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If insufficiently treated, Lyme borreliosis can evolve into an inflammatory disorder affecting skin, joints, and the CNS. Early innate immunity may determine host responses targeting infection. Thus, we sought to characterize the immediate cytokine storm associated with exposure of PBMC to moderate levels of live Borrelia burgdorferi. Since Th17 cytokines are connected to host defense against extracellular bacteria, we focused on interleukin (IL)-17 and IL-22. Here, we report that, despite induction of inflammatory cytokines including IL-23, IL-17 remained barely detectable in response to B. burgdorferi. In contrast, T cell-dependent expression of IL-22 became evident within 10 h of exposure to the spirochetes. This dichotomy was unrelated to interferon-gamma but to a large part dependent on caspase-1 and IL-1 bioactivity derived from monocytes. In fact, IL-1beta as a single stimulus induced IL-22 but not IL-17. Neutrophils display antibacterial activity against B. burgdorferi, particularly when opsonized by antibodies. Since neutrophilic inflammation, indicative of IL-17 bioactivity, is scarcely observed in Erythema migrans, a manifestation of skin inflammation after infection, protective and antibacterial properties of IL-22 may close this gap and serve essential functions in the initial phase of spirochete infection.
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.
Interleukin (IL)-18, formerly known as interferon (IFN)-γ-inducing factor, is a crucial mediator of host defence and inflammation. Control of IL-18 bioactivity by its endogenous antagonist IL-18 binding protein (IL-18BP) is a major objective of immunoregulation. IL-18BP is strongly up-regulated by IFN-γ, thereby establishing a negative feedback mechanism detectable in cell culture and in vivo. Here we sought to investigate in D.L. Dexter (DLD) colon carcinoma cells molecular mechanisms of IL-18BP induction under the influence of IFN-γ. Mutational analysis revealed that a proximal γ-activated sequence (GAS) at the IL-18BP promoter is of pivotal importance for expression by IFN-γ-activated cells. Use of siRNA underscored the essential role of the signal transducer and activator of transcription (STAT)-1 in this process. Indeed, electrophoretic mobility shift assay and chromatin immunoprecipitation analysis proved STAT1 binding to this particular GAS site. Maximal expression of IL-18BP was dependent on de novo protein synthesis but unaffected by silencing of interferon regulatory factor-1. Altogether, data presented herein indicate that direct action of STAT1 on the IL-18BP promoter at the proximal GAS element is key to IL-18BP expression by IFN-γ-stimulated DLD-1 colon carcinoma cells.
Acetaminophen [paracetamol, N-acetyl-p-aminophenol (APAP)]-induced acute liver injury (ALI) not only remains a persistent clinical challenge but likewise stands out as well-characterized paradigmatic model of drug-induced liver damage. APAP intoxication associates with robust hepatic necroinflammation the role of which remains elusive with pathogenic but also pro-regenerative/-resolving functions being ascribed to leukocyte activation. Here, we shine a light on and put forward a unique role of the interleukin (IL)-1 family member IL-18 in experimental APAP-induced ALI. Indeed, amelioration of disease as previously observed in IL-18-deficient mice was further substantiated herein by application of the IL-18 opponent IL-18-binding protein (IL-18BPd:Fc) to wild-type mice. Data altogether emphasize crucial pathological action of this cytokine in APAP toxicity. Adding recombinant IL-22 to IL-18BPd:Fc further enhanced protection from liver injury. In contrast to IL-18, the role of prototypic pro-inflammatory IL-1 and tumor necrosis factor-α is controversially discussed with lack of effects or even protective action being repeatedly reported. A prominent detrimental function for IL-18 in APAP-induced ALI as proposed herein should relate to its pivotal role for hepatic expression of interferon-γ and Fas ligand, both of which aggravate APAP toxicity. As IL-18 serum levels increase in patients after APAP overdosing, targeting IL-18 may evolve as novel therapeutic option in those hard-to-treat patients where standard therapy with N-acetylcysteine is unsuccessful. Being a paradigmatic experimental model of ALI, current knowledge on ill-fated properties of IL-18 in APAP intoxication likewise emphasizes the potential of this cytokine to serve as therapeutic target in other entities of inflammatory liver diseases.
Background: The transcription factor T-bet is pivotal for initiation of Th1-related immunoactivation. Identification of novel genes directly regulated by T-bet is crucial.
Results: Genome-wide analysis and subsequent experiments revealed that T-bet up-regulates IL-36γ/IL-1F9 in myeloid cells.
Conclusion: IL-1-related IL-36γ is a direct T-bet target in myeloid cells.
Significance: Observations suggest that IL-36γ , besides IFNγ, contributes to T-bet functions in immunopathology
By concerted action in dendritic (DC) and T cells, T-box expressed in T cells (T-bet, Tbx21) is pivotal for initiation and perpetuation of Th1 immunity. Identification of novel T-bet-regulated genes is crucial for further understanding the biology of this transcription factor. By combining siRNA technology with genome-wide mRNA expression analysis, we sought to identify new T-bet-regulated genes in predendritic KG1 cells activated by IL-18. One gene robustly dependent on T-bet was IL-36γ, a recently described novel IL-1 family member. Promoter analysis revealed a T-bet binding site that, along with a κB site, enables efficient IL-36γ induction. Using knock-out animals, IL-36γ reliance on T-bet was extended to murine DC. IL-36γ expression by human myeloid cells was confirmed using monocyte-derived DC and M1 macrophages. The latter model was employed to substantiate dependence of IL-36γ on endogenous T-bet in human primary cells. Ectopic expression of T-bet likewise mediated IL-36γ production in HaCaT keratinocytes that otherwise lack this transcription factor. Additional experiments furthermore revealed that mature IL-36γ has the capability to establish an inflammatory gene expression profile in human primary keratinocytes that displays enhanced mRNA levels for TNFα, CCL20, S100A7, inducible NOS, and IL-36γ itself. Data presented herein shed further light on involvement of T-bet in innate immunity and suggest that IL-36γ, besides IFNγ, may contribute to functions of this transcription factor in immunopathology.
Cytokine regulation of high-output nitric oxide (NO) derived from inducible NO synthase (iNOS) is critically involved in inflammation biology and host defense. Herein, we set out to characterize the role of type I interferon (IFN) as potential regulator of hepatic iNOS in vitro and in vivo. In this regard, we identified in murine Hepa1-6 hepatoma cells a potent synergism between pro-inflammatory interleukin-β/tumor necrosis factor-α and immunoregulatory IFNβ as detected by analysis of iNOS expression and nitrite release. Upregulation of iNOS by IFNβ coincided with enhanced binding of signal transducer and activator of transcription-1 to a regulatory region at the murine iNOS promoter known to support target gene expression in response to this signaling pathway. Synergistic iNOS induction under the influence of IFNβ was confirmed in alternate murine Hepa56.1D hepatoma cells and primary hepatocytes. To assess iNOS regulation by type I IFN in vivo, murine acetaminophen (APAP)-induced sterile liver inflammation was investigated. In this model of acute liver injury, excessive necroinflammation drives iNOS expression in diverse liver cell types, among others hepatocytes. Herein, we demonstrate impaired iNOS expression in type I IFN receptor-deficient mice which associated with diminished APAP-induced liver damage. Data presented indicate a vital role of type I IFN within the inflamed liver for fine-tuning pathological processes such as overt iNOS expression.
Background Production of interferon (IFN)-gamma is key to efficient anti-tumor immunity. The present study was set out to investigate effects of IFNgamma on the release of the potent pro-angiogenic mediator IL-8 by human A549 lung carcinoma cells.
Methods A549 cells were cultured and stimulated with interleukin (IL)-1beta alone or in combination with IFNgamma. IL-8 production by these cells was analyzed with enzyme linked immuno sorbent assay (ELISA). mRNA-expression was analyzed by real-time PCR and RNase protection assay (RPA), respectively. Expression of inhibitor-kappaBalpha, cellular IL-8, and cyclooxygenase-2 was analyzed by Western blot analysis.
Results Here we demonstrate that IFNgamma efficiently reduced IL-8 secretion under the influence of IL-1beta. Surprisingly, real-time PCR analysis and RPA revealed that the inhibitory effect of IFNgamma on IL-8 was not associated with significant changes in mRNA levels. These observations concurred with lack of a modulatory activity of IFNgamma on IL-1beta-induced NF-kappaB activation as assessed by cellular IkappaB levels. Moreover, analysis of intracellular IL-8 suggests that IFNgamma modulated IL-8 secretion by action on the posttranslational level. In contrast to IL-8, IL-1beta-induced cyclooxygenase-2 expression and release of IL-6 were not affected by IFNgamma indicating that modulation of IL-1beta action by this cytokine displays specificity.
Conclusions Data presented herein agree with an angiostatic role of IFNgamma as seen in rodent models of solid tumors and suggest that increasing T helper type 1 (Th1)-like functions in lung cancer patients e.g. by local delivery of IFNgamma may mediate therapeutic benefit via mechanisms that potentially include modulation of pro-angiogenic IL-8.
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 5-h exposure of endotoxemic mice to 4°C reduces body temperature by 5.0° and enhances splenic and colonic il22 gene expression. In contrast, tumor necrosis factor-α 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 nuclear factor of activated T cells (NFAT)-c2. Notably, NFAT antagonism by cyclosporin 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 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.
Murine acetaminophen-induced acute liver injury (ALI) serves as paradigmatic model for drug-induced hepatic injury and regeneration. As major cause of ALI, acetaminophen overdosing is a persistent therapeutic challenge with N-acetylcysteine clinically used to ameliorate parenchymal necrosis. To identify further treatment strategies that serve patients with poor N-acetylcysteine responses, hepatic 3′mRNA sequencing was performed in the initial resolution phase at 24 h/48 h after sublethal overdosing. This approach disclosed 45 genes upregulated (≥5-fold) within this time frame. Focusing on C5aR1, we observed in C5aR1-deficient mice disease aggravation during resolution of intoxication as evidenced by increased liver necrosis and serum alanine aminotransferase. Moreover, decreased hepatocyte compensatory proliferation and increased caspase-3 activation at the surroundings of necrotic cores were detectable in C5aR1-deficient mice. Using a non-hypothesis-driven approach, herein pro-regenerative/-resolving effects of C5aR1 were identified during late acetaminophen-induced ALI. Data concur with protection by the C5a/C5aR1-axis during hepatectomy and emphasize the complex role of inflammation during hepatic regeneration and repair.
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.