NK Cells Regulate CD4~+T Cell Subsets And Pulmonary Macrophage Polarization In Chlamydial Lung Infection

Part one:Natural killer cells regulate CD4+T cell subsets in chlamydial lung infectionObjectiveChlamydica is a strict intracellular parasitic prokaryotic cell microbes that can cause a variety of human diseases.Chlamydia trachomatis(C.truchomatis)is the most common pathogen of sexually transmitted diseases and can cause neonatal chlamydial pneumonia.Chloamydia pneumoniae(C.pneumoniae)is the third major pathogen of community-acquired pneumonia after Streptococcus pneumoniae and Haemophilus influenzae.which causes acute atypical pneumonia in children,adults.especially the elderly.Although a variety of broad-spectrum antibiotics can be used to treat chlamydial infection,but due to the different clinical manifestations of individuals.drug resistance and other factors affect the timely diagnosis and effective treatment of'chlamydial disease.Currently,there is no effective vaccine for the prevention of chlamydial disease.Therefore,it is necessary to study the immune response and immune regulation mechanism in order to further control chlamydial infection.Numerous studies have shown that innate and adaptive immune response play an important role in host defense chlamydial infection.Chlamydial infection can activate innate immune cells including DC(dendritic cells),macrophages(macrophages,M?),NK cells(nature killer cells)and so on.These cells not only induce an innate immune response but also affect the adaptive immune response.Cellular-mediated immune response is the main adaptive immunity in host defense chlamydial infection.CD4+T cell immune response(IFN-?;Th1)for the host to clear the chlamydial infection plays a primary protective role.Th17 cells play an important role in the clearance of chlamydial infection by producing cytokines such as IL-17 and IL-22.Previous studies have found that human and mouse chlamydial infection induced Tregs response increased in local tissue and associated with histopathological damage.NK cells are an important member of innate lymphocytes and play an important role in the body's first line of defense against pathogen invasion.NK cells without sensitization can directly identify and kill infected cells,in addition to cytotoxicity,more attention has been paid to NK cell immune regulation role.More and more studies have shown that NK cells can regulate T cell responses in infectious and inflammatory diseases.At present,studies have shown that NK cells play a protective role in host defense against chlamydial infection,the reported studies on the immunoregulation role of NK cell in modulating CD4+T cell subset are limited.BALB/c mice chlamydial respiratory tract infection model has been widely used to study the immunoregulatory effect of NK cells on the important CD4+T cell subsets.In current study.we used a NK cell-specific antibody(anti-asialo GM1)to specifically eliminate NK cells to investigate the eff-ect of NK cell on the clearing ability of infected chlamydial and histopathological damage.The immunoregulation effect of NK cells on different CD4+T cell subsets(Th1.Th17 and Tregs)in infected local(lung tissue)and immune organs(spleen and mediastinal lymph nodes)were detected by ELISA,intracellular cytokine staining and real-time quantitative PCR.Furthermore,we analyzed the effect of NK cells on the maintenance of CD4+T cell subsets immune balance.Methods1.The effect of NK cells depletion on chlamydial diseases1.1 Chlamydia:Hep-2 cells were cultured and infected with C.muridarum.Glass bead method to collect infected cells,then ultrasonically broken to release chlamydia,centrifugal removal of cell debris.After ultra-centrifugation purification EBs were stored at-80 ?.1.2 Establishment of mice lung infection model:For mouse infection,1×103 inclusion-forming units(IFUs)of live C.muridarum organisms in 40 ?l SPG buffer were used to inoculate mice(6-8 weeks,female)intranasally.1.3 NK cell depletion in vivo:Mice received an intravenous tail-vein injection of 20?l anti-asialo GM1 or control normal rabbit IgG antibody(isotype)in 50 ?l PBS 1 day before and 1 day after C..muridarum infection,then every 3 or 5 day injected 10?l anti-asialo GM1 or isotype in 50 ?l PBS until the end of the test.Flow cytometry was used to detect NK cell clearance efficiency in lung.1.4 Assessment of mice disease:Body weights of mice were monitored daily.Lung injury was assessed by HE staining(hematoxylin-eosin staining).The lung C.muridarum burden was assessed by immunostaining of chlamydial inclusions(IFUs).2.NK cells regulate CD4+T cell subsets immune response2.1 Effects of NK cells on cytokines secreted by Th1,Th17 and Tregs cells:At predetermined days after inoculation(day 6 and 12).the mice were euthanized under light anesthesia with isoflurane.The supernatant of spleen.lung and mediastinal lymph nodes cells was harvested after 72h culture,and the production of cytokines(IFN-?,IL-17A,IL-22,IL-10)was measured by ELISA(enzyme-linked immunosorbent assay).Cytokines(IFN-?.IL-17 and IL-22)levels in serum were assayed by ELISA.2.2 Effects of NK cells on Th1,Th17 and Tregs cell populations:Splenocytes,lung mononuclear cells and mediastinal lymph node cells were incubated and intracellular cytokine stained.The percentage and number of CD3+CD4+IFN-?+T(Th1).CD3+CD4+IL-17A+T(Th17)and CD4+CD25+Foxp3+T(Treg)cells in the spleen,lung and mediastinal lymph nodes were detected by flow cytometry.3.NK cells regulate Th1/Treg and Th17/Treg balance3.1 Effects of NK cells on the expression of transcription factor in T cell subsets:RNA was extracted from mouse splenocytes and lung mononuclear cells.The expression of Th1(T-bet),Th2(GATA3),Th17(ROR?T)and Treg(Foxp3)transcription factor mRN A expression was detected by real-time quantitative PCR.3.2 Effects of NK cells on T cell differentiation related cytokines:At day 6 and 12 after infection,the supernatant of lung and mediastinal lymph nodes cells was harvested after 72h culture.and the production of cytokines(IL-6,TGF-? and IL-12p40)were assayed by ELISA.3.3 Effects of NK cells on Th1/Treg and Th17/Treg ratio:Th1/Treg and Th17/Treg ratio in splenocytes,lung mononuclear cells and mediastinal lymph node cells were calculated by the numbers of Th1 or Th17 cells by the number of Treg cells,respectively.Results1.NK cell-depleted mice show more severe disease to chlamydial lung infection 1.1 Anti-asialo-GM1 antibody deplete NK cells specifically:The percentage of NK cells in the lungs was measured at day 4 after chlamydial infection.The results showed that the number and proportion of NK cells in the lungs of mice injected with anti-asialo-GM1 antibody were significantly decreased,and the clearance rate was about 90%.1.2 NK cell-depleted mice show more severe disease:Following chlamydial lung infection,as compared to isotype control mice.NK cell-depleted mice showed greater body weight loss and slower recovery,higher chlamydial loads(IFUs)in the lung and more intense pathologic changes.2.NK cells enhance Th1 and Th17 responses but inhibit Tregs response2.1 NK cell promote IFN-?,IL-17 and IL-22 secretion with chlamydial lung infection:IFN-? is mainly secreted by Thl cells,whereas Th17 cells are characterized by cytokines such as IL-17 and IL-22.Use the ELISA method to detect the expression of IFN-?,IL-17 and IL-22 in spleen,lung,mediastinal lymph nodes and serum at day 6 and 12.The results showed that the levels of IFN-?,IL-17 and IL-22 in the above organs were significantly decreased after NK cells depleted,and the serum concentration was also decreased.2.2 NK cell-depletion lead to decreased Th1 and Th17 cells populations:Th1 immune response plays a key role in host defense against chlamydial infection,Th17 cells synergistically promote Thl cell response.Flow cytometry was used to analyze the effect of NK cell-depletion on the ratio and absolute number of CD3+CD4+IFN-?+T(Th1)and CD3+CD4+IL-17A+T(Th17)cells.The results showed that the proportion and absolute number of Th1 and Th17 cells in the spleen,lung and mediastinal lymph nodes of NK cells depleted group were significantly lower than those of the control group at day6 and day 12.Indicating that NK cells promote the immune response of Th1 and Th17 cells in local(lung)and immune organs(spleen and mediastinal lymph nodes).2.3 NK cell-depletion lead to significant increase proportion and absolute number of Tregs:To observe the effect of NK cell removal on Tregs response in mouse lung infection,intracellular cytokine staining and low cytometry analysis the proportion and absolute number of(CD4+CD25+Foxp3+T)Tregs.The results showed that the proportion and absolute number of Tregs in the lung,spleen and mediastinal lymph nodes of NK cells depleted mice were significantly higher than control mice,especially in the early stage of chlamydial infection(6 days).It was proved that NK cells had inhibitory effect on Tregs response in Chlamydial infection.especially in the early stages of infection(6 days).3.Effect of NK cell on Th1/Treg,Th17/Treg balance:3.1 NK cells promote Thl and Th17 cell differentiation but inhibit Tregs differentiation:Real-time quantitative PCR detect the expression of T cell subsets specific transcription factors in mouse spleen and lung cells.The results showed that the expression of T-bet(Th1)and ROR?T(Th17)was decreased and the expression of Foxp3(Tregs)and GATA3(Th2)was increased in NK cells depleted mice.3.2 NK cell depleted mice shown lower IL-12p40,IL-6 and higher TGF-?expression:The expression of T cell subsets differentiation related cytokines was observed in the supernatant of local lung and mediastinal lymph nodes cells by ELISA.The results showed that the secretion of IL-12p40(Th1)and IL-6(Th17)was decreased and the expression of TGF-?(Tregs)was increased in the lung and lymph node cells of NK cells depleted mice.3.3 NK cell depletion leads to imbalanced Th1/Treg and Th17/Treg responses:The absolute number of T cell subsets was analyzed and the Th1/Treg and Th17/Treg ratios were calculated.The results showed that the removal of NK cells resulted in decreased Th1/Treg and Th17/Treg ratios in the lung,spleen and mediastinal lymph nodes at day6 after chlamydial infection.At day 12,the ratios of Th1/Treg and Th17/Treg was low in the isotype control group,and the ratio of NK cell depleted group was still significantly lower than that of the control group.Conclusions and Significance1.NK cells play an important protective role in BALB/c mice against chlamydial pulmonary infection.NK Cells contribute to BALB/c mice to clear the chlamydial in the lungs,reduce lung tissue pathology and accelerate disease recovery.2.We demonstrated that NK cells can inhibit Tregs responses and help to enhance the protective immune response in chlamydial infection.The results are consistent in the the local infection site of(lung)and lymphoid organs(spleen and lymph nodes).3.NK cells can inhibit the Tregs response,promoting Thl and Th17 responses,and maintain the immune balance of Th1/Treg and Th17/Treg,which is beneficial to the rapid and effective chlamydial infection and reduce the pathological damage.The above study systematically studied the immunoregulatory effect of NK cells on CD4+T cell subsets in chlamydial pulmonary infection,especially revealed the significant inhibitory effect of NK cells on Tregs response for the first time.We found NK cells play an important protective role in the host defense against chlamydial pulmonary infection by maintaining Th1/Treg and Th17/Treg balance.This study provides a new insight into the mechanism of NK cell immune regulation.Part two:Natural killer cells regulate pulmonary macrophage polarization in chlamydial lung infectionObjectiveMacrophages are important innate immune cells,which play an important role in maintaining immune homeostasis and resisting pathogen infection.Macrophages have a strong plasticity.and polarization state is the key to play a different function,Macrophages have at least two different polarization state,classically activated Ml macrophages and alternatively activated M2 macrophages.M1 macrophages can be activated by IFN-?.PAMPs.TNF? and so on.M1 produce proinflammatory cytokines and overexpress iNOS.thus contributing to clearing pathogens.On the other hand,M2 was activated by Th2 type cytokines such as IL-4,DAMPs and TGF-?,characterized by the relatively high expression of arginase-1,YM-1 and CD206 which are involved in promoting phagocytosis and clearance of parasites,participate in immunomodulation and promote tissue remodeling and matrix precipitation the repair or remodeling of tissues.Similar to Th1/Th2 polarization,the conversion between macrophages M1 and M2 phenotype is also controlled by specific transcription factors.It is known that molecules such as C/EBP-?,PU.1,NF-?B(P65/P50),Statl,IRF5 and HIFa are involved in the regulation of Ml macrophage activation;Stat3,Stat6,IRF4 and SOCS1 Signaling molecules are involved in the regulation of macrophage M2 type polarization.The polarization mechanism of macrophages is complex and has been the focus of recent research.Some research works showed that macrophages involved in host defense and clear pathogen infection,and its role in the control of chlamydial infection has a certain understanding.M1 macrophages can limit the proliferation of chlamydial in the cell,and enhance the innate immune and adaptive immune response through cytokines secretion,which will help the body to quickly remove the infection;Chlamydial can grow normally in the M2 macrophages and further infect other organs.Therefore,induction of modest M1 macrophage response is conducive to clear chlamydial infection.NK cells can act on cytokine microenvironment and interact with other immune cells to control their cytotoxicity or positive immune regulation of macrophages and play a different role in different pathogen infections.In chlamydial lung infection,wheher NK cells can regulate macrophages,especially lung local macrophages are not clear.MicroRNAs(miRNAs)are endogenous non-coding RNAs that involved in the regulation of gene expression at the post-translational level.miRNAs can regulate the transcription of key molecules in macrophage polarization and influence macrophage polarization.Whether or not miRNAs are involved in NK cell-induced macrophage polarization in chlamydial infection is still unclear.This study aimed to investigate the effect of NK cells on pulmonary macrophage polarization and its possible mechanism in chlarmydial lung infection.BALB/c mice were treated with anti-asialo GM1 antibody to specifically eliminate NK cells in vivo,The expression of miRNAs and their target molecules in lung macrophages were detected.The macrophage polarization status and the expression of miRNAs and their target molecules in lung macrophages were analyzed.Methods1.Purification and flow cytometry analysis of macrophages1.1 Lung macrophage purification:BALB/c mice inhale chlamydial intranasally,experimental group as same as the first part.Mice were sacrificed on day 6 after infection.Lung tissue was digested with collagenase ?(10 mg/ml)and centrifuged at percoll density gradient to prepare mononuclear cells.Anti-F4/80 magnetic beads antibody was used to isolate and purify lung macrophages.1.2 lung macrophage purity analysis:5 × 105 purified lung macrophages were used for analysis the percentage of F4/80+CD11 b+cells by flow cytometry.2.Effects of NK cells on macrophage polarization2.1 Effects of NK cells on the expression of polarized cytokines in lung macrophages:The total RNA was extracted from lung macrophage of isotype control and NK cell depleted mice.The mRNA expression of TNF-?,IL-6(M1-type cytokines)and IL-10(M2-type cytokine)were detected by real-time quantitative PCR.2.2 Effects of NK cells on the polarization of M1/M2 macrophages:The mRNA and protein level of M1(iNOS.IRF5)and M2 macrophage markers(Arg-1,IRF4)were detected by real-time quantitative PCR and western blot.The ratio of iNOS/Arg-1 and IRF5/IRF4 were calculated according to the relative expression level of mRNA and protein.2.3 Effects of NK cells on the percentage of M1 and M2 cells in lungs:The percentages of M1(CD45+F4/80+iNOS+)and M2(CD45+F4/80+CD206+)were detected in lung mononuclear cells by flow cytometry.3.The mechanism of NK cells regulate macrophage polarization3.1 NK cells affect the expression of miR-155 and miR-223:The lung macrophages were isolated and purified by magnetic beads at day 6 after chlamydial infection from isotype control and NK cells depleted mice.Total miRNA was extracted and reverse transcribed into cDNA using miR-155 and miR-223 specific reverse transcription primers.The expression of miR-155 and miR-223 was detected by real-time quantitative PCR.3.2 Effects of NK cells on miRNA target expression:Total RNA was extracted from lung macrophages,and real-time quantitative PCR was used to detect the expression of SOCS1(target of miR-155)and pknoxl(target of miR-223).Results1.NK cells promote M1 but inhibit M2 macrophage polarization in chlamydial infection1.1 Lung macrophage purity:We isolated purified lung macrophages by using anti-F4/80 magnetic beads.Flow cytometry analysis results showed that the percentage of F4/80+CD11b+ macrophages was more than 96%after magnetic bead sorting.1.2 NK cells promote M1 cytokines but inhibit M2 cytokines in lung macrophage:The expression of TNF-?,IL-6(M1-type cytokine)and IL-10(M2-type cytokine)mRNA in lung macrophages on the sixth day after Chlamydial infection were detected by real-time quantitative PCR.The results showed that the expression of TNF-? and IL-6 in lung macrophages was significantly decreased and the expression of IL-10 was significantly increased in NK cell depleted mice.1.3 NK cell depletion leads to decrease iNOS/Arg-1 and IRF5/IRF4 ratios in chlamydial lung infection:Real-time quantitative PCR and western blot were used to detect the expression of iNOS,Arg-1,IRF5 and IRF4 in lung macrophages or lung tissues.The results showed that the expression of iNOS and IRF5 decreased slightly and the expression of Arg-1 and IRF4 was significantly increased in NK cell depleted group in both mRNA and protein levels.Furthermore,NK cell depletion leaded to the significantly decreased ratios of iNOS/Arg-1 and IRF5/IRF4 both in nRNA and protein level.1.4 NK cells depletion induced increased M2 but decreased M2 macrophages in chlamydial lung infection:was used to the percentage of M1 and M2 macrophages in the lungs of mice were analyze by flow cytometry.The results showed that the proportion of M1 cells(CD45+F4/80+iNOS+)was significantly lower(P<0.05).and the proportion of M2 macrophages(CD45+F4/80+CD206+)significantly increased in the lungs of NK cells depleted mice.2.miR-155 and miR-223 involved in NK cell-induced pulmonary macrophage polarization during chlamydial lung infection2.1 NK cells depletion induced increased miR-223 but decreased miR-155 expression in lung macrophages during chlamydial infection:Real-time quantitative PCR was used to detect the expression of miRNAs in lung macrophages in isotype control mice and NK cells depleted mice after chlamydial infection.The results showed that the expression of lung macrophages miR-155 was significantly decreased and miR-223 was significantly higher in NK cell-depleted mice than those in control group.2.2 NK cells depletion induced increased SOCS1 but decreased pknoxl in lung macrophages during chlamydial infection:The expression of SOCS1(target of miR-155)and pknox1(target of miR-223)was detected by Real-time quantitative PCR.The results showed that NK cells depletion induced increased SOCS1 but decreased pknox1 expression in lung macrophages during chlamydial infection.Conclusions and Significance1.In chlamydial lung infection,NK cells have a regulation effect on the polarization of lung macrophages.NK cells can induce M1 macrophage polarization but inhibit M2 macrophage polarization.2.NK cell depletion induced decreased mir-155 and increased expression of its target SOSC1.but increased expression of miR-223 and decreased expression of its target pknox1 in pulmonary macrophages of C.muridarum infected mice.These results suggested that miR-155.miR-223 and their target molecules are involved in NK cell-induced pulmonary macrophage polarization process.The findings of current study provide the first report,to our knowledge,on the immunoregulatory effect and possible mechanism of NK cells in macrophages polarization in chlamydial lung infection.We found that miR-155 and miR-223 are involved in the regulation of pulmonary macrophage polarization.Our findings provide a new experimental basis for elucidating the immune regulation between NK cells and lung macrophages.