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Unresolved inflammation maintained by release of danger‐associated molecular patterns, particularly high‐mobility group box‐1 (HMGB1), is crucial for hepatocellular carcinoma (HCC) pathogenesis. To further characterize interactions between leucocytes and necrotic cancerous tissue, a cellular model of necroinflammation was studied in which murine Raw 264.7 macrophages or primary splenocytes were exposed to necrotic lysates (N‐lys) of murine hepatoma cells or primary hepatocytes. In comparison to those derived from primary hepatocytes, N‐lys from hepatoma cells were highly active—inducing in macrophages efficient expression of inflammatory cytokines like C‐X‐C motif ligand‐2 , tumor necrosis factor‐α, interleukin (IL)‐6 and IL‐23‐p19. This activity associated with higher levels of HMGB1 in hepatoma cells and was curbed by pharmacological blockage of the receptor for advanced glycation end product (RAGE)/HMGB1 axis or the mitogen‐activated protein kinases ERK1/2 pathway. Analysis of murine splenocytes furthermore demonstrated that N‐lys did not comprise of functionally relevant amounts of TLR4 agonists. Finally, N‐lys derived from hepatoma cells supported inflammatory splenic Th17 and Th1 polarization as detected by IL‐17, IL‐22 or interferon‐γ production. Altogether, a straightforward applicable model was established which allows for biochemical characterization of immunoregulation by HCC necrosis in cell culture. Data presented indicate a remarkably inflammatory capacity of necrotic hepatoma cells that, at least partly, depends on the RAGE/HMGB1 axis and may shape immunological properties of the HCC microenvironment.

Interferon (IFN)-γ-inducing interleukin (IL)-18 is a crucial inflammatory cytokine systemically provided by monocytes. It is counteracted by IL-18 binding protein (IL-18BP), a decoy receptor that displays IFNγ-inducibility thus curbing inflammation by negative feedback. Since IL18BP inducibility is pronounced in human epithelial cells but diminished in monocytes, differential IL18BP regulation was investigated herein in both types of cells. Interestingly, DNA-demethylating 5-aza-2'-deoxycytidine enhanced IFNγ-induced IL-18BP only in monocytic but not in epithelial cells. Subsequent promoter analysis brought into focus a specific CpG (coined CpG2) neighboring a γ-activated site responsible for IL18BP induction. CpG2 was consistently methylated in monocytic but unmethylated in epithelial cells. Notably, demethylation by 5-aza-2'-deoxycytidine treatment of monocytic cells impeded methyl-CpG-binding protein-2 (MeCP2) interaction with CpG2, increased adjacent histone H3K9-acetylation, and enhanced RNA-polymerase-II recruitment to the nearby IL18BP transcriptional start. Both latter observations are indicative of a gene locus displaying augmented transcriptional activity. Data suggest that epigenetic silencing by single CpG methylation determines differential IL18BP inducibility in monocytic versus epithelial cells. This regulatory principle should serve and control pivotal IL-18-related cell type-specific (patho)-physiological functions. Whereas epithelial IL-18BP evidently counteracts pathological inflammation at biological barriers, retarded IL18BP inducibility in monocytes may be key to combat blood-borne infections in IL-18-dependent manner.

Interleukin (IL)-22 activates STAT (signal transducer and activator of transcription) 3 and antiapoptotic and proproliferative pathways; but beyond this, the molecular mechanisms by which IL-22 promotes carcinogenesis are poorly understood. Characterizing the molecular signature of IL-22 in human DLD-1 colon carcinoma cells, we observed increased expression of 26 genes, including NNMT (nicotinamide N-methyltransferase, ≤10-fold) and CEA (carcinoembryonic antigen, ≤7-fold), both known to promote intestinal carcinogenesis. ERP27 (endoplasmic reticulum protein-27, function unknown, ≤5-fold) and the proinflammatory ICAM1 (intercellular adhesion molecule-1, ≤4-fold) were also increased. The effect on CEA was partly STAT3-mediated, as STAT3-silencing reduced IL-22-induced CEA by ≤56%. Silencing of CEA or NNMT inhibited IL-22-induced proliferation/migration of DLD-1, Caco-2, and SW480 colon carcinoma cells. To validate these results in primary tissues, we assessed IL-22-induced gene expression in organoids from human healthy colon and colon cancer patients, and from normal mouse small intestine and colon. Gene regulation by IL-22 was similar in DLD-1 cells and human and mouse healthy organoids. CEA was an exception with no induction by IL-22 in organoids, indicating the 3-dimensional organization of the tissue may produce signals absent in 2D cell culture. Importantly, augmentation of NNMT was 5-14-fold greater in human cancerous compared to normal organoids, supporting a role for NNMT in IL-22-mediated colon carcinogenesis. Thus, NNMT and CEA emerge as mediators of the tumor-promoting effects of IL-22 in the intestine. These data advance our understanding of the multifaceted role of IL-22 in the gut and suggest the IL-22 pathway may represent a therapeutic target in colon cancer.

By generating biologically active factors luminal microbiota shape the intestinal micro-milieu thereby regulating pathological processes such as inflammation and carcinogenesis. Preclinical data suggest that bacterial-derived butyrate and the signal transducer and activator of transcription (STAT)-3 activating cytokine interleukin (IL)-22 display concordant protective properties at the inflamed colonic epithelium. Herein, biochemical cooperation between the short-chain fatty acid butyrate and IL-22 was investigated by focusing on human Caco2 colon epithelial/carcinoma cells. We report that physiological levels of butyrate enhance IL-22 signaling thereby enforcing expression of the prototypic STAT3-downstrean target genes α1-antichymotrypsin and suppressor of cytokine signaling (SOCS)-3. A dual mode of butyrate action on the IL-22/STAT3 axis was identified. Butyrate acted by upregulating IL-22R1, the decisive chain of the heterodimeric IL-22 receptor, and, independent from that, has the potential to directly amplify STAT3-mediated gene activation as detected by chromatin immunoprecipitation analysis of STAT3 binding to the SOCS3 promoter. Since trichostatin A acted similarly, inhibition of histone deacetylases is likely at the root of these butyrate biological properties. The mutual benefit gained from interactions between the host and commensal intestinal bacteria-derived factors is an expanding field of research beginning to affect clinical practice. Data presented herein propose a supportive and fine-tuning role for butyrate in IL-22 signaling that might be therapeutically exploited by local butyrate administration or by increasing its bacterial production in the context of a fiber-rich diet. © 2016 BioFactors, 43(2):283-292, 2017.

Cytokine networks initiated by means of innate immunity are regarded as a major determinant of host defence in response to acute infection by bacteria including Borrelia burgdorferi. Herein, we demonstrate that interferon (IFN)-α, either endogenously produced after exposure of cells to toll-like receptor-9-activating CpG oligonucleotides or provided as recombinant cytokine, weakens activation of the anti-bacterial interleukin (IL)-1/IL-22 axis in human peripheral blood mononuclear cells exposed to viable B. burgdorferi. As IFN-α has been related to pathological dissemination of the spirochaete, data suggest an immunoregulatory role of type I IFN in this context that is able to significantly modify cytokine profiles thereby possibly determining early course of B. burgdorferi infection.

Disturbed homeostasis as a result of tissue stress can provoke leukocyte responses enabling recovery. Since mild hypothermia displays specific clinically relevant tissue-protective properties and interleukin (IL)-22 promotes healing at host/environment interfaces, effects of lowered ambient temperature on IL-22 were studied. We demonstrate that a 5h exposure of endotoxemic mice to 4°C reduces body temperature by 5.0 degrees and enhances splenic and colonic il22 gene expression. In contrast, tumor necrosis factor (TNF)-a and IL-17A were not increased. In vivo data on IL-22 were corroborated using murine splenocytes and human peripheral blood mononuclear cells (PBMC) cultured upon 33°C and polyclonal T cell activation. Upregulation by mild hypothermia of largely T-cell-derived IL-22 in PBMC required monocytes and associated with enhanced nuclear T-cell NFATc2. Notably, nuclear factor of activated T cells (NFAT) antagonism by cyclosporine A or FK506 impaired IL-22 upregulation at normothermia and entirely prevented its enhanced expression upon hypothermic culture conditions. Data suggest that intact NFAT signaling is required for efficient IL-22 induction upon normothermic and hypothermic conditions. Hypothermia furthermore boosted early signal transducer and activator of transcription (STAT)-3 activation by IL-22 and shaped downstream gene expression in epithelial-like cells. Altogether, data indicate that hypothermia supports and fine-tunes IL-22 production/action which may contribute to regulatory properties of low ambient temperature.

By generating biologically active factors luminal microbiota shape the intestinal micro-milieu thereby regulating pathological processes such as inflammation and carcinogenesis. Preclinical data suggest that bacterial-derived butyrate and the signal transducer and activator of transcription (STAT)-3 activating cytokine interleukin (IL)-22 display concordant protective properties at the inflamed colonic epithelium. Herein, biochemical cooperation between the short-chain fatty acid butyrate and IL-22 was investigated by focusing on human Caco2 colon epithelial/carcinoma cells. We report that physiological levels of butyrate enhance IL-22 signaling thereby enforcing expression of the prototypic STAT3-downstrean target genes α1-antichymotrypsin and suppressor of cytokine signaling (SOCS)-3. A dual mode of butyrate action on the IL-22/STAT3 axis was identified. Butyrate acted by upregulating IL-22R1, the decisive chain of the heterodimeric IL-22 receptor, and, independent from that, has the potential to directly amplify STAT3-mediated gene activation as detected by chromatin immunoprecipitation analysis of STAT3 binding to the SOCS3 promoter. Since trichostatin A acted similarly, inhibition of histone deacetylases is likely at the root of these butyrate biological properties. The mutual benefit gained from interactions between the host and commensal intestinal bacteria-derived factors is an expanding field of research beginning to affect clinical practice. Data presented herein propose a supportive and fine-tuning role for butyrate in IL-22 signaling that might be therapeutically exploited by local butyrate administration or by increasing its bacterial production in the context of a fiber-rich diet. © 2016 BioFactors, 43(2):283-292, 2017.

Acetaminophen (APAP, N-acetyl-p-aminophenol, or paracetamol) overdosing is a prevalent cause of acute liver injury. While clinical disease is initiated by overt parenchymal hepatocyte necrosis in response to the analgetic, course of intoxication is substantially influenced by associated activation of innate immunity. This process is supposed to be set in motion by release of danger associated molecular patterns (DAMPs) from dying hepatocytes and is accompanied by an inflammatory cytokine response. Murine models of APAP-induced liver injury emphasize the complex role that DAMPs and cytokines play in promoting either hepatic pathogenesis or resolution and recovery from intoxication. Whereas the function of key inflammatory cytokines is controversially discussed, a subclass of specific cytokines capable of efficiently activating the hepatocyte signal transducer and activator of transcription (STAT)-3 pathway stands out as being consistently protective in murine models of APAP intoxication. Those include foremost interleukin (IL)-6, IL-11, IL-13, and IL-22. Above all, activation of STAT3 under the influence of these cytokines has the capability to drive hepatocyte compensatory proliferation, a key principle of the regenerating liver. Herein, the role of these specific cytokines during experimental APAP-induced liver injury is highlighted and discussed in a broader perspective. In hard-to-treat or at-risk patients standard therapy may fail and APAP intoxication can proceed towards a fatal condition. Focused administration of recombinant STAT3-activating cytokines may evolve as novel therapeutic approach under those ill-fated conditions.

Interleukin (IL)-22 is a STAT3-activating cytokine displaying characteristic AU-rich elements (ARE) in the 3′-untranslated region (3′-UTR) of its mRNA. This architecture suggests gene regulation by modulation of mRNA stability. Since related cytokines undergo post-transcriptional regulation by ARE-binding tristetraprolin (TTP), the role of this destabilizing protein in IL-22 production was investigated. Herein, we demonstrate that TTP-deficient mice display augmented serum IL-22. Likewise, IL-22 mRNA was enhanced in TTP-deficient splenocytes and isolated primary T cells. A pivotal role for TTP is underscored by an extended IL-22 mRNA half-life detectable in TTP-deficient T cells. Luciferase-reporter assays performed in human Jurkat T cells proved the destabilizing potential of the human IL-22-3′-UTR. Furthermore, overexpression of TTP in HEK293 cells substantially decreased luciferase activity directed by the IL-22-3′-UTR. Transcript destabilization by TTP was nullified upon cellular activation by TPA/A23187, an effect dependent on MEK1/2 activity. Accordingly, IL-22 mRNA half-life as determined in TPA/A23187-stimulated Jurkat T cells decreased under the influence of the MEK1/2 inhibitor U0126. Altogether, data indicate that TTP directly controls IL-22 production, a process counteracted by MEK1/2. The TTP-dependent regulatory pathway described herein likely contributes to the role of IL-22 in inflammation and cancer and may evolve as novel target for pharmacological IL-22 modulation.

Interleukin (IL)-22 is a cytokine displaying tissue protective and pro-regenerative functions in various preclinical disease models. Anti-bacterial, pro-proliferative, and anti-apoptotic properties mediated by activation of the transcription factor signal transducer and activator of transcription (STAT)-3 are key to biological functions of this IL-10 family member. Herein, we introduce RINm5F insulinoma cells as rat β-cell line that, under the influence of IL-22, displays activation of STAT3 with induction of its downstream gene targets Socs3, Bcl3, and Reg3b. In addition, IL-22 also activates STAT1 in this cell type. To refine those observations, IL-22 biological activity was evaluated using ex vivo cultivated murine pancreatic islets. In accord with data on RINm5F cells, islet exposure to IL-22 activated STAT3 and upregulation of STAT3-inducible Socs3, Bcl3, and Steap4 was evident under those conditions. As these observations supported the hypothesis that IL-22 may exert protective functions in toxic β-cell injury, application of IL-22 was investigated in murine multiple-low-dose streptozotocin (STZ)-induced diabetes. For that purpose, recombinant IL-22 was administered thrice either immediately before and at disease onset (at d4, d6, d8) or closely thereafter (at d8, d10, d12). These two IL-22-treatment periods coincide with two early peaks of β-cell injury detectable in this model. Notably, none of the two IL-22-treatment strategies affected diabetes incidence or blood glucose levels in STZ-treated mice. Moreover, pathological changes in islet morphology analyzed 28 days after disease induction were not ameliorated by IL-22 administration. Taken together, despite being active on rat RINm5F insulinoma cells and murine pancreatic islets, recombinant IL-22 fails to protect pancreatic β-cells in the tested protocols from toxic effects of STZ and thus is unable to ameliorate disease in the widely used model of STZ-induced diabetes.

Interleukin (IL)-22 is a STAT3-activating cytokine displaying characteristic AU-rich elements (ARE) in the 3′-untranslated region (3′-UTR) of its mRNA. This architecture suggests gene regulation by modulation of mRNA stability. Since related cytokines undergo post-transcriptional regulation by ARE-binding tristetraprolin (TTP), the role of this destabilizing protein in IL-22 production was investigated. Herein, we demonstrate that TTP-deficient mice display augmented serum IL-22. Likewise, IL-22 mRNA was enhanced in TTP-deficient splenocytes and isolated primary T cells. A pivotal role for TTP is underscored by an extended IL-22 mRNA half-life detectable in TTP-deficient T cells. Luciferase-reporter assays performed in human Jurkat T cells proved the destabilizing potential of the human IL-22-3′-UTR. Furthermore, overexpression of TTP in HEK293 cells substantially decreased luciferase activity directed by the IL-22-3′-UTR. Transcript destabilization by TTP was nullified upon cellular activation by TPA/A23187, an effect dependent on MEK1/2 activity. Accordingly, IL-22 mRNA half-life as determined in TPA/A23187-stimulated Jurkat T cells decreased under the influence of the MEK1/2 inhibitor U0126. Altogether, data indicate that TTP directly controls IL-22 production, a process counteracted by MEK1/2. The TTP-dependent regulatory pathway described herein likely contributes to the role of IL-22 in inflammation and cancer and may evolve as novel target for pharmacological IL-22 modulation.

Overdosing of the analgesic acetaminophen (APAP, paracetamol) is a major cause of acute liver injury. Whereas toxicity is initiated by hepatocyte necrosis, course of disease is regulated by mechanisms of innate immunity having the potential to serve in complex manner pathogenic or pro-regenerative functions. Interleukin (IL)-36γ has been identified as novel IL-1-like cytokine produced by and targeting epithelial (-like) tissues. Herein, we investigated IL-36γ in acute liver disease focusing on murine APAP-induced hepatotoxicity. Enhanced expression of hepatic IL-36γ and its prime downstream chemokine target CCL20 was detected upon liver injury. CCL20 expression coincided with the later regeneration phase of intoxication. Primary murine hepatocytes and human Huh7 hepatocellular carcinoma cells indeed displayed enhanced IL-36γ expression when exposed to inflammatory cytokines. Administration of IL-36 receptor antagonist (IL-36Ra) decreased hepatic CCL20 in APAP-treated mice. Unexpectedly, IL-36Ra likewise increased late phase hepatic injury as detected by augmented serum alanine aminotransferase activity and histological necrosis which suggests disturbed tissue recovery upon IL-36 blockage. Finally, we demonstrate induction of IL-36γ in inflamed livers of endotoxemic mice. Observations presented introduce IL-36γ as novel parameter in acute liver injury which may contribute to the decision between unleashed tissue damage and initiation of liver regeneration during late APAP toxicity.