Inhibition of long myosin light-chain kinase activation alleviates intestinal damage after binge ethanol exposure and burn injury

Laboratory evidence suggests that intestinal permeability is elevated following either binge ethanol exposure or burn injury alone, and this barrier dysfunction is further perturbed when these insults are combined. We and others have previously reported a rise in both systemic and local proinflammatory cytokine production in mice after the combined insult. Knowing that long myosin light-chain kinase (MLCK) is important for epithelial barrier maintenance and can be activated by proinflammatory cytokines, we examined whether inhibition of MLCK alleviated detrimental intestinal responses seen after ethanol exposure and burn injury. To accomplish this, mice were given vehicle or a single binge ethanol exposure followed by a sham or dorsal scald burn injury. Following injury, one group of mice received membrane permeant inhibitor of MLCK (PIK). At 6 and 24 h postinjury, bacterial translocation and intestinal levels of proinflammatory cytokines were measured, and changes in tight junction protein localization and total intestinal morphology were analyzed. Elevated morphological damage, ileal IL-1β and IL-6 levels, and bacterial translocation were seen in mice exposed to ethanol and burn injury relative to either insult alone. This increase was not seen in mice receiving PIK after injury. Ethanol-exposed and burn-injured mice had reduced zonula occludens protein-1 and occludin localization to the tight junction relative to sham-injured mice. However, the observed changes in junctional complexes were not seen in our PIK-treated mice following the combined insult. These data suggest that MLCK activity may promote morphological and inflammatory responses in the ileum following ethanol exposure and burn injury.     

Estrogen prevents intestinal inflammation after trauma-hemorrhage via downregulation of angiotensin II and angiotensin II subtype I receptor

Although angiotensin II (Ang II) plays a key role in development of organ ischemia-reperfusion injury, it remains unclear whether it is involved in development of intestinal injury following trauma-hemorrhage (T-H). Studies have shown that 17beta-estradiol (E2) administration following T-H improves small intestinal blood flow; however, it is unclear whether Ang II plays a role in this E2-mediated salutary effect. Male Sprague-Dawley rats underwent laparotomy and hemorrhagic shock (removal of 60% total blood volume, fluid resuscitation after 90 min). At onset of resuscitation, rats were treated with vehicle, E2, or E2 and estrogen receptor antagonist ICI 182,780 (ICI). A separate group of rats was treated with Ang II subtype I receptor (AT1R) antagonist losartan. At 24 h after T-H, plasma Ang II, IL-6, TNF-alpha, intercellular adhesion molecule (ICAM)-1, cytokine-induced neutrophil chemoattractant (CINC)-1 and CINC-3 levels, myeloperoxidase (MPO) activity, and AT1R expression were determined. T-H significantly increased plasma and intestinal Ang II, IL-6, TNF-alpha levels, intestinal ICAM-1, CINC-1, CINC-3 levels, MPO activity, and AT1R protein compared with shams. E2 treatment following T-H attenuated increased intestinal MPO activity, Ang II level, and AT1R protein expression. ICI administration abolished the salutary effects of E2. In contrast, losartan administration attenuated increased MPO activity without affecting Ang II and AT1R levels. Thus Ang II plays a role in producing small intestine inflammation following T-H, and the salutary effects of E2 on intestinal inflammation are mediated in part by Ang II and AT1R downregulation.     

Trauma-hemorrhage inhibits splenic dendritic cell proinflammatory cytokine production via a mitogen-activated protein kinase process

Although splenic dendritic cell (DC) functions are markedly altered following trauma-hemorrhage, the mechanism(s) responsible for the altered DC functions remains unknown. We hypothesized that trauma-hemorrhage inhibits DC function via suppressing toll-like receptor 4 (TLR4) expression and mitogen-activated protein kinases (MAPKs). To examine this, male C3H/HeN (6-8 wk) mice were randomly assigned to sham operation or trauma-hemorrhage. Trauma-hemorrhage was induced by midline laparotomy and approximately 90 min of hypotension [blood pressure (BP) 35 mmHg], followed by fluid resuscitation (4x the shed blood volume in the form of Ringer lactate). Two hours later, mice were euthanized, splenic DCs were isolated, and the changes in their MAPK activation, TLR4-MD-2 expression, and ability to produce cytokines were measured. The results indicate that trauma-hemorrhage downregulated the lipopolysaccharide (LPS)-induced MAPK activation in splenic DCs. In addition to the decrease in MAPK activation, surface expression of TLR4-MD-2 was suppressed following trauma-hemorrhage. Furthermore, LPS-induced cytokine production from splenic DCs was also suppressed following trauma-hemorrhage. These findings thus suggest that the decrease in TLR4-MD-2 and MAPK activation may contribute to the LPS hyporesponsiveness of splenic DCs following trauma-hemorrhage. Hyporesponsiveness of splenic DCs was also found after stimulation with the TLR2 agonist zymosan. Our results may thus explain the profound immunosuppression that is known to occur under those conditions.     

Mechanism of estrogen-mediated intestinal protection following trauma-hemorrhage: p38 MAPK-dependent upregulation of HO-1

p38 MAPK has been reported to regulate the inflammatory response in various cell types via extracellular stimuli. p38 MAPK activation also results in the induction of heme oxygenase (HO)-1, which exerts potent anti-inflammatory effects. Although studies have shown that 17beta-estradiol (E(2)) prevented organ dysfunction following trauma-hemorrhage, it remains unknown whether p38 MAPK/HO-1 plays any role in E(2)-mediated attenuation of intestinal injury under those conditions. To study this, male rats underwent trauma-hemorrhage (mean blood pressure approximately 40 mmHg for 90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were treated with vehicle, E(2) (1 mg/kg body wt), the p38 MAPK inhibitor SB-203580 (2 mg/kg body wt) or E(2) plus SB-203580. Two hours thereafter, intestinal myeloperoxidase (MPO) activity and lactate, TNF-alpha, IL-6, ICAM-1, cytokine-induced neutrophil chemoattractant (CINC)-1, and macrophage inflammatory protein (MIP)-2 levels were measured. Intestinal p38 MAPK and HO-1 protein levels were also determined. Trauma-hemorrhage led to an increase in intestinal MPO activity and lactate, TNF-alpha, IL-6, ICAM-1, CINC-1, and MIP-2 levels. This was accompanied with a decrease in intestinal p38 MAPK activity and increase in HO-1 expression. Administration of E(2) normalized all the above parameters except HO-1, which was further increased following trauma-hemorrhage. Administration of SB-203580 with E(2) abolished the E(2)-mediated restoration of the above parameters as well as the increase in intestinal HO-1 expression following trauma-hemorrhage. These results suggest that the p38 MAPK/HO-1 pathway plays a critical role in mediating the salutary effects of E(2) on shock-induced intestinal injury.     

First Report of bla CTX-M-15-Type ESBL-Producing Klebsiella pneumoniae in Wild Migratory Birds in Pakistan

EcoHealth - We investigated wild migratory birds faecal swabs for extended-spectrum β-lactamases-producing Klebsiella pneumoniae (ESBL-K. pneumoniae) from wetland habitats in Pakistan. ESBL-K....     

Tissue compartment-specific role of estrogen receptor subtypes in immune cell cytokine production following trauma-hemorrhage.

Although 17beta-estradiol administration following trauma-hemorrhage attenuates plasma cytokines and alteration in immune cell cytokine production, it is not known whether the salutary effects are mediated via estrogen receptor (ER)-alpha or ER-beta. Accordingly, we examined which ER subtype predominantly mediates the salutary effects of 17beta-estradiol on systemic inflammatory response/immune cell cytokine production in various tissues following trauma-hemorrhage. Male rats underwent trauma-hemorrhage (mean blood pressure: 40 mmHg for 90 min) and fluid resuscitation. The ER-alpha agonist propyl pyrazole triol (PPT; 5 microg/kg), the ER-beta agonist diarylpropionitrile (DPN; 5 microg/kg), 17beta-estradiol (50 microg/kg), or vehicle (10% DMSO) was injected subcutaneously during resuscitation, and various measurements were made 24 h thereafter. 17beta-Estradiol or PPT administration following trauma-hemorrhage prevented the increase in plasma IL-6 and IL-10 levels that were observed in vehicle-treated animals. IL-6 and TNF-alpha production by Kupffer cells increased; however, splenic macrophages (SMPhi), alveolar macrophages (AMPhi), and peripheral blood mononuclear cells (PBMC) had decreased release of these cytokines after trauma-hemorrhage. IL-10 production, however, increased in all macrophage populations. Administration of 17beta-estradiol following trauma-hemorrhage prevented all of these alterations. PPT had the same effects as 17beta-estradiol on IL-6 and TNF-alpha production by Kupffer cells and SMPhi, and DPN had the same effects on AMPhi and PBMC. The same effects as 17beta-estradiol on IL-10 production were observed by PPT on Kupffer cells and DPN on PBMC. Both agonists were equally effective on SMPhi and AMPhi. Thus ER subtypes have tissue compartment-specific roles in mediating the effects of 17beta-estradiol on immune cell functions following trauma-hemorrhage.     

Alteration in intestine tight junction protein phosphorylation and apoptosis is associated with increase in IL-18 levels following alcohol intoxication and burn injury

Intestinal mucosal barrier is the first line of defense against bacteria and their products originating from the intestinal lumen. We have shown a role for IL-18 in impaired gut barrier function following acute alcohol (EtOH) intoxication combined with burn injury. To further delineate the mechanism, this study examined whether IL-18 alters intestine tight junction proteins or induces mucosal apoptosis under these conditions. To accomplish this, rats were gavaged with EtOH (3.2 g/kg) prior to ~ 12.5% total body surface area burn or sham injury. One day after injury, EtOH combined with burn injury resulted in a significant decrease in total occludin protein and its phosphorylation in small intestine compared to either EtOH or burn injury alone. There was no change in claudin-1 protein content but its phosphorylation on tyrosine was decreased following EtOH and burn injury. This was accompanied with an increase in mucosal apoptosis (p < 0.05). The treatment of rats with anti-IL-18 antibody at the time of burn injury prevented intestine apoptosis and normalized tight junction proteins following EtOH and burn injury. Altogether, these findings suggest that IL-18 modulates tight junction proteins and cause apoptosis leading to impaired intestinal mucosal integrity following EtOH intoxication combined with burn injury.