Reciprocal interactions between commensal bacteria and gamma delta intraepithelial lymphocytes during mucosal injury

Abstract

The intestinal mucosal surface is in direct contact with a vast beneficial microbiota. The symbiotic nature of this relationship is threatened when the surface epithelium is injured, yet little is known about how mucosal surfaces maintain homeostasis with commensal microbes following damage. Gammadelta intraepithelial lymphocytes (gammadelta IEL) reside at the gut epithelial surface, where they stimulate mucosal healing following acute injury. A genome-wide analysis of the gammadelta IEL response to dextran sulfate sodium-induced colonic damage revealed induction of a complex transcriptional program, including coordinate regulation of cytoprotective, immunomodulatory, and antibacterial factors. Studies in germfree mice demonstrated that commensal microbiota regulate key components of this transcriptional program, thus revealing a dialogue between commensal bacteria and gammadelta IEL in injured epithelia. Analysis of TCRdelta-deficient mice indicated that gammadelta T cells are essential for controlling mucosal penetration of commensal bacteria immediately following dextran sulfate sodium-induced damage, suggesting that a key function of gammadelta IEL is to maintain host-microbial homeostasis following acute mucosal injury. Taken together, these findings disclose a reciprocal relationship between gammadelta T cells and intestinal microbiota that promotes beneficial host-microbial relationships in the intestine.

FIGURE 1

Colonic γδ IEL exhibit a complex transcriptional response to DSS-induced mucosal injury. A, Affymetrix Mouse Genome 430 2.0 arrays were used to compare transcript abundance between γδ IEL from untreated and DSS-treated colons. Differentially expressed transcripts were identified, as outlined in Materials and Methods, revealing 272 transcriptional changes between the DSS-treated and untreated groups. The differentially regulated genes are displayed as a heatmap in which expression level is defined by Z-score (defined in Materials and Methods). See Table S1 for a list of all 272 differentially regulated genes and their associated signal intensities. B, Key functional groups were delineated using Gene Ontology terminology, and are displayed as heatmaps.

FIGURE 2

DSS treatment elicits coordinate expression of cytoprotective, immunomodulatory, and antibacterial factors in γδ IEL. A, RNA was isolated from sorted γδ IEL and amplified, and Q-PCR analysis was performed to quantitate expression of βig-h3, which stimulates epithelial proliferation during wound healing (15). Assays were performed on γδ IEL isolated from pooled colons (n ≥ 5 mice), run in triplicate, and are shown as mean values normalized to GAPDH. Relative expression levels were calculated in relation to untreated samples. Results are representative of two independent experiments, unt, Untreated; DSS, DSS treated. Error bars, ±SEM. Q-PCR quantitation of proinflammatory cytokine expression (B) and antimicrobial factor expression (C) was conducted as in A. D, Flow cytometry was performed on total IEL populations from untreated and DSS-treated colons. Intracellular staining was conducted with Abs directed against lysozyme and RegIIIγ, as well as a control rabbit polyclonal Ab. Gated γδ IEL populations are shown, and percentages of the gated populations are given. SSC, side scatter.

FIGURE 3

Commensal bacteria govern a component of the γδ IEL response to mucosal injury, γδ IEL from germfree-untreated and DSS-treated mice were analyzed by microarray. Signal intensities of the 272 transcripts that were differentially regulated in DSS-treated conventional mice were compared with the corresponding signal intensities from germ-free γδ IEL. A, Unsupervised hierarchical clustering of the signal intensity data revealed a subset of transcripts that require commensal bacteria for enrichment after DSS-induced mucosal injury. B, Bacteria-regulated transcripts that encode cytoprotective, immunomodulatory, and antibacterial factors. See Fig. S1 for the complete list of 68 bacteria-regulated transcripts. Heatmap color is keyed to gene expression level as defined by Z-score.

FIGURE 4

Commensal bacteria direct antibacterial and proinflammatory responses in γδ IEL following mucosal injury. A, βig-h3 levels were quantitated by Q-PCR of amplified γδ IEL mRNAs, revealing that bacterial signals are not required for expression of this cell proliferation-inducing factor. In contrast, Q-PCR analysis revealed that commensal bacteria are required for DSS-induced expression of proinflammatory cytokines (B) and the antibacterial lectin RegIIIγ (C). Q-PCR analysis additionally revealed MyD88-dependent and MyD88-independent regulation of proinflammatory cytokines (B) and MyD88-independent regulation of RegIIIγ (C). These responses were reconstituted in adult germfree mice conventionalized with a normal microflora (B and C). All Q-PCR analyses were conducted on amplified mRNAs from pooled colons, as described in the Fig. 2 legend, and results are representative of two independent experiments. conv-L, Conventional from birth; conv-D, conventionalized for 72 h; gf, germfree.

FIGURE 5

γδ T cells limit opportunistic penetration of commensal bacteria following mucosal injury. A and B, Wild-type and TCRδ^-/- mice were treated with 2% DSS over a time course of 5 days. Numbers of MLN bacteria (A) or luminal bacteria (B) were quantitated in treated and untreated wild-type and TCRδ^-/- mice. Error bars represent SEM, and results are pooled from three independent experiments. *, p < 0.05; **, p < 0.01.

FIGURE 6

Interactions between commensal bacteria and γδ IEL during colonic mucosal injury. γδ IEL are situated between epithelial cells and are located on the basolateral side of tight junctions, which restrict paracellular penetration of luminal bacteria. Upon injury, commensal bacteria stimulate γδ IEL expression of antimicrobial factors, such as RegIIIγ, and of chemotactic cytokines (KC, CXCL-9, IL-1β, MIP2α). It is not yet clear whether this involves direct stimulation of γδ IEL by bacteria or bacterial products, or whether bacteria act indirectly through other cells (e.g., epithelial cells). γδ T cells function to limit bacterial penetration of mucosal surfaces specifically during the early stages following epithelial injury. We propose that bacteria-induced expression of directly antimicrobial proteins and chemotactic cytokines may account for the protective function of γδ T cells during injury. In combination with prior studies showing that γδ IEL stimulate healing of damaged intestinal epithelia (1), our findings suggest a multifaceted role for γδ IEL in restoring homeostasis following mucosal damage. γδ, γδ IEL; TJ, tight junction.