Meaghan E. Killeen

Prolonged treatment with N-acetylcystine delays liver recovery from acetaminophen hepatotoxicity

IntroductionAcetaminophen (APAP) toxicity is the most common cause of acute liver failure in the US and Europe. Massive hepatocyte necrosis is the predominant feature of APAP-induced acute liver injury (ALI). Liver regeneration is a vital process for survival after a toxic insult, it occurs at a relative late time point after the injurious phase. Currently, N-acetylcysteine (NAC), a glutathione precursor, is the antidote for acetaminophen overdose. However, NAC is effective only for patients who present within hours of an acute overdose, and is less effective for late-presenting patients. It is possible that in delayed patients, previously reduced endogenous glutathione (GSH) level has restored and prolonged treatment with NAC might be toxic and impair liver regeneration. Therefore, we hypothesize that prolonged treatment with NAC impairs liver regeneration in ALI induced by APAP.MethodsALI was induced in C57BL/6 male mice by a single dose of APAP (350 mg/kg) by intraperitoneal injection. After two hours of APAP challenge, the mice were given 100 mg/kg NAC dissolved in 0.6 mL saline, or saline treatment every 12 hours for a total of 72 hours.ResultsSeventy-two hours after APAP challenge, compared with saline treatment, NAC treatment significantly increased serum transaminases (alanine transaminase/aspartate aminotransferase), induced evident hepatocyte vacuolation in the periportal area and delayed liver regeneration seen in histopathology. This detrimental effect was associated with reduced hepatic nuclear factor (NF)-κB DNA binding and decreased expression of cell cycle protein cyclin D1, two important factors in liver regeneration.ConclusionsProlonged treatment with NAC impairs liver regeneration in ALI induced by APAP.

Signaling through Purinergic Receptors for ATP Induces Human Cutaneous Innate and Adaptive Th17 Responses: Implications in the Pathogenesis of Psoriasis

Human cutaneous dendritic cells (DCs) have the ability to prime and bias Th17 lymphocytes. However, the factors that stimulate cutaneous DCs to induce Th17 responses are not well known. Alarmins, such as ATP, likely play a pivotal role in the induction and maintenance of cutaneous immune responses by stimulating DC maturation, chemotaxis, and secretion of IL-1β and IL-6, Th17-biasing cytokines. In this study, using a well-established human skin model, we have demonstrated that signaling purinergic receptors, predominantly the P2X7 receptor (P2X7R), via an ATP analog initiate innate proinflammatory inflammation, DC17 differentiation, and the subsequent induction of Th17-biased immunity. Moreover, our results suggest a potential role for P2X7R signaling in the initiation of psoriasis pathogenesis, a Th17-dependent autoimmune disease. In support of this, we observed the increased presence of P2X7R in nonlesional and lesional psoriatic skin compared with normal healthy tissues. Interestingly, there was also a P2X7R variant that was highly expressed in lesional psoriatic skin compared with nonlesional psoriatic and normal healthy skin. Furthermore, we demonstrated that psoriatic responses could be initiated via P2X7R signaling in nonlesional skin following treatment with a P2X7R agonist. Mechanistic studies revealed a P2X7R-dependent mir-21 angiogenesis pathway that leads to the expression of vascular endothelial growth factor and IL-6 and that may be involved in the development of psoriatic lesions. In conclusion, we have established that purinergic signaling in the skin induces innate inflammation, leading to the differentiation of human Th17 responses, which have implications in the pathogenesis and potential treatment of psoriasis.

Bile high-mobility group box 1 contributes to gut barrier dysfunction in experimental endotoxemia

Lipopolysaccharide (LPS) is an important factor in sepsis. LPS given by intraperitoneal injection induces intestinal hyperpermeability and bacterial translocation in animals and stimulates hepatic Kupffer cells to release TNF-alpha into the bile. This study aims to test the hypothesis that in response to LPS stimulation, hepatic Kupffer cells and extrahepatic macrophages release a large amount of the inflammatory cytokine high-mobility group box 1 (HMGB1) into the bile and that bile containing HMGB1 contributes to gut barrier dysfunction in experimental endotoxemia. To test this, rat common bile ducts were catheterized and bile flow rate was monitored before and during the LPS administration. Eight hours after LPS challenge, anti-HMGB1 neutralizing antibody or nonimmune (sham) IgG was injected into the duodenal lumen of endotoxemic rats; normal mice were also gavaged with normal or endotoxemic rat bile (bile collected from LPS-treated rats). We found that after LPS challenge, the bile flow rate in rats was significantly decreased at the 4- to 12-h time points, TNF-alpha concentration in the bile was markedly elevated at the 3- to 4-h time points, and bile HMGB1 levels were significantly increased at the 8- to 12-h time points. Duodenal injection with anti-HMGB1 antibody reversed LPS-induced gut barrier dysfunction in rats. In addition, feeding endotoxemic rat bile to normal mice significantly increased both mucosal permeability and bacterial translocation. The increase in permeability and bacterial translocation was reversible following removal of HMGB1 from the endotoxemic rat bile. These findings document that bile HMGB1 mediates gut barrier dysfunction in experimental endotoxemia.

Ethyl pyruvate ameliorates liver injury secondary to severe acute pancreatitis.

BACKGROUND Ethyl pyruvate (EP) is capable of significantly decreasing serum alanine aminotransferase and reducing hepatic necrosis in a murine model of severe acute pancreatitis (SAP); however, the working mechanism is still unclear. This study aims to elucidate the underlying mechanism of EP solution ameliorating SAP-induced liver injury and provide a new therapeutic agent to treat liver injury. MATERIALS AND METHODS Acute necrotizing pancreatitis was induced in C57Bl/6 male mice by feeding the animals a choline-deficient diet supplemented with 0.5% ethionine for 24 h; then the animals were challenged with 7 hourly 50 mug/kg cerulein i.p. injections and a single i.p. injection of Escherichia coli lipopolysaccharide (4 mg/kg). Two hours after the injection of lipopolysaccharide, 40 mg/kg EP, the same volume of Ringers lactate solution (RLS), or saline solution were i.p. injected to animals of EP, RLS, and control groups every 6 h for a total 48-h period. RESULTS When mice were treated with EP, hepatic mRNA expression of tumor necrosis factor-alpha, interleukin-6, inducible nitric oxide synthase, and cyclooxygenase-2 was significantly lower than that in pancreatitis mice treated with RLS. Compared to RLS treatment, treatment with EP significantly decreased the number of inflammatory cell infiltration and markedly inhibited hepatic nuclear factor-kappa B DNA binding; EP therapy dramatically inhibited high motility group B1 release from inflamed hepatic tissue and significantly decreased the concentration of hepatic tissue malondialdehyde, an oxidative stress parameter. EP treatment also significantly improved body circulating blood volume. CONCLUSION EP is a potent anti-inflammatory and anti-oxidative agent to ameliorate hepatic local inflammatory response and resultantly decreases liver injury secondary to SAP.

MCPIP1/Regnase-1 Restricts IL-17A– and IL-17C–Dependent Skin Inflammation

The IL-17 family cytokines IL-17A and IL-17C drive the pathogenesis of psoriatic skin inflammation, and anti–IL-17A Abs were recently approved to treat human psoriasis. Little is known about mechanisms that restrain IL-17 cytokine-mediated signaling, particularly IL-17C. In this article, we show that the endoribonuclease MCP-1–induced protein 1 (MCPIP1; also known as regnase-1) is markedly upregulated in human psoriatic skin lesions. Similarly, MCPIP1 was overexpressed in the imiquimod (IMQ)-driven mouse model of cutaneous inflammation. Mice with an MCPIP1 deficiency (Zc3h12a+/−) displayed no baseline skin inflammation, but they showed exacerbated pathology following IMQ treatment. Pathology in Zc3h12a+/− mice was associated with elevated expression of IL-17A– and IL-17C–dependent genes, as well as with increased accumulation of neutrophils in skin. However, IL-17A and IL-17C expression was unaltered, suggesting that the increased inflammation in Zc3h12a+/− mice was due to enhanced downstream IL-17R signaling. Radiation chimeras demonstrated that MCPIP1 in nonhematopoietic cells is responsible for controlling skin pathology. Moreover, Zc3h12a+/−Il17ra−/− mice given IMQ showed almost no disease. To identify which IL-17RA ligand was essential, Zc3h12a+/−Il17a−/− and Zc3h12a+/−Il17c−/− mice were given IMQ; these mice had reduced but not fully abrogated pathology, indicating that MCPIP1 inhibits IL-17A and IL-17C signaling. Confirming this hypothesis, Zc3h12a−/− keratinocytes showed increased responsiveness to IL-17A and IL-17C stimulation. Thus, MCPIP1 is a potent negative regulator of psoriatic skin inflammation through IL-17A and IL-17C. Moreover, to our knowledge, MCPIP1 is the first described negative regulator of IL-17C signaling.

Extracellular activation of arginase-1 decreases enterocyte inducible nitric oxide synthase activity during systemic inflammation

Liver dysfunction secondary to severe inflammation is associated with the release of enzymes normally sequestered within hepatocytes. The purpose of these studies was to test the hypothesis that these enzymes are released, at least in part, to modulate potentially deleterious inflammatory processes in distant tissues like the gut. Human Caco-2(BBe) enterocyte-like cells were exposed to cytomix (IFN-gamma, TNF-alpha, and IL-1beta) in the absence or presence of human liver cytosol (LC). Nitric oxide (NO(*)) and inducible nitric oxide synthase (iNOS) protein production were measured by the Griess assay and Western analysis, respectively. Cytomix induced the expression of iNOS and release of NO(*). LC protein (400 microg/ml) added to the basal compartment but not apical compartment completely blocked the release of NO(*) but only slightly decreased the magnitude of iNOS protein induction. Ultrafiltration and ultracentrifugation studies demonstrated that microsome-associated arginase-1 activity was the iNOS-suppressing activity in LC. Liver arginase required activation by a <10-kDa factor that was present in supernatants of cytomix-stimulated cells. The selective iNOS inhibitor l-N(6)-(1-iminoethyl)-lysine.2HCl prevented production of this factor. The biotin switch assay detected increased S-nitrosylation of arginase-1 after incubation with supernatants from immunostimulated Caco-2 cells. Serum from endotoxemic mice contained significantly greater arginase activity compared with serum from control mice. Furthermore, the ratio of mucosal monomeric to dimeric iNOS increased in endotoxemic mice compared with controls. Thus reciprocal activation of arginase-1 and modulation of mucosal iNOS activity may be protective because it would be expected to decrease NO(*)-dependent intestinal barrier dysfunction on that basis.

Electrophilic nitro-fatty acids suppress allergic contact dermatitis in mice.

Reactions between nitric oxide (NO), nitrite ( NO2− ), and unsaturated fatty acids give rise to electrophilic nitro‐fatty acids (NO2‐FAs), such as nitro oleic acid (OA‐NO2) and nitro linoleic acid (LNO2). Endogenous electrophilic fatty acids (EFAs) mediate anti‐inflammatory responses by modulating metabolic and inflammatory signal transduction reactions. Hence, there is considerable interest in employing NO2‐FAs and other EFAs for the prevention and treatment of inflammatory disorders. Thus, we sought to determine whether OA‐NO2, an exemplary nitro‐fatty acid, has the capacity to inhibit cutaneous inflammation.

Extracellular ATP and IL-23 Form a Local Inflammatory Circuit Leading to the Development of a Neutrophil-Dependent Psoriasiform Dermatitis

Psoriasis is a chronic inflammatory skin disease dependent on the IL-23/IL-17 axis, a potent inflammatory pathway involved in pathogen clearance and autoimmunity. Several triggers have been proposed as initiators for psoriasis, including alarmins such as adenosine triphosphate. However, the role of alarmins in psoriasis pathogenesis and cutaneous inflammation has not been well addressed. Studies show that signaling through the P2X7 receptor (P2X7R) pathway underlies the development of psoriasiform inflammation. In this regard, psoriasiform dermatitis induced by IL-23 is dependent on P2X7R signaling. Furthermore, direct activation of the P2X7R is sufficient to induce a well-characterized psoriasiform dermatitis. Mechanistic studies determined that P2X7R-induced inflammation is largely dependent on the IL-1β/NLRP3 inflammasome pathway and neutrophils. In conclusion, this work provides basic mechanistic insight into local inflammatory circuits induced after purinergic P2X7R signaling that are likely involved in the pathogenesis of many inflammatory diseases, such as psoriasis.

Topical electrophilic nitro-fatty acids potentiate cutaneous inflammation

ABSTRACT Endogenous electrophilic fatty acids mediate anti‐inflammatory responses by modulating metabolic and inflammatory signal transduction and gene expression. Nitro‐fatty acids and other electrophilic fatty acids may thus be useful for the prevention and treatment of immune‐mediated diseases, including inflammatory skin disorders. In this regard, subcutaneous (SC) injections of nitro oleic acid (OA‐NO2), an exemplary nitro‐fatty acid, inhibit skin inflammation in a model of allergic contact dermatitis (ACD). Given the nitration of unsaturated fatty acids during metabolic and inflammatory processes and the growing use of fatty acids in topical formulations, we sought to further study the effect of nitro‐fatty acids on cutaneous inflammation. To accomplish this, the effect of topically applied OA‐NO2 on skin inflammation was evaluated using established murine models of contact hypersensitivity (CHS). In contrast to the effects of subcutaneously injected OA‐NO2, topical OA‐NO2 potentiated hapten‐dependent inflammation inducing a sustained neutrophil‐dependent inflammatory response characterized by psoriasiform histological features, increased angiogenesis, and an inflammatory infiltrate that included neutrophils, inflammatory monocytes, and &ggr;&dgr; T cells. Consistent with these results, HPLC‐MS/MS analysis of skin from psoriasis patients displayed a 56% increase in nitro‐conjugated linoleic acid (CLA‐NO2) levels in lesional skin compared to non‐lesional skin. These results suggest that nitro‐fatty acids in the skin microenvironment are products of cutaneous inflammatory responses and, in high local concentrations, may exacerbate inflammatory skin diseases. Graphical abstract Figure. No caption available. HighlightsOA‐NO2 applied to sites of cutaneous inflammation potentiates that inflammation.The inflammatory response induced by OA‐NO2 is dependent on neutrophils.Human psoriatic lesions have an increase in nitrated fatty acids.

Glycolytic Bias in mTORC2 Deficient Dendritic Cells Potentiates Antigen-Specific Immunity and Accelerates Graft Rejection

Background/Hypothesis The mechanistic target of rapamycin (mTOR) is known to function in two discrete complexes: mTOR complex 1 (mTORC1) and mTORC2. The function of mTORC1 in dendritic cells (DCs) has been studied extensively using rapamycin (RAPA) as an inhibitor. RAPA inhibition of mTORC1 prevents DC maturation, leading to decreased T effector cell proliferation and increased regulatory T cell (Treg) differentiation. Our group has recently demonstrated that mTORC2 deletion in DCs leads to both an enhanced pro-inflammatory DC phenotype and Th1/Th17 allogeneic T cell polarization and proliferation. However, the underlying mechanism has not been resolved. In addition, the role of mTORC2 in DCs in the context of transplantation has not been defined. We hypothesized that ablation of mTORC2 in DCs would alter metabolic activity, resulting in augmented antigen-specific T cell responses and accelerated graft rejection. Methods To assess the role of mTORC2 in DCs in transplantation, we used models of non-MHC mismatched skin transplantation in which either the graft donor or recipient was deficient in mTORC2 specifically in conventional CD11c+ DC (TORC2DC-/-). Graft survival was monitored; Banff rejection scoring was performed by a blinded pathologist. T cell infiltration and collagen degradation in the graft were determined by immunohistochemistry. To ascertain whether skin-resident TORC2DC-/- could augment inflammatory responses, we performed a cell-mediated delayed-type hypersensitivity assay. As mTORC2 has been implicated in cytoskeletal dynamics, we measured DC migration into secondary lymphoid tissue. To elucidate the role of mTORC2 in regulating DC metabolism we analyzed glycolytic capacity and mitochondrial respiratory activity of wild-type (WT) DC and TORC2DC-/- using a Seahorse XF Bioanalyzer. Mitochondrial mass and activity were determined via flow cytometric analysis of MitoTracker Green and TMRE uptake, respectively. ATP production was assessed using a luciferase-based luminescence assay. Results/Conclusions We demonstrate for the first time, that TORC2DC-/- deficiency in either skin graft donors or recipients accelerates immune-mediated rejection. TORC2DC-/- mice also exhibit enhanced T cell and inflammatory monocytic infiltration in the course of delayed-type hypersensitivity responses. These effects are not due to differential migration of TORC2DC-/- to secondary lymphoid tissue. TORC2DC-/- utilize an altered metabolic program, wherein glycolytic function is enhanced as compared to WT DCs. This metabolic phenotype corresponds with increased viability of TORC2DC-/- after stimulation, which may allow these TORC2DC-/- to persist in secondary lymphoid tissue longer than WT DCs. These findings reveal a novel role for mTORC2 in regulating DC immunometabolism, and may provide a basis for therapeutic targeting of DC metabolism to regulate immune responses in transplantation. NIH T32 AI74490.