Investigation of GS1 and GS2 C. concisus adaptation in the human gastrointestinal tract and their effects on PD-L1 expression in human intestinal epithelial cells

Abstract

Campylobacter concisusis a Gram-negative bacterium with a singular polar flagellum and curved morphology that enables it to traverse the mucus layer and directly contact the intestinal epithelium, providing the opportunity to trigger intestinal inflammation and facilitate the progression of inflammatory diseases to cancer. This thesis reports novel findings regarding the adaptation of different genomospecies of C. concisus to the human gastrointestinal tract and the potential for C. concisus to affect the PD-1 and PD-L1 pathway and thus the development of inflammatory-driven cancer.

C. concisus was previously reported to be associated with inflammatory bowel disease (IBD). It has two genomospecies (GS) that carry distinct and specific genes, suggesting they may have different pathogenic potentials. Previous studies have found that GS2 C. concisus strains are invasive to intestinal epithelial cells, suggesting that they may contribute to the initiation of IBD. However, no clinical studies to date have demonstrated GS2 strains to be more associated with IBD.

In Chapter 2, the colonization of the human gastrointestinal tract by GS1 and GS2 C. concisus is systematically examined in patients with IBD and in healthy controls. This chapter provides novel information regarding the adaptation of different genomospecies of C. concisus to the human gastrointestinal tract. Two new PCR methods were developed and validated for detecting and quantifying C. concisus strains, based on polymorphisms of the 23S rRNA gene. These PCR methods were used to evaluate GS1 and GS2 C. concisus prevalence in 56 oral and enteric samples. Quantitative PCR showed significantly higher level of GS2 C. concisus than GS1 C. concisus in samples collected from the upper and lower gastrointestinal tracts of both patients with IBD and healthy controls, indicating that GS2 C. concisus is better adapted to the human gastrointestinal tract. A meta-analysis of the compositions of GS1 and GS2 C. concisus strains isolated in previous studies was also conducted, and showed similar findings except that a significantly lower number of GS2 strains were isolated from faecal samples of healthy individuals; this suggests a potential difference between healthy controls and patients with gastrointestinal diseases in either colonizing strains or the enteric environment.

Overall, the human gastrointestinal tract harbours a complex community of bacteria, some of which are pathogens capable of establishing chronic infections. These microbes may be involved in the development of inflammation-driven cancer. The immune system uses various effector cells and molecules to control and eradicate infectious agents and cancer cells. In particular, cytotoxic T cells (CTL) are major effector cells in anti-tumour immune responses. However, the functions of these effector cells can be inhibited by immune checkpoint proteins and molecules in tumour environments; this inhibition contributes to cancer cell immune evasion. Accordingly, the blockade of immune checkpoint proteins and molecules, such as PD-1 and its ligand PD-L1, has shown great promise in cancer treatment.

Previous studies have shown that some microbes capable of establishing chronic infections in humans, such as Helicobacter pylori, also increase host PD-L1 and PD-1 expression on both mRNA and protein level; these microbes might be involved in the development of inflammation-driven cancer through assisting immune evasion of cancer cells. However, whether other bacterial species associated with chronic inflammation in the human gastrointestinal tract have effects on the PD-1 and PD-L1 pathway remains to be investigated.

In the case of C. concisus, its curved morphology and flagellum enable it to pass through the intestinal mucus layer and contact intestinal epithelial cells. Previous studies have shown that colonization of the intestinal tract by C. concisus can induce epithelial production of proinflammatory cytokines such as IFN-γ and TNF-α. Interestingly, IFN-γ and TNF-α were previously reported to play important roles in upregulating PD-L1 mRNA and protein expression in intestinal cells. Evidence of T cell infiltration at the jejunum and colon after infection with C. concisus in mice suggests that C. concisus might be involved in the development of cancers through shaping interactions between PD-1 and PD-L1.

In Chapter 3, the effects of GS1 and GS2 C. concisus strains on PD-L1 expression in human epithelial cells are investigated. Briefly, the expression levels of PD-L1 mRNA and protein in human epithelial cells were analysed after C. concisus infection. The results suggest that human epithelial cells do express PD-L1 mRNA but barely expressed PD-L1 protein on their surfaces. Treatment of these cells with IFN-γ can induce PD-L1 protein expression. Also, some C. concisus strains can regulate PD-L1 expression at the mRNA level, but effects depended upon strain rather than genomospecies. In contrast, PD-L1 protein expression was not statistically different for different infecting strains or genomospecies.

In conclusion, this thesis reports novel findings regarding the adaptation of different genomospecies of C. concisus to the human gastrointestinal tract. Furthermore, it provides information regarding the effects of different genomospecies of C. concisus on PD-L1 expression in human intestinal epithelial cells. These data may provide a better understanding of the pathogenicity of this opportunistic bacterium.