Lipopolysaccharide- and p38 MAPK-mediated signaling of the Heme oxygenase-1 (HO-1) gene in macrophages

Monocytes or macrophages are crucial regulators of inflammation. Intracellular signaling in macrophages is tightly regulated to ensure that they do not undergo excessive activation, which may lead to chronic inflammation accompanied with potential damage to the host tissue. Induction of HO-1 in macrophages has been shown to have potent immunomodulatory properties. In this thesis two different signalling mechanisms are described which lead to the induction of HO-1 in macrophages. In the first part the signalling pathway leading to the induction of HO-1 via toll-like receptor (TLR)4 by the classical pro-inflammatory stimulus lipopolysaccharide (LPS) is described. The second part of this thesis deals with the induction of HO-1 caused by the inhibition of the mitogen activated protein kinase (MAPK) p38. Induction of HO-1 by LPS in macrophages has been described earlier, but the exact regulatory mechanisms of this pathway are not well understood. In this thesis it is shown that Bruton’s tyrosine kinase (Btk) mediates the LPS-induction of HO-1. LPS-dependent induction of HO-1 was blocked in macrophages treated with the Btk inhibitor, LFM-A13 or in Btk alveolar macrophages. Promoter studies and quantitative real time PCR studies revealed a transcriptional regulatory mechanism. Btk was shown to mediate the production of ROS and activation of the transcription factor Nrf2. p38 MAPK inhibition is shown to increase the expression of HO-1. This was rather surprising, because activation of p38 MAPK has earlier been shown to mediate HO-1 induction caused by various stimuli. This increase in HO-1 expression was also observed in p38mouse embryonic fibroblasts. Further analysis revealed that p38 inhibition leads to an increased production of reactive oxygen species (ROS) and activation of Nrf2. Furthermore, ERK MAPK was also shown to be involved in this pathway. Taken together, this thesis demonstrates that signaling to HO-1 in macrophages is primarily mediated by the transcription factor Nrf2. Further studies to unravel the regulation of this gene may help to develop novel strategies for clinical intervention in inflammatory disorders.Monozyten und Makrophagen spielen eine bedeutsame Rolle für die Regulation von Entzündungsreaktionen. Die intrazelluläre signaltransduktion in Makrophagen wird streng reguliert, um eine überschießende Aktivierung dieser Zellen zu vermeiden, die zu einer chronischen Entzündung mit möglicher Gewebeschädigung führen könnte. Für die Induktion der Hämoxygenase (HO)-1 in Makrophagen konnte eine wirksame immunmodulatorische Wirkung gezeigt werden. In der vorliegenden Arbeit werden zwei verschiedene Signalübertragungwege beschrieben, die zu einer Induktion der HO-1 in Makrophagen führen. Im ersten Teil der Arbeit ist ein Signalweg beschrieben, der zu einer Induktion der HO-1 über eine Aktivierung von Toll-like Rezeptor (TLR)-4 durch den klassischen pro-inflammatorischen Stimulus Lipopolysaccharid (LPS) führt. Der zweite Teil der Arbeit beschäftigt sich mit der Induktion der HO-1 durch die Inhibition der Mitogen-aktivierten Proteinkinase (MAPK) p38. Die Induktion der HO-1 durch LPS wurde bereits früher erstmals beschrieben, aber die genauen regulatorischen Mechanismen sind nicht genau bekannt. In der vorliegenden Arbeit wurde gezeigt, dass die Bruton-Tyrosinkinase (Btk) die LPS-abhängige Induktion der HO-1 vermittelt. Die HO-1 Induktion durch LPS wurde durch Behandlung von Makrophagen mit dem Btk Inhibitor LFM-A13 oder in Btk-/- Alveolarmakrophagen blockiert. Promoterstudien und quantitative real time PCR Studien zeigten einen zugrundeliegenden transkriptionalen Mechanismus. Weiterhin konnte gezeigt werden, dass die Inhibition der p38 MAPK die HO-1 Expression heraufreguliert. Dieser Befund war unerwartet, weil in früheren Untersuchungen gezeigt worden war, dass p38 Aktivierung die Induktion der HO-1 vermittelt hatte. Diese HO-1 Induktion konnte auch in p38-/- murinen embryonalen Fibroblasten beobachtet werden. Weitere Untersuchungen zeigten, dass die Inhibition von p38 zu einer gesteigerten Produktion von reaktiven Sauerstoffspezies (ROS) und einer Aktivierung von Nrf2 führte. Außerdem konnte gezeigt werden, dass die MAPK ERK an dieser Regulation beteiligt war. Zusammenfassend zeigt diese Arbeit, dass die signaltransduktion zum HO-1 Gen in Makrophagen in erster Linie durch den Transkriptionsfaktor Nrf2 vermittelt wird. Weitere Studien zur Aufklärung der genauen Regulation dieses Genes könnten dabei helfen, neue Behandlungsstrategien bei der entzündlichen Erkrankungen zu entwickeln

Heme oxygenase-1 as a therapeutic target in inflammatory disorders of the gastrointestinal tract

Heme oxygenase (HO)-1 is the inducible isoform of the first and rate-limiting enzyme of heme degradation. HO-1 not only protects against oxidative stress and apoptosis, but has received a great deal of attention in recent years because of its potent anti-inflammatory functions. Studies with HO-1 knockout animal models have led to major advances in the understanding of how HO-1 might regulate inflammatory immune responses, although little is known on the underlying mechanisms. Due to its beneficial effects the targeted induction of this enzyme is considered to have major therapeutic potential for the treatment of inflammatory disorders. This review discusses current knowledge on the mechanisms that mediate anti-inflammatory protection by HO-1. More specifically, the article deals with the role of HO-1 in the pathophysiology of viral hepatitis, inflammatory bowel disease, and pancreatitis. The effects of specific HO-1 modulation as a potential therapeutic strategy in experimental cell culture and animal models of these gastrointestinal disorders are summarized. In conclusion, targeted regulation of HO-1 holds major promise for future clinical interventions in inflammatory diseases of the gastrointestinal tract

Human and murine macrophages exhibit differential metabolic responses to lipopolysaccharide - A divergent role for glycolysis

International audienceMacrophages adopt different phenotypes in response to microenvironmental changes, which can be principally classified into inflammatory and anti-inflammatory states. Inflammatory activation of macrophages has been linked with metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis. In contrast to mouse macrophages, little information is available on the link between metabolism and inflammation in human macrophages. In the current report it is demonstrated that lipopolysaccharide (LPS)-activated human peripheral blood monocyte-derived macrophages (hMDMs) fail to undergo metabolic reprogramming towards glycolysis, but rely on oxidative phosphorylation for the generation of ATP. By contrast, activation by LPS led to an increased extracellular acidification rate (glycolysis) and decreased oxygen consumption rate (oxidative phosphorylation) in mouse bone marrow-derived macrophages (mBMDMs). Mitochondrial bioenergetics after LPS stimulation in human macrophages was unchanged, but was markedly impaired in mouse macrophages. Furthermore, treatment with 2-deoxyglucose, an inhibitor of glycolysis, led to cell death in mouse, but not in human macrophages. Finally, glycolysis appeared to be critical for LPS-mediated induction of the anti-inflammatory cytokine interleukin-10 in both human and mouse macrophages. In summary, these findings indicate that LPS-induced immunometabolism in human macrophages is different to that observed in mouse macrophages

TLR4 activation alters labile heme levels to regulate BACH1 and heme oxygenase-1 expression in macrophages

International audienceHeme oxygenase (HO)-1, a stress-inducible enzyme that converts heme into carbon monoxide (CO), iron and biliverdin, exerts important anti-inflammatory effects in activated macrophages. HO-1 expression is mainly governed by a mutual interplay between the transcriptional factor NRF2 and the nuclear repressor BTB and CNC homology 1 (BACH1), a heme sensor protein. In the current study we hypothesized that alterations in the levels of intracellular labile heme in macrophages stimulated by lipopolysaccharide (LPS), a prototypical pro-inflammatory Toll-like receptor (TLR)4 agonist, are responsible for BACH1-dependent HO-1 expression. To this end, labile heme was determined in both mouse bone marrow-derived macrophages (mBMDMs) and human monocyte-derived macrophages (hMDMs) using an apo-horseradish peroxidase-based assay. We found that LPS raised the levels of labile heme, depressed BACH1 protein and up-regulated HO-1 in mBMDMs. In contrast, in hMDMs LPS decreased labile heme levels while increasing BACH1 expression and down-regulating HO-1. These effects were abolished by the TLR4 antagonist TAK-242, suggesting that TLR4 activation triggers the signaling cascade leading to changes in the labile heme pool. Studies using mBMDMs from BACH1-/- and NRF2-/- mice revealed that regulation of HO-1 and levels of labile heme after LPS stimulation are strictly dependent on BACH1, but not NRF2. A strong interplay between BACH1-mediated HO-1 expression and intracellular levels of labile heme was also confirmed in hMDMs with siRNA knockdown studies and following inhibition of de novo heme synthesis with succinylacetone. Finally, CORM-401, a compound that liberates CO, counteracted LPS-dependent down-regulation of HO-1 and restored levels of labile heme in hMDMs. In conclusion, alterations of labile heme levels in macrophages following TLR4 stimulation play a crucial role in BACH1-mediated regulation of HO-1 expression

Le déficit génétique en BACH1 altère la fonction mitochondriale et augmente l'activation de l'inflammasome NLRP3 dans les macrophages de souris

International audienceBTB-and-CNC homologue 1 (BACH1), a heme-regulated transcription factor, mediates innate immune responses via its functional role in macrophages. BACH1 has recently been shown to modulate mitochondrial metabolism in cancer cells. In the current study, we utilized a proteomics approach and demonstrate that genetic deletion of BACH1 in mouse macrophages is associated with decreased levels of various mitochondrial proteins, particularly mitochondrial complex I. Bioenergetic studies revealed alterations of mitochondrial energy metabolism in BACH1− /− macrophages with a shift towards increased glycolysis and decreased oxidative phosphorylation. Moreover, these cells exhibited enhanced mitochondrial membrane potential and generation of mitochondrial reactive oxygen species (mtROS) along with lower levels of mitophagy. Notably, a higher inducibility of NLRP3 inflammasome activation in response to ATP and nigericin following challenge with lipopolysaccharide (LPS) was observed in BACH1-deficient macrophages compared to wild-type cells. Mechanistically, pharmacological inhibition of mtROS markedly attenuated inflammasome activation. In addition, it is shown that inducible nitric oxide synthase and cyclooxygenase-2, both of which are markedly induced by LPS in macrophages, are directly implicated in BACH1-dependent regulation of NLRP3 inflammasome activation. Taken together, the current findings indicate that BACH1 is critical for immunomodulation of macrophages and may serve as a target for therapeutic approaches in inflammatory disorders

HLA class II antibodies induce necrotic cell death in human endothelial cells via a lysosomal membrane permeabilization-mediated pathway

Abstract Antibody-mediated rejection (AMR) is the major cause of allograft loss after solid organ transplantation. Circulating donor-specific antibodies against human leukocyte antigen (HLA), in particular HLA class II antibodies are critical for the pathogenesis of AMR via interactions with endothelial cells (ECs). To investigate the effects of HLA class II antibody ligation to the graft endothelium, a model of HLA-DR antibody-dependent stimulation was utilized in primary human ECs. Antibody ligation of HLA class II molecules in interferon-γ-treated ECs caused necrotic cell death without complement via a pathway that was independent of apoptosis and necroptosis. HLA-DR-mediated cell death was blocked by specific neutralization of antibody ligation with recombinant HLA class II protein and by lentiviral knockdown of HLA-DR in ECs. Importantly, HLA class II-mediated cytotoxicity was also induced by relevant native allele-specific antibodies from human allosera. Necrosis of ECs in response to HLA-DR ligation was mediated via hyperactivation of lysosomes, lysosomal membrane permeabilization (LMP), and release of cathepsins. Notably, LMP was caused by reorganization of the actin cytoskeleton. This was indicated by the finding that LMP and actin stress fiber formation by HLA-DR antibodies were both downregulated by the actin polymerization inhibitor cytochalasin D and inhibition of Rho GTPases, respectively. Finally, HLA-DR-dependent actin stress fiber formation and LMP led to mitochondrial stress, which was revealed by decreased mitochondrial membrane potential and generation of reactive oxygen species in ECs. Taken together, ligation of HLA class II antibodies to ECs induces necrotic cell death independent of apoptosis and necroptosis via a LMP-mediated pathway. These findings may enable novel therapeutic approaches for the treatment of AMR in solid organ transplantation

Inhibition of Heme Oxygenase-1 Activity Enhances Wilms Tumor-1-Specific T-Cell Responses in Cancer Immunotherapy

Wilms tumor protein-1 (WT1) is an attractive target for adoptive T-cell therapy due to its expression in solid tumors and hematologic malignancies. However, T cells recognizing WT1 occur in low frequencies in the peripheral blood of healthy donors, limiting potential therapeutic possibilities. Tin mesoporphyrin (SnMP) is known to inhibit heme oxygenase-1 (HO-1), which has been shown to boost the activation and proliferation of human virus-specific T cells. We analyzed the influence of this effect on the generation of WT1-specific T cells and developed strategies for generating quantities of these cells from healthy donors, sufficient for adoptive T-cell therapies. HO-1 inhibition with SnMP increased WT1-specific T-cell frequencies in 13 (26%) of 50 healthy donors. To assess clinical applicability, we measured the enrichment efficiency of SnMP-treated WT1-specific T cells in response to a WT1-specific peptide pool and a HLA-A*02:01-restricted WT1 peptide by cytokine secretion assay. SnMP treatment resulted in a 28-fold higher enrichment efficacy with equal functionality. In conclusion, pharmacological inhibition of HO-1 activity with SnMP results in more efficient generation of functionally active WT1-specific T cells. This study demonstrates the therapeutic potentials of inhibiting HO-1 with SnMP to enhance antigen-specific T-cell responses in the treatment of cancer patients with WT1-positive disease

Heme Oxygenase-1 Inhibits HLA Class I Antibody-Dependent Endothelial Cell Activation

Antibody-mediated rejection (AMR) is a key limiting factor for long-term graft survival in solid organ transplantation. Human leukocyte antigen (HLA) class I (HLA I) antibodies (Abs) play a major role in the pathogenesis of AMR via their interactions with HLA molecules on vascular endothelial cells (ECs). The antioxidant enzyme heme oxygenase (HO)-1 has anti-inflammatory functions in the endothelium. As complement-independent effects of HLA I Abs can activate ECs, it was the goal of the current study to investigate the role of HO-1 on activation of human ECs by HLA I Abs. In cell cultures of various primary humanmacro- and microvascular ECs treatment with monoclonal pan-and allele-specific HLA I Abs up-regulated the expression of inducible proinflammatory adhesion molecules and chemokines (vascular cell adhesion molecule-1 [VCAM-1], intercellular cell adhesion molecule-1 [ICAM-1], interleukin-8 [IL-8] and monocyte chemotactic protein 1 [MCP-1]). Pharmacological induction of HO-1 with cobalt-protoporphyrin IX reduced, whereas inhibition of HO-1 with either zinc-protoporphyrin IX or siRNA-mediated knockdown increased HLA I Ab-dependent up-regulation of VCAM-1. Treatment with two carbon monoxide (CO)-releasing molecules, which liberate the gaseous HO product CO, blocked HLA I Ab-dependent EC activation. Finally, in an in vitro adhesion assay exposure of ECs to HLA I Abs led to increased monocyte binding, which was counteracted by up-regulation of HO-1. In conclusion, HLA I Ab-dependent EC activation is modulated by endothelial HO-1 and targeted induction of this enzyme may be a novel therapeutic approach for the treatment of AMR in solid organ transplantation