Il - 9 ~ +, Cd4 ~ + T Fine/il - 9 And The Research Of H. Pylori Infection And Gamma Delta T Cells In The Role Of The Host Response Of Staphylococcus Aureus Pneumonia

Helicobacter pylori （H. pylori） is a helix-shaped, Gram-negative bacterium found inthe stomach and duodenum. Recently, it has been well identified that H. pylori can causeacute or chronic gastritis, even peptic ulcers and gastric cancer in human. It is subdividedinto cagA-positive （type Ⅰ） and cagA-negative strains （typeⅡ）. In most clinical cases,atrophic gastritis is caused by typeⅠH. pylori and is highly associated with gastric cancer.The cytotoxin associated protein （CagA） is delivered into host gastric epithelial cellsthrough type IV secretion system, and phosphorylated, and then it activates downstreamcell signal pathways and causes the gastric epithelial cells shaped into hummingbird cellsand more severe infectious symptoms. The infected epithelial cells release large amounts ofcytokines and chemokines and induce the the host’s cell immune responses. It’s proved thatCD4^<sup>+T cells play important roles in H. pylori infection. IL-9is a T cell growth factor found inTh2cell lines by Uyttenhove in1988. IL-9can function as either a positive or a negativeregulator of immune responses. In2011, Stephens et al found that IL-9secreted by Th17cells can indirectly inhibit Th17cells’ immune activities through stimulating proliferationof mucosa mast cells. They propose that whether IL-9functioning as a proinflammatory orantiinflammatory cytokine is depended on the microenviroment caused by the diseases.Recently, a lot pf papers have reported the dual functions of IL-9. On the one hand, IL-9promotes inflammatory and allergy through activating macrophage, mast cell andeosinophilic granulocyte; on the other hand, IL-9negatively reguates inflammatory inducedby viruses and inhances nTreg cells’ immune inhibition activity. In a mouse model ofasthma, Arnold reveled that H. pylori infection can reduce the severity of asthma byinducing iTreg cells. It showed that H. pylori infection may have a negative correlation withasthma. In summary, CD4⁺T cells are involved in the host’s immune response, but itremains unclear whether IL-9is involved in the immune response and what regulateryfunction it plays.In this study, we use H. pylori/B0strain to infect wide type （WT） BALB/c mice and IL-9knock-out （IL-9ko） mice, in order to investigate the relationship between IL-9⁺CD4⁺Tcells/IL-9and H. pylori infection. It will provide new guides and thoughts for the study ofIL-9⁺CD4⁺T cells’ roles in the natural process of H. pylori infection.Methods1.Cytokine test in blood samples of H.pylori infected personsSerum samples of clinical donors were collected and divided into H.pylori infectionpositive and negative groups based on the results of H.pylori-specific antibody test. Thenthe IL-9protein levels in the serum samples were tested by ELISA.IL-9detection in H.pylori infected mouse model（1） Establishment of H.pylori/B0infected BALB/c and IL9ko mouse model.（2） IL-9detection in H.pylori/B0infected mouse model.3. Preliminary function study of IL-9in H.pylori infected mouse model（1） Bacteria loads detection in stomach of mouse model.（2）Inflammation scoring and analysis of histopathological changes （H.E staining） ofgastric tissue.4.Cell resources of IL-9in H.pylori/B0infected mouse model（1） Inducing differentiation of IL-9-producing T cells in peripheral blood of H.pyloriinfected persons.（2）Inducing differentiation of IL-9-producing T cells in mouse spleen.Results1.Serum IL-9levels were significantly higher in H.pylori-negative group than inH.pylori-positive group.2.Compared to uninfected control group, bacteria loads in stomachs of H.pyloriinfected WT mice were increased at any time points after infection.It indicated thesuccessful establishment of H.pylori/B0strain infected mouse model. On day28, mRNAexpression levels of IL-17, especially of IFN-γ and IL-10,were significantly increased inH.pylori infected group than control group.While the mRNA expression level of IL-9issignificantly lower in infected group.3.Bacteria loads in stomachs of H.pylori infected BALB/c mice reached maximum onday28after infection, and maintained high a week before and after this day, and then began to decrease on day35. Bacteria loads in stomachs of H.pylori infected IL-9ko mice thaninfected BALB/c mice and also reached peak on day28. Pathological changes of stomachsections under the same experiment condition were analyzed and it shows thatinflammatory cell infiltration in laminae propria was higher in IL-9ko group than WTgroup.4.IL-9⁺CD4⁺T cells’percentage in PBMC of H.pylori infection negative and positivepersons was1.9％and0.914％, respectively. The result was validated in mice, too.IL-9⁺CD4⁺T cells’percentage in mononuclear cells of mice splenocytes was0.7％and0.31％, respectively. When adding the cytokines into the PBMC or mononuclear cells ofH.pylori infection for unspecific activation, IL-9⁺CD4⁺T cells’percentage were notablelyincreased.ConclusionsBoth mRNA expression and cellular production of IL-9were decreased in H.pyloriinfected persons and mice. In H.pylori/B0infected WT and IL9ko mouse model, bacterialoads of H.pylori/B0were negatively related to inflammatory injury.In host immune to H.pylori/B0strain, CD4⁺T cells decreased IL-9secretion indicatedthat IL-9⁺CD4⁺T cells could inhibition of host Th9cell immune response. Staphylococcus aureus （S.aureus） is a flagellum, non-endospore, non-capsulated,Gram-positive, extracellular bacterium. The shape under the microscope is spheroidal, thediameter is0.8μm, and thyrsiform. Staphylococcus aureus is important pathogenicbacterium causing that a lot of infections ranging from skin and soft tissue infections,including pneumonia, osteomyelitis, septic arthritis, bacteremia, endocarditic and cellulitis.S.aureus-induced pneumonia accounts for25.5%of community-acquired pneumonia and20-50%nosocomial pneumonia, which could cause especially severe pulmonary infectionand is associated with high morbidity and mortality. Pathogenicity of S.aureus isprincipally depended on multiple toxin and invasive enzyme, such as plasma-coagulase,Staphyolysin, Leukocidin, Enterotoxin, Epidermolytic toxin, Toxic shock syndrome toxin1,Staphylokinase. In recent years, S. aureus infections and methicillin susceptible S. aureus（MSSA） caused community-acquired methicillin-resistant S. aureus （CA-MRSA）. It couldresist all kinds of human Beta-lactam antibiotics and clinical treatment was became toodifficult.In innate immune responses, according to the different structure of TCR’s doublechain, Tcells are divided into αβ T cells and γδ T cells. γδ T cells are Lower expression inhuman and animal,and are distributed mainly over skin, respiratory passages, alimentarytract, genital system. Majority γδ T cells surface do not express adaptor molecule of CD8and CD4through MHC I and MHC II molecule self recognition. γδ Tcell is worked as abridge between inherent immunity and acquired immunity, which have function such asimmune surveillance, immunoloregulation, Tumor cell recognition. In host immune, γδTcell play an important role as the first protector against the entry of microorganisms.According to the different cytokine secretory, γδ Tcell are divided into Vγ1⁺cell andVγ4⁺cell subsets.Staphylococcus aureus can revoke innate immune responses in host, the entry ofmicroorganisms induce abundant neutrophil to the site of infection for removal of pathogenic bacteria. In spite of γδ Tcells in lung tissue were occupied a small part of innateimmune cell, γδ Tcell worked as the first protector against the entry of microorganisms.According to the study, k.pneumoniae, Mycobacterium tuberculosis, streptococcuspneumoniae induced pulmonary infection, γδ Tcell can decrease the host immuneprotection function and lead to more serious inflammatory lung injury. γδ Tcell play animportant role in Staphylococcus aureus-induced skin disease. In recent years, the role ofimmune response and mechanism of γδ Tcell has not been reported in the lung tissueinflammation of Staphylococcus aureus infection.In order to study the effect of γδ Tcells in acute Staphylococcus aureus pneumonia, inthis work, we establish the Staphylococcus aureus strains infection model of pneumonia inC57BL/6mice and TCR-δ^-/-mice. In order to γδ Tcells as the research object, we havepreliminary study on Staphylococcus aureus pneumonia gamma delta T cells of the hostimmune response and mechanism research.Methods:1.Establishment of Staphylococcus aureus infected C57BL/6and TCR-δ^-/-micemodel.2. Detection index in lung tissue2.1The total number of bacterial load evaluation.2.2Lung tissue pathological section hematoxylin-eosin staining （hematoxylin-eosin,HE）.2.3To detect the expression of cytokines in lung tissue of mice Real-time by PCRmRNA.2.4Flow cytometry were used to study the cytokine.Results:1. In S.aures/75strain infection pneumonia of mice lung tissue, γδ Tcells are increaseand the number of V γ4⁺cell subsets aremore than V γ1⁺subsets.2. TCR-δ^-/-mice can lead to host the bacterial clearance severity weaken and acutelung injury.3. Infection of S.aures/75, in the TCR-δ^-/-mice infected site, showed neutrophilrecruitment was inhibited and cytokine production to reduce the phenomenon. 4. S.aures/75strain infection can be localized by γδ Tcell immune response in the lungtissue of mice induced by early WT, production of cytokines IL-17. But in the TCR-δ^-/-mice infected group, lack of γδ Tcells leading to the generation of IL-17deletion.5. Lack of γδ T cells caused by inhibition of neutrophil recruitment.Conclusions:1. In the lung tissue infected with S.aures/75strain, γδ Tcells increased inflammatoryinjury of lung tissue and helped the bacterial clearance in the host.2. γδ Tcells are a major source of cytokines IL-17, and S.aures/75strain infection inearly may influence neutrophil recruitment.