In adult mice, myocardial infarction (MI) activates the cardiac lymphatics, which undergo sprouting angiogenesis (lymphangiogenesis), drain interstitial fluid and traffic macrophages to mediastinal lymph nodes (MLNs). This prevents edema and reduces inflammatory/fibrotic immune cell content to improve cardiac function. Here we investigated the role of cardiac lymphatics and macrophage clearance across the neonatal mouse regenerative window. The response to injury revealed limited lymphangiogenesis and clearance of macrophages from postnatal day 1 compared to postnatal day 7 infarcted hearts. This coincides with the maturation of lymphatic endothelial cell junctions from impermeable to permeable and with altered signaling between lymphatic endothelial cells and macrophages. Mice lacking the lymphatic endothelial receptor-1 (LYVE-1), where macrophage lymphatic trafficking is impaired in adults, experienced worse long-term outcomes after MI induced at postnatal day 1, suggesting an alternative role for LYVE-1 in macrophages. Macrophage-specific deletion of Lyve1 during neonatal heart injury impaired heart regeneration. This study demonstrates that immature cardiac lymphatics are impermeable to clearance in early neonates, ensuring retention of pro-regenerative LYVE-1-dependent macrophages.

Neisseria gonorrhoeae is a human-specific pathogen that causes the important sexually transmitted infection, gonorrhoea, an inflammatory condition of the genitourinary tract. The bacterium is closely related to the meningococcus, a leading cause of bacterial meningitis. Both these invasive bacterial species undergo autolysis when in the stationary phase of growth. Autolysis is a form of programmed cell death (PCD) which is part of the life cycle of remarkably few bacteria and poses an evolutionary conundrum as altruistic death provides no obvious benefit for single-celled organisms. Here, we searched for genes present in these 2 invasive species but not in other members of the Neisseria genus. We identified a ~3.4 kb horizontally acquired region, we termed the nap island, which is largely restricted to the gonococcus and meningococcus. The nap island in the gonococcus encodes 3 cationic, bacteriocin-like peptides which have no detectable antimicrobial activity. Instead, the gonococcal Neisseria autolysis peptides (Naps) promote autolytic cell death when bacteria enter the stationary phase of growth. Furthermore, strains lacking the Naps exhibit reduced autolysis in assays of PCD. Expression of Naps is likely to be phase variable, explaining how PCD could have arisen in these important human pathogens. NapC also induces lysis of human cells, so the peptides are likely to have multiple roles during colonisation and disease. The acquisition of the nap island contributed to the emergence of PCD in the gonococcus and meningococcus and potentially to the appearance of invasive disease in Neisseria spp.

Biased signaling in G protein-coupled receptors offers therapeutic promise, yet rational design of biased ligands remains challenging due to limited mechanistic understanding. Here, we report a molecular framework for controlling signaling bias at the immunometabolic receptor GPR84. We identified three structurally-matched ligands (OX04529, OX04954, and OX04539) with varying steric profiles that exhibit comparable Gi protein activation but dramatically different β-arrestin recruitment capacities. A high-resolution cryo-EM structure of GPR84-Gi in complex with OX04529, complemented by molecular dynamics simulations and targeted mutagenesis, revealed that steric interactions between ligand substituents and Leu3366.52 and Phe1875.47 indirectly disrupt a critical polar network involving Tyr3326.48, Asn1043.36 and Asn3627.45 essential for β-arrestin recruitment. Based on these insights, we developed a steric-dependent model that enabled rational design of G protein-biased agonists with predictable β-arrestin recruitment profiles. This mechanistic framework provides a blueprint for designing biased agonists with customized signaling profiles at GPR84 and potentially other class A GPCRs.

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.

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.

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.

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-kB 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/GSK3b 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.

Abstract Almost all patients with or at high risk of atherosclerosis are treated with statin therapy. However, there remains a high burden of cardiovascular risk in these patients. Indeed, it is now clear that residual inflammatory risk is a stronger predictor of cardiovascular events, cardiovascular death, and all-cause death than residual cholesterol risk (as measured by low-density lipoprotein cholesterol). Therefore, in this study we sought to better understand the origin of inflammatory residual risk associated with previous exposure to high cholesterol. Tissue resident macrophages and bone marrow derived macrophages (BMDM) from mice which have been exposed to high cholesterol display an augmented polarisation profile. BMDM’s generated ex vivo are grown under standard condition so any effects come from in vivo exposure to high cholesterol. BMDMs from high cholesterol mice display a heighten M1 and blunted M2-like phenotype. Metabolomic analysis of M(IL-4) macrophages reveal they have breaks in the TCA cycle resulting in reduced capacity to utilise OxPhos which is needed for full M(IL-4) polarisation. Macrophages from chimeric mice generated by bone marrow transfer of high cholesterol bone marrow into irradiated normo-cholesterol mice retain the same phenotype as macrophages from high-cholesterol mice. Critically we demonstrate that there are genome wide irreversible epigenetic changes in bone marrow cells and tissue resident macrophages as a result of exposure to high cholesterol. Mice from a high cholesterol background have an increased number of total HSC (lin-ckit+sca1+) within the bone marrow, however, there is a decreased in the total number of long term (LT)-HSC; critically, this phenotype is not reversible when lipids levels are normalised using monoclonal antibody therapy against PCSK9. This is coupled with an irreversible myeloid skewing in the bone marrow immune cell composition. Newly recruited macrophages within the adipose also retain a heighten M1 and blunted M2-like phenotype despite lipid normalisation conferring a negative systemic metabolic phenotype. Critically, these data show that cholesterol induces long term epigenetic and metabolic changes to HSC and macrophages, which may account for residual inflammatory risk.

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 β-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 β-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.

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-1β (IL-1β) 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.

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.

SummaryThe adrenal glands are hormone secreting glands that sit on top of the kidneys. Adrenal glands produce glucocorticoids, mineralocorticoids, and catecholamines, and are therefore critical regulators of the stress response, the immune response, metabolism, and blood pressure. Despite being identified for more that 30 years, our understanding of adrenal macrophages remains incomplete. In numerous other tissues, macrophages carry out a plethora of physiological and homeostatic roles in addition to their classical immune functions. The aim of this study was to characterise the macrophage compartment of the adrenal gland and assess its contribution to adrenal function. Using anin vivoapproach, we herein describe two morphologically and spatially distinct subsets of adrenal macrophages – dendritic-like macrophages that are present throughout the gland in young and old mice, and “foamy” lipid-laden macrophages that accumulate in the murine adrenal cortex in an age and diet-dependent manner. Furthermore, we present data showing that these foamy-like macrophages accumulate cholesterol and thereby regulate adrenal hormonal output, at steady state and in the context of obesity. We hereby provide novel insights into the physiological roles of macrophages in the adrenal gland and the mechanisms by which adrenal hormone production is regulated.

The NLRP3 inflammasome mediates the response to monosodium urate (MSU) crystals and is responsible for the debilitating symptoms of gout. ASC is the essential inflammasome adaptor that bridges NLRP3 to caspase-1 and inflammasome assembly is via sequential homotypic PYRIN domain (PYD)-PYD and caspase recruitment domain (CARD)-CARD interactions between NLRP3 and ASC and ASC and caspase-1, respectively. While controlled inflammasome activation is essential for host defense and wound healing, dysregulated and excessive inflammasome responses cause inflammatory diseases. Hence, proper control is key for a balanced inflammasome response. Humans, but not mice, encode a family of 3 small CARD-only proteins (COPs): CARD16, CARD17 and CARD18, but their role in controlling inflammasome responses in vivo is unknown, which is the focus of our study. COP transgenic mice show reduced MSU crystal induced release of IL-1b and consequently ameliorated gout symptoms. Transgenic macrophages and COP expressing THP-1 cells show impaired MSU crystal-mediated NLRP3 inflammasome and caspase-1 activation, cytokine release and pyroptosis, while COP KO results in a hyper response to MSU. Also, COPs show unique binding pattern to the CARD of caspase-1 and ASC in naïve, primed and MSU-activated macrophages and interfere with the essential CARD-CARD-mediated caspase-1 self interaction and the ASC-caspase-1 interaction by a competitive binding mechanism. We demonstrate that COPs are functional in mice and inhibit NLRP3 inflammasome assembly by a competitive binding mechanism that prevents cytokine release and pyroptotis and ameliorates inflammatory disease. Altogether, our findings illustrate that the COPs have a role in maintaining homeostasis. National Institutes of Health. (AI099009 and AR064349 to C.S, AI134030, AI140702 and AI120625 to C.S. and A.D.)

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 challenge remaining is to follow the effects of biased signalling to the physiological functions of innate immune cell types.

The poxvirus lumpy skin disease virus (LSDV) is the etiological agent of lumpy skin disease (LSD), a severe disease of cattle and water buffalo that is characterised by numerous necrotic cutaneous nodules. LSD is a rapidly emerging disease, spreading into and across the Middle East, eastern Europe, and Asia in the past decade. The disease causes substantial production and economic losses in rural communities and affected regions. LSDV is mechanically transmitted by haematophagous arthropods including stable flies ( Stomoxys calcitrans ), however our understanding of this mechanical transmission method is sparse. A secreted saliva collection methodology using a modified artificial membrane feeding system was optimised for S. calcitrans and used to collect and characterise secreted S. calcitrans saliva. Saliva was mixed with LSDV and shown not to affect virus growth in primary bovine fibroblasts. S. calcitrans saliva or spot-feeding by S. calcitrans was then incorporated into a bovine in vivo experimental model of LSD to determine if either influenced disease pathogenesis. S. calcitrans saliva resulted in fewer animals developing disease, however this difference was not statistically significant. Spot-feeding with S. calcitrans prior to inoculation did not alter the number of animals that developed disease or the overall severity of disease however disease progression was accelerated as demonstrated by the appearance of cutaneous nodules, detection of viral DNA in the blood stream, and production of neutralising antibodies. This shows that S. calcitrans influence disease kinetics through co-incident bite trauma and/or saliva deposition. This increases our understanding of LSDV pathogenesis and highlights the overlooked importance of mechanical vectors in pathogen transmission. Author summary Insect vectors are important conduits for the transmission of pathogens that cause diseases such as Zika, dengue, malaria, and lumpy skin disease. Biological vector-borne transmission incorporates a replication phase for the pathogen in the insect, whereas no replication occurs in the vector during mechanical transmission. When the insect bites the host it inoculates a pathogen whilst also delivering arthropod-derived factors such as saliva components and causing tissue trauma through biting and probing. Arthropod saliva and/or bite trauma have been shown to enhance the speed and severity of disease following inoculation with a range of biologically transmitted viruses. This study examined if this was true also for the mechanically transmitted pathogen lumpy skin disease virus (LSDV). LSDV is a neglected pathogen that causes severe systemic disease in cattle and is transmitted mechanically by the stable fly Stomoxys calcitrans . Using an experimental bovine model of LSD, we found that disease occurred more rapidly when virus was delivered in association with the bites of uninfected flies. This work has increased our knowledge of lumpy skin disease virus transmission, and the discovery that disease outcome can be impacted by previously overlooked mechanical insect vectors should prompt further investigation into this mechanism of transmission.

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.