Search

Apoptosis is traditionally considered to be an immunologically silent form of cell death. Multiple mechanisms exist to ensure that apoptosis does not stimulate the immune system to cause inflammation or autoimmunity. Against this expectation, we now report epithelia are programmed to provoke, rather than suppress, inflammation in response to apoptosis. We found that an acute inflammatory response led by neutrophils occurs in zebrafish and cell culture when apoptotic epithelial cells cannot be expelled from the monolayer by apical extrusion. This reflects an intrinsic circuit where ATP released from apoptotic cells stimulates epithelial cells in the immediate vicinity to produce IL-8. As the epithelial barrier is compromised when apical extrusion fails, this juxta-apoptotic proinflammatory pathway may represent an early-response mechanism at sites of potential microbial ingress. Conversely, apical extrusion prevents inappropriate epithelial inflammation by physically eliminating apoptotic cells before they can activate this proinflammatory circuit.

The immune system must be able to respond to a myriad of different threats, each requiring a distinct type of response. Here, we demonstrate that the cytoplasmic lysine deacetylase HDAC7 in macrophages is a metabolic switch that triages danger signals to enable the most appropriate immune response. Lipopolysaccharide (LPS) and soluble signals indicating distal or far-away danger trigger HDAC7-dependent glycolysis and proinflammatory IL-1β production. In contrast, HDAC7 initiates the pentose phosphate pathway (PPP) for NADPH and reactive oxygen species (ROS) production in response to the more proximal threat of nearby bacteria, as exemplified by studies on uropathogenic Escherichia coli (UPEC). HDAC7-mediated PPP engagement via 6-phosphogluconate dehydrogenase (6PGD) generates NADPH for antimicrobial ROS production, as well as D-ribulose-5-phosphate (RL5P) that both synergizes with ROS for UPEC killing and suppresses selective inflammatory responses. This dual functionality of the HDAC7-6PGD-RL5P axis prioritizes responses to proximal threats. Our findings thus reveal that the PPP metabolite RL5P has both antimicrobial and immunomodulatory activities and that engagement of enzymes in catabolic versus anabolic metabolic pathways triages responses to different types of danger for generation of inflammatory versus antimicrobial responses, respectively.

TLRs reprogram macrophage metabolism, enhancing glycolysis and promoting flux through the tricarboxylic acid cycle to enable histone acetylation and inflammatory gene expression. The histone deacetylase (HDAC) family of lysine deacetylases regulates both TLR-inducible glycolysis and inflammatory responses. Here, we show that the TLR4 agonist LPS, as well as agonists of other TLRs, rapidly increase enzymatic activity of the class IIa HDAC family (HDAC4, 5, 7, 9) in both primary human and murine macrophages. This response was abrogated in murine macrophages deficient in histone deacetylase 7 (Hdac7), highlighting a selective role for this specific lysine deacetylase during immediate macrophage activation. With the exception of the TLR3 agonist polyI:C, TLR-inducible activation of Hdac7 enzymatic activity required the MyD88 adaptor protein. The rapid glycolysis response, as assessed by extracellular acidification rate, was attenuated in Hdac7-deficient mouse macrophages responding to submaximal LPS concentrations. Surprisingly however, reconstitution of these cells with either wild-type or an enzyme-dead mutant of Hdac7 enhanced LPS-inducible glycolysis, whereas only the former promoted production of the inflammatory mediators Il-1β and Ccl2. Thus, Hdac7 enzymatic activity is required for TLR-inducible production of specific inflammatory mediators, whereas it acts in an enzyme-independent fashion to reprogram metabolism in macrophages responding to submaximal LPS concentrations. Hdac7 is thus a bifurcation point for regulated metabolism and inflammatory responses in macrophages. Taken together with existing literature, our findings support a model in which submaximal and maximal activation of macrophages via TLR4 instruct glycolysis through distinct mechanisms, leading to divergent biological responses.

Significance While increasing evidence associates the disruption of circadian rhythms with pathologic conditions, including obesity, type 2 diabetes, and nonalcoholic fatty liver diseases (NAFLD), the involved mechanisms are still poorly described. Here, we show that, in both humans and mice, the pathogenesis of NAFLD is associated with the disruption of the circadian clock combined with perturbations of the growth hormone and sex hormone pathways. However, while this condition protects mice from the development of fibrosis and insulin resistance, it correlates with increased fibrosis in humans. This suggests that the perturbation of the circadian clock and its associated disruption of the growth hormone and sex hormone pathways are critical for the pathogenesis of metabolic and liver diseases.

Histone deacetylases (HDACs) catalyse removal of acetyl groups from lysine residues on both histone and non-histone proteins to control numerous cellular processes. Of the 11 zinc-dependent classical HDACs, HDAC4, 5, 7 and 9 are class IIa HDAC enzymes that regulate cellular and developmental processes through both enzymatic and non-enzymatic mechanisms. Over the last two decades, HDAC7 has been associated with key roles in numerous physiological and pathological processes. Molecular, cellular, in vivo and disease association studies have revealed that HDAC7 acts through multiple mechanisms to control biological processes in immune cells, osteoclasts, muscle, the endothelium and epithelium. This HDAC protein regulates gene expression, cell proliferation, cell differentiation and cell survival and consequently controls development, angiogenesis, immune functions, inflammation and metabolism. This review focuses on the cell biology of HDAC7, including the regulation of its cellular localisation and molecular mechanisms of action, as well as its associative and causal links with cancer and inflammatory, metabolic and fibrotic diseases. We also review the development status of small molecule inhibitors targeting HDAC7 and their potential for intervention in different disease contexts.

BACKGROUND AND PURPOSE: Chronic liver diseases feature excessive collagen and matrix protein deposition or crosslinking that characterises fibrosis, leads to scar tissue, and disrupts liver functions. There is no effective treatment. This study investigated whether treatment with selective histone deacetylase (HDAC) inhibitors might specifically reduce type 2 inflammation in the injured liver, thereby attenuating fibrogenesis in mice. EXPERIMENTAL APPROACH: Thioacetamide (TAA) was used to induce hepatic inflammation, fibrosis, and liver damage in female C57BL/6 mice, similar to the clinical features of chronic human liver disease. We used eight inhibitors of different human HDAC enzymes to probe histological (IHC and TUNEL), biochemical and immunological changes (flow cytometry, qPCR, Legendplex, and elisa) in pathology, fibrosis, hepatic immune cell flux, and inflammatory cytokine expression. KEY RESULTS: Inhibitors of class I, but not class II, HDAC enzymes potently suppressed chronic hepatic inflammation and fibrosis in mice, attenuating accumulation and activation of IL‐33‐dependent, but not IL‐25‐dependent, group 2 innate lymphoid cells (ILC2) and inhibiting type 2 inflammation that drives hepatic stellate cells to secrete excessive collagen and matrix proteins. CONCLUSIONS AND IMPLICATIONS: The results show that potent and selective inhibitors of class I only HDAC enzymes profoundly inhibit hepatocyte death and type 2 inflammation to prevent TAA‐induced liver fibrosis in mice. The specific HDAC enzymes identified here may be key promoters of inflammation in chronic liver fibrosis.

Mitochondria have a multitude of functions, including energy generation and cell signaling. Recent evidence suggests that mitochondrial dynamics (i.e. the balance between mitochondrial fission and fusion) also regulate immune functions. Here, we reveal that lipopolysaccharide (LPS) stimulation increases mitochondrial numbers in mouse bone marrow‐derived macrophages (BMMs) and human monocyte‐derived macrophages. In BMMs, this response requires Toll‐like receptor 4 (Tlr4) and the TLR adaptor protein myeloid differentiation primary response 88 (MyD88) but is independent of mitochondrial biogenesis. Consistent with this phenomenon being a consequence of mitochondrial fission, the dynamin‐related protein 1 (Drp1) GTPase that promotes mitochondrial fission is enriched on mitochondria in LPS‐activated macrophages and is required for the LPS‐mediated increase in mitochondrial numbers in both BMMs and mouse embryonic fibroblasts. Pharmacological agents that skew toward mitochondrial fusion also abrogated this response. LPS triggered acute Drp1 phosphorylation at serine 635 (S635), followed by sustained Drp1 dephosphorylation at serine 656 (S656), in BMMs. LPS‐induced S656 dephosphorylation was abrogated in MyD88‐deficient BMMs, suggesting that this post‐translational modification is particularly important for Tlr4‐inducible fission. Pharmacological or genetic targeting of Tlr4‐inducible fission had selective effects on inflammatory mediator production, with LPS‐inducible mitochondrial fission promoting the expression and/or secretion of a subset of inflammatory mediators in BMMs and mouse embryonic fibroblasts. Thus, triggering of Tlr4 results in MyD88‐dependent activation of Drp1, leading to inducible mitochondrial fission and subsequent inflammatory responses in macrophages., Here, we demonstrate that lipopolysaccharide (LPS) acts via a Tlr4–MyD88–Drp1 axis to initiate mitochondrial fission and selective inflammatory responses in macrophages. We also show that LPS promotes sequential dynamin‐related protein 1 (Drp1) serine phosphorylation (S635) and dephosphorylation (S656), with the latter modification requiring the adaptor molecule myeloid differentiation primary response 88 (MyD88). We did not observe LPS‐inducible mitochondrial DNA replication in the same cells, suggesting that LPS‐inducible mitochondrial fission can occur independently of mitochondrial DNA replication.

Hyaluronan (HA), a major extracellular matrix glycosaminoglycan, is a biomarker for cirrhosis. However, little is known about the regulatory and downstream mechanisms of HA overproduction in liver fibrosis. Hepatic HA and HA synthase 2 (HAS2) expression was elevated in both human and murine liver fibrosis. HA production and liver fibrosis were reduced in mice lacking HAS2 in hepatic stellate cells (HSCs), whereas mice overexpressing HAS2 had exacerbated liver fibrosis. HAS2 was transcriptionally up-regulated by transforming growth factor-β through Wilms tumor 1 to promote fibrogenic, proliferative, and invasive properties of HSCs via CD44, Toll-like receptor 4 (TLR4), and newly identified downstream effector Notch1. Inhibition of HA synthesis by 4-methylumbelliferone reduced HSC activation and liver fibrosis in mice. Our study provides evidence that HAS2 actively synthesizes HA in HSCs and that it promotes HSC activation and liver fibrosis through Notch1. Targeted HA inhibition may have potential to be an effective therapy for liver fibrosis.

Summary: Histone deacetylases (HDACs) drive innate immune cell-mediated inflammation. Here we identify class IIa HDACs as key molecular links between Toll-like receptor (TLR)-inducible aerobic glycolysis and macrophage inflammatory responses. A proteomic screen identified the glycolytic enzyme pyruvate kinase M isoform 2 (Pkm2) as a partner of proinflammatory Hdac7 in murine macrophages. Myeloid-specific Hdac7 overexpression in transgenic mice amplifies lipopolysaccharide (LPS)-inducible lactate and promotes a glycolysis-associated inflammatory signature. Conversely, pharmacological or genetic targeting of Hdac7 and other class IIa HDACs attenuates LPS-inducible glycolysis and accompanying inflammatory responses in macrophages. We show that an Hdac7-Pkm2 complex acts as an immunometabolism signaling hub, whereby Pkm2 deacetylation at lysine 433 licenses its proinflammatory functions. Disrupting this complex suppresses inflammatory responses in vitro and in vivo. Class IIa HDACs are thus pivotal intermediates connecting TLR-inducible glycolysis to inflammation via Pkm2. : Das Gupta et al. show that HDAC7 and other class IIa HDAC enzymes control macrophage metabolism. They initiate TLR-inducible glycolysis in these cells and interact with the glycolytic enzyme PKM2 to drive inflammatory responses in vitro and in vivo. Class IIa HDAC inhibitors may have potential for attenuating immunometabolism-linked inflammation. Keywords: glycolysis, histone deacetylases, immunometabolism, inflammation, lysine acetylation, macrophage, post-translational modification, pyruvate kinase, toll-like receptor

Chronic liver disease (CLD) is associated with tissue-destructive fibrosis. Considering that common mechanisms drive fibrosis across etiologies, and that steatosis is an important cofactor for pathology, we performed RNA sequencing on liver biopsies of patients with different fibrosis stages, resulting from infection with hepatitis C virus (HCV) (with or without steatosis) or fatty liver disease. In combination with enhanced liver fibrosis score correlation analysis, we reveal a common set of genes associated with advanced fibrosis, as exemplified by those encoding the transcription factor ETS-homologous factor (EHF) and the extracellular matrix protein versican (VCAN). We identified 17 fibrosis-associated genes as candidate EHF targets and demonstrated that EHF regulates multiple fibrosis-associated genes, including VCAN, in hepatic stellate cells. Serum VCAN levels were also elevated in advanced fibrosis patients. Comparing biopsies from patients with HCV with or without steatosis, we identified a steatosis-enriched gene set associated with advanced fibrosis, validating follistatin-like protein 1 (FSTL1) as an exemplar of this profile. In patients with advanced fibrosis, serum FSTL1 levels were elevated in those with steatosis (versus those without). Liver Fstl1 mRNA levels were also elevated in murine CLD models. We thus reveal a common gene signature for CLD-associated liver fibrosis and potential biomarkers and/or targets for steatosis-associated liver fibrosis.

Interferon regulatory factor (IRF) family members impart cell-type specificity to toll-like receptor (TLR) signalling, and we recently identified a role for IRF6 in TLR2 signalling in epithelial cells. TLR3 has a well-characterized role in wound healing in the skin, and here, we examined TLR3-dependent IRF6 functions in human keratinocytes. Primary keratinocytes responded robustly to the TLR3 agonist poly(IC) with upregulation of mRNAs for interferon-β (IFN-β), the interleukin-12 (IL-12) family member IL-23p19 and the chemokines IL-8 and chemokine (C-C motif) ligand 5 (CCL5). Silencing of IRF6 expression enhanced poly(IC)-inducible IFN-β mRNA levels and inhibited poly(IC)-inducible IL-23p19 mRNA expression in primary keratinocytes. Consistent with these data, co-transfection of IRF6 increased poly(IC)-inducible IL-23p19 promoter activity, but inhibited poly(IC)-inducible IFN-β promoter activity in reporter assays. Surprisingly, poly(IC) did not regulate IL-12p40 expression in keratinocytes, suggesting that TLR3-inducible IL-23p19 may have an IL-23-independent function in these cells. The only other IL-12 family member that was strongly poly(IC) inducible was EBI3, which has not been shown to heterodimerize with IL-23p19. Both co-immunoprecipitation and proximity ligation assays revealed that IL-23p19 and EBI3 interact in cells. Co-expression of IL-23p19 and EBI3, as compared with IL-23p19 alone, resulted in increased levels of secreted IL-23p19, implying a functional role for this heterodimer. In summary, we report that IRF6 regulates a subset of TLR3 responses in human keratinocytes, including the production of a novel IL-12 family heterodimer (p19/EBI3). We propose that the TLR3-IRF6-p19/EBI3 axis may regulate keratinocyte and/or immune cell functions in the context of cell damage and wound healing in the skin.

Members of the Interferon Regulatory Factor (IRF) family of transcription factors have essential roles in developmental pathways, as well as immune function and host defence. IRF6 has a well-characterized role in epithelial cell differentiation, but has not been studied in the context of immune function. In this thesis, I investigated IRF6 involvement in Toll-like Receptor (TLR) responses in epithelial cells. In contrast to the ubiquitous and myeloid-restricted expression patterns of IRF3 and IRF8, respectively, IRF6 was selectively expressed in a range of human epithelial cell types, particularly keratinocytes. Keratinocyte differentiation also increased IRF6 mRNA and protein expression. Primary human keratinocytes, as well as the keratinocyte cell line HaCaT, responded weakly to most TLR ligands, with the exception of the TLR3 agonist poly(IC). Poly(IC) robustly upregulated mRNA expression of IFN-β, as well as the chemokines IL-8, CCL5 and CCL20 in both of these cell types. Surprisingly, poly(IC) also selectively upregulated mRNAs encoding IL-23p19 and EBI3, two members of the IL-12 family that have not been shown to heterodimerize. Although IRF6 enhanced poly(IC)-inducible IFN-β promoter activity in HEK-TLR3 cells, silencing IRF6 expression did not decrease poly(IC)-inducible IFN-β mRNA expression in primary keratinocytes. Several other inducible genes were also unaffected, however knock-down of IRF6 did impair poly(IC)-inducible IL-23p19 mRNA expression in primary keratinocytes. Conversely, ectopic expression of IRF6 in the bladder epithelial cell line T24 conferred poly(IC)-inducible IL-23p19 mRNA expression. To investigate the mechanisms responsible, the IL-23p19 promoter was cloned and transient transfection analyses were performed. These experiments indicated that IRF6 enhanced IL-23p19 promoter activity upon poly(IC) stimulation. Poly(IC) also trigged nuclear translocation of IRF6 in keratinocytes. However, IL-23p19 protein was not detected in supernatants or cell lysates from poly(IC)-activated keratinocytes. Therefore, the effect of inflammatory stimuli, like IFN-γ, on keratinocyte responses to poly(IC) was examined. IFN-γ priming enhanced poly(IC)-inducible gene expression but still did not license IL-23p19 secretion. Interestingly, keratinocyte differentiation increased poly(IC)-inducible IL-23p19 mRNA expression, but decreased poly(IC)-inducible IFN-β mRNA expression upon IFN-γ priming. Collectively, these data suggest that IRF6 promotes a sub-set of TLR3 responses in human keratinocytes. Studies were also undertaken to determine whether IRF6 is involved in MyD88-dependent signalling responses in keratinocytes. Most MyD88-dependent TLR agonists were generally ineffective at triggering inflammatory responses in these cells. Investigation of other MyD88-dependent ligands revealed that the TLR7 agonist imiquimod and the TLR2/6 agonist FSL-1 did upregulate inflammatory chemokine expression in keratinocytes. Knock-down studies suggested that IRF6 contributed to TLR7-inducible CCL5 and IFN-β mRNA expression in these cells. Given the modest effects of most MyD88-dependent TLR agonists on inflammatory responses in keratinocytes, factors that may enhance these responses were also investigated. Keratinocyte differentiation increased imiquimod-inducible CCL5 mRNA expression in keratinocytes, whereas there was no clear effect of IFN-γ priming. The role of IRF6 in promoting TLR2/6 responses in keratinocytes remains unresolved. In total, my findings posit IRF6 as a transducer of a sub-set of MyD88-dependent and -independent TLR responses in keratinocytes, implicating it as a sentinel transcription factor in pathogen sensing by the skin.

In addition to their established roles in host defence, Toll-like Receptors (TLRs) have emerging roles in control of homeostasis, injury and wound repair. The dsRNA-sensing receptor, TLR3, has been particularly implicated in such processes in several different tissues including the skin, intestine and liver, as well as in the control of reparative mechanisms in the brain, heart and kidneys, following ischemia reperfusion injury. In this review, we provide an overview of TLR3 signalling and functions in inflammation, tissue damage and repair processes, as well as therapeutic opportunities that may arise in the future from knowledge of such pathways.

Neutrophil extrusion of neutrophil extracellular traps (NETs) and concomitant cell death (NETosis) provides host defense against extracellular pathogens, whereas macrophage death by pyroptosis enables defense against intracellular pathogens. We report the unexpected discovery that gasdermin D (GSDMD) connects these cell death modalities. We show that neutrophil exposure to cytosolic lipopolysaccharide or cytosolic Gram-negative bacteria (Salmonella ΔsifA and Citrobacter rodentium) activates noncanonical (caspase-4/11) inflammasome signaling and triggers GSDMD-dependent neutrophil death. GSDMD-dependent death induces neutrophils to extrude antimicrobial NETs. Caspase-11 and GSDMD are required for neutrophil plasma membrane rupture during the final stage of NET extrusion. Unexpectedly, caspase-11 and GSDMD are also required for early features of NETosis, including nuclear delobulation and DNA expansion; this is mediated by the coordinate actions of caspase-11 and GSDMD in mediating nuclear membrane permeabilization and histone degradation. In vivo application of deoxyribonuclease I to dissolve NETs during murine Salmonella ΔsifA challenge increases bacterial burden in wild-type but not in Casp11 -/- and Gsdmd -/- mice. Our studies reveal that neutrophils use an inflammasome- and GSDMD-dependent mechanism to activate NETosis as a defense response against cytosolic bacteria.