These data support the hypothesis that Hh signaling is an important epithelial modulator of inflammatory signaling in the small intestinal lamina propria. The transcriptional response of isolated E18.5 mesenchyme to both Shh and Ihh revealed immune response genes and inflammatory pathways among the top downregulated processes. Moreover, exogenous Hh ligand alone can reduce the strongly pro-inflammatory response observed during culture of isolated mesenchyme, indicating that epithelial Hh plays a major role in modifying inflammatory pathways. In accordance with this hypothesis, analysis of adult VFHhip animals demonstrates that chronic reduction of Hh signals in the adult intestine leads to villus loss, spontaneous inflammation, and death. Taken together, these data provide the first direct evidence implicating epithelial Hh signals in modulation of inflammation in the small intestine, and provide insight into the role of this developmental signaling pathway in immune homeostasis in the gut.
One important trend emerging from the microarray experiments is the observation that Hh signals stimulate the expression of genes involved in the development and function of smooth muscle cells, including Myocd, Igf1
, and Fgfr2
. This is an important observation in concert with extensive data indicating that Hh signals are crucial in smooth muscle development in the GI tract8-10
, and vasculature23
. In addition, recent data from our group indicate that Hh signals stimulate development of villus and muscularis mucosa smooth muscle through direct activation of the SRF co-activator, Myocardin (Zacharias et al.
, submitted). The fact that Myocardin and several other smooth muscle-related genes were also identified in our microarray experiments emphasizes the importance of Hh signals in the specification of smooth muscle cells.
The other major trend of our microarray studies was the unexpected identification of Hh ligands as key regulators of immune pathways in the intestine. Given the role of Hh in fate determination in multiple systems, it is tempting to speculate that the expression changes that we interpret as modification of inflammatory signaling actually represent modulation of the fate or activity of myofibroblast or myeloid cell lineages. Modulation of myeloid cell fate or phenotype would explain why addition of Hh, typically a ligand that promotes gene activation, down-regulates so many pro-inflammatory genes. The major pathways identified in our expression data are clearly associated with myeloid cell innate immunity, and our data demonstrate that myeloid cells respond directly to Hh signaling in the small intestine. Myeloid cells are key determinants of inflammation in the intestine3
, exerting regulatory control and communicating with other cell types to maintain proper immune homeostasis. Differential populations of myeloid cells are pro- or anti-inflammatory and these phenotypes can be modulated during response to external signals4
. One intriguing possibility is that Hh signals help to create or maintain a proper balance of tolerogenic versus pro-inflammatory myeloid populations in the small intestine; this possibility will need to be formally explored in future studies. Additionally, myofibroblasts have been shown to secrete inflammatory mediators24
, and are responsive to Hh signaling throughout life7
. Reduction in Hh signaling in late gestation8
or postnatally (Zacharias et al.
, submitted) leads to mislocalization of myofibroblasts. Such changes in localization or function stimulated by reduced Hh may provoke altered inflammatory signaling from this regulatory population. Together, the myeloid and myofibroblast populations provide the best candidates for the cellular targets that receive Hh signals intended to regulate immune response and inflammation.
Recent studies in the colon suggest that reduction in Hh signal transduction predisposes to inflammation in both mouse and human6
. Our results here emphasize the point that functional Hh signals are required to maintain a tolerogenic milieu in the context of the mammalian small intestine. The stochastic nature of the development of significant inflammatory disease in VFHhip suggests that an unknown stimulus is needed to begin the inflammatory process. This stimulus may be the aberrant response to normal trauma in the absence of functional Hh signaling, a barrier breach due to villus loss, or another trigger. Regardless of the precise etiology, Hh signaling is clearly required to protect from such an event as single transgenic littermates of VFHhip animals survive without inflammatory disease.
Hh has now been implicated in both colonic6
and small intestinal inflammatory regulation. This is intriguing in light of significant recent studies demonstrating that some human susceptibility loci are associated with celiac disease as well as Ulcerative Colitis (e.g., polymorphism in the IL2/IL21 region of the genome)25
. Strikingly, the inflammatory phenotype seen in VFHhip animals shares phenotypic similarities with both human Crohn's disease and celiac disease. The inflammation is patchy and can be transmural, characteristics similar to Crohn's. VFHhip animals also exhibit villus atrophy, crypt hyperplasia, and profound inflammation, mirroring a celiac-like phenotype. Moreover, the wasting disease experienced by older VFHhip animals may be the result of malnutrition secondary to lost absorptive surface after villus loss and inflammation; reduced absorptive surface is a hallmark of celiac disease in humans. Additionally, the prevalence of dermatitis in VFHhip animals mirrors the high incidence of dermatitis herpetiformis in human celiac disease patients14
. While the dermatitis in VFHhip animals may be a result of malabsorption or malnutrition, some VFHhip skin lesions demonstrate IgA deposition, a key finding in celiac-related dermatitis in humans. Finally, loss of smooth muscle may be a first step in the development of inflammation in VFHhip animals. Likewise, in humans with celiac disease, smooth muscle populations are affected; anti-smooth muscle antibodies are often found in celiac patients and may help identify a subset of those patients who are particularly susceptible to advanced disease26
Our analysis of small intestinal immune cells that respond to Hh signaling indicates that CD11b+
cells respond in vivo and in vitro to Hh ligand. Sorted populations of CD11b cells are functionally capable of responding to Hh signals by down-regulation of IL-6 protein and mRNA. Interestingly, there appear to be at least two different morphological subsets of these cells that demonstrate different levels of Gli expression. The Gli1High
expressing cells have the morphology of dendritic cells. Thymic dendritic cells have also been shown to express Gli1 and respond functionally to Hh27
. Interestingly, although early studies indicated that T cells are also Hh responding cells28,29,30
, ablation of Hh signaling by knockout of Smoothened in DN4 T cells has no effect on the differentiation or expansion of CD4 or CD8 populations30
. Our finding that small intestinal T cells do not express Gli1 are in accord with these latter functional studies.
Overall, the data presented here provide novel evidence that Hh signaling is an important anti-inflammatory signal in the small intestine. The inflammatory milieu in the small intestine is specialized, and many studies have shown that tolerogenic signals from both the stroma and epithelium are critical in modulating the tolerogenic response of the small intestine innate immune system3,31,32
. The emerging role of the Hh signaling pathway as important modulator of inflammation identifies an additional cellular signaling molecule from the epithelium as an important factor in balancing the inflammatory response of the mesenchyme. Hh signaling may cooperate with other intestinal tolerogenic signals (e.g., TSLP) to pattern a proper intestinal inflammatory response, but these microarray studies show that Hh alone can dramatically alter inflammatory signaling in isolated mesenchyme. Reduction in this homeostatic Hh influence may predispose to a disordered inflammatory response even in the presence of an otherwise normal immune system and may contribute to human gastrointestinal disease.