Graft-versus-host disease propagation depends on increased intestinal epithelial tight junction permeability

Abstract

Graft-versus-host disease (GVHD) is a complication of hematopoietic stem cell transplantation (HSCT) that affects multiple organs. GVHD-associated intestinal damage can be separated into two distinct phases, initiation and propagation, which correspond to conditioning-induced damage and effector T cell activation and infiltration, respectively. Substantial evidence indicates that intestinal damage induced by pretransplant conditioning is a key driver of GVHD initiation. Here, we aimed to determine the impact of dysregulated intestinal permeability on the subsequent GVHD propagation phase. The initiation phase of GVHD was unchanged in mice lacking long MLCK (MLCK210), an established regulator of epithelial tight junction permeability. However, MLCK210-deficient mice were protected from sustained barrier loss and exhibited limited GVHD propagation, as indicated by reduced histopathology, fewer CD8+ effector T cells in the gut, and improved overall survival. Consistent with these findings, intestinal epithelial MLCK210 expression and enzymatic activity were similarly increased in human and mouse GVHD biopsies. Intestinal epithelial barrier loss mediated by MLCK210 is therefore a key driver of the GVHD propagation. These data suggest that inhibition of MLCK210-dependent barrier regulation may be an effective approach to limiting GVHD progression.

Keywords: Gastroenterology; Oncology; Stem cell transplantation; T cells; Tight junctions.

Figure 1. MLCK210 expression and myosin light chain phosphorylation are increased within the intestinal epithelium of patients with GVHD.

(A) Representative histopathology of human small intestinal biopsies. Arrowhead denotes an apoptotic cell. Scale bars: 50 μm (top), 10 μm (bottom). (B) Human small intestinal biopsies from healthy controls (left) or GVHD patients (right) were immunostained for ZO-1 (red), β-actin (white), and Hoechst 33342 (blue). Scale bar: 10 μm. Representative images are shown. (C) Biopsies from healthy controls (left) or GVHD patients (right) were immunostained for MLCK210 (brown), counterstained with hematoxylin, and scored from 0 to 3. Each point represents an individual patient. Representative images from n = 9 patients and n = 8 controls are shown. *P < 0.05, Mann-Whitney U test. Scale bar: 10 μm. (D) Biopsies from healthy controls (left) or GVHD patients (right) were immunostained for phosphorylated myosin light chain (pMLC, green) and E-cadherin (ECAD, red). Arrowhead denotes MLC phosphorylation at the perijunctional actomyosin ring. Stains were scored from 0 to 3, with each point representing an individual patient. Representative images from n = 9 patients and n = 8 controls are shown. *P < 0.05, Mann-Whitney U test. Scale bar: 10 μm.

Figure 2. MLCK210 expression and activity as well as cytokines associated with MLCK210 upregulation are elevated in minor mismatch experimental GVHD.

B6 WT recipients were lethally irradiated followed by a syngeneic (B6) or allogeneic (129) BMT. Mice were sacrificed 14 days after BMT. (A) Representative histopathology of the small intestine. Arrowheads denote apoptotic epithelial cells. Scale bars: 50 μm (top), 10μm (bottom). (B) Jejunal segments were immunostained for MLCK210 (green) and E-cadherin (red). Arrowheads indicate the location of the perijunctional actomyosin ring. Images are representative of >3 independent experiments. Quantitative analysis is shown in D. Scale bar: 10 μm. (C) Jejunal segments were immunostained for phosphorylated myosin light chain (pMLC, green) and E-cadherin (red). Arrowheads indicate the location of the perijunctional actomyosin ring. Images are representative of >3 independent experiments. Quantitative analysis is shown in D. Scale bars: 50 μm (top), 10 μm (bottom). (D) MLCK210 expression and MLC phosphorylation were determined morphometrically. Each point represents an average of 4 fields from one segment of tissue from a single mouse. Two segments were analyzed per mouse. Data are normalized to the mean of mice that did not receive BMT. MLCK210 mRNA was determined by quantitative PCR (qPCR) in purified epithelial cells. Each point represents an individual mouse. Data are normalized to the mean of mice that did not receive BMT. *P < 0.05, 2-tailed t test (B6→WT vs. 129→WT). (E) Jejunal cytokines were determined by ELISA. Each point represents an individual mouse. *P < 0.05, 2-tailed t test (B6→WT vs. 129→WT).

Figure 3. Allogeneic BMT fails to induce barrier dysfunction in MLCK^–/– mice.

(A) Probes used to measure MLCK210-dependent barrier loss. (B) Intestinal permeability to fluorescein was evaluated on d14 after syngeneic (B6→WT) or allogeneic (129→WT or MLCK^–/–) BMT. Values are normalized to the mean of WT mice that did not receive BMT. Each point represents an individual mouse. *P < 0.05, 2-tailed t test. (C and D) Intestinal permeability to 4-kDa dextran on d14 (C) or d35 (D) after BMT, normalized to the mean of WT mice without BMT. Each point represents an individual mouse. *P < 0.05, 2-tailed t test. (E and F) Jejunum harvested on d35 and immunostained for phosphorylated myosin light chain (pMLC, green) and E-cadherin (ECAD, red). Arrowheads denote perijunctional actomyosin ring. Note the preservation of myosin light chain phosphorylation within villus smooth muscle in MLCK^–/– mice. Each point represents the average of 4 fields from one segment of tissue; 2 segments were analyzed per mouse. Data are normalized to the mean of WT mice without BMT. Scale bars: 50 μm (top), 10 μm (bottom). **P < 0.01, ANOVA with Bonferroni’s correction (vs. all other conditions). (G and H) 35 days after BMT, mice were injected with Alexa Fluor 647–BSA and sacrificed 30 minutes later. Confocal imaging demonstrates bright intravascular signal, indicative of retained dye, in WT and MLCK^–/– mice without BMT or after syngeneic (B6→B6) BMT. In contrast, intravascular signal is markedly diminished in both WT and MLCK^–/– mice after allogeneic (129→B6) BMT. Note the sharp paracellular pattern in MLCK^–/– mice, consistent with preservation of the tight junction barrier, relative to the diffuse signal over the epithelium of WT mice (arrowheads). Scale bar: 25 μm. Each point represents an average of 6 regions of lamina propria from one mouse. Data are normalized to mean of WT mice without BMT. **P < 0.01, ANOVA with Bonferroni’s correction (vs. other conditions for each genotype; matched conditions across genotypes were not significantly different).

Figure 4. GVHD severity is markedly reduced in MLCK^–/– mice.

B6 WT or MLCK^–/– recipients were lethally irradiated, followed by a syngeneic (B6) or allogeneic (129) BMT. (A) Relative weight and (B) disease activity scores (n = 8–12/group). *P < 0.05, 2-tailed t tests on d35 (129→WT vs. 129→MLCK^–/–). (C) Serum TNF as determined by ELISA, 35 days after BMT. Each point represents an individual mouse. **P < 0.01, 2-tailed t test (129→WT vs. 129→MLCK^–/–). (D) Survival (n = 6–8/group). **P < 0.01, Kaplan-Meier log-rank test (129→WT vs. 129→MLCK^–/–). (E and F) Liver, skin, and jejunum were harvested 35 days after BMT and sections immunostained for CD8 (green) and E-cadherin (ECAD, red). (E) Representative images of biliary and squamous (skin) epithelium from WT (left panels) and MLCK^–/– (right panels) mice are shown. Arrowheads denote infiltration by CD8⁺ cells. Scale bars: 20μm. (F) Representative images of jejunum from WT (left) and MLCK^–/– (right) mice. Arrowheads denote direct contact of CD8⁺ cells with the epithelium. Scale bar: 50 μm. (G) Jejunum harvested 35 days after BMT was immunostained for CD3, CD4, Foxp3, and T-bet. Graphs show CD3⁺, CD4⁺Foxp3⁺, or CD4⁺T-bet⁺ T cells. Each point represents an individual mouse. **P < 0.01, 2-tailed t test.

Figure 5. GVHD severity is modulated by intestinal epithelial MLCK210.

B6 recipients of the indicated genotypes were lethally irradiated, followed by a 129 BMT. (A) Relative weight and (B) disease activity scores (n = 6–9/group). *P < 0.05, ANOVA with Bonferroni’s correction vs. all other conditions. (C) Survival (n = 8–15/group). *P < 0.05, Kaplan-Meier log-rank test, MLCK^–/– compared with all other groups. (D) Sections of jejunum were immunostained for CD8 (green) and granzyme B (red) 35 days after BMT. Representative images are shown. Arrowheads indicate colocalization. Scale bar: 20 μm. Each point represents an individual mouse. **P < 0.01, ANOVA with Bonferroni’s correction for MLCK210-KO mice vs. all other conditions (except syngeneic BMT) on d35. (E) Histopathology of jejunum (arrowheads denote apoptotic epithelial cells), liver (arrowheads denote lymphocytes infiltrating biliary epithelium), and skin (arrowheads denote apoptotic squamous cells, asterisks indicate preserved pilosebaceous units) on d35. Scale bars: intestine, 100 μm, 20 μm; liver, 300 μm, 50 μm; skin, 300 μm. (F) Total pathology scores (sum of jejunum, liver, and skin scores) 35 days after BMT. Each point represents an individual mouse. **P < 0.01, ANOVA with Bonferroni’s correction (vs. all other conditions). (G) Gross photos of 2 mice/group 35 days after BMT. Arrowheads point to hair loss and skin ulcers. Scale bar: 1 cm. (H) Correlation between intestinal permeability to fluorescein at 2 weeks after BMT and GVHD severity 5 weeks after BMT. Each point represents an individual mouse. Colors correspond to those for each condition shown in F. r = 0.75, P < 0.01 by Pearson’s correlation coefficient and degrees of freedom.

Figure 6. GVHD is diminished in MLCK^–/– mice following major mismatch BMT.

B6 WT or MLCK^–/– recipients were lethally irradiated, followed by BALB/c BMT. (A) Gross photos of 3 mice/group 35 days after BMT. Arrowhead points to hair loss and skin ulcers. Scale bar: 0.5 cm. (B) Representative histopathology of jejunum (arrowheads denote apoptotic epithelial cells; arrows indicate intraepithelial lymphocytes), liver (arrowhead indicates intraepithelial lymphocyte; asterisk denotes neutrophils, i.e., active pericholangitis), and skin (asterisk is adjacent to a damaged pilosebaceous gland) on d35. Scale bars: intestine, 200 μm, 20 μm; liver, 200 μm, 20 μm; skin, 100 μm. (C) Total pathology scores (jejunum, liver, and skin) 35 days after BMT. Each point represents an individual mouse. **P < 0.01, 2-tailed t test. (D) Disease activity scores at 35 days after BMT. Each point represents an individual mouse. *P < 0.01, 2-tailed t test. (E) Survival (n = 9 per group). *P < 0.05, Kaplan-Meier log-rank test, MLCK^–/– compared with WT. (F) MLCK210 mRNA was analyzed by qPCR in purified jejunal epithelial cells. Each point represents an individual mouse. Data are normalized to the mean of mice that did not receive BMT. **P < 0.01, 2-tailed t test. (G and H) Jejunum was harvested 35 days after BMT and sections immunostained for phosphorylated myosin light chain (pMLC, green) and E-cadherin (ECAD, red). Arrowheads denote perijunctional actomyosin ring. **P < 0.01, 2-tailed t test. Scale bar: 25 μm. (I) Jejunum was harvested 35 days after BMT and immunostained for CD8, granzyme B, and E-cadherin. Left: quantification of CD8⁺granzyme B⁺ cells/mm² of tissue. **P < 0.01, 2-tailed t test. Right: representative images of CD8 (green) infiltration. Arrowheads in low-power views denote intraepithelial CD8⁺ cells. Scale bars: 50 μm, 10 μm.

Figure 7. Genetic MLCK210 inhibition reduces effector CD8+ T cell accumulation within mesenteric, but not systemic, lymph nodes.

B6 mOVA^Tg (n = 14), B6 mOVA^TgMLCK^–/– (n = 11), and B6 WT controls (n = 4) were lethally irradiated, followed by an OT-I BMT consisting of 5 million WT bone marrow cells, 10 million WT splenocytes, and 2 million OT-I splenocytes. (A) Weight and (B) disease activity scores. **P < 0.01, 2-tailed t tests on d35. (C) Pathology scores of GVHD target organs (jejunum, liver, and skin) and representative small intestinal histopathology 35 days after BMT. Each point represents an individual mouse. *P < 0.05, 2-tailed t test. Arrows denote intraepithelial lymphocytes; arrowheads denote apoptotic bodies. Scale bars: 50 μm; inset: 10 μm. (D) Small intestine samples from day 35 after BMT were immunostained for CD3 (green), ZO-1 (red), E-cadherin (ECAD, blue), and Hoechst 33342 (white). Representative images are shown. Scale bar: 50μm. Graph shows CD3⁺ infiltration in the small intestine 35 days after BMT; each point represents an individual mouse. *P < 0.05, 2-tailed t test. (E) Flow cytometry analysis of cells isolated from spleen, mesenteric lymph nodes (MLN), and axillary/brachial lymph nodes (A/B) on d35 after BMT. The proportions of CD8⁺ T cells relative to live cells are shown. (F and G) CD8⁺ T cell intracellular granzyme B expression as evaluated by flow cytometry 35 days after BMT. (F) Representative flow cytometry plots and (G) quantification. Each point represents an individual sample. *P < 0.05, 2-tailed t test.