An atlas of human vector-borne microbe interactions reveals pathogenicity mechanisms.

Vector-borne diseases are a leading cause of death worldwide and pose a substantial unmet medical need. Pathogens binding to host extracellular proteins (the "exoproteome") represents a crucial interface in the etiology of vector-borne disease. Here, we used bacterial selection to elucidate host-microbe interactions in high throughput (BASEHIT)-a technique enabling interrogation of microbial interactions with 3,324 human exoproteins-to profile the interactomes of 82 human-pathogen samples, including 30 strains of arthropod-borne pathogens and 8 strains of related non-vector-borne pathogens. The resulting atlas revealed 1,303 putative interactions, including hundreds of pairings with potential roles in pathogenesis, including cell invasion, tissue colonization, immune evasion, and host sensing. Subsequent functional investigations uncovered that Lyme disease spirochetes recognize epidermal growth factor as an environmental cue of transcriptional regulation and that conserved interactions between intracellular pathogens and thioredoxins facilitate cell invasion. In summary, this interactome atlas provides molecular-level insights into microbial pathogenesis and reveals potential host-directed targets for next-generation therapeutics.

Combining Cellular Immunology With RNAseq to Identify Novel Chlamydia T-Cell Subset Signatures.

Chlamydia trachomatis serovars A-L cause important diseases of the eyes and reproductive tract by infecting epithelium lining those organs. A major hurdle for vaccine trials is finding a surrogate biomarker for protective immunity. Investigational data argues for T-cell biomarker(s) reflecting mucosal adaption, cytokine polarization, B-cell help, antibacterial effector mechanisms, or some combination thereof. A human investigation and 2 mouse studies link IL-13 to protection from infection/immunopathology. We performed RNAseq on T cells resident in spleens and genital tracts of naturally immune mice. CD4 signatures were consistent with helper function that differed by site including a genital tract-specific Fgl2 signal. The genital tract CD8 signature featured IL-10 and promotion of healing/scarring with a unique transcription of granzyme A. The RNAseq data was used to refine previously published CD4γ13 and CD8γ13 transcriptomes derived from protective T-cell clones, potentially identifying practicable T-cell subset signatures for assessing Chlamydia vaccine candidates.

Comparison of Chlamydia outer membrane complex to recombinant outer membrane proteins as vaccine.

The Chlamydial outer membrane complex (COMC) from the elementary body (EB) is a protein rich insoluble outer membrane shell from which cytosolic proteins have been extracted with detergent. In this study we conducted mass spectrometry experiments to detect proteins in the COMC prepared from C. muridarum EB. Proteomic analysis showed that the COMC contained 75 proteins that included 10 outer membrane proteins (OMPs) such as the major outer membrane protein (MOMP) and polymorphic membrane proteins (Pmps) that were previously identified as CD4 T cell vaccine candidates. We tested the vaccine efficacy of COMC in comparison to individual or combination of recombinant OMPs formulated with Th1 polarizing adjuvant DDA/MPL in two murine genital tract models (C. muridarum and C. trachomatis) by measuring organismal shedding, tubal pathology and immune responses including neutralizing antibodies. In the C. muridarum model, the COMC vaccine generated broadly reactive immune responses against multiple outer membrane proteins, high levels of EB binding and neutralizing antibody and exhibited superior protection against genital infection and pathology when compared to the recombinant PmpG vaccine. Denaturing the COMC by boiling significantly reduced protection. In the C. trachomatis model, the COMC vaccine also conferred greater protection compared to individual or multiple recombinant outer membrane proteins. Immunization with multiple COMCs from C. trachomatis serovars D, F and J generated neutralizing antibodies against multiple C. trachomatis serovars. We conclude that broader immunogenicity and generation of neutralizing antibody may explain the superior efficacy of COMC vaccine. The study suggests that conformationally intact proteins will be necessary for a successful recombinant OMP vaccine.

A Class II-Restricted CD8γ13 T-Cell Clone Protects During Chlamydia muridarum Genital Tract Infection.

BACKGROUND: The T-cell response to chlamydia genital tract infections in humans and mice is unusual because the majority of antigen-specific CD8 T cells are not class I restricted (referred to here as "unrestricted" or "atypical"). We previously reported that a subset of unrestricted murine chlamydia-specific CD8 T cells had a cytokine polarization pattern that included interferon (IFN)-γ and interleukin (IL)-13. METHODS: In this study, we investigated the transcriptome of CD8γ13 T cells, comparing them to Tc1 clones using microarray analysis. That study revealed that CD8γ13 polarization included IL-5 in addition to IFN-γ and IL-13. Adoptive transfer studies were performed with Tc1 clones and a CD8γ13 T-cell clone to determine whether either influenced bacterial clearance or immunopathology during Chlamydia muridarum genital tract infections. RESULTS: To our surprise, an adoptively transferred CD8γ13 T-cell clone was remarkably proficient at preventing chlamydia immunopathology, whereas the multifunctional Tc1 clone did not enhance clearance or significantly alter immunopathology. Mapping studies with major histocompatibility complex (MHC) class I- and class II-deficient splenocytes showed our previously published chlamydia-specific CD8 T-cell clones are MHC class II restricted. CONCLUSIONS: The MHC class II-restricted CD8 T cells may play an important role in protection from intracellular pathogens that limit class I antigen presentation or diminish CD4 T-cell numbers or impair their function.

Discordance in the Epithelial Cell-Dendritic Cell Major Histocompatibility Complex Class II Immunoproteome: Implications for Chlamydia Vaccine Development.

BACKGROUND: Chlamydia trachomatis and Chlamydia muridarum are intracellular bacterial pathogens of mucosal epithelial cells. CD4 T cells and major histocompatibility complex (MHC) class II molecules are essential for protective immunity against them. Antigens presented by dendritic cells (DCs) expand naive pathogen-specific T cells (inductive phase), whereas antigens presented by epithelial cells identify infected epithelial cells as targets during the effector phase. We previously showed that DCs infected by C trachomatis or C muridarum present epitopes from a limited spectrum of chlamydial proteins recognized by Chlamydia-specific CD4 T cells from immune mice. METHODS: We hypothesized that Chlamydia-infected DCs and epithelial cells present overlapping sets of Chlamydia-MHC class II epitopes to link inductive and effector phases to generate protective immunity. We tested that hypothesis by infecting an oviductal epithelial cell line with C muridarum, followed by immunoaffinity isolation and sequencing of MHC class I- and II-bound peptides. RESULTS: We identified 26 class I-bound and 4 class II-bound Chlamydia-derived peptides from infected epithelial cells. We were surprised to find that none of the epithelial cell class I- and class II-bound chlamydial peptides overlapped with peptides presented by DCs. CONCLUSIONS: We suggest the discordance between the DC and epithelial cell immunoproteomes has implications for delayed clearance of Chlamydia and design of a Chlamydia vaccine.

B Cell Presentation of Chlamydia Antigen Selects Out Protective CD4γ13 T Cells: Implications for Genital Tract Tissue-Resident Memory Lymphocyte Clusters.

Surveillance and defense of the enormous mucosal interface with the nonsterile world are critical to protecting the host from a wide range of pathogens. Chlamydia trachomatis is an intracellular bacterial pathogen that replicates almost exclusively in the epithelium of the reproductive tract. The fallopian tubes and vagina are poorly suited to surveillance and defense, with limited immune infrastructure positioned near the epithelium. However, a dynamic process during clearing primary infections leaves behind new lymphoid clusters immediately beneath the epithelium. These memory lymphocyte clusters (MLCs) harboring tissue-resident memory (Trm) T cells are presumed to play an important role in protection from subsequent infections. Histologically, human Chlamydia MLCs have prominent B cell populations. We investigated the status of genital tract B cells during C. muridarum infections and the nature of T cells recovered from immune mice using immune B cells as antigen-presenting cells (APCs). These studies revealed a genital tract plasma B cell population and a novel genital tract CD4 T cell subset producing both gamma interferon (IFN-γ) and interleukin-13 (IL-13). A panel of CD4 T cell clones and microarray analysis showed that the molecular fingerprint of CD4γ13 T cells includes a Trm-like transcriptome. Adoptive transfer of a Chlamydia-specific CD4γ13 T cell clone completely prevented oviduct immunopathology without accelerating bacterial clearance. Existence of a CD4γ13 T cell subset provides a plausible explanation for the observation that human peripheral blood mononuclear cell (PBMC) Chlamydia-specific IFN-γ and IL-13 responses predict resistance to reinfection.

Pediatric Kawasaki Disease and Adult Human Immunodeficiency Virus Kawasaki-Like Syndrome Are Likely the Same Malady.

Background. Pediatric Kawasaki disease (KD) and human immunodeficiency virus (HIV)+ adult Kawasaki-like syndrome (KLS) are dramatic vasculitides with similar physical findings. Both syndromes include unusual arterial histopathology with immunoglobulin (Ig)A+ plasma cells, and both impressively respond to pooled Ig therapy. Their distinctive presentations, histopathology, and therapeutic response suggest a common etiology. Because blood is in immediate contact with inflamed arteries, we investigated whether KD and KLS share an inflammatory signature in serum. Methods. A custom multiplex enzyme-linked immunosorbent assay (ELISA) defined the serum cytokine milieu in 2 adults with KLS during acute and convalescent phases, with asymptomatic HIV+ subjects not taking antiretroviral therapy serving as controls. We then prospectively collected serum and plasma samples from children hospitalized with KD, unrelated febrile illnesses, and noninfectious conditions, analyzing them with a custom multiplex ELISA based on the KLS data. Results. Patients with KLS and KD subjects shared an inflammatory signature including acute-phase reactants reflecting tumor necrosis factor (TNF)-α biologic activity (soluble TNF receptor I/II) and endothelial/smooth muscle chemokines Ccl1 (Th2), Ccl2 (vascular inflammation), and Cxcl11 (plasma cell recruitment). Ccl1 was specifically elevated in KD versus febrile controls, suggesting a unique relationship between Ccl1 and KD/KLS pathogenesis. Conclusions. This study defines a KD/KLS inflammatory signature mirroring a dysfunctional response likely to a common etiologic agent. The KD/KLS inflammatory signature based on elevated acute-phase reactants and specific endothelial/smooth muscle chemokines was able to identify KD subjects versus febrile controls, and it may serve as a practicable diagnostic test for KD.

Tissue-Resident T Cells as the Central Paradigm of Chlamydia Immunity.

For almost 2 decades, results from Chlamydia pathogenesis investigations have been conceptualized using a cytokine polarization narrative. Recent viral immunity studies identifying protective tissue-resident memory T cells (Trm) suggest an alternative paradigm based on localized immune networks. As Chlamydia vaccines enter the preclinical pipeline and, in the case of an attenuated trachoma vaccine, are given to human subjects, it may be useful to ask whether cytokine polarization is the appropriate framework for understanding and evaluating vaccine efficacy. In this review, we revisit C. trachomatis pathogenesis data from mice and humans using a Trm narrative and note a comfortable concordance with the Chlamydia pathogenesis literature.

Modeling the transcriptome of genital tract epithelial cells and macrophages in healthy mucosa versus mucosa inflamed by Chlamydia muridarum infection.

Chlamydia trachomatis urogenital serovars are intracellular bacteria that parasitize human reproductive tract epithelium. As the principal cell type supporting bacterial replication, epithelial cells are central to Chlamydia immunobiology initially as sentries and innate defenders, and subsequently as collaborators in adaptive immunity-mediated bacterial clearance. In asymptomatic individuals who do not seek medical care a decisive struggle between C. trachomatis and host defenses occurs at the epithelial interface. For this study, we modeled the immunobiology of epithelial cells and macrophages lining healthy genital mucosa and inflamed/infected mucosa during the transition from innate to adaptive immunity. Upper reproductive tract epithelial cell line responses were compared to bone marrow-derived macrophages utilizing gene expression microarray technology. Those comparisons showed minor differences in the intrinsic innate defenses of macrophages and epithelial cells. Major lineage-specific differences in immunobiology relate to epithelial collaboration with adaptive immunity including an epithelial requirement for inflammatory cytokines to express MHC class II molecules, and a paucity and imbalance between costimulatory and coinhibitory ligands on epithelial cells that potentially limits sterilizing immunity (replication termination) to Chlamydia-specific T cells activated with limited or unconventional second signals.

An atypical CD8 T-cell response to Chlamydia muridarum genital tract infections includes T cells that produce interleukin-13.

Chlamydia trachomatis urogenital serovars D-K are intracellular bacterial pathogens that replicate almost exclusively in human reproductive tract epithelium. In the C. muridarum mouse model for human Chlamydia genital tract infections CD4 T helper type 1 cell responses mediate protective immunity while CD8 T-cell responses have been associated with scarring and infertility. Scarring mediated by CD8 T cells requires production of tumour necrosis factor-α (TNF-α); however, TNF-α is associated with protective immunity mediated by CD4 T cells. The latter result suggests that TNF-α in-and-of itself may not be the sole determining factor in immunopathology. CD8 T cells mediating immunopathology presumably do something in addition to producing TNF-α that is detrimental during resolution of genital tract infections. To investigate the mechanism underlying CD8 immunopathology we attempted to isolate Chlamydia-specific CD8 T-cell clones from mice that self-cleared genital tract infections. They could not be derived with antigen-pulsed irradiated naive splenocytes; instead derivation required use of irradiated immune splenocyte antigen-presenting cells. The Chlamydia-specific CD8 T-cell clones had relatively low cell surface CD8 levels and the majority were not restricted by MHC class Ia molecules. They did not express Plac8, and had varying abilities to terminate Chlamydia replication in epithelial cells. Two of the five CD8 clones produced interleukin-13 (IL-13) in addition to IL-2, TNF-α, IL-10 and interferon-γ. IL-13-producing Chlamydia-specific CD8 T cells may contribute to immunopathology during C. muridarum genital tract infections based on known roles of TNF-α and IL-13 in scar formation.

Perforin is detrimental to controlling [corrected] C. muridarum replication in vitro, but not in vivo.

CD4 T cells are critical for clearing experimental Chlamydia muridarum genital tract infections. Two independent in vitro CD4 T cell mechanisms have been identified for terminating Chlamydia replication in epithelial cells. One mechanism, requiring IFN-γ and T cell-epithelial cell contact, terminates infection by triggering epithelial production of nitric oxide to chlamydiacidal levels; the second is dependent on T cell degranulation. We recently demonstrated that there are two independent in vivo clearance mechanisms singly sufficient for clearing genital tract infections within six weeks; one dependent on iNOS, the other on Plac8. Redundant genital tract clearance mechanisms bring into question negative results in single-gene knockout mice. Two groups have shown that perforin-knockout mice were not compromised in their ability to clear C. muridarum genital tract infections. Because cell lysis would be detrimental to epithelial nitric oxide production we hypothesized that perforin was not critical for iNOS-dependent clearance, but posited that perforin could play a role in Plac8-dependent clearance. We tested whether the Plac8-dependent clearance was perforin-dependent by pharmacologically inhibiting iNOS in perforin-knockout mice. In vitro we found that perforin was detrimental to iNOS-dependent CD4 T cell termination of Chlamydia replication in epithelial cells. In vivo, unexpectedly, clearance in perforin knockout mice was delayed to the end of week 7 regardless of iNOS status. The discordant in vitro/in vivo results suggest that the perforin's contribution to bacterial clearance in vivo is not though enhancing CD4 T cell termination of Chlamydia replication in epithelial cells, but likely via a mechanism independent of T cell-epithelial cell interactions.

Pentoxifylline, inflammation, and endothelial function in HIV-infected persons: a randomized, placebo-controlled trial.

BACKGROUND: Untreated HIV may increase the risk of cardiovascular events. Our preliminary in vitro and in vivo research suggests that pentoxifylline (PTX) reduces vascular inflammation and improves endothelial function in HIV-infected persons not requiring antiretroviral therapy. METHODS: We performed a randomized, placebo-controlled trial of PTX 400 mg orally thrice daily for 8 weeks in 26 participants. The primary endpoint was change in flow-mediated dilation (FMD) of the brachial artery after 8 weeks. Nitroglycerin-mediated dilation (NTGMD) and circulating markers of inflammation, cellular immune activation, coagulation, and metabolism were also assessed. RESULTS: The difference in mean absolute change (SD) in FMD after 8 weeks between the placebo [-1.06 (1.45)%] and PTX [-1.93 (3.03)%] groups was not significant (P = 0.44). No differences in NTGMD were observed. The only significant between-group difference in the changes in biomarkers from baseline to week 8 was in soluble tumor necrosis factor receptor-1 (sTNFRI) [-83.2 pg/mL in the placebo group vs. +65.9 pg/mL in the PTX group; P = 0.03]. PTX was generally well-tolerated. CONCLUSIONS: PTX did not improve endothelial function and unexpectedly increased the inflammatory biomarker sTNFRI in HIV-infected participants not requiring antiretroviral therapy. Additional interventional research is needed to reduce inflammation and cardiovascular risk in this population. TRIAL REGISTRATION: ClinicalTrials.gov NCT00796822.

Nearly Fatal Case of Whipple's Disease in a Patient Mistakenly on Anti-TNF Therapy.

Whipple's disease is a rare cause of chronic diarrhea and abdominal pain that may be confused with inflammatory bowel disease. We report a Whipple's case misdiagnosed as Crohn's disease in which treatment with anti-tumor necrosis factor (anti-TNF) therapy led to nearly fatal progression. Lymph node tissue obtained during laparotomy for suspected bowel necrosis stained dramatically with periodic acid-Schiff (PAS), and electron microscopy showed a bacterium consistent with Trophyrema whipplei. The patient made a remarkable recovery complicated only by cholestatic hepatitis, which was likely a treatment-associated inflammatory response. This case serves as a reminder that all granulomatous infections should be considered prior to initiation of anti-TNF therapies.

PmpG303-311, a protective vaccine epitope that elicits persistent cellular immune responses in Chlamydia muridarum-immune mice.

Urogenital Chlamydia serovars replicating in reproductive epithelium pose a unique challenge to host immunity and vaccine development. Previous studies have shown that CD4 T cells are necessary and sufficient to clear primary Chlamydia muridarum genital tract infections in the mouse model, making a protective CD4 T cell response a logical endpoint for vaccine development. Our previous proteomics studies identified 13 candidate Chlamydia proteins for subunit vaccines. Of those, PmpG-1 is the most promising vaccine candidate. To further that work, we derived a PmpG(303-311)-specific multifunctional Th1 T cell clone, designated PmpG1.1, from an immune C57BL/6 mouse and used it to investigate the presentation of the PmpG(303-311) epitope by infected epithelial cells. Epithelial presentation of the PmpG(303-311) epitope required bacterial replication, occurred 15 to 18 h postinfection, and was unaffected by gamma interferon (IFN-γ) pretreatment. Unlike epitopes recognized by other Chlamydia-specific CD4 T cell clones, the PmpG(303-311) epitope persisted on splenic antigen-presenting cells (APC) of mice that cleared primary genital tract infections. PmpG1.1 was activated by unmanipulated irradiated splenocytes from immune mice without addition of exogenous Chlamydia antigen, and remarkably, activation of PmpG1.1 by unmanipulated immune splenocytes was stronger 6 months postinfection than it was 3 weeks postinfection. Enhanced presentation of PmpG(303-311) epitope on splenic APC 6 months postinfection reflects some type of "consolidation" of a protective immune response. Understanding the antigen-presenting cell populations responsible for presenting PmpG(303-311) early (3 weeks) and late (6 months) postinfection will likely provide important insights into stable protective immunity against Chlamydia infections of the genital tract.

Plac8-dependent and inducible NO synthase-dependent mechanisms clear Chlamydia muridarum infections from the genital tract.

Chlamydia trachomatis urogenital serovars replicate predominantly in genital tract epithelium. This tissue tropism poses a unique challenge for host defense and vaccine development. Studies utilizing the Chlamydia muridarum mouse model have shown that CD4 T cells are critical for clearing genital tract infections. In vitro studies have shown that CD4 T cells terminate infection by upregulating epithelial inducible NO synthase (iNOS) transcription and NO production. However, this mechanism is not critical, as iNOS-deficient mice clear infections normally. We recently showed that a subset of Chlamydia-specific CD4 T cell clones could terminate replication in epithelial cells using an iNOS-independent mechanism requiring T cell degranulation. We advance that work using microarrays to compare iNOS-dependent and iNOS-independent CD4 T cell clones. Plac8 was differentially expressed by clones having the iNOS-independent mechanism. Plac8-deficient mice had delayed clearance of infection, and Plac8-deficient mice treated with the iNOS inhibitor N-monomethyl-l-arginine were largely unable to resolve genital tract infections over 8 wk. These results demonstrate that there are two independent and redundant T cell mechanisms for clearing C. muridarum genital tract infections: one dependent on iNOS, and the other dependent on Plac8. Although T cell subsets are routinely defined by cytokine profiles, there may be important subdivisions by effector function, in this case CD4(Plac8).

Chlamydia-specific CD4 T cell clones control Chlamydia muridarum replication in epithelial cells by nitric oxide-dependent and -independent mechanisms.

Chlamydia trachomatis serovars D-K are sexually transmitted intracellular bacterial pathogens that replicate in epithelial cells lining the human reproductive tract. It is clear from knockout mice and T cell depletion studies using Chlamydia muridarum that MHC class II and CD4 T cells are critical for clearing bacteria from the murine genital tract. It is not clear how CD4 T cells interact with infected epithelial cells to mediate bacterial clearance in vivo. Previous work using an epithelial tumor cell line showed that a Chlamydia-specific CD4 T cell clone was able to inhibit C. muridarum replication in vitro via induction of epithelial NO production. We have previously shown that Chlamydia-specific CD4 T cell clones can recognize and be activated by infected reproductive tract epithelial cells and block Chlamydia replication in them. We extend those observations by investigating the mechanism used by a panel of CD4 T cell clones to control Chlamydia replication in epithelial cells. We found that Chlamydia-specific CD4 T cell clones were cytolytic, but that cytolysis was not likely critical for controlling C. muridarum replication. For one, CD4 T cell clone-induced epithelial NO production was critical for controlling replication; however, the most potent CD4 T cell clones were dependent on T cell degranulation for replication control with only a minor additional contribution from NO production. We discuss our data as they relate to existing knockout mouse studies addressing mechanisms of T cell-mediated control of Chlamydia replication and their implications for intracellular epithelial pathogens in mouse models.

The Chlamydia muridarum-induced IFN-ß response is TLR3-dependent in murine oviduct epithelial cells.

Epithelial cells lining the murine genital tract act as sentinels for microbial infection, play a major role in the initiation of the early inflammatory response, and can secrete factors that modulate the adaptive immune response when infected with Chlamydia. C. muridarum-infected murine oviduct epithelial cells secrete the inflammatory cytokines IL-6 and GM-CSF in a TLR2-dependent manner. Further, C. muridarum infection induces IFN-ß synthesis in the oviduct epithelial cells in a TRIF-dependent manner. Because murine oviduct epithelial cells express TLR3 but not TLRs 4, 7, 8, or 9, we hypothesized that TLR3 or an unknown TRIF-dependent pattern recognition receptor was the critical receptor for IFN-ß production. To investigate the role of TLR3 in the Chlamydia-induced IFN-ß response in oviduct epithelial cells, we used small interfering RNA, dominant-negative TLR3 mutants, and TLR3-deficient oviduct epithelial cells to show that the IFN-ß secreted during C. muridarum infection requires a functional TLR3. Interestingly, we demonstrate that the TLR3 signaling pathway is not required for IFN-ß synthesis in C. muridarum-infected macrophages, suggesting that there are alternate and redundant pathways to Chlamydia-induced IFN-ß synthesis that seem to be dependent upon the cell type infected. Finally, because there is no obvious dsRNA molecule associated with Chlamydia infection, the requirement for TLR3 in Chlamydia-induced IFN-ß synthesis in infected oviduct epithelial cells implicates a novel ligand that binds to and signals through TLR3.