The Effect Of Mycobacterium Tuberculosis On Regulating Neutrophil Function And Expression Co-stimulatory Molecules And Its Underlying Mechanisms

Tuberculosis (TB) is a chronic infectious disease and a kind of respiratoryinfection disease, caused by the Mycobacterium tuberculosis (Mtb) infection. SinceTB is mainly a result of tissue damage mediated by the body’s immune system, TBpatients tend to have Th1immune response inhibition, Th2immune response levelhigher abnormal phenomenon，TB is also considered to be an immune disorderdisease. Direction and level of immune response determines largely the developmentand outcome of TB.Neutrophils as an important innate immune effector cells, which is the firstline of defense against pathogenic microorganism infection. Earlier studies aggredthat neutrophils as phagocytes participate in the innate immune response of TB in theearly stages of infection. Nearly studies suggest that neutrophils also participated ininduction and regulation of adaptive immunity,which have important influences onthe development of TB immune.Co-stimulatory molecule is a kind of proteins expressed on immune cells andimmune related cells, which mediated signal is the costimulatory signal.Costimulatory signal regulate immune response is bidirectional, through positive andnegative costimulatory signals to the body to maintain the regulation of immunebalance. Positive costimulatory molecules such as CD28, CD80, CD86, etc. canactivate T cells to proliferation, differentiation into effector cells, enhance immuneresponses, resistance to invading pathogens; Negative costimulatory molecules suchas programmed death factor1(PD-1),Ligand of Programmed death1(PD-L1)inhibited T cell proliferation and cytokine production, inhibition of immune responseand prevent excessive immune injury and autoimmune diseases, help maintain thebody’s immune homeostasis. In recent years, studies have shown that neutrophils alsoexpress costimulatory molecules, suggesting that neutrophils express costimulatorymolecules may impact anti-TB immunity.In this study, comparative analysis of active tuberculosis patients and healthyhuman peripheral blood neutrophils differences in levels of costimulatory molecules and functions to explore TB patients neutrophil expression of costimulatorymolecules and cell function characteristic changes. And validated Mtb have an effecton neutrophil function and expression of costimulatory molecules in vitro by themethod of Mtb infection.On this basis,this study we use specific monoclonal antibodyto block PD-1/PD-L1pathway,and explore the role of negative costimulatory signalsregulating neutrophil function.Part I The function and expression characteristics of co-stimulatory molecule ofneutrophils on the peripheral blood of tuberculosis patientsObjective:To analysis the level of costimulatory molecules and function of TB patients，neutrophils, and explore the relationship between the abnormal immune function andco-stimulatory molecule of TB patients.Methods:Collected30cases of patients with active pulmonary tuberculosis and20casesof healthy peripheral blood, expression levels of costimulatory molecules,ROS andCD62L in neutrophils were measured by flowcytometry;NO production in neutrophilswere measured by nitric acid deoxidize enzyme meathod; Neutrophils chemotacticability of IL-8were measured by Transwell Chamber.Results:Statistical analysis showed that,compared with the healthy control group,peripheral blood neutrophils of TB patients have the following characteristics:(1)Expression of CD86significantly lower(P <0.05) and PD-L1significantlyincreased(P <0.01). But expression of CD28、CD80、PD-1and HLA-DR were nosignificant difference between two groups(P>0.05).(2)After PMA stimulation,the amount of ROS and NO production in neutrophilssignificantly increased(P <0.01).(3) CD62L expression in neutrophils has no significant change(P>0.05).(4) Neutrophils chemotactic ability of IL-8significantly lower(P <0.05).Conclusion:The positive co-stimulatory molecule of CD86expression on neutrophils of tuberculosis patients was significant lower. On the contrary, negative co-stimulatorymolecule of PD-L1was significant higher. Oxidative burst in neutrophils significantlyincreased and chemotactic activity of neutrophils decreased,thus prompt deregulateco-stimulatory molecule expression of neutrophils may affect the function of thecells.Part II Effect of Mtb infection on neutrophil function andexpression of costimulatory moleculesObjective:To analysis the level of costimulatory molecules and function of Mtb infectedneutrophils, and investigate the effect of Mtb infection on neutrophil function andexpression of costimulatory molecules.Methods:Collected20cases of healthy peripheral blood, expression levels of costimu-latory molecules,ROS,CD62L and apoptosis rate in Mtb infected neutrophils weremeasured by flowcytometry; NO production in Mtb infected neutrophils weremeasured by nitric acid deoxidize enzyme meathod; Mtb infected neutrophilschemotactic ability of IL-8were measured by Transwell Chamber;Neutrophilsbactericidal capacity of Mtb was measured by CFU.Results:Statistical analysis showed that, compared with the control group, Mtb infectedneutrophils have the following characteristics:(1)①Expression of CD28、CD80、CD86、PD-1and PD-L1significantlyincreased(P <0.01) But expression of HLA-DR were no significant differencebetween two groups(P>0.05).②Amount of ROS and NO production in neutrophils significantly increased(P<0.01).③Expression of CD62L significantly lower(P <0.01)④Neutrophils chemotactic ability of IL-8significantly increased (P <0.05).(2) MS and Mtb infection can significantly increase the level of neutrophilapoptosis, but compared with MS groups, Mtb infected neutrophils has lower apoptosis rate（P <0.01）。（3）Neutrophils can effectively kill nonpathogenic MS, but not toxic Mtb，which lead to Mtb replicate.Conclusion:1. Mtb infection can effectively activate neutrophils, accompanied by a series ofchanges in the expression of costimulatory molecules, but these changes are related toneutrophil function need for further research.2. Mtb infected neutrophils express costimulatory molecules changes,thesechanges are not inconsistent with TB patient peripheral blood neutrophilscostimulatory molecule expression patterns. After analysis of the specific reasons yetto be determined by detecting neutrophils of infectious location in TB patients.Part III Blocking PD-1/PD-L1pathway influence function ofneutrophils in vitroObjective:To block PD-1/PD-L1pathway in vitro,explore the negative co-stimulationsignal regulate the function of neutrophils.Methods:Collected15cases of healthy peripheral blood. The whole blood were incubatedwith anti-PD-L1monoclonal antibody,thereafter adding Mtb,incubate for30min.Oxidative burst and expression of CD62L in neutrophils were measured byflowcytometry. Neutrophils chemotactic ability of IL-8were measured by TranswellChamber.Results:Statistical analysis showed that,compared with the control group, the amount ofROS production in Mtb combined anti-PD-L1group significantly increased(P <0.05),CD62L expression significantly lower(P <0.05)),chemotaxis of neutrophilssignificantly increased(P <0.05).Conclusion:Oxidative burst,cell activation and chemotactic activity of neutrophils enhancedby blocking PD-1/PD-L1pathway in vitro, thus prompt the negative co-stimulation signal suppress neutrophil anti-TB immunity. This may open a new way of clinicaltreatment of tuberculosis...