Infection of Immunocompetent Conventional Mice with Shiga Toxin-Producing E. coli: The DSS + STEC Model.

Previous methods of infecting mice with Shiga toxin-producing E. coli (STEC) required suppression of host immune function or ablation of the gut microbiota to induce susceptibility to gastrointestinal colonization. Consequently, many pathogen-host interactions occurring in immunocompetent hosts during STEC infection and Shiga toxicosis have remained unclear. The following protocol describes the use of dextran sulfate sodium (DSS) to induce a mild colitis in immunocompetent conventional C57BL/6 mice to facilitate susceptibility to STEC infection by oral gavage. STEC colonization in infected mice was confirmed by recovery of live STEC via fecal cultures and quantified via quantitative polymerase chain reaction of fecal DNA for the STEC-specific gene eae. DSS colitis is well established, broadly reproducible, and does not require specialized equipment or high levels of technical proficiency to be a useful method of inducing susceptibility to gastrointestinal STEC colonization. The DSS + STEC mouse model provides a novel and useful tool for the exploration of local STEC-host interactions in the gut environment and the pathogenesis of Shiga toxicosis.

Copper primes adaptation of uropathogenic Escherichia coli to superoxide stress by activating superoxide dismutases.

Copper and superoxide are used by the phagocytes to kill bacteria. Copper is a host effector encountered by uropathogenic Escherichia coli (UPEC) during urinary tract infection in a non-human primate model, and in humans. UPEC is exposed to higher levels of copper in the gut prior to entering the urinary tract. Effects of pre-exposure to copper on bacterial killing by superoxide has not been reported. We hypothesized that copper-replete E. coli is more sensitive to killing by superoxide in vitro, and in activated macrophages. We utilized wild-type UPEC strain CFT073, and its isogenic mutants lacking copper efflux systems, superoxide dismutases (SODs), regulators of a superoxide dismutase, and complemented mutants to address this question. Surprisingly, our results reveal that copper protects UPEC against killing by superoxide in vitro. This copper-dependent protection was amplified in the mutants lacking copper efflux systems. Increased levels of copper and manganese were detected in UPEC exposed to sublethal concentration of copper. Copper activated the transcription of sodA in a SoxR- and SoxS-dependent manner resulting in enhanced levels of SodA activity. Importantly, pre-exposure to copper increased the survival of UPEC within RAW264.7 and bone marrow-derived murine macrophages. Loss of SodA, but not SodB or SodC, in UPEC obliterated copper-dependent protection from superoxide in vitro, and from killing within macrophages. Collectively, our results suggest a model in which sublethal levels of copper trigger the activation of SodA and SodC through independent mechanisms that converge to promote the survival of UPEC from killing by superoxide. A major implication of our findings is that bacteria colonizing copper-rich milieus are primed for efficient detoxification of superoxide.

Transient neonatal antibiotic exposure increases susceptibility to late-onset sepsis driven by microbiota-dependent suppression of type 3 innate lymphoid cells.

Extended early antibiotic exposure in the neonatal intensive care unit is associated with an increased risk for the development of late-onset sepsis (LOS). However, few studies have examined the mechanisms involved. We sought to determine how the neonatal microbiome and intestinal immune response is altered by transient early empiric antibiotic exposure at birth. Neonatal mice were transiently exposed to broad-spectrum antibiotics from birth for either 3- (SE) or 7-days (LE) and were examined at 14-days-old. We found that mice exposed to either SE or LE showed persistent expansion of Proteobacteria (2 log difference, P < 0.01). Further, LE mice demonstrated baseline translocation of E. coli into the liver and spleen and were more susceptible K. pneumoniae-induced sepsis. LE mice had a significant and persistent decrease in type 3 innate lymphoid cells (ILC3) in the lamina propria. Reconstitution of the microbiome with mature microbiota by gavage in LE mice following antibiotic exposure resulted in an increase in ILC3 and partial rescue from LOS. We conclude that prolonged exposure to broad spectrum antibiotics in the neonatal period is associated with persistent alteration of the microbiome and innate immune response resulting in increased susceptibility to infection that may be partially rescued by microbiome reconstitution.

Prenatal arsenic exposure interferes in postnatal immunocompetence despite an absence of ongoing arsenic exposure.

Arsenic (As) readily crosses the placenta and exposure of the fetus may cause adverse consequences later in life, including immunomodulation. In the current study, the question was asked how the immune repertoire might respond in postnatal life when there is no further As exposure. Here, pregnant mice (Balb/c [H-2d]) were exposed to arsenic trioxide (As2O3) through their drinking water from time of conception until parturition. Their offspring, 4-week-old mice who had not been exposed again to As, were used for functional analyses of innate, humoral and cellular immunity. Compared to cells from non-As-exposed dam offspring, isolated peritoneal macro-phages (Mϕ) displayed no differences in T-cell stimulating ability. Levels of circulating IgG2a but not IgG1 were decreased in As-exposed dam offspring as compared to control offspring counterparts. Mixed-leukocyte reactions (MLR) indicated that CD4+ T-cells from the prenatal As-exposed mice were significantly less responsive to allogenic stimulation as evidenced by decreases in interferon (IFN)-γ and IL-2 production and in expression of CD44 and CD69 (but not CD25) activation markers. Interestingly, the Mϕ from the prenatal As-exposed mice were capable of stimulating normal allogenic T-cells, indicating that T-cells from these mice were refractory to allogenic signals. There was also a significant decrease in absolute numbers of splenic CD4+ and CD8+ T-cells due to prenatal As exposure (as compared to control). Lastly, the impaired immune function of the prenatal As-exposed mice was correlated with a very strong susceptibility to Escherichia coli infection. Taken together, the data from this study clearly show that in utero As exposure may continue to perpetuate a dampening effect on the immune repertoire of offspring, even into the early stages of postnatal life.

Outcome of children with Shiga toxin-associated haemolytic uraemic syndrome treated with eculizumab: a matched cohort study.

BACKGROUND: Treatment with eculizumab in Shiga toxin-associated haemolytic and uraemic syndrome (STEC-HUS) remains controversial despite its increasing utilization. The aim of our study was to evaluate the outcomes of children treated with eculizumab for STEC-HUS in a single-centre matched cohort study. METHODS: Data were retrospectively collected from medical records of children diagnosed with STEC-HUS. The outcomes of patients treated with eculizumab for STEC-HUS were compared with those of a control group of untreated patients matched for age, sex and severity of acute kidney injury with a 1:2 matching scheme. RESULTS: Eighteen children (median age 40.6 months) with STEC-HUS treated with eculizumab were compared with 36 matched control patients (median age 36.4 months) who did not receive eculizumab. All patients survived in the two groups. Within 1 month of HUS onset, the evolution of haematological and renal parameters did not differ between the two groups. At 12 months of follow-up, renal outcome was not significantly different between the two groups. At the last follow-up, the prevalence of decreased glomerular filtration rate in the eculizumab group (27%) was not statistically different from that in controls (38%), as was the prevalence of proteinuria and high blood pressure. Children who received eculizumab more often had extrarenal sequelae during follow-up. Eculizumab treatment appeared to be safe in children with STEC-HUS. CONCLUSION: The benefit of eculizumab on renal and extrarenal outcomes in STEC-HUS could not be established based on our findings. However, efficacy and safety are not best assessed by the observational design and small sample size of our study. Randomized controlled trials are thus required to determine the efficacy of eculizumab in this indication.

Pro- and anti-inflammatory effects of sulforaphane on placental cytokine production.

Placental inflammation increases the risk of adverse pregnancy outcomes and possibly neurodevelopmental disorders in the offspring. Previous research suggests it may be possible to modulate the placental immune response to bacteria to favor an anti-inflammatory phenotype with dietary factors. Sulforaphane (SFN) is a dietary supplement with known anti-inflammatory activities, however, its effects on placental cytokine production are unclear. Therefore, we evaluated the effects of SFN on biomarkers of inflammation and neurodevelopment under basal conditions and a setting of mild infection. Placental explant cultures were established and treated with up to 10 µM SFN in the presence and absence of 107 CFU/ml heat-killed E. coli. Concentrations of IL-1ß, TNF-α, IL-6, sgp130, HO-1 and BDNF in conditioned medium were quantified by immunoassay. SFN increased antioxidant HO-1 expression in the absence, but not the presence, of infection. SFN inhibited IL-1ß and IL-10, but tended to promote, TNF-α production by bacteria-stimulated cultures. IL-6 and BDNF were inhibited by SFN irrespective of co-treatment with E.coli. A negative regulator of IL-6 signaling, sgp130, was increased by SFN under basal conditions, but not in E. coli-stimulated cultures. These results suggest that SFN has mixed effects on the placenta inhibiting both pro-inflammatory (IL-1ß) and anti-inflammatory factors (IL-10) but promoting regulators of oxidative stress and inflammation (HO-1 and sgp130) in an infection-dependent manner.

Angiotensin-(1-7) protects from brain damage induced by shiga toxin 2-producing enterohemorrhagic Escherichia coli.

Shiga toxin 2 (Stx2)-producing enterohemorrhagic induced brain damage. Since a cerebroprotective action was reported for angiotensin (Ang)-(1-7), our aim was to investigate whether Ang-(1-7) protects from brain damage induced by Stx2-producing enterohemorrhagic Escherichia coli The anterior hypothalamic area of adult male Wistar rats was injected with saline solution or Stx2 or Stx2 plus Ang-(1-7) or Stx2 plus Ang-(1-7) plus A779. Rats received a single injection of Stx2 at the beginning of the experiment, and Ang-(1-7), A779, or saline was administered daily in a single injection for 8 days. Cellular ultrastructural changes were analyzed by transmission electron microscopy. Stx2 induced neurodegeneration, axonal demyelination, alterations in synapse, and oligodendrocyte and astrocyte damage, accompanied by edema. Ang-(1-7) prevented neuronal damage triggered by the toxin in 55.6 ± 9.5% of the neurons and the Stx2-induced synapse dysfunction was reversed. In addition, Ang-(1-7) blocked Stx2-induced demyelination in 92 ± 4% of the axons. Oligodendrocyte damage caused by Stx2 was prevented by Ang-(1-7) but astrocytes were only partially protected by the peptide (38 ± 5% of astrocytes were preserved). Ang-(1-7) treatment resulted in 50% reduction in the number of activated microglial cells induced by Stx2, suggesting an anti-inflammatory action. All these beneficial effects elicited by Ang-(1-7) were blocked by the Mas receptor antagonist and thus it was concluded that Ang-(1-7) protects mainly neurons and oligodendrocytes, and partially astrocytes, in the central nervous system through Mas receptor stimulation.

Intra-articular steroid injection for osteoarthritis of the hip prior to total hip arthroplasty : is it safe? a systematic review.

AIMS: Using a systematic review, we investigated whether there is an increased risk of post-operative infection in patients who have received an intra-articular corticosteroid injection to the hip for osteoarthritis prior to total hip arthroplasty (THA). METHODS: Studies dealing with an intra-articular corticosteroid injection to the hip and infection following subsequent THA were identified from databases for the period between 1990 to 2013. Retrieved articles were independently assessed for their methodological quality. RESULTS: A total of nine studies met the inclusion criteria. Two recommended against a steroid injection prior to THA and seven found no risk with an injection. No prospective controlled trials were identified. Most studies were retrospective. Lack of information about the methodology was a consistent flaw. CONCLUSIONS: The literature in this area is scarce and the evidence is weak. Most studies were retrospective, and confounding factors were poorly defined or not addressed. There is thus currently insufficient evidence to conclude that an intra-articular corticosteroid injection administered prior to THA increases the rate of infection. High quality, multicentre randomised trials are needed to address this issue. Cite this article: Bone Joint J 2016;98-B:1027-35.

Effect of crofelemer extract on severity and consistency of experimentally induced enterotoxigenic Escherichia coli diarrhea in newborn Holstein calves.

The objective of this study was to evaluate the effect of a standardized botanical extract of Croton lechleri, named crofelemer extract, on fecal dry matter and fecal scores on diarrheic newborn Holstein bull calves induced by enterotoxigenic Escherichia coli. A double-blinded randomized clinical trial was performed in which 60 newborn Holstein bull calves were clean caught and transported to an isolation facility where calves were individually housed and randomly assigned to 1 of 3 treatment groups: placebo (control), enteric-coated formulation of crofelemer extract (ECROF), and nonenteric-coated formulation of crofelemer extract (CROF). Diarrhea was induced at first feeding with an inoculum of the enterotoxigenic Escherichia coli (ATCC 31616) administered with a third of the recommended dose of a colostrum replacer. All calves enrolled in this study received treatments starting on the second feeding (diarrhea onset) and treatments were administered before feeding time (0600 and 1600h) for 6 feedings consecutively. All calves in this study had failure of passive transfer. The only cause of death in this study was due to septicemia, accounting for 1 death out of each treatment group. All the calves were examined twice daily, within 2h after feeding, from d 1 (prechallenge) until 10, on d 15, and a last examination on d 25 of life. Five parameters were evaluated during each examination; rectal temperature, clinical assessment of dehydration status, fecal scores, attitude, and appetite. No differences were observed between treatment groups for rectal temperature, attitude, and appetite. Fecal dry matter was analyzed as prechallenge fecal dry matter, dry matter during treatment, and fecal dry matter after treatment cessation. No difference in prechallenge fecal dry matter was observed and prechallenge fecal dry matter was used as a covariate in the models. Fecal dry matter during treatment was significantly higher for ECROF calves when compared with control calves and CROF calves. Additionally, ECROF fecal dry matter after treatment cessation had a statistical tendency to be higher when compared with control calves. Together, these results suggest that enteric-coated formulation of the standardized crofelemer extract, a natural-product with antisecretory properties, can significantly increase fecal dry matter of neonatal calves with experimentally induced enterotoxigenic Escherichia coli diarrhea. More research is needed to test the efficacy of enteric-coated crofelemer on incidence and severity of secretory diarrhea on calves under natural challenge conditions.

Prolonged antibiotic therapy increases risk of infection after transrectal prostate biopsy: a case report after pancreasectomy and review of the literature.

Infection due to prostate biopsy afflicted more than 5% of patients and is the most common reason for hospitalization. A large series from US SEER-Medicare reported that men undergoing biopsy were 2.26 times more likely to be hospitalized for infectious complications within 30 days compared with randomly selected controls. The factors predicting a higher susceptibility to infection remain largely unknown but some authors have higlighted in the etiopathogenesis the importance of the augmented prevalence of ciprofloxacin resistant variant of bacteria in the rectum flora. We present one case of sepsis after transrectal prostate biopsy in a patient with history of pancreatic surgery. Based on our experience patients candidated to prostate biopsy with transrectal technique with history of recent major surgery represent an high risk category for infective complication. Also major pancreatic surgery should be consider an high risk category for infection. A transperineal approach and preventive measures (such as rectal swab) should be adopted to reduce biopsy driven infection.

Cellulitis lesions in broiler chickens are induced by Escherichia coli Vacuolating Factor (ECVF).

Escherichia coli Vacuolating Factor (ECVF) is a heat-labile, vacuolating cytotoxin produced by avian pathogenic E. coli (APEC) isolated from avian cellulitis lesions. In this report, we intend to demonstrate that purified ECVF induces the inflammatory process of cellulitis. Our group is the first to demonstrate the effect of ECVF in a histological analysis by in situ inoculation of broiler chickens with purified ECVF. The animals were inoculated with the APEC AC53 and with purified ECVF subcutaneously on their ventral surface (in the sternum region). The histological analysis showed different grades of an acute inflammatory response in the epidermis, dermis and panniculus. An increase in mRNA expression of the proinflammatory cytokine TNF-α was also demonstrated in the inflamed tissue. When ECVF was systemically administered, increased levels of TNF-α and IL-10 were observed in the serum. These results suggest that ECVF plays a key role in the inflammatory process associated with cellulitis that is mainly mediated by TNF-α. In addition, this inflammation can be downregulated by the anti-inflammatory cytokine IL-10.

Enteropathogenic e.coli sustains iodoacetamide-induced ulcerative colitis-like colitis in rats: modulation of IL-1ß, IL-6, TNF-α, COX-2, and apoptosisi.

Pathogenic or non-pathogenic bacteria from flora may play a key role in inflammatory bowel disease (IBD) pathogenesis. However, a specific infectious agent causing IBD has not been identified. This study assessed the impact of enteropathogenic E. coli (EPEC) on the modulation of IL-1beta, IL-6, TNF- alpha, COX-2, BAX and Bcl-2 expression, in sustaining inflammation of a rat colitis model. Two hundred male Sprague-Dawley rats (4 groups) were inoculated weekly or bi-weekly for 70 days, with 1 percent methylcellulose (MC), (b) 6 percent iodoacetamide (IA) in 1 percent MC, (c) 4x108 CFU of EPEC, and (d) IA+EPEC. After a month, treatment was stopped in half of the animals in each group. IL-1beta, IL-6, TNF-alpha, COX-2, BAX and Bcl-2 expression were measured in colonic mucosa scrapings. IL-1beta, IL-6, TNF-alpha, and COX-2 were significantly increased in colonic mucosa of the IA+EPEC group and to a lesser but significant level in the IA group compared to controls, or EPEC alone, both in continued and discontinued treatment groups. Additionally, the BAX/Bcl-2 ratio decreased, indicating less apoptosis in the IA+EPEC group which exhibited more necrosis. These effects increased with experiment duration. This work provides new arguments favouring the role of bacteria in IBD pathogenesis.

Microparticle generation and leucocyte death in Shiga toxin-mediated HUS.

BACKGROUND: Shiga toxin-induced haemolytic uraemic syndrome (STEC-HUS) is an acute multisystem disorder characterized by renal failure, neurological dysfunction, haemolysis and intravascular thrombosis. Circulating microparticles originating from a number of cell types including thrombocytes and leucocytes are elevated in paediatric patients. In vitro data also suggest modification of leucocyte death by Shiga toxin. Here, we investigated microparticle generation and leucocyte cell death in vivo in adult STEC-HUS patients during acute disease and recovery. METHODS: Multi-colour flow cytometry and immunofluorescence were used to assess microparticle concentration and provenience thrombocyte microparticle seeding to leucocytes and leucocyte cell death in adult STEC-HUS patients treated at a tertiary care centre during the STEC-HUS outbreak in Germany in 2011. RESULTS: Plasma microparticle concentrations of both platelet and leucocyte origin were elevated during acute STEC-HUS. Platelet microparticles (MP) were detected on a high proportion of monocytes and granulocytes. Among therapeutic interventions, plasma exchange reduced platelet marker expression on leucocytes, inhibition of complement had only moderate impact on the number of circulating MP and did not alter platelet microparticle binding to leucocytes. Numbers of apoptotic and necrotic monocytes and granulocytes were significantly increased in patients with STEC-HUS compared to healthy controls. Complement inhibition significantly increased the number of circulating apoptotic cells. Monocyte apoptosis on admission was significantly higher in patients subsequently assigned to plasma exchange or admitted to the intensive care unit. CONCLUSIONS: In STEC-HUS, elevated numbers of circulating MP and dead leucocytes were detected. Monocyte and granulocyte deaths are novel markers of acute STEC-HUS that may actively contribute to tissue destruction by liberation of pro-inflammatory enzymes and cytokines.

Maternal/fetal mortality and fetal growth restriction: role of nitric oxide and virulence factors in intrauterine infection in rats.

OBJECTIVE: The mechanism of infection-related deaths of pregnant rats and intrauterine growth restriction are not understood. We assessed whether nitric oxide (NO) has differential effects on infection with Escherichia coli Dr/Afa mutants that lack either AfaE or AfaD invasins. STUDY DESIGN: Sprague-Dawley rats were infected intrauterinally with the clinical strain of E coli AfaE(+)D(+) or 1 of its isogenic mutants in the presence or absence of the NO synthesis inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). Maternal/fetal mortality rates, fetoplacental weight, and infection rates were evaluated. RESULTS: Maternal and/or fetal death was associated with the presence of at least 1 virulence factor (AfaE(+)D(+)>AfaE(+)D(-)>AfaE(-)D(+)) and was increased by L-NAME treatment. The fetal and placental weights were lower than controls and were further reduced by L-NAME treatment. CONCLUSION: These results demonstrate that NO enhanced AfaE- and AfaD-mediated virulence and plays an important role in Dr/Afa(+)E coli gestational tropism.

Microcirculatory effects of fluid therapy and dopamine, associated or not to fluid therapy, in endotoxemic hamsters.

Classically septic shock treatment takes into consideration only systemic parameters but failure in retaining arteriolar blood flow and functional capillary density (FCD) during sepsis worsens the outcome. Thus, we have investigated the effects of vigorous volume resuscitation (VR), two doses of dopamine and their combination upon the microcirculation during endotoxemia to evaluate if improvement on FCD and arteriolar blood flow would increase survival time. Sixty-seven adult male hamsters were studied using the window chamber model. Animals were randomized 1 h after the intravenous injection of 1 mg/kg of E. coli lipopolysaccharide (LPS) in LPS, no treatment; LPS/dopamine 3.0 µg/kg/min; LPS/dopamine 7.5 µg/kg/min; LPS/VR 20 ml/kg in 30 min followed by 20 ml/kg/h of saline; LPS/VR/Dopa 3.0, 20 ml/kg in 30 min followed by 20 ml/kg/h of saline associated to dopamine 3.0 µg/kg/min; LPS/VR/Dopa 7.5 (n = 6), 20 ml/kg in 30 min followed by 20 ml/kg/h of saline associated to dopamine 7.5 µg/kg/min and compared them to a Control group, no LPS. When present, treatment lasted 5 h. VR improved FCD and arteriolar blood flow score while dopamine did not. In conclusion, (1) improvement of FCD and arteriolar blood flow improved survival time; (2) VR recovered FCD and arteriolar blood flow and (3) in combination to VR, both dopamine doses reduced tissue perfusion (its low dose yielded the worst result).

Preserved cerebral microcirculation in early stages of endotoxemia in mechanically-ventilated rabbits.

The purpose of our study was to evaluate changes of the cerebral microcirculation during the early stages of endotoxemia in mechanically-ventilated rabbits using Sidestream dark-field (SDF) imaging. Images were obtained using SDF imaging from the surface of the brain via craniotomy before and after rapid administration of a high dose of endotoxin or saline (control group). Although endotoxin shock was successfully induced, we have not found any significant alteration of the cerebral microcirculation during the shock. We speculate that either the model of sepsis with a rapid high dose of endotoxin does not reflect the usual progression of septic encephalopathy or some components other than cerebral microcirculatory alteration play a role at the early stage of septic encephalopathy and the cerebral microcirculation is still preserved. Further studies are needed to clarify our findings.

Severe complication after a doxorubicin-eluting-bead embolization: surgical management and pathological findings.

Doxorubicin-eluting-bead embolization (DEB) is considered a safe and efficient treatment of hepatocellular carcinoma (HCC) with a low complication rate and an increased tumor response compared with conventional transarterial chemoembolization. We describe a case of a 69-year-old patient who underwent DEB for HCC and who developed a liver abscess requiring urgent left liver lobectomy. Despite this severe complication, efficacy of DEB embolization was histologically proved as a large ischemic zone with complete tumor necrosis.

Rhabdomyolysis due to Escherichia coli sepsis in three pediatric patients with acute lymphoblastic leukemia.

Rhabdomyolysis with myoglobinuria is an uncommon complication of bacterial sepsis. The authors describe three pediatric acute lymphoblastic leukemia patients who developed rhabdomyolysis during a neutropenic sepsis episode due to Escherichia coli. All of the patients needed hemodynamic supportive treatment because of septic shock. Broad-spectrum antibiotics, alkalinization, and intravenous fluid therapy was given. One patient with renal insufficiency died, despite aggressive treatment. Muscle pain and dark urine color should alert physicians to the possibility of rhabdomyolysis in immunocompromised patients with sepsis. Early and appropriate treatment is critical in these patients to prevent renal failure and shock, and for a better outcome.

Acute alcohol intake impairs lung inflammation by changing pro- and anti-inflammatory mediator balance.

Previous studies have shown that alcohol (ethanol [EtOH]) intoxication impairs lung immunity by affecting cytokines pivotal to the inflammatory process. The objective of this study was to test the hypothesis that acute alcohol intoxication impairs lung innate immunity by downregulating the expression of proinflammatory mediators while simultaneously upregulating anti-inflammatory mediators. EtOH was administered to the mice 0.5h prior to an intratracheal injection of Escherichia coli lipopolysaccharide (LPS). The animals were killed either 4 or 24h after LPS to recover plasma, lungs, and bronchoalveolar lavage fluid. Lung inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1beta), IL-6, macrophage inhibitory factor (MIF), IL-10, TGF-beta, and receptors for TNF-alpha, IL-1beta, IL-6, and TGF-beta as well as glycoprotein (gp)130 and corticosterone (CS) levels were evaluated at mRNA and protein level. While the mRNA expression and the soluble TNF-Rp55 levels were significantly upregulated by EtOH, LPS-induced TNF-alpha activity, TNF-Rp55 mRNA expression, and soluble TNF-Rp55 levels were significantly suppressed. The LPS-induced expression of IL-1beta, IL-6, MIF, gp130, and receptors IL-1RI, IL-1RII, and IL-6Ralpha were also significantly impaired by EtOH. EtOH increased significantly the basal IL-10 activity at 3h, which continued to remain elevated even at 24h. The EtOH effect on IL-10 activity persisted even in LPS-challenged mice. EtOH and LPS augmented lung CS levels independently of each other. EtOH suppressed upregulation of TGF-beta1 mRNA expression by LPS and blocked completely LPS-induced TGF-beta1 secretion. In conclusion, the data suggest that the suppression of acute lung inflammation by EtOH intoxication is largely due to impairment by EtOH of proinflammatory cytokine signaling at the levels of cytokine expression and secretion as well as receptor expression and soluble receptor activity. The augmentation by EtOH of anti-inflammatory mediators' secretion most likely shifts the cytokine balance in the anti-inflammatory direction.

Acute alcohol intoxication suppresses the pulmonary ELR-negative CXC chemokine response to lipopolysaccharide.

Alcohol abuse impairs the pulmonary immune response to infection and increases the morbidity and mortality of bacterial pneumonia. Acute alcohol intoxication suppresses lung expression of CXC chemokines bearing the Glu-Leu-Arg motif (ELR+) following lipopolysaccharide (LPS) challenge, but its effect on the structurally related ELR- CXC chemokines, which attract T cells, is unknown. We therefore investigated the effect of acute alcohol intoxication on the pulmonary response to intratracheal (i.t.) LPS challenge for the ELR- CXC chemokines monokine induced by gamma (MIG or CXCL9), interferon-inducible protein 10 (IP-10 or CXCL10), and interferon-inducible T cell alpha chemoattractant (I-TAC or CXCL11). Male C57BL/6 or C3H/HeN mice were given an intraperitoneal injection of ethanol (3.0 g/kg) or phosphate buffered saline 30 min before i.t. LPS challenge. Chemokine mRNA transcripts were measured at 0, 2, 6, and 16 h. Acute alcohol intoxication inhibited the lung's expression of all three chemokine genes in response to LPS. Lung IFN-gamma mRNA was also inhibited by acute intoxication over the same time course. The in vitro effect of ethanol on chemokine secretion was further studied in the MH-S alveolar macrophage cell line. IP-10, MIG, and I-TAC in response to LPS were enhanced by exogenous interferon (IFN)-gamma, and these responses were blunted by exposure to ethanol. Alcohol exposure did not affect MH-S cell nuclear factor kappa beta p65 nuclear localization during challenge, despite dose-dependent inhibition of Erk 1/2 phosphorylation. In addition, phospho-signal transduction and activator of transcription 1 was not decreased in the presence of acute ethanol, thereby indicating that acute intoxication does not affect IFN-gamma signaling in MH-S cells. Recruitment of CD3+ T cells into the alveolar space 4 days after LPS challenge was moderately impaired by acute ethanol intoxication. These results implicate acute ethanol intoxication as a significant inhibitor of lymphocyte chemoattractant expression during pulmonary inflammation.