In humans, infection with Chlamydia trachomatis can result in chronic infection with severe reproductive consequences. The immune response elicited by natural infection fails to protect against reinfection and can contribute to tissue inflammation and damage. CD4+ T cells are key mediators of protection against C. trachomatis. However, these same cells, together with neutrophils, also contribute to tissue pathology. The identity and effector functions of the CD4+ T cells that contribute to protection, pathology, or both remain poorly defined. Notably, C. trachomatis pathology is serovar specific. Infection with serovar D induces severe tissue inflammation in the female upper genital tract, whereas infection with serovar L2 does not. Using a murine model of genital infection, we found that infection with serovar D selectively drives the polarization of naïve CD4+ T cells into inflammatory Th17 cells through the induction of Th17-polarizing cytokines, such as IL-1β, IL-6, and IL-23, compared with serovar L2 infection. Single-cell RNA sequencing of CD4+ T cells from the uteri of serovar D-infected mice revealed a Th17-skewed response with transcriptional features of an inflammatory phenotype, including upregulation of Bhlhe40 and Il1r1. Th17 cells have been reported to contribute to pathology through secretion of pro-inflammatory cytokines that recruit neutrophils and promote tissue damage. Together, these findings demonstrate that serovar D promotes pro-inflammatory CD4+ T-cell responses that could contribute to immunopathology in C. trachomatis infection, in contrast to the response elicited by serovar L2. They also underscore the importance of developing a vaccine that elicits protective immunity while minimizing harmful inflammatory responses.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that if left untreated can cause reproductive harm. Failure of natural adaptive immunity results in chronic and repeat infections. In efforts to understand the failure of adaptive immunity, we have previously discovered that CD8+ T cells, normally integral for controlling intracellular pathogen infections, are misprogrammed by PD-1/PD-L1 signaling during in vivo C. trachomatis infection and fail to mount a protective response. Seeking to uncover the pathways and host factors involved in PD-L1 upregulation that may lead to CD8+ T-cell inhibition, we discovered that C. trachomatis triggers the secretion of host type I interferons (IFNs) that are necessary and sufficient to upregulate PD-L1 in vitro. Additionally, secretion of type I IFNs is dependent on C. trachomatis development and its type III secretion system. We have also validated that type I IFNs contribute to upregulation of PD-L1 during C. trachomatis infection in vivo using a mouse model of infection. Overall, these findings reveal that C. trachomatis induction of this host pathway may contribute to adaptive immune evasion.

The nasal cavity serves as a critical barrier, filtering airborne pathogens to prevent them from reaching the lungs. Immune cells in the nasal mucosa neutralize these pathogens, protecting against local infection. However, the composition, origins, and functions of these immune populations remain largely unexplored. Our research identified a significant population of extravascular neutrophils (EVNs) in the nasal mucosa during homeostasis. Using cell surface markers and mRNA profiles, these EVNs were categorized into three subsets: N1, N2, and N3. Through parabiosis and imaging, we found that N1 neutrophils reside in bone marrow adjacent to the nasal cavity and likely migrate directly into the mucosa via bone conduits. By contrast, N2 neutrophils are derived from the bloodstream and differentiate into N3 neutrophils within 5–6 days. Functionally, N2 neutrophils express genes characteristic of conventional neutrophils and are the primary subset responsible for pathogen phagocytosis. Meanwhile, N3 neutrophils upregulate genes associated with antigen-presenting cells, engulf material from surrounding cells under steady-state conditions, and present antigens to CD8+ T cells in vitro. In summary, the nasal mucosa harbors a diverse population of EVNs with distinct origins, phenotypes, and specialized functions. These findings highlight the complexity of the nasal immune landscape and its essential role in host defense. Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

ABSTRACT The lack of effective adaptive immunity against Chlamydia trachomatis leads to chronic or repeated infection and serious disease sequelae. Dendritic cells (DCs) are professional antigen-presenting cells that are crucial for the activation of T cells during C. trachomatis infection. cDC1s and cDC2s are the two main DC subsets responsible for T cell priming, but little is known about how C. trachomatis affects their ability to prime T cells. Using a mouse model of infection, we found that C. trachomatis uptake reduced the viability of cDC1s and cDC2s both in vitro and in vivo , with cDC1s experiencing more death. DC death was mainly due to apoptosis and is alleviated in Casp3/7 or Bak1/Bax knockout DCs. In addition, we observed that C. trachomatis -specific CD8+ T cells were preferentially activated by cDC1s. Reduction in DC viability by C. trachomatis impaired the ability of infected DCs to activate T cells upon co-culture, although in the case of CD8+ T cell priming, controlling for viability was insufficient to fully rescue the defect. RNA sequencing of DCs from infected mice showed upregulation of cell death pathways, supporting our observations of DC death caused by C. trachomatis . Finally, we validated our findings with human DCs in vitro , observing C. trachomatis -induced cell death. These results indicate that C. trachomatis may evade the adaptive immune system by directly inducing cell death in DCs.

ABSTRACT The nasal mucosa (NM) has several critical functions, including as a chemosensory organ, as a filter and conditioning surface of inhaled air for the lower airways, and as a first line of defense against airborne infections. Owing to its constant exposure to ever-changing environments, the NM is arguably the most frequently infected tissue in mammals. Consequently, vertebrates harbor an intricate network of subepithelial immune cells that are dispersed throughout the NM. However, the origin, composition, and function of nasal immune cells and their pathophysiological role are poorly understood. Here, we show that murine steady-state NM harbors a prominent population of extravascular neutrophils (EVN) that are abundant in both conventional and germ-free mice, suggesting that their presence is not driven by microbial stimuli. Nasal EVN can be subdivided into three phenotypically distinct subsets: one population that we have termed nN1 is CD11b int Ly6G int , while the other two subsets are both CD11b hi Ly6G hi and distinguishable by the absence (nN2) or presence (nN3) of CD11c and SiglecF. nN1 EVN originate in bone marrow (BM) within osseous structures in the skull. These locally produced neutrophils appear to access the adjacent NM via conduits that connect BM cavities to the submucosal lamina propria. nN2 cells reach the NM via the blood and readily engulf infectious microbes. In the absence of infection, nN2 cells differentiate into the nN3 subset, which does not capture microbes but assumes phenotypic and functional features of antigen-presenting cells, including the capacity to cross-present exogenous antigens to CD8 T cells. These findings indicate that steady-state mammalian NM harbors a unique innate cellular immune environment that is unlike any other barrier tissue.

Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infection in both men and women. Immunity to C. trachomatis involves many cell types, but CD4+ + T cells play a key role in protecting the host during natural infection. Specifically, IFN-g g production by CD4+ + T cells is the main effector responsible for bacterial clearance, yet the exact mechanism by which IFN-g g confers protection is poorly defined. In our efforts to define the specific mechanisms for bacterial clearance, we now show that IFN-g g upregulates expression of MHC class II (MHCII) on nonhematopoietic cells during C. trachomatis infection in vivo. We also find that MHCII expression on epithelial cells of the upper genital tract contributes to the efficient clearance of bacteria mediated by pathogen-specific CD4+ + Th1 cells. As we further cataloged the protective mechanisms of C. trachomatis-specific- specific CD4+ + T cells, we found that the T cells also express granzyme B (GzmB) when coincubated with infected cells. In addition, during C. trachomatis infection of mice, primed activated-naive CD4+ + Th1 cells displayed elevated granzyme transcripts ( GzmA, GzmB, GzmM, GzmK, GzmC) ) compared with memory CD4+ + T cells in vivo. Finally, using intracellular cytokine staining and a GzmB-/-- /- mouse strain, we show that C. trachomatis-specific- specific CD4+ + Th1 cells express GzmB upon Ag stimulation, and that this correlates with Chlamydia clearance in vivo. Together these results have led us to conclude that Chlamydia-specific specific CD4+ + Th1 cells develop cytotoxic capacity through engagement with nonhematopoietic MHCII, and this correlates to C. trachomatis clearance. The Journal of Immunology, , 2024, 213: 328-338.- 338.

Borrelia burgdorferi, the causative agent of Lyme disease, establishes a long-term infection inside its mammalian hosts. Despite the continued presence of the bacteria in animal models of disease, inflammation is transitory and resolves spontaneously. Exhausted T cells are present in many long-term infections and mediate a balance between pathogen clearance and preventing tissue damage resulting from excess inflammation. Indeed, we have found that CD4+ T cells in the draining inguinal and popliteal lymph nodes, as well as the heart and ankle joint, significantly upregulate the immunoinhibitory molecule PD-1 following B. burgdorferi infection. While PD-1 expression on T cells is a strong indication of CD4+ T cell exhaustion, we have extended our findings by assessing expression of a ligand for PD-1, PD-L1, on antigen presenting cells in vitro and in vivo following B. burgdorferi infection. We have shown that not only is PD-L1 is significantly upregulated on both bone marrow derived macrophages (BMDMs) and dendritic cells (BMDCs) following B. burgdorferi stimulation, but is also upregulated on macrophages and a subset of conventional dendritic cells (cDC2s) in the draining lymph nodes following in vivo infection. We are currently testing if mice deficient in either PD-1 or PD-L1 exhibit changes in ankle joint or heart inflammation compared to wild-type mice, which would suggest that the PD-1/PD-L1 axis is critical for regulating inflammation during B. burgdorferi infection. Together, these data will allow us to determine how this immune checkpoint regulates inflammation during B. burgdorferi infection. Supported by grants from the NIH (R01 AI150157), the Harvard Medical School Fairbairn Family Lyme Research Initiative, and the Tufts University School of Medicine Natalie Zucker Research Center for Women's Scholars Award.

The Lyme disease bacterial pathogen, Borrelia burgdorferi, establishes a long-term infection inside its mammalian hosts. Despite the continued presence of the bacteria in animal models of disease, inflammation is transitory and resolves spontaneously. T cells with limited effector functions and the inability to become activated by antigen, termed exhausted T cells, are present in many long-term infections. These exhausted T cells mediate a balance between pathogen clearance and preventing tissue damage resulting from excess inflammation. Exhausted T cells express a variety of immunoinhibitory molecules, including the molecule PD-1. Following B. burgdorferi infection, we found that PD-1 and its ligand PD-L1 are significantly upregulated on CD4+ T cells and antigen presenting cell subsets, respectively. Using mice deficient in PD-1, we found that the PD-1/PD-L1 pathway did not impact bacterial clearance but did impact T cell expansion and accumulation in the ankle joint and popliteal lymph nodes without affecting B cell populations or antibody production, suggesting that the PD-1/PD-L1 pathway may play a role in shaping the T cell populations present in affected tissues.

The nasal mucosa is a versatile tissue where functions such as olfaction and conditioning of air take place. A particularly relevant and less explored function of this tissue relates to its capacity to respond to insult. Air-borne pathogens can use the nasal mucosa as a portal of entry to further disseminate into deeper tissues and little is known about the immune cell populations these pathogens encounter upon infection. We asked what immune cell populations are present in the murine nasal mucosa and unexpectedly found extravascular neutrophils to be a constitutive and prominent population in the steady state. These neutrophils were found in comparable numbers in conventional vs. germ-free mice and can be divided into three well-defined populations based on the expression of specific cell surface markers and mRNA transcript profiles. One of the populations was traced to nasal mucosa-associated pockets of bone marrow and determined to contribute to the nasal mucosa neutrophil extravascular pool. A second population was found to preferentially phagocytose bacteria during infection. In the absence of infection, this population differentiates into a third population, which interacts with other cells of the nasal mucosa in a manner that resembles trogocytosis. Lastly, we found extravascular granulocyte hematopoietic precursors in the nasal mucosa that likely contribute to the extravascular neutrophil pool. Thus, the nasal mucosa encompasses a constitutive population of extravascular neutrophils that show diverse origins and specialization of function. These findings contribute to our understanding of the composition and development of the microenvironment that air-borne pathogens encounter upon infection in the nasal mucosa.

ABSTRACT Chlamydia trachomatis is the most commonly reported sexually transmitted infection in the United States. The high prevalence of infection and lack of a vaccine indicate a critical knowledge gap surrounding the host's response to infection and how to effectively generate protective immunity. The immune response to C. trachomatis is complex, with cells of the adaptive immune system playing a crucial role in bacterial clearance. Here, we discuss the CD4+ and CD8+ T cell response to Chlamydia, the importance of antigen specificity and the role of memory T cells during the recall response. Ultimately, a deeper understanding of protective immune responses is necessary to develop a vaccine that prevents the inflammatory diseases associated with Chlamydia infection.

Sticholysins (Sts) I and II (StI and StII) are pore-forming proteins (PFPs), purified from the Caribbean Sea anemone Stichodactyla helianthus. StII encapsulated into liposomes induces a robust antigen-specific cytotoxic CD8+ T lymphocytes (CTL) response and in its free form the maturation of bone marrow-derived dendritic cells (BM-DCs). It is probable that the latter is partially supporting in part the immunomodulatory effect on the CTL response induced by StII-containing liposomes. In the present work, we demonstrate that the StII's ability of inducing maturation of BM-DCs is also shared by StI, an isoform of StII. Using heat-denatured Sts we observed a significant reduction in the up-regulation of maturation markers indicating that both PFP's ability to promote maturation of BM-DCs is dependent on their conformational characteristics. StII-mediated DC maturation was abrogated in BM-DCs from toll-like receptor (TLR) 4 and myeloid differentiation primary response gene 88 (MyD88)-knockout mice but not in cells from TLR2-knockout mice. Furthermore, the antigen-specific CTL response induced by StII-containing liposomes was reduced in TLR4-knockout mice. These results indicate that StII, and probably by extension StI, has the ability to induce maturation of DCs through a TLR4/MyD88-dependent pathway, and that this activation contributes to the CTL response generated by StII-containing liposomes.

Stan Falkow looked at the world with his eyes peering from the outer membrane of a gram-negative bacterium. It was a great vantage point from which to dream about the possible superpowers these organisms might have. He mused about these dreams with his trainees who sometimes found through rigorous scientific exploration that the superpowers really were there! Stan also realized that bacterial pathogenesis by definition was a two-way street, and that masterful understanding of bacterial virulence factors also required a masterful understanding of host cell processes against which the virulence factors were deployed. In my own scientific journey, I have sought to explore bacterial-host interactions that result in subtle alterations of the host's adaptive immune response. Here, as an example, I describe an interaction between Chlamydia trachomatis and host T cells that may contribute to the establishment of persistent infection.

Memory antigen-specific CD4+ T cells against Chlamydia trachomatis are necessary for protection against secondary genital tract infection. While it is known that naïve antigen-specific CD4+ T cells can traffic to the genital tract in an antigen-specific manner, these T cells are not protective during primary infection. Here, we sought to compare the differences between memory and naïve antigen-specific CD4+ T cells in the same mouse following secondary infection using transgenic CD4+ T cells (NR1 T cells). Using RNA sequencing, we found that there were subtle but distinct differences between these two T cell populations. Naïve NR1 T cells significantly upregulated cell cycle genes and were more proliferative than memory NR1 T cells in the draining lymph node. In contrast, memory NR1 T cells were more activated than naïve NR1 T cells and were enriched in the genital tract. Together, our data provide insight into the differences between memory and naïve antigen-specific CD4+ T cells during C. trachomatis infection.

Chlamydia trachomatisis an important mucosal pathogen that is the leading cause of sexually transmitted bacterial infections in the United States. Despite this, there is no vaccine currently available. In order to develop such a vaccine, it is necessary to understand the components of the immune response that can lead to protection against this pathogen. It is well known that antigen-specific CD4+T cells are critical forChlamydiaclearance, but the contexts in which they are protective or not protective are unknown. Here, we aimed to characterize the importance of gamma interferon production and sensing by T cells and the effects on the immune response toC. trachomatis. Our work here helps to define the contexts in which antigen-specific T cells can be protective, which is critical to our ability to design an effective and protective vaccine againstC. trachomatis.

The invasion of Chlamydia trachomatis, an obligate intracellular bacterium, into epithelial cells is driven by a complex interplay of host and bacterial factors. To comprehensively define the host genes required for pathogen invasion, we undertook a fluorescence-activated cell sorting (FACS)-based CRISPR screen in human cells. A genome-wide loss-of-function library was infected with fluorescent C. trachomatis and then sorted to enrich for invasion-deficient mutants. The screen identified heparan sulfate, a known pathogen receptor, as well as coatomer complex I (COPI). We found that COPI, through a previously unappreciated role, promotes heparan sulfate cell surface presentation, thereby facilitating C. trachomatis attachment. The heparan sulfate defect does not fully account for the resistance of COPI mutants. COPI also promotes the activity of the pathogen's type III secretion system. Together, our findings establish the requirement for COPI in C. trachomatis invasion and the utility of FACS-based CRISPR screening for the elucidation of host factors required for pathogen invasion.

Many non-mucosal vaccines are poorly protective against mucosal pathogens, presumably because they do not generate mucosa-tropic memory cells. Few mucosal vaccines are in clinical use because live vaccine vectors pose safety risks and killed or molecular antigens (Ags) are weak immunogens when applied to intact mucosa. Adjuvants can potentially overcome this poor immunogenicity, however, conventional mucosal adjuvants possess unfavorable safety profiles. We have developed an adjuvanted vaccine against Chlamydia trachomatis. Genital Ct infection induced protective immunity that depended on interferon-γ (IFN-γ) producing CD4 T-cells, whereas mucosal exposure to UV-inactivated Ct (UV-Ct) generated tolerogenic Ct-specific regulatory T-cells, resulting in exacerbated bacterial burden upon Ct challenge. However, mucosal immunization with UV-Ct complexed with charge-switching synthetic adjuvant particles (cSAP) did not exert the tolerogenic effect of UV-Ct alone but elicited long-lived protection. This differential effect of UV-Ct-cSAP versus UV-Ct was because the former was presented by immunogenic CD11b+CD103–dendritic cells (DCs), while the latter was acquired by tolerogenic CD11b–CD103+ DCs. Genital protection was achieved after intrauterine or intranasal, but not subcutaneous vaccination and was inducible in conventional and humanized mice. Regardless of vaccination route, UV-Ct-cSAP induced robust systemic memory cells. However, only mucosal vaccination induced a wave of Ct-specific effector T-cells that seeded the mucosa during the first week and established resident memory T cells (TRM). Without TRM, mice were suboptimally protected, even when circulating memory cells were abundant. For optimal Ct clearance, both early seeding by TRM and infection-induced recruitment of a second wave of circulating memory cells were required. Thus, using a novel mucosal vaccine platform, we demonstrate that protection against Ct depends on synergistic actions of two memory T cell subsets with distinct migratory properties.

Antigen-specific CD4+ T cells against Chlamydia are crucial for driving bacterial clearance and mediating protection against reinfection. Although the Chlamydia trachomatis protein Cta1 has been identified to be a dominant murine CD4+ T cell antigen, its level of expression during the bacterial developmental cycle and precise localization within the host cell are unknown. Newly developed tools for Chlamydia genetic manipulation have allowed us to generate a C. trachomatis strain expressing a heterologous CD4+ T cell epitope from ovalbumin (OVA) consisting of OVA residues 323 to 339 (OVA323-339). By tagging proteins expressed in C. trachomatis with OVA323-339, we can begin to understand how protein expression, developmental regulation, and subcellular compartmentalization affect the potential of those proteins to serve as antigens. When OVA323-339 was expressed as a fusion with green fluorescent protein, we found that we were able to elicit an OT-II T cell response in an antigen-dependent manner, but surprisingly, these T cells were unable to reduce bacterial burden in mice. These data suggest that the subcellular localization of antigen, the level of antigen expression, or the timing of expression within the developmental cycle of Chlamydia may play a crucial role in eliciting a protective CD4+ T cell response.

Chlamydia trachomatis is the most commonly reported bacterial sexually transmitted infection in the United States. Modeling infection in animals can be challenging, as mice naturally clear C. trachomatis when it is deposited in the lower genital tract. However, C. trachomatis can productively infect mice when the lower genital tract is bypassed and bacteria are deposited directly into the upper genital tract via transcervical inoculation. Interestingly, the mouse-adapted Chlamydia species C. muridarum can infect mice both by transcervical inoculation and by natural ascension if introduced into the vaginal vault. In this study, we investigated whether the route of infection plays a role in the downstream immune responses to C. muridarum infection. We found that transcervical infection with C. muridarum results in higher bacterial burdens in the upper genital tract at earlier time points, correlating with levels of innate immune cells. When bacterial burdens were equivalent in intravaginally and transcervically infected mice at later time points, we observed substantially higher levels of adaptive immune cells in transcervically infected mice. Our data suggest that different routes of infection with the same organism can elicit different immune responses in the same tissue.

Gut-innervating nociceptor sensory neurons respond to noxious stimuli by initiating protective responses including pain and inflammation; however, their role in enteric infections is unclear. Here, we find that nociceptor neurons critically mediate host defense against the bacterial pathogen Salmonella enterica serovar Typhimurium (STm). Dorsal root ganglia nociceptors protect against STm colonization, invasion, and dissemination from the gut. Nociceptors regulate the density of microfold (M) cells in ileum Peyer's patch (PP) follicle-associated epithelia (FAE) to limit entry points for STm invasion. Downstream of M cells, nociceptors maintain levels of segmentous filamentous bacteria (SFB), a gut microbe residing on ileum villi and PP FAE that mediates resistance to STm infection. TRPV1+ nociceptors directly respond to STm by releasing calcitonin gene-related peptide (CGRP), a neuropeptide that modulates M cells and SFB levels to protect against Salmonella infection. These findings reveal a major role for nociceptor neurons in sensing and defending against enteric pathogens.