The first, and arguably best, opportunity for the immune system to limit infection by orally acquired microbial pathogens is in the gastrointestinal tract. In this study, we demonstrate that mice deficient for MyD88 have a diminished capacity to kill L. monocytogenes in the lumen of the distal small intestine, a region that expresses high levels of antimicrobial peptides and proteins. MyD88-deficient mice express normal complements of cryptdins in the intestinal mucosa, but markedly diminished levels of RegIIIγ, a bactericidal lectin that selectively kills Gram-positive bacteria. We show that antibody- mediated depletion of RegIIIγ from the intestinal lumen of wt mice compromises killing of L. monocytogenes to an extent similar to that seen in MyD88−/− mice. Complementing MyD88-deficient mice with recombinant RegIIIγ, on the other hand, enhances intestinal killing of L. monocytogenes to nearly wt levels. These results strongly support the conclusion that RegIIIγ is a major effector molecule in defense against oral L. monocytogenes infection. Furthermore, these findings demonstrate, for the first time, that MyD88-mediated signals are essential for the induction of RegIIIγ, and that these innate immune signals mediate killing of micro organisms before invasion of the intestinal mucosa.
Several models can be proposed for MyD88-mediated defense against L. monocytogenes
infection in the small intestine. The simplest model is that L. monocytogenes
invades the intestinal epithelium, in the process triggering TLRs in the gut mucosa and inducing synthesis and secretion of RegIIIγ into the bowel lumen and subsequent microbial killing. We believe this model is unlikely for several reasons. First, we see high levels of RegIIIγ associated with the bowel mucosa in uninfected wt mice ( and not depicted), suggesting that commensal bacteria have induced a high level of expression before infection with L. monocytogenes
. Second, induction of RegIIIγ mRNA is minimal after intestinal infection with L. monocytogenes
(). Third, killing of attenuated L. monocytogenes
strains that are incapable of invading the intestinal epithelium and wt bacteria is similar, indicating that bacterial invasion is not required for the MyD88-mediated enhancement of L. monocytogenes
killing. An alternative model, therefore, is that MyD88-mediated signals, presumably triggered by commensal organisms, induce RegIIIγ production in the distal small bowel, thereby conditioning the bowel and enhancing resistance to subsequent exposure to pathogenic bacteria. In support of this model, previous studies have demonstrated that commensal bacteria induce RegIIIγ in the distal ileum, and germ-free mice have markedly reduced expression of RegIIIγ (24
). Not all intestinal bacteria are equivalent at inducing RegIIIγ, however. The Gram-negative bacterium B. thetaiotaomicron
induces expression of RegIIIγ in intestinal epithelial cells, whereas the Gram-positive bacterium B. longum
suppresses RegIIIγ expression (25
). These findings suggest that resistance or susceptibility to enteric L. monocytogenes
infection may be highly influenced by the composition of the commensal microbial flora.
MyD88- and RegIIIγ-mediated killing of pathogenic microbes in the intestinal lumen is likely to have beneficial consequences for the mammalian host. Indeed, clear correlations between the likelihood of systemic infection and the size of the bacterial inoculum have been demonstrated for most intestinal bacterial pathogens. Although it is conventional to think that microbes disseminating from the bowel transit through regional MLNs before entry into the blood stream, recent studies suggest that bacterial replication in the bowel lumen results in direct hematogenous spread of bacteria to spleen and liver (37
). A mechanism that kills bacteria in the gut lumen, thus, could be predicted to significantly reduce the risk of bacterial dissemination. Although RegIIIγ likely provides such a mechanism, it is not the only protective mechanism. For example, the probiotic bacterium Lactobacillus salivarius
UCC118 expresses a bacteriocin that kills L. monocytogenes
in the gut lumen, thereby limiting systemic infection (38
). Our finding that luminal clearance of L. monocytogenes
was normal in mice lacking both TNF and IFN-γ, two cytokines that individually play essential roles in systemic immunity to this pathogen and are positively regulated by MyD88-mediated signals, illustrates how distinct mucosal and systemic innate immune defenses can be.
Nod2-deficient mice are more susceptible to intestinal L. monocytogenes
infection, a phenotype that has been attributed to diminished cryptdin expression in the mouse intestine (17
). Our results demonstrating that Rip2-deficient mice have normal clearance of L. monocytogenes
from the lumen of the distal small intestine () suggest that Nod2/Rip2-mediated antimicrobial activities are either temporally or anatomically distinct from those mediated by MyD88/RegIIIγ. Further studies will be required to precisely define the timing and localization of Nod2/Rip2-mediated inhibition of L. monocytogenes
dissemination from the mouse intestine.
Several recent studies have demonstrated that intestinal dendritic cells can extend dendrites into the intestinal lumen and, in some circumstances, can also phagocytose bacteria and carry them to spleen and liver (34
). Because dendritic cells express a broad array of innate immune receptors, it is plausible that MyD88-mediated signaling in BM-derived intestinal DCs might contribute to the induction of RegIIIγ in the small intestine. Recent studies have also demonstrated a role for MyD88-mediated signaling in epithelial regeneration (15
) after treatment with DSS. In this system, MyD88-mediated signals in macrophages underlying the epithelium (40
) promote the repositioning of intestinal mesenchymal cells and their focal production of prostaglandin E2, which enhances the activity of colonic epithelial progenitor cells (41
). Our experiments with BM chimeras, however, demonstrated that RegIIIγ induction in the small intestine is exclusively induced by MyD88-mediated signals in non–BM-derived cells, presumably intestinal epithelial cells. It remains unclear, however, whether MyD88-mediated induction of RegIIIγ occurs directly in Paneth cells, or whether innate immune signals initiated in intestinal epithelial cells near the lumen are transmitted to Paneth cells in intestinal crypts.
Our studies demonstrate an important, previously undescribed role for MyD88-mediated innate immune signaling in defense against infection by an intestinal pathogen. Although innate immune responses are typically considered to be rapid, at times even immediate, in this case, innate immune activation by commensal organisms occurs preemptively, before exposure to the pathogen. Importantly, the preemptive induction of RegIIIγ is not associated with overt evidence inflammation, but rather reflects a homeostatic, noninflammatory state mediated by commensal organisms. It is likely that MyD88-mediated expression of RegIIIγ influences the composition of the commensal flora, with downstream implications for mucosal homeostasis and susceptibility to Gram-positive intestinal pathogens.