Recent studies have revealed that antibiotic treatment compromises the innate immune system of the intestinal mucosa by depleting commensal microbes that normally stimulate epithelial cells to produce homeostatic levels of AMPs [9
]. Here, we asked whether the TLR5 ligand flagellin could restore innate immune deficits caused by treatment with broad-spectrum antibiotics. We show that flagellin administration induces RegIIIγ in epithelial cells along the entire length of the small intestine of antibiotic-treated mice. The extensive upregulation of RegIIIγ was likely caused by systemic stimulation of TLR5 and secretion of IL-22. In contrast, RegIIIγ expression is limited to the ileum under steady-state conditions due to the higher density of bacteria in the distal small intestine compared to more proximal regions [9
]. Our results also show that TLR5-expressing hematopoietic cells mediate flagellin-induced RegIIIγ expression. In accordance, we found that IL-22 is also required for the response, suggesting that flagellin-stimulated hematopoietic cells produce IL-22, which signals intestinal epithelial cells to express RegIIIγ. Consistent with previous studies demonstrating that oral LPS induces RegIIIγ in the small intestine and enhances resistance to VRE infection, we demonstrate that systemically administered flagellin reduces susceptibility to VRE colonization in antibiotic-treated mice.
Although we have previously demonstrated that antibody-mediated blockade of RegIIIγ significantly diminishes in vivo killing of both VRE and Listeria monocytogenes
], RegIIIγ may not solely mediate flagellin’s protective effects. RegIIIβ is also upregulated in response to flagellin administration. Although RegIIIβ has no reported bactericidal activity, it binds peptidoglycan and may contribute to flagellin-mediated protection against VRE [9
]. Flagellin induces several cytokines that may promote VRE clearance. In addition, MyD88-mediated signals in the gut promote repair and maintenance of the mucosal barrier [11
]. Together these responses may also contribute to flagellin-mediated resistance to VRE colonization.
Both hematopoietic and non-hematopoietic cells of the intestine respond to flagellin via the TLR5-MyD88-mediated pathway [23
]. In the case of hematopoietic cells, the high level of TLR5 expression in the small intestine has been attributed to a subset of CD11chi
lamina propria dendritic cells important for IgA production and Th17 development [22
]. Given that RegIIIγ expression under normal conditions depends on direct detection of commensal bacteria by intestinal epithelial cells via the TLR-MyD88-mediated pathway [9
], we were surprised to find that TLR5 activation of hematopoietic cells is required for flagellin-mediated RegIIIγ expression. Our results using systemic flagellin administration support an alternative pathway of RegIIIγ induction. IL-22-mediated RegIIIγ expression induced by the presence of TLR ligands within subepithelial tissues, as opposed to stimulation from the luminal side of the epithelium, may play a critical role in alerting epithelial cells to the loss of mucosal integrity or presence of systemic infection. While RegIIIγ expression regulated by apical TLR stimulation is tightly controlled [18
], IL-22 expression may be more indiscriminant with respect to stimulation by a variety of microbe-derived molecules once the epithelial barrier has been breached [29
]. Further work is needed to identify the hematopoietic cell subsets responsible for flagellin-induced RegIIIγ expression. The candidates for the source of IL-22 include Th17 cells, γδ-T cells, NK cells, and lymphoid tissue-inducer cells [34
The protective effects of flagellin against numerous challenges, including lethal irradiation, chemical damage, and infectious agents, have been well described [19
]; however, the mechanism of TLR5-mediated protection has remained undefined. Prophylactic systemic administration of a TLR5 agonist results in dramatic survival after lethal irradiation [21
]. High-dose ionizing radiation causes massive cell loss in the hematopoietic system and intestinal mucosa, which leads to invasion by commensal bacteria and fatal septicemia [39
]. NFκB activation protects against lethal irradiation by initiating anti-apoptotic pathways in radiosensitive tissue [41
]. The radioprotective effects of flagellin are thought to be mediated by this mechanism [21
]; however, based on our study, it is also possible that RegIIIγ expression induced by TLR5 activation bolsters the impaired mucosal barrier by directly killing invading bacteria.
The therapeutic use of TLR ligands has been approached cautiously due to the potential to stimulate undesired inflammatory responses. Most efforts to manipulate the immune system have targeted the adaptive arm, with a focus on enhancing long-term immunity. The innate immune system, however, can exert rapid and broad defense against invading organisms, and, if properly timed, might be exploited as an approach to ameliorate several clinical problems. For example, TLR ligands have been used as adjuvants in a variety of vaccines including two hepatitis B virus vaccines which use TLR4 agonists to induce a robust memory response [42
]. One challenge, however, to the therapeutic use of flagellin is that repeated administration induces antibodies that eventually block TLR5 activation [43
]. Therefore, targeting TLR5 might require the development agonists that do not stimulate neutralizing antibody responses.
Moderate stimulation of TLR5 has not been shown to induce severe sepsis; however, this does not exclude the possibility that flagellin administration may induce inflammation. In controlled trials, flagellin has been administered to humans with little side effects [44
]. In spite of this, it is possible that certain subsets of patients will experience adverse reactions to flagellin treatment. Flagellin-mediated IL-22 induction may exacerbate skin plaques in psoriasis patients [47
]. Also, stimulation of TLR5 is suspected to play a role in the pathogenesis of inflammatory bowel disease [48
]. Thus, the therapeutic use of flagellin will require extensive clinical study with a particular focus on potential complications resulting from accentuated inflammatory responses.
The commensal flora of the gut plays a critical role in the development and maintenance of a healthy intestinal mucosa. Treatment with broad-spectrum antibiotics greatly diminishes the intestinal microbial flora leading to increased susceptibility to a variety of bacterial infections, including VRE and Clostridium difficile
]. The depletion of commensal bacteria results in diminished innate immune defenses, most notably RegIIIγ, due to reduced activation of Toll-like receptors [9
]. Our experiments provide further evidence for the critical role of TLRs in RegIIIγ expression and for the ability of TLR activation to re-establish AMP expression which antibiotics have impaired. Our results suggest that flagellin may have therapeutic potential and may prevent intestinal invasion with resistant microbes in patients treated with broad-spectrum antibiotics. RegIIIγ induction along the small intestine may provide an approach to restrict potentially pathogenic bacteria in the intestinal lumen, thereby limiting colonization and dissemination, both within and between individuals.