The Role Of MyD88, IL-12and LIGHT In The Process Of Chlamydia Muridarum Infection And Immunity In Mice

Chlamydia trachomatis is a leading cause of sexually transmitted diseases, whichcan induce urethritis, epididymitis and prostatitis among men and cervicitis,endometritis, salpingitis, and pelvic inflammatory disease among women, if untreated,which may lead to serious complications, including ectopic pregnancy, tubal infertility,infertility and so on. C. trachomatis infection can also increase the chance of cervicalcancer, ovarian cancer and HIV infection. But till now, the precise pathogenic andimmunologic mechanisms remain unclear and no effective vaccine is licenced. Sostuding on chlamydial pathogenesis and immunologic mechanisms and searching fortherapeutic targets will be of great importance.C. trachomatis is an obligate intracellular parasites, host anti-C. trachomatisimmune mechanism including adaptive and innate immunity, among which the CD4+Th1cell-dependent immunity represents a major protection mechanism. However, thesame immunity response may also contribute to the chlamydia-induced inflammatorypathologies. MyD88is a critical signaling adaptor molecule required by many Toll-likereceptors and plays important roles in innate immunity. LIGHT is a costimulatory factorfor T cells activation, which can enhance Th1cells differentiation, proliferation andTh1-type cytokines production after interacted with its receptor. Interleukin-12(IL-12)is an important Th1cytokine which can promote Th1cells differentiation andproliferation. So these immune molecules may develop significant effects in controllingthe intracellular C. trachomatis infection.Because mice only showed slight inflammation in lower genital tract after infectedwith human C. trachomatis strains, and mice intravaginally infected with a murine biovar of C. trachomatis (known as mouse pneumonitis [MoPn] agent and nowclassified as a new species, Chlamydia muridarum) organisms can developinflammatory pathologies including endometritis and hydrosalpinx similar to those inhumans. So MoPn has been used to study the mechanisms of C. trachomatispathogenesis and immunity in a mouse genital infection model.ObjectiveIn this research, mice deficient in MyD88, IL-12p35, IL-12p40and LIGHT wasused to intravaginally infected with MoPn. Numbers of Chlamydia inclusion bodies invaginal swab, pathologies of mice urogenital tract and host immune response werecompared with wild type mice to investigate the role of MyD88, IL-12and LIGHTplayed in MoPn urogenital infection. These study will lay the foundation for thepathogenesis research of C. trachomatis.MethodsEach of MyD88KO, IL-12p35KO, IL-12p40KO, LIGHT KO and wild type micewas inoculated intravaginally with1×104IFUs of live MoPn organisms. For some mice,a secondary infection was similarly carried out at two weeks later when MoPn wasnegative in vaginal swab after primary infection. To monitor live organism shedding,vaginal swabs were taken on different days after intravaginal infection (once every3to4days for the first50days and once per week thereafter). Each swab was suspended in500μl of SPG followed by sonication on ice and the released organisms were titrated onHeLa cell monolayers.On day80or143after primary infection, mice were sacrificed after anesthetized,and serum were collected for titrating and isotyping anti-MoPn antibodies by indirectimmuno-fluorescence assay. After mice spleen was isolated, splenocytes were harvestedand cultured in24-well plates at a density of5×106cells/well with or without stimulation with UV-inactivated MoPn elementary bodies at a final concentration of1×106IFU/ml. After culture for72hours, the culture supernatants were collected forIL-4, IL-5, IL-17and IFN-γ measurements with ELISA.Urogenital tract tissues and kidneys were harvested from mice after sacrificed.Before the tissues were removed from mouse bodies, an in situ gross examination wasperformed for evidence of hydrosalpinx and enlarged, swollen or necrotic kidneys orany other related abnormalities of uterine horn, oviducts and kidneys. The excisedtissues, after photographing, were fixed in10%neutral formalin and embedded inparaffin and serially sectioned longitudinally. The sections were stained withhematoxylin and eosin (H&E). The H&E stained sections were assessed by pathologistsblinded to mouse treatment and scored for severity of inflammation and pathologies. Ifthe difference of urogenital tract tissues and kidneys was significant between theknockout and wild type mice, the chlamydial Ags in tissues sections were detected withimmunohistochemisty. And the whole kidney, cevix, uterine horn and oviduct tissueswere homogenized separately in SPG and briefly sonicated. The released organismswere titrated on HeLa cell monolayers and the chlamydia inclusion bodies were counted.The kidney homogenates also were serially diluted and plated on blood agar plates fortitrating bacteria.For MyD88knockout mice group, Mφs were collected from peritoneal cavity, and2×105cells were added to each well of48-well plates. After washing away nonadherentcells, the adherent Mφs were infected with MoPn at a multiplicity of infection of5. Theinfected cultures were incubated for48h at37°C in a CO2incubator and measuredIL-1α, IL-6, TNF-α and MIP-2by ELISA.Results1. The chlamydia shedding time of MyD88KO mice was similar to wild type mice, butboth the gross appearance and the dilation and inflammation scores under microscope of the isolated genital tracts showed that the genital tract pathology especiallyhydrosalpinx degree from the MyD88-deficient mice was much more severe thanwild type mice (P＜0.01). After titrating the chlamydia IFUs from genital tract tissuehomogenates, we found that genital tissues from both wild-type and MyD88KO micecontained similar amounts of live organisms on day10. However, by day23, liveorganisms were detected in the upper genital tract tissues (uterine horn and oviduct)of the MyD88KO but not wild-type mice. The similar result also showed in directdetection of chlamydial Ags in mouse genital tissue sections. There weresignificantly higher numbers of chlamydial inclusions in the cervix, uterine horn, andoviduct tissue sections of MyD88KO than wild-type mice on day10and day23post-infection (P＜0.05).The proinflammatory cytokines IL-1a, IL-6, TNF-a and MIP-2in the urogenitaltract or macrophage supernatant from the MyD88KO mice were much lower thanthat from wild type mice (P＜0.05). Splenocytes from wild-type mice produced highlevels of IFN-γ and IL-17but low levels of IL-4and IL-5upon restimulation withchlamydial Ags. However, splenocytes from the MyD88KO mice produced onlyminimal levels of IFN-γ or IL-17but significantly high levels of IL-4and IL-5. Allmice developed robust anti-C. muridarum organism IgG antibody, but the ratio ofChlamydia specific IgG2a versus IgG1is＞1(indicative of Th1dominance) inwild-type and＜1(reflective of Th2dominance) in MyD88KO mice.2. The chlamydia shedding time of LIGHT KO mice was similar to wild type mice,which cleared the organisms within28days after primary infection, and acquiredprotective immunity against MoPn reinfection. All mice regardless of genotypesdeveloped severe upper genital tract pathology and showed no significant differencebetween LIGHT KO and wild type mice. All mice developed robust anti-MoPnorganism IgG antibody responses and the ratios of IgG2a versus IgG1showed nosignificant difference between LIGHT KO and wild type mice. Splenocytes fromMoPn-infected LIGHT KO and wild type mice produced high levels of IFN-γ and IL-17, but IL-4and IL-5couldn’t be detected.3. Compared with wild type mice, mice deficient in either IL-12p35or p40bothdeveloped similar but prolonged infection time courses after primary infection, butsimilar time couses after secondary infection. All mice regardless of genotypesdeveloped severe upper genital tract pathology, and no significant difference wasfound among the mouse groups.Almost all kidneys from IL-12p40KO mice and some IL-12p35KO mice wereabnormal, which exhibited pathology of pyelonephritis, but no obvious abnormalitywas noted in any of the kidneys from wild type mice. The number of mice withabnormal kidneys in the p40KO group was significantly higher than that of wildtype mice after both primary and secondary infections and than that of p35KO groupafter primary infection. Neither the age-matched IL-12p35KO nor p40KO micedeveloped any gross kidney pathology in the absence of chlamydial infection. MoPninclusions could be detected only in kidneys with gross pathology from eitherIL-12p35or p40KO groups, while no inclusion was ever detected in kidneys fromwild type mice. The number of chlamydial inclusions of p40KO mice was also muchhigher than that from the p35KO mice. Live bacteria were detected in mice deficientin either p35or p40KO mice but not wild type mice. However, there was nosignificant difference in the number of live bacteria recovered from p35versus p40KO mice.All mice regardless of genotypes developed robust anti-MoPn organism IgGantibody responses during the course of both primary and secondary infections. Theratios of IgG2a versus IgG1revealed that wild type mice had a significantly higherratio than either IL-12p35or p40KO mice. Splenocytes from MoPn-infected wildtype mice produced high levels of IFN-γ upon restimulation with chlamydialantigens while mice deficient in either IL-12p35or p40failed to do so. Splenocytesfrom both wild type and IL-12p35KO mice produced significantly high amounts ofIL-17while the splenocytes from IL-12p40KO mice failed to do so. IL-4and IL-5 couldn’t be detected.Conclusions1. MyD88is dispensable for protection against MoPn urogenital tract infection, but isnecessary for inflammatory cytokine production in the early phase of host response tochlamydial infection, preventing urogenital tract pathology and limitting theorganisms to ascend into the upper genital tract following intravaginal infection.2. LIGHT signal pathway may be not correlated with protection against MoPnurogenital tract infection and urogenital tract pathology induced by MoPn.3. Protection against MoPn urogenital tract primary infection rely on IL-12dependentTh1immune response, and MoPn induced urogenital tract pathology is notcompletely dependent on IL-12and IL-23. IL-12and IL-23can limit intravaginallyinfected MoPn to spread into kidneys, and chlamydia infection may correlated withnephritis when IL-12or IL-23is deficient.