The Anti-Inflammatory Effects of INSL5 in a DSS-Induced Model of Ulcerative Colitis

Abstract

Insulin-like peptide 5 (INSL5) is a novel gastrointestinal (GI) hormone, primarily expressed in the distal colon and rectum. Its cognate receptor is the G-protein coupled receptor (GPCR) relaxin family peptide receptor 4 (RXFP4), which is expressed in a variety of tissues and cells, including the vagus nerve, colon, liver, heart, kidney, and gonads. Current research suggests that INSL5 is involved in satiety, glucose homeostasis, colonic propulsion, the male and female reproductive systems, and the progression of cancer. Our lab has recently found RXFP4 expression in all murine immune organs and some innate immune cells, implying that RXFP4 and therefore INSL5 may be involved in the immune system. Due to the high levels of INSL5 in the distal gut, it may play a homeostatic role in conditions of chronic inflammation, such as inflammatory bowel disease (IBD). The gut is intimately linked to the spleen via the spleen-gut axis and it is well documented that hyposplenism (splenic dysfunction) is correlated with GI disorders. The first objective of this study is to determine if DSS treatment induces inflammation in the spleen. The second objective is to determine if INSL5 influences immune cell responses in the murine spleen. I hypothesize that dextran sodium sulfate (DSS)-induced colitis will also induce hyposplenism. Additionally, I hypothesize that INSL5 exerts anti-inflammatory effects via RXFP4 expressed on splenic macrophages in a mouse model. To test these hypotheses, eight C57BL/6 strain male mice aged eight to twelve weeks were exposed to 2% DSS in their drinking water for seven days, while eight mice of the same strain and age were given regular drinking water as a control. Following euthanasia, blood samples were taken, the spleens were extracted, weighed, and some sections were kept for histological or molecular analysis while the rest were dissociated into RPMI cell culture media to generate primary culture of splenocytes. Histological preparations of whole spleen were stained with Prussian blue to test for thrombosis while blood smears were analysed for the presence of Howell-Jolly bodies and hypochromic erythrocytes, markers of hyposplenism. RNA was extracted from splenic macrophages of eight control and eight DSS-treated mice for qPCR analyses, and RNA from the spleens of four control and four DSS-treated mice was sent for RNA-sequencing. For the transcriptomic analysis, k-means clustering was performed to identify gene clusters associated with the largest standard deviation in gene expression across samples and the list of genes falling into each cluster were tested for enrichment using the GO Biological Process database. Subsequently, differentially expressed genes (DEGs) were identified from the transcriptomic data using an FDR<0.1 and log fold change > 1.5; the list of DEG’s was used for a gene enrichment analysis using the EnrichR database. Meanwhile, the aliquots of dissociated splenocytes were enriched for macrophages and then exposed to one of four treatments; control (no treatment), lipopolysaccharide (1.0 μg/ml LPS from Escherichia coli per well for 24 hours), co-treatment (100 nM INSL5 and 1.0 μg/ml LPS for 24 hours), and INSL5 pre-treatment (100 nM INSL5 hormone for 12 hours followed by 1.0 μg/ml LPS for 24 hours). Cytokine profiles and other macrophage markers were measured via qPCR and ELISA assay. Our histological, hematological, and qPCR results showed that DSS-induced colitis resulted in hyposplenism via the spleen-gut axis. According to the RNA seq data, DSS-treatment resulted in the upregulation of 146 genes and the downregulation of 21 genes, most of which were associated with the immune system or erythrocyte development. The results of the experiment on macrophage enriched splenocytes indicated that INSL5 played a minor role in alleviating inflammation by reducing the expression of some pro-inflammatory cytokines. However, the results of the cell culture experiment from the DSS-exposed murine spleens indicated that they experienced hyposplenism too extreme for LPS or INSL5 treatment to cause any significant change in immune gene expression. Unfortunately, the ELISA data did not show any significant effect of INSL5 in the spleen, however this may be a result of human error. Overall, our results indicate a strong relationship between the disorders of the gut and the spleen, however INSL5 appears to have a minimal role on immunological role of the spleen. Rather, the RNA-Seq data showed that DSS played a greater role in affecting its erythropoietic function. Further research is warranted, especially into the role of INSL5 in the gut, where it is most highly expressed, and where data mining indicate that its cognate receptor RXFP4 is found on diverse immune cell subsets. Research into the immunological role of INSL5 may be helpful for treating GI disorders such as ulcerative colitis.