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1.
Figure 1

Figure 1. Genome-wide microarray analysis shows C. trachomatis elicits robust Type 2 immunity.. From: Human Female Genital Tract Infection by the Obligate Intracellular Bacterium Chlamydia trachomatis Elicits Robust Type 2 Immunity.

Compared to expression in uninfected controls, endometrial tissue from women with existing endometrial Chlamydia infection displayed 15-fold, 13-fold, and 11-fold increases in the expression of MMP-10, IL-24, and IL-13Rα2, respectively. These genes, each with biological activity linked to Type 2 immunity, were 3 of the 4 most dramatically upregulated genes in Chlamydia-infected tissue. Significance of differences between groups was determined by use of Dunn’s test (see Methods section for further details regarding statistical considerations). Open circles indicate samples from uninfected controls (n = 10); gray circles indicate samples from women with existing endometrial Chlamydia infection (n = 12) (horizontal bars indicate median values for each group).

Rodolfo D. Vicetti Miguel, et al. PLoS One. 2013;8(3):e58565.
2.
Figure 3

Figure 3. TH2-type immunity dominates host response to C. trachomatis infection.. From: Human Female Genital Tract Infection by the Obligate Intracellular Bacterium Chlamydia trachomatis Elicits Robust Type 2 Immunity.

PBMC were isolated from women with no history of Chlamydia infection (n = 7) and women with an existing endocervical or endometrial Chlamydia infection (n = 14) at enrollment and again from the latter women 1 and 4 months after initiating an anti-chlamydial antimicrobial. Flow cytometric analysis of intracellular cytokine staining (ICS) allowed comparison of EB-stimulated (A) CD3+CD4+ and (B) CD3+CD4- T cells that proliferated and produced IFN-γ, TNF, IL-4, or IL-17 (calculation described in Methods section). The adjusted percentages of cytokines that were produced in response to EB stimulation among uninfected and infected women were compared using Kruskal-Wallis’ test and Dunn’s post-hoc test (horizontal bars indicate medians). Grey boxes indicate pairs considered in the comparison for each p value displayed, and significant p values are indicated in bold characters.

Rodolfo D. Vicetti Miguel, et al. PLoS One. 2013;8(3):e58565.
3.
Figure 4

Figure 4. Endometrial Chlamydia infection is associated with the presence of CD4+ T cell aggregates and high expression of the TH2 transcription factor GATA-3.. From: Human Female Genital Tract Infection by the Obligate Intracellular Bacterium Chlamydia trachomatis Elicits Robust Type 2 Immunity.

Sequential sections of paraffin-embedded endometria from women with no identified C. trachomatis, N. gonorrhoeae, or T. vaginalis lower or upper genital tract infection (n = 4) or with endometrial C. trachomatis infection (n = 6) were used to immunohistochemically evaluate T-bet or GATA-3 expression (both DAB), and the presence of CD4+ mononuclear cells (Vector Red) as described in Methods section. Aggregates of GATA-3+ (but not T-bet+) and CD4+ mononuclear cells were seen in endometrial stroma of Chlamydia-infected tissue (representative micrographs shown at X200 magnification). Moreover, only a few CD4+ mononuclear cells were present in uninfected endometrial tissue even tough GATA-3 was expressed at high levels in both instances. Right panels show images displaying DAB or Vector Red staining and hematoxylin as counterstain, while left panels show DAB or Vector Red layer alone.

Rodolfo D. Vicetti Miguel, et al. PLoS One. 2013;8(3):e58565.
4.
Figure 2

Figure 2. The ability of peripheral T cells from women with existing or treated Chlamydia infection to proliferate in response to stimulation with C. trachomatis elementary bodies (EB) decreased 4 months after antimicrobial administration.. From: Human Female Genital Tract Infection by the Obligate Intracellular Bacterium Chlamydia trachomatis Elicits Robust Type 2 Immunity.

Peripheral blood mononuclear cells (PBMC) isolated from women at enrollment and at 1 and 4 m follow-up visits were cultured 96 h in presence of inactivated EB or media alone. Proliferation of (A) CD3+CD4+ and (B) CD3+CD4- cells was assessed by flow cytometry using stimulation indexes calculated as described in Methods section. Stratification of Chlamydia-infected women by time since diagnosis and treatment of infection showed T cell proliferation was higher 1 month after treatment compared to enrollment, and that proliferative capacity diminished 4 months after treatment. Stimulation indexes of samples from Chlamydia-infected women (n = 14) at indicated visits were compared to those from women with no known history of infection (n = 7) using one-way ANOVA and Dunnett’s multiple comparison test (horizontal bars indicate means).

Rodolfo D. Vicetti Miguel, et al. PLoS One. 2013;8(3):e58565.
5.
Figure 5

Figure 5. Endometrial Chlamydia infection causes infiltration of CD4+ T cells expressing GATA-3.. From: Human Female Genital Tract Infection by the Obligate Intracellular Bacterium Chlamydia trachomatis Elicits Robust Type 2 Immunity.

Sections of paraffin-embedded endometria from women with no identified C. trachomatis, N. gonorrhoeae, or T. vaginalis lower or upper genital tract infection (n = 4) or with endometrial C. trachomatis infection (n = 6) were utilized to simultaneously detect the expression of GATA-3 (DAB) and CD4 (Vector Red) using immunohistochemistry, as described in Methods section. (A) In uninfected endometrial tissue, we observed scarce numbers of CD4+ cells coexpressing GATA-3, however, in endometrial tissue from Chlamydia-infected women the presence of aggregates of GATA-3+ CD4+ mononuclear cells was patent (representative micrographs shown at X200 magnification). Upper left panels show images displaying Vector Red staining, while upper right panels show images displaying DAB staining as defined by spectral analysis. Lower right panels show original images used in analysis, and lower left panels show images in which GATA-3 and CD4 colocalization areas have been digitally highlighted (light blue). Circles delineate areas of highest colocalization in images shown. (B) Colocalization of CD4+ areas within GATA-3+ areas increases dramatically with Chlamydia infection, indicating that endometrial Chlamydia infection drives the infiltration of GATA-3+CD4+ T cells that form aggregates. Each symbol represents the percentage of colocalization observed in a single field. Matching colors indicate all the fields evaluated from one specimen. Comparison was performed using a two-tailed Mann-Whitney test (horizontal bars indicate medians).

Rodolfo D. Vicetti Miguel, et al. PLoS One. 2013;8(3):e58565.
6.
Figure 6

Figure 6. Endometrial Chlamydia infection promotes alternative activation of macrophages.. From: Human Female Genital Tract Infection by the Obligate Intracellular Bacterium Chlamydia trachomatis Elicits Robust Type 2 Immunity.

Endometrial tissue from women with no identified C. trachomatis, N. gonorrhoeae, or T. vaginalis lower or upper genital tract infection (n = 4), or from women with endocervical or endometrial C. trachomatis infection (n = 14 for Panel A; n = 12 for Panel B) were processed for flow cytometric analysis as described in Methods section. Macrophages were identified as FSC-AintSSC-AintCD45+CD15-CD14+HLA-DR+ live cells (as depicted in Figure S3), and 2 monoclonal antibody panels were used to interrogate macrophage differentiation and activation. Panel (A) evaluated expression of CD163, CD209, CD200R and CD206, while panel (B) evaluated expression of CD64, CD80, CD40 and CD86. Comparisons were done using unpaired one-tailed Student t-tests with Welch’s correction (horizontal bars indicate mean values for each group and significant p values are indicated in bold characters). Open circles indicate samples from uninfected controls; light gray circles indicate samples from women with cervical Chlamydia infection; and dark gray circles indicate samples from women with endometrial Chlamydia infection. Representative contour plots of CD200R, CD206, CD40 and CD80 expression by endometrial macrophages are displayed next to figures. For CD200R and CD206 expression evaluation (A), representative flow plots from peripheral blood monocytes treated with IL-4 (100 U/ml) for 24 hours and the corresponding untreated control are also shown.

Rodolfo D. Vicetti Miguel, et al. PLoS One. 2013;8(3):e58565.

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