucosa. Although the direct antiviral activity of IFN-ε was thought to be weak compared with IFN-α, IFN-ε controls Chlamydia muridarum and herpes simplex virus 2 in mice, possibly through modulation of immune response. We show here that IFN-ε induces an antiviral state in human macrophages that blocks HIV-1 replication. IFN-ε had little or no protective effect in activated CD4+ T cells or transformed cell lines unless activated CD4+ T cells were infected with replication-competent HIV-1 at a low MOI. The block to HIV infection of macrophages was maximal after 24 hours of treatment and was reversible. IFN-ε acted on early stages of the HIV life cycle, including viral entry, reverse transcription, and nuclear import. The protection did not appear to operate through known type I IFN-induced HIV host restriction factors, such as APOBEC3A and SAMHD1. IFN-ε-stimulated immune mediators and pathways had the signature of type I IFNs but were distinct from IFN-α in macrophages. IFN-ε induced significant phagocytosis and ROS, which contributed to the block to HIV replication. These findings indicate that IFN-ε induces an antiviral state in macrophages that is mediated by different factors than those induced by IFN-α. Understanding the mechanism of IFN-ε-mediated HIV inhibition through immune modulation has implications for prevention.

CCL19-CCR7-dependent reverse transendothelial migration of myeloid cells clears Chlamydia muridarum from the arterial intima.

Regions of the normal arterial intima predisposed to atherosclerosis are sites of ongoing monocyte trafficking and also contain resident myeloid cells with features of dendritic cells. However, the pathophysiological roles of these cells are poorly understood. Here we found that intimal myeloid cells underwent reverse transendothelial migration (RTM) into the arterial circulation after systemic stimulation of pattern-recognition receptors (PRRs). This process was dependent on expression of the chemokine receptor CCR7 and its ligand CCL19 by intimal myeloid cells. In mice infected with the intracellular pathogen Chlamydia muridarum, blood monocytes disseminated infection to the intima. Subsequent CCL19-CCR7-dependent RTM was critical for the clearance of intimal C. muridarum. This process was inhibited by hypercholesterolemia. Thus, RTM protects the normal arterial intima, and compromised RTM during atherogenesis might contribute to the intracellular retention of pathogens in atherosclerotic lesions.

IFNγ is Required for Optimal Antibody-Mediated Immunity against Genital Chlamydia Infection.

Defining the mechanisms of immunity conferred by the combination of antibody and CD4+ T cells is fundamental to designing an efficacious chlamydial vaccine. Using the Chlamydia muridarum genital infection model of mice, which replicates many features of human C. trachomatis infection and avoids the characteristic low virulence of C. trachomatis in the mouse, we previously demonstrated a significant role for antibody in immunity to chlamydial infection. We found that antibody alone was not protective. Instead, protection appeared to be conferred through an undefined antibody-cell interaction. Using gene knockout mice and in vivo cellular depletion methods, our data suggest that antibody-mediated protection is dependent on the activation of an effector cell population in genital tract tissues by CD4+ T cells. Furthermore, the CD4+ T cell-secreted cytokine gamma interferon (IFN-γ) was found to be a key component of the protective antibody response. The protective function of IFN-γ was not related to the immunoglobulin class or to the magnitude of the Chlamydia-specific antibody response or to recruitment of an effector cell population to genital tract tissue. Rather, IFN-γ appears to be necessary for activation of the effector cell population that functions in antibody-mediated chlamydial immunity. Our results confirm the central role of antibody in immunity to chlamydia reinfection and demonstrate a key function for IFN-γ in antibody-mediated protection.

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.