Bacteriophage Expansion Linked to Exacerbation of Colitis
by Deborah Vennesa Subbiah
The genetic composition of bacteriophages of the gastrointestinal microbiota differs significantly in healthy controls compared to individuals diseased with inflammatory bowel disease (IBD). A striking distinction occurs in the order of bacteriophages, Caudovirales, within the intestine of Crohn’s disease (CD) and ulcerative colitis (UC) patients. Although not directly infecting mammalian host cells, these viruses possess a range of molecules that may be implicated in stimulation of the immune system. However, the capacity of bacteriophages to stimulate the mammalian immune system and the concurrent effect on mammalian disease remains indefinite.
The mammalian gastrointestinal tract (GIT) is colonized with microorganisms from all three domains of life that have sizeable influence on health and immunity. Within the domains, a greater quantity of the microbiota remains unclassified. However, while viruses of the intestine boast members directly infecting eukaryotic cells, they are predominantly made up of bacteriophages who infect and parasitize bacterial cells. Bacteriophages are genetically highly diverse and are estimated at 1031 on the planet, exceeding bacteria almost tenfold in many ecosystems. Bacteriophages inhabit the mucosal layer of the intestinal tract as free phage virions. This phenomenon has been shown in several studies to promote optimal intestinal barrier function, conferring protection against invasive bacteria. Alterations to the normal gut microbiota, often referred to as dysbiosis, are suspected to be the cause of various diseases such as colorectal cancer and IBD. Based on this and the rapid evolution of antibiotic resistance, phage modulation of bacterial responses has resurfaced as a viable option that is being currently studied in various clinical and experimental settings as a treatment option for diseases associated with the gut microbiota.
To carry out the study, fecal samples from an IBD clinic belonging to individual’s diagnosed with Crohn’s disease were obtained. Escherichia coli NC101 was used as an enrichment host to isolate the bacteriophages. Bacteriophages were subjected to a series of experiments which included flow cytometry, RT-qPCR, and ELISA to obtain the data.
The activation of the immune system by bacteriophages is context specific and physiologically relevant. Many animal models report that in infectious settings, targeting of specific bacteria by bacteriophages can favorably reduce bacterial burden and hence improve disease. In this study, E. coli-specific bacteriophages substantially increased the survival of animals predisposed to the development of colorectal cancer by markedly reducing colonization by carcinogenic bacteria. This was additionally demonstrated in APCmin mice treated with bacteriophages in the absence of E. coli, where tumor burden showed no reduction, suggesting that in this setting, immunity stimulated by bacteriophages is not adequate to mount potent anti-tumor immunity. Furthermore, the study found that the expansion of bacteriophages during chronic inflammatory diseases may be detrimental. This was supported by recent reports stipulating that the abundance of bacteriophages within the order Caudovirales was significantly increased in patients suffering from CD. Therefore, given that bacteriophages can confer distinctive functions to bacterial populations, it may be interesting to postulate that diseased individuals exhibit distinctive bacteriophage populations. A recent study found an enrichment of Enterobacteriaceae-specific phages in mice with colitis, proving this theory.
Moreover, the existence of bacteriophages inflates intestinal immunological responses in a TLR9-dependent way, suggesting that these organisms may be functionally relevant during intestinal illness. In agreement with the findings, a positive correlation between the production of mucosal IFN-y and the total viral abundance was identified. Furthermore, in vitro studies revealed that virus-like particles (VLP) set apart from actively diseased UC patients elicited robust IFN-y responses. IFN-y is produced by multiple cells including CD8+ T-cells, natural killer T-cells (NKT), natural killer cells (NK) and innate lymphoid cells (ILC). Phage-treated germ-free (GF) mice demonstrated an expansion in CD8+ T-cells, implying that multiple cell types are influenced by bacteriophages. As most of these cells express TLR9, it is likely that bacteriophages activate these cells in a TLR9-dependent pathway, contributing to the exacerbation of colitis. Incidentally, individuals that failed to respond to a fecal microbiota transplant (FMT) were significantly enriched with bacteriophages of the order Caudovirales further reinforcing that Caudovirales bacteriophages may indicate the necessity for additional maintenance FMT delivery or therapy escalation in the event of FMT failure.
The data generated from this study is particularly relevant to human health as bacteriophage virions populate the intestinal mucus in copious amounts where they can be sampled by immune cells. The study highlights how bacteriophages could be a double-edged sword in the gut’s inflammatory milieu. Because bacteriophages promote immunity in part through TLR9, inhibiting this pathway during phage therapy may reduce inflammatory responses to the phage. Furthermore, the specificity of bacteriophages for their bacterial targets and the influence this may have on the mammalian host governs the need for future work to understand the nature of these complex interactions and how they can be manipulated for therapeutic benefit.
Gogokhia, L., Buhrke, K., Bell, R., Hoffman, B., Brown, D., & Hanke-Gogokhia, C. et al. (2019). Expansion of Bacteriophages Is Linked to Aggravated Intestinal Inflammation and Colitis. Cell Host & Microbe, 25(2), 285-299.e8. doi: 10.1016/j.chom.2019.01.008