The Characteristics And Role Of γδT Cells In Helicobacter Pylori Infection

Helicobacter pylori （H. pylori） are a Gram-negative, microaerophilic bacterium thatspecifically resides in the surface mucous layer of gastric mucosa. Epidemiological datademonstrate that there are more than50%of the population worldwide infected with H.pylori, which may cause chronic active gastritis, leading to peptic ulcer disease,mucosa-associated lymphoid tissue lymphoma and gastric adenocarcinoma. Although H.pylori infection elicits specific humoral and cellular immune responses, the bacteriumpersists and the inflammation continues for decades.T lymphocyte plays a critical role in the pathogenesis of persistent H. pylori infection.Traditionally, two subsets of T cells have been defined: β T cells and γ T cells. To date,studies of immune responses to H. pylori have largely focused on β⁺CD4⁺T helper cells.However, innate immune response of γ T cells to H. pylori infection has yet to beelucidated. γ T cells are a minor T cell subset present in the spleen and lymph nodes, butrepresent a major subset within the epithelia of intestine and skin. γ T cells play animportant role in the recognition of unrestricted major histocompatibility complex （MHC）molecules and contribute to mucosal defense against invading pathogens.H pylori have a particular tropism for the gastric epithelium, and γ T cells are mainlylocalized in mucosal tissues and abundant in the gastrointestinal tract. Thus, the relationshipbetween development and differentiation of γ T cells and the progression of H. pyloriinfection raise great concerns. Previous study reported that the percentage of γ T cellssignificantly increased in the H. pylori positive group in comparison with healthy controls.After H. pylori eradication therapy, the γ T cell count significantly decreased. However,the characteristics and role of γ T cell response in H. pylori infection have not beenelucidated. In addition, the detailed mechanism through which γ T cell regulates andaffects the system immune response is not clear.【Objectives】1. Try to characterize γ T cell responses to H. pylori shortly after infection of a C57BL/6mouse model.2. Aim to elucidate the role of γ T cells in the early phase of H. pylori infection3. To explore the mechanism through which γ T cell regulates and affects the immuneresponse to H. pylori infection4. To analyze human peripheral blood γ T cells response in H. pylori-infected patients【Methods】1. C57BL/6mice were orogastrically challenged with H. pylori and lymphocytes ofspleen, Peyer’s patches（PP）, mesenteric lymph node（MLN） and intestinal intraepitheliallymphocytes（iIEL） were collected at the indicated times post-infection. γ T cells responseswere examined to analyze the characteristics, subsets and cytokine profiles by flowcytometry.2. Wild-type （WT） C57BL/6and γ T cell-deficient （TCR--/-） mice was infected withH. pylori orally. The bacteria load in gastric tissue was measured with real-time PCR todetect the16s rDNA of H. pylori and the gastric inflammation was evaluated by H&Estaining. In addition, γ T cells were expanded from splenocytes in vitro and were i.v.transferred into TCR--/-mice before H. pylori infection to assay the effects of γ T cells onthe colonization and inflammation.3. Wild-type （WT） C57BL/6and γ T cell-deficient （TCR--/-） mice was infected withH. pylori orally. Splenic lymphocytes were collected for analysis of specific Th1, Th2,Th17and Th22response by intracellular staining. Moreover, cytokines, chemokines andMMPs mRNA expression was detected by real-time PCR. Serum IgG and mucosal IgAwere evaluated by ELISA.4. The peripheral blood mononuclear cells were isolated from25H. pylori-positivepatients with chronic antral gastritis and18H. pylori-negative healthy controls. To detectand analyze CD3, TCR γ, IFN-γ, IL-17and IL-22using flow cytometry.【Results】1. The characteristics of γ T cell response to H. pylori infection1.1Following H. pylori challenge, the proportion of γ T cell in the lymphocytes ofspleen, Peyer’s patches, mesenteric lymph node and intraepithelial lymphocytes wassignificantly increased. In the spleen and MLN, γ T cell was expanded from24h pi and reach a peak at48h pi, whereas in the PP and iIEL γ T cell reach its peak at24h pi. Thetime course of γ T cell responses in different organs suggests that PP and iIEL may be twoearlier sites of γ T cell priming during H. pylori infection.1.2Purified spleen CD3⁺T cells were co-cultured with viable H. pylori or fixedbacteria. Only viable H. pylori induce the expansion of γ T cell （P <0.05）. The lack ofCagA or UreB could not show the obvious influences on γ T cell proliferation （P>0.05）.1.3Following H. pylori challenge, γ T cell exhibit increased CD69, CD44and CD25expression （P <0.05）.1.4Analysis of the γ T cell subsets in the spleen demonstrated that Vγ4⁺cells seemedto be responsible for the rapid increase in the population of γ T cell （P <0.05）. In thegastric mucosa, Vγ7were the dominant γ T cell population. After H. pylori infection, theexpression of the Vγ4gene increased.1.5Effect of H. pylori infection on cytokine profiles of γ T cells was investigated,IL-4-, IL-10-and IL-22-producing γ T cells comprise a very small part of γ T cells and nosignificant change was observed after H. pylori infection. IFN-γ-and IL-17-producing γ Tcells were the dominant population. IFN-γ⁺γ T cells did not change significantly （P>0.05）,whereas the percentage of IL-17⁺γ T cells significantly was increased at24h pi and reacha peak at48h, even persisted for the follow-up time points（P <0.01）. At the early stage ofH. pylori infection, IL-17⁺γ T cells were the major source of IL-17. In addition,IL-23-mediated signaling induced IL-17production by γ T cells.2. The protective role of γ T cells in H. pylori infection.2.1The colonization of H. pylori and the inflammation of gastric mucosa in the γ Tcell-deficient were significantly higher than that in the WT mice （P <0.05）, suggesting thatdeletion of γ T cells might impair host defense against H. pylori infection.2.2γ T-cell-transferred mice showed significantly lower copies of H. pylori thanthose from HBSS-treated mice （P <0.05）. In addition, the mean microscopical score of γT-cell-transferred mice were significantly lower compared to HBSS-treated mice （P <0.05）. These results indicate that γ T cells might increase the resistance to H. pyloriinfection.3. The effect of γ T cells on host immune response to H. pylori infection3.1Splenic lymphocytes from TCR--/-mice showed significantly higher Th17and Th22cell responses than those from WT mice when stimulated with H. pylori whole cellprotein or PMA/ionomycin. On the contrary, Th1and Th2cell responses were significantlyattenuated. In addition, at the early stage of infection, expression of IL-17mRNA waslower in gastric mucosa from TCR--/-mice than those of from WT mice, whereas at28d piIL-17mRNA was expressed at a higher level. IFN_-γmRNA was expressed at a lower levelin the gastric mucosa of TCR--/-mice than those in WT mice. These results suggest thatlack of γ T cells might have an effect on specific Th cell responses to H. pylori infection.3.2Decreased expression of neutrophil-inducing cytokine/chemokine in the stomachof infected TCR--/-mice3.3Deletion of γ T cell led to attenuated production of gastric mucosal H. pylorispecific IgA （P <0.05）, but it had no significant effect on the level of serum IgG （P>0.05）.3.4H. pylori infected TCR--/-mice showed significantly increase of CCL2, CXCL1and CCL25mRNA expression compared with those of infected WT mice （P <0.05）.Whereas, CCL28mRNA was expressed at a lower level in comparison with that of infectedWT mice （P <0.05）. Other detected chemokines, such as CCL5, CCL20and CXCL2wasnot significantly changed.3.5Lack of γ T cells led to increased expression of MMP-9mRNA and reducedexpression of MMP-7（P <0.05）. No significant change of other MMPs （MMP-2, MMP-3and MMP-13） was observed. These results imply that γ T cells may exert its effect on theH. pylori infection by enhancing or attenuating expression of chemokines and MMPs.4. The frequency of γ T cells in H. pylori-positive patients was not significantlydifferent compared to those of healthy controls （P>0.05）. However, we observed thatIL-17-producing γ T cells were one important source of IL-17in response to H. pyloriinfection. Moreover, the subsets of IL-17-and IL-22-producing, not IFN-γ-producing γ Tcells markedly increased （P <0.05）.【Conclusion】1. H. pylori infection induced rapidly γ T cells response in the C57BL/6mousemodel. The γ T cells responses occurred earlier in the PP and iIEL than in the spleen orMLN. Vγ4⁺γ T cells were responsible for the increased population of γ T cell. Moreover,IL-17produced by γ T cells was the dominant source of IL-17at the early stage of H.pylori infection. IL-23-mediated signaling induced IL-17production by γ T cells. 2. γ T cells play a protective role in H. pylori infection.3. The role of γ T cells on H. pylori infection might be associated with alteredspecific adaptive immune response and the expression of cytokines/chemokines and MMPs.4. γ T cells might involve human immune response to H. pylori infection via production ofIL-17.【significance】Previous studies largely focused on the adaptive immune response to H. pyloriinfection. Innate immune response to H. pylori infection has yet to elucidate. As a bridgebetween the innate and the adaptive immune system, γ T cells played an indispensible rolein pathogen elimination and immune regulation. A better understanding of thecharacteristics, function and regulation of γ T cells responses to H. pylori infection mayhelp us to explore novel and effective immunotherapies for gastric diseases induced by thisorganism.