Ly6Chi Monocytes Are Metabolically Reprogrammed in the Blood during Inflammatory Stimulation and Require Intact OxPhos for Chemotaxis and Monocyte to Macrophage Differentiation.

Acute inflammation is a rapid and dynamic process involving the recruitment and activation of multiple cell types in a coordinated and precise manner. Here, we investigate the origin and transcriptional reprogramming of monocytes using a model of acute inflammation, zymosan-induced peritonitis. Monocyte trafficking and adoptive transfer experiments confirmed that monocytes undergo rapid phenotypic change as they exit the blood and give rise to monocyte-derived macrophages that persist during the resolution of inflammation. Single-cell transcriptomics revealed significant heterogeneity within the surface marker-defined CD11b+Ly6G-Ly6Chi monocyte populations within the blood and at the site of inflammation. We show that two major transcriptional reprogramming events occur during the initial six hours of Ly6Chi monocyte mobilisation, one in the blood priming monocytes for migration and a second at the site of inflammation. Pathway analysis revealed an important role for oxidative phosphorylation (OxPhos) during both these reprogramming events. Experimentally, we demonstrate that OxPhos via the intact mitochondrial electron transport chain is essential for murine and human monocyte chemotaxis. Moreover, OxPhos is needed for monocyte-to-macrophage differentiation and macrophage M(IL-4) polarisation. These new findings from transcriptional profiling open up the possibility that shifting monocyte metabolic capacity towards OxPhos could facilitate enhanced macrophage M2-like polarisation to aid inflammation resolution and tissue repair.

Deficiency of factor-inhibiting HIF creates a tumor-promoting immune microenvironment.

Hypoxia signaling influences tumor development through both cell-intrinsic and -extrinsic pathways. Inhibiting hypoxia-inducible factor (HIF) function has recently been approved as a cancer treatment strategy. Hence, it is important to understand how regulators of HIF may affect tumor growth under physiological conditions. Here we report that in aging mice factor-inhibiting HIF (FIH), one of the most studied negative regulators of HIF, is a haploinsufficient suppressor of spontaneous B cell lymphomas, particular pulmonary B cell lymphomas. FIH deficiency alters immune composition in aged mice and creates a tumor-supportive immune environment demonstrated in syngeneic mouse tumor models. Mechanistically, FIH-defective myeloid cells acquire tumor-supportive properties in response to signals secreted by cancer cells or produced in the tumor microenvironment with enhanced arginase expression and cytokine-directed migration. Together, these data demonstrate that under physiological conditions, FIH plays a key role in maintaining immune homeostasis and can suppress tumorigenesis through a cell-extrinsic pathway.

Drug repurposing screen identifies novel anti-inflammatory activity of sunitinib in macrophages.

Inflammation is a driver of human disease and an unmet clinical need exists for new anti-inflammatory medicines. As a key cell type in both acute and chronic inflammatory pathologies, macrophages are an appealing therapeutic target for anti-inflammatory medicines. Drug repurposing - the use of existing medicines for novel indications - is an attractive strategy for the identification of new anti-inflammatory medicines with reduced development costs and lower failure rates than de novo drug discovery. In this study, FDA-approved medicines were screened in a murine macrophage NF-κB reporter cell line to identify potential anti-inflammatory drug repurposing candidates. The multi-tyrosine kinase inhibitor sunitinib was found to be a potent inhibitor of NF-κB activity and suppressor of inflammatory mediator production in murine bone marrow derived macrophages. Furthermore, oral treatment with sunitinib in mice was found to reduce TNFα production, inflammatory gene expression and organ damage in a model of endotoxemia via inhibition of NF-κB. Finally, we revealed sunitinib to have immunomodulatory effects in a model of chronic cardiovascular inflammation by reducing circulating TNFα. This study validates drug repurposing as a strategy for the identification of novel anti-inflammatory medicines and highlights sunitinib as a potential drug repurposing candidate for inflammatory disease via inhibition of NF-κB signalling.

OxPhos in adipose tissue macrophages regulated by BTK enhances their M2-like phenotype and confers a systemic immunometabolic benefit in obesity.

Bruton's tyrosine kinase (BTK) is a non-receptor bound kinase involved in pro-inflammatory signalling in activated macrophages, however, its role within adipose tissue macrophages remains unclear. We have demonstrated that BTK signalling regulates macrophage M2-like polarisation state by up-regulating subunits of mitochondrially encoded electron transport chain Complex I (ND4 and NDL4) and Complex IV (mt-CO1, mt-CO2 and mt-CO3) resulting in an enhanced rate of oxidative phosphorylation (OxPhos) in an NF-κB independent manner. Critically, BTK expression is elevated in adipose tissue macrophages from obese individuals with diabetes, while key mitochondrial genes (mtC01, mtC02 and mtC03) are decreased in inflammatory myeloid cells from obese individuals. Inhibition of BTK signalling either globally (Xid mice) or in myeloid cells (LysMCreBTK), or therapeutically (Acalabrutinib) protects HFD-fed mice from developing glycaemic dysregulation by improving signalling through the IRS1/Akt/GSK3ß pathway. The beneficial effects of acalabrutinib treatment are lost in macrophage ablated mice. Inhibition of BTK signalling in myeloid cells but not B-cells, induced a phenotypic switch in adipose tissue macrophages from a pro-inflammatory M1-state to a pro-resolution M2-like phenotype, by shifting macrophage metabolism towards OxPhos. This reduces both local and systemic inflammation and protected mice from the immunometabolic consequences of obesity. Therefore, in BTK we have identified a macrophage specific, druggable target that can regulate adipose tissue polarisation and cellular metabolism that can confer systematic benefit in metabolic syndrome.

Development of Highly Potent, G-Protein Pathway Biased, Selective, and Orally Bioavailable GPR84 Agonists.

Orphan G-protein-coupled receptor 84 (GPR84) is a receptor that has been linked to cancer, inflammatory, and fibrotic diseases. We have reported DL-175 as a biased agonist at GPR84 which showed differential signaling via Gαi/cAMP and ß-arrestin, but which is rapidly metabolized. Herein, we describe an optimization of DL-175 through a systematic structure-activity relationship (SAR) analysis. This reveals that the replacement of the naphthalene group improved metabolic stability and the addition of a 5-hydroxy substituent to the pyridine N-oxide group, yielding compounds 68 (OX04528) and 69 (OX04529), enhanced the potency for cAMP signaling by 3 orders of magnitude to low picomolar values. Neither compound showed detectable effects on ß-arrestin recruitment up to 80 µM. Thus, the new GPR84 agonists 68 and 69 displayed excellent potency, high G-protein signaling bias, and an appropriate in vivo pharmacokinetic profile that will allow investigation of GPR84 biased agonist activity in vivo.

Biased agonists of GPR84 and insights into biological control.

GPR84 was first identified as an open reading frame encoding an orphan Class A G protein coupled receptor in 2001. Gpr84 mRNA is expressed in a limited number of cell types with the highest levels of expression being in innate immune cells, M1 polarised macrophages and neutrophils. The first reported ligands for this receptor were medium chain fatty acids with chain lengths between 9 and 12 carbons. Subsequently, a series of synthetic agonists that signal via the GPR84 receptor were identified. Radioligand binding assays and molecular modelling with site-directed mutagenesis suggest the presence of three ligand binding sites on the receptor, but the physiological agonist(s) of the receptor remain unidentified. Here, we review the effects of GPR84 agonists on innate immune cells following a series of chemical discoveries since 2001. The development of highly biased agonists has helped to probe receptor function in vitro, and the remaining challenge is to follow the effects of biased signalling to the physiological functions of innate immune cell types. LINKED ARTICLES: This article is part of a themed issue GPR84 Pharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.10/issuetoc.

Kinetic insights into agonist-dependent signalling bias at the pro-inflammatory G-protein coupled receptor GPR84.

GPR84 is an orphan G-protein coupled receptor (GPCR) linked to inflammation. Strategies targeting GPR84 to prevent excessive inflammation in disease are hampered by a lack of understanding of its precise functional role. We have developed heterologous cell lines with low GPR84 expression levels that phenocopy the response of primary cells in a label-free cell electrical impedance (CEI) sensing system that measures cell morphology and adhesion. We then investigated the signalling profile and membrane localisation of GPR84 upon treatment with 6-OAU and DL-175, two agonists known to differentially influence immune cell function. When compared to 6-OAU, DL-175 was found to exhibit a delayed impedance response, a delayed and suppressed activation of Akt, which together correlated with an impaired ability to internalise GPR84 from the plasma membrane. The signalling differences were transient and occurred only at early time points in the low expressing cell lines, highlighting the importance of receptor number and kinetic readouts when evaluating signalling bias. Our findings open new ways to understand GPR84 signalling and evaluate the effect of newly developed agonists.

High Throughput Fluorescence-Based In Vitro Experimental Platform for the Identification of Effective Therapies to Overcome Tumour Microenvironment-Mediated Drug Resistance in AML.

The interactions between Acute Myeloid Leukaemia (AML) leukemic stem cells and the bone marrow (BM) microenvironment play a critical role during AML progression and resistance to drug treatments. Therefore, the identification of novel therapies requires drug-screening methods using in vitro co-culture models that closely recreate the cytoprotective BM setting. We have developed a new fluorescence-based in vitro co-culture system scalable to high throughput for measuring the concomitant effect of drugs on AML cells and the cytoprotective BM microenvironment. eGFP-expressing AML cells are co-cultured in direct contact with mCherry-expressing BM stromal cells for the accurate assessment of proliferation, viability, and signaling in both cell types. This model identified several efficacious compounds that overcome BM stroma-mediated drug resistance against daunorubicin, including the chromosome region maintenance 1 (CRM1/XPO1) inhibitor KPT-330. In silico analysis of genes co-expressed with CRM1, combined with in vitro experiments using our new methodology, also indicates that the combination of KPT-330 with the AURKA pharmacological inhibitor alisertib circumvents the cytoprotection of AML cells mediated by the BM stroma. This new experimental model and analysis provide a more precise screening method for developing improved therapeutics targeting AML cells within the cytoprotective BM microenvironment.

CARD-only proteins regulate in vivo inflammasome responses and ameliorate gout.

Inflammatory responses are crucial for controlling infections and initiating tissue repair. However, excessive and uncontrolled inflammation causes inflammatory disease. Processing and release of the pro-inflammatory cytokines interleukin-1ß (IL-1ß) and IL-18 depend on caspase-1 activation within inflammasomes. Assembly of inflammasomes is initiated upon activation of cytosolic pattern recognition receptors (PRRs), followed by sequential polymerization of pyrin domain (PYD)-containing and caspase recruitment domain (CARD)-containing proteins mediated by homotypic PYD and CARD interactions. Small PYD- or CARD-only proteins (POPs and COPs, respectively) evolved in higher primates to target these crucial interactions to limit inflammation. Here, we show the ability of COPs to regulate inflammasome activation by modulating homotypic CARD-CARD interactions in vitro and in vivo. CARD16, CARD17, and CARD18 displace crucial CARD interactions between caspase-1 proteins through competitive binding and ameliorate uric acid crystal-mediated NLRP3 inflammasome activation and inflammatory disease. COPs therefore represent an important family of inflammasome regulators and ameliorate inflammatory disease.

Drug repurposing in cardiovascular inflammation: Successes, failures, and future opportunities.

Drug repurposing is an attractive, pragmatic approach to drug discovery that has yielded success across medical fields over the years. The use of existing medicines for novel indications enables dramatically reduced development costs and timescales compared with de novo drug discovery and is therefore a promising strategy in cardiovascular disease, where new drug approvals lag significantly behind that of other fields. Extensive evidence from pre-clinical and clinical studies show that chronic inflammation is a driver of pathology in cardiovascular disease, and many efforts have been made to target cardiovascular inflammation therapeutically. This approach has been met with significant challenges however, namely off-target effects associated with broad-spectrum immunosuppression, particularly in long-term conditions such as cardiovascular disease. Nevertheless, multiple anti-inflammatory medicines have been assessed for efficacy in cardiovascular clinical trials, with most of these being repurposed from their original indications in autoimmune conditions like rheumatoid arthritis. In this review, we discuss the mixed successes of clinical trials investigating anti-inflammatory drugs in cardiovascular disease, with examples such as anti-cytokine monoclonal antibodies, colchicine, and methotrexate. Looking to the future, we highlight potential new directions for drug repurposing in cardiovascular inflammation, including the emerging concepts of drug re-engineering and chrono-pharmacology.

A Human CD68 Promoter-Driven Inducible Cre-Recombinase Mouse Line Allows Specific Targeting of Tissue Resident Macrophages.

Current genetic tools designed to target macrophages in vivo often target cells from all myeloid lineages. Therefore, we sought to generate a novel transgenic mouse which has a tamoxifen inducible Cre-recombinase under the control of the human CD68 promoter (hCD68-CreERT2). To test the efficiency and specificity of the of Cre-recombinase activity we crossed the hCD68-CreERT2 mice with a loxP-flanked STOP cassette red fluorescent protein variant (tdTomato) mouse. We established that orally dosing mice with 2 mg of tamoxifen for 5 consecutive days followed by a 5-day induction period resulted in robust expression of tdTomato in CD11b+ F4/80+ tissue resident macrophages. Using this induction protocol, we demonstrated tdTomato expression within peritoneal, liver and spleen macrophages and blood Ly6Clow monocytes. Importantly there was limited or no inducible tdTomato expression within other myeloid cells (neutrophils, monocytes, dendritic cells and eosinophils), T cells (CD4+ and CD8+) and B cells (CD19+). We also demonstrated that the level of tdTomato expression can be used as a marker to identify different populations of peritoneal and liver macrophages. We next assessed the longevity of tdTomato expression in peritoneal macrophages, liver and splenic macrophages and demonstrated high levels of tdTomato expression as long as 6 weeks after the last tamoxifen dose. Importantly, hCD68-CreERT2 expression is more restricted than that of LysM-Cre which has significant expression in major myeloid cell types (monocytes and neutrophils). To demonstrate the utility of this novel macrophage-specific Cre driver line we demonstrated tdTomato expression in recruited CD11b+CD64+F4/80+ monocyte-derived macrophages within the atherosclerotic lesions of AAV8-mPCSK9 treated mice, with limited expression in recruited neutrophils. In developing this new hCD68-CreERT2 mouse we have a tool that allows us to target tissue resident macrophages, with the advantage of not targeting other myeloid cells namely neutrophils and inflammatory monocytes.

NF-κB Signaling and Inflammation-Drug Repurposing to Treat Inflammatory Disorders?

NF-κB is a central mediator of inflammation, response to DNA damage and oxidative stress. As a result of its central role in so many important cellular processes, NF-κB dysregulation has been implicated in the pathology of important human diseases. NF-κB activation causes inappropriate inflammatory responses in diseases including rheumatoid arthritis (RA) and multiple sclerosis (MS). Thus, modulation of NF-κB signaling is being widely investigated as an approach to treat chronic inflammatory diseases, autoimmunity and cancer. The emergence of COVID-19 in late 2019, the subsequent pandemic and the huge clinical burden of patients with life-threatening SARS-CoV-2 pneumonia led to a massive scramble to repurpose existing medicines to treat lung inflammation in a wide range of healthcare systems. These efforts continue and have proven to be controversial. Drug repurposing strategies are a promising alternative to de novo drug development, as they minimize drug development timelines and reduce the risk of failure due to unexpected side effects. Different experimental approaches have been applied to identify existing medicines which inhibit NF-κB that could be repurposed as anti-inflammatory drugs.

Epithelial Mesenchymal Transition (EMT) and Associated Invasive Adhesions in Solid and Haematological Tumours.

In solid tumours, cancer cells that undergo epithelial mesenchymal transition (EMT) express characteristic gene expression signatures that promote invasive migration as well as the development of stemness, immunosuppression and drug/radiotherapy resistance, contributing to the formation of currently untreatable metastatic tumours. The cancer traits associated with EMT can be controlled by the signalling nodes at characteristic adhesion sites (focal contacts, invadopodia and microtentacles) where the regulation of cell migration, cell cycle progression and pro-survival signalling converge. In haematological tumours, ample evidence accumulated during the last decade indicates that the development of an EMT-like phenotype is indicative of poor disease prognosis. However, this EMT phenotype has not been directly linked to the assembly of specific forms of adhesions. In the current review we discuss the role of EMT in haematological malignancies and examine its possible link with the progression towards more invasive and aggressive forms of these tumours. We also review the known types of adhesions formed by haematological malignancies and speculate on their possible connection with the EMT phenotype. We postulate that understanding the architecture and regulation of EMT-related adhesions will lead to the discovery of new therapeutic interventions to overcome disease progression and resistance to therapies.

Bruton's TK regulates myeloid cell recruitment during acute inflammation.

BACKGROUND AND PURPOSE: Bruton's TK (BTK) is a non-receptor kinase best known for its role in B lymphocyte development that is critical for proliferation and survival of leukaemic cells in B-cell malignancies. However, BTK is expressed in myeloid cells, particularly neutrophils, monocytes and macrophages where its inhibition has been reported to cause anti-inflammatory properties. EXPERIMENTAL APPROACH: We explored the role of BTK on migration of myeloid cells (neutrophils, monocytes and macrophages), in vitro using chemotaxis assays and in vivo using zymosan-induced peritonitis as model systems. KEY RESULTS: Using the zymosan-induced peritonitis model of sterile inflammation, we demonstrated that acute inhibition of BTK prior to zymosan challenge reduced phosphorylation of BTK in circulating neutrophils and monocytes. Moreover, pharmacological inhibition of BTK with ibrutinib specifically inhibited neutrophil and Ly6Chi monocytes, but not Ly6Clo monocyte recruitment to the peritoneum. X-linked immunodeficient (XID) mice, which have a point mutation in the Btk gene, had reduced neutrophil and monocyte recruitment to the peritoneum following zymosan challenge. Pharmacological or genetic inhibition of BTK signalling substantially reduced human monocyte and murine macrophage chemotaxis, to a range of clinically relevant chemoattractants (C5a and CCL2). We also demonstrated that inhibition of BTK in tissue resident macrophages significantly decreases chemokine secretion by reducing NF-κB activity and Akt signalling. CONCLUSION AND IMPLICATIONS: Our work has identified a new role of BTK in regulating myeloid cell recruitment via two mechanisms, reducing monocyte/macrophages' ability to undergo chemotaxis and reducing chemokine secretion, via reduced NF-κB and Akt activity in tissue resident macrophages.

Tissue-resident macrophages regulate lymphatic vessel growth and patterning in the developing heart.

Macrophages are components of the innate immune system with key roles in tissue inflammation and repair. It is now evident that macrophages also support organogenesis, but few studies have characterized their identity, ontogeny and function during heart development. Here, we show that the distribution and prevalence of resident macrophages in the subepicardial compartment of the developing heart coincides with the emergence of new lymphatics, and that macrophages interact closely with the nascent lymphatic capillaries. Consequently, global macrophage deficiency led to extensive vessel disruption, with mutant hearts exhibiting shortened and mis-patterned lymphatics. The origin of cardiac macrophages was linked to the yolk sac and foetal liver. Moreover, the Cx3cr1+ myeloid lineage was found to play essential functions in the remodelling of the lymphatic endothelium. Mechanistically, macrophage hyaluronan was required for lymphatic sprouting by mediating direct macrophage-lymphatic endothelial cell interactions. Together, these findings reveal insight into the role of macrophages as indispensable mediators of lymphatic growth during the development of the mammalian cardiac vasculature.

How Have Leukocyte In Vitro Chemotaxis Assays Shaped Our Ideas about Macrophage Migration?

Macrophage chemotaxis is crucial during both onset and resolution of inflammation and unique among all leukocytes. Macrophages are able to switch between amoeboid and mesenchymal migration to optimise their migration through 3D environments. This subtle migration phenotype has been underappreciated in the literature, with macrophages often being grouped and discussed together with other leukocytes, possibly due to the limitations of current chemotaxis assays. Transwell assays were originally designed in the 1960s but despite their long-known limitations, they are still one of the most popular methods of studying macrophage migration. This review aims to critically evaluate transwell assays, and other popular chemotaxis assays, comparing their advantages and limitations in macrophage migration studies.

X-Linked Immunodeficient Mice With No Functional Bruton's Tyrosine Kinase Are Protected From Sepsis-Induced Multiple Organ Failure.

We previously reported the Bruton's tyrosine kinase (BTK) inhibitors ibrutinib and acalabrutinib improve outcomes in a mouse model of polymicrobial sepsis. Now we show that genetic deficiency of the BTK gene alone in Xid mice confers protection against cardiac, renal, and liver injury in polymicrobial sepsis and reduces hyperimmune stimulation ("cytokine storm") induced by an overwhelming bacterial infection. Protection is due in part to enhanced bacterial phagocytosis in vivo, changes in lipid metabolism and decreased activation of NF-κB and the NLRP3 inflammasome. The inactivation of BTK leads to reduced innate immune cell recruitment and a phenotypic switch from M1 to M2 macrophages, aiding in the resolution of sepsis. We have also found that BTK expression in humans is increased in the blood of septic non-survivors, while lower expression is associated with survival from sepsis. Importantly no further reduction in organ damage, cytokine production, or changes in plasma metabolites is seen in Xid mice treated with the BTK inhibitor ibrutinib, demonstrating that the protective effects of BTK inhibitors in polymicrobial sepsis are mediated solely by inhibition of BTK and not by off-target effects of this class of drugs.

20 Years an Orphan: Is GPR84 a Plausible Medium-Chain Fatty Acid-Sensing Receptor?

GPR84 is an inflammation-induced receptor highly expressed on immune cells, yet its endogenous ligand is still unknown. This makes any interpretation of its physiological activity in vivo difficult. However, experiments with potent synthetic agonists have highlighted what the receptor can do, namely, enhance proinflammatory signaling and macrophage effector functions such as phagocytosis. Developing drugs to block these effects has attracted interest from the scientific community with the aim of decreasing disease activity in inflammatory disorders or enhancing inflammation resolution. In this review, we critically reassess the widely held belief that the major role of GPR84 is that of being a medium-chain fatty acid (MCFA) receptor. While MCFAs have been shown to activate GPR84, it remains to be demonstrated that they are present in relevant tissues at appropriate concentrations. In contrast to four other "full-time" free fatty acid receptor subtypes, GPR84 is not expressed by enteroendocrine cells and has limited expression in the gastrointestinal tract. Across multiple tissues and cell types, the highest expression levels of GPR84 are observed hours after exposure to an inflammatory stimulus. These factors obscure the relationship between ligand and receptor in the human body and do not support the exclusive physiological pairing of MCFAs with GPR84. To maximize the chances of developing efficacious drugs for inflammatory diseases, we must advance our understanding of GPR84 and what it does in vivo.

Inhibition of Bruton's TK regulates macrophage NF-κB and NLRP3 inflammasome activation in metabolic inflammation.

BACKGROUND AND PURPOSE: There are no medications currently available to treat metabolic inflammation. Bruton's tyrosine kinase (BTK) is highly expressed in monocytes and macrophages and regulates NF-κB and NLRP3 inflammasome activity; both propagate metabolic inflammation in diet-induced obesity. EXPERIMENTAL APPROACH: Using an in vivo model of chronic inflammation, high-fat diet (HFD) feeding, in male C57BL/6J mice and in vitro assays in primary murine and human macrophages, we investigated if ibrutinib, an FDA approved BTK inhibitor, may represent a novel anti-inflammatory medication to treat metabolic inflammation. KEY RESULTS: HFD-feeding was associated with increased BTK expression and activation, which was significantly correlated with monocyte/macrophage accumulation in the liver, adipose tissue, and kidney. Ibrutinib treatment to HFD-fed mice inhibited the activation of BTK and reduced monocyte/macrophage recruitment to the liver, adipose tissue, and kidney. Ibrutinib treatment to HFD-fed mice decreased the activation of NF-κB and the NLRP3 inflammasome. As a result, ibrutinib treated mice fed HFD had improved glycaemic control through restored signalling by the IRS-1/Akt/GSK-3ß pathway, protecting mice against the development of hepatosteatosis and proteinuria. We show that BTK regulates NF-κB and the NLRP3 inflammasome specifically in primary murine and human macrophages, the in vivo cellular target of ibrutinib. CONCLUSION AND IMPLICATIONS: We provide "proof of concept" evidence that BTK is a novel therapeutic target for the treatment of diet-induced metabolic inflammation and ibrutinib may be a candidate for drug repurposing as an anti-inflammatory agent for the treatment of metabolic inflammation in T2D and microvascular disease.

Macrophages directly contribute collagen to scar formation during zebrafish heart regeneration and mouse heart repair.

Canonical roles for macrophages in mediating the fibrotic response after a heart attack include extracellular matrix turnover and activation of cardiac fibroblasts to initiate collagen deposition. Here we reveal that macrophages directly contribute collagen to the forming post-injury scar. Unbiased transcriptomics shows an upregulation of collagens in both zebrafish and mouse macrophages following heart injury. Adoptive transfer of macrophages, from either collagen-tagged zebrafish or adult mouse GFPtpz-collagen donors, enhances scar formation via cell autonomous production of collagen. In zebrafish, the majority of tagged collagen localises proximal to the injury, within the overlying epicardial region, suggesting a possible distinction between macrophage-deposited collagen and that predominantly laid-down by myofibroblasts. Macrophage-specific targeting of col4a3bpa and cognate col4a1 in zebrafish significantly reduces scarring in cryoinjured hosts. Our findings contrast with the current model of scarring, whereby collagen deposition is exclusively attributed to myofibroblasts, and implicate macrophages as direct contributors to fibrosis during heart repair.