Investigating the Effect of Cytokines on the Proliferation and Metabolism of Intraepithelial Lymphocytes

Abstract

The gut is exposed constantly to invading bacterial populations and dietary antigens. To protect it from harm, a complex and diverse network of patrolling immune cells can be found spread throughout the gut and in specialized lymphoid structures. One immune cells type playing a key role in the protection and homeostasis of the gut, are the intraepithelial T-lymphocytes (IELs). This population of T cells, reside within the epithelial layer of the gut and are primarily known for their protective role against invading pathogens, but when overactivated these cells can also be harmful to the intestine (as exemplified by inflammatory bowel disease). A feature specific to these cells is their semi-activated state that distinguishes them from other immune cells in the circulation. This semi-activated state is characterized by expressing a mixture of different factors, from activation markers to effector proteins (such as granzymes), as well as by its high expression of inhibitory receptors and of a state of metabolic arrest. In this study I evaluated the role that different gut-inflammation related cytokines (e.g. IL-15, IL-18, IL-21, IL-33) can have on the activation of IELs, with a specific focus on the effect of these cytokines on IEL mitochondrial regulatory mechanisms. I used flow-cytometry to identify changes in cell size, proliferation and survival; the MitoQC mouse model, that expressed a mitochondria-targeted fluorescent mitophagy reporter, to measure changes in the rate of mitophagy; Oxygraphy to assess mitochondrial respiratory capacity; and finally immunoblotting to evaluate expression of specific mitochondrial proteins. Overall findings showed that IELs have a better respiratory capacity compared to LN lymphocytes, and more fission related mitochondria-shaping proteins (Drp1, Fis1 & Mff). Furthermore, when exposed to the cytokine IL-15, IELs show increased of the fusion-related isoform of the mitochondrial protein of Opa1 (s-Opa1), that may potentiate mitochondrial function. Finally, I showed that while the addition of IL-18 or IL-21 on their own had minimal effects on IELs, the combination of IL-15/18/21 in IELs resulted in highly increased cell growth, production of IFN-γ and reduction in mitophagy, along with an additional reduction in fission related proteins (Mff & pdrp1(s616)). These findings highlight the potential of cytokines to drive IEL activation, at least to some extent through their effects on mitochondrial homeostasis.

Date of Award	2022
Original language	English
Supervisor	Mahima Swamy (Supervisor)