Effect Of Interleukin-7 On Abnormal Bone Marrow Hematopoiesis Induced By Persistent Mycobacterium Tuberculosis Antigen Stimulation

Part 1 Effect of interleukin-7 on abnormal bone marrow hematopoiesis induced by persistent Mycobacterium tuberculosis antigen stimulationHematopoietic stem cells and hematopoietic progenitor cells generate immune cells such as lymphocytes to support the anti-infection immunity function through self-renewal,proliferation,and differentiation.When the body is infected,inflammatory cytokines such as IFN-γand TNF-αare produced to regulate the proliferation and differentiation of hematopoietic stem cells.In the early stage of infection,inflammatory cytokines will promote the proliferation of hematopoietic stem cells and promote myeloid differentiation to supplement the monocytes and granulocytes consumed in peripheral blood;however,if the infection persists for too long and cannot be cleared,the long-term effects of inflammatory cytokines Stimulation can lead to dysfunction and even failure of hematopoietic stem cells,resulting in bone marrow hematopoietic dysfunction and a decrease in the number of whole blood cells in peripheral blood.Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis（M.tuberculosis）.Patients with severe tuberculosis show different degrees of lymphopenia.In addition to the death of peripheral lymphocytes caused by M.tuberculosis infection,the mechanism of lymphopenia is also related to the abnormal bone marrow hematopoiesis.Preliminary studies in our laboratory have found that high-dose Bacille Calmette-Guérin（BCG）infection and continuous stimulation of Mycobacterium tuberculosis antigens can lead to increased expression of myeloid differentiation-related transcription factors in bone marrow hematopoietic stem cells and hematopoietic progenitor cells,and decreased expression of lymphoid differentiation-related transcription factors,the number of peripheral blood lymphocytes decreased.IL-2 treatment can promote the proliferation of hematopoietic stem cells,increase the expression of lymphoid differentiation transcription factors,and increase the number of peripheral blood lymphocytes.In order to further understand the regulatory factors of abnormal bone marrow hematopoiesis in severe tuberculosis,this study investigated the regulatory effects of interleukin-7（IL-7）and berberine on lymphopenia and bone marrow hematopoiesis.IL-7 is a key cytokine in lymphocyte development in the bone marrow,involved in bone marrow lymphopoiesis and maintains T cell homeostasis.Berberine is a small molecule drug with broad anti-inflammatory activity,which can effectively inhibit the inflammatory response.Objective:Using the M.tuberculosis antigen stimulation model to study the intervention effect of IL-7 and berberine on Mycobacterium tuberculosis antigen-induced lymphopenia,and to analyze whether the intervention of IL-7 and berberine can inhibit the inflammation caused by persistent tuberculosis antigen stimulation,improve the abnormal differentiation of bone marrow hematopoietic stem cells and hematopoietic progenitor cells,and reverse peripheral blood lymphopenia.Methods:（1）C57BL/6 mice were injected with large doses of BCG（5×10~7CFU/mice）into the tail vein and subcutaneously injected with M.tuberculosis antigens stimulation,which caused low peripheral blood lymphocytes and bone marrow hematopoietic dysfunction.After antigen stimulation,the following indicators were used to reflect the effect of antigen stimulation on bone marrow hematopoietic function and lymphocytes:（1）Detection of the number of lymphocytes in peripheral blood;（2）ELISA to detect the levels of IFN-γ,TNF-αand IL-7 in serum and bone marrow;（3）Bone marrow LK（Lineage^-c-kit⁺）cells were sorted by magnetic beads and the expression of differentiation-related transcription factors Batf2,Irf8 and E2a was detected by RT-PCR;（4）Flow cytometry was used to detect the proportion of lymphoid progenitor cells（Lineage^-c-kit⁺CD127⁺）in bone marrow.（2）The M.tuberculosis antigen-stimulated model was treated with r AAV-IL-7,and r AAV-EGFP was used as a control.The detection index is the same as above.（3）The M.tuberculosis antigen-stimulated model was treated with berberine at doses of 20,40,and 80 mg/kg,and the expression of inhibitory receptor PD-1 on the surface of spleen T lymphocytes was detected by flow cytometry,and the rest of the detection indicators were the same as above.Results:（1）To establish the pathological model of bone marrow hematopoietic hypofunction caused by persistent M.tuberculosis antigen stimulation:high-dose BCG infection and persistent M.tuberculosis antigen stimulation can cause a strong inflammatory response in mice,and the levels of IFN-γand TNF-αin serum increase,and the level of IL-7 decreases,the level of IFN-γin bone marrow increased;the expression of myeloid differentiation-related transcription factors Batf2 and Irf8 in bone marrow LK cells increased,and the expression of lymphoid differentiation-related transcription factor E2a decreased.Compared with the PBS control group（2.29±0.68?）,the proportion of lymphoid progenitor cells in the bone marrow cells in the persistent antigen stimulation group（0.56±0.11?）was significantly lower（p<0.01）.At the same time,compared with the number of peripheral blood lymphocytes in the PBS control group（7.32±0.88×10~9/L）,the number of peripheral blood lymphocytes in the persistent antigen stimulation group（1.67±0.39×10~9/L）was significantly decreased（p<0.01）.（2）Treatment of r AAV-IL-7 on M.tuberculosis antigen stimulation model:r AAV-IL-7 treatment can increase the level of IL-7 in serum,reduce the levels of IFN-γand TNF-αin serum and promote the expression of lymphoid differentiation-related transcription factor E2a in bone marrow LK cells.The proportion of lymphoid progenitor cells in the bone marrow of the r AAV-IL-7 treatment group（4.46±1.40?）was significantly higher than that of the persistent antigen stimulation group（0.56±0.11?,p<0.01）.The number of peripheral blood lymphocytes in the r AAV-IL-7 treatment group（3.64±1.15×10~9/L）was significantly higher than that in the persistent antigen stimulation group（1.67±0.39×10~9/L,p<0.01）.（3）Treatment of berberine on M.tuberculosis antigen stimulation model:40mg/kg berberine treatment can reduce the level of IFN-γin serum and bone marrow,and the ratio of CD4⁺PD-1⁺cells in splenocytes in berberine treatment group（0.94±0.13%）was significantly lower than that in the persistent antigen stimulation group（3.05±0.88%,p<0.01）.The proportion of CD8⁺PD-1⁺cells in splenocytes of the berberine treatment group（0.45±0.12%）was significantly lower than that of the persistent antigen stimulation group（1.34±0.52%,p<0.01）.The expression of myeloid differentiation-related transcription factors Batf2 and Irf8 in bone marrow LK cells decreased,and the proportion of lymphoid progenitor cells in bone marrow increased.The number of peripheral blood lymphocytes in the berberine treatment group（4.32±1.10×10~9/L）was significantly higher than that in the persistent antigen stimulation group（1.67±0.39×10~9/L,p<0.01）.Conclusions:（1）High-dose BCG infection and continuous stimulation of Mycobacterium tuberculosis antigens can induce the increase of IFN-γin the serum and bone marrow of mice,promote the increase of myeloid differentiation transcription factors in the bone marrow LK cells,and the decrease of lymphoid progenitor cells in the bone marrow.The number of peripheral blood lymphocytes decreased.（2）r AAV-IL-7 intervention can promote the increase of serum IL-7,decrease the levels of IFN-γand TNF-αin serum,promote the increase of lymphoid progenitor cells in bone marrow,and increase the number of lymphocytes in peripheral blood.（3）Berberine intervention decreased the levels of IFN-γin serum and bone marrow,decreased the expression of PD-1 in spleen T lymphocytes,and increased the number of peripheral blood lymphocytes.Part 2 Large-scale purification of fusion protein LT29 of Mycobacterium tuberculosisSubunit vaccines against tuberculosis are a class of vaccines constructed by selecting the protective antigens of M.tuberculosis.Compared with attenuated vaccines and inactivated vaccines,protein subunit vaccines have clear components and fewer side effects.Our research group screened the M.tuberculosis antigens ESAT6 and Rv2626 c in the early stage,and fused the two to construct the M.tuberculosis fusion protein LT29,which was passed through a 5ml column volume hydrophobic chromatography column Butyl Sepharose High Performance and an ion chromatography column DEAE Sepharose Fast Flow purification yields a protein with a purity greater than 95%.The immunogenicity of LT29 was evaluated,and it was found that the fusion protein could induce strong cellular and humoral immune responses,and was a good candidate for tuberculosis subunit vaccine.Objective: Based on the small-scale preparation method of M.tuberculosis fusion protein LT29 established earlier,a larger-scale purification system was established to meet the requirements of industrial production of fusion protein.Methods:（1）Exploration of large-scale purification methods:（1）The fusion protein LT29 was expressed by using the Escherichia coli BL21 expression system;（2）For the second step of purification,using the AKTA avant protein purifier,the impurity protein and the target protein were washed with different proportions of PB buffer and high-salt PB buffer after adsorption with a 50 ml column volume of hydrophobic chromatography column Butyl Sepharose High Performance;（3）For the second step of purification,using the AKTA avant protein purifier,the impurity protein and the target protein were washed with different proportions of PB buffer and high-salt PB buffer after adsorption with a 50 ml column volume of hydrophobic chromatography column Butyl Sepharose High Performance;（4）Finally,the target protein was washed with different proportions of PB buffer and high-salt PB buffer after adsorption by ion chromatography column DEAE Sepharose Fast Flow with a column volume of 25ml;（5）The protein purity was determined by SDS-PAGE electrophoresis.（2）Preliminary evaluation of vaccine immune protection efficiency: The C57BL/6 mice were subcutaneously immunized with LT29/DPC three times in the groin at weeks on 0-4-12,and 12 weeks after the last immunization,mice were challenged by tail vein injection of the attenuated M.tuberculosis strain H37 Ra strain（5×106 CFU/mice）.The protective effect of fusion protein LT29 on M.tuberculosis infection was reflected by the bacterial load in the spleen and lung of the mice three weeks after the strain challenge.Results:（1）Escherichia coli BL21 can express the fusion protein LT29 with high efficiency;LT29 was preliminarily purified by salting out with 30% saturated ammonium sulfate;after adsorption on the hydrophobic chromatography column Butyl Sepharose High Performance,the impurities protein were washed with 100% PB buffer,dd H2 O was used to elute the target protein for the second step purification;after adsorption on the ion chromatography column DEAE Sepharose Fast Flow,the target protein was eluted with 80% PB buffer plus 20% high-salt PB buffer for the third step purification.The fusion protein LT29 with a purity greater than 95% can be obtained,and a large-scale purification method has been successfully established.（2）The challenge experiment of the attenuated H37 Ra strain of M.tuberculosis confirmed that the fusion protein LT29 has a certain protective effect.Conclusions: A large-scale purification method for LT29 was successfully established,and the M.tuberculosis fusion protein LT29 with a purity greater than 95% was obtained by three steps of salting out,hydrophobic chromatography and ion chromatography.