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. Author manuscript; available in PMC: 2016 Jun 1.
Published in final edited form as: Gastroenterology. 2015 Mar 4;148(7):1417–1426. doi: 10.1053/j.gastro.2015.02.053

γδ Intraepithelial Lymphocyte Migration Limits Transepithelial Pathogen Invasion and Systemic Disease in Mice

Karen L Edelblum 1, Gurminder Singh 1, Matthew A Odenwald 1, Amulya Lingaraju 1, Kamal El Bissati 2, Rima McLeod 2, Anne I Sperling 3,4, Jerrold R Turner 1,3
PMCID: PMC4685713  NIHMSID: NIHMS740675  PMID: 25747597

Abstract

Background & Aims

Intraepithelial lymphocytes that express the γδ T cell receptor (γδ IELs) limit pathogen translocation across the intestinal epithelium by unknown mechanisms. We investigated whether γδ IEL migration and interaction with epithelial cells promote mucosal barrier maintenance during enteric infection.

Methods

Salmonella typhimurium or Toxoplasma gondii were administered to γδ T cell-deficient (Tcrd KO), CD103-deficient (CD103 KO), or control TcrdEGFP C57BL/6 reporter mice. Intravital microscopy was used to visualize migration of GFP-tagged γδ T cells within the small intestinal mucosa of mice infected with DsRed-labeled S typhimurium. Mixed bone marrow chimeras were generated to assess the effects of γδ IEL migration on early pathogen invasion and chronic systemic infection.

Results

Morphometric analyses of intravital video microscopy data showed that γδ IELs rapidly localized to and remained near epithelial cells in direct contact with bacteria. Within 1 hr, greater numbers of T gondii or S typhimurium were present within mucosae of mice with migration-defective occludin KO γδ T cells, compared with controls. Pathogen invasion in Tcrd KO mice was quantitatively similar to that in mice with occludin-deficient γδ T cells, whereas invasion in CD103 KO mice, which have increased migration of γδ T cells into the lateral intercellular space, was reduced by 63%. Consistent with a role of γδ T cell migration in early host defense, systemic salmonellosis developed more rapidly and with greater severity in mice with occludin-deficient γδ IELs, relative to those with wild-type or CD103 KO γδ IELs.

Conclusions

In mice, intraepithelial migration to epithelial cells in contact with pathogens is essential to γδ IEL surveillance and immediate host defense. γδ IEL occludin is required for early surveillance that limits systemic disease.

Keywords: cell, intestinal epithelium, host defense, tight junction

Intraepithelial lymphocytes (IEL) are localized to epithelial barriers and are most abundant within the intestine. Approximately half of small intestinal IELs express the nonconventional γδ T cell receptor, and are thought to bridge innate and adaptive immunity1. Although the precise function of intestinal γδ IELs is incompletely understood, in their absence, translocation of both commensal bacteria and enteric pathogens, such as Salmonella typhimurium and Toxoplasma gondii, is enhanced2-5. While responses to individual pathogens differ, γδ T cells secrete the anti-microbial protein RegIIIγ via intestinal epithelial MyD88-dependent signaling in response to commensal and pathogenic bacteria5. These and other data indicate that γδ IEL interactions with intestinal epithelia are involved in host defense.

We have recently reported that intestinal γδ IELs are highly motile6. γδ IELs actively migrate along the basement membrane and into the lateral intercellular space between adjacent epithelial cells at speeds up to 7.7 μm/min. As a direct result of this migration, each villous epithelial cell is contacted by a γδ IEL ~4 times each hour. This may explain the ability of a relatively small number of IELs to provide defense over a large epithelial surface. γδ IEL motility requires expression of the transmembrane tight junction protein occludin by both γδ IELs and enterocytes. In mice with specific deletion of occludin in γδ IELs, epithelial cells were contacted by a γδ IEL less than once per hour. Further, occludin-deficient γδ IELs failed to migrate efficiently into the lateral intercellular space. Conversely, binding of CD103 (αEβ7 integrin) on γδ T cells to E-cadherin expressed on the epithelial basolateral surface7, 8 may stabilize intercellular interactions and limit motility, as CD103 deficiency increases migration within the epithelium by reducing the length of time a γδ IEL is retained between adjacent epithelial cells6. Despite the profound effects of occludin or CD103 deficiency on intestinal γδ IEL motile behavior, the impact of these perturbations the host response to pathogens has not been investigated.

Here we tested the hypothesis that γδ IEL migration is a critical component of innate immune surveillance. We show that GFP-labeled γδ IELs are concentrated near epithelial cells in direct contact with DsRed-labeled Salmonella typhimurium using intravital confocal microscopy of the intestinal mucosa. Further, γδ IELs are retained within the lateral intercellular space when in proximity to a bacterial-adherent cell. In contrast, occludin-deficient γδ IELs failed to fully migrate into the lateral intercellular space and their epithelial retention was not affected by S. typhimurium infection. Impaired γδ IEL migration was accompanied by a marked increase in S. typhimurium or T. gondii invasion into the intestinal lamina propria in mice to a similar extent as in γδ T-cell-deficient mice. As a result, earlier and more severe salmonellosis developed in mice with occludin-deficient γδ IELs. Conversely, CD103 deletion in γδ T cells enhanced γδ IEL migration into the lateral intercellular space and reduced pathogen invasion. These data indicate that γδ IEL motility and interactions with intestinal epithelia are critical for immediate innate defense against invasive pathogens.

Materials and Methods

Animals

All mice were used at 8-14 wk of age and maintained on a C57BL/6 background. Wildtype, CD103 KO9 and Tcrd KO10 mice were obtained from The Jackson Laboratories. TcrdH2BeGFP (TcrdEGFP) mice11 were crossed to either occludin KO mice12 backcrossed onto a C57BL/6 background for 10 generations provided by M. Neville (University of Colorado, Denver, CO) or to CD103 KO mice. All studies were conducted in an Association of the Assessment and Accreditation of Laboratory Animal Care (AALAC)-accredited facility according to protocols approved by the University of Chicago Institutional Animal Care and Use Committee.

Permeability assay

Following fasting for 3 h, mice were gavaged with 16 mg FITC-dextran 4kD in 0.2ml water. Blood was collected via the retro-orbital route after 3 hours. Serum FITC fluorescence was determined by plate reader at 495 nm excitation/525 nm emission.

Generation of bone marrow chimeras

Mice were lethally irradiated with 11Gy γ-irradiation and reconstituted 24 h later by i.v. injection of 4 × 106 Tcrd KO bone marrow cells and 1 × 106 of either wildtype TcrdEGFP, occludin KO;TcrdEGFP or CD103 KO;TcrdEGFP bone marrow. Experiments were performed 8 weeks post-engraftment.

Pathogen infection

DsRed-labeled S. typhimurium (strain SL3201) was generously provided by A. Neish (Emory University, Atlanta, GA). Mice were anesthetized, a 3-4 cm loop of jejunum or ileum exposed and the luminal surface of the intestine was exposed as described previously. 108 CFU DsRed-SL3201 was applied directly to the exposed luminal surface for the times indicated, after which mice were sacrificed and the loop of intestine was fixed for analysis by fluorescence microscopy. For live imaging, the intestine was bathed in 108 CFU SL3201 diluted in Hank's balanced salt solution (HBSS) containing 1 mM Alexa Fluor 633 to a final concentration of 2.5 × 107 CFU. Systemic infection of S. typhimurium was assessed by oral gavage of mixed bone marrow chimeras with 107 CFU DsRed-SL3201. Antibiotics, such as streptomycin, were not administered prior to infection since we have observed changes in γδ IEL migratory behavior after antibiotic-induced alteration of the intestinal microbiota (data not shown). Mice were sacrificed 6-10 days post-infection, based on the severity of clinical scores or if an individual mouse lost more than 20% of its initial body weight. Organs were harvested and either fixed in 10% neutral buffered formalin or 1% paraformaldehyde for further immunohistochemical analysis. Clinical scores were determined on the basis of fur texture, posture and activity on a scale of 0-2, with a potential combined score of 6. Histological scores were determined on a scale of 0-2 based on the following criteria: crypt dilation, distortion and elongation as well as the number of aberrant crypts and goblet cells, with a potential combined score of 10.

The ME49 strain of Toxoplasma gondii was maintained as tachyzoites by serial passage in human foreskin fibroblasts as previously described13. 10-12 week old female HLA-B*0702 transgenic mice13 were infected intraperitoneally with 1 × 104 ME49 type II tachyzoites. Tissue cysts were isolated from the brains of these mice 22 days post-infection and quantified14. Mice were gavaged with 10 cysts in 100 ml sterile PBS and euthanized after one hour to assess parasite translocation.

Live imaging experiments

Imaging was performed as previously described6, 15, 16, in which a multi-photon inverted confocal microscope (SP5; Leica) with a 40x 0.8 N.A. water immersion objective was used. EGFP was imaged using an Argon laser with a spectral emission of 491–580 nm, DsRed was imaged using a laser (DPSS 561) with a spectral emission of 589–727, and Alexa Fluor 633 was imaged with a spectral emission of 640–769 nm. Pinholes of 134 μm were used for these three channels. Hoechst dye was imaged using a multiphoton laser and a pinhole of 540 μm. Images were acquired by taking 15 μm Z-stacks at 1.5 μm spacing for a total time of 60-90 s between acquisition of Z-stacks. Three-dimensional rendering and image analysis was performed using Imaris (v.7.3.1; Bitplane) and ImageJ (NIH). IEL localization was determined by generating surfaces for both the IELs and the lumen and performing a distance transformation to determine the distance of the IEL relative to the lumen. Distances less than 15 μm from the lumen were determined to be within the lateral intercellular space between adjacent epithelial cells based on the average height of a columnar epithelial cell. In infected mice, “bacterial adjacent” epithelia were defined as enterocytes with an identifiable bacterium that had either invaded, or was associated with the apical surface, within 10 μm of the lateral intercellular space studied.

Immunofluorescence and image analysis

Mouse intestine was fixed in 1% paraformaldehyde for 2 h, washed with 50mM NH4Cl and cryoprotected in 30% sucrose (w/v) at 4°C overnight. Tissue was then embedded in Optimal Cutting Temperature (OCT) medium, snap-frozen and stored at −80°C. Frozen sections were immunostained as previously described6 using primary antibodies, rabbit anti-E-cadherin (Cell Signaling), rabbit anti-claudin-15, Alexa Fluor-594-conjugated mouse anti-occludin, rabbit anti-ZO-1 (Invitrogen), rabbit anti-CD3 (Abcam) or rabbit LDH1 antiserum17 followed by appropriate secondary antibodies and Hoechst 33342 dye (Invitrogen). Slides were mounted with Prolong Gold (Invitrogen) and visualized on a DMI6000 inverted epifluorescence microscope equipped with a Rolera EMC2 CCD camera (Q-imaging), 20x/0.50 PH2, 40x/0.60CORR/PH, or 63x/0.70CORR dry objectives and Metamorph 7 acquisition software (Molecular Devices). Images were deconvolved for 10 iterations using Autodeblur (Media Cybernetics).

Morphometric analysis of S. typhimurium and T. gondii was quantified as the number of organisms that had invaded into or across, i.e. into the lamina propria, an epithelial cell. Invasion into an epithelial cell required that the organism be localized basal to the perijunctional actomyosin ring, as defined by phalloidin staining. Organisms apical to the perijunctional actomyosin ring were considered to be luminal or associated with the brush border, and were not counted. To avoid artifact induced by deconvolution, unprocessed images were used for quantitative analyses. For each mouse, 6-8 fields were analyzed, each containing approximately 100 mm2 of epithelial-covered villus mucosa within each field. Data are reported as number of organisms per 0.1 mm2 tissue. The observer was blinded for the analysis.

Statistical Analysis

All data are presented as ± SEM. P values of direct comparisons between two independent samples were determined by a two-tailed Student t test and considered to be significant if P ≤ 0.05. In cases in which the data were not normally distributed, Mann-Whitney Rank Sum tests were performed. Comparisons between two independent variables at multiple time points were determined by two-way ANOVA. Comparisons between multiple independent variables were determined by one-way ANOVA and in cases in which the data were not normally distributed, Kruskal-Wallis one-way ANOVA on Ranks was performed and Dunn's Method was used for multiple pairwise comparisons. Fisher's exact test was used to compare proportions between two independent variables. Kaplan Meier Logrank test was used to compare survival between two independent populations.

Results

γδ IELs provide immediate innate defense against mucosal pathogens

To investigate the contributions of γδ IELs to immediate innate responses to enteric pathogens, mice were infected orally with the intracellular protozoan parasite T. gondii, which transmigrates across the intestinal epithelium18. Parasites were detected by immunostaining for lactate dehydrogenase-1 (LDH1), an enzyme involved in T. gondii cell cycle regulation17 (Figure 1A). Infection of γδ T-cell-deficient (Tcrd KO) mice resulted in the increased translocation of parasites into the lamina propria, relative to wildtype mice, within one hour of exposure (Figure 1B). CD103 deficiency enhances γδ IEL migration within the intestinal epithelium6, likely explaining the marked protection of CD103 (αE integrin)-deficient mice from parasite translocation, with a 63% reduction in invasive parasites compared to wildtype.

Figure 1. Mice deficient in γδ T cells exhibit increased susceptibility to enteric pathogen translocation.

Figure 1

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(A) LDH1 staining of T. gondii in the intestinal lamina propria (LP). Individual epithelial cells (ep) and basement membrane are outlined in a white or yellow dashed line, respectively. Scale bar, 10 μm. (B) Morphometric analysis of parasite translocation after 1 h in WT, Tcrd KO and CD103 KO mice. n=4-8 mice in two independent experiments. Mean ± SEM is shown. * P<0.001, ** P=0.01 compared to WT. (C) Morphometric analysis of S. typhimurium invasion in WT, Tcrd KO and CD103 KO mice at the time points indicated. n=6-10 mice from at least 2 independent experiments. Approximately 300 villi were counted for each condition. Mean ± SEM is shown. Two-way ANOVA shows the differences in invasion between the three genotypes. *P=0.04 **P<0.001. (D) Upper row. Low magnification micrographs of S. typhimurium (red, arrows)-infected small intestine from WT, Tcrd KO and CD103 KO mice. Nuclei are labeled with Hoechst (blue) and f-actin is shown in green. Scale bar, 20 μm. Lower row. Representative high magnification fields from infected WT, Tcrd KO and CD103 KO mice. Translocation of S. typhimurium is indicated (white arrows), bacteria not counted (yellow arrowheads). Scale bar, 5 μm. (E) Occludin, ZO-1, E-cadherin or claudin-15 (green) were immunolabeled in jejunum from WT, Tcrd KO and CD103 KO mice. Nuclei are labeled with Hoechst (blue) and f-actin is shown in red. Scale bar, 5 μm. (F) Paracellular flux of 4kD FITC-dextran in WT, Tcrd KO and CD103 KO mice is shown.

To determine whether Tcrd KO and CD103 KO mice have divergent immediate innate responses to other enteric pathogens, mice were infected with the Gram-negative bacterium S. typhimurium (SL3201). Increased bacterial translocation was apparent in Tcrd KO mice within 15 min and persisted for at least 1 hour (Figure 1C, D). Similar to the protection observed against T. gondii infection, bacterial invasion was reduced in CD103 KO mice. All further studies were performed at the 30 min timepoint to ensure sufficient bacterial exposure to the mucosal surface while still eliciting an immediate immune response. In contrast to previous reports3, tight and adherens junction organization and position were unaffected in Tcrd or CD103 KO mice (Figure 1E). Further, these mice exhibited no increase in intestinal permeability to FITC-4kD dextran (Figure 1F), indicating that the observed differences in susceptibility are not due to effects on intestinal epithelial barrier integrity or structure.

Based on our findings that loss of γδ T cells resulted in pathogen translocation almost immediately following exposure to S. typhimurium or T. gondii, and that CD103 deficiency prevents bacterial translocation, we hypothesized that these effects may be attributed to γδ T cell migration or interactions with the intestinal epithelium. Therefore, we next assessed whether γδ IEL migration is altered in the presence of an enteric pathogen.

γδ IEL migration is altered in the presence of Salmonella

We have previously used GFP γδ T-cell reporter mice (TcrdEGFP)11 and intravital confocal microscopy to show that γδ IELs migrate continuously along the basement membrane and into lateral intercellular spaces6. This dynamic behavior allows the small number of γδ IELs to interact with nearly all of the villous epithelium over short intervals6. Based on the early pathogen protection conferred by γδ T cells and the observed surveillance-like migratory phenotype of these cells, we hypothesized that γδ IEL migration might be rapidly modified in response to S. typhimurium infection.

Intravital confocal microscopy during S. typhimurium infection showed a marked increase in γδ IEL localization to epithelial cells in close proximity to bacteria (Figure 2A, Supplementary Movie 1). In addition, there was a significant increase in number of γδ IELs within the lateral intercellular space (the first 15 μm from the lumen) in infected, relative to uninfected mice (Figure 2B). As we have previously reported, CD103-deficient γδ IELs migrate more frequently into the lateral intercellular space; however, Salmonella exposure did not dramatically enhance CD103 KO γδ IEL migration (Figure 2B). Although the small change in γδ IEL migration between infected and uninfected CD103 KO mice is statistically significant due to increased power as a result of a large sample size, this difference is unlikely to be biologically meaningful.

Figure 2. γδ IELs migrate more frequently into and remain longer within the lateral intercellular space in the presence of Salmonella.

Figure 2

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(A) Maximum projection of γδ IEL (green) migration over the course of 30 min in uninfected (left) or DsRed-labeled S. typhimurium-infected (cyan arrowheads, right) TcrdEGFP (WT) mice. The small regions of green signal present in the lumen (red) are artifacts of the projection. A yellow dashed line represents the basement membrane. Scale bar = 30 μm. (B) Frequency of γδ IELs within the lateral intercellular space (first 15 μm from the intestinal lumen) in uninfected and infected WT or uninfected and infected CD103 KO mice. n=6,969, 5,402, 14,259 and 3,104 γδ T cells, respectively. Mean ± SEM is shown. P<0.001. (C) Maximum speed of γδ IELs in uninfected and infected WT or uninfected and infected CD103 KO mice. n=860 and 1,056, 411 and 599 tracks, respectively. *P<0.05. (D) Duration of γδ IEL retention within the lateral intercellular space. Salmonella-infected mice are indicated as (+S. typhimurium). γδ IELs near bacterial-adjacent epithelial cells (see Materials and Methods) are indicated as (+ bacterial-adj). In contrast, (− bacterial-adj) indicates γδ IELs in which flanking epithelium was not in contact with bacteria. *P=0.001. (E) Average number of times an epithelial cell is contacted by a γδ IEL over the course of an hour in uninfected and infected WT or CD103 KO mice. *P<0.001. For panels C, D, and E 4-6 mice (total of 25-30 villi) were independently imaged for each experimental condition. In panels D and E, each point represents a single microscopic field (1-2 fields per mouse).

Concomitant with Salmonella-induced increases in localization to the lateral intercellular space, the maximum migratory speed of WT γδ IELs was decreased in infected mice (4.2 ± 0.1 vs. 3.5 ± 0.1 μm/min, WT uninfected vs. infected)(Figure 2C). This was due to increased dwell time for WT γδ IELs within the lateral intercellular space at sites close to bacteria (Figure 2D). While CD103 KO γδ IELs migrated more rapidly than WT γδ IELs, S. typhimurium did not affect the migratory speed of CD103 KO γδ IELs (9.6 ± 0.1 vs. 9.4 ± 0.2 μm/min)(Figure 2C). Although the dwell time of CD103 KO γδ IELs was increased by infection, this was not restricted to γδIELs near bacterial-adjacent epithelia (Figure 2D). However, increased retention reduced the number of contacts between epithelia and CD103-deficient γδ IELs following infection (Figure 2E). Taken together, S. typhimurium infection promoted migration and prolonged residence of WT γδ IELs into the lateral intercellular space at sites of infection, whereas only retention was increased for CD103-deficient γδ IELs.

γδ IEL migration into the lateral intercellular space is critical for immediate host defense

We reported that efficient migration into the lateral intercellular space requires γδ IEL expression of the tight junction protein occludin6. This observation provided a tool that allowed us to ask whether the γδ IEL migration into the lateral intercellular space contributed to host defense. TcrdEGFP mice were crossed with occludin KO mice, and these mice were used to generate mixed bone marrow chimeras by engrafting 20% TcrdEGFP occludin KO and 80% Tcrd KO bone marrow into lethally-irradiated wildtype recipients (occludin KOGFPγδ chimeras). This resulted in mice expressing GFP+ occludin KO γδ T cells while maintaining occludin expression in other cell types. Mice engrafted with 20% wildtype TcrdEGFP and 80% Tcrd KO bone marrow (WTGFPγδ chimeras) served as controls. Morphometric analysis of GFP expression and FACS analysis confirmed that similar numbers of γδ T cells were present in the small intestine 8 weeks after transfer (Supplementary Figure 1). These chimeric mice were challenged orally with T. gondii, and quantitative analysis showed 6.2-fold as many parasites in the lamina propria in occludin KOGFPγδ chimeras as in occludin-sufficient WTGFPγδ chimera controls (Figure 3A). The number of invasive parasites detected within the lamina propria of occludin KOGFPγδ chimeras was dramatically increased compared to controls, and was remarkably similar to mice completely lacking γδ T cells (Figure 1B). Challenge of occludin KOGFPγδ chimeras with S. typhimurium also resulted in a significant increase in bacterial invasion relative to WTGFPγδ chimeras (Figure 3B). These data show that the protective effect afforded by γδ IEL occludin expression is not specific to a single pathogen type. Thus, occludin expression by γδ IELs is necessary for their protective function against enteric pathogens.

Figure 3. γδ IEL occludin expression is required to prevent enteric pathogen transmigration across the intestinal epithelium.

Figure 3

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(A) Morphometric analysis of parasite translocation 1h post-infection in wildtype and occludin KO γδ IELs. n=9-11. Mean ± SEM of 2 independent experiments is shown. *P<0.001. (B) Morphometric analysis of S. typhimurium invasion after 30 min in wildtype, occludin KO γδ IEL chimeras. n=6-14. Mean ± SEM of at least 2 independent experiments is shown. *P=0.002, **P=0.04. (C) Distance of WTGFPγδ and occludin KOGFPγδ IELs from the intestinal lumen (μm). n=3,132 and 1,560 WTGFPγδ and occludin KOGFPγδ T cells, respectively. Mean ± SEM is shown. *P<0.001. (D) Number of γδ IELs contacting a single epithelial cell over the course of an hour in WTGFPγδ or occludin KOGFPγδ chimeras. Open circles represent areas near bacterial-adherent cells, filled circles represent areas in which no bacteria are observed. Mean ± SEM is shown. *P=0.02. (E) Duration of WTGFPγδ or occludin KOGFPγδ IEL retention in the lateral intercellular space. Open circles represent areas near bacterial-adherent cells, filled circles represent areas in which no bacteria are observed. Mean ± SEM is shown. *P=0.04, **P=0.02.

To determine whether the requirement for occludin expression is due to effects on γδ IEL migration and not another undefined function of occludin, we took advantage of the accelerated migration of CD103 KO γδ IELs6. CD103 KO mixed bone marrow chimeras were generated to restrict CD103 deletion to γδ T cells, and similar to our observations of T. gondii and S. typhimurium translocation in CD103 KO mice (Figure 1B and C), loss of γδ IEL CD103 expression reduced S. typhimurium invasion (Figure 3B).

In addition to the increased pathogen numbers observed in the lamina propria of occludin KOGFPγδ chimeras, we also found that the majority of occludin KOGFPγδ IELs remained beneath the epithelium along the basement membrane (more than 16 μm from the lumen) even after bacterial challenge (Figure 3C). While 18 ± 6.3% of WTGFPγδ IELs migrated within the lateral intercellular space, only 6.3 ± 2.5% of occludin KOGFPγδ IELs were present within the lateral intercellular space following Salmonella infection. Further, the reduced motility of occludin KOGFPγδ IELs resulted in a nearly 50% reduction in the number of γδ IEL/epithelial contacts relative to WTGFPγδ IELs (Figure 3D). Similar to TcrdEGFP mice, IELs in WTGFPγδ chimeras exhibited increased dwell time when localized near bacterial-adherent epithelial cells (6.8 ± 1.4 vs. 2.7 ± 0.9 min), whereas S. typhimurium infection did not prolong retention of the few occludin KOGFPγδ IELs that did enter the lateral intercellular space (Figure 3E). Therefore, occludin expression by γδ IELs not only facilitates migration and entry into the lateral intercellular space, but also is necessary to promote a sustained interaction between γδ IELs and bacterial-adherent enterocytes. The data suggest that this occludin-dependent γδ IEL migration and retention within lateral intercellular space is critical to host defense, and support the hypothesis that the antimicrobial effector response is most efficient at this distinctive site.

Loss of γδ IEL occludin increases susceptibility to systemic S. typhimurium infection

Salmonella infection is initiated by invasion across the intestinal epithelium, resulting in the development of either self-limited gastrointestinal inflammation or fatal disseminated disease19. Our data show that occludin-dependent γδ IEL migration limits pathogen translocation, as does loss of CD103 expression in γδ IELs. To determine whether the extent of early pathogen invasion is a marker of disease progression at later time points, WTGFPγδ, occludin KOGFPγδ or CD103 KOGFPγδ chimeras were infected with S. typhimurium.

Occludin KOGFPγδ chimeras succumbed to systemic infection more rapidly than WTGFPγδ chimeras (Figure 4A), and exhibited more severe clinical signs of disease (Figure 4B). In contrast, CD103 KOGFPγδ chimeras developed systemic disease at a rate similar to WTGFPγδ chimeras (Figure 4A,B), suggesting that the modest reduction in S. typhimurium invasion at early times did not provide long-term benefit beyond that afforded by systemic immune responses. Histopathologic analysis of colon confirmed more severe disease (Figure 4C and D) as well as increased CD3+ T cell infiltration (Figure 4E) in occludin KOGFPγδ chimeras following infection. In contrast, and consistent with the clinical data, there was no significant difference in histopathology between S. typhimurium-infected WTGFPγδ and CD103GFPγδ chimeras. Taken together, these data indicate that increased bacterial translocation during the initial phase of infection results in the acceleration of systemic disease in the absence of γδ IEL occludin expression.

Figure 4. Increased bacterial translocation in occludin KOGFPγδ chimeras results in increased susceptibility to systemic Salmonella infection.

Figure 4

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(A) Survival curve *P=0.05 and (B) clinical scores of WTGFPγδ, occludin KOGFPγδ, CD103 KOGFPγδ chimeras at the date of death following oral gavage with 107 CFU SL3201. n=14-22 mice over at least 2 independent experiments. Mean ± SEM is shown is shown *P<0.001. (C) Representative histological scores of colons from SL3201-infected of WTGFPγδ and occludin KOGFPγδ chimeras sacrificed at 9 d post-infection. n=7-8 mice. Mean ± SEM is shown *P<0.001. (D) H&E micrographs of colons from SL3201-infected WTGFPγδ and occludin KOGFPγδ chimeras. Scale bar = 40 μm. (E) Quantification of CD3+ immunostaining in small intestine and colon sections of SL3201-infected WTGFPγδ, occludin KOGFPγδ, CD103 KOGFPγδ. n=5-10 mice. Mean is shown *P<0.001.

Discussion

Although we have previously shown that γδ IELs are highly motile resulting in direct interactions with the majority of the villous epithelium, a functional role for γδ IEL migration remained unclear. γδ T cells are implicated in providing the first line of defense in various epithelia due to their ability to bridge innate and adaptive immune responses20. Based on the close proximity of γδ IELs to the intestinal lumen and direct interactions with neighboring epithelial cells, γδ IELs are well positioned to provide an immediate response to invasion of enteric pathogens.

Our data demonstrate that the critical contribution of γδ IELs to immediate innate defense depends on continuous γδ IEL surveillance of the intestinal epithelium, recruitment of γδ IELs into the lateral intercellular space at sites of infection, and increased intraepithelial dwell times once a bacterial-adherent epithelial cell has been identified. Inhibition of γδ IEL motility through loss of occludin expression allows increased pathogen translocation, fails to support increases in γδ IEL interactions with bacterial-adherent epithelial cells and results in more severe systemic salmonellosis. Conversely, increased γδ IEL surveillance through the disruption of CD103/E-cadherin interactions prevents pathogen translocation, thus demonstrating that γδ IEL migration is an essential component of the immediate innate host defense response.

We show that epithelial barrier function is not impaired in mice deficient in γδ T cells (Figure 1D,E). Nevertheless, we note that these mice were recently reported to exhibit mild epithelial defects, including increased goblet cell numbers and altered mucin production without significant changes in the mucus layer21. While these differences may contribute to increased pathogen translocation in γδ T-cell-deficient mice, our studies using mixed bone marrow chimeras indicate that γδ IEL migration is required for rapid protection against pathogen translocation.

γδ IEL-mediated protection against T. gondii has been attributed to direct effects on epithelial occludin distribution3, 22. However, we found no change in localization of epithelial occludin or other junctional proteins in γδ T-cell-deficient mice (Figure 1D). Rather, interactions between γδ IEL and epithelial occludin promote γδ IEL migration and retention at sites of pathogen invasion. In contrast, loss of epithelial E-cadherin interaction with CD103 on γδ T cells results in enhanced IEL migration within the intraepithelial compartment and reduced pathogen translocation. This was unexpected as loss of CD103 was reported to reduce overall IEL numbers9. However, we did not observe significant alterations in γδ IEL number in CD103 KO mice, likely because CD103 deficiency more profoundly affects the αβ IEL population.

Taken together, these data indicate that occludin and CD103 function as positive or negative regulators of γδ IEL migration into the lateral intercellular space, respectively, and that this localization directly contributes to host defense against invasive pathogens. It is possible that resistance to pathogen translocation may reflect activation of signaling pathways downstream of occludin or CD103/E-cadherin, since several receptor/ligand interactions between γδ IELs and enterocytes have recently been shown to contribute to intestinal mucosal homeostasis23, 24. However, based on our observation that γδ IEL migration has a profound effect on the invasion of two distinct enteric pathogens, and that γδ IEL retention within the epithelium is increased near bacterial-adherent enterocytes, it is more likely that γδ IEL migration is a key factor in innate immune surveillance.

The data indicate that development of a more rapid systemic disease in S. typhimurium-infected occludin KOGFPγδ chimeras results, at least in part, from the initial increase in bacterial translocation during infection. However, we cannot exclude another occludin-dependent function within γδ IELs, either at initial stages or later in the course of infection. While the data showing that occludin- and CD103-deficient γδ IELs display inverse phenotypes, further study will be needed to assess other potential mechanisms of host defense. For example, it is possible that γδ IEL production of growth factors25, 26 or anti-inflammatory cytokines27-29 is occludin-dependent.

Previous studies showed that Salmonella infection likely elicits epithelial IL-23-dependent IL-22 production by γδ IELs to promote Paneth cell expression of the anti-microbial protein angiogenin-4 within 4 hours of infection29. However, in vitro studies showed that direct γδ IEL contact with intestinal epithelial cells was not necessary to induce this response. In contrast, we demonstrate γδ T-cell-dependent responses to Salmonella occur within 15 min following exposure. These rapid kinetics are not consistent with a transcriptional response to infection.

Therefore, we propose a model in which mucosal pathogen adherence triggers events that promote γδ IEL migration toward the affected epithelial cell and into the lateral intercellular space, perhaps resulting in interaction between the γδ TCR with stress antigens expressed on the surface of epithelial cells20, 30. This could induce the release of pre-formed anti-microbial peptides5, 29, 31 or other metabolites32 from γδ IELs or epithelial cells. Interactions between γδ IELs and the epithelium may then trigger subsequent innate or adaptive immune responses involved in later stages of infection. While the model is hypothetical, we hope that future work will address the contributions of the proposed mechanisms. These studies nevertheless provide a foundation to understand the means by which migration and direct association of γδ IELs with the intestinal epithelium provides a crucial first line of defense against enteric pathogens.

Supplementary Material

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Acknowledgements

We thank R. Jones for thoughtful discussions regarding experimental design, I. Prinz for kindly providing TcrdH2BEGFP mice, Y. Zhou for technical assistance, A. Neish for the DsRed-labeled S. typhimurium, V. Bindokas and the University of Chicago Integrated Light Microscopy Core Facility for confocal microscopy and image analysis support, the University of Chicago Flow Cytometry Facility and A-C. France for assistance with statistical analysis.

Grant support: This work was supported by National Institute of Health Grants K01DK093627 and F32DK084859 (KLE), U01AI77887 (RM), R01DK61931 and R01DK68271 (JRT), R01HL118758, U19A1095230, R21A1094408 (AIS); The University of Chicago Digestive Disease Research Core Center Grant Pilot & Feasibility Award (KLE) P30DK42086; Gastro-intestinal Research Fund Associates’ Board Award (KLE); Crohn's and Colitis Foundation of America and Broad Medical Research Foundation (JRT); The Cornwell and Mann Family Foundation; and donations from S. Wilson, S. Powers and the Engel, Taub, Musillami, Morel, Samuel, Harris, Pritzker, Rooney-Alden, Cussen and Kapnick families (RM); The University of Chicago Comprehensive Cancer Center (P30CA14599), The University of Chicago Institute for Translational Medicine (UL1RR024999), and The University of Chicago Digestive Disease Research Core Center (P30DK042086).

Footnotes

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The authors have nothing to disclose.

Author contributions: K.L.E. conceived the project, designed and performed experiments and prepared the manuscript. G.S., A.L., M.O. performed key experiments. K.E.B. performed T. gondii experiments and provided feedback on the manuscript. R.M. discussed experimental data and provided feedback on the manuscript. A.I.S. and J.R.T. equally contributed to the development of the project, experimental design, discussion of experimental data and manuscript preparation.

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