Role of Adhesion G Protein-Coupled Receptors in Immune Dysfunction and Disorder.

Disorders of the immune system, including immunodeficiency, immuno-malignancy, and (auto)inflammatory, autoimmune, and allergic diseases, have a great impact on a host's health. Cellular communication mediated through cell surface receptors, among different cell types and between cell and microenvironment, plays a critical role in immune responses. Selective members of the adhesion G protein-coupled receptor (aGPCR) family are expressed differentially in diverse immune cell types and have been implicated recently in unique immune dysfunctions and disorders in part due to their dual cell adhesion and signaling roles. Here, we discuss the molecular and functional characteristics of distinctive immune aGPCRs and their physiopathological roles in the immune system.

Molecular and cellular regulation of psoriatic inflammation.

This review highlights the molecular and cellular mechanisms underlying psoriatic inflammation with an emphasis on recent developments which may impact on treatment approaches for this chronic disease. We consider both the skin and the musculoskeletal compartment and how different manifestations of psoriatic inflammation are linked. This review brings a focus to the importance of inflammatory feedback loops that exist in the initiation and chronic stages of the condition, and how close interaction between the epidermis and both innate and adaptive immune compartments drives psoriatic inflammation. Furthermore, we highlight work done on biomarkers to predict the outcome of therapy as well as the transition from psoriasis to psoriatic arthritis.

TMEM16A/ANO1 calcium-activated chloride channel as a novel target for the treatment of human respiratory syncytial virus infection.

INTRODUCTION: Human respiratory syncytial virus (HRSV) is a common cause of respiratory tract infections (RTIs) globally and is one of the most fatal infectious diseases for infants in developing countries. Of those infected, 25%-40% aged ≤1 year develop severe lower RTIs leading to pneumonia and bronchiolitis, with ~10% requiring hospitalisation. Evidence also suggests that HRSV infection early in life is a major cause of adult asthma. There is no HRSV vaccine, and the only clinically approved treatment is immunoprophylaxis that is expensive and only moderately effective. New anti-HRSV therapeutic strategies are therefore urgently required. METHODS: It is now established that viruses require cellular ion channel functionality to infect cells. Here, we infected human lung epithelial cell lines and ex vivo human lung slices with HRSV in the presence of a defined panel of chloride (Cl-) channel modulators to investigate their role during the HRSV life-cycle. RESULTS: We demonstrate the requirement for TMEM16A, a calcium-activated Cl- channel, for HRSV infection. Time-of-addition assays revealed that the TMEM16A blockers inhibit HRSV at a postentry stage of the virus life-cycle, showing activity as a postexposure prophylaxis. Another important negative-sense RNA respiratory pathogen influenza virus was also inhibited by the TMEM16A-specific inhibitor T16Ainh-A01. DISCUSSION: These findings reveal TMEM16A as an exciting target for future host-directed antiviral therapeutics.

IL-36 and IL-1/IL-17 Drive Immunity to Oral Candidiasis via Parallel Mechanisms.

Protection against microbial infection by the induction of inflammation is a key function of the IL-1 superfamily, including both classical IL-1 and the new IL-36 cytokine families. Candida albicans is a frequent human fungal pathogen causing mucosal infections. Although the initiators and effectors important in protective host responses to C. albicans are well described, the key players in driving these responses remain poorly defined. Recent work has identified a central role played by IL-1 in inducing innate Type-17 immune responses to clear C. albicans infections. Despite this, lack of IL-1 signaling does not result in complete loss of immunity, indicating that there are other factors involved in mediating protection to this fungus. In this study, we identify IL-36 cytokines as a new player in these responses. We show that C. albicans infection of the oral mucosa induces the production of IL-36. As with IL-1α/ß, induction of epithelial IL-36 depends on the hypha-associated peptide toxin Candidalysin. Epithelial IL-36 gene expression requires p38-MAPK/c-Fos, NF-κB, and PI3K signaling and is regulated by the MAPK phosphatase MKP1. Oral candidiasis in IL-36R-/- mice shows increased fungal burdens and reduced IL-23 gene expression, indicating a key role played by IL-36 and IL-23 in innate protective responses to this fungus. Strikingly, we observed no impact on gene expression of IL-17 or IL-17-dependent genes, indicating that this protection occurs via an alternative pathway to IL-1-driven immunity. Thus, IL-1 and IL-36 represent parallel epithelial cell-driven protective pathways in immunity to oral C. albicans infection.

Cathepsin S is the major activator of the psoriasis-associated proinflammatory cytokine IL-36γ.

The proinflammatory cytokine IL-36γ is highly expressed in epithelial cells and is a pivotal mediator of epithelial inflammation. In particular, IL-36γ is strongly associated with the inflammatory skin disease psoriasis. As with other IL-1 cytokines, IL-36γ is expressed as an inactive precursor and must be processed by specific proteases to become bioactive. Our aim therefore was to identify protease/s capable of IL-36γ activation and explore the importance of this activation in psoriasis. Using a keratinocyte-based activity assay in conjunction with small-molecule inhibitors and siRNA gene silencing, cathepsin S was identified as the major IL-36γ-activating protease expressed by epithelial cells. Interestingly, cathepsin S activity was strongly up-regulated in samples extracted from psoriasis patients relative to healthy controls. In addition, IL-36γ-Ser18, identified as the main product of cathepsin S-dependent IL-36γ cleavage, induced psoriasiform changes in human skin-equivalent models. Together, these data provide important mechanistic insights into the activation of IL-36γ and highlight that cathepsin S-mediated activation of IL-36γ may be important in the development of numerous IL-36γ-driven pathologies, in addition to psoriasis.

Cell-type-restricted anti-cytokine therapy: TNF inhibition from one pathogenic source.

Overexpression of TNF contributes to pathogenesis of multiple autoimmune diseases, accounting for a remarkable success of anti-TNF therapy. TNF is produced by a variety of cell types, and it can play either a beneficial or a deleterious role. In particular, in autoimmunity pathogenic TNF may be derived from restricted cellular sources. In this study we evaluated the feasibility of cell-type-restricted TNF inhibition in vivo. To this end, we engineered MYSTI (Myeloid-Specific TNF Inhibitor)--a recombinant bispecific antibody that binds to the F4/80 surface molecule on myeloid cells and to human TNF (hTNF). In macrophage cultures derived from TNF humanized mice MYSTI could capture the secreted hTNF, limiting its bioavailability. Additionally, as evaluated in TNF humanized mice, MYSTI was superior to an otherwise analogous systemic TNF inhibitor in protecting mice from lethal LPS/D-Galactosamine-induced hepatotoxicity. Our results suggest a novel and more specific approach to inhibiting TNF in pathologies primarily driven by macrophage-derived TNF.

International Union of Basic and Clinical Pharmacology. XCIV. Adhesion G protein-coupled receptors.

The Adhesion family forms a large branch of the pharmacologically important superfamily of G protein-coupled receptors (GPCRs). As Adhesion GPCRs increasingly receive attention from a wide spectrum of biomedical fields, the Adhesion GPCR Consortium, together with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification, proposes a unified nomenclature for Adhesion GPCRs. The new names have ADGR as common dominator followed by a letter and a number to denote each subfamily and subtype, respectively. The new names, with old and alternative names within parentheses, are: ADGRA1 (GPR123), ADGRA2 (GPR124), ADGRA3 (GPR125), ADGRB1 (BAI1), ADGRB2 (BAI2), ADGRB3 (BAI3), ADGRC1 (CELSR1), ADGRC2 (CELSR2), ADGRC3 (CELSR3), ADGRD1 (GPR133), ADGRD2 (GPR144), ADGRE1 (EMR1, F4/80), ADGRE2 (EMR2), ADGRE3 (EMR3), ADGRE4 (EMR4), ADGRE5 (CD97), ADGRF1 (GPR110), ADGRF2 (GPR111), ADGRF3 (GPR113), ADGRF4 (GPR115), ADGRF5 (GPR116, Ig-Hepta), ADGRG1 (GPR56), ADGRG2 (GPR64, HE6), ADGRG3 (GPR97), ADGRG4 (GPR112), ADGRG5 (GPR114), ADGRG6 (GPR126), ADGRG7 (GPR128), ADGRL1 (latrophilin-1, CIRL-1, CL1), ADGRL2 (latrophilin-2, CIRL-2, CL2), ADGRL3 (latrophilin-3, CIRL-3, CL3), ADGRL4 (ELTD1, ETL), and ADGRV1 (VLGR1, GPR98). This review covers all major biologic aspects of Adhesion GPCRs, including evolutionary origins, interaction partners, signaling, expression, physiologic functions, and therapeutic potential.

IL-36γ has proinflammatory effects on human endothelial cells.

Interleukin-36 cytokines are predominantly expressed by epithelial cells. Significant upregulation of epidermal IL-36 is now a recognised characteristic of psoriatic skin inflammation. IL-36 is known to induce inflammatory responses in dendritic cells, fibroblasts and epithelial cells. Although vascular alterations are a hallmark of psoriatic lesions and dermal endothelial cells are well known to play a critical role in skin inflammation, the effects of IL-36 on endothelial cells are unexplored. We here show that endothelial cells including dermal microvascular cells express a functionally active IL-36 receptor. Adhesion molecules VCAM-1 and ICAM-1 are upregulated by IL-36γ stimulation, and this is reversed by the presence of the endogenous IL-36 receptor antagonist. IL-36γ-stimulated endothelial cells secrete the proinflammatory chemokines IL-8, CCL2 and CCL20. Chemotaxis assays showed increased migration of T-cells following IL-36γ stimulation of endothelial cells. These results suggest a role for IL-36γ in the dermal vascular compartment, and it is likely to enhance psoriatic skin inflammation by activating endothelial cells and promoting leucocyte recruitment.

Effective delivery and selective insecticidal activity of double-stranded RNA via complexation with diblock copolymer varies with polymer block composition.

BACKGROUND: Chemical insecticides are an important tool to control damaging pest infestations. However, lack of species specificity, the rise of resistance and the demand for biological alternatives with improved ecotoxicity profiles means that chemicals with new modes of action are required. RNA interference (RNAi)-based strategies using double-stranded RNA (dsRNA) as a species-specific bio-insecticide offer an exquisite solution that addresses these issues. Many species, such as the fruit pest Drosophila suzukii, do not exhibit RNAi when dsRNA is orally administered due to degradation by gut nucleases and slow cellular uptake pathways. Thus, delivery vehicles that protect and deliver dsRNA are highly desirable. RESULTS: In this work, we demonstrate the complexation of D. suzukii-specific dsRNA for degradation of vha26 mRNA with bespoke diblock copolymers. We study the ex vivo protection of dsRNA against enzymatic degradation by gut enzymes, which demonstrates the efficiency of this system. Flow cytometry then investigates the cellular uptake of Cy3-labelled dsRNA, showing a 10-fold increase in the mean fluorescence intensity of cells treated with polyplexes. The polymer/dsRNA polyplexes induced a significant 87% decrease in the odds of survival of D. suzukii larvae following oral feeding only when formed with a diblock copolymer containing a long neutral block length (1:2 cationic block/neutral block). However, there was no toxicity when fed to the closely related Drosophila melanogaster. CONCLUSION: We provide evidence that dsRNA complexation with diblock copolymers is a promising strategy for RNAi-based species-specific pest control, but optimisation of polymer composition is essential for RNAi success. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Orai1 and CRAC channel dependence of VEGF-activated Ca2+ entry and endothelial tube formation.

RATIONALE: Orai1 and the associated calcium release-activated calcium (CRAC) channel were discovered in the immune system. Existence also in endothelial cells has been suggested, but the relevance to endothelial biology is mostly unknown. OBJECTIVE: The aim of this study was to investigate the relevance of Orai1 and CRAC channels to vascular endothelial growth factor (VEGF) and endothelial tube formation. METHODS AND RESULTS: In human umbilical vein endothelial cells, Orai1 disruption by short-interfering RNA or dominant-negative mutant Orai1 inhibited calcium release-activated (store-operated) calcium entry, VEGF-evoked calcium entry, cell migration, and in vitro tube formation. Expression of exogenous wild-type Orai1 rescued the tube formation. VEGF receptor-2 and Orai1 partially colocalized. Orai1 disruption also inhibited calcium entry and tube formation in endothelial progenitor cells from human blood. A known blocker of the immune cell CRAC channel (3-fluoropyridine-4-carboxylic acid (2',5'-dimethoxybiphenyl-4-yl)amide) was a strong blocker of store-operated calcium entry in endothelial cells and inhibited calcium entry evoked by VEGF in 3 types of human endothelial cell. The compound lacked effect on VEGF-evoked calcium-release, STIM1 clustering, and 2 types of transient receptor potential channels, TRPC6 and TRPV4. Without effect on cell viability, the compound inhibited human endothelial cell migration and tube formation in vitro and suppressed angiogenesis in vivo in the chick chorioallantoic membrane. The compound showed 100-fold greater potency for endothelial compared with immune cell calcium entry. CONCLUSIONS: The data suggest positive roles for Orai1 and CRAC channels in VEGF-evoked calcium entry and new opportunity for chemical modulation of angiogenesis.

Adhesion-GPCRs: emerging roles for novel receptors.

The G protein-coupled receptor (GPCR) family comprises the largest class of cell surface receptors found in metazoan proteomes. Within the novel GPCR subfamily of adhesion-GPCRs, approximately 150 distinct orthologues, from invertebrates to mammals, have been identified to date. All members of this family contain a large extracellular region, often containing common protein modules, coupled to a seven-transmembrane domain via a stalk region that seems to be crucial for functionality. Owing to their unique structure, restricted expression profile and involvement in several human diseases, adhesion-GPCRs have long been proposed to have vital dual roles in cellular adhesion and signalling. More recent studies have provided structural, evolutionary, developmental and immunological insights in relation to the adhesion-GPCR family.

VelcroVax: a "Bolt-On" Vaccine Platform for Glycoprotein Display.

Having varied approaches to the design and manufacture of vaccines is critical in being able to respond to worldwide needs and newly emerging pathogens. Virus-like particles (VLPs) form the basis of two of the most successful licensed vaccines (against hepatitis B virus [HBV] and human papillomavirus). They are produced by recombinant expression of viral structural proteins, which assemble into immunogenic nanoparticles. VLPs can be modified to present unrelated antigens, and here we describe a universal "bolt-on" platform (termed VelcroVax) where the capturing VLP and the target antigen are produced separately. We utilize a modified HBV core (HBcAg) VLP with surface expression of a high-affinity binding sequence (Affimer) directed against a SUMO tag and use this to capture SUMO-tagged gp1 glycoprotein from the arenavirus Junín virus (JUNV). Using this model system, we have solved the first high-resolution structures of VelcroVax VLPs and shown that the VelcroVax-JUNV gp1 complex induces superior humoral immune responses compared to the noncomplexed viral protein. We propose that this system could be modified to present a range of antigens and therefore form the foundation of future rapid-response vaccination strategies. IMPORTANCE The hepatitis B core protein (HBc) forms noninfectious virus-like particles, which can be modified to present a capturing molecule, allowing suitably tagged antigens to be bound on their surface. This system can be adapted and provides the foundation for a universal "bolt-on" vaccine platform (termed VelcroVax) that can be easily and rapidly modified to generate nanoparticle vaccine candidates.

F4/80 and the related adhesion-GPCRs.

The F4/80 monoclonal antibody was first reported in this journal 30 years ago (Eur. J. Immunol. 1981. 11: 805-815). F4/80 has become a widely used marker for monocytes and many, but not all, tissue macrophages in the mouse. F4/80 is a member of the EGF-TM7 family of leukocyte plasma membrane heptahelical molecules, which includes CD97 and EMR2. This Viewpoint summarises current knowledge of the expression, structure and functions of the EGF-TM7 family, as part of a larger family of tissue adhesion-GPCRs.

GPR97 triggers inflammatory processes in human neutrophils via a macromolecular complex upstream of PAR2 activation.

Neutrophils play essential anti-microbial and inflammatory roles in host defense, however, their activities require tight regulation as dysfunction often leads to detrimental inflammatory and autoimmune diseases. Here we show that the adhesion molecule GPR97 allosterically activates CD177-associated membrane proteinase 3 (mPR3), and in conjugation with several protein interaction partners leads to neutrophil activation in humans. Crystallographic and deletion analysis of the GPR97 extracellular region identified two independent mPR3-binding domains. Mechanistically, the efficient binding and activation of mPR3 by GPR97 requires the macromolecular CD177/GPR97/PAR2/CD16b complex and induces the activation of PAR2, a G protein-coupled receptor known for its function in inflammation. Triggering PAR2 by the upstream complex leads to strong inflammatory activation, prompting anti-microbial activities and endothelial dysfunction. The role of the complex in pathologic inflammation is underscored by the finding that both GPR97 and mPR3 are upregulated on the surface of disease-associated neutrophils. In summary, we identify a PAR2 activation mechanism that directs neutrophil activation, and thus inflammation. The PR3/CD177/GPR97/PAR2/CD16b protein complex, therefore, represents a potential therapeutic target for neutrophil-mediated inflammatory diseases.

The macrophage F4/80 receptor is required for the induction of antigen-specific efferent regulatory T cells in peripheral tolerance.

We show that the mouse macrophage-restricted F4/80 protein is not required for the development and distribution of tissue macrophages but is involved in the generation of antigen-specific efferent regulatory T (T reg) cells that suppress antigen-specific immunity. In the in vivo anterior chamber (a.c.)-associated immune deviation (ACAID) model of peripheral tolerance, a.c. inoculation of antigen into F4/80(-/-) mice was unable to induce efferent T reg cells and suppress delayed-type hypersensitivity (DTH) responses. Moreover, the use of anti-F4/80 mAb and F4/80(-/-) APCs in an in vitro ACAID model showed that all APC cells in the culture must be able to express F4/80 protein if efferent T reg cells were to be generated. In a low-dose oral tolerance model, WT but not F4/80(-/-) mice generated an efferent CD8(+) T reg cell population that suppressed an antigen-specific DTH response. Peripheral tolerance was restored in F4/80(-/-) mice by adoptive transfer of F4/80(+) APCs in both peripheral tolerance models, indicating a central role for the F4/80 molecule in the generation of efferent CD8(+) T reg cells.

Role of ADGRG1/GPR56 in Tumor Progression.

Cellular communication plays a critical role in diverse aspects of tumorigenesis including tumor cell growth/death, adhesion/detachment, migration/invasion, angiogenesis, and metastasis. G protein-coupled receptors (GPCRs) which constitute the largest group of cell surface receptors are known to play fundamental roles in all these processes. When considering the importance of GPCRs in tumorigenesis, the adhesion GPCRs (aGPCRs) are unique due to their hybrid structural organization of a long extracellular cell-adhesive domain and a seven-transmembrane signaling domain. Indeed, aGPCRs have been increasingly shown to be associated with tumor development by participating in tumor cell interaction and signaling. ADGRG1/GPR56, a representative tumor-associated aGPCR, is recognized as a potential biomarker/prognostic factor of specific cancer types with both tumor-suppressive and tumor-promoting functions. We summarize herein the latest findings of the role of ADGRG1/GPR56 in tumor progression.

The Immunological Impact of IL-1 Family Cytokines on the Epidermal Barrier.

The skin barrier would not function without IL-1 family members, but their physiological role in the immunological aspects of skin barrier function are often overlooked. This review summarises the role of IL-1 family cytokines (IL-1α, IL-1ß, IL-1Ra, IL-18, IL-33, IL-36α, IL-36ß, IL-36γ, IL-36Ra, IL-37 and IL-38) in the skin. We focus on novel aspects of their interaction with commensals and pathogens, the important impact of proteases on cytokine activity, on healing responses and inflammation limiting mechanisms. We discuss IL-1 family cytokines in the context of IL-4/IL-13 and IL-23/IL-17 axis-driven diseases and highlight consequences of human loss/gain of function mutations in activating or inhibitory pathway molecules. This review highlights recent findings that emphasize the importance of IL-1 family cytokines in both physiological and pathological cutaneous inflammation and emergent translational therapeutics that are helping further elucidate these cytokines.

Structural insight into Pichia pastoris fatty acid synthase.

Type I fatty acid synthases (FASs) are critical metabolic enzymes which are common targets for bioengineering in the production of biofuels and other products. Serendipitously, we identified FAS as a contaminant in a cryoEM dataset of virus-like particles (VLPs) purified from P. pastoris, an important model organism and common expression system used in protein production. From these data, we determined the structure of P. pastoris FAS to 3.1 Å resolution. While the overall organisation of the complex was typical of type I FASs, we identified several differences in both structural and enzymatic domains through comparison with the prototypical yeast FAS from S. cerevisiae. Using focussed classification, we were also able to resolve and model the mobile acyl-carrier protein (ACP) domain, which is key for function. Ultimately, the structure reported here will be a useful resource for further efforts to engineer yeast FAS for synthesis of alternate products.

Immunity and adhesion-GPCRs.

Adhesion-GPCRs are unusual, owing to their unique structure, comprising a large and complex extracellular domain composed of various common protein modules. Adhesion-GPCR family members are expressed ubiquitously; however the expression of each receptor is highly regulated and often restricted to specific cell types. The EGF-TM7 adhesion-GPCR subfamily members are predominantly expressed by leukocytes and involved in coordinating both the innate and acquired immune responses. Here we highlight some immunological insights in relation to EGF-TM7 proteins and other members of the adhesion-GPCR family.