{"paper_id":"28052bc9-9e96-4367-802f-34f59315beaa","body_text":"Sexually transmitted infections (STIs) remain a major health challenge worldwide. Recently WHO estimated that about 1 million new STIs are acquired daily. In 2016, about 376 million new STIs were reported world-wide, of which 127 million were caused by  Chlamydia  [ 1 ]. In the US,  Chlamydia  spp. continue to be the leading cause of STIs, representing 1.7 million cases of approximately 2.3 million STIs reported in 2017 [ 2 ]. The highest rates of new chlamydial infections occur in young adults, especially young women of reproductive age. In about 70–80% of cases, chlamydial infections in women are asymptomatic and as such may go untreated. About 15% of untreated chlamydial infections progress to PID [ 3 – 5 ]. If left untreated, PID may result in infertility, ectopic pregnancy, and chronic pelvic pain [ 6 ]. Pregnant women infected with  Chlamydia  can pass the infection to their infants, potentially resulting in neonatal ophthalmia and pneumonia. Infection with  Chlamydia  also increases the risk for HIV transmission and HPV-associated cervical cancer [ 7 ,  8 ]. With specific regard to  Chlamydia , to date no effective vaccines have been licensed for use in humans.  Chlamydia  spp. are obligatory intracellular bacterial pathogens that undergo a biphasic developmental cycle, thus they exist in two forms: the infectious elementary bodies (EBs) and the non-infectious reticulate bodies (RBs) [ 9 ]. Host infection is initiated when EBs first infect epithelial cells that line mucosal surfaces (e.g. FRT). Within epithelial cells, EBs enclose themselves within an endocytic vesicle termed inclusion, where EBs transform into RBs. Within an inclusion, RBs divide by binary fission and then transform back into the infectious EB form. EBs are released following cell lysis or inclusion extrusion, and then go on to infect other neighboring cells.  C .  muridarum  is a mouse-specific pathogen and does not infect humans, although it shares near genomic synteny with human-specific  C .  trachomatis  [ 10 ]. However,  C .  muridarum  has been widely used as a model to study both pathogenesis of and immunity to human-specific  C .  trachomatis . This is because intravaginal infections of mice with  C .  muridarum  mimic  C .  trachomatis  pathology in humans, which may result in hydrosalpinx and infertility [ 11 – 14 ]. Human-specific  C .  trachomatis  strains have been used for per-vaginal (PV) infections of mice. However, such infections do not recapitulate the pathology observed in humans as they cause a mild, short-lived FRT infection and no post-infection upper FRT pathology [ 15 ]. Human specific chlamydial strains produce upper FRT pathologies in mice only when animals are inoculated with large doses of  Chlamydia  directly in the uterus or ovaries [ 16 ], which does not represent physiological conditions of human infection.\nIn addition to infecting the FRT, in both humans and animals,  Chlamydia  spp. infect and persist in the GI tract without causing inflammation or pathology. GI tract  Chlamydia  are more resistant to azithromycin treatment and represent a potential reservoir for recurring FRT infections [ 17 – 19 ]. Treatment failure rates for rectal  Chlamydia  in men and women range between 6% and 23% [ 20 ,  21 ]. In humans,  C .  trachomatis  infection of the GI tract may occur via oral or anal intercourse however, it remains unknown whether  C .  trachomatis  disseminates from the FRT to the GI tract internally and independent of sexual behavior. In mice, following FRT infection  C .  muridarum  reaches the GI tract via an internal, non-oral, non-rectal route [ 22 ]. Detection of  C .  muridarum  genomes in the FRT, GI tract, liver, spleen, heart, and lung 4 weeks after infection, indicates a systemic spread of the pathogen [ 22 ]. Moreover, when administered intravenously (IV)  C .  muridarum  infects the GI tract [ 23 ] and its genome copies are detected systemically for up to 14 d after IV infection, suggesting that  C .  muridarum  survives in the circulation. However, live  Chlamydia  was not detected systemically after day 7 of infection, which coincided with the peak of GI tract live  Chlamydia  loads, leading authors to suggest a temporal correlation between blood-borne and GI tract  Chlamydia  [ 23 ].\nConsidering that  Chlamydia  spp. are obligate intracellular pathogens we hypothesized that following FRT infection  Chlamydia  spp. rely on cell transport in order to reach the GI tract. Here we confirm our hypothesis and show that inhibition of cell migration abrogates  Chlamydia  spread from the FRT to the GI tract. In our experimental model in which coprophagy and grooming are precluded, we show that  Chlamydia  infection of the spleen is a critical step for  Chlamydia  dissemination to the GI tract. Finally, we show that carriage of  Chlamydia  by CD11c +  DCs from the FRT to the draining ILNs represents the first step in  Chlamydia  systemic dissemination. This work will improve our understanding of the mechanisms that govern FRT to ILN cell migration and antigen transport. Further elucidating the mechanisms of and the importance of  Chlamydia  transport and systemic dissemination in long-term FRT pathology will be critical for the development of vaccination and therapeutic approaches.\n\nIn female mice, PV infection with  C .  muridarum  causes tubal fibrosis and hydrosalpinx pathology similar to long-term disease sequelae caused by  C .  trachomatis  in humans. For these reasons, the mouse PV model of infection is extensively used. In addition, in both humans and mice  C .  trachomatis  and  C .  muridarum  respectively, infect the GI tract without causing pathology. Whether and how  Chlamydia  spp. may spread internally remains unknown. However, others have suggested that in mice  Chlamydia  spreads from the FRT to the GI tract via blood circulation [ 23 ]. Considering that  C .  muridarum  (like  C .  trachomatis ) is an intracellular pathogen, we hypothesized that its dissemination from the FRT to the GI tract depends on cell transport. To begin addressing this hypothesis, C57BL/6 mice were PV infected with 10 3 , 10 4 , 10 5 , 10 6  or 10 7  inclusion forming units (IFU) of  C .  muridarum . At 0, 3, 7 and 14 days post-infection (dpi), ILNs, spleen, liver, and cecal scrapings were collected for determination of  Chlamydia  titers. In a set of studies mice were infected IV with 10 5  IFU of  C .  muridarum  in order to investigate the dynamics of systemic  Chlamydia  clearance. To preclude GI tract infection via grooming or coprophagy, PV-infected mice were singly housed on wire-mesh bottom cages and fitted with lightweight neck collars. In IV-infected mice, live  Chlamydia  was detected in ILNs by 3 dpi and was cleared by 9 dpi ( Fig 1A ). As expected, IV administered  Chlamydia  was captured in the spleen, reached peak titer by 6 dpi and was mostly cleared by 9 dpi ( Fig 1D ). In contrast, PV-infected mice had high  Chlamydia  ILN titers even at 14 dpi, possibly indicating a delayed clearance due to a continuous  Chlamydia  supply to the ILN from the infected FRT ( Fig 1B and 1C ). In PV-infected mice,  Chlamydia  was detected in the spleen by 7 dpi regardless of the dose of infection (10 3 −10 7  IFU), ( Fig 1E and 1F  and  S1B Fig ). However, it is important to note that mice PV infected with 10 3 , 10 4  or 10 5  IFU of  Chlamydia  exhibited lower ILN, spleen, and cecal titers compared to mice infected with 10 6  or 10 7  IFU of  Chlamydia  ( S1A–S1C Fig ). In addition, ceca of 2/5, 2/5 and 1/5 mice infected with 10 3 , 10 4 , or 10 5 , respectively, were negative for  Chlamydia  at 7 dpi ( S1C Fig ). This finding indicates that low dose may delay, but not preclude  Chlamydia  systemic spread. By 14 dpi, most animals cleared  Chlamydia  from the spleen and  Chlamydia  clearance was slower in mice infected with higher doses ( Fig 1E and 1F ). Finally, in all infected mice (IV or PV)  Chlamydia  colonized the ceca by 7 dpi, and at high PV doses (10 6  and 10 7  IFU) 100% of mice had cecal  Chlamydia  titers at 7 dpi, which were not significantly different from cecal titers observed at 14 dpi ( Fig 1G–1I ). To ensure that animals were infected with the intended dose of  Chlamydia , vaginal swabs were collected and titered ( S1D Fig ). In addition, all mice were infected from one common  Chlamydia  stock that was prepared and titered in advance. Unlike some other bacterial pathogens (e.g.  Salmonella  spp.),  Chlamydia  does not infect the liver, as only 1/80 PV-infected mice had detectable  Chlamydia  titers in the liver, regardless of the dose of PV infection ( S1E Fig ). Similarly, no  Chlamydia  was found in gallbladder/bile, plasma, or blood cell fractions tested at different times after PV infection ( S1E Fig ).  Chlamydia  tissue titers shown here are from the first passage. Although all samples were passaged 6 times, all negative samples remained negative even after 6 passages. These data indicate that  Chlamydia  disseminates systemically in a stepwise manner, by first infecting the FRT-draining ILNs (by 3 dpi), then the spleen, and the GI tract (by 7 dpi).\nChlamydia  titers in ILNs (A-C), spleens (D-F) and ceca (G-I) of mice at 0, 3, 6, 9 or 0, 3, 7, 14 days post IV or PV infections, respectively.  Chlamydia  titers are expressed as log 10  number of IFU per ILNs, per g of spleen, or per cecum. Data are expressed as the mean ± SD. Group means were separated using Tukey’s multiple comparison test and declared significantly different at p<0.05 (n = 5 mice per time point). Group means that do not share superscript are significantly different (p<0.05).\nFollowing an infection, tissue DCs capture the antigen, carry it to the lymph nodes and present it to T cells for induction of adaptive immune responses. Upon pathogen encounter tissue DC (e.g. skin DC) mature, upregulate the expression of CD80/CD86 and the chemokine receptor CCR7 [ 24 ,  25 ]. The interaction of CCR7 with CCL21/CCL19 ligands mediates DC migration to local draining lymph nodes, where DCs present captured antigens to T cells, thus activating adaptive immunity [ 24 – 27 ]. CCL21 and CCL19 are expressed in both lymphatic endothelial cells and lymph nodes [ 28 ], therefore the CCR7:CCL21/CCL19 axis is essential for intravasation of tissue DCs into lymphatics and for DC migration to local draining lymph nodes. To investigate the role of CCR7-mediated cell migration in  Chlamydia  dissemination, CCR7 -/-  and C57BL/6 mice (controls) were PV infected with 1x10 6  IFU of  Chlamydia . We chose to use a higher dose, since this dose allows for 100% infection of the GI tract by 7 dpi. We reasoned that if  Chlamydia  dissemination is inhibited at a high dose of PV infection, the inhibition would be more drastic for lower infectious doses. We found that systemic dissemination of  Chlamydia  was significantly inhibited in CCR7 -/-  mice, resulting in reduced titers in ILN (p<0.01), spleen (p<0.01), and ceca (p<0.01) at 7 dpi compared to C57BL/6 controls ( Fig 2 ). At 14 dpi ILN and cecal  Chlamydia  titers in CCR7 -/-  mice were significantly lower compared to 14 dpi controls (p<0.02 and p<0.001, respectively). However, there were no differences in spleen titers between CCR7 -/-  and C57BL/6 mice at 14 dpi (p<0.5) ( Fig 2B ). Overall this data shows that CCR7:CCL19/CCL21 signaling axis is important for  Chlamydia  dissemination, and that the delayed  Chlamydia  spread to the ILNs is likely due to impaired CCR7-mediated cell migration. In addition, the delayed infection of the ILN, spleen, and the GI tract in CCR7 -/-  mice indicates that in the absence of CCR7, signaling via other receptor/chemokine pairs such as CCR8:CCL1/CCL8, CXCR4/CXCL12, CX3CR1/CX3CL1 and/or S1PR: S1P might direct cell migration and thus allow for  Chlamydia  systemic dissemination, albeit less efficiently.\nChlamydia  titers in ILNs (A), spleen (B) and ceca (C) of C57BL/6 (control) and CCR7 -/-  mice at 7 and 14 dpi PV with 10 6  IFU of  Chlamydia .  Chlamydia  titers are expressed as log 10  number of IFU per ILNs, per g of spleen, or per cecum. Data are representative of two experiments and are expressed as the mean ± SD. Group means were separated using Student’s t-test (n = 10 mice per time point). Group means that do not share a superscript are significantly different from each other (p<0.05).\nIn addition to CCR7, mature DC upregulate the expression of S1P receptors (S1PR) which allows them to migrate by sensing higher S1P concentrations in lymph and blood [ 29 ]. In vivo, S1P-mediated cell migration can be inhibited by FTY720, an S1P analog [ 30 ]. We reasoned that if S1P mediates the FRT to ILN and ILN to spleen cell migration,  Chlamydia  would not disseminate systemically. To test this hypothesis, FTY720 was provided to mice via drinking water for the duration of the studies, starting at 3 days prior to PV infection with 10 6  IFU of  Chlamydia . Interestingly, S1P signaling does not appear to be essential for FRT-ILN cell migration as  Chlamydia  ILN titers of FTY720-treated mice were not significantly different from controls at 7 dpi (p<0.2) ( Fig 3A ). However, S1P signaling is essential for cell egress from the ILNs and thus for  Chlamydia  transport to the spleen and the GI tract ( Fig 3B and 3C ). Compared to controls, FTY720-treated mice had significantly reduced  Chlamydia  loads in the spleen (p<0.002 and p<0.06) and ceca (p<0.001 and p<0.001) at 7 and 14 dpi, respectively ( Fig 3B and 3C ).\nChlamydia  titers in ILNs (A), spleen (B), and ceca (C) of C57BL/6 (control) and FTY720-treated mice at 7 and 14 dpi PV with 10 6  IFU of  Chlamydia .  Chlamydia  titers are expressed as log 10  number of IFU per ILNs, per g of spleen, or per cecum. Data are representative of two experiments and are expressed as the mean ± SD. Group means were separated using Student’s t-test (n = 10 mice per time point). Group means that do not share a superscript are significantly different from each other (p<0.05).\nThe results of CCR7 -/-  and FTY720-treated mice show that CCR7 signaling is more important for the FRT-ILN cell migration and thus the first step of  Chlamydia  transport, while S1P signaling is indispensable for cell egress from ILNs and therefore  Chlamydia  transport from the ILN to the spleen and the GI tract. We then hypothesized that infection of the spleen is the second important step for  Chlamydia  dissemination to the GI tract. To test this hypothesis splenectomized and control mice were PV infected with 10 6  IFU of  Chlamydia . We found that removal of the spleen significantly diminished GI tract infection by  Chlamydia  at 7 and 14 dpi ( Fig 4B ). Although ceca of 5/16 splenectomized mice were positive for  Chlamydia , their titers were 2 to 3-fold lower compared to controls ( Fig 4B ). Due to intimate anatomical connection between the mesenteric lymph nodes (MLNs) and the GI tract, we considered that infection of the MLNs, caused by circulating infected cells might be a source of cecal  Chlamydia . Analysis of MLN  Chlamydia  titers revealed that MLNs become infected, likely by circulating cells however, the low frequency of infection and low MLN titers do not support this notion. In IV-infected mice  Chlamydia  MLN titers peaked at 6 dpi and diminished by 9 dpi ( S2A Fig ). Similarly, in PV-infected mice MLN titers peaked at 7 dpi and show clearance dynamics similar to the spleen ( S2B and S2C Fig  and  Fig 1 ). There were no significant differences in MLN  Chlamydia  titers among time points in IV-infected mice (p<0.08), or mice infected PV with 10 6  (p<0.2) or 10 7  (p<0.1) IFU of  Chlamydia  ( S2A–S2C Fig ). Moreover,  Chlamydia  is present in MLNs of CCR7 -/-  mice only at 14 dpi and the frequency of  Chlamydia  MLN infection and MLN titers are lower compared to the frequency of infection and titers in the spleen ( S2D Fig  and  Fig 2B ). Similarly, MLNs of FTY720-treated mice were not infected ( S2E Fig ) and only 0/8 and 1/8 splenectomized mice had MLN titers at 7 and 14 dpi, respectively ( S2F Fig ). Interestingly, splenectomized mice exhibit  Chlamydia  titers in the liver ( S1E Fig ), indicating that in the absence of the spleen,  Chlamydia -harboring cells migrate to the liver, which then contributes to the infection of the GI tract, although less effectively compared to the spleen. While 5/16 (about 30%) of splenectomized mice were positive for  Chlamydia  in liver and ceca (at 7 and 14 dpi) ( Fig 4B ,  S2E Fig ), the incidence of liver infection in controls with intact spleens was only about 1% ( S1E Fig ). There were no differences in  Chlamydia  titers in vaginal swabs of control and splenectomized mice at 0, 6, 9, or 12 dpi ( Fig 4C ), indicating that the lack of the spleen does not affect FRT  Chlamydia  clearance in this timeframe. We also examined the gallbladder and blood fractions (plasma and cells separately) for presence of infection and of 46 and 64 samples respectively, we found no samples that were positive for live  Chlamydia  ( S1E Fig ).\nChlamydia  titers in ILNs (A) and ceca (B) of C57BL/6 (control) and splenectomized mice at 7 and 14 dpi PV with 10 6  IFU of  Chlamydia . (C)  Chlamydia  titers in vaginal swabs of C57BL/6 (control) and splenectomized mice at 0, 6, 9 and 12 dpi PV with 10 6  IFU of  Chlamydia .  Chlamydia  titers are expressed as log 10  number of IFU per ILNs, per cecum, or per vaginal swab. Data are representative of two experiments and are expressed as the mean ± SD. Group means were separated using Student’s t-test (n = 8 mice per time point). Group means that do not share a superscript are significantly different from each other (p<0.05).\nAlthough systemic dissemination of  Chlamydia  was inhibited in splenectomized, CCR7 -/- , and FTY720-treated mice compared to controls (Figs  2 – 4 ), there were no differences in  Chlamydia  titers in vaginal swabs among these groups and controls at any time point (0, 3, 6, 9 or 12 dpi) ( Fig 5A ). Systemic dissemination of  Chlamydia  however, is critical for induction of adaptive immunity, as splenectomized, CCR7 -/- , and FTY720-treated mice exhibited no (splenectomized, FTY720-treated), or significantly reduced (CCR7 -/- )  Chlamydia -specific serum IgG antibodies compared to C57BL/6 controls ( Fig 5B ). While all PV infected C57BL/6 mice had high serum IgG titers by 14 dpi regardless of the dose (10 6  or 10 7  IFU), all splenectomized and FTY720-treated mice were negative for  Chlamydia -specific IgG, and only 3/11 CCR7 -/-  mice had  Chlamydia -specific IgG titers (albeit low), reflecting the observed delayed  Chlamydia  dissemination in CCR7 -/-  mice ( Fig 5B ). In addition, ascension of  Chlamydia  in the upper FRT is not critical for its systemic dissemination, as there were no differences in  Chlamydia  titers in ILNs (p<0.4), spleen (p<0.3), or ceca (p<0.8) between controls and uterectomized mice ( Fig 5C–5E ) at 7 dpi.\n(A) Vaginal swab titers of splenectomized, FTY720-treated, CCR7 -/- , and C57BL/6 control mice at 0, 3, 6, 9 and 12 dpi PV with 10 6  IFU of  Chlamydia . (B) Serum IgG titers of splenectomized, FTY720-treated, CCR7 -/- , and C57BL/6 mice at 14 dpi PV with 10 6  (and 10 7  controls) IFU of  Chlamydia . IgG titers are expressed as log 10  titer values, with the titer being the highest dilution having an absorbance value twice that of the background. (C-E)  Chlamydia  titers in ILNs, spleen and ceca of control and uterectomized mice at 7 dpi PV with 10 6  IFU of  Chlamydia . Data are representative of two experiments and are expressed as the mean ± SD. Group means were separated using Student’s t-test. Group means that do not share a superscript are significantly different from each other (p<0.05).\nAt 7 dpi there was a significant increase in the ILN size ( S3A and S3B Fig ), likely caused by induced immune responses, increased cell recruitment and proliferation following ILN infection. Moreover, live  Chlamydia  was present in CD11c +  DCs isolated from ILNs of PV-infected mice at 3 and 7 dpi ( Fig 6A ). We then set out to examine whether CD11c +  DCs of the FRT mediate  Chlamydia  transport from the FRT to the draining ILNs. To address this question, control (C57BL/6 background that lack high affinity diphtheria toxin (DTx) receptor) and CD11c-DTR mice were injected intra-peritoneally (IP) with DTx at 0 or 3 dpi for selective depletion of CD11c +  DCs. At 3 or 7 dpi ILNs, spleen, and cecal scrapings were collected and their  Chlamydia  loads determined. Depletion of CD11c +  DCs prior to infection completely abrogated infection of the ILNs at 3 dpi ( Fig 6B ) with ILNs of all 10 mice (across two separate studies) being negative for  Chlamydia  ( Fig 6B ). As expected, no  Chlamydia  was recovered from spleens and ceca of control or CD11c-DTR mice at 3 dpi ( S3D and S3E Fig ). These results indicate that CD11c +  DCs transport  Chlamydia  from the FRT to the ILNs ( Fig 6B ). In addition to being negative for  Chlamydia , ILNs of CD11c-DTR mice were of normal size, much like the ILNs of control mice ( S3A Fig ). The lack of CD11c +  DCs did not affect  Chlamydia  loads at 3 dpi in vaginal swabs, as there were no differences in titers between control and CD11c-DTR mice ( Fig 6C ). Depletion of CD11c +  DCs was confirmed by FACS analysis of CD11c +  DC proportions in the lymph nodes and spleens of control and CD11c-DTR mice at 24h post-DTx treatment ( Fig 6D and 6E ). We then examined whether CD11c +  DC depletion prior to and after infection of ILNs (at 0 or 3 dpi) would affect  Chlamydia  burden in the spleen and the GI tract at 7 dpi. DTx treatment had no effect on FRT  Chlamydia  titers, as no differences in  Chlamydia  loads of vaginal swabs were observed among DTx-treated controls or CD11c-DTR mice at 3 (p<0.8) or 6 dpi (p<0.8) ( Fig 6F ). At 7 dpi, we saw no significant differences in  Chlamydia  titers in ILNs (p<0.18) and spleens (p<0.4) among C57BL/6 controls and CD11c-DTR mice following DTx treatment at 0 or 3 dpi, although CD11c-DTR mice had numerically lower  Chlamydia  loads in these tissues ( Fig 6G and 6H ). However, depletion of CD11c +  DCs at 0 or 3 dpi abrogated  Chlamydia  dissemination to the GI tract, as at 7 dpi ceca of all CD11c-DTR mice (treated with DTx at 0 or 3 dpi) were negative for  Chlamydia  and DTx-treated control mice exhibited significantly higher cecal  Chlamydia  titers (p<0.001) ( Fig 6I ). Presence of  Chlamydia  in spleens, but not ceca of DTx-treated CD11c-DTR mice at 7 dpi again indicates that  Chlamydia  infects the spleen before spreading to the GI tract. In addition, there were no differences in cecal  Chlamydia  titers between DTx-treated and untreated controls ( S3C Fig ). Taken together, our findings may be summed up as follows: 1) After FRT infection,  Chlamydia  reaches the GI tract in stages, by first infecting ILNs, then the spleen, and the GI tract; 2) Low dose PV infection delays, but does not abrogate  Chlamydia  dissemination to the spleen and the GI tract; 3) Ascension of  Chlamydia  to the upper FRT is not essential for its systemic dissemination; 4) Inhibition of cell migration in CCR7 -/-  and FTY720-treated mice delays or abrogates  Chlamydia  systemic dissemination; and 5) CD11c +  DCs mediate the first step of  Chlamydia  systemic dissemination, from the FRT to the draining ILNs.\n(A) Recovery of  Chlamydia  from CD11c +  DCs isolated from ILNs of uninfected (control) and PV-infected mice at 0, 3, and 7 dpi PV with 10 6  IFU of  Chlamydia . (B)  Chlamydia  titers in ILNs and (C) vaginal swabs of C57BL/6 (control) and CD11c-DTR mice at 3 dpi following treatment with DTx at 0 dpi (n = 10 mice/group). (D, E) Flow cytometry analysis of CD11c +  DC proportions in LNs and spleens of CD11c-DTR mice at 24h post-treatment with PBS (control) or DTx. (F) Vaginal swab titers of control (C57BL/6, white bars) or CD11c-DTR mice PV infected with 10 6  IFU of  Chlamydia  and treated with DTx at 0 or 3 dpi (black or striped bars, respectively). (G-I)  Chlamydia  titers at 7 dpi in ILNs (G), spleen (H), and ceca (I) of C57BL/6 (control) and CD11c-DTR mice treated with DTx at 0 or 3 dpi PV with 10 6  IFU of  Chlamydia . Data are representative of two experiments and are expressed as the mean ± SD. Group means were separated using Student’s t-test or Tukey’s multiple comparison procedure. Group means that do not share a superscript are significantly different from each other (p<0.05).\n\nC .  trachomatis  is a human-specific pathogen that can cause long-term disease sequela such as PID, ectopic pregnancy, endometriosis, tubal fibrosis, and infertility [ 31 ]. The long-term FRT pathology (PID) occurs in about 10% of women, likely because infections remain undetected and untreated [ 4 ].  C .  trachomatis  is also routinely found in the GI tract of women. However, it remains unknown whether systemic dissemination of  C .  trachomatis  plays a role in GI tract infection. In mice, under experimental settings in which coprophagy and grooming are precluded,  Chlamydia  disseminates from the FRT to the GI tract via an internal, non-oral, non-rectal route [ 22 ], although no mechanism of transit has been proposed. Blood-borne  C .  muridarum  can establish a long-lasting infection in the GI tract, leading to the suggestion that  C .  muridarum  disseminates via the circulation [ 23 ]. Since IV infection is not an appropriate model for PV  C .  trachomatis  infection in women, we used the PV  C .  muridarum  mouse model of infection. Using this model we show that  Chlamydia  dissemination from the FRT to the GI tract depends on active cell migration and consists of three distinct steps: 1) DC-mediated  Chlamydia  transport from the FRT to the draining ILNs; 2) S1P-dependent  Chlamydia  transport from ILNs to the spleen; and 3)  Chlamydia  transport from the spleen to the GI tract via yet to be determined mechanisms. Based on presented data we propose the following model of systemic  Chlamydia  dissemination ( Fig 7 ). After initially infecting epithelial cells of the FRT,  Chlamydia  EBs are internalized by lamina propria CD11c +  DCs (1). This triggers DC maturation and increased expression of CCR7, which allows DCs to sense CCL21, enter local lymphatics (2) and migrate to the draining ILNs (3). In the ILNs,  Chlamydia  undergoes EB-RB-EB differentiation and proliferation. In the ILNs, other and yet to be identified cells become infected, enter the circulation by sensing the higher S1P gradients, and migrate to the spleen (4) where  Chlamydia  infects other cells and proliferates before being ferried to the GI tract.\nChlamydia  first infect epithelial cells of the FRT. CD11c +  DCs capture  Chlamydia  EBs in the lamina propria (LP) of the FRT, which triggers DC migration to the FRT-draining ILNs. In the ILNs, immune responses to  Chlamydia  are initiated, leading to lymphocyte activation, recruitment, and proliferation. The second step of  Chlamydia  transport from ILN to the spleen (by yet to be identified host cells) depends on S1P-mediated cell migration, which is inhibited by FTY720 treatment.\nDCs are potent antigen presenting cells and T cell activators that also play important roles in antigen capture and transport, cytokine secretion, and thus induction of immunity and tolerance [ 32 – 34 ]. Most evidence showing a significant role of mucosal DCs in pathogen uptake and transport has been gathered from HIV studies. In explanted human FRT and intestinal tissues, DCs capture and transport HIV through the mucosa [ 35 ,  36 ], leading to the hypothesis that DCs capture the virus and carry it to the lymph nodes resulting in the establishment of a systemic infection. Although HIV and  Chlamydia  spp. are very different pathogens, similar mechanisms appear to mediate their systemic spread following FRT infection. Depletion of CD11c +  DCs prior to infection abrogates  Chlamydia  spread to the ILNs, indicating that DCs carry  Chlamydia  to the ILNs, much like the proposed HIV transport by DCs. Although signaling cues that direct DC egress from the FRT during  Chlamydia  infection have not been previously examined, CCR7 signaling is a critical mediator of DC migration from the skin [ 37 ], intestinal mucosa [ 38 ] and the lungs [ 39 ]. The lack of FTY720 treatment effect in the FRT-ILN  Chlamydia  spread is in line with findings that immature DCs do not migrate to S1P [ 29 ]. Moreover, migration of DCs to draining lymph nodes in mice that lack S1P in lymphatic fluid is not different from DC migration in wild-type mice [ 40 ] which indicates that S1P signaling is dispensable for guiding FRT DC migration and thus  Chlamydia  transport to ILNs.\nThe decreased  Chlamydia  transport to ILNs in CCR7 -/-  mice indicates that CCR7:CCL19/CCL21 signaling axis is critical for FRT to ILN DC migration. CCR7 chemokine agonist CCL21 is expressed in lymphatic endothelial cells and lymph nodes. It is thus essential for DC entry into local lymphatics and for DC migration to and within LNs [ 28 ,  41 ,  42 ]. Indeed, in CCR7 -/-  mice tissue DC migration is reduced by about 90% under inflammatory conditions [ 27 ,  43 ], which may explain the delayed FRT to ILN  Chlamydia  spread. The delayed  Chlamydia  spread in CCR7 -/-  mice may also indicate that in the absence of CCR7 other signaling receptor/chemokine pairs (such as CCR8:CCL1/CCL8, CXCR4/CXCL12 and CX3CR1/CX3CL1) direct DC migration [ 44 – 46 ]. The essentiality of CCR7 for migration of both DCs and Langerhans cells was confirmed in irradiated wild-type mice that were reconstituted with wild type and CCR7 -/-  bone marrow. In this model, the expression of CCR7 in wild-type DCs conferred a strong competitive advantage for accumulation of migratory DCs in draining lymph nodes [ 47 ]. However, since CCR7 -/-  mice exhibit anomalies in lymph node architecture and cell distribution [ 27 ], studies using bone marrow chimeras are warranted to confirm the definitive role of CCR7 signaling in FRT DC migration and  Chlamydia  transport.\nInhibition of the second step of  Chlamydia  transport, namely from the ILNs to the spleen by FTY720, indicates that S1P signaling is essential for egress of  Chlamydia -infected cells from the ILNs. Within ILNs  Chlamydia  was recovered from isolated CD11c +  DCs at 3 and 7 dpi. Infected DCs may be of FRT origin, or may be local ILN DCs that become infected by incoming FRT DCs. DCs also appear to be a significant  Chlamydia  reservoir in the ILNs, as their depletion at 3 dpi lowers ILN and spleen  Chlamydia  loads and abrogates GI tract infection at 7 dpi. While there is abundant evidence that tissue DCs transport antigen to the local draining lymph nodes, there is no evidence that once in the lymph nodes, tissue DCs re-enter the circulation to reach distant sites, such as the spleen. Rather, once in the lymph nodes, tissue-migratory DCs lose their motility [ 48 ] and most die [ 49 ]. Other cell types that migrate towards S1P in vivo such as T cells, NK cells [ 50 ,  51 ], and monocytes [ 52 ] may also contribute to  Chlamydia  dissemination. However, DCs have been shown to pass HIV infection to T cells either directly via transfer of surface virions (trans-infection) [ 53 ] or via the exosomes [ 54 ]. It is thus possible that during antigen presentation in the ILNs DCs pass  Chlamydia  EBs to T lymphocytes, which then become effector T cells, enter the circulation in responses to higher S1P concentration and ferry  Chlamydia  initially to the spleen and then to the GI tract. Alternatively, EBs might not need to productively infect T cells, but merely bind to T cell surface receptors in order to be carried to the spleen and the GI tract. This possibility is in line with our finding that FTY720 inhibits ILN to spleen/GI tract, but not FRT to ILN  Chlamydia  spread. Our finding that removal of the spleen significantly diminishes  Chlamydia  infection of the GI tract provides additional support to this notion. Furthermore, our finding that DC depletion at 0 and 3 dpi does not preclude spleen infection at 7 dpi, but does preclude GI tract infection, indicates that infection of the spleen is an important step in  Chlamydia  dissemination to the GI tract.\nWe did consider the possibility that mesenteric lymph nodes (MLN) may be the source of GI tract  Chlamydia  and analyzed  Chlamydia  titers in MLNs of IV and PV-infected mice. Although MLNs can become infected (likely by circulating  Chlamydia -infected cells), they do not appear to be the source of GI tract  Chlamydia . Our finding that splenectomized and FTY-treated mice have lower  Chlamydia  titers in the ILNs at 14 dpi might indicate that recirculating infected effector T cells that develop during protective immune responses home to the sites of infection (ILNs), and thus might contribute to the maintenance of  Chlamydia  loads in the ILNs.\nWhile inhibition of systemic  Chlamydia  dissemination in splenectomized, CCR7 -/- , or FTY720-treated mice did not affect vaginal shedding (for up to 14 dpi), it significantly hindered the induction of  Chlamydia -specific serum IgG. Women with tubal infertility present with high titers of  Chlamydia -specific IgG antibodies [ 14 ,  55 ,  56 ] therefore this finding sheds an important light on the possible connection between systemic  C .  trachomatis  dissemination and long-term FRT pathology in humans. In both mice and humans, CD4 +  T cells are essential for the resolution of chlamydial infections. However, the role of  Chlamydia -specific antibodies [ 31 ,  57 ] and CD8 +  T cells remains unclear [ 58 ]. Depletion of CD8 +  T cells was shown to significantly reduce hydrosalpinx and  Chlamydia  spread to the GI tract in mice, leading to the suggestion that the GI tract infection may promote hydrosalpinx by inducing “pathogenic” CD8 +  T cells [ 59 ].\nBased on current knowledge gathered from clinical and animal studies a “two-hit model” of chlamydial pathogenesis was proposed [ 60 ], according to which the 1 st  hit is caused by ascension of  Chlamydia  to the upper FRT that leads to tissue damage and formation of MHC: Chlamydia  peptide complexes. The 2 nd  hit that results in long-term pathology of fibrosis and hydrosalpinx is then delivered by recruited “pathogenic” CD8 +  T cells, induced in the GI tract following  Chlamydia  dissemination. Based on our findings described here, we propose a revised hypothesis, according to which the 1 st  hit is the  Chlamydia  spread to the spleen, where  Chlamydia -specific “pathogenic” CD8 +  T cells are induced. The 2 nd  hit occurs when  Chlamydia  reaches the upper FRT (by ascension or by recirculating infected cells), leading to recruitment of  Chlamydia -specific CD8 +  T cells, which contribute to the long-lasting FRT pathology.\nThis work will be important for understanding the signaling mechanisms that govern cell migration and antigen transport from the FRT during an infection. In addition, further elucidating in vivo  Chlamydia  cell tropism, as well as delineating the roles of lymphoid tissues (e.g. the spleen) in generation of “pathogenic”  Chlamydia -specific CD8 +  T cells, will be critical for identifying targets for therapeutic intervention and vaccine development.\n\nHuman cervical carcinoma epithelial cells (HeLa 229, ATCC CCL-2) were grown at 37°C with 5% CO 2  in Dulbecco’s modified Eagle medium (DMEM) that was supplemented with 10% fetal bovine serum (DMEM-10) and 50 μg/mL gentamicin.  Chlamydia muridarum  strain Nigg was propagated in HeLa cells and purified as described previously [ 61 ]. Briefly, confluent cell monolayers were infected by centrifugation for 1 h at 545 x  g , or rocking for 2 h at 37°C and 5% CO 2 . Infected cells were then incubated in DMEM-10 supplemented with 1x nonessential amino acids, and 1 μg/mL cyclohexamide for 40 h, after which HeLa cells were ruptured by sonication and cell debris pelleted by centrifugation (500 x  g ). Collected supernatant harboring EBs was then centrifuged at 10,000 x  g , following which pelleted EBs were collected and purified on Percoll gradient by centrifugation at 30,000 x  g , as described previously [ 62 ]. All  Chlamydia  EB stocks were stored at -80°C in sucrose-phosphate buffered glutamic acid (SPG) until used.\nFemale 6–8 week-old C57BL/6, CCR7 -/- , and CD11c-DTR mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and housed under specific pathogen free conditions.\nStudies were conducted in strict accordance with recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal protocol (Protocol # 18–041) was approved by the Southern Illinois University Institutional Animal Care and Use Committee. Mice were anesthetized with 1–3% isoflurane delivered in a stream of oxygen by a controlled precision vaporizer. To ensure that animals were anesthetized, the respiration rate was monitored and a toe pinch was performed. At the end of the studies animals were euthanized using CO 2 . Cervical dislocation was additionally performed to ensure that the animal was deceased.\nMice were PV infected with either 10 3 , 10 4 ,10 5 , 10 6  or 10 7  IFU of  Chlamydia  in a 10 μL volume of SPG. Five days prior to PV infections mice were sub-cutaneously (SC) injected with 2.5 mg medroxyprogesterone acetate (Henry Schein) in order to synchronize their estrus cycles. To exclude the possibility of  Chlamydia  transmission via coprophagy or grooming, mice were housed individually in wire-mesh bottom cages and fitted with custom-made, light-weight neck collars. In one study mice were infected IV with 10 5  IFU of  Chlamydia  in 200 μl of sterile saline solution via a lateral tail vein. To consistently infect animals with intended doses of  Chlamydia , a single stock of renographin-purified EBs with a known titer was used. At pre-determined times following PV or IV infections, mice were euthanized and tissues were collected for cell isolation, determination of  Chlamydia  titers, and  Chlamydia -specific antibodies.\nFTY720 (Sigma) was dissolved in drinking water (1.85 mg/L) supplied ad libitum to C57BL/6 mice 3 d prior to infection and for the reminder of the studies (for up to 14 dpi).\nAt 3, 7, and 14 d post PV or 3, 6 and 9 d post IV infection, mice were euthanized and blood was collected via cardiac puncture. Plasma and blood cells were separated by centrifugation. From each mouse at any given time point 4x10 6  cells were resuspended in 400 μL of SPG, sonicated twice (for 5 sec) and cell debris pelleted by centrifugation. Collected supernatant was used to infect fresh HeLa monolayers (2 replicates, 200 μL each). For titering samples of plasma, HeLa monolayers were overlaid with 200 μl of plasma that was either undiluted, diluted 1:2, or 1:4 in SPG. ILNs, spleen, liver, gallbladder, MLNs and ceca were aseptically excised and placed in sterile tubes containing SPG. Liver and spleen tissue samples were weighed in order to determine  Chlamydia  titers per g of tissue. The ceca were dissected longitudinally and their contents were gently removed by washing with PBS containing 0.1 mg/mL gentamicin and 2.5 μg/mL Fungizone. Cecal epithelium was then gently scraped with a scalpel blade in SPG and deposited in a sterile Eppendorf tube [ 63 ]. Collected cecal scrapings, ILNs, liver, and spleen tissues were vortexed for 1 minute, homogenized, and then sonicated briefly. Cell debris was pelleted by centrifugation 425 x  g  for 5 min at 4°C. Collected supernatant was diluted in DMEM (1:2 ratio) and used to infect HeLa monolayers in 48-well plates in duplicate. At 40 h post-infection, bacteria were harvested and serial dilutions (10 −1  to 10 −7 ) were used to infect duplicates of confluent cell monolayers in 96-well plates by centrifugation for 1 h at 545 x  g  for titering.  Chlamydia -negative cultures were expanded for six passages to ensure that very low  Chlamydia  titers could be detected. If no live  Chlamydia  were detected after six passages, tissues were considered  Chlamydia -negative. All titers shown here are from the first passage and all samples that were negative in the first passage remained negative for  Chlamydia  for up to six passages.\nAt 24 h post-infection monolayers were fixed with ice-cold methanol for 10 min at room temperature. Methanol was then aspirated and cells were incubated with an anti- Chlamydia  monoclonal antibody conjugated to fluorescein at 37°C for 30 minutes (Pathfinder,  Chlamydia  Culture Confirmation System, BioRad). Cells were then washed 3 times with ddH 2 O and mounted with 90% glycerol. Fluorescent inclusions were counted at 400X magnification using a Leica DMIL microscope. For each replicate of each serial dilution, 20 random views were counted. The total number of live organisms in a given sample was calculated as described by others [ 22 ] and titers are expressed as log 10  IFU per tissue (ILNs, MLNs), per g of tissue (spleen, liver), per vaginal swab, or per cecum.\nAt the end of each study (14 dpi) mice were euthanized and blood was collected by cardiac puncture. Flat-bottomed 96-well plates were coated with renografin-purified  Chlamydia  EBs at a protein concentration of 10 μg/ml in coating buffer (0.02 M Na 2 CO3/0.07 MNaHCO 3  in H 2 O, pH 9.6). ELISA assays were conducted as described previously [ 64 – 67 ]. Antibody titers are expressed as log 10  value of the highest reciprocal dilution that yielded an OD value twice that of a negative control.\nMice were anesthetized with 1–3% isoflurane delivered in a stream of oxygen by a controlled precision vaporizer. To ensure that animals were anesthetized, the respiration rate was monitored and a toe pinch was performed before and during surgical procedures. Ophthalmic ointment was applied to the eyes of anesthetized animals using a sterile swab in order to prevent corneal drying. Preemptive analgesia (Meloxicam, 2 mg/kg body weight) was administered SC per recommendations of a veterinarian. The fur surrounding the surgical site was shaved using an electric razor and the surgical site was wiped free from hair with 70% ethanol. The skin was then disinfected with 3 applications of Betadine and 70% ethanol. For splenectomy the animal was placed on a surgical stage laying on its right side and draped with sterile gauze. The peritoneum was exposed by a 1–2 cm incision in the skin parallel to the edge of the rib cage, midway between the last rib and the hip joint. A second incision was made in the peritoneal wall, exposing the spleen. The spleen was exteriorized using forceps and the connective tissues were cut and blood vessels cauterized using a Bovie cautery pen in order to separate the spleen from the body. For removal of uteri a 2–2.5 cm ventral midline incision was made into the skin and then through the linea alba of the abdomen, exposing the FRT. The uterus and connected adipose tissue was exteriorized and the uterine horns and ovaries identified. A Bovie cautery pen was used to dissect through the mesometrial membrane and blood vessels to separate the uterus from the posterior body wall. The Bovie pen and/or small dissecting scissors were used to separate the uterine horn from the ovary, leaving the ovary and oviduct intact. A ligature using 5–0 absorbable suture was made at the base of each uterine horn and both uteri were severed anterior to the suture knot and removed from the body. Following uterectomy or splenectomy, the abdominal cavity was closed using a simple interrupted pattern with 5–0 absorbable polyglycolic acid suture. The skin incision was closed using 6–0 nylon suture in a simple interrupted pattern. Following surgery, topical antibiotic was applied to the incision daily and animals were fitted with light weight neck collars until the incision was fully healed (few days). Animals were allowed to recover for 2–3 weeks before being used for further studies.\nCD11c-DTR mice and controls lacking the high-affinity DTR (C57BL/6 background) were IP injected with 4 ng/g body weight dose of DTx (Sigma) 10 h prior to PV infection (0 dpi) or 3 dpi with 10 6  IFU of  Chlamydia . At 3 or 7 dpi respectively, ILNs, MLNs, spleens and cecal scrapings were collected for determining  Chlamydia  titers. To confirm that CD11c +  DCs were depleted following DTx treatment, single cell suspensions of spleens and lymph nodes of control and CD11c-DTR mice (with and without DTx depletion) were analyzed by flow cytometry as described previously [ 68 ]. Cell suspensions were analyzed using monoclonal antibodies specific for CD11c and MHC class II (BioLegend).\nAt day 0, 3 or 7 post-PV infection with 10 6  IFU of  Chlamydia , single cell suspensions of ILNs isolated from infected and control mice were prepared and CD11c +  DCs were isolated using MACS MicroBeads kit (Miltenyi). Briefly, isolated DCs were sonicated on ice for 5 sec (2x) and cell debris was pelleted by centrifugation. Collected supernatant was used to infect HeLa monolayers for titering using Pathfinder as described above.\nData were analyzed using ANOVA procedures and SAS software. Population means were separated using Tukey’s multiple comparison procedures or Student’s t-test and were declared significantly different at p<0.05. Data are expressed as the mean ± SD of the mean. As an alternative to Student’s t-test and in cases when normal distribution assumption was not met data were analyzed using Wicoxon/Kruskal-Wallis tests.\n\n(A-C)  Chlamydia  titers in ILNs, spleen and ceca of mice PV infected with 10 3 , 10 4  or 10 5  IFU of  Chlamydia . (D) Vaginal swab titers of mice PV-infected with 10 3 , 10 4 , 10 5 , 10 6  or 10 7  IFU of  Chlamydia  at 0, 3, 6, 9 and 12 dpi. (E) Incidence of  Chlamydia -positive samples of liver, gallbladder, blood plasma or cell fractions. (F) Vaginal swab titers of control and uterectomized mice at 0, 3, and 6 dpi. Data are expressed as the mean ± SD. Group means were separated using Tukey’s multiple comparison test or Student’s t-test and declared significantly different at p<0.05 (n = 5 (A-D) or n = 8 (F) mice per time point). Group means that do not share superscript are significantly different (p<0.05).\n(TIF)\nClick here for additional data file.\n(A-C)  Chlamydia  titers in MLNs at 0, 3, 6, and 9 or 0, 3, 7 and 14 dpi IV with 10 5  (A) or PV (B, C) with 10 6  or 10 7  IFU of  Chlamydia . (D-F)  Chlamydia  titers in MLNs of CCR7 -/- , FTY720-treated, and splenectomized mice at 7 and 14 dpi PV with 10 6  IFU of  Chlamydia . (G) MLN  Chlamydia  titers at 7 dpi in mice treated with DTx at 0 or 3 dpi PV with 10 6  IFU of  Chlamydia . Data are expressed as the mean ± SD. Group means were separated using Tukey’s multiple comparison test and declared significantly different at p<0.05 (n = 5 mice per time point (A-C), or n = 8–10 mice per time point from two separate studies (D-G). Group means that do not share superscript are significantly different (p<0.05).\n(TIF)\nClick here for additional data file.\n(A, B) ILNs of control (A) or  Chlamydia -infected (B) mice at 7dpi PV with 10 6  IFU of  Chlamydia . (C) Cecal  Chlamydia  titers in control C57BL/6 mice with or without DTx treatment. (D, E)  Chlamydia  titers at 3 dpi PV with 10 6  IFU of  Chlamydia  in spleen and ceca of control and CD11c-DTR mice treated with DTx at 0 dpi.\n(TIF)\nClick here for additional data file.","source_license":"CC-BY-4.0","license_restricted":false}