High-mobility group box 1 (HMGB1), a cytokine-like proinflammatory protein, is secreted by activated macrophages and released by necrotic cells. We hypothesized that immunostimulated enterocytes might be another source for this mediator. Accordingly, Caco-2 cells or primary mouse intestinal epithelial cells (IECs) were incubated with "cytomix" (a mixture of TNF, IL-1beta, and IFN-gamma) for various periods. HMGB1 in cell culture supernatants was detected by Western blot analysis and visualized in Caco-2 cells with the use of fluorescence confocal and immunotransmission electron microscopy. Caco-2 cells growing on filters in diffusion chambers were stimulated with cytomix for 48 h in the absence or presence of anti-HMGB1 antibody, and permeability to fluorescein isothiocyanate-dextran (average molecular mass, 4 kDa; FD4) was assessed. Cytomix-stimulated Caco-2 cells secreted HMGB1 into the apical but not the basolateral compartments of diffusion chambers. Although undetectable at 6 and 12 h after the start of incubation with cytomix, HMGB1 was present in supernatants after 24 h of incubation. HMGB1 secretion by Caco-2 monolayers also was induced when the cells were exposed to FSL-1, a Toll-like receptor (Tlr)-2 agonist, or flagellin, a Tlr5 agonist, but not lipopolysaccharide, a Tlr4 agonist. Cytomix also induced HMGB1 secretion by primary IECs. Cytoplasmic HMGB1 is localized within vesicles in Caco-2 cells and is secreted, at least in part, associated with exosomes. Incubating Caco-2 cells with cytomix increased FD4 permeation, but this effect was significantly decreased in the presence of anti-HMGB1 antibody. Collectively, these data support the view that HMGB1 is secreted by immunostimulated enterocytes. This process may exacerbate inflammation-induced epithelial hyperpermeability via an autocrine feedback loop.
Human cutaneous 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. Here, utilizing a well-established human skin model we have demonstrated that signaling purinergic receptors, predominantly P2X7R, via an ATP analog initiates 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 non-lesional and lesional psoriatic skin compared to normal healthy tissues. Interestingly, there was also a P2X7R variant (P2X7RB) that was highly expressed in lesional psoriatic skin compared to non-lesional psoriatic and normal healthy skin. Furthermore, we demonstrated that psoriatic responses could be initiated via P2X7R signaling in non-lesional skin following treatment with a P2X7R agonist. Mechanistic studies revealed a P2X7R-dependent mir-21 angiogenesis pathway that leads to the expression of VEGF and IL-6, and which 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.
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.
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. To date, little is known about mechanisms that restrain IL-17 cytokine-mediated signaling, particularly IL-17C. Here, we show that the endoribonuclease 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 and also 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 non-hematopoietic 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 both 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, MCPIP1 is the first described negative regulator of IL-17C signaling.
The diuretic ethacrynic acid (EA) has been shown to inhibit signaling by the proinflammatory transcription factor nuclear factor-B (NF-B). Accordingly, we sought to determine whether this compound is capable of inhibiting the release of cytokines [interleukin (IL)-6 and IL-10] and NO from RAW 264.7 murine macrophage-like cells stimulated with lipopolysaccharide (LPS). Additionally, we sought to determine whether EA can inhibit secretion of high-mobility group box 1 (HMGB1), a nuclear protein that is secreted by immunostimulated macrophages and functions in the extracellular milieu as a proinflammatory mediator. In a concentration-dependent manner, EA inhibited secretion of IL-6, IL-10, nitric oxide, and HMGB1. As expected, EA inhibited NF-B DNA binding in LPS-stimulated RAW 264.7 cells. Treating these cells with pyrrolidine dithiocarbamate, SN50 (amino acid sequence AAVALLPAVLLAL-LAPVQRKRQKLMP) or 5-(thien-3-yl)-3-aminothiophene-2-carboxamide (SC-514) also inhibited LPS-induced NF-B DNA binding, but these compounds failed to inhibit LPS-induced HMGB1 secretion. These findings suggested that inhibition of HMGB1 secretion by EA might occur via a mechanism unrelated to the NF-B signaling pathway. Because EA is an electrophilic compound that is known to be capable of inducing expression of so-called phase 2 proteins, we sought to determine whether two other phase 2 enzyme inducers, oltipraz and DL-sulforaphane, also are capable of inhibiting HMGB1 release from immunostimulated macrophages. Incubating RAW 264.7 cells with either oltipraz or DL-sulforaphane inhibited LPS-induced HMGB1 secretion. Moreover, both EA and DL-sulforaphane inhibited relocalization of nuclear HMGB1 into the cytoplasm of LPS-stimulated RAW 264.7 cells. These data suggest that phase 2 inducers may exert anti-inflammatory effects by inhibiting secretion of the cytokine-like nuclear protein HMGB1.High-mobility group proteins are small DNA binding proteins that serve an important role in transcriptional regulation (Bustin et al., 1990). When present in the extracellular milieu, one of these proteins, high-mobility group box 1 (HMGB1), also functions as a cytokine-like molecule. For example, HMGB1 promotes TNF release from mononuclear cells (Andersson et al., 2001) and induces DNA binding by the proinflammatory transcription factor NF-B and inducible nitric-oxide synthase expression in Caco-2 human enterocyte-like cells (Sappington et al., 2002). Additionally, HMGB1 promotes adhesion molecule expression on cultured human umbilical venular endothelial cells (Treutiger et al., 2003). HMGB1 is actively secreted by immunostimulated macrophages (Wang et al., 1999;Gardella et al., 2002;Bonaldi et al., 2003;Rendon-Mitchell et al., 2003) and is also released by necrotic but not apoptotic cells (Scaffidi et al., 2002). Extracellular HMGB1 has been implicated in the pathogenesis of inflammatory diseases in both experimental ani- ABBREVIATIONS: HMGB1, high-mobility group box 1; TNF, tumor necrosis factor; NF-B, nuclear factor-B; LPS, lipopolysaccharid...
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