The Role Of NMAAP1 In Promoting M1 Polarization Of Macrophages And The Antitumor Effect Of BCG

Bacillus Calmette–Guérin（BCG）can be used in immuno-infusion therapy for patients with non-muscular invasive bladder cancer,greatly improving survival.It was discovered that while T and B cells had no effect on BCG’s tumor killing effect,eliminating macrophages resulted in the disappearance of BCG’s anti-tumor effect,indicating that macrophages could not be replaced in BCG’s anti-tumor immune mechanism.Macrophages are extremely dynamic and can be classified into various subgroups.It primarily focuses on M1 and M2 activation modes.Interferon-γ（IFN-γ）and bacterial Lipopolysaccharide（LPS）activate M1-type macrophages,allowing them to play an anti-infection and tumor-killing role.Some studies have shown that BCG can activate macrophages to M1 type via TLR2 and TLR4,thereby killing tumors,but the mechanism is still unknown.Early research teams discovered that BCG activated macrophages（BAM）expressed a new molecular,which we call new macrophage activation associated protein 1（NMAAP1）.This protein had 547 amino acids,and data analysis predicted that it contained nearly 90%of the Mab-21 domain sequence.NMAAP1 has been linked to several diseases,including colon cancer,glioma and non-small cell lung cancer,Alzheimer’s disease,and autism.Current research indicates that macrophages overexpressing NMAAP1 are polarized towards the M1 type,with increased expression of M1 genes（i NOS,TNF-α,IL-6,IL-12,IL-1β）and decreased expression of some M2 genes.In addition,NMAAP1 can significantly improve macrophage phagocytosis,implying that NMAAP1 can promote macrophage adhesion and clearance activities.However,the role of NMAAP1 in BCG’s promotion of M1macrophage differentiation and antitumor effect remained unclear.Research purpose:The effect of NMAAP1 on M1-type macrophages stimulated by BCG was studied both in vivo and in vitro.Lys M^Cre+/-NMAAP1^Flox/Flox conditional knockout mice（called CKO mice）and Lys M^Cre-/-NMAAP1^Flox/Flox control mice were used in this study（called Flox mice）.Research methods:1.The absence of macrophage NMAAP1 inhibits the antitumor effect of BCGIn order to explore the effect of NMAAP1 on tumor growth in the process of BCG promoting macrophage polarization,CKO and Flox tumor-bearing mice received three intraperitoneal injections of BCG at D7,D17,and D19,Mouse tumor tissues were obtained and weighed at D28,and H&E staining was done.2.The effect of NMAAP1 loss in macrophages on the number of T and B cells in mouse lymph nodes and spleen2.1 In order to investigate the effect of NMAAP1 deletion on B cells,CD4⁺T cells and CD8⁺T cells in lymph nodes and spleen,flow cytometry was used to detect the number of above immune cells in lymph nodes and spleen.2.2 To investigate the effects of NMAAP1 on B cells and T cells in the lymph nodes and spleen of mouse peripheral immune organs in a tumor setting,the cells in peripheral immune organs of tumor-bearing mice were subjected to flow cytometry analysis.3.Effect of NMAAP1 on BCG’s promotion of macrophage M1 polarization3.1.In the first part of the results,we have observed that the absence of NMAAP1 can inhibit the anti-tumor growth effect of BCG stimulating macrophage production,but how NMAAP1 affects the polarization of macrophages in this process is hard to ascertain.Therefore,flow cytometry and immunofluorescence staining of macrophages in tumor tissues of tumor-bearing mice were performed.3.2.To measure whether the absence of NMAAP1 affects BCG stimulation of BMDM towards M1 polarization in vitro,BCG was used to stimulate BMDM in CKO and Flox mice,and macrophage polarization was detected by flow cytometry,q PCR,and ELISA,respectively.3.3.To determine whether the absence of NMAAP1 affects the tumor killing effect of BCG stimulation on BMDM,we added the BMDM culture supernatant of BCG stimulation on NMAAP1 deletion into MCA207 cell culture system,and detected the death of tumor cells by lactate dehydrogenase（LDH）kit.Research results:1.The absence of macrophage NMAAP1 inhibits the antitumor effect of BCGAfter NMAAP1 deletion,the mice’s tumor weight considerably increased.Following BCG stimulation,the CKO group had greater tumor weights than mice in the Flox group.H&E staining of tumor tissues showed that the structure of tumor cells was not complete after BCG stimulation,the cells shrank,and local cell degeneration and necrosis could be seen,among which the inflammatory cell infiltration was reduced compared with the control group.2.The effect of NMAAP1 loss in macrophages on the number of T and B cells in mouse lymph nodes and spleen2.1 Under physiological settings,the conditional deletion of macrophage NMAAP1 had no influence on the number of T and B cells.Flow cytometry showed that the number of B cells,CD4⁺T cells,and CD8⁺T cells in the lymph nodes and spleen were unaffected by the absence of NMAAP1.2.2 The impact of macrophage NMAAP1 loss on the number of T and B cells in mouse lymph nodes and spleen under tumor-bearing conditionFlow cytometry revealed no notable changes in the B cell and CD8⁺T cell infiltration in the lymph nodes and spleen.Moreover,the proportion of CD4⁺T cell infiltration in lymph nodes of Flox+BCG group was higher than that of Flox+NS group,but there was no significant difference in CD4⁺T cell infiltration in lymph nodes of CKO+NS group and CKO+BCG group.Meanwhile,the proportion of CD4⁺T cells in spleen and lymph nodes of Flox+BCG group was higher than that of CKO+BCG group.3.Effect of NMAAP1 on BCG’s promotion of macrophage M1 polarization3.1.NMAAP1 deletion reduces the number of M1 macrophages in mouse tumorsFlow cytometry showed that the proportion of MHC-II labeled M1 macrophages in Flox mice was higher than that in CKO mice after BCG injection,while the proportion of M2 macrophage marker CD206 mice showed no significant trend.At the same time,immunofluorescence results of tumor tissues also found that the proportion of F4/80⁺MHC-II⁺macrophages in Flox mice after BCG stimulation was much higher than that in CKO mice,on the contrary,more F4/80⁺CD206⁺macrophages were observed in tumor tissues of NMAAP1 knockout mice.3.2.NMAAP1 loss inhibited BCG-induced BMDM polarization to M1.The gene level results revealed that the expressions of Tnf-α、Il-1β、i NOS and other pro-inflammatory factors were down-regulated in the absence of NMAAP1,while the gene expression of anti-inflammatory factor Il-10 was up-regulated,but there was no significant effect on Tgf-βand Arg-1.Besides that,we also found the expression of TNF-αand IL-1βdecreased at the protein level.3.3.NMAAP1 loss inhibited BCG-stimulated BMDM killing of MCA207 tumor cellsThe results showed that NMAAP1 loss reduced the amount of LDH released in the supernatant of tumor cell culture,indicating that NMAAP1 loss would inhibit the effect of BCG stimulating BMDM to kill MCA207 tumor cells.Research conclusion:1.NMAAP knockout in macrophages inhibited the antitumor effect of BCG.2.NMAAP knockout in macrophages does not affect T cells and B cells in lymph nodes and spleen of mice under physiological condition,but only affects CD4⁺T cells under tumor-bearing condition.3.NMAAP1 may regulate BCG’s promotion of macrophage polarization to M1type and play a role in killing MCA207 fibrosarcoma cells.