Tumor Suppressor Caliban Regulates Intestinal Innate Immune Response To Bacterial Infection

The changes of immune system function play a key role in the occurrence and development of tumors.Currently,it is believed that tumors are formed due to defects in immune monitoring or increased tolerance to cancer antigens^[1].Defects of the immune system not only lead to the occurrence and development of tumors,but also lead to adverse reactions to tumor treatment.In recent years,tumor immunotherapy is a great breakthrough in the field of tumor therapy.Among them,the Immune checkpoint inhibitors（ICIs）,which target the immunoregulatory molecules on the surface of T cells（or their ligands）,enhance the anti-tumor Immune response,and have improved the long-term survival and efficacy in the treatment of tumors^[2].The results of the application of ICIS reveal the ability of the immune system to fight and possibly overcome disease.Therefore,immune-related research has become a hot spot of research at present.The intestinal flora is closely related to the innate and acquired immune response.With the deepening of research,it has been gradually recognized that intestinal flora plays an important role in tumor immunotherapy through innate and adaptive immunity^[3].Recent studies have shown that the intestinal flora affects the anti-tumor efficacy of ICIs^[4-5].And in some studies,it has been found that changes in intestinal flora can be used to predict the efficacy of tumor immunotherapy^[6],suggesting that it may become a new target for predicting the efficacy of tumor immunotherapy.We currently have some understanding about the influence of intestinal flora on tumors and immunity.However,the relevant mechanism of how intestinal flora affects intestinal immunity is currently unclear,and further research is still needed.The Caliban（clbn）gene of Drosophila has carried out a lot of basic research in our group.Studies have shown that as a nuclear export mediator of the transcription factor Prospero,Caliban of Drosophila melanogaster is homologous with sdccag1（seriologically defined colon cancer antigen 1）or NY-CO-1（New-York colon 1）protein and has the function of tumor suppressor in human non small cell lung cancer（NSCLC）^[7].Studies have shown that Drosophila clbn and human sdccag1 are excellent candidate genes for gene therapy of lung cancer and other possible human cancers^[9].Subsequently,it was found that clbn knockout Drosophila was highly sensitive to irradiation,and tumor-like black massive tissue appeared after irradiation,which was considered as a manifestation of congenital immunodeficiency^[10].Recent studies have found that clbn knockout flies have shorter lifespan,intestinal injury and abnormal proliferation of intestinal stem cells^[11].These evidences suggest that clbn gene may be involved in immune regulation.Drosophila,as a model organism for studying intestinal innate immunity,has unparalleled advantages.Because the reproductive cycle of Drosophila is short,and their mucosal structure and immune signals are highly conserved with those of mammals,so the mechanism of intestinal immunity in mammals can be inferred through the study of Drosophila.The immune deficiency（IMD）pathway provides the main local defense for the intestine of Drosophila.The diaminopimelic acid（DAP）type peptidon（PGN）of gram-negative bacteria is recognized by the PGN recognition receptor-LC（PGN recognition receptor-LC,PGRP-LC）on the cell membrane or PGN recognition receptor-LE（PGRP-LE）on the cytoplasm,which initiates the IMD signaling cascade and translocates Relish into the nucleus.Finally,it leads to the production of antimicrobial peptides（AMPs）^[12].In this study,we investigated whether tumor suppressor Caliban is involved in the regulation of Drosophila innate immune response and its potential molecular mechanism.First,by detecting the intestinal bacterial load of Drosophila under physiological conditions,it was found that compared with wild type Drosophila,the intestinal bacterial load of clbn mutant Drosophila was significantly increased.Then,after feeding Drosophila with pathogenic bacteria Ecc-15,the intestinal infection model was established.After dissecting the intestinal tract,the intestinal pathogen load level of Drosophila was detected.It was found that the intestinal pathogen load of clbn mutant Drosophila was significantly higher than that of wild type Drosophila.Next,the pathogenic bacteria Ecc-15 was used to feed Drosophila,and the intestinal tract was dissected.RT-q PCR was used to detect the expression level of antimicrobial peptides in the intestinal tract of Drosophila clbn mutant,and it was found that the expression level of antimicrobial peptides in the intestinal tract of Drosophila clbn mutant was higher than that of wild type Drosophila.In addition,after feeding Drosophila with pathogenic bacteria Ecc-15 again and dissecting the intestinal tract,RT-q PCR method was applied to detect the expression levels of related genes in the natural immune IMD signaling pathway of Drosophila intestines.It was found that compared with wild type Drosophila,the expression level of negative regulatory gene PGRP-LB upstream of natural immune IMD signaling pathway of clbn mutant Drosophila was decreased.Finally,pathogenic bacteria Ecc-15 were used to feed Drosophila,and the intestinal tract was dissected.RT-q PCR was used to detect the expression levels of related genes in the TNF signaling pathway in the intestinal tract of Drosophila,and it was found that the clbn mutant flies eiger gene expression changed.Therefore,this study suggested that,the intestinal bacterial load and the expression level of antimicrobial peptides were significantly increased after clbn mutation infection in Drosophila,suggesting that clbn gene may be involved in the intestinal immune process of Drosophila.After infection with clbn mutation,the expression level of PGRP-LB,a negative regulator of IMD signaling pathway,was decreased and the expression of TNF signaling pathway was altered,suggesting that these may be ones of the mechanisms by which clbn gene participates in the intestinal immune process of Drosophila.All these indicate that the regulation of innate immune IMD signaling pathway requires the involvement of clbn,and clbn could be necessary to prevent the overactivation of IMD signaling pathway.