I/R of the small intestine occurs in clinical settings and animal models of hemorrhagic shock, for example, through superior mesenteric artery occlusion (SMAO). I/R leads to the systemic inflammatory response syndrome, sepsis, and multiple organ failure (1, 2). The underlying pathomechanisms proposed are hypoxia- and reoxygenation-induced microvascular permeability increase and mucosal barrier dysfunction, leading to systemic translocation of intestinal bacteria and endotoxin (3 and references therein). An additional pathomechanism is thought to be gut ischemia triggering an intestinal proinflammatory response that turns the gut into a source of cytokines and tissue injury factors (4-6). However, the molecular mechanisms and networks promoting the dysfunction of the gut barrier after reperfusion are still poorly understood.
The Janus kinase (Jak) and signal transducer and activator of transcription (STAT) pathways are activated by various cytokines and growth factors (7). The Jak family of nonreceptor tyrosine kinases includes Jak1, Jak2, Jak3, and Tyk2 (7, 8). The best-known substrates of Jaks are one or more of the seven members of the STAT protein family. STATs are activated by phosphorylation of tyrosine and serine residues (8, 9). Activated homodimerized or heterodimerized STATs translocate to the nucleus and bind to DNA elements of STAT-responsive genes.
Jak-STAT signaling is involved in I/R injury, albeit its contribution is reportedly contradictory in various I/R models, that is, either deleterious (10-12) or protective (12-14). So far, the role of Jak-STAT members in intestinal I/R injury has not been addressed. Tyk2, STAT1, type I interferons (IFN-α/IFN-β), and, to a lower degree, type 2 IFN (IFN-γ) are important for the progression of endotoxin shock (15, 16). Tyk2−/− mice are partially deficient in IFN-α/IFN-β and IFN-γ responses (17-19). STAT1−/− mice lack responses to both types of IFNs (20, 21). Here, we demonstrate that Tyk2 and STAT1 substantially contribute to intestinal I/R-caused pathophysiology. This is shown with gene-targeted compared with wild-type (WT) mice in vivo by increased survival, on the cellular level by the lack of destruction of intestinal structures and decreased tissue neutrophil infiltration, and on the molecular level by decreased expression of adhesion proteins and proteases known to promote inflammatory processes.
MATERIALS AND METHODS
Tyk2−/− mice were (17) back-crossed for more than 10 generations to C57BL/6. STAT1−/− mice were on mixed background 129S2/Sv × C57BL/6 (20) and kindly provided by D.E. Levy (New York University School of Medicine, New York, NY). Interferon α/β receptor chain 1 (IFNAR1)−/− mice (22) on C57BL/6 genetic background were kindly provided by U. Kalinke (Paul Ehrlich Institute, Langen, Germany), and their use was limited only to the survival study (data not shown; "Discussion"). For WT controls, nongene-targeted littermates from the respective heterozygous crosses were used. All animals were housed under specific pathogen-free conditions according to Federation of European Laboratory Animal Science Associations guidelines. Before the experiment, animals were fasted overnight but allowed water ad libitum. All animal experiments were carried out in accordance with protocols approved by the Austrian Laws and European Directives.
SMAO and experimental design
Mice were anesthetized with an i.p. application of ketamine/xylazine (200 mg kg−1 per 8 mg kg−1 body weight) and subjected to SMAO as previously described (23). Briefly, a median laparotomy was performed, and the SMA was isolated from the surrounding connective tissue near its aortic origin and occluded with an atraumatic microclip. The intestines remained pale during the occlusion, and there was no venous stasis or congestion. After 45 min of intestinal ischemia, the microclip was removed to allow reperfusion of the small intestine, and the abdomen was closed in two layers. During this procedure, the intestinal tract was maintained at 37°C by placing it between gauze pads soaked with warmed 0.9% NaCl. The animals were then placed in their cages with free access to water and standardized laboratory food, and survival was monitored for 24 h. The experimental groups for the survival study were as follows: (1) mice of the respective genotypes (n = 30) were subjected to SMAO; and (2) mice of the respective genotypes (n = 30) were subjected to identical surgical procedure except the artery was not occluded and the mice were maintained under anesthesia until the end of the experiment (sham control). In separate experiments, mice were killed at 1 and 4h after reperfusion and used (1) for histology and immunohistochemistry (n = 4) or (2) for assaying myeloperoxidase (MPO; n = 6).
Microscopy and histological scoring of injury
For light microscopy, tissue samples of the small intestine were taken immediately from the killed animals, fixed in 4% buffered formalin, and embedded in paraffin. Serial sections (3-μm thick) were cut for evaluation of the intestinal morphology using routine hematoxylin-eosin staining. The sections were scored for evidence of histological injury as described (24): 0, normal histology; 1, slight disruption of the surface epithelium; 2, epithelial cell loss at the villus tip; 3,mucosal vasocongestion, hemorrhage, and focal necrosis, with loss of less than one half of villi; and 4, damage extending to more than one half of villi. The scores from two experiments and two separate segments were averaged (n = 8). For electron microscopy, tissue samples of the small intestine were fixed in3% glutaraldehyde, postfixed with 2% osmium tetroxide in Soerensen buffer (pH 7.4), dehydrated, and then embedded in Epon resin. Semithin intestinal sections were stained with toluidine blue. Ultrathin sections were stained in 2% uranyl acetate and 2.7% lead citrate and examined under a transmission electron microscope (EM900; Zeiss, Jena, Germany).
The primary antibodies included rat monoclonal anti-intracellular adhesion molecule (ICAM) 1 (clone KAT-1, R and D Systems, Oxfordshire, U.K.; 1:100), goat polyclonal anti-P-selectin (CD62P; Santa Cruz Biotechnology, Santa Cruz, Calif; 1:100), rat monoclonal antimouse neutrophil (clone 7/4; ImmunoKontact, Bioggio Lugano, Switzerland; 1:500), and rabbit polyclonal antimatrix metalloproteinase (anti-MMP; all from NeoMarkers, Fremont, Calif) MMP-2 (1:200), MMP-9 (1:800), and MMP-14 (1: 400) antibodies. To detect ICAM-1 and neutrophils, formalin-fixed and paraffin-embedded tissue sections were heated in citrate buffer (0.01M; pH 6.0) for 3 × 5 min in a microwave oven for antigen retrieval. For the detection of P-selectin, an EDTA solution (1 mM; pH 8.0) was used for heat denaturation. Biotinylated rat (mouse adsorbed) and goat antibody (Vectastain; Vector, Torrance, Calif) were used as secondary antibodies. Diaminobenzidine with 0.03% H202 in TRIS buffer (0.05 M; pH 7.4) was used as chromogen. Sections were counterstained with Mayers Hemalumn. Negative controls were performed by omitting the primary antibody.
Myeloperoxidase content was assayed as described previously (25). Intestinal tissue (50 mg) was homogenized in 2mL of homogenization buffer (3.4 mM KH2HPO4, 16 mM Na2HPO4; pH 7.4). After centrifugation for 20 min at 10,000g, 10 volumes of resuspension buffer (43.2 mM KH2HPO4, 6.5 mM Na2HPO4, 10 mM EDTA, 0.5% hexadecyltrimethylammonium; pH 6.0) was added to the pellet, and the sample was sonicated for 10 s. After heating for 2 h at 60°C, the supernatant was reacted with 3,3′, 3, 5′-tetramethylbenzidine (Sigma-Aldrich, Vienna, Austria), and the optical density was measured at 655 nm.
Differences in survival were tested between individuals in SMAO-operated WT groups versus SMAO-operated gene-target groups, and the total number of survivorswas tested for significant deviation from equality between groups using the Fisher exact test (or probability test) assuming a hypergeometric distribution. With theFisher method (26), individual P values were combined. Histological injury score and MPO activity of Tyk2−/−, STAT1−/−, and WT mice were analyzed with ANOVA.
Tyk2- and STAT1-deficient mice show increased resistance to intestinal I/R injury
To determine the role of Tyk2 and STAT1 in an intestinal ischemia model, the SMA of WT and mutant mice was occluded for 45 min followed by 24 h of reperfusion. Most WT mice succumbed to intestinal I/R injury, whereas Tyk2−/− and STAT1−/− mice showed significantly increased survival rates of 60% and 80%, respectively (Table 1). Sham-operated mice survived the entire 24 h observation period irrespective of their genotype. The survival data of the mutant mice clearly demonstrate the involvement of Tyk2 and STAT1 in the progression of intestinal I/R shock.
Intact intestinal tissue architecture upon I/R in Tyk2- and STAT1-deficient mice
Histological sections of WT intestines showed severe I/R-induced damage. Already in the early stage of reperfusion (1h), mucosal slough at the villus tips together with capillary congestion was prominent (Fig. 1, A and B). After 4 h of reperfusion, the destruction ranged from extended subepithelial space with detached epithelial cell sheets at the tips of the villi to denuded villi with exposed lamina propria. In some cases, complete disintegration of the intestinal villi was observed (Fig. 1, A and B). In contrast, jejunum sections of Tyk2-deficient mice did not show I/R-induced injury and had normal architecture of the villus even after 4 h of reperfusion (Fig. 1A). STAT1-deficient mice were similarly protected from intestinal damage (Fig. 1B). Normal intestinal villi were detected in both groups of sham-operated animals (Fig. 1, A and B). The degree of I/R-induced mucosal damage expressed as a microscopic injury score is shown in the respective lower panels of Figure 1, which coincides with our light microscopic observations. Thus, the integrity of the intestinal barrier observed in the gene-targeted mice indicates the contribution of Tyk2 and STAT1 to the I/R-associated gut barrier dysfunction.
Tyk2 and STAT1 contribute to the intestinal epithelial cell destruction during I/R injury
Electron microscopy showed an irregular and disintegrated brush border at the luminal surface of the villus epithelium in WT mice after 1 h of reperfusion (Fig. 2). Whole enterocyte cell layers were detached from the basal membrane and showed clear indications of destruction, that is, balloon-like subnuclear blebbing and loss of ribosomes (Fig. 2). In Tyk2-deficient mice, the epithelial cells remained intact and showed a regular brush border (Fig. 2). The enterocytes were firmly attached to the basal membrane (Fig. 2). Similar observations were made for STAT1−/− mice (data not shown). Normal intestinal epithelial cells were detected in both groups of sham-operated animals (data not shown). Thus, the deterioration of the intestinal epithelial cells during I/R injury is Tyk2- and STAT1-dependent.
Reduced expression of MMPs in the reperfused intestine of Tyk2- and STAT1-deficient mice
The biological activity of MMPs, especially of MMP-2, MMP-9, and MMP-14, facilitates the transmigration of activated cells into inflamed tissues (27). The breakdown of the intestinal tissue integrity upon I/R is also at least in part attributed to MMP activity (28, 29). After 1 h of reperfusion in intestinal immunostained sections of WT mice, a strong expression of MMP-2, MMP-9, and MMP-14 was found in the intestinal epithelial cells (Fig. 3, A and B). In intestinal sections from Tyk2-deficient mice, MMP staining could not be detected (Fig. 3A). A similar situation was found in STAT1-deficient mice (Fig. 3B). Intestinal sections from both groups of sham-operated mice did not reveal any MMP expression (data not shown). These data demonstrate that Tyk2 and STAT1 are required for MMP-2, MMP-9, and MMP-14 expressions during intestinal I/R injury.
P-selectin and ICAM-1 induction upon intestinal I/R is dependent on Tyk2 and STAT1
The adhesion molecules ICAM-1 and P-selectin have been shown to be central and up-regulated in several I/R models (30, 31). Thus, their expression was monitored in the reperfused intestines by immunohistochemistry. After 4 h of reperfusion, a strong staining for P-selectin and ICAM-1 in the endothelial cells of postcapillary venules of WT mice was found (Fig. 4). The levels of both proteins were especially induced during the late I/R because less immunostaining could be detected after 1 h of reperfusion (data not shown). In contrast, intestinal sections from Tyk2- and STAT1-deficient mice did not reveal any up-regulation, although basal expression of P-selectin and ICAM-1 was detectable (Fig. 4). Thus, Tyk2 and STAT1 are required for the induction of both adhesion molecules upon intestinal I/R injury.
Tyk2 and STAT1 increase neutrophil recruitment in the reperfused intestine
Consistent with the induced high expression of adhesion molecules, WT mice showed a significant increase in neutrophils (measured by MPO content) in the intestine after intestinal ischemia and 4 h of reperfusion. Tyk2- (Fig. 5A) and STAT1-deficient (Fig. 5B) mice showed a significant reduction of MPO content upon I/R. This demonstrates the participation of Tyk2 and STAT1 in the complex mechanisms leading to neutrophil migration into postischemic intestine and in the consequent tissue MPO accumulation.
To our knowledge, this is the first report of an involvement of the Jak-Stat axis components Tyk2 and STAT1 in the progression of intestinal I/R-induced shock. Other studies also place Jaks and/or STATs as critical signaling molecules in the context of ischemic injury, albeit their contribution to the outcome of I/R-induced shock is reportedly conflicting. Concerning the Jak family, Jak2 was the only member studied and found to promote not only inflammation (32) and apoptosis (10) but also cytoprotection (13) in heart and cerebral ischemia. These studies were performed with small organic kinase inhibitors. Hence, our study provides the first in vivo genetic evidence for a Jak family member driving ischemic pathomechanisms and adds Tyk2 to the list of molecules involved. With respect to STATs, several studies in gene-deficient mice have been performed. In analogy to our findings, a deleterious role of STAT1 in brain I/R (11) has been found. STAT6 is reported to contribute to renal I/R (12), whereas STAT3 and STAT4 functions protect from cardiac and renal I/R, respectively (12, 14). A contrasting role for STAT1 (deleterious) and STAT3 (protective) in myocardium I/R injury has been further established with mice expressing constitutively active STAT3 and with extensive tissue culture work on STAT1 (33, 34). Thus, Tyk2 and STATs 1/3/4 are proposed as promising targets for therapeutic intervention in I/R conditions, especially the results emerging from studies with genetically engineered mice.
The survival of Tyk2- and STAT1-null mice is facilitated by the virtual absence of intestinal tissue and cell lesions observed upon I/R in WT mice. Up-regulation of MMP-2, MMP-9, and MMP-14 (MT1-MMP) has been shown in I/R models, including hind limb, cerebrum, myocardium, and lung (29, 35-37). Consistent with their damage-promoting functions, these MMPs were barely detectable in Tyk2−/− and STAT1−/− mice upon intestinal I/R. In general, MMP production and secretion is up-regulated in vivo during systemic shock conditions (38). IFN α/β and IFN-γ were reported to inhibit MMP-2, MMP-9, and MMP-14 expression in a variety of inflammatory settings (39-41). For MMP-9, the IFN-induced transcriptional down-regulation was shown to be STAT1-dependent (40, 41). On the other hand, systemic administration of Toll-like receptor 9 ligand to IFN-γ−/− mice demonstrated the dependence of MMP-9 transcriptional activation on IFN-γ (42). Our data clearly demonstrate the requirement of the IFN-α/IFN-β and IFN-γ signaling components Tyk2 and STAT1 as positive regulators of MMP-2, MMP-9, and MMP-14 expression during intestinal I/R. A novel prominent positive regulatory role of IFN-α/IFN-β in MMP activation and progression of I/R shock is further supported by preliminary studies of IFNAR1-deficient mice in our model. Interferon α/β receptor chain 1−/− mice are unresponsive to IFN-α/IFN-β and showed a 100% survival rate after intestinal I/R injury (data not shown). Taken together, type 1 and type 2 IFNs seem to have a dual role during inflammatory processes in that they are necessary for the initial up-regulation of MMPs but are also involved in counterregulatory activities.
P-selectin and ICAM-1 are major players in endothelium-regulating neutrophil trafficking. During inflammatory responses, including the early phase of reperfusion, P-selectin is released and allows the leukocytes to roll along the endothelium (43). ICAM-1, which is constitutively expressed on the surface of endothelial cells, is then involved in neutrophil adhesion. Many inflammatory mediators, including LPS, H2O2, ILs, TNF-α, and IFN-γ, have been shown to increase P-selectin (44, 45) and ICAM-1 expression (46, 47). Here, we demonstrate the requirement of Tyk2 and STAT1 in the signaling cascade(s) leading to the expression and/or up-regulation of P-selectin and ICAM-1 during intestinal I/R. As a consequence of the absent adhesion molecules in the mutant mice, neutrophil infiltration into the lamina propria of the postischemic intestine was reduced.
Understanding of cellular and molecular signaling events underlying I/R injury is crucial for the development of new therapeutic strategies. In mice, it is clear that Tyk2 and STAT1 are critical players in the progression of ischemic injury. This provides the rationale for the search for selective blockers of Tyk2 and/or STAT1 as potential therapeutic adjuncts in protecting ischemic tissues. Our studies and studies from another laboratory with an endotoxin-induced shock model suggest a mechanistic role of Tyk2 and STAT1 in both the production of and/or response to type 1 and type 2 IFNs during pathogenesis (15, 16). Here, the survival studies with IFNAR1−/− mice provide preliminary evidence for a previously unrecognized central role of type I IFN response in ischemic injury. Further studies will focus on the relative importance of type 1 and type 2 IFNs and their signaling components in I/R pathomechanisms.
The authors thank Robert Lajko for his technical support. M.M. is supported by the FWF SFB F28, the Austrian Federal Ministry for Science and Research (BM_WFa GZ200.112/1-VI/1/2004), and the Viennese Foundation for Science, Research and Technology (WWTF grant LS133).
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