<i>Chlamydia</i>-specific T_H17 immunity promoted genital tract damage.

<p>Groups of mice described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162445#pone.0162445.g004" target="_blank">Fig 4C</a> were euthanized at 21 dpc, and UGT processed for histological evaluation. (A) Representative results from H&E staining shows pervasive genital tract damage in mice treated with antibody blocking IFN-γ signaling characterized by UGT inflammation and intra-abdominal adhesions; (scale bar top row, 500 μm; scale bar lower rows, 50 μm). (B) Semi-quantitative scoring system identifies significant UGT inflammation in mice administered antibody blocking IFN-γ signaling. (C) In separate studies, DLN were excised at 5 dpc from controls or mice administered antibodies blocking IFN-γ, IL-17, or IFN-γ and IL-17 signaling concomitant with challenge, and processed into single-cell suspensions. Flow cytometry was used to quantify intracellular accumulation of IFN-γ and IL-17 by CD4⁺ T cells that responded to <i>Chlamydia</i> EB stimulation; (percentages of cytokine-producing CD4⁺ T cells are displayed) (bars indicate means). In other studies, flow cytometry was used to characterize UGT inflammation in uninfected controls, mice at 5 dpc, and mice at 5 dpc that were administered antibodies blocking IFN-γ signaling concomitant with the ivag challenge infection. (D) Representative contour plots for UGT macrophages and inflammatory monocytes displayed; (numbers denote percentage of the myeloid cell populations). (E) Heightened IL-17 secretion by CD4⁺ T cells induced by IFN-γ signaling blockade significantly increased numbers of eosinophils, inflammatory monocytes, polymorphonuclear neutrophils, and macrophages in the UGT at 5 dpc (values are mean ±SD). Results displayed in Fig 5 are representative of 2 independent experiments (n = 5 per group).</p

Primary ivag <i>C</i>. <i>trachomatis</i> infection elicits robust endometrial inflammation.

<p>As denoted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162445#pone.0162445.g001" target="_blank">Fig 1</a>, Balb/cJ mice were infected with 10⁴ IFU of <i>C</i>. <i>trachomatis</i> serovar D. Animals were euthanized at indicated dpi, and UGT tissue used to characterize inflammation by H&E staining (upper panels) or IHC of CD45⁺ cell infiltrates; (lower panels) (n = 5–10 per group). Representative images indicate endometrial leukocyte infiltration was particularly intense at 7 dpi; (scale bar, 50 μm) (displayed results are representative of 2–3 independent experiments).</p

Intravaginal (ivag) <i>C</i>. <i>trachomatis</i> infection of mice ascends to the upper genital tract (UGT).

<p>6–8 week old female Balb/cJ mice were injected s.c. with 1 mg DMPA 5 days prior to ivag infection with 10⁴ IFU of <i>C</i>. <i>trachomatis</i> (<i>Ct</i>) serovar D (mice were infected daily for 3 consecutive days). On indicated dpi, cervicovaginal lavage (CVL) specimens were collected to assess <i>Chlamydia</i> clearance via (A) IFU assays (B) RT-qPCR that measured <i>Chlamydia</i> DNA levels and (C) ELISA that quantitated C<i>hlamydia</i> lipopolysaccharide (LPS) levels; (mean ±SD, n = 5 per group). (D) in separate studies, mice were euthanized on specified dpi, UGT excised, and <i>Chlamydia</i> DNA and host DNA quantified via RT-qPCR; (median ± range, n = 5 per time point) (ND, non-detectable) (displayed results representative of 2–3 independent experiments).</p

Intravaginal <i>Chlamydia trachomatis</i> Challenge Infection Elicits T_H1 and T_H17 Immune Responses in Mice That Promote Pathogen Clearance and Genital Tract Damage

<div><p>While ascension of <i>Chlamydia trachomatis</i> into the upper genital tract of women can cause pelvic inflammatory disease and Fallopian tube damage, most infections elicit no symptoms or overt upper genital tract pathology. Consistent with this asymptomatic clinical presentation, genital <i>C</i>. <i>trachomatis</i> infection of women generates robust T_H2 immunity. As an animal model that modeled this response would be invaluable for delineating bacterial pathogenesis and human host defenses, herein we explored if pathogen-specific T_H2 immunity is similarly elicited by intravaginal (ivag) infection of mice with oculogenital <i>C</i>. <i>trachomatis</i> serovars. Analogous to clinical infection, ascension of primary <i>C</i>. <i>trachomatis</i> infection into the mouse upper genital tract produced no obvious tissue damage. Clearance of ivag challenge infection was mediated by interferon (IFN)-γ-producing CD4⁺ T cells, while IFN-γ signaling blockade concomitant with a single ivag challenge promoted tissue damage by enhancing <i>Chlamydia</i>-specific T_H17 immunity. Likewise, IFN-γ and IL-17 signaling blockade or CD4⁺ T cell depletion eliminated the genital pathology produced in untreated controls by multiple ivag challenge infections. Conversely, we were unable to detect formation of pathogen-specific T_H2 immunity in <i>C</i>. <i>trachomatis</i>-infected mice. Together, our work revealed <i>C</i>. <i>trachomatis</i> infection of mice generates T_H1 and T_H17 immune responses that promote pathogen clearance and immunopathological tissue damage. Absence of <i>Chlamydia</i>-specific T_H2 immunity in these mice newly highlights the need to identify experimental models of <i>C</i>. <i>trachomatis</i> genital infection that more closely recapitulate the human host response.</p></div

Exogenous sex steroids regulate genital epithelial barrier function in female rhesus macaques.

There is concern that using depot-medroxyprogesterone acetate (DMPA) for pregnancy prevention heightens HIV susceptibility. While no clinical data establishes causal link between HIV acquisition and use of this injectable progestin, prior work from our laboratory showed that DMPA comparably lowers genital levels of the cell-cell adhesion molecule desmoglein-1 (DSG1) and weakens genital epithelial barrier function in female mice and women. We likewise saw DMPA increase mouse susceptibility to multiple genital pathogens including HIV. Herein, we sought to confirm and extend these findings by comparing genital epithelial barrier function in untreated rhesus macaques (RM) vs. RM treated with DMPA or DMPA and estrogen (E). Compared to controls, genital tissue from RM with pharmacologically relevant serum levels of medroxyprogesterone acetate displayed significantly lower DSG1 levels and greater permeability to low molecular mass molecules. Conversely, DMPA-mediated effects on genital epithelial integrity and function were obviated in RM administered DMPA and E. These data corroborate the diminished genital epithelial barrier function observed in women initiating DMPA and identify RM as a useful preclinical model for defining effects of exogenous sex steroids on genital pathogen susceptibility. As treatment with E averted DMPA-mediated loss of genital epithelial barrier function, our results also imply that contraceptives releasing progestin and E may be less likely to promote transmission of HIV and other sexually transmitted pathogens than progestin-only compounds

<i>C</i>. <i>trachomatis</i>-specific CD4⁺ T cells displayed T_H1 and T_H17 effector function, but control of ivag <i>C</i>. <i>trachomatis</i> challenge is mainly mediated by T_H1 immunity.

<p>At 60 dpi, mice were ivag challenged with 10⁶ IFU of <i>C</i>. <i>trachomatis</i> serovar D. At 5 dpc, animals were euthanized, DLN excised and processed into single-cell suspensions, and cells incubated with inactivated <i>Chlamydia</i> EB or media alone. (A) Representative contour plots from the intracellular cytokine staining (ICS) assay used to quantify secretion of IFN-γ, TNF, IL-17, and IL-4 by CD4⁺ and CD8⁺ T cells that responded to stimulation with <i>Chlamydia</i> EB; (quadrant numbers indicate percentages of cytokine-producing cells). (B) Percentages of cytokine-producing CD4⁺ and CD8⁺ T cells; (n = 13) (bars indicate medians). (C) Using additional Balb/cJ mice at 60 dpi, 10⁶ IFU of <i>C</i>. <i>trachomatis</i> serovar D was ivag administered to untreated controls or mice treated with antibodies blocking IFN-γ, IL-17, or IFN-γ and IL-17 signaling concomitant with challenge. At various days after challenge, CVL specimens were collected to measure <i>Chlamydia</i> DNA levels via RT-qPCR. AUC analysis for bacterial clearance revealed that <i>Chlamydia</i> challenge was primarily controlled by T_H1 immunity; (n = 5 per group) (values are means ±SD) (results representative of 2 independently performed studies) (ND, non-detectable).</p

CD4⁺ T cells controlled ivag <i>C</i>. <i>trachomatis</i> challenge infection.

<p>Uninfected Balb/cJ mice were ivag infected with <i>C</i>. <i>trachomatis</i> as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162445#pone.0162445.g001" target="_blank">Fig 1</a> or remained uninfected. (A) Uninfected mice or mice 90 dpi were ivag challenged with 10⁶ IFU of <i>C</i>. <i>trachomatis</i> serovar D. At indicated dpi or dpc, mice were euthanized and UGT processed for H&E staining or analysis of CD3ε⁺ cells by IHC; representative images reveal intense mononuclear CD3ε⁺ infiltrate at 5 dpc (scale bar, 50 μm). (B) Quantification of CD3ε⁺ cell infiltrates with ImageJ software (as described in Methods) showed no statistically significant differences between mice at dpi 90 and dpc 21 (n = 15 per group) (bars indicate means). (C) <i>Chlamydia</i> DNA levels were determined by RT-qPCR mice in CVL specimens collected from mice during primary ivag infection, after ivag challenge, and after ivag challenge of mice administered antibodies depleting CD4⁺ or CD8⁺ T cells 1 day prior to challenge and every other day until euthanasia. Areas under the curve (AUC) for bacterial clearance were compared as described in Methods, and this revealed CD4⁺ T cells were the T cell subset that controlled <i>Chlamydia</i> challenge; (n = 5 per group) (values are mean ±SD) (results representative of 2 independent experiments) (ND, non-detectable).</p