C5aR plus MEK inhibition durably targets the tumor milieu and reveals tumor cell phagocytosis.

Plexiform neurofibromas (PNFs) are nerve tumors caused by loss of NF1 and dysregulation of RAS-MAPK signaling in Schwann cells. Most PNFs shrink in response to MEK inhibition, but targets with increased and durable effects are needed. We identified the anaphylatoxin C5a as increased in PNFs and expressed largely by PNF m acrophages. We defined pharmacokinetic and immunomodulatory properties of a C5aR1/2 antagonist and tested if peptide antagonists augment the effects of MEK inhibition. MEK inhibition recruited C5AR1 to the macrophage surface; short-term inhibition of C5aR elevated macrophage apoptosis and Schwann cell death, without affecting MEK-induced tumor shrinkage. PNF macrophages lacking C5aR1 increased the engulfment of dying Schwann cells, allowing their visualization. Halting combination therapy resulted in altered T-cell distribution, elevated Iba1+ and CD169+ immunoreactivity, and profoundly altered cytokine expression, but not sustained trumor shrinkage. Thus, C5aRA inhibition independently induces macrophage cell death and causes sustained and durable effects on the PNF microenvironment.

Complement activation and cellular inflammation in Fabry disease patients despite enzyme replacement therapy.

Defective α-galactosidase A (AGAL/GLA) due to missense or nonsense mutations in the GLA gene results in accumulation of the glycosphingolipids globotriaosylceramide (Gb3) and its deacylated derivate globotriaosylsphingosine (lyso-Gb3) in cells and body fluids. The aberrant glycosphingolipid metabolism leads to a progressive lysosomal storage disorder, i. e. Fabry disease (FD), characterized by chronic inflammation leading to multiorgan damage. Enzyme replacement therapy (ERT) with agalsidase-alfa or -beta is one of the main treatment options facilitating cellular Gb3 clearance. Proteome studies have shown changes in complement proteins during ERT. However, the direct activation of the complement system during FD has not been explored. Here, we demonstrate strong activation of the complement system in 17 classical male FD patients with either missense or nonsense mutations before and after ERT as evidenced by high C3a and C5a serum levels. In contrast to the strong reduction of lyso-Gb3 under ERT, C3a and C5a markedly increased in FD patients with nonsense mutations, most of whom developed anti-drug antibodies (ADA), whereas FD patients with missense mutations, which were ADA-negative, showed heterogenous C3a and C5a serum levels under treatment. In addition to the complement activation, we found increased IL-6, IL-10 and TGF-ß1 serum levels in FD patients. This increase was most prominent in patients with missense mutations under ERT, most of whom developed mild nephropathy with decreased estimated glomerular filtration rate. Together, our findings demonstrate strong complement activation in FD independent of ERT therapy, especially in males with nonsense mutations and the development of ADAs. In addition, our data suggest kidney cell-associated production of cytokines, which have a strong potential to drive renal damage. Thus, chronic inflammation as a driver of organ damage in FD seems to proceed despite ERT and may prove useful as a target to cope with progressive organ damage.

Platelets regulate ischemia-induced revascularization and angiogenesis by secretion of growth factor-modulating factors.

In ischemic tissue, platelets can modulate angiogenesis. The specific factors influencing this function, however, are poorly understood. Here, we characterized the complement anaphylatoxin C5a-mediated activation of C5a receptor 1 (C5aR1) expressed on platelets as a potent regulator of ischemia-driven revascularization. We assessed the relevance of the anaphylatoxin receptor C5aR1 on platelets in patients with coronary artery disease as well as those with peripheral artery disease and used genetic mouse models to characterize its significance for ischemia and growth factor-driven revascularization. The presence of C5aR1-expressing platelets was increased in the hindlimb ischemia model. Ischemia-driven angiogenesis was significantly improved in C5aR1-/- mice but not in C5-/- mice, suggesting a specific role of C5aR1. Experiments using the supernatant of C5a-stimulated platelets suggested a paracrine mechanism of angiogenesis inhibition by platelets by means of antiangiogenic CXC chemokine ligand 4 (CXCL4, PF4). Lineage-specific C5aR1 deletion verified that the secretion of CXCL4 depends on C5aR1 ligation on platelets. Using C5aR1-/-CXCL4-/- mice, we observed no additional effect in the revascularization response, underscoring a strong dependence of CXCL4 secretion on the C5a-C5aR1-axis. We identified a novel mechanism for inhibition of neovascularization via platelet C5aR1, which was mediated by the release of antiangiogenic CXCL4.

Autoimmune pre-disease.

Approximately 5% of the world-wide population is affected by autoimmune diseases. Overall, autoimmune diseases are still difficult to treat, impose a high burden on patients, and have a significant economic impact. Like other complex diseases, e.g., cancer, autoimmune diseases develop over several years. Decisive steps in the development of autoimmune diseases are (i) the development of autoantigen-specific lymphocytes and (often) autoantibodies and (ii) potentially clinical disease manifestation at a later stage. However, not all healthy individuals with autoantibodies develop disease manifestations. Identifying autoantibody-positive healthy individuals and monitoring and inhibiting their switch to inflammatory autoimmune disease conditions are currently in their infancy. The switch from harmless to inflammatory autoantigen-specific T and B-cell and autoantibody responses seems to be the hallmark for the decisive factor in inflammatory autoimmune disease conditions. Accordingly, biomarkers allowing us to predict this progression would have a significant impact. Several factors, such as genetics and the environment, especially diet, smoking, exposure to pollutants, infections, stress, and shift work, might influence the progression from harmless to inflammatory autoimmune conditions. To inspire research directed at defining and ultimately targeting autoimmune predisease, here, we review published evidence underlying the progression from health to autoimmune predisease and ultimately to clinically manifest inflammatory autoimmune disease, addressing the following 3 questions: (i) what is the current status, (ii) what is missing, (iii) and what are the future perspectives for defining and modulating autoimmune predisease.

Differential expression of C5aR1 and C5aR2 in innate and adaptive immune cells located in early skin lesions of bullous pemphigoid patients.

Bullous pemphigoid (BP), the by far most frequent autoimmune subepidermal blistering disorder (AIBD), is characterized by the deposition of autoantibodies against BP180 (type XVII collagen; Col17) and BP230 as well as complement components at the dermal-epidermal junction (DEJ). The mechanisms of complement activation in BP patients, including the generation of C5a and regulation of its two cognate C5aRs, i.e., C5aR1 and C5aR2, are incompletely understood. In this study, transcriptome analysis of perilesional and non-lesional skin biopsies of BP patients compared to site-, age-, and sex-matched controls showed an upregulated expression of C5AR1, C5AR2, CR1, and C3AR1 and other complement-associated genes in perilesional BP skin. Of note, increased expressions of C5AR2 and C3AR1 were also observed in non-lesional BP skin. Subsequently, double immunofluorescence (IF) staining revealed T cells and macrophages as the dominant cellular sources of C5aR1 in early lesions of BP patients, while C5aR2 mainly expressed on mast cells and eosinophils. In addition, systemic levels of various complement factors and associated molecules were measured in BP patients and controls. Significantly higher plasma levels of C3a, CD55, and mannose-binding lectin-pathway activity were found in BP patients compared to controls. Finally, the functional relevance of C5aR1 and C5aR2 in BP was explored by two in vitro assays. Specific inhibition of C5aR1, resulted in significantly reduced migration of human neutrophils toward the chemoattractant C5a, whereas stimulation of C5aR2 showed no effect. In contrast, the selective targeting of C5aR1 and/or C5aR2 had no effect on the release of reactive oxygen species (ROS) from Col17-anti-Col17 IgG immune complex-stimulated human leukocytes. Collectively, this study delineates a complex landscape of activated complement receptors, complement factors, and related molecules in early BP skin lesions. Our results corroborate findings in mouse models of pemphigoid diseases that the C5a/C5aR1 axis is pivotal for attracting inflammatory cells to the skin and substantiate our understanding of the C5a/C5aR1 axis in human BP. The broad expression of C5aRs on multiple cell types critical for BP pathogenesis call for clinical studies targeting this axis in BP and other complement-mediated AIBDs.

Complement receptor C5aR1 on osteoblasts regulates osteoclastogenesis in experimental postmenopausal osteoporosis.

In recent years, evidence has accumulated that the complement system, an integral part of innate immunity, may be involved in the regulation of bone homeostasis as well as inflammatory bone loss, for example, in rheumatoid arthritis and periodontitis. Complement may also contribute to osteoporosis development, but investigation of the mechanism is limited. Using mice with a conditional deletion of the complement anaphylatoxin receptor C5aR1, we here demonstrated that C5aR1 in osteoblasts (C5aR1 Runx2-Cre mice) or osteoclasts (C5aR1 LysM-Cre mice) did not affect physiological bone turnover or age-related bone loss in either sex, as confirmed by micro-computed tomography, histomorphometry, and biomechanical analyses of the bone and by the measurement of bone turnover markers in the blood serum. When female mice were subjected to ovariectomy (OVX), a common model for postmenopausal osteoporosis, significant bone loss was induced in C5aR1 fl/fl and C5aR1 LysM-Cre mice, as demonstrated by a significantly reduced bone volume fraction, trabecular number and thickness as well as an increased trabecular separation in the trabecular bone compartment. Confirming this, the osteoclast number and the receptor activator of nuclear factor k-B (RANK) ligand (RANKL) serum level were significantly elevated in these mouse lines. By contrast, C5aR1 Runx2-Cre mice were protected from bone loss after OVX and the serum RANKL concentration was not increased after OVX. These data suggested that bone cell-specific C5aR1 may be redundant in bone homeostasis regulation under physiological conditions. However, C5aR1 on osteoblasts was crucial for the induction of bone resorption under osteoporotic conditions by stimulating RANKL release, whereas C5aR1 on osteoclasts did not regulate OVX-induced bone loss. Therefore, our results implicate C5aR1 on osteoblasts as a potential target for treating postmenopausal osteoporosis.

C-X-C Motif Chemokine Ligand 9 and Its CXCR3 Receptor Are the Salt and Pepper for T Cells Trafficking in a Mouse Model of Gaucher Disease.

Gaucher disease is a lysosomal storage disease, which happens due to mutations in GBA1/Gba1 that encodes the enzyme termed as lysosomal acid ß-glucosidase. The major function of this enzyme is to catalyze glucosylceramide (GC) into glucose and ceramide. The deficiency of this enzyme and resultant abnormal accumulation of GC cause altered function of several of the innate and adaptive immune cells. For example, augmented infiltration of T cells contributes to the increased production of pro-inflammatory cytokines, (e.g., IFNγ, TNFα, IL6, IL12p40, IL12p70, IL23, and IL17A/F). This leads to tissue damage in a genetic mouse model (Gba19V/-) of Gaucher disease. The cellular mechanism(s) by which increased tissue infiltration of T cells occurs in this disease is not fully understood. Here, we delineate role of the CXCR3 receptor and its exogenous C-X-C motif chemokine ligand 9 (CXCL9) in induction of increased tissue recruitment of CD4+ T and CD8+ T cells in Gaucher disease. Intracellular FACS staining of macrophages (Mϕs) and dendritic cells (DCs) from Gba19V/- mice showed elevated production of CXCL9. Purified CD4+ T cells and the CD8+ T cells from Gba19V/- mice showed increased expression of CXCR3. Ex vivo and in vivo chemotaxis experiments showed CXCL9 involvement in the recruitment of Gba19V/- T cells. Furthermore, antibody blockade of the CXCL9 receptor (CXCR3) on T cells caused marked reduction in CXCL9- mediated chemotaxis of T cells in Gba19V/- mice. These data implicate abnormalities of the CXCL9-CXCR3 axis leading to enhanced tissue recruitment of T cells in Gaucher disease. Such results provide a rationale for blockade of the CXCL9/CXCR3 axis as potential new therapeutic targets for the treatment of inflammation in Gaucher disease.

The C5a/C5a receptor 1 axis controls tissue neovascularization through CXCL4 release from platelets.

Platelets contribute to the regulation of tissue neovascularization, although the specific factors underlying this function are unknown. Here, we identified the complement anaphylatoxin C5a-mediated activation of C5a receptor 1 (C5aR1) on platelets as a negative regulatory mechanism of vessel formation. We showed that platelets expressing C5aR1 exert an inhibitory effect on endothelial cell functions such as migration and 2D and 3D tube formation. Growth factor- and hypoxia-driven vascularization was markedly increased in C5ar1-/- mice. Platelet-specific deletion of C5aR1 resulted in a proangiogenic phenotype with increased collateralization, capillarization and improved pericyte coverage. Mechanistically, we found that C5a induced preferential release of CXC chemokine ligand 4 (CXCL4, PF4) from platelets as an important antiangiogenic paracrine effector molecule. Interfering with the C5aR1-CXCL4 axis reversed the antiangiogenic effect of platelets both in vitro and in vivo.In conclusion, we identified a mechanism for the control of tissue neovascularization through C5a/C5aR1 axis activation in platelets and subsequent induction of the antiangiogenic factor CXCL4.

C3 Drives Inflammatory Skin Carcinogenesis Independently of C5.

Nonmelanoma skin cancer such as cutaneous squamous cell carcinoma (cSCC) is the most common form of cancer and can occur as a consequence of DNA damage to the epithelium by UVR or chemical carcinogens. There is growing evidence that the complement system is involved in cancer immune surveillance; however, its role in cSCC remains unclear. Here, we show that complement genes are expressed in tissue from patients with cSCC, and C3 activation fragments are present in cSCC biopsies, indicating complement activation. Using a range of complement-deficient mice in a two-stage mouse model of chemically-induced cSCC, where a subclinical dose of 7,12-dimethylbenz[a]anthracene causes oncogenic mutations in epithelial cells and 12-O-tetradecanoylphorbol-13-acetate promotes the outgrowth of these cells, we found that C3-deficient mice displayed a significantly reduced tumor burden, whereas an opposite phenotype was observed in mice lacking C5aR1, C5aR2, and C3a receptor. In addition, in mice unable to form the membrane attack complex, the tumor progression was unaltered. C3 deficiency did not affect the cancer response to 7,12-dimethylbenz[a]anthracene treatment alone but reduced the epidermal hyperplasia during 12-O-tetradecanoylphorbol-13-acetate-induced inflammation. Collectively, these data indicate that C3 drives tumorigenesis during chronic skin inflammation, independently of the downstream generation of C5a or membrane attack complex.

GM-CSF and IL-33 Orchestrate Polynucleation and Polyploidy of Resident Murine Alveolar Macrophages in a Murine Model of Allergic Asthma.

Allergic asthma is a chronical pulmonary disease with high prevalence. It manifests as a maladaptive immune response to common airborne allergens and is characterized by airway hyperresponsiveness, eosinophilia, type 2 cytokine-associated inflammation, and mucus overproduction. Alveolar macrophages (AMs), although contributing to lung homeostasis and tolerance to allergens at steady state, have attracted less attention compared to professional antigen-presenting and adaptive immune cells in their contributions. Using an acute model of house dust mite-driven allergic asthma in mice, we showed that a fraction of resident tissue-associated AMs, while polarizing to the alternatively activated M2 phenotype, exhibited signs of polynucleation and polyploidy. Mechanistically, in vitro assays showed that only Granulocyte-Macrophage Colony Stimulating Factor and interleukins IL-13 and IL-33, but not IL-4 or IL-5, participate in the establishment of this phenotype, which resulted from division defects and not cell-cell fusion as shown by microscopy. Intriguingly, mRNA analysis of AMs isolated from allergic asthmatic lungs failed to show changes in the expression of genes involved in DNA damage control except for MafB. Altogether, our data support the idea that upon allergic inflammation, AMs undergo DNA damage-induced stresses, which may provide new unconventional therapeutical approaches to treat allergic asthma.

Complement C5a Induces Pro-inflammatory Microvesicle Shedding in Severely Injured Patients.

Initially underestimated as platelet dust, extracellular vesicles are continuously gaining interest in the field of inflammation. Various studies addressing inflammatory diseases have shown that microvesicles (MVs) originating from different cell types are systemic transport vehicles carrying distinct cargoes to modulate immune responses. In this study, we focused on the clinical setting of multiple trauma, which is characterized by activation and dysfunction of both, the fluid-phase and the cellular component of innate immunity. Given the sensitivity of neutrophils for the complement anaphylatoxin C5a, we hypothesized that increased C5a production induces alterations in MV shedding of neutrophils resulting in neutrophil dysfunction that fuels posttraumatic inflammation. In a mono-centered prospective clinical study with polytraumatized patients, we found significantly increased granulocyte-derived MVs containing the C5a receptor (C5aR1, CD88) on their surface. This finding was accompanied by a concomitant loss of C5aR1 on granulocytes indicative of an impaired cellular chemotactic and pro-inflammatory neutrophil functions. Furthermore, in vitro exposure of human neutrophils (from healthy volunteers) to C5a significantly increased MV shedding and C5aR1 loss on neutrophils, which could be blocked using the C5aR1 antagonist PMX53. Mechanistic analyses revealed that the interaction between C5aR1 signaling and the small GTPase Arf6 acts as a molecular switch for MV shedding. When neutrophil derived, C5a-induced MV were exposed to a complex ex vivo whole blood model significant pro-inflammatory properties (NADPH activity, ROS and MPO generation) of the MVs became evident. C5a-induced MVs activated resting neutrophils and significantly induced IL-6 secretion. These data suggest a novel role of the C5a-C5aR1 axis: C5a-induced MV shedding from neutrophils results in decreased C5aR1 surface expression on the one hand, on the other hand it leads to profound inflammatory signals which likely are both key drivers of the neutrophil dysfunction which is regularly observed in patients suffering from multiple traumatic injuries.

C5aR1 Activation Drives Early IFN-γ Production to Control Experimental Toxoplasma gondii Infection.

Toxoplasma gondii (T. gondii) is a parasite infecting animals and humans. In intermediate hosts, such as humans or rodents, rapidly replicating tachyzoites drive vigorous innate and adaptive immune responses resulting in bradyzoites that survive within tissue cysts. Activation of the innate immune system is critical during the early phase of infection to limit pathogen growth and to instruct parasite-specific adaptive immunity. In rodents, dendritic cells (DCs) sense T. gondii through TLR11/12, leading to IL-12 production, which activates NK cells to produce IFN-γ as an essential mechanism for early parasite control. Further, C3 can bind to T. gondii resulting in limited complement activation. Here, we determined the role of C5a/C5aR1 axis activation for the early innate immune response in a mouse model of peritoneal T. gondii infection. We found that C5ar1-/- animals suffered from significantly higher weight loss, disease severity, mortality, and parasite burden in the brain than wild type control animals. Severe infection in C5ar1-/- mice was associated with diminished serum concentrations of IL-12, IL-27, and IFN-γ. Importantly, the serum levels of pro-inflammatory cytokines, including IL-1α, IL-6, and TNF-α, as well as several CXC and CC chemokines, were decreased in comparison to wt animals, whereas anti-inflammatory IL-10 was elevated. The defect in IFN-γ production was associated with diminished Ifng mRNA expression in the spleen and the brain, reduced frequency of IFN-γ+ NK cells in the spleen, and decreased Nos2 expression in the brain of C5ar1-/- mice. Mechanistically, DCs from the spleen of C5ar1-/- mice produced significantly less IL-12 in response to soluble tachyzoite antigen (STAg) stimulation in vivo and in vitro. Our findings suggest a model in which the C5a/C5aR1 axis promotes IL-12 induction in splenic DCs that is critical for IFN-γ production from NK cells and subsequent iNOS expression in the brain as a critical mechanism to control acute T. gondii infection.

Allergen-Induced C5a/C5aR1 Axis Activation in Pulmonary CD11b+ cDCs Promotes Pulmonary Tolerance through Downregulation of CD40.

Activation of the C5/C5a/C5a receptor 1 (C5aR1) axis during allergen sensitization protects from maladaptive T cell activation. To explore the underlying regulatory mechanisms, we analyzed the impact of C5aR1 activation on pulmonary CD11b+ conventional dendritic cells (cDCs) in the context of house-dust-mite (HDM) exposure. BALB/c mice were intratracheally immunized with an HDM/ovalbumin (OVA) mixture. After 24 h, we detected two CD11b+ cDC populations that could be distinguished on the basis of C5aR1 expression. C5aR1- but not C5aR1+ cDCs strongly induced T cell proliferation of OVA-reactive transgenic CD4+ T cells after re-exposure to antigen in vitro. C5aR1- cDCs expressed higher levels of MHC-II and CD40 than their C5aR1+ counterparts, which correlated directly with a higher frequency of interactions with cognate CD4+ T cells. Priming of OVA-specific T cells by C5aR1+ cDCs could be markedly increased by in vitro blockade of C5aR1 and this was associated with increased CD40 expression. Simultaneous blockade of C5aR1 and CD40L on C5aR1+ cDCs decreased T cell proliferation. Finally, pulsing with OVA-induced C5 production and its cleavage into C5a by both populations of CD11b+ cDCs. Thus, we propose a model in which allergen-induced autocrine C5a generation and subsequent C5aR1 activation in pulmonary CD11b+ cDCs promotes tolerance towards aeroallergens through downregulation of CD40.

Distinct roles of the anaphylatoxin receptors C3aR, C5aR1 and C5aR2 in experimental meningococcal infections.

The complement system is pivotal in the defense against invasive disease caused by Neisseria meningitidis (Nme, meningococcus), particularly via the membrane attack complex. Complement activation liberates the anaphylatoxins C3a and C5a, which activate three distinct G-protein coupled receptors, C3aR, C5aR1 and C5aR2 (anaphylatoxin receptors, ATRs). We recently discovered that C5aR1 exacerbates the course of the disease, revealing a downside of complement in Nme sepsis. Here, we compared the roles of all three ATRs during mouse nasal colonization, intraperitoneal infection and human whole blood infection with Nme. Deficiency of complement or ATRs did not alter nasal colonization, but significantly affected invasive disease: Compared to WT mice, the disease was aggravated in C3ar-/- mice, whereas C5ar1-/- and C5ar2-/- mice showed increased resistance to meningococcal sepsis. Surprisingly, deletion of either of the ATRs resulted in lower cytokine/chemokine responses, irrespective of the different susceptibilities of the mice. This was similar in ex vivo human whole blood infection using ATR inhibitors. Neutrophil responses to Nme were reduced in C5ar1-/- mouse blood. Upon stimulation with C5a plus Nme, mouse macrophages displayed reduced phosphorylation of ERK1/2, when C5aR1 or C5aR2 were ablated or inhibited, suggesting that both C5a-receptors prime an initial macrophage response to Nme. Finally, in vivo blockade of C5aR1 alone (PMX205) or along with C5aR2 (A8Δ71-73) resulted in ameliorated disease, whereas neither antagonizing C3aR (SB290157) nor its activation with a "super-agonist" peptide (WWGKKYRASKLGLAR) demonstrated a benefit. Thus, C5aR1 and C5aR2 augment disease pathology and are interesting targets for treatment, whereas C3aR is protective in experimental meningococcal sepsis.

A Novel Role for C5a in B-1 Cell Homeostasis.

B-1 cells constitute a unique subpopulation of lymphocytes residing mainly in body cavities like the peritoneal cavity (PerC) but are also found in spleen and bone marrow (BM). As innate-like B cells, they mediate first line immune defense through low-affinity natural IgM (nIgM) antibodies. PerC B-1 cells can egress to the spleen and differentiate into nIgM antibody-secreting plasma cells that recognize conserved exogenous and endogenous cellular structures. Homing to and homeostasis within the PerC are regulated by the chemokine CXCL13 released by PerC macrophages and stroma cells. However, the exact mechanisms underlying the regulation of CXCL13 and B-1 homeostasis are not fully explored. B-1 cells play important roles in the inflammatory response to infection, autoimmunity, ischemia/reperfusion injury, obesity, and atherosclerosis. Remarkably, this list of inflammatory entities has a strong overlap with diseases that are regulated by complement suggesting a link between B-1 cells and the complement system. Interestingly, up to now, no data exist regarding the role of complement in B-1 cell biology. Here, we demonstrate for the first time that C5a regulates B-1 cell steady-state dynamics within the peritoneum, the spleen, and the BM. We found decreased B-1a cell numbers in the peritoneum and the spleen of C5aR1-/- mice associated with increased B1-a and B1-b numbers in the spleen and high serum titers of nIgM antibodies directed against phosphorylcholine and several pneumococcal polysaccharides. Similarly, peritoneal B-1a cells were decreased in the peritoneum and splenic B-1a and B-1b cells were increased in C5aR2-/- mice. The decrease in peritoneal B-1 cell numbers was associated with decreased peritoneal CXCL13 levels in C5aR1-/- and C5aR2-/- mice. In search for mechanisms, we found that combined TLR2 and IL-10 receptor activation in PerC macrophages induced strong CXCL13 production, which was significantly reduced in cells from C5aR1- and C5aR2-deficient mice and after combined C5aR-targeting. Such stimulation also induced marked local C5 production by PerC macrophages and C5a generation. Importantly, peritoneal in vivo administration of C5a increased CXCL13 production. Taken together, our findings suggest that local non-canonical C5 activation in PerC macrophages fuels CXCL13 production as a novel mechanism to control B-1 cell homeostasis.

The C5a/C5aR1 axis controls the development of experimental allergic asthma independent of LysM-expressing pulmonary immune cells.

C5a regulates the development of maladaptive immune responses in allergic asthma mainly through the activation of C5a receptor 1 (C5aR1). Yet, the cell types and the mechanisms underlying this regulation are ill-defined. Recently, we described increased C5aR1 expression in lung tissue eosinophils but decreased expression in airway and pulmonary macrophages as well as in pulmonary CD11b+ conventional dendritic cells (cDCs) and monocyte-derived DCs (moDCs) during the allergic effector phase using a floxed green fluorescent protein (GFP)-C5aR1 knock-in mouse. Here, we determined the role of C5aR1 signaling in neutrophils, moDCs and macrophages for the pulmonary recruitment of such cells and the importance of C5aR1-mediated activation of LysM-expressing cells for the development of allergic asthma. We used LysM-C5aR1 KO mice with a specific deletion of C5aR1 in LysMCre-expressing cells and confirmed the specific deletion of C5aR1 in neutrophils, macrophages and moDCs in the airways and/or the lung tissue. We found that alveolar macrophage numbers were significantly increased in LysM-C5aR1 KO mice. Induction of ovalbumin (OVA)-driven experimental allergic asthma in GFP-C5aR1fl/fl and LysM-C5aR1 KO mice resulted in strong but similar airway resistance, mucus production and Th2/Th17 cytokine production. In contrast, the number of airway but not of pulmonary neutrophils was lower in LysM-C5aR1 KO as compared with GFP-C5aR1fl/fl mice. The recruitment of macrophages, cDCs, moDCs, T cells and type 2 innate lymphoid cells was not altered in LysM-C5aR1 KO mice. Our findings demonstrate that C5aR1 is critical for steady state control of alveolar macrophage numbers and the transition of neutrophils from the lung into the airways in OVA-driven allergic asthma. However, C5aR1 activation of LysM-expressing cells plays a surprisingly minor role in the recruitment and activation of such cells and the development of the allergic phenotype in OVA-driven experimental allergic asthma.

Monitoring C3aR Expression Using a Floxed tdTomato-C3aR Reporter Knock-in Mouse.

C3a exerts multiple biologic functions through activation of its cognate C3a receptor. C3-/- and C3aR-/- mice have been instrumental in defining important roles of the C3a/C3aR axis in the regulation of acute and chronic inflammatory diseases, including ischemia/reperfusion injury, allergic asthma, autoimmune nephritis, and rheumatoid arthritis. Surprisingly little is known about C3aR expression and function in immune and stromal cells. To close this gap, we generated a floxed tandem-dye Tomato (tdTomato)-C3aR reporter knock-in mouse, which we used to monitor C3aR expression in cells residing in the lung, airways, lamina propria (LP) of the small intestine, brain, visceral adipose tissue, bone marrow (BM), spleen, and the circulation. We found a strong expression of tdTomato-C3aR in the brain, lung, LP, and visceral adipose tissue, whereas it was minor in the spleen, blood, BM, and the airways. Most macrophage and eosinophil populations were tdTomato-C3aR+ Interestingly, most tissue eosinophils and some macrophage populations expressed C3aR intracellularly. BM-derived dendritic cells (DCs), lung-resident cluster of differentiation (CD) 11b+ conventional DCs (cDCs) and monocyte-derived DCs, LP CD103+, and CD11b+ cDCs but not pulmonary CD103+ cDCs and splenic DCs were tdTomato-C3aR+ Surprisingly, neither BM, blood, lung neutrophils, nor mast cells expressed C3aR. Similarly, all lymphoid-derived cells were tdTomato-C3aR-, except some LP-derived type 3 innate lymphoid cells. Pulmonary and LP-derived epithelial cells expressed at best minor levels of C3aR. In summary, we provide novel insights into the expression pattern of C3aR in mice. The floxed C3aR knock-in mouse will help to reliably track and conditionally delete C3aR expression in experimental models of inflammation.

Novel insights into the expression pattern of anaphylatoxin receptors in mice and men.

The anaphylatoxins (AT) C3a and C5a play important roles as mediators of inflammation. Further, they regulate and control multiple innate and adaptive immune responses through binding and activation of their cognate G protein-coupled receptors, i.e. C3a receptor (C3aR), C5a receptor 1 (C5aR1) and C5a receptor 2 (C5aR2), although the latter lacks important sequence motifs for G protein-coupling. Based on their pleiotropic functions, they contribute not only to tissue homeostasis but drive, perpetuate and resolve immune responses in many inflammatory diseases including infections, malignancies, autoimmune as well as allergic diseases. During the past few years, transcriptome expression data provided detailed insights into AT receptor tissue mRNA expression. In contrast, our understanding of cellular AT receptor expression in human and mouse tissues under steady and inflammatory conditions is still sketchy. Ligand binding studies, flow cytometric and immunohistochemical analyses convincingly demonstrated tissue-specific C5aR1 expression in various cells of myeloid origin. However, a detailed map for C3aR or C5aR2 expression in human or mouse tissue cells is still lacking. Also, reports about AT expression in lymphoid cells is still controversial. To understand the multiple roles of the ATs in the innate and adaptive immune networks, a detailed understanding of their receptor expression in health and disease is required. Recent findings obtained with novel GFP or tdTomato AT-receptor knock-in mice provide detailed insights into their expression pattern in tissue immune and stroma cells. Here, we will provide an update about our current knowledge of AT receptor expression pattern in humans and mice.

Complement drives glucosylceramide accumulation and tissue inflammation in Gaucher disease.

Gaucher disease is caused by mutations in GBA1, which encodes the lysosomal enzyme glucocerebrosidase (GCase). GBA1 mutations drive extensive accumulation of glucosylceramide (GC) in multiple innate and adaptive immune cells in the spleen, liver, lung and bone marrow, often leading to chronic inflammation. The mechanisms that connect excess GC to tissue inflammation remain unknown. Here we show that activation of complement C5a and C5a receptor 1 (C5aR1) controls GC accumulation and the inflammatory response in experimental and clinical Gaucher disease. Marked local and systemic complement activation occurred in GCase-deficient mice or after pharmacological inhibition of GCase and was associated with GC storage, tissue inflammation and proinflammatory cytokine production. Whereas all GCase-inhibited mice died within 4-5 weeks, mice deficient in both GCase and C5aR1, and wild-type mice in which GCase and C5aR were pharmacologically inhibited, were protected from these adverse effects and consequently survived. In mice and humans, GCase deficiency was associated with strong formation of complement-activating GC-specific IgG autoantibodies, leading to complement activation and C5a generation. Subsequent C5aR1 activation controlled UDP-glucose ceramide glucosyltransferase production, thereby tipping the balance between GC formation and degradation. Thus, extensive GC storage induces complement-activating IgG autoantibodies that drive a pathway of C5a generation and C5aR1 activation that fuels a cycle of cellular GC accumulation, innate and adaptive immune cell recruitment and activation in Gaucher disease. As enzyme replacement and substrate reduction therapies are expensive and still associated with inflammation, increased risk of cancer and Parkinson disease, targeting C5aR1 may serve as a treatment option for patients with Gaucher disease and, possibly, other lysosomal storage diseases.