Christian Ehlting

The macrophage response towards LPS and its control through the p38MAPK–STAT3 axis

In macrophages detection of gram-negative bacteria particularly involves binding of the outer-wall component lipopolysaccharide (LPS) to its cognate receptor complex, comprising Toll like receptor 4 (TLR4), CD14 and MD2. LPS-induced formation of the LPS receptor complex elicits a signaling network, including intra-cellular signal-transduction directly activated by the TLR4 receptor complex as well as successional induction of indirect autocrine and paracrine signaling events. All these different pathways are integrated into the macrophage response towards an inflammatory stimulus by a highly complex cross-talk of the pathways engaged. This also includes a tight control by several intra- and inter-cellular feedback loops warranting an inflammatory response sufficient to battle invading pathogens and to avoid non-essential tissue damage caused by an overwhelming inflammatory response. Several evidences indicate that the reciprocal cross-talk between the p38(MAPK)-pathway and signal transducer and activator of transcription (STAT)3-mediated signal-transduction forms a critical axis successively activated by LPS. The balanced activation of this axis is essential for both induction and propagation of the inflammatory macrophage response as well as for the control of the resolution phase, which is largely driven by IL-10 and sustained STAT3 activation. In this context regulation of suppressor of cytokine signaling (SOCS)3 expression and the recently described divergent regulatory roles of the two p38(MAPK)-activated protein kinases MK2 and MK3 for the regulation of LPS-induced NF-κB- and IRF3-mediated signal-transduction and gene expression, which includes the regulation of IFNβ, IL-10 and DUSP1, appears to play an important role.

Regulation of Suppressor of Cytokine Signaling 3 (SOCS3) mRNA Stability by TNF-α Involves Activation of the MKK6/p38MAPK/MK2 Cascade

The potential of some proinflammatory mediators to inhibit gp130-dependent STAT3 activation by enhancing suppressor of cytokine signaling (SOCS) 3 expression represents an important molecular mechanism admitting the modulation of the cellular response toward gp130-mediated signals. Thus, it is necessary to understand the mechanisms involved in the regulation of SOCS3 expression by proinflammatory mediators. In this study, we investigate SOCS3 expression initiated by the proinflammatory cytokine TNF-α. In contrast to IL-6, TNF-α increases SOCS3 expression by stabilizing SOCS3 mRNA. Activation of the MAPK kinase 6 (MKK6)/p38MAPK-cascade is required for TNF-α-mediated stabilization of SOCS3 mRNA and results in enhanced SOCS3 protein expression. In fibroblasts or macrophages deficient for MAPK-activated protein kinase 2 (MK2), a downstream target of the MKK6/p38MAPK cascade, basal SOCS3-expression is strongly reduced and TNF-α-induced SOCS3-mRNA stabilization is impaired, indicating that MK2 is crucial for the control of SOCS3 expression by p38MAPK-dependent signals. As a target for SOCS3 mRNA stability-regulating signals, a region containing three copies of a pentameric AUUUA motif in close proximity to a U-rich region located between positions 2422 and 2541 of the 3′ untranslated region of SOCS3 is identified. One factor that could target this region is the zinc finger protein tristetraprolin (TTP), which is shown to be capable of destabilizing SOCS3 mRNA via this region. However, data from TTP-deficient cells suggest that TTP does not play an irreplaceable role in the regulation of SOCS3 mRNA stability by TNF-α. In summary, these data indicate that TNF-α regulates SOCS3 expression on the level of mRNA stability via activation of the MKK6/p38MAPK cascade and that the activation of MK2, a downstream target of p38MAPK, is important for the regulation of SOCS3 expression.

Distinct Functions of the Mitogen-activated Protein Kinase-activated Protein (MAPKAP) Kinases MK2 and MK3 MK2 MEDIATES LIPOPOLYSACCHARIDE-INDUCED SIGNAL TRANSDUCERS AND ACTIVATORS OF TRANSCRIPTION 3 (STAT3) ACTIVATION BY PREVENTING NEGATIVE REGULATORY EFFECTS OF MK3

In LPS-treated macrophages, activation of STAT3 is considered to be crucial for terminating the production of inflammatory cytokines. By analyzing the role of MAPK-activated protein kinase (MK) 2 and MK3 for LPS-induced STAT3 activation in macrophages, the present study provides evidence that MK2 is crucial for STAT3 activation in response to LPS because it prevents MK3 from impeding IFNβ gene expression. Accordingly, LPS-induced IFNβ gene expression is down-regulated in MK2-deficient macrophages and can be reconstituted by additional ablation of the MK3 gene in MK2/3−/− macrophages. This is in contrast to LPS-induced IL-10 expression, which essentially requires the presence of MK2. Further analysis of downstream signaling events involved in the transcriptional regulation of IFNβ gene expression suggests that, in the absence of MK2, MK3 impairs interferon regulatory factor 3 protein expression and activation and inhibits nuclear translocation of p65. This inhibition of p65 nuclear translocation coincides with enhanced expression and delayed degradation of IκBβ, whereas expression of IκBα mRNA and protein is impaired in the absence of MK2. The observation that siRNA directed against IκBβ is able to reconstitute IκBα expression in MK2−/− macrophages suggests that enhanced expression and delayed degradation of IκBβ and impaired NFκB-dependent IκBα expression are functionally linked. In summary, evidence is provided that MK2 regulates LPS-induced IFNβ expression and downstream STAT3 activation as it restrains MK3 from mediating negative regulatory effects on NFκB- and interferon regulatory factor 3-dependent LPS signaling.

Bile acids PKA-dependently induce a switch of the IL-10/IL-12 ratio and reduce proinflammatory capability of human macrophages

That cholestatic conditions are accompanied by an enhanced susceptibility to bacterial infection in human and animal models is a known phenomenon. This correlates with the observation that bile acids have suppressive effects on cells of innate and adaptive immunity. The present study provides evidence that in human macrophages, bile acids inhibit the LPS‐induced expression of proinflammatory cytokines without affecting the expression of the anti‐inflammatory cytokine IL‐10. This results in a macrophage phenotype that is characterized by an increased IL‐10/IL‐12 ratio. Correspondingly, bile acids suppress basal phagocytic activity of human macrophages. These effects of bile acids can be mimicked by cAMP, which is presumably induced TGR5‐dependently. The data provided further suggest that in primary human macrophages, modulation of the macrophage response toward LPS by bile acids involves activation of CREB, disturbed nuclear translocation of NF‐κB, and PKA‐dependent enhancement of LPS‐induced cFos expression. The increase in cFos expression is paralleled by an enhanced formation of a protein complex comprising cFos and the p65 subunit of NF‐κB. In summary, the data provided suggest that in human macrophages, bile acids induce an anti‐inflammatory phenotype characterized by an increased IL‐10/IL‐12 ratio via activation of PKA and thereby, prevent their activation as classically activated macrophages. This bile acid‐induced modulation of macrophage function may also be responsible for the experimentally and clinically observed anti‐inflammatory and immunosuppressive effects of bile acids.

TNF-α Induces Tyrosine Phosphorylation and Recruitment of the Src Homology Protein-Tyrosine Phosphatase 2 to the gp130 Signal-Transducing Subunit of the IL-6 Receptor Complex

Recently, it has been demonstrated that TNF-α and LPS induce the expression of suppressor of cytokine signaling 3 (SOCS3) and inhibit IL-6-induced STAT3 activation in macrophages. Inhibitor studies suggested that both induction of SOCS3 and inhibition of IL-6-induced STAT3 activation depend on the activation of p38 mitogen-activated protein kinase. Since recruitment of the tyrosine phosphatase Src homology protein tyrosine phosphatase 2 (SHP2) to the signal-transducing receptor subunit gp130 attenuates IL-6-mediated STAT-activation, we were interested in whether TNF-α also induces the association of SHP2 to the gp130 receptor subunit. In this study we demonstrate that stimulation of macrophages and fibroblast cell lines with TNF-α causes the recruitment of SHP2 to the gp130 signal-transducing subunit and leads to tyrosine phosphorylation of SHP2 and gp130. In this context the cytoplasmic SHP2/SOCS3 recruitment site of gp130 tyrosine 759 is shown to be important for the inhibitory effects of TNF-α, since mutation of this residue completely restores IL-6-stimulated activation of STAT3 and, consequently, of a STAT3-dependent promoter. In this respect murine fibroblasts lacking exon 3 of SHP2 are not sensitive to TNF-α, indicating that functional SHP2 and its recruitment to gp130 are key events in inhibition of IL-6-dependent STAT activation by TNF-α. Furthermore, activation of p38 mitogen-activated protein kinase is shown to be essential for the inhibitory effect of TNF-α on IL-6 signaling and TNF-α-dependent recruitment of SHP2 to gp130.

Glucocorticoids increase interleukin‐6–dependent gene induction by interfering with the expression of the suppressor of cytokine signaling 3 feedback inhibitor

Glucocorticoids are known to be potent regulators of inflammation and have been used pharmacologically against inflammatory, immune, and lymphoproliferative diseases for more than 50 years. Due to their possible and well‐documented side effects, it is crucial to understand the molecular mechanisms and targets of glucocorticoid action in detail. Several modes of action have been discussed; nevertheless, none of them fully explain all the functions of glucocorticoids. Therefore, we analyzed the cross‐talk between glucocorticoids and interleukin‐6 (IL‐6) in the liver. IL‐6 exerts pro‐inflammatory as well as anti‐inflammatory properties and is a main inducer of the acute‐phase response. The balance between the proinflammatory and anti‐inflammatory activities of IL‐6 is tightly regulated by suppressor of cytokine signaling 3 (SOCS3), a well‐known feedback inhibitor of IL‐6 signaling. Here, it is demonstrated that glucocorticoids enhance IL‐6–dependent γ‐fibrinogen expression. Studying of the underlying mechanism revealed prolonged activation of signal transducer and activator of transcription 3 (STAT3) caused by down‐regulation of SOCS3 protein expression. Consequently, in SOCS3‐deficient cells glucocorticoids do not affect IL‐6–induced signal transduction. Moreover, in hepatocytes lacking the SOCS3 recruiting motif within gp130, IL‐6–dependent γ‐fibrinogen expression is not influenced by glucocorticoid treatment. Conclusion: Glucocorticoids interfere with IL‐6–induced expression of the feedback inhibitor SOCS3, thereby leading to enhanced expression of acute‐phase genes in hepatocytes. This mechanism contributes to the explanation of how glucocorticoids affect inflammation and acute‐phase gene induction. (HEPATOLOGY 2012;55:256–266)

BMP-9 interferes with liver regeneration and promotes liver fibrosis

Objective Bone morphogenetic protein (BMP)-9, a member of the transforming growth factor-β family of cytokines, is constitutively produced in the liver. Systemic levels act on many organs and tissues including bone and endothelium, but little is known about its hepatic functions in health and disease. Design Levels of BMP-9 and its receptors were analysed in primary liver cells. Direct effects of BMP-9 on hepatic stellate cells (HSCs) and hepatocytes were studied in vitro, and the role of BMP-9 was examined in acute and chronic liver injury models in mice. Results Quiescent and activated HSCs were identified as major BMP-9 producing liver cell type. BMP-9 stimulation of cultured hepatocytes inhibited proliferation, epithelial to mesenchymal transition and preserved expression of important metabolic enzymes such as cytochrome P450. Acute liver injury caused by partial hepatectomy or single injections of carbon tetrachloride (CCl4) or lipopolysaccharide (LPS) into mice resulted in transient downregulation of hepatic BMP-9 mRNA expression. Correspondingly, LPS stimulation led to downregulation of BMP-9 expression in cultured HSCs. Application of BMP-9 after partial hepatectomy significantly enhanced liver damage and disturbed the proliferative response. Chronic liver damage in BMP-9-deficient mice or in mice adenovirally overexpressing the selective BMP-9 antagonist activin-like kinase 1-Fc resulted in reduced deposition of collagen and subsequent fibrosis. Conclusions Constitutive expression of low levels of BMP-9 stabilises hepatocyte function in the healthy liver. Upon HSC activation, endogenous BMP-9 levels increase in vitro and in vivo and high levels of BMP-9 cause enhanced damage upon acute or chronic injury.

MAPKAP kinase 2 regulates IL-10 expression and prevents formation of intrahepatic myeloid cell aggregates during cytomegalovirus infections

BACKGROUND & AIMS The kinase p38(MAPK) and its downstream target MAPKAP kinase (MK) 2 are critical regulators of inflammatory responses towards pathogens. To date, the relevance of MK2 for regulating IL-10 expression and other cytokine responses towards cytomegalovirus (CMV) infection and the impact of this pathway on viral replication in vitro and in vivo is unknown and the subject of this study. METHODS The effect of MK2, interferon-α receptor (IFNAR)1, tristetraprolin (TTP) and IL-10 on mouse (M)CMV virus titres, cytokine expression, signal transduction, transcript stability, liver enzymes release, immune cell recruitment and aggregation in response to MCMV infection were studied ex vivo in hepatocytes and macrophages, as well as in vivo. RESULTS MK2 is critical for MCMV-induced production of IL-10, IFN-α2 and 4, IFN-β, IL-6, and TNF-α but not for IFN-γ. The MCMV-induced IL-10 production requires activation of IFNAR1 and is further regulated by MK2 and TTP-dependent stabilization of IL-10 transcripts. MK2(-/-) mice are able to control acute MCMV replication, despite deregulated cytokine production. This may be related to the observation that MCMV-infected MK2(-/-) mice show enhanced formation of focal intrahepatic lymphocyte infiltrates resembling intrahepatic myeloid cell aggregates of T cell expansion (iMATEs), which were also observed in MCMV-infected IL-10(-/-) mice but are almost absent in MCMV-infected wild-type controls. CONCLUSIONS The data suggest that MK2 is critical for regulating cytokine responses towards acute MCMV infection, including that of IL-10 via IFNARI-mediated circuits. MCMV stimulates expression of MK2-dependent cytokines, in particular IL-10 and thereby prevents enhanced formation of intrahepatic iMATE-like cellular aggregates.

Model-Based Characterization of Inflammatory Gene Expression Patterns of Activated Macrophages.

Macrophages are cells with remarkable plasticity. They integrate signals from their microenvironment leading to context-dependent polarization into classically (M1) or alternatively (M2) activated macrophages, representing two extremes of a broad spectrum of divergent phenotypes. Thereby, macrophages deliver protective and pro-regenerative signals towards injured tissue but, depending on the eliciting damage, may also be responsible for the generation and aggravation of tissue injury. Although incompletely understood, there is emerging evidence that macrophage polarization is critical for these antagonistic roles. To identify activation-specific expression patterns of chemokines and cytokines that may confer these distinct effects a systems biology approach was applied. A comprehensive literature-based Boolean model was developed to describe the M1 (LPS-activated) and M2 (IL-4/13-activated) polarization types. The model was validated using high-throughput transcript expression data from murine bone marrow derived macrophages. By dynamic modeling of gene expression, the chronology of pathway activation and autocrine signaling was estimated. Our results provide a deepened understanding of the physiological balance leading to M1/M2 activation, indicating the relevance of co-regulatory signals at the level of Akt1 or Akt2 that may be important for directing macrophage polarization.

Crucial role of the MAPKAP kinases 2 and 3 for pathogen-induced inflammation and their relevance for the immune response of the liver

The liver plays an important role in innate and adaptive immunity and in particular in induction of tolerance. The liver is frequently exposed to pathogens like gut-derived food antigens, environmental toxins and bacterial or viral products reaching the liver via the blood flow. It is central for production of acute phase proteins, among others including protease inhibitors, soluble pattern-recognition receptors and components of the complement system, which are important constituents of innate immunity. The liver harbors the largest pool of sessile tissue macrophages within the body, mainly localized within the periportal area of the sinusoids at a strategically important position. These resident tissue macrophages play a key role for the elimination of opsonized immune complexes by phagocytosis and are important sources of inflammatory cytokines, which induce acute phase protein production in hepatocytes and further trigger innate immunity and subsequent formation of adaptive immunity. Within macrophages the MAPKAP kinases (MK)2 and 3, which represent downstream targets of the MAP kinase family member p38MAPK, are known to own key functions in the coordination of the inflammatory response. Thereby, their role for the regulation of the expression of inflammatory as well as anti-inflammatory cytokines, such as TNF-α, IL-1β, IL-6, IFN-γ and IL-10 has been mainly investigated in the context of bacterial components and in particular in the context of lipopolysaccharide (LPS). In this context MK2 and MK3 have been suggested to act in a co-operative manner as the expression of these cytokines is abrogated upon deletion of MK2 and is further diminished by the additional deletion of MK3. However, while investigating the role of MK2 and MK3 for the regulation of LPS-induced IFN-β production our group recently provided evidence that MK2 and MK3 are also able to exert rather distinct than co-operative regulatory effects on gene expression [1]. At the current stage the data suggest that these distinct regulatory effects of MK2 and MK3 become apparent, if regulation of respective target gene expression by these two kinases exclusively occurs at the level of transcription and does not involve post-transcriptional regulatory mechanisms such as regulation of transcript stability. Contrariwise, if regulation of gene expression by MK2 and MK3 occurs at the level of transcript stability these two kinases mainly seem to act co-operatively. Thus, MK2 and to a lesser extent MK3 are critical for regulation of TNF-α, IL-6 and IL-10 in response to LPS, where they are involved in control of transcript stability or translation. This is in contrast to the regulation of IFN-β gene expression by MK2 and MK3 where MK2 controls IFN-β gene expression by neutralizing inhibitory effects of MK3, which in the absence of MK2 inhibits transcriptional activation of IFN-β gene expression by impeding IRF-3 protein expression as well as LPS-induced nuclear translocation of NFκB [1]. Of note, unlike for example regulation of LPS-induced IL-10 expression, which essentially requires MK2 for stabilization of the IL-10 transcript, the stability of the IFN-β transcript does not require the presence of MK2. Extended analysis of LPS-induced gene expression in macrophages derived from wild-type animals or from animals deficient for MK2 or MK2 and MK3 using “whole genome microarrays” suggested that there is a larger group of genes, which are controlled by MK2 and MK3 in a way that is comparable to that of IFN-β. Interestingly, these studies further revealed that the deletion of MK2 or of both MK2 and MK3 results in massive alterations of the inflammatory response of macrophages towards LPS, which also includes that approximately 30% of genes are only regulated by LPS if MK2 or MK2 and MK3 are absent. This indicates that apart from being critically involved in positive or negative regulation of gene expression in response to LPS MK2 and MK3 are also required to prevent a larger group of genes from being regulated in response to LPS. The role of MK2 is comparatively well studied in the context of bacterial infections and the inflammatory host response towards bacteria-derived pathogens such as LPS [2] whereas its role for viral infections and virus-host interactions is less well characterized. Apart from other inflammatory mediators cytomegalovirus (CMV) induces the expression of the anti-inflammatory cytokine IL-10 that dampens activation of Th1 cells, NK cells and macrophages, which are required for optimal pathogen clearance, but also contribute to tissue damage during infection. Furthermore the expression of IL-10 limits immune cell activation and suppresses IFN-γ-induced MHC class I and II surface expression, thereby escaping anti-viral mechanisms and diminishing the pro-inflammatory response. This is of particular importance for the prevention of CMV-induced liver pathogenesis as suggested from studies on IL-10-deficient mice, which upon infection with murine (M)CMV develop an exaggerated pro-inflammatory cytokine response and enhanced liver injury, characterized by the increased induction of apoptotic cell death and enlarged cellular infiltration. However, the molecular mechanisms involved in CMV-induced IL-10 expression are unclear. Our studies reported herein propose a relevance of MK2 for MCMV-infection in vivo and characterizes its role for virus-induced IL-10 expression in vitro and in vivo. The data presented indicate that in macrophages MK2 is crucial for MCMV-induced IFN-β expression, which in turn represents an important mediator for sustained IL-10 production in response to MCMV infection as suggested from studies using macrophages isolated from interferon alpha receptor (IFNAR)1-deficient mice or wild-type macrophages treated with antagonizing antibodies specific for IFNAR1. These data suggest that type I IFN driven feedback loops are critical for virus-induced production of IL-10. In addition, MK2 is further essential for the stabilization of the IL-10 transcript. Thereby, MK2 stabilizes the IL-10 transcript most likely by preventing destabilizing effects of tristetraproline (TTP), which is expressed in the host cell upon MCMV-infection to exert a negative feedback regulation on IL-10 expression. Apart from IL-10 and IFN-β MK2 is further demonstrated to be necessary for MCMV-induced production of other cytokines such as IL-6 and TNF-α, whereas it is dispensable for the production of IFN-γ. As a consequence this results in an increased IFN-γ/IL-10 ratio, which may be responsible for the enhancement of MHC class I and II expression observed in the liver tissue of MCMV-infected MK2-deficient mice. Enhanced expression of MHC proteins in turn may be involved in the observed formation of intrahepatic CD11b+ and CD8+ mononuclear cell aggregates and in an enhancement of apoptotic cell death observable in the liver tissue of MCMV-infected MK2-deficient mice. The latter may contribute to the fact that despite of profound alterations of cytokine expression, including impaired expression of IFN-β, IL-6, IL-10 and TNF-α in response to MCMV infection, viral clearance is not impaired in MK2-deficient mice.