Reduced spreading of <i>Chlamydia muridarum</i> from the oviduct/ovary into uterine horn after intrabursal injection.

<p>The wild type <i>C</i>. <i>muridarum</i> organisms were intrabursally inoculated into the right oviduct/ovary of female C57BL/6J mice with 2 x 10⁵ inclusion forming units (IFUs) per mouse. At various time points post inoculation as indicated along the X-axis, groups of mice (n = 4 to 5) were sacrificed for harvesting the right oviduct/ovary (RO, solid bar) and right uterine horn (RU, open) respectively. The tissue samples were homogenized for titrating <i>C</i>. <i>muridarum</i> live organisms, and the titers were expressed as Log10 IFUs displayed along the Y-axis. Please note that titers of live organisms recovered from RO that received direct injection were always significantly higher than those from RU that is on the opposite side of the uterotubal junction (**p<0.01, *p<0.05, Wilcoxon), suggesting a functional barrier between the oviduct and uterine horn. The lower recovery during the first 24h is consistent with the concept that the inoculum needs time to replicate and to differentiate the replicating but non-infectious reticulate bodies into the infectious elementary bodies.</p

Evaluating the effect of MyD88 or STING pathways on <i>C</i>. <i>muridarum</i> ascending to the oviduct following a transcervical inoculation.

<p>Groups of C57BL/6J (panels a & b, n = 5), STING-deficient (c & d, STNG-/-, n = 5) and MyD88-deficient (e & f, MyD88-/-, n = 5) female mice were each inoculated with 2 x 10⁵ IFUs transcervically and 4 (a, c & e) or 14 (b, d & f) days after the inoculation, the entire uterine horn (both right and left) and oviduct/ovary (both right and left) tissues from each mouse were harvested as listed along the X-axis. The tissues were homogenized for titrating infectious organisms as displayed in Log10 IFUs along the Y-Axis. Please note that C57BL/6J mice displayed significant differences in log10 IFUs between uterine and oviduct/ovary tissues (a & b, **p<0.01, Wilcoxon) but neither STING-deficient nor MyD88-deficient mice exhibited any significant differences.</p

Comparing gross pathology in the oviducts of mice with or without deficiency in STING following a transcervical inoculation.

<p>Groups of C57BL/6J (n = 10) and STING-deficient (n = 10) female mice were each inoculated with 2 x 10⁵ IFUs transcervically as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183189#pone.0183189.g006" target="_blank">Fig 6</a> legend and 70 days after the inoculation, mice were sacrificed for evaluating gross pathology. (A) A representative image is shown from each group (A, panels a & b respectively). Hydrosalpinges were marked with red arrows while the severity of hydrosalpinx was scored based on the semi-quantitative scheme described in the materials and the scores were marked with white numbers. (B) Summary of both the hydrosalpinx incidence and severity. The incidence of mice with unilateral or bilateral hydrosalpinges was counted separately while the severity was scored based on total hydrosalpinges. Please note that only 10% the wild type C57BL/6J mice developed bilateral hydrosalpinx while 80% of the STING-deficient mice did so (**p<0.01, Fisher Exact). The STING-deficient mice also developed more severe hydrosalpinx than the wild type mice (**p<0.01, Wilcoxon).</p

Comparing <i>C</i>. <i>muridarum</i> recoveries from vaginal swabs of mice with or without deficiency in STING following a transcervical inoculation.

<p>Groups of C57BL/6J (solid bar or, solid square, n = 10) and STING-deficient (open bar or open square, n = 10) female mice were each inoculated with 2 x 10⁵ IFUs transcervically and at various times after the inoculation as indicated along the X-axis, vaginal swabs were taken for titrating infectious organisms as displayed in Log10 IFUs (left) or % of mice with positive IFU (right) along the Y-Axis. Please note that there is no significant difference in either log10 IFUs (Wilcoxon) or % of mice with positive IFUs (Fisher’s Exact) between the wild type and STING-deficient mice (data obtained from two independent experiments), indicating that the STING-deficiency did not affect the descending of the <i>C</i>. <i>muridarum</i> organisms from the endocervcal compartments into the ectocervical and vaginal compartments or the replication of the <i>C</i>. <i>muridarum</i> organisms in these compartments.</p

Comparing inflammatory pathology in the oviducts of mice with or without deficiency in STING following a transcervical inoculation.

<p>The genital tract tissues from the same mice as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183189#pone.0183189.g007" target="_blank">Fig 7</a> legend were subjected to H&E staining and evaluation of inflammatory infiltrates in both oviducts. (A) A representative H&E staining image is shown from each group (panels a & b from wild type C57BL/6J mice and c & d from STING-/- mice respectively under 10x objective lens). To visualize the infiltrating cells, examples of a 100x objective lens view are also provided as marked with a1 to d1. The inflammatory cells are marked with red arrows. (B) The H&E stained sections were semi-quantitatively scored based on the scheme described in the materials and method section, and the scores are summarized. Please note that the STING-deficient mice developed more severe inflammatory infiltration in the oviduct tissues (p<0.05, Wilcoxon).</p

Comparing <i>C</i>. <i>muridarum</i> recoveries from 9 different sections of the mouse genital tract following inoculation into the right bursa.

<p>A group of 5 C57BL/6J female mice were each inoculated with 2 x 10⁵ IFUs at the right bursa (as indicated with a red arrow) and 3 days after the inoculation, segments of the entire genital tract tissues were harvested as shown along with the X-axis, from the right oviduct/ovary (RO), right uterine horn distal region (RU_d), middle (RU_m) or the proximal region (RU_p), cervico-vagina (CV), left side uterine horn proximal region (RU_p), middle (LU_m) and distal (RU_d) to the left oviduct/ovary (LO). The tissues were homogenized for titrating infectious organisms as displayed in Log10 IFUs along the Y-Axis. Please note that significant differences in log10 IFUs were observed only between RO and RU_d (*p<0.05, Wilcoxon) and between LU_d and LO (**p<0.01, Wilcoxon) but not other adjacent tissue sections.</p

Evaluating the effect of MyD88 or STING pathways on the spread of <i>C</i>. <i>muridarum</i> in the genital tract following an inoculation to the right bursa.

<p>Groups of C57BL/6J (panel a, n = 5), STING deficient (b, STING-/-, n = 5) and MyD88 deficient (c, MyD88-/-, n = 5) female mice were each inoculated with 2 x 10⁵ IFUs at the right bursa (RO, as indicated with a red arrow) and 3 days after the inoculation, segments of the genital tract tissues were harvested as listed along the X-axis, from RO, RU (entire right uterine horn), CV, LU (entire left uterine horn) to LO. The tissues were homogenized for titrating infectious organisms as displayed in Log10 IFUs along the Y-Axis. Please note that C57BL/6J mice displayed significant differences in log10 IFUs between RO and RU (*p<0.05, Wilcoxon) and between LU and LO (**p<0.01, Wilcoxon) respectively but neither STING-deficient nor MyD88-deficient mice were able to maintain the differences.</p

Comparing <i>C</i>. <i>muridarum</i> with or without plasmid for their ability to spread to the GI tracts of CBA/1J mice following an intravaginal inoculation.

<p>The experiments were carried out and the results were presented as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177691#pone.0177691.g001" target="_blank">Fig 1</a> legend above. The plasmid-competent <i>C</i>. <i>muridarum</i> organisms were inoculated into 5 while the plasmid-deficient to 10 mice. The data were collected from 2 independent experiments. Plasmid-deficient <i>C</i>. <i>muridarum</i> displayed significantly longer shedding from the mouse genital tracts (panel a, p<0.05, days to clearance, Wilcoxon rank sum) but the overall shedding courses were similar between plasmid-deficient and -competent <i>C</i>. <i>muridarum</i> (panels a & b, p>0.05, area under the curve, Wilcoxon rank sum). However, plasmid-deficient <i>C</i>. <i>muridarum</i> developed significantly delayed/reduced shedding courses in the GI tracts in terms of both the level of shedding (panel c, **p<0.01, area under the curve, Wilcoxon rank sum) and number of mice remaining positive for shedding (panel d, *p<0.05, areas under the curve, Wilcoxon rank sum).</p

Comparing <i>C</i>. <i>muridarum</i> with or without plasmid for their ability to spread to the GI tracts of C57BL/6J mice following an intravaginal inoculation.

<p>The plasmid-competent (Filled bar or square; n = 6) or -deficient (open bar or square; n = 5) <i>C</i>. <i>muridarum</i> organisms were intravaginally inoculated into female C57BL/6J mice with 2 x 10⁵ inclusion forming units (IFUs) per mouse. At various time points post inoculation as indicated along the X-axis, both vaginal (panels a & b) and rectal (c & d) swabs were taken for titrating live organisms and the recovered live organisms were expressed as Log₁₀ IFUs per swab as displayed along the Y-axis (panels a & c) and % of mice detected positive for IFUs at a given time point were plotted in panels b & d. The data were collected from two independent experiments. Note that both plasmid-deficient and -competent <i>C</i>. <i>muridarum</i> displayed similar live organism shedding courses from the genital tract (panels a & b, p>0.05, area under the curve or days to clearance, Wilcoxon rank sum). However, plasmid-deficient <i>C</i>. <i>muridarum</i> developed a significantly delayed/reduced shedding course from the same mouse GI tracts in terms of both the level of shedding (panel c, **p<0.01, area under the curve, Wilcoxon rank sum) and number of mice remaining positive for shedding (panel d, *p<0.05, area under the curve, Wilcoxon rank sum).</p

Comparing <i>C</i>. <i>muridarum</i> with or without plasmid for their ability to spread to the GI tracts of C57BL/6J mice following an intravenous inoculation.

<p>The plasmid-competent (solid bar; n = 4) or -deficient (open bar; n = 7) <i>C</i>. <i>muridarum</i> organisms were inoculated into female C57BL/6J mice with 2 x 10⁶ IFUs per mouse via a retro-orbital injection. At various time points post inoculation as indicated along the X-axis, blood drops (panel a), rectal (b) and vaginal (c) swabs were taken for quantitating <i>C</i>. <i>muridarum</i> genomes or titrating live organisms. The recovered genome copies and live organisms were expressed as Log₁₀ genomes per 10μl blood (a) or Log₁₀ IFUs per swab (b & c) as displayed along the Y-axis. The data were obtained from two experiments. Note that although both plasmid-competent and -deficient <i>C</i>. <i>muridarum</i> displayed similar genome recovery time courses from the blood (panel a, p>0.05, area under the curve, Wilcoxon rank sum), plasmid-deficient <i>C</i>. <i>muridarum</i> developed a significantly delayed/reduced shedding course from the GI tracts (b, *p<0.05, areas under the curves, Wilcoxon rank sum). No live organisms were detected in the genital tracts of any mice (c).</p