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

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 knockout (KO) mice lacking either the T cell receptor δ chain (Tcrd) or CD103, or control TcrdEGFP C57BL/6 reporter mice. Intravital microscopy was used to visualize migration of green fluorescent protein (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 hour, 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: Host Defense; Intestinal Epithelium; T cell; Tight Junction.

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

(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.

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

(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).

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

(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 WT^GFPγδ and occludin KO^GFPγδ IELs from the intestinal lumen (μm). n=3,132 and 1,560 WT^GFPγδ and occludin KO^GFPγδ 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 WT^GFPγδ or occludin KO^GFPγδ 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 WT^GFPγδ or occludin KO^GFPγδ 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.

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

(A) Survival curve *P=0.05 and (B) clinical scores of WT^GFPγδ, occludin KO^GFPγδ, CD103 KO^GFPγδ 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 WT^GFPγδ and occludin KO^GFPγδ 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 WT^GFPγδ and occludin KO^GFPγδ chimeras. Scale bar = 40 μm. (E) Quantification of CD3⁺ immunostaining in small intestine and colon sections of SL3201-infected WT^GFPγδ, occludin KO^GFPγδ, CD103 KO^GFPγδ. n=5-10 mice. Mean is shown *P<0.001.