IL-10 Downregulates CD44 Expression And Hinders The Development Of Effector Memory Th1 Cells In BCG-vaccinated Mice

Background:Tuberculosis(TB)is caused by Mycobacterium tuberculosis(M.tb).TB has many manifestations,affecting bone,the central nervous system,and many other organ systems,but it is primarily a pulmonary disease that is initiated by the deposition of M.tb,contained in aerosol droplets,onto lung alveolar surfaces.Approximately one third of the world’s population is infected with M.tb.TB remains one of the world’s top 10 causes of death from infectious disease.According to the WHO,there were about 10.1 million new TB cases worldwide in 2017,including 5.8 million men,3.2 million women and 1 million children.Bacillus calmette-guerin(BCG)is the only vaccine that can be used to prevent TB in humans,but it has no protective effect on adults.Therefore,improvement of the protective efficiency of BCG vaccine is the urgent task for TB prevention.Whether immune memory is successfully developed by a vaccine is crucial for prevention against infectious diseases.Effector memory T(TEM)cells quickly response to the re-exposure to antigens,and play a key role in a recall immune response in the local tissues.Therefore,promotion of TEM cells might be an attractive strategy to improve the vaccine efficacy.Objective:We aim to investigate the factors that influence the development of memory cells during BCG vaccination.In the current study,we focus on IL-10 effects on development of CD4+TEM cells in the mice immunized with BCG.Methods:The IL-10-/-(or WT)mice were immunized with BCG,the CD4+memory T cells in spleens and lungs were measured by flow cytometry(FCM).In some experiments,BCG-immunized WT mice were injected with pcDNA-sIL-l0Ra to block the IL-10 in vivo,and then the CD4+memory T cells were determined.The IL-10,IFN-y,IL-4 and IL-17A production by CD4+T cells from BCG-immunized mice were determined by FCM.RT-qPCR,western blot,ELISA and FCM were used to measure the CD44 expression on CD4+T cells.RNA second-generation sequencing were employed to look for the candidate genes involved in CD44-mediated regulation of CD4+memory T cells.RT-qPCR and Western blot were used to validate the candidate genes and molecules.Results:The higher levels of CD4+TEM cells in spleen and lung were increased in BCG-immunized IL-10-/-mice than those in BCG-WT mice.IFN-y production by CD4+T cells were increased by re-exposure to BCG in BCG-IL-10-/-mice compared with BCG-WT mice.These results indicate that IL-10 hindered the development of CD4+TEM cells,especially BCG-specific Th1 memory immune response.After 30 days of BCG immunization,the IL-10 production by CD4+T cells in WT mice reached its peak,indicating that IL-10 production by CD4+T cells might contribute to downregulating the development of CD4+TEM cells.Treatment with pcDNA3.1-sIL-10Ra or anti-IL-10Ra Ab to block IL-10 function enhanced the CD4+TEM cells in spleens and lungs from BCG-immunized WT mice.These results further confirmed that IL-10 hindered the development of CD4+TEM cells.The CD44 expression on CD4+T cells was upregulated in BCG-IL-10-/-mice.During BCG immunization,pcDNA3.1-sIL-10Ra treatment did not promote CD4+T cells to product IFN-γ in CD44-/-mice upon re-exposure to BCG.These results indicated that IL-10 downregulated the IFN-γ Th1 memory cells via CD44.IL-10 might promote the CD44 degradation via enhancement of Lys48(K48)-linked ubiquitination of CD44.Using RNA second-generation sequencing,RT-qPCR and Western blot,proteoglycan(Prg2)was identified as a related molecule to both IL-10 and CD44.Both IL-10 and CD44 deficiency downregulated the Prg2 expression.The ACTG(actin-γ)then was identified as Prg2-binding protein by pulldown and liquid chromatography-mass spectrometry(LC-MS)/MS analysis.Conclusion:In the current study,we demonstrated that IL-10 inhibited the development of BCG-induced CD4+TEM cells,especially IFNγ+Th1 memory cells.The suppression of BCG-induced IFNγ+Th1 memory cells depended on IL-10-mediated downregulation of CD44 expression.IL-10 might promote the ubiquitin-proteasome system pathway to degrade CD44 molecule.The Prg2 and ACTG might be involved in IL-10-mediated and CD44 dependent regulation of IFNy+Th1 memory cells,but the mechanism needed to be clarified in the future study.