A vasculature niche orchestrates stromal cell phenotype through PDGF signaling: Importance in human fibrotic disease.

SignificanceTissue fibrotic diseases, for example of the liver and lung, represent a huge unmet medical need. In this study, using single-cell RNA sequencing, cytometry by time of flight (CyTOF), tissue imaging, and functional assays, we identify a complex vascular niche in Dupuytren's disease (DD), a common localized fibrotic condition of the palm, where early-disease-stage tissue can be accessed readily. We uncover a population of myofibroblast precursors within the pericyte compartment and demonstrate that the endothelium instructs the differentiation of functionally distinct stromal cells, thereby orchestrating discrete microenvironments in the fibrotic milieu. Together, these findings provide a basis for the concept of targeting blood vessel signaling to control the progression of human fibrosis.

COVID-19 therapeutics: Challenges and directions for the future.

The emergence of SARS-CoV-2 triggering the COVID-19 pandemic ranks as arguably the greatest medical emergency of the last century. COVID-19 has highlighted health disparities both within and between countries and will leave a lasting impact on global society. Nonetheless, substantial investment in life sciences over recent decades has facilitated a rapid scientific response with innovations in viral characterization, testing, and sequencing. Perhaps most remarkably, this permitted the development of highly effective vaccines, which are being distributed globally at unprecedented speed. In contrast, drug treatments for the established disease have delivered limited benefits so far. Innovative and rapid approaches in the design and execution of large-scale clinical trials and repurposing of existing drugs have saved many lives; however, many more remain at risk. In this review we describe challenges and unmet needs, discuss existing therapeutics, and address future opportunities. Consideration is given to factors that have hindered drug development in order to support planning for the next pandemic challenge and to allow rapid and cost-effective development of new therapeutics with equitable delivery.

IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses.

Polymorphisms in the gene encoding the transcription factor IRF5 that lead to higher mRNA expression are associated with many autoimmune diseases. Here we show that IRF5 expression in macrophages was reversibly induced by inflammatory stimuli and contributed to the plasticity of macrophage polarization. High expression of IRF5 was characteristic of M1 macrophages, in which it directly activated transcription of the genes encoding interleukin 12 subunit p40 (IL-12p40), IL-12p35 and IL-23p19 and repressed the gene encoding IL-10. Consequently, those macrophages set up the environment for a potent T helper type 1 (T(H)1)-T(H)17 response. Global gene expression analysis demonstrated that exogenous IRF5 upregulated or downregulated expression of established phenotypic markers of M1 or M2 macrophages, respectively. Our data suggest a critical role for IRF5 in M1 macrophage polarization and define a previously unknown function for IRF5 as a transcriptional repressor.

Identifying collagen VI as a target of fibrotic diseases regulated by CREBBP/EP300.

Fibrotic diseases remain a major cause of morbidity and mortality, yet there are few effective therapies. The underlying pathology of all fibrotic conditions is the activity of myofibroblasts. Using cells from freshly excised disease tissue from patients with Dupuytren's disease (DD), a localized fibrotic disorder of the palm, we sought to identify new therapeutic targets for fibrotic disease. We hypothesized that the persistent activity of myofibroblasts in fibrotic diseases might involve epigenetic modifications. Using a validated genetics-led target prioritization algorithm (Pi) of genome wide association studies (GWAS) data and a broad screen of epigenetic inhibitors, we found that the acetyltransferase CREBBP/EP300 is a major regulator of contractility and extracellular matrix production via control of H3K27 acetylation at the profibrotic genes, ACTA2 and COL1A1 Genomic analysis revealed that EP300 is highly enriched at enhancers associated with genes involved in multiple profibrotic pathways, and broad transcriptomic and proteomic profiling of CREBBP/EP300 inhibition by the chemical probe SGC-CBP30 identified collagen VI (Col VI) as a prominent downstream regulator of myofibroblast activity. Targeted Col VI knockdown results in significant decrease in profibrotic functions, including myofibroblast contractile force, extracellular matrix (ECM) production, chemotaxis, and wound healing. Further evidence for Col VI as a major determinant of fibrosis is its abundant expression within Dupuytren's nodules and also in the fibrotic foci of idiopathic pulmonary fibrosis (IPF). Thus, Col VI may represent a tractable therapeutic target across a range of fibrotic disorders.

Histone H3K27me3 demethylases regulate human Th17 cell development and effector functions by impacting on metabolism.

T helper (Th) cells are CD4+ effector T cells that play a critical role in immunity by shaping the inflammatory cytokine environment in a variety of physiological and pathological situations. Using a combined chemico-genetic approach, we identify histone H3K27 demethylases KDM6A and KDM6B as central regulators of human Th subsets. The prototypic KDM6 inhibitor GSK-J4 increases genome-wide levels of the repressive H3K27me3 chromatin mark and leads to suppression of the key transcription factor RORγt during Th17 differentiation. In mature Th17 cells, GSK-J4 induces an altered transcriptional program with a profound metabolic reprogramming and concomitant suppression of IL-17 cytokine levels and reduced proliferation. Single-cell analysis reveals a specific shift from highly inflammatory cell subsets toward a resting state upon demethylase inhibition. The root cause of the observed antiinflammatory phenotype in stimulated Th17 cells is reduced expression of key metabolic transcription factors, such as PPRC1. Overall, this leads to reduced mitochondrial biogenesis, resulting in a metabolic switch with concomitant antiinflammatory effects. These data are consistent with an effect of GSK-J4 on Th17 T cell differentiation pathways directly related to proliferation and include regulation of effector cytokine profiles. This suggests that inhibiting KDM6 demethylases may be an effective, even in the short term, therapeutic target for autoimmune diseases, including ankylosing spondylitis.

TNF receptor 2 signaling prevents DNA methylation at the Foxp3 promoter and prevents pathogenic conversion of regulatory T cells.

Regulatory T (Treg) cells expressing the transcription factor Foxp3 play an important role in maintaining immune homeostasis. Chronic inflammation is associated with reduced Foxp3 expression, function, and loss of phenotypic stability. Previous studies have established the importance of TNF receptor 2 (TNFR2) in the generation and/or activation of Treg cells. In this study, we assess the importance of TNFR2 in healthy mice and under inflammatory conditions. Our findings reveal that, in health, TNFR2 is important not only for the generation of Treg cells, but also for regulating their functional activity. We also show that TNFR2 maintains Foxp3 expression in Treg cells by restricting DNA methylation at the Foxp3 promoter. In inflammation, loss of TNFR2 results in increased severity and chronicity of experimental arthritis, reduced total numbers of Treg cells, reduced accumulation of Treg cells in inflamed joints, and loss of inhibitory activity. In addition, we demonstrate that, under inflammatory conditions, loss of TNFR2 causes Treg cells to adopt a proinflammatory Th17-like phenotype. It was concluded that TNFR2 signaling is required to enable Treg cells to promote resolution of inflammation and prevent them from undergoing dedifferentiation. Consequently, TNFR2-specific agonists or TNF1-specific antagonists may be useful in the treatment of autoimmune disease.

Fully reduced HMGB1 accelerates the regeneration of multiple tissues by transitioning stem cells to GAlert.

A major discovery of recent decades has been the existence of stem cells and their potential to repair many, if not most, tissues. With the aging population, many attempts have been made to use exogenous stem cells to promote tissue repair, so far with limited success. An alternative approach, which may be more effective and far less costly, is to promote tissue regeneration by targeting endogenous stem cells. However, ways of enhancing endogenous stem cell function remain poorly defined. Injury leads to the release of danger signals which are known to modulate the immune response, but their role in stem cell-mediated repair in vivo remains to be clarified. Here we show that high mobility group box 1 (HMGB1) is released following fracture in both humans and mice, forms a heterocomplex with CXCL12, and acts via CXCR4 to accelerate skeletal, hematopoietic, and muscle regeneration in vivo. Pretreatment with HMGB1 2 wk before injury also accelerated tissue regeneration, indicating an acquired proregenerative signature. HMGB1 led to sustained increase in cell cycling in vivo, and using Hmgb1-/- mice we identified the underlying mechanism as the transition of multiple quiescent stem cells from G0 to GAlert HMGB1 also transitions human stem and progenitor cells to GAlert Therefore, exogenous HMGB1 may benefit patients in many clinical scenarios, including trauma, chemotherapy, and elective surgery.

Novel CBG Derivatives Can Reduce Inflammation, Pain and Obesity.

Interest in CBG (cannabigerol) has been growing in the past few years, due to its anti-inflammatory properties and other therapeutic benefits. Here we report the synthesis of three new CBG derivatives (HUM-223, HUM-233 and HUM-234) and show them to possess anti-inflammatory and analgesic properties. In addition, unlike CBG, HUM-234 also prevents obesity in mice fed a high-fat diet (HFD). The metabolic state of the treated mice on HFD is significantly better than that of vehicle-treated mice, and their liver slices show significantly less steatosis than untreated HFD or CBG-treated ones from HFD mice. We believe that HUM-223, HUM-233 and HUM-234 have the potential for development as novel drug candidates for the treatment of inflammatory conditions, and in the case of HUM-234, potentially for obesity where there is a huge unmet need.

Glatiramer Acetate Enhances Myeloid-Derived Suppressor Cell Function via Recognition of Paired Ig-like Receptor B.

Glatiramer acetate (GA; Copaxone) is a copolymer therapeutic that is approved by the Food and Drug Administration for the relapsing-remitting form of multiple sclerosis. Despite an unclear mechanism of action, studies have shown that GA promotes protective Th2 immunity and stimulates release of cytokines that suppress autoimmunity. In this study, we demonstrate that GA interacts with murine paired Ig-like receptor B (PIR-B) on myeloid-derived suppressor cells and suppresses the STAT1/NF-κB pathways while promoting IL-10/TGF-ß cytokine release. In inflammatory bowel disease models, GA enhanced myeloid-derived suppressor cell-dependent CD4+ regulatory T cell generation while reducing proinflammatory cytokine secretion. Human monocyte-derived macrophages responded to GA by reducing TNF-α production and promoting CD163 expression typical of alternative maturation despite the presence of GM-CSF. Furthermore, GA competitively interacts with leukocyte Ig-like receptors B (LILRBs), the human orthologs of PIR-B. Because GA limited proinflammatory activation of myeloid cells, therapeutics that target LILRBs represent novel treatment modalities for autoimmune indications.

Inhibition of histone H3K27 demethylases selectively modulates inflammatory phenotypes of natural killer cells.

Natural killer (NK) cells are innate lymphocytes, important in immune surveillance and elimination of stressed, transformed, or virus-infected cells. They critically shape the inflammatory cytokine environment to orchestrate interactions of cells of the innate and adaptive immune systems. Some studies have reported that NK cell activation and cytokine secretion are controlled epigenetically but have yielded only limited insight into the mechanisms. Using chemical screening with small-molecule inhibitors of chromatin methylation and acetylation, further validated by knockdown approaches, we here identified Jumonji-type histone H3K27 demethylases as key regulators of cytokine production in human NK cell subsets. The prototypic JMJD3/UTX (Jumonji domain-containing protein 3) H3K27 demethylase inhibitor GSK-J4 increased global levels of the repressive H3K27me3 mark around transcription start sites of effector cytokine genes. Moreover, GSK-J4 reduced IFN-γ, TNFα, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-10 levels in cytokine-stimulated NK cells while sparing their cytotoxic killing activity against cancer cells. The anti-inflammatory effect of GSK-J4 in NK cell subsets, isolated from peripheral blood or tissue from individuals with rheumatoid arthritis (RA), coupled with an inhibitory effect on formation of bone-resorbing osteoclasts, suggested that histone demethylase inhibition has broad utility for modulating immune and inflammatory responses. Overall, our results indicate that H3K27me3 is a dynamic and important epigenetic modification during NK cell activation and that JMJD3/UTX-driven H3K27 demethylation is critical for NK cell function.

IRF5 controls both acute and chronic inflammation.

Whereas the importance of macrophages in chronic inflammatory diseases is well recognized, there is an increasing awareness that neutrophils may also play an important role. In addition to the well-documented heterogeneity of macrophage phenotypes and functions, neutrophils also show remarkable phenotypic diversity among tissues. Understanding the molecular pathways that control this heterogeneity should provide abundant scope for the generation of more specific and effective therapeutics. We have shown that the transcription factor IFN regulatory factor 5 (IRF5) polarizes macrophages toward an inflammatory phenotype. IRF5 is also expressed in other myeloid cells, including neutrophils, where it was linked to neutrophil function. In this study we explored the role of IRF5 in models of acute inflammation, including antigen-induced inflammatory arthritis and lung injury, both involving an extensive influx of neutrophils. Mice lacking IRF5 accumulate far fewer neutrophils at the site of inflammation due to the reduced levels of chemokines important for neutrophil recruitment, such as the chemokine (C-X-C motif) ligand 1. Furthermore we found that neutrophils express little IRF5 in the joints and that their migratory properties are not affected by the IRF5 deficiency. These studies extend prior ones suggesting that inhibiting IRF5 might be useful for chronic macrophage-induced inflammation and suggest that IRF5 blockade would ameliorate more acute forms of inflammation, including lung injury.

Indoleamine 2,3-dioxygenase-1 is protective in atherosclerosis and its metabolites provide new opportunities for drug development.

Atherosclerosis is the major cause of cardiovascular disease (CVD), the leading cause of death worldwide. Despite much focus on lipid abnormalities in atherosclerosis, it is clear that the immune system also has important pro- and antiatherogenic functions. The enzyme indoleamine-2,3-dioxygenase (IDO) catalyses degradation of the essential amino acid tryptophan into immunomodulatory metabolites. How IDO deficiency affects immune responses during atherogenesis is unknown and we explored potential mechanisms in models of murine and human atherosclerosis. IDO deficiency in hypercholesterolemic ApoE(-/-) mice caused a significant increase in lesion size and surrogate markers of plaque vulnerability. No significant changes in cholesterol levels were observed but decreases in IL-10 production were found in the peripheral blood, spleen and lymph node B cells of IDO-deficient compared with IDO-competent ApoE(-/-) mice. 3,4,-Dimethoxycinnamoyl anthranilic acid (3,4-DAA), an orally active synthetic derivative of the tryptophan metabolite anthranilic acid, but not l-kynurenine, enhanced production of IL-10 in cultured splenic B cells. Finally, 3,4-DAA treatment reduced lesion formation and inflammation after collar-induced arterial injury in ApoE(-/-) mice, and reduced cytokine and chemokine production in ex vivo human atheroma cell cultures. Our data demonstrate that endogenous production of tryptophan metabolites via IDO is an essential feedback loop that controls atherogenesis and athero-inflammation. We show that the IDO pathway induces production of IL-10 in B cells in vivo and in vitro, suggesting that IDO may induce immunoregulatory functions of B cells in atherosclerosis. The favorable effects of anthranilic acid derivatives in atherosclerosis indicate a novel approach toward therapy of CVD.

OX40L blockade is therapeutic in arthritis, despite promoting osteoclastogenesis.

An immune response is essential for protection against infection, but, in many individuals, aberrant responses against self tissues cause autoimmune diseases such as rheumatoid arthritis (RA). How to diminish the autoimmune response while not augmenting infectious risk is a challenge. Modern targeted therapies such as anti-TNF or anti-CD20 antibodies ameliorate disease, but at the cost of some increase in infectious risk. Approaches that might specifically reduce autoimmunity and tissue damage without infectious risk would be important. Here we describe that TNF superfamily member OX40 ligand (OX40L; CD252), which is expressed predominantly on antigen-presenting cells, and its receptor OX40 (on activated T cells), are restricted to the inflamed joint in arthritis in mice with collagen-induced arthritis and humans with RA. Blockade of this pathway in arthritic mice reduced inflammation and restored tissue integrity predominantly by inhibiting inflammatory cytokine production by OX40L-expressing macrophages. Furthermore, we identify a previously unknown role for OX40L in steady-state bone homeostasis. This work shows that more targeted approaches may augment the "therapeutic window" and increase the benefit/risk in RA, and possibly other autoimmune diseases, and are thus worth testing in humans.

Anti-TNF therapy: past, present and future.

While for a century therapeutics has been dominated by small molecules, i.e. organic chemicals of ~400Da absorbable via the gut, this is no longer the case. There are now a plethora of important medicines which are proteins and injectable, which have dramatically improved the therapy of many inflammatory diseases and of cancer. Most of these are monoclonal antibodies, some are receptor Ig Fc fusion proteins, others are cytokines or enzymes. The key to this new aspect of therapeutics has been the filling of unmet needs, and the consequent commercial success, which promoted further research and development. The first 'biologic' for a common disease, rheumatoid arthritis (RA), was a monoclonal antibody, infliximab, to human tumour necrosis factor (TNF). This was based on our work, which is described in this review, summarizing how TNF was defined as a good target in RA, how it was developed is described here, as well as future indications for anti-TNF and related agents. Biologics are now the fastest growing sector of therapeutics.

Unraveling the signaling pathways promoting fibrosis in Dupuytren's disease reveals TNF as a therapeutic target.

Dupuytren's disease is a very common progressive fibrosis of the palm leading to flexion deformities of the digits that impair hand function. The cell responsible for development of the disease is the myofibroblast. There is currently no treatment for early disease or for preventing recurrence following surgical excision of affected tissue in advanced disease. Therefore, we sought to unravel the signaling pathways leading to the development of myofibroblasts in Dupuytren's disease. We characterized the cells present in Dupuytren's tissue and found significant numbers of immune cells, including classically activated macrophages. High levels of proinflammatory cytokines were also detected in tissue from Dupuytren's patients. We compared the effects of these cytokines on contraction and profibrotic signaling pathways in fibroblasts from the palmar and nonpalmar dermis of Dupuytren's patients and palmar fibroblasts from non-Dupuytren's patients. Exogenous addition of TNF, but not other cytokines, including IL-6 and IL-1ß, promoted differentiation into specifically of palmar dermal fibroblasts from Dupuytren's patients in to myofibroblasts. We also demonstrated that TNF acts via the Wnt signaling pathway to drive contraction and profibrotic signaling in these cells. Finally, we examined the effects of targeted cytokine inhibition. Neutralizing antibodies to TNF inhibited the contractile activity of myofibroblasts derived from Dupuytren's patients, reduced their expression of α-smooth muscle actin, and mediated disassembly of the contractile apparatus. Therefore, we showed that localized inflammation in Dupuytren's disease contributes to the development and progression of this fibroproliferative disorder and identified TNF as a therapeutic target to down-regulate myofibroblast differentiation and activity.

Stimulation of the α7 nicotinic acetylcholine receptor protects against neuroinflammation after tibia fracture and endotoxemia in mice.

Surgery and critical illness often associate with cognitive decline. Surgical trauma or infection can lead independently to learning and memory impairments via similar, but not identical, cellular signaling of the innate immune system that promotes neuroinflammation. In this study we explored the putative synergism between aseptic orthopedic surgery and infection, the latter reproduced by postoperative lipopolysaccharide (LPS) administration. We observed that surgery and LPS augmented systemic inflammation up to postoperative d 3 and this was associated with further neuroinflammation (CD11b and CD68 immunoreactivity) in the hippocampus in mice compared with those receiving surgery or LPS alone. Administration of a selective α7 subtype nicotinic acetylcholine receptor (α7 nAChR) agonist 2 h after LPS significantly improved neuroinflammation and hippocampal-dependent memory dysfunction. Modulation of nuclear factor-kappa B (NF-κB) activation in monocytes and regulation of the oxidative stress response through nicotinamide adenine dinucleotide phosphate (NADPH) signaling appear to be key targets in modulating this response. Overall, these results suggest that it may be conceivable to limit and possibly prevent postoperative complications, including cognitive decline and/or infections, through stimulation of the cholinergic antiinflammatory pathway.

Development of anti-TNF therapy for rheumatoid arthritis.

The aetiology of systemic, autoimmune, chronic inflammatory diseases--such as rheumatoid arthritis--is not known, and their pathogenesis is complex and multifactorial. However, progress in the characterization of intercellular mediators--proteins that are now known as cytokines--has led to the realization that one cytokine, tumour-necrosis factor (TNF; previously known as TNF-alpha), has an important role in the pathogenesis of rheumatoid arthritis. This discovery heralded a new era of targeted and highly effective therapeutics for rheumatoid arthritis and, subsequently, other chronic inflammatory diseases.