HIF-1 mediates pathogenic inflammatory responses to intestinal ischemia-reperfusion injury

Abstract

Acute lung injury (ALI) and the development of the multiple organ dysfunction syndrome (MODS) are major causes of death in trauma patients. Gut inflammation and loss of gut barrier function as a consequence of splanchnic ischemia-reperfusion (I/R) have been implicated as the initial triggering events that contribute to the development of the systemic inflammatory response, ALI, and MODS. Since hypoxia-inducible factor (HIF-1) is a key regulator of the physiological and pathophysiological response to hypoxia, we asked whether HIF-1 plays a proximal role in the induction of gut injury and subsequent lung injury. Utilizing partially HIF-1α-deficient mice in a global trauma hemorrhagic shock (T/HS) model, we found that HIF-1 activation was necessary for the development of gut injury and that the prevention of gut injury was associated with an abrogation of lung injury. Specifically, in vivo studies demonstrated that partial HIF-1α deficiency ameliorated T/HS-induced increases in intestinal permeability, bacterial translocation, and caspase-3 activation. Lastly, partial HIF-1α deficiency reduced TNF-α, IL-1β, cyclooxygenase-2, and inducible nitric oxide synthase levels in the ileal mucosa after T/HS whereas IL-1β mRNA levels were reduced in the lung after T/HS. This study indicates that prolonged intestinal HIF-1 activation is a proximal regulator of I/R-induced gut mucosal injury and gut-induced lung injury. Consequently, these results provide unique information on the initiating events in trauma-hemorrhagic shock-induced ALI and MODS as well as potential therapeutic insights.

Fig. 1.

Histological criteria used to grade villous damage following trauma hemorrhagic shock (T/HS). 0 = normal mucosa, 1 = development of subepithelial Gruenhagen's space and vacuolization at the villus tip, 2 = extension of the subepithelial space with moderate lifting of epithelial layer from the lamina propria, 3 = massive subepithelial lifting/ sloughing and increased vacuolization from the tip to midportion of villi, 4 = epithelial lifting and vacuolization from the tip to lower portion of villi, and 5 = mucosal ulceration and disintegration of the lamina propria. The shorter arrows depict the presence of vacuoles, the longer arrows depict the subepithelial and epithelial lifting, and the stars depict mucosal ulceration. Magnification ×400.

Fig. 2.

Induction of hypoxia-inducible factor (HIF)-1α- and HIF-1-dependent genes in the ileal mucosa after T/HS. Wild-type (WT) and HIF-1α^+/− mice were subjected to T/HS or sham shock (T/SS) for 60 min and 3 h reperfusion. A: relative levels of HIF-1α, GLUT1, and cyclooxygenase-2 (COX-2) mRNA levels in the ileal mucosa were determined by real-time PCR using the ΔΔCt method by using a log₂ scale. Mean values ± SE are shown (n = 4–15 mice/group). B: whole cell extracts (WCEs) prepared from mucosal scrapings from the distal ileum were examined for HIF-1α, inducible nitric oxide synthase (iNOS), and total MAPK expression by Western blot analysis. A nonspecific (NS) band (70 kDa) recognized by iNOS antibody also confirmed equal loading of our samples. Numbers refer to individual mice.

Fig. 3.

Partial HIF-1α deficiency attenuates T/HS-induced gut injury. WT and HIF-1α^+/− mice were subjected to T/HS or T/SS for 60 min and 3 h reperfusion. A: representative sections of hematoxylin and eosin (H&E) staining of the distal ileum (×200). Arrows depict subepithelial lifting sloughing and mucosal edema in the villi. B: percentage of villous injury. C: histological scoring of villous damage. Values in B and C are expressed as means ± SE (n = 7–11 mice/group). D: MPO activity (U/mg of protein) was measured in the ileal mucosa. Values are expressed as means ± SE (n = 5–10 mice/group).

Fig. 4.

Partial HIF-1α deficiency attenuates T/HS-induced lung injury. WT and HIF-1α^+/− mice were subjected to T/HS or T/SS for 60 min and 3 h reperfusion. A: representative section of H&E staining of the lung. B: lung injury score. C: number of infiltrating polymorphonuclear cells (PMNs) per high-power field (hpf). D: MPO activity (U/mg of protein) was measured in the lung. Values in B–D are expressed as means ± SE (n = 6–13 mice/group).

Fig. 5.

HIF-1 directly impairs gut barrier function. A: in vivo intestinal permeability was assessed in WT and HIF-1α^+/− mice subjected to T/SS or T/HS for 60 min and 3 h reperfusion by quantifying 4.4-kDa fluorescein isothiocyanate-dextran (FD-4) concentration (μg/ml) in the serum. Values are expressed as means ± SE (n = 5–8 mice/group). B: bacterial translocation to the mesenteric lymph nodes (MLN) was measured in WT and HIF-1α^+/− mice subjected to T/SS or T/HS. Number of colony forming units of bacteria per gram MLN (CFU/g) and values are expressed as means ± SD (n = 6–8 mice/group).

Fig. 6.

HIF-1 promotes apoptosis during gut ischemia-reperfusion (I/R) injury. A and B: WCEs were prepared from ileal mucosa of WT and HIF-1α^+/− mice subjected to T/HS or T/SS for 60 min and 3 h reperfusion. A: cleaved caspase-3 and total p42/p44 expression was determined by Western blotting. B: densitometry was performed to quantify cleaved caspase-3 and total p42/p44 expression. Data are represented as means ± SE (4–6 mice/group). C: caspase-3 activity is expressed as absorbance at 405 nm per 50 μg of protein. Values are expressed as means ± SE (n = 4–5/group). D: caspase-3/7 activity assay using WCEs derived from HIF-1α and control (Ctl) Caco-2 transfectants exposed to normoxia (N), hypoxia (H) for 3 h, or H followed by reoxygenation for 3 h (H/R). RFU, relative fluorescence units. Mean values ± SE are shown (n = 3–4/condition).

Fig. 7.

HIF-1 differentially regulates proinflammatory cytokines in the ileal mucosa and lung after T/HS. RNA was prepared from ileal mucosa and lung of WT and HIF-1α^+/− mice subjected to T/HS or T/SS for 60 min and 3 h reperfusion. Relative mRNA expression for TNF-α, IL-1β, IL-6, and IL-10 was quantified by real-time PCR using the ΔΔCt method using a log₂ scale. Mean values ± SE are shown (n = 4–7 mice/group).