Thirty-eight-negative kinase 1 mediates trauma-induced intestinal injury and multi-organ failure

Abstract

Dysregulated intestinal epithelial apoptosis initiates gut injury, alters the intestinal barrier, and can facilitate bacterial translocation leading to a systemic inflammatory response syndrome (SIRS) and/or multi-organ dysfunction syndrome (MODS). A variety of gastrointestinal disorders, including inflammatory bowel disease, have been linked to intestinal apoptosis. Similarly, intestinal hyperpermeability and gut failure occur in critically ill patients, putting the gut at the center of SIRS pathology. Regulation of apoptosis and immune-modulatory functions have been ascribed to Thirty-eight-negative kinase 1 (TNK1), whose activity is regulated merely by expression. We investigated the effect of TNK1 on intestinal integrity and its role in MODS. TNK1 expression induced crypt-specific apoptosis, leading to bacterial translocation, subsequent septic shock, and early death. Mechanistically, TNK1 expression in vivo resulted in STAT3 phosphorylation, nuclear translocation of p65, and release of IL-6 and TNF-α. A TNF-α neutralizing antibody partially blocked development of intestinal damage. Conversely, gut-specific deletion of TNK1 protected the intestinal mucosa from experimental colitis and prevented cytokine release in the gut. Finally, TNK1 was found to be deregulated in the gut in murine and porcine trauma models and human inflammatory bowel disease. Thus, TNK1 might be a target during MODS to prevent damage in several organs, notably the gut.

Keywords: Apoptosis; Gastroenterology; Inflammation; Inflammatory bowel disease; Tight junctions.

Figure 1. Expression of TNK1 causes animal distress, systemic inflammation, and rapid lethality.

(A) By an inducible cassette exchange (ICE) approach (60), Myc-tagged Tnk1 was targeted to a specific conditionally regulated locus by Cre-lox recombination. (B) Behavior analysis shows that expression of TNK1 impairs locomotion of the animals (n = 6 per group). (C and D) TNK1-expressing mice show signs of cachexia, as demonstrated by decreased food intake (C) and body weight (D) (n = 12 per group, t = 24 hours). (E) Mice exhibit a drop in body temperature upon TNK1 expression (n = 5 per group, t = 24 hours). (F) TNK-expressing mice exhibit hypoglycemia (left) and hypercortisolism (right) (n = 4 per group). (G) Differential blood count suggests systemic bacterial inflammation (n = 8–10 per group, t = 24 hours), as designated by an increase in the total number of white blood cells (WBC), granulocytes, and monocytes and a decrease of lymphocytes. (H) TNK1-expressing mice show a significant decrease in red blood cell (RBC) number, declined hematocrit (HCT), and hemoglobin (HGB). (I–K) TNK1-expressing mice also exhibit a dysregulation of coagulation, as indicated by diminished platelets (n = 9 per group) (I) and rotational thromboelastometry analysis (n = 3–4 per group) (J and K) (t = 24 hours). Prolonged EXTEM (J) and FIBTEM (K) clotting time (CT) and reduced α angle specify abnormal clot formation. A prolonged EXTEM clot formation time (CFT) and reduced EXTEM maximum clot firmness (MCF) indicate abnormal clot formation in TNK1-expressing mice (J, right 2 graphs). (L) Kaplan-Meier analysis shows decreased survival upon TNK1 expression (n = 12). MS, median survival. Data are expressed as mean ± SEM. Differences were tested by parametric 2-tailed, unpaired Student’s t tests (*P = 0.01–0.05; **P = 0.001–0.01; ***P = 0.0001–0.001; ****P < 0.0001).

Figure 2. TNK1 expression impairs the functionality of the intestinal barrier.

(A and B) TNK1-expressing mice show bloody diarrhea and significant colon shrinkage (A) and erythema (B, arrows) 24 hours after doxycycline administration (n = 6 per group). Macroscopically the colon also appears distended. (C) Expression of TNK1 perturbs normal intestinal architecture. Mice show severe disruption of the mucosal architecture in small and large intestine. Representative histological images of the large and small intestine of Rosa26rtTA/+, Hrpt Myc-Tnk1tg 24 hours after doxycycline or saline administration are shown. Expression of TNK1 affects the integrity of both adherens junctions and tight junctions as demonstrated by a substantial reduction in E-cadherin (middle panels) and claudin-1 (bottom panels) immunoreactivity in the epithelium of the small and large intestine of TNK1-expressing animals. (D) Myeloperoxidase (MPO) elevation in gut tissues of TNK1-expressing mice points to neutrophil infiltration. Neutrophil accumulation contributes to local tissue destruction (n = 4 per group). (E and F) Impairment of intestinal barrier is accompanied by bacterial translocation to the intestinal wall (E) and to the mesenteric lymph nodes (F) (arrows indicate Gram⁺ bacteria). Data are expressed as mean ± SEM. Differences were tested by parametric 2-tailed, unpaired Student’s t tests (**P = 0.001–0.01; ***P = 0.0001–0.001; ****P < 0.0001). Scale bars: 50 μm (F); 100 μm (C and E); 10 μm (IF images).

Figure 3. Mice with forced TNK1 expression show massive apoptosis at the base of the intestinal crypts.

(A–C) Graphs display elevated mRNA levels of proapoptotic (Bax) and prosurvival (Xiap, Bcl2) genes in the small and large intestine (n = 10 per group) of TNK1-expressing mice. (D) Western blots show expression of cC3 and its substrate cPARP in the small intestine and colon (n = 3). (E and F) Representative images of the murine small and large intestine show immunoreactivity for the apoptotic marker cC3 (arrowheads point to cC3-positive cells). Corresponding charts depict positional quantification of apoptosis (n = 6 per group), which was done according to Buczacki et al. (61) by counting of cells at the crypt (stem cell compartment) and villi. Cells were counted at the base of the crypt (from positions 0 to +4, counting from the bottom of the crypt to the transit-amplifying progenitor cells and the villus area). (G) Expression of the stem cell marker LGR5 in the small and large intestine is also reduced in Tnk1-expressing mice as indicated by quantitative reverse transcriptase PCR (RT-qPCR) analysis (n = 5 per group). (H) Representative images of the crypt-base stem cell compartment positive for cC3 (top panel: white arrows indicate stem and reserve stem [position +4] cells positive for apoptotic marker) or the Ki67 proliferative marker (bottom panel). (I) Crypt phenotype was quantified by counting of regenerating crypts, which are defined as containing at least 5 adjacent Ki67⁺ cells (arrows) contained within a crypt-like structure (34) (n = 10–12 per group). All analyses were performed 24 hours after doxycycline or saline treatment. Data are expressed as mean ± SEM. Differences were tested by parsametric 2-tailed, unpaired Student’s t tests. ANOVA test was applied for multiple-comparison analysis. The mean of each column was compared with the mean of a control column by Dunnett’s multiple-comparisons test. (*P = 0.01–0.05; **P = 0.001–0.01; ***P = 0.0001–0.001; ****P < 0.0001.) Scale bars: 50 (H); 100 μm (E and F).

Figure 4. TNK1-expressing mice show liver, pancreas, and lung damage.

The liver is grossly enlarged (A and B) and shows discoloration (A) due to fat deposits (D). TNK1-expressing mice also show histopathological changes in the liver (n = 10 per group). (A–E) Hepatocyte ballooning, degeneration, and cell death besides inflammatory infiltrates are observed in murine liver samples 24 hours after doxycycline administration. Representative images of H&E- and cC3-stained liver are shown (A). (F–H) TNK1-expressing mice exhibit signs of mild edematous pancreatitis (F, left panels) with localized areas of tissue apoptosis (F, middle panels) (n = 10 per group). There is also an increase and apical accumulation of zymogen granules in acinar cells (F, right panel, electron micrograph). (G and H) Application of a pancreatitis score confirms higher interstitial edema (G) and inflammatory infiltrates (H). (I and J) Signs of emphysema, pulmonary edema, congestion, and cell death accompanied by inflammation are observed in lungs after 24 hours of doxycycline exposure (n = 10 per group). The corresponding chart depicts scoring of the lung damage. (K) Recombinant TNK1 is not expressed in the lung, as compared with high expression in the intestine. Representative Western blot is shown (n = 3). All experimental procedures were conducted 24 hours after exposure of mice to saline or doxycycline. Data are expressed as mean ± SEM. Differences were tested by parametric 2-tailed, unpaired Student’s t tests (*P = 0.01–0.05; **P = 0.001–0.01; ***P = 0.0001–0.001; ****P < 0.0001). Scale bars: 100 μm.

Figure 5. TNK1 expression is dysregulated in response to stress across various species.

(A–C) TNK1 is expressed in the small and large intestine in response to polytrauma. Analysis of TNK1 was assessed by RT-qPCR at the mRNA level (A and B) and by immunohistochemistry at the protein level (C) (n = 10–12 per group). HS, hemorrhagic shock; PT, polytrauma. (D and E) TNK1 protein and transcript levels are also increased in the ileal tissue under septic conditions (cecal ligation and puncture [CLP] model). (D) Representative images of ileal sections from CLP mice show increased immunoreactivity for TNK1. (E) Corresponding charts depict quantification of the stained area or TNK1 mRNA level in the ileal tissue of sham and septic mice (n = 8 per group). (F and G) TNK1 expression was detectable in the gut of pigs subjected to hemorrhagic shock. Representative images of porcine gut sections stained against TNK1 (F) and corresponding quantification graphs (G) are shown (n = 6–8 per group). (H and I) TNK1 is also elevated in patients suffering from Crohn’s disease (CD) as shown by immunohistochemistry. Representative images (H) and quantification (I) show TNK1 protein expression or its absence in epithelial cells of ileum and colon from healthy individuals and CD patients. Three independent patients per group were investigated, leading to total n = 6 per disease state. Data are expressed as mean ± SEM. Differences were tested by parametric 2-tailed, unpaired Student’s t tests. ANOVA test was applied for multiple-comparison analysis. The mean of each column was compared with the mean of a control column by Dunnett’s multiple-comparisons test. (*P = 0.01–0.05; **P = 0.001–0.01; ****P < 0.0001.) Scale bars: 50 μm (C, D, and F); 100 μm (H).

Figure 6. TNK1 expression results in activation of transcription factors STAT3 and NF-κB.

(A) Graphs show elevated IL6 transcript (n = 8–10 per group) in the small and large intestine and a massive increase of plasma IL-6 (n = 5 per group). (B) Charts show increased levels of Tnfa in the colon and small intestine of TNK1-expressing mice (n = 8–10 per group). (C–E) TNK1 expression results in phosphorylation and activation of transcription factor STAT3. (C) Representative images of small and large intestine stained for STAT3 and phosphorylated STAT3 (p-STAT3) are shown. Arrows point to nuclear localization of p-STAT3. (D) Representative Western blots show p-STAT3 expression in the small and large intestine 24 hours after TNK1 expression (n = 3). (E) STAT3 phosphorylation is a direct consequence of expression of constitutively active TNK1 (TNK1wt) as indicated by in vitro kinase assay (wt, wild type; kd, kinase dead) (n = 3). (F and G) TNK1 expression also leads to activation and nuclear translocation of NF-κB/p65 subunit. (F) Representative images display nuclear translocation of NF-κB/p65 subunit upon TNK1 expression. (G) Cellular/tissue fractioning shows NF-κB/p65 subunit increase in nuclear fraction upon TNK1 expression (n = 3). Arrows point to the nuclear location of p-STAT3 or NF-κB/p65 subunit. Data are expressed as mean ± SEM. Differences were tested by parametric 2-tailed, unpaired Student’s t tests. ANOVA test was applied for multiple-comparison analysis. The mean of each column was compared with the mean of a control column by Dunnett’s multiple-comparisons test. (*P = 0.01–0.05; **P = 0.001–0.01.) Scale bars: 50 μm (C, small intestine, and F), 100 μm (C, colon).

Figure 7. Infliximab abolishes TNK1-induced apoptosis in the small intestine and decreases programmed cell death in the colon.

(A) Schematic representation of the infliximab/doxycycline treatment time frame. Infliximab (IFX; 6.25 μg/g) was administered (day 1) i.v. into the tail vein of 8-week-old animals with matched genotype. Twenty-four hours (day 3) after IFX treatment, doxycycline (Dox; 50 μg/g) or saline was administered i.p. Mice were sacrificed on the fourth day after the start of the treatment. (B) IFX-pretreated mice revealed an improved clinical picture with a significantly lower weight loss (n = 6–10 per group). (C) IFX pretreatment protects architecture of small intestine from the TNK1-induced damage as indicated by H&E staining. Colonic mucosa is only partially preserved under the same treatment condition. The TNK1 expression is not affected by TNF-α neutralization. (D) IFX pretreatment protects intestinal barrier as indicated by the normal distribution of the tight and adherens junction proteins E-cadherin and claudin-1. (E) TNF-α neutralization impairs TNK1-induced apoptosis. Representative images of the murine small and large intestine show immunoreactivity for the apoptotic marker cleaved caspase-3 (indicated by arrowheads). (F) Corresponding charts depict quantification of cell death (n = 6–10 per group). Apoptotic cells were counted at the base of the crypt (Cr) and villi (Vi). (G) IFX pretreatment prevents stem cell loss as indicated by RT-qPCR for the stem cell marker LGR5 (n = 10 per group). (H) TNF-α induction upon TNK1 expression was strongly attenuated in small intestine and colon (n = 8–10 per group). Data are expressed as mean ± SEM. Differences were tested by parametric 2-tailed, unpaired Student’s t tests. ANOVA test was applied for multiple-comparison analysis. The mean of each column was compared with the mean of a control column by Dunnett’s multiple-comparisons test. (*P = 0.01–0.05; **P = 0.001–0.01; ***P = 0.0001–0.001; ****P < 0.0001.) Scale bars: 50 μm (E, insets); 100 μm (C and E); 10 μm (IF images).

Figure 8. TNK1-deficient mice (VilCreTNK1^–/–) are less susceptible to DSS-induced colitis.

(A) Scheme of the knockout approach. (B and C) TNK1 knockout abolishes TNK1 expression, as shown in mini–gut organoids (A, bottom) and intestine from VilCreTnk1^–/– mice (mRNA [B] and protein [C]). In response to DSS-induced colitis, TNK1^fl/fl mice show increased TNK1 expression as compared with untreated TNK1^fl/fl mice (n = 8–10 per group): (B) mRNA and (C) protein. (D) Representative images of H&E-stained colonic sections of TNK1^fl/fl mice with colitis display increased wall thickness, distortion of the crypt architecture, formation of crypt abscess with the loss of goblet cells, and diffuse infiltration with mononuclear cells. In contrast, sections of VilCreTnk1^–/– display less severe acute colonic pathology with focal leukocyte infiltration. (E and F) Corresponding graphs represent scoring and grading of inflammation-associated histological changes (E) and quantification of mucin-positive goblet cells (F) (n = 5–10 per group). Leukocyte infiltration was confirmed by CD45 staining (D, bottom) and (G) quantification of the stained area (n = 5–10 per group). (H) TNK1^fl/fl mice with colitis show impaired intestinal barrier as indicated by E-cadherin and claudin-1 staining. (I and J) Consistently with the lower histological score, VilCreTnk1^–/– mice show decreased levels of the proinflammatory cytokines IL6 and Tnfa in colonic tissue (n = 8–10 per group). Data are expressed as mean ± SEM. Differences were tested by parametric 2-tailed, unpaired Student’s t tests. ANOVA test was applied for multiple-comparison analysis. The mean of each column was compared with the mean of a control column by Dunnett’s multiple-comparisons test. (*P = 0.01–0.05; **P = 0.001–0.01; ***P = 0.0001–0.001; ****P < 0.0001.) Scale bars: 50 μm (C); 100 μm (D); 10 μm (IF images).