The gut microbiome as a target for regulatory T cell-based immunotherapy: induction of regulatory lymphocytes by oral administration of anti-LPS enriched colostrum alleviates immune mediated colitis

The gastrointestinal tract contains a large and diverse population of commensal bacteria and is also one of the primary sites of exposure to pathogens. Intestinal microbes play a role in both the immune system and in maintaining metabolic homeostasis [17]. In a healthy host the immune system is tolerant to gut bacteria and does not mount an effector response to bacteria-derived antigens [18]. Thus, the intestinal immune system is instructed by the microbes to limit responses to luminal antigens [17]. Gut microbes can inhibit the transport of both commensal and pathogenic bacteria from the lumen to the mesenteric lymph nodes (MLNs) [17].

Breakdown of tolerance at the gut level may contribute to the development of IBD [19]. This breakdown may be associated with bowel infection. The compartmentalization of systemic and gut mucosal immunity apparatuses focuses adaptive immunity on gut microbes specifically at the level of the bowel. In circumstances of increased gut microbial exposure due to elevated gut epithelial permeability resulting from genetic deficiencies in local defense mechanisms or during gastrointestinal pathogenic infections, this paradigm is broken and a systemic immune response towards gut microbes is induced [20]. The loss of tolerance to commensals during bowel infections leads to activation of microbiome-specific T cells which differentiate into inflammatory effector cells [19]. These T cells may go on to form memory cells that are pathogen-specific. The response to commensals during infection in the bowel parallels the immune response to pathogenic microbes, suggesting that the adaptive responses against commensals are an integral component of mucosal immunity [19]. In the absence of Myd88 or with antibiotic-induced dysbiosis, non-invasive bacteria were trafficked to MLNs in a CCR7-dependent manner, inducing an inflammatory response [17].

The data of the present study supports a possible correlation between specific E. Coli strains and IBD. Following enterocyte stress, translocation of E. coli strains C25 and HBTEC-1 across Caco-2 monolayers was markedly stimulated and accompanied by an increase in internalization into the enterocytes. The data suggested that E. coli strains capable of translocation may comprise a separate group of E. coli strains [21]. Enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) strains of E. coli use a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection. This system enables bacterial colonization while interfering with the antimicrobial host response [22]. In a recent study it was shown that a T3SS effector NleB1 from EPEC binds to host cell death-domain-containing proteins and thereby inhibits death receptor signaling [22]. NleB1 prevented Fas ligand or TNF-induced formation of the canonical death-inducing signaling complex (DISC) and proteolytic activation of caspase-8, an essential step in death-receptor-induced apoptosis. NleB activity suggests that EPEC antagonize death-receptor-induced apoptosis of infected cells, thereby blocking a major antimicrobial host response [22]. These findings may be of importance for a possible association between specific strains of E. Coli and IBD. Increased gut permeability caused by the disruption of intracellular tight junctions in the intestine correlates with a high prevalence of small intestine bacterial overgrowth [23, 24]. Translocation of normally non-pathogenic bacteria across the gut may be a driving force of inflammatory responses associated with IBD [21]. Translocation may not be purely passive, but may occur via transcellular pathways activated in enterocytes by inflammatory or metabolic stress.

It was suggested that in human patients with a higher than average prevalence of small intestinal bacterial overgrowth there is an increased bacterial translocation [25]. Dysregulation of the intestinal immune response may increase the risk of exposure, something that may lead them to develop Crohn’s disease [25]. Serum lipopolysaccharide-binding protein (LBP) is a marker of disease activity in CD with a similar accuracy as that of the highly sensitive-CRP. LBP served as an independent predictor for 1-year clinical flare-up in these patients [26]. Studies have demonstrated that patients with Crohn’s disease have a shorter bowel length which may predispose them to the development of the disease via alterations of intestinal motility and changes in intestinal flora [25].

In the present study oral administration of hyperimmune colostrum preparations enriched with anti LPS effectively alleviated immune mediated colitis in mice and was associated with a significant induction of CD4 + CD25+ and CD4 + Foxp3+ regulatory T cells in the spleens of treated mice. Promotion of Tregs by oral administration of Imm124-E also improved insulin resistance, liver enzymes, hepatic fat accumulation and serum lipid profiles in mice and in humans [14, 15].

Tregs are critical for maintaining immune homeostasis and establishing tolerance to foreign, non-pathogenic antigens including those found in commensal bacteria and food [27]. Absence of Tregs or defective Treg function correlate with disease severity [28]. Tregs have suppressive mechanisms and control the threshold for peripheral antigen recognition via tonic down-regulation of dendritic cell (DC) co-stimulation. They are also implicated in maintaining the tolerogenic function of DCs [29]. Defects in Treg number or function lower the activation threshold, allowing proliferation and differentiation of self-reactive CD4 cells. Failure to maintain the tolerogenic commitment of DCs exposed to commensal microbes and allergens results in IBD [29]. Tregs were suggested to be beneficial in the context of IBD [27].

The human gut microbiome is important for interactions at both the intestinal level and the colonic epithelial cells level, with dendritic cells, and T and B immune cells [30]. These interactions affect gut barrier and defense mechanisms. Microbiotic composition may determine T effector- and Tregs balance and thus plays a role in the pathogenesis of IBD [30]. Alterations in the gut’s microbiome, acting via Tregs, were responsible for increased incidence of IBD [29]. Treg-based immunotherapy could potentially be custom-designed ad hoc to specifically suit each patient with limited side effects based on their individual microbial signatures [31].

A synergistic effect between anti-LPS antibodies and adjuvants in the colostrum is suggested by the results of the present study. This notion is based on the effect of the colostrums enriched with anti LPS antibodies compared to that of the control group that received antibody-free colosturm. Bovine colostrum (BC) treatment has been shown to be associated with an increased number of Tregs [15, 32, 33]. CD25 expression on peripheral blood monocytes (PBMCs) was enhanced by treatment with BC-derived IL-1β, TNF-β and IFN-γ [34]. Exposure to BC increased the percentage of cells expressing CD11a, CD11c, and CD43 and decreased the percentage of cells expressing CD62L, which facilitates lymphocyte trafficking [33, 35]. BC also contains defensin proteins, osteopontin, exosomes, TLRs, cathelicidin, eosinophil-derived neurotoxin LL-37 and high levels of β-glycosphingolipids (BGS) [15, 33, 36]. Some of these mediators serve as mucosal adjuvants, enhancing the interaction between subsets of antigen presenting cells and Tregs in the bowel mucosa [37, 38].

The present data suggests that oral antibodies have an active effect on the adaptive immune system. These results further support the possibility of altering the adaptive immune system by feeding of antibodies. Oral administration of anti-CD3 both in mice and humans altered the systemic immune response via presentation of antibodies at the gut level [6, 39, 40].

In summary, oral administration of Imm124E promoted Tregs and alleviated inflammation of the bowel in the immune-mediated colitis model. The data supports an association between gut bacterial translocation and Tregs, and suggests that the microbiome may serve as a target for Tregs-based immunotherapy.