Immunoprotective role of dendritic cells in Chlamydia muridarum respiratory infection / 中华微生物学和免疫学杂志

Objective:To investigate the role of dendritic cells (DC) in Chlamydia muridarum ( Cm) respiratory infection and their effect on adaptive immune response. Methods:C57BL/6 mice were exposed to 1×10 3 inclusion-forming units (IFU) of Cm through inhalation to establish the mouse model of Cm respiratory infection. The proportion of CD11c + MHCⅡ + DC and the expression of costimulatory molecules (CD40, CD80 and CD86) in spleen tissues were detected by flow cytometry on 0, 3 and 7 d after infection. The expression of IL-12p40, IL-10 and IL-6 at mRNA level in spleen tissues was detected by qPCR. Mouse splenic DC isolated on 7 d after Cm infection were sorted by magnetic beads and then transferred to recipient mice. Th1 response in the recipient mice was measured using intracellular cytokine staining 14 d after infection. Results:Cm respiratory infection induced massive infiltration of DC and promoted the expression of costimulatory molecules on splenic DC. The expression of IL-12 and IL-10 at mRNA level in splenic DC reached the peak on 3 d after infection. Transferring the splenic DC of Cm-infected mice into the recipient mice could alleviate the disease condition in the recipient mice after Cm infection with reduced Cm inclusion-forming units in lung tissues and significantly increased proportion of Th1 cells in lung and spleen tissues. Conclusions:Cm respiratory infection could induce the maturation and activation of DC, which promoted Th1 immune response. DC played an important role in Cm infection.

Chlamydia muridarum respiratory infection induces macrophage infiltration and polarization toward M1 phenotype / 中华微生物学和免疫学杂志

Objective:To investigate the infiltration and polarization of macrophages in mice during Chlamydia muridarum ( Cm) respiratory infection. Methods:C57BL/6 mice were intranasally infected with 1×10 3 inclusion-forming units (IFU) of Cm to establish the mouse model of Cm respiratory tract infection. The percentages of CD45 + F4/80 + cells and the macrophages expressing CD86, major histocompatibility complex Ⅱ (MHC), inducible nitric oxide synthase (iNOS) and CD206 were detected by flow cytometry. Expression of iNOS, CD206 and CCL2 at mRNA level was detected by real-time quantitative PCR. Results:Cm respiratory tract infection induced the increase of macrophages in mouse lung tissues. Compared with uninfected group, CD45 + F4/80 + macrophages were increased significantly from day 3 and reached the peak on day 7 after Cm infection. Moreover, the expression of CD86, MHCⅡ and CCL2 was increased, and the macrophages were polarized to M1 phenotype. However, the expression of M2 macrophage marker CD206 was decreased gradually. Further studies showed that iNOS expression, the indicator of M1 macrophage activation, was increased after Cm infection and reached to the top on day 7. Conclusions:Cm respiratory infection could induce the infiltration of macrophages in lung tissues and promote the polarization of macrophages to M1 phenotype.

Chlamydia muridarum respiratory infection induces infiltration and polarization of alveolar macrophages and pulmonary interstitial macrophages / 中华微生物学和免疫学杂志

Objective:To investigate the effects of Chlamydia muridarum ( Cm) respiratory tract infection on the infiltration and polarization of alveolar macrophages (AMs) and pulmonary interstitial macrophages (IMs). Methods:A C57BL/6 mouse model of Cm respiratory tract infection was established through nasal inhalation. Flow cytometry was used to detect AMs (CD45 + F4/80 + CD11c + ) and IMs (CD45 + F4/80 + CD11c -) in lung tissues at 0, 3, 7 and 14 d after Cm respiratory tract infection. The proportions of M1 (CD80 + , CD86 + , MHCⅡ + , iNOS + ) and M2 (CD206 + , Arg1 + ) macrophages in AMs and IMs were also detected. Results:(1) Cm respiratory tract infection induced the infiltration of AMs and IMs. Compared with the uninfected group (0 d), the proportions and the numbers of AMs and IMs of were significantly increased 3 d after infection ( P<0.05, P<0.01). The numbers of AMs and IMs reached the peak 7 d after infection ( P<0.001). (2) Compared with the uninfected group, the proportions of CD80 + and CD86 + cells in AMs were significantly up-regulated 3 d after infection ( P<0.05, P<0.01); the proportion of MHCⅡ + cells in AMs increased after infection and reached the peak at 14 d ( P<0.05), while the proportion of CD206 + cells decreased after infection ( P<0.05). (3) Compared with the uninfected group, the proportions of CD80 + and CD86 + cells in IMs were increased 3 d after infection ( P<0.05, P<0.001) and the proportion of MHCⅡ + cells was significantly increased 14 d after infection ( P<0.01), while there was no significant change in the proportion of CD206 + cells. (4) In AMs, the proportion of iNOS + cells increased continuously after infection ( P<0.01), while the proportion of Arg1 + cells decreased continuously after infection, especially at 7 d and 14 d ( P<0.05). In IMs, the proportion of iNOS + cells reached the peak at 7 d ( P<0.001), but the proportion of Arg1 + cells showed no significant change after infection. Conclusions:Cm respiratory tract infection induced the infiltration of AMs and IMs, stimulated the polarization of AMs and IMs towards the M1 phenotype and weakened the polarization of AMs to M2 macrophages, but had no significant influence on the polarization of IMs towards the M2 phenotype.

Detection of Chlamydia in multiple visceral organs of mice inoculated with Chlamydia through different routes / 中华皮肤科杂志

Objective To certify that Chlamydia can spread from the genital tract to the gastrointestinal tract for long-lasting colonization.Methods Totally,120 female C57BL/6J mice aged 5-6 weeks were divided into 4 experimental groups to be inoculated with purified Chlamydia muridarum (C.muridarum) elementary bodies in the vagina (n =35),gastric area (n =30),anus and rectum (n =30),retro-orbital venous plexus (n =5) respectively.Moreover,corresponding negative groups inoculated with sucrose phosphate glutamate buffer (n =5) were set up for each experimental group.On days 3,7,and every 7 days,vaginal and rectal discharges were collected with swabs from the mice,and the number of live C muridarum orgnisms in exfoliated cells infected with C muridarum in the swabs was determined.Indirect immunofluorescence assay and quantitative PCR (qPCR) were performed to determine the number of live chlamydial organisms and the copy number of chlamydial genomes in the mouse genital tract (vagina,uterus,oviduct and ovary),gastrointestinal tract (stomach,small intestine,cecum,colon,rectum)and parenteral tissues (heart,liver,spleen,lung,kidney) on days 7,14,28,56 and 105 after the inoculation.The number of live chlamydial organisms and copy number of chlamydial genomes were transformed logarithmically with a base of 10.The degree of hydrosalpinx and inflammation in the genital tract,and histopathological changes of the gastrointestinal tract were observed.The infectivity and virulence of C.muridarum in the genital tract and gastrointestinal tract were evaluated in the intragastric inoculation group and intra-anal and intrarectal inoculation group on days 28 and 56 after the inoculation.Blood samples were obtained from the mouse caudal vein in the retro-orbital venous plexus inoculation group on days 3,5,7,10 and 14 after the inoculation,the number of live chlamydial organisms and the copy number of chlamydial genomes in the blood samples were determined,and chlamydial infectivity in the genital tract and gastrointestinal tract was evaluated on day 56.Results On day 7 after the inoculation in the vagina,both C.muridarum live organisms and genomes were detected in the genital tract,gastrointestinal tract and parenteral tissues of all the mice.The largest common logarithm of the number of C.muridarum inclusion forming units (IFU) was observed in the vagina (6.26 ± 0.56),with the common logarithm of the copy number of chlamydial genomes in the vagina being 7.30 ± 0.23,and the common logarithms of the number of Chlamydia IFU and genomic copy were 2.60 ± 1.95 and 4.87 ± 0.09 respectively in the rectum.On day 28,no live Chlamydia was detected in the heart,lung or other parenteral tissues,while live Chlamydia could be found in the genital tract and gastrointestinal tract.The common logarithms of the number of Chlamydia IFU and genomic copy were 3.47 ± 1.06 and 5.80 ± 1.49 respectively in the vagina,and 4.00 ±0.35 and 5.14 ± 0.81 respectively in the rectum.On day 56,live Chlamydia could only be detected in the gastrointestinal tract.On day 105,live Chlamydia and its genomes could be still detected in the gastrointestinal tract,and the common logarithms of the number of Chlamydia IFU and genomic copy could be up to 2.60 ± 0.65 and 4.29 ± 0.57 respectively in the rectum.On days 28 and 56 after the inoculation,both live Chlamydia and its genomes could be detected in the gastrointestinal tract of all the mice in the intragastric inoculation group and intra-anal and intrarectal inoculation group.Chlamydia could survive in the blood for about 14 days in the retro-orbital venous plexus inoculation group,and live Chlamydia was detected in anal-rectal swabs in all the mice on day 14.On day 56 after the intravaginal inoculation with C.muridarum,severe hydrosalpinx,chronic inflammation and oviduct dilation occurred in the genital tract of 5 mice,but there was no obvious infiltration of inflammatory cells in the gastrointestinal tract,and inflammatory pathological changes were not observed in the gastrointestinal tract of mice after inoculation with Chlamydia through other routes either.Conclusion The infection with Chlamydia in the genital tract can lead to systemic dissemination,and Chlamydia can be spread to the gastrointestinal tract,and colonize and survive in the gastrointestinal tract for a long time.

Overexpression of TLR2,TLR4 and MyD88 is associated with inflammation in C3H mice during Chlamydia respiratory infection / 中国免疫学杂志

Objective:To investigate the mechanism of excessive inflammation in the lung of C3H/HeN(C3H) mice following Chlamydia muridarum(Cm) airway infection.Methods:Chlamydial pneumonitis was induced in C3H and C57BL/6(C57) mice by intranasal inoculation with 1×103IFU (inclusion forming unites) of Cm strains.The expression of TLR2,TLR4 and MyD88 mRNA in the lung at different time point post-infection was measured by RT-PCR.Results:Cm infection induced Toll-like receptors expression in two strains of mice.The expression of TLR2 and TLR4 mRNA,especially TLR2 mRNA(P<0.001 or P<0.05),were significantly higher in highly susceptible C3H mice on day 7 and day 14 d post-infection compared with C57 mice.Further studies showed that the expression of MyD88 mRNA was also significantly higher in C3H mice on day 7 post-infection,and maintained high expression untill the day 14.Conclusion:Cm lung infection induced high level of TLR2,TLR4 and MyD88 mRNA expression in C3H mice,which may associate with excessive inflammation in C3H mice.

Inhibitory effects of the Chlamydiaphage phiCPG1 capsid protein Vp1 on Chlamydia psittaci strain guinea pig inclusion conjunctivitis and Chlamydia trachomatis serovar E / 中华皮肤科杂志

Objective To evaluate inhibitory effects of the Chlamydiaphage phiCPG1 capsid protein Vp1 on Chlamydia psittaci strain guinea pig inclusion conjunctivitis (GPIC) and Chlamydia trachomatis (Ct) serovar E,and to provide new ideas for the treatment of Ct infection.Methods The Chlamydiaphage phiCPG1 capsid protein Vp1 was expressed in Escherichia coli BL21 transfected with the recombinant plasmid Vp1-pET30a (+),identified by Western blot analysis and purified by using dialysis bags.Bicinchonininc acid (BCA) assay was performed to determine the concentration of Vp1 protein.GPIC and Ct serovar E strains were both classified into 4 groups to be firstly incubated with Vp1 protein (Vp1 group),Tris-glycine solution (Tris group),S protein (S group) or Dulbecco's Modified Eagle Medium (DMEM,DMEM group) at room temperature for 3 hours,then were used to infect Hela cells followed by 72-hour (GPIC) or 48-hour (Ct serovar E) culture with the presence of Vp 1 protein (Vp 1 group),Tris-glycine solution (Tris group),S protein (S group) or DMEM (DMEM group).Subsequently,immunofluorescence staining was conducted to observe and count chlamydial inclusions.Results The number of GPIC inclusions was significantly different between the 4 groups after 72-hour culture (F=476.632,P＜ 0.05),and lower in the Vp1 group (5.0 ± 1.5) than in the Tris group (24 ± 1.2,P＜ 0.05),S group (25 ± 1.7,P＜ 0.05) and DMEM group (25 ± 1.5,P＜ 0.05),but insignificantly different between the latter 3 groups (P ＞ 0.05).Compared with the DMEM group,the Vp1 group showed a significant decrease of 80.2％ ± 3.99％ and 77.2％ ± 1.79％ in the number of GPIC and Ct serovar E inclusions respectively,with no significant difference in the inhibitory effect of Vp1 on GPIC versus Ct serovar E (t =2.057,P ＞ 0.05).Conclusion The phiCPG1 capsid protein Vp1 can obviously inhibit GPIC and Ct serovar E infections to a similar degree.

Expression of exogenous genes in Chlamydia muridarum by transfection with shuttle vectors / 中华临床感染病杂志

Objective To add an open reading frame in the shuttle vector of pGFP ∷ CM for transfection of exogenous genes into Chlamydia muridarum.Methods The sequence of plasmid pGFP ∷ CM and new open reading frame (including promoter of pgp4,mCherry gene of red fluorescence protein and transcription termination sequence of Chlamydia trachomatis CT579) were amplified by polymerase chain reaction (PCR),and the products were transfected into Stellar competent cells.The recombinant plasmids were identified by PCR,enzyme digestion and sequencing.Then the recombinant plasmid was transfected into plasmid-free strain CMUT3,and the GFP-and mCherry-positive inclusions were observed under the fluorescence microscope.After the ampicillin selection and plaque purification,the purified CMUT3-pGFP-mCherry-CM was identified by indirect immunofluorecesent stain using anti-pgp3 and anti-glgA antibodies.Results The correct recombinant plasmid after sequencing identification,enzyme digestion and PCR amplification was successfully transfected into CMUT3,and the GFP-and mCherry-positive inclusions were observed.The transfected strain CMUT3-pGFP-mCherry-CM was purified after ampicillin selection and plaque purification.The expression of pgp3 and glgA protein in CMUT3-pGFP-mCherry-CM was similar to that in CMUT3-pGFP ∷ CM.Conclusion An open reading frame is successfully added in the plasmid pGFP ∷ CM,and the new plasmid can be transfected into CMUT3 and express exogenous protein,which can be used for further study on the function of single chlamydial protein.

Mice deficient in IL-12p35 or IL-12p40 develop renal lesions during Chlamydia muridarum urogenital infection / 中华微生物学和免疫学杂志

Abstract] Objective To study the roles of IL-12 and IL-23 in the development of protective im-munity and pathological changes during chlamydial urogenital infection.Methods C57BL/6J wild type (wt) mice and mice deficient in IL-12p35 (IL-12p35 KO) or IL-12p40 (IL-12p40 KO)were inoculated in-travaginally with 1×104 IFU of live Chlamydia muridarum ( C.muridarum) organisms.Half mice of each group were reinfected on day 114 after primary infection.Vaginal swabs were taken every 3 or 4 days to mo-nitor live organism shedding.The mice were sacrificed after 114 or 143 days of primary infection and the va-ginal tract and kidney samples were collected for pathological analysis.The numbers of chlamydial inclusion bodies and bacteria in kidney homogenates were titrated after 100 days of primary infection.Results The infection time courses of mice deficient in either IL-12p35 or IL-12p40 were similar after primary infection, but were prolonged as compared with the wild type mice.All mice regardless of genotypes developed severe pathological damages in upper genital tracts with no significant difference among different groups.Almost all IL-12p40 KO mice and some IL-12p35 KO mice showed pathological changes in kidney samples.No obvious abnormality was observed in any of the kidneys from wild type mice.Neither the age-matched IL-12p35 KO nor IL-12p40 KO mice developed any gross pathological changes in kidney in the absence of chlamydial in-fection.C.muridarum inclusions were detected in kidney samples with gross pathological damages from IL-12p35 KO mice and IL-12p40 KO mice.No inclusions were ever detected in kidneys from the wild type mice.The numbers of chlamydial inclusions in the IL-12p40 KO mice were much higher than those of the IL-12p35 KO mice.Live bacteria were detected in mice deficient in either IL-12p35 or IL-12p40, but not in the wild type mice.No significant difference with the number of live bacteria was found between IL-12p35 KO mice and IL-12p40 KO mice.Conclusion IL-12 and IL-23 could inhibit the spread of C.muridarum in-fection from genital tract to kidney.The deficiency of IL-12 or IL-23 might relate to the renal lesions induced by Chlamydia infection.

IFN-γup-regulates Th17/IL-17 response against Chlamydia trachomatis lung infection / 中华微生物学和免疫学杂志

Objective To investigate the regulation of IFN-γ to Th17 response in Chlamydia muridarum (Cm) lung infection in mice. Methods A murine model of pneumonia induced by intranasal inoculation of Cm was used for this study. Anti-mouse IFN-γ McAbs were used to neutralize endogenous IFN-γfollowing Cm lung infection. Control group received the same dose of isotype antibody (IgG2a). Mice were sacrificed at day 7 postinfection. Chlamydial growth in the lung was assessed by immunoenzyme technique.IL-17 and IL-23 mRNA expression in the lung was assayed by RT-PCR and the proliferation of IL-17 + CD4 +T cells in the spleen was assayed by intracellular cytokine staining. Results IFN-γ-neutralized mice exhibited serious disease course, include greater body weight loss, higher organism growth and much more severe pathological changes in the lung compared with control mice. The mRNA expression of IL-17 and IL-23 in the lung and the proliferation of IL-17 + CD4 + T cells in the spleen significantly decreased in the IL-17- neutralized mice. Conclusion IFN-γ was protective in Cm lung infection through up-regulating the antigen specific Th17 responses.

Cloning, expression and identification of phage's capsid Vp3 protein of guinea pig inclusion conjunctivitis chlamydia / 中华传染病杂志

Objective To get phagc's capsid Vp3 gene and protein of guinea pig inclusion conjunctivitis (GPIC) chlamydia. Methods The genome DNA was extracted from the φCPG1 phage.The full sequence of Vp3 gene was amplified by polymerase chain reaction (PCR) from the above genome DNA. The Vp3 gene was digested by restriction endonuclease and then inserted into prokaryotic plasmid vector pET30a (+). The recombinant plasmid was transformed into E. coil BL21, and was identified by restriction endonuclease, PCR and sequencing. The E. coil BL21 with expected recombinant plasmid was induced and the expressed recombinant Vp3 protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, then purified by agarose gel. Results The recombinant gene was sequenced and proved to be 447 bp which was consistent with the φCPG1 Vp3 gene sequence in GenBank. A 25 000 capsid protein was expressed and confirmed by SDS-PAGE and Western blot. The purified protein was obtained. Conclusion The capsid Vp3 protein of φCPG1 is successfully expressed and purified, which is helpful for the further study on its mechanism and clinical applications.