Investigating the role of 5-HT and MCH in the regulation of human immune response

Yusoff, Syaratul Dalina (2020). Investigating the role of 5-HT and MCH in the regulation of human immune response. University of Birmingham. Ph.D.

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Abstract

The involvement of neurotransmitters and neuropeptides as mediators in the dysregulation of the immune response leading to conditions such as chronic inflammation, autoimmune diseases and haematological cancers has been reported. Indeed, different types of innate and adaptive immune cells have been shown to express neurotransmitter/neuropeptide receptors on their surface membrane and some are even able to produce the neural factors under different conditions. This project was designed to investigate the role of the monoamine serotonin (5-HT) and also that of the orexigenic neuropeptide melanin-concentrating hormone (MCH) in human immune cell functions, specifically T lymphocytes, one of the key players in adaptive immune and inflammatory responses. To investigate the effects of agonist and antagonists targeting 5-HT receptors on the CD4\(^+\) and CD8\(^+\) T cell response to stimulus, human peripheral blood mononuclear cells were stimulated to induce proliferation in a serum-free environment. It was observed that inhibition of 5-HT synthesis enzyme tryptophan hydroxylase 1 (TPH1) with para-chlorophenylalanin (pCPA; 0.1-1mM) suppressed proliferation of both CD4 and CD8 T lymphocytes with significant inhibition observed at 1mM (p<0.01), yet somehow cell survival was maintained in stimulated cultures. However, the addition of 0.1mM 5-HTP was not able to reverse the suppressive effect of pCPA. Next, we demonstrated that exogenous 5-HT (10nM – 10\(\mu\)) has no impact on neither CD4 nor CD8 T cell proliferation. Stimulated PBMCs when treated with 5-HT (10nM-10\(\mu\)M) has the same cell survival compared with control. Treating the PBMCs with the 5-HT2A/2C receptor antagonist, ritanserin or the 5-HT7 receptor antagonist SB285719 had no effect on proliferation

We then assessed MCHR1 receptor expression in immune cell lines Jurkat and K562 using Western blot. Both cells showed an MCHR1 protein band at 55kDa comparable to that in the neuroblastoma cell line SH-SY5Y. In normal human PBMC, we saw a two-fold increase in MCHR1 receptor expression in activated CD3 T lymphocytes while CD19 B lymphocytes exhibited a two-fold decrease (n=3, p<0.01) in expression level upon stimulation as detected by flow cytometer. The differential expression was significantly observed in naïve CD8 T lymphocytes (n=5, p<0.05) where MCHR1 has upregulated in the CD69\(^+\) population. MCH-treated, isolated naïve T cell subsets had a non-significant increase in percentage of proliferated cells which was not seen in PBMC culture. However, MCH (100nM) failed to induce ERK1/2 signalling protein phosphorylation in Jurkat cells endogenously expressing MCHR1.

Immunohistochemistry analysis on inflamed colonic tissue sections obtained from four IBD patients (3 Crohn’s, 1 ulcerative colitis) were compared with non-inflamed control tissue, and results showed there to be MCHR1 immunoreactivity on colonic epithelial cells as well as on some mucosal infiltrating immune cells. A few of those cells were CD11c\(^+\) while CD14\(^+\) cells do not co-express MCHR1. Using the pfaffl method, real time RT-qPCR displayed elevated pMCH gene transcripts but not MCHR1 and MCHR2 in IBD colon tissue samples. Overall the results indicate that 5-HT appears not to have a significant influence in regulating the activity of T lymphocytes while MCH displays a possibly under-appreciated role in specific subsets of immune cells which could be related to the pathogenesis of inflammatory bowel diseases.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):