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Complement C5a protein has been shown to play a major role in tissue regeneration through interaction with its receptor (C5aR) on target cells. Expression of this receptor has been reported in the nervous system which, upon injury, has no treatment to restore the lost functions. This work aimed at investigating the Complement C5a effect on axonal growth after axotomy in vitro. Primary hippocampal neurons were isolated from embryonic Wistar rats. Cell expression of C5aR mRNA was verified by RT-PCR while its membrane expression, localization, and phosphorylation were investigated by immunofluorescence. Then, the effects of C5a on injured axonal growth were investigated using a 3D-printed microfluidic device. Immunofluorescence demonstrated that the primary cultures contained only mature neurons (93%) and astrocytes (7%), but no oligodendrocytes or immature neurons. Immunofluorescence revealed a co-localization of NF-L and C5aR only in the mature neurons where C5a induced the phosphorylation of its receptor. C5a application on injured axons in the microfluidic devices significantly increased both the axonal growth speed and length. Our findings highlight a new role of C5a in regeneration demonstrating an enhancement of axonal growth after axotomy. This may provide a future therapeutic tool in the treatment of central nervous system injury. [ABSTRACT FROM AUTHOR]

The complement system is critically involved in the pathogenesis of sepsis. In particular, complement anaphylatoxin C5a is generated in excess during sepsis, leading to cellular dysfunction. Recent studies have shown that excessive C5a impairs adrenomedullary catecholamine production release and induces apoptosis in adrenomedullary cells. Currently, the mechanisms by which C5a impacts adrenal cell function are poorly understood. The PC12 cell model was used to examine the cellular effects following treatment with recombinant rat C5a. The levels of caspase activation and cell death, protein kinase signaling pathway activation, and changes in inflammatory protein expression were examined following treatment with C5a. There was an increase in apoptosis of PC12 cells following treatment with high-dose C5a. Ten inflammatory proteins, primarily involved in apoptosis, cell survival, and cell proliferation, were upregulated following treatment with high-dose C5a. Five inflammatory proteins, involved primarily in chemotaxis and anti-inflammatory functions, were downregulated. The ERK/MAPK, p38/MAPK, JNK/MAPK, and AKT protein kinase signaling pathways were upregulated in a C5aR-dependent manner. These results demonstrate an apoptotic effect and cellular signaling effect of high-dose C5a. Taken together, the overall data suggest that high levels of C5a may play a role in C5aR-dependent apoptosis of adrenal medullary cells in sepsis. [ABSTRACT FROM AUTHOR]

Ischemia/reperfusion (I/R) is inevitable during kidney transplantation and causes acute kidney injury (AKI), which affects immediate outcome and leads to chronic changes such as fibrotic remodeling of the graft. We investigated pro-fibrotic signaling after I/R, focusing on the complement component and receptor C5a/C5aR1 and macrophage/tubule crosstalk. Male Dark Agouti rats were subjected to I/R and their kidneys were harvested 10 min, 6 h, 24 h, 3 days, 5 days and 8 weeks after reperfusion. The development of renal fibrosis was assessed by the detection of Vimentin (VIM), α-smooth muscle actin (α-SMA) and collagen by immunohistochemistry and Sirius Red staining, respectively. The characterization of C5a/C5aR1 activity and C5aR1+ cells was performed by multiplex mRNA analysis, ELISA, immunofluorescence flow cytometry and in situ hybridization in animal models and cell culture analyses. In the cell culture experiments, we focused on macrophage/tubule cell crosstalk in co-culture experiments and mimicked in vivo conditions by hypoxia/reoxygenation and supplementation with C5a. Already 6–24 h after the induction of I/R in the rat model, C5a concentration in the plasma was significantly increased compared to the control. The matrix components VIM and α-SMA peaked on day 5 and declined after 8 weeks, when an increase in collagen was detected using Sirius Red. In contrast to early I/R-induced C5a activation, renal C5ar1 expression was maximal at day 5 and C5 expression increased until week 8, indicating that the renal upregulation of expression is not required for early complement activation. C5aR1 mRNA was detected in neutrophils and macrophages, but not in proximal tubular cells in the injured kidneys. The macrophage/tubular cell co-culture experiments showed that macrophages were mainly responsible for the increased expression of fibrosis-associated genes in tubule cells (ACTA2, VIM, SNAI1, TGFB1 and FGF-2), and hypoxia/reoxygenation had a partially enhancing effect. A direct pro-fibrotic effect of C5a was not observed. Increased TGF-ß levels were dependent on the differentiation of macrophages to the M2 subtype. In conclusion, the early activation of mesenchymal markers in tubular epithelial cells leads to long-term fibrotic remodeling characterized by VIM expression and driven by TGF-ß-dependent macrophage/tubular crosstalk. The chemoattractive properties of complement C5a may contribute to the recruitment of pro-fibrotic macrophages. [ABSTRACT FROM AUTHOR]

Complement C5a protein has been shown to play a major role in tissue regeneration through interaction with its receptor (C5aR) on target cells. Expression of this receptor has been reported in the nervous system which, upon injury, has no treatment to restore the lost functions. This work aimed at investigating the Complement C5a effect on axonal growth after axotomy in vitro. Primary hippocampal neurons were isolated from embryonic Wistar rats. Cell expression of C5aR mRNA was verified by RT-PCR while its membrane expression, localization, and phosphorylation were investigated by immunofluorescence. Then, the effects of C5a on injured axonal growth were investigated using a 3D-printed microfluidic device. Immunofluorescence demonstrated that the primary cultures contained only mature neurons (93%) and astrocytes (7%), but no oligodendrocytes or immature neurons. Immunofluorescence revealed a co-localization of NF-L and C5aR only in the mature neurons where C5a induced the phosphorylation of its receptor. C5a application on injured axons in the microfluidic devices significantly increased both the axonal growth speed and length. Our findings highlight a new role of C5a in regeneration demonstrating an enhancement of axonal growth after axotomy. This may provide a future therapeutic tool in the treatment of central nervous system injury.

Background and Objectives: Dental caries is one of the most common human pathological conditions resulting from the invasion of bacteria into the dentin. Current treatment options are limited. In many cases, endodontic therapy leads to permanent pulp tissue loss. Dentin-pulp complex regeneration involves dental pulp stem cells (DPSCs) that differentiate into odontoblast-like cells under an inflammatory context. However, limited information is available on how DPSC differentiation processes are affected under inflammatory environments. We identified the crucial role of complement C5a and its receptor C5aR in the inflammation-induced odontoblastic DPSC differentiation. Methodology: Here, we further investigated the role of a second and controversial C5a receptor, C5L2, in this process and explored the underlying mechanism. Human DPSCs were examined during 7-, 10-, and 14-day odontogenic differentiation treated with TNFα, C5L2 CRISPR, and tyrosine receptor kinase B (TrkB) antagonist [cyclotraxin-B (CTX-B)]. Results: Our data demonstrate that C5L2 CRISPR knockout (KO) enhances mineralization in TNFα-stimulated differentiating DPSCs. We further confirmed that C5L2 CRISPR KO significantly enhances dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) expression after 14-day odontoblastic DPSC differentiation, and treatment with CTX-B abolished the TNFα/C5L2 CRISPR KOinduced DSPP and DMP-1 increase, suggesting TrkB's critical role in this process. Conclusion and Key applications: Our data suggest a regulatory role of C5L2 and TrkB in the TNFα-induced odontogenic DPSC differentiation. This study may provide a useful tool to understand the mechanisms of the role of inflammation in dentinogenesis that is required for successful DPSC engineering strategies. [ABSTRACT FROM AUTHOR]

Ischemia/reperfusion (I/R) is inevitable during kidney transplantation and causes acute kidney injury (AKI), which affects immediate outcome and leads to chronic changes such as fibrotic remodeling of the graft. We investigated pro-fibrotic signaling after I/R, focusing on the complement component and receptor C5a/C5aR1 and macrophage/tubule crosstalk. Male Dark Agouti rats were subjected to I/R and their kidneys were harvested 10 min, 6 h, 24 h, 3 days, 5 days and 8 weeks after reperfusion. The development of renal fibrosis was assessed by the detection of Vimentin (VIM), α-smooth muscle actin (α-SMA) and collagen by immunohistochemistry and Sirius Red staining, respectively. The characterization of C5a/C5aR1 activity and C5aR1+ cells was performed by multiplex mRNA analysis, ELISA, immunofluorescence flow cytometry and in situ hybridization in animal models and cell culture analyses. In the cell culture experiments, we focused on macrophage/tubule cell crosstalk in co-culture experiments and mimicked in vivo conditions by hypoxia/reoxygenation and supplementation with C5a. Already 6–24 h after the induction of I/R in the rat model, C5a concentration in the plasma was significantly increased compared to the control. The matrix components VIM and α-SMA peaked on day 5 and declined after 8 weeks, when an increase in collagen was detected using Sirius Red. In contrast to early I/R-induced C5a activation, renal C5ar1 expression was maximal at day 5 and C5 expression increased until week 8, indicating that the renal upregulation of expression is not required for early complement activation. C5aR1 mRNA was detected in neutrophils and macrophages, but not in proximal tubular cells in the injured kidneys. The macrophage/tubular cell co-culture experiments showed that macrophages were mainly responsible for the increased expression of fibrosis-associated genes in tubule cells (ACTA2, VIM, SNAI1, TGFB1 and FGF-2), and hypoxia/reoxygenation had a partially enhancing effect. A direct pro-fibrotic effect of C5a was not observed. Increased TGF-ß levels were dependent on the differentiation of macrophages to the M2 subtype. In conclusion, the early activation of mesenchymal markers in tubular epithelial cells leads to long-term fibrotic remodeling characterized by VIM expression and driven by TGF-ß-dependent macrophage/tubular crosstalk. The chemoattractive properties of complement C5a may contribute to the recruitment of pro-fibrotic macrophages.

Background and Objectives: Dental caries is one of the most common human pathological conditions resulting from the invasion of bacteria into the dentin. Current treatment options are limited. In many cases, endodontic therapy leads to permanent pulp tissue loss. Dentin–pulp complex regeneration involves dental pulp stem cells (DPSCs) that differentiate into odontoblast-like cells under an inflammatory context. However, limited information is available on how DPSC differentiation processes are affected under inflammatory environments. We identified the crucial role of complement C5a and its receptor C5aR in the inflammation-induced odontoblastic DPSC differentiation.Methodology: Here, we further investigated the role of a second and controversial C5a receptor, C5L2, in this process and explored the underlying mechanism. Human DPSCs were examined during 7-, 10-, and 14-day odontogenic differentiation treated with TNFα, C5L2 CRISPR, and tyrosine receptor kinase B (TrkB) antagonist [cyclotraxin-B (CTX-B)].Results: Our data demonstrate that C5L2 CRISPR knockout (KO) enhances mineralization in TNFα-stimulated differentiating DPSCs. We further confirmed that C5L2 CRISPR KO significantly enhances dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) expression after 14-day odontoblastic DPSC differentiation, and treatment with CTX-B abolished the TNFα/C5L2 CRISPR KO-induced DSPP and DMP-1 increase, suggesting TrkB’s critical role in this process.Conclusion and Key applications: Our data suggest a regulatory role of C5L2 and TrkB in the TNFα-induced odontogenic DPSC differentiation. This study may provide a useful tool to understand the mechanisms of the role of inflammation in dentinogenesis that is required for successful DPSC engineering strategies.

EP67 is a second-generation, human C5a-derived decapeptide agonist of C5a receptor 1 (C5aR1/CD88) that selectively activates mononuclear phagocytes over neutrophils to potentiate protective innate and adaptive immune responses while potentially minimizing neutrophil-mediated toxicity. Pro7 and N-methyl-Leu8 (Me-Leu8) amino acid residues within EP67 likely induce backbone structural changes that increase potency and selective activation of mononuclear phagocytes over neutrophils versus first-generation EP54. The low coupling efficiency between Pro7 and Me-Leu8 and challenging purification by HPLC, however, greatly increase scale-up costs of EP67 for clinical use. Thus, the goal of this study was to determine whether replacing Pro7 and/or Me-Leu8 with large-scale amenable amino acid residues predicted to induce similar structural changes (cyclohexylalanine7 and/or leucine8) sufficiently preserves EP67 activity in primary human mononuclear phagocytes and neutrophils. We found that EP67 analogues had similar potency, efficacy, and selective activation of mononuclear phagocytes over neutrophils. Thus, replacing Pro7 and/or Me-Leu8 with large-scale amenable amino acid residues predicted to induce similar structural changes is a suitable strategy to overcome scale-up challenges with EP67.

Traumatic spinal cord injury (SCI) brings numerous inflammatory cells, including macrophages, from the circulating blood to lesions, but pathophysiological impact resulting from spatiotemporal dynamics of macrophages is unknown. Here, we show that macrophages centripetally migrate toward the lesion epicenter after infiltrating into the wide range of spinal cord, depending on the gradient of chemoattractant C5a. However, macrophages lacking interferon regulatory factor 8 (IRF8) cannot migrate toward the epicenter and remain widely scattered in the injured cord with profound axonal loss and little remyelination, resulting in a poor functional outcome after SCI. Time-lapse imaging and P2X/YRs blockade revealed that macrophage migration via IRF8 was caused by purinergic receptors involved in the C5a-directed migration. Conversely, pharmacological promotion of IRF8 activation facilitated macrophage centripetal movement, thereby improving the SCI recovery. Our findings reveal the importance of macrophage centripetal migration via IRF8, providing a novel therapeutic target for central nervous system injury.

Abstract Background Thrombotic events cannot be completely prevented by antithrombotics, implicating a therapeutic gap due to inflammation, a not yet sufficiently addressed mechanism. Neutrophil extracellular traps (NETs) are an essential interface between inflammation and thrombosis, but exactly how the NETotic process is initiated and maintained during arterial thrombosis remains incompletely understood. Methods and results We found that the plasma concentrations of C5a were higher in patients with ST-elevation myocardial infarction (STEMI) than in patients with angina and higher in mice with left common carotid artery (LCCA) thrombosis induced by FeCl3 than in control mice. We observed that the thrombus area and weight were decreased and that NET formation in the thrombi was reduced in the group treated with the selective C5aR1 receptor inhibitor PMX53 compared with the NaCl group. In vitro, NETosis was observed when C5a was added to neutrophil cultures, and this effect was reversed by PMX53. In addition, our data showed that C5a increased the production of mitochondrial reactive oxygen species (ROS) and that the promotion of NET formation by C5a was mitochondrial ROS (Mito-ROS) dependent. Furthermore, we found that C5a induced the production of Mito-ROS by inhibiting mitochondrial STAT3 activity. Conclusions By inhibiting mitochondrial STAT3 to elicit Mito-ROS generation, C5a triggers the generation of NETs to promote the development of arterial thrombosis. Hence, our study identifies complement C5a as a potential new target for the treatment and prevention of thrombosis.

Global bursts in free intracellular calcium (Ca2+) are among the most conspicuous signaling events in immune cells. To test the common view that Ca2+ bursts mediate rearrangement of the actin cytoskeleton in response to the activation of G protein-coupled receptors, we combined single-cell manipulation with fluorescence imaging and monitored the Ca2+ concentration in individual human neutrophils during complement-mediated chemotaxis. By decoupling purely chemotactic pseudopod formation from cell-substrate adhesion, we showed that physiological concentrations of anaphylatoxins, such as C5a, induced nonadherent human neutrophils to form chemotactic pseudopods but did not elicit Ca2+ bursts. By contrast, pathological or supraphysiological concentrations of C5a often triggered Ca2+ bursts, but pseudopod protrusion stalled or reversed in such cases, effectively halting chemotaxis, similar to sepsis-associated neutrophil paralysis. The maximum increase in cell surface area during pseudopod extension in pure chemotaxis was much smaller-by a factor of 8-than the known capacity of adherent human neutrophils to expand their surface. Because the measured rise in cortical tension was not sufficient to account for this difference, we attribute the limited deformability to a reduced ability of the cytoskeleton to generate protrusive force in the absence of cell adhesion. Thus, we hypothesize that Ca2+ bursts in neutrophils control a mechanistic switch between two distinct modes of cytoskeletal organization and dynamics. A key element of this switch appears to be the expedient coordination of adhesion-dependent lock or release events of cytoskeletal membrane anchors.

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Our prior study indicates a close relationship between alternative complement pathway activation, galactose-deficient IgA1 (Gd-IgA1) concentration and clinical severity of IgA nephropathy (IgAN). Nonetheless, the relationship between complement factors and the updated Oxford classification of IgAN remains unclear. This study enrolled eighty-four previously untreated, biopsy-diagnosed IgAN patients. The clinical and laboratory findings were collected at the time of biopsy. Plasma levels of complement factor C5a, factor Ba and Gd-IgA1 were measured and analyzed. It was found that the levels of proteinuria positively correlated with the updated Oxford classification of mesangial hypercellularity (M), endocapillary hypercellularity (E), tubular atrophy/interstitial fibrosis (T) and crescents (C). In addition, plasma Gd-IgA1 titer was significantly elevated in IgAN patients with tubular atrophy/interstitial fibrosis (T). In separate multivariable logistic regression models, both Gd-IgA1 and factor Ba independently predict higher T scores. The results indicate that both the levels of Gd-IgA1 antibody and biomarkers of the alternative complement pathway activation reflect the Oxford classification of IgAN. Whether these biomarkers can be used to guide therapeutic decisions requires further study. [ABSTRACT FROM AUTHOR]

Recent studies have implicated the complement system in the pathogenesis of antineutrophil cytoplasmic antibody-associated vasculitis (AAV), prompting the development of novel therapeutic agents to target the complement system accordingly. The pivotal role of complement component C5a, in particular, has been the subject of a number of phase II and phase III clinical trials. Indeed, the US Food and Drug Administration has already approved avacopan, an oral C5a receptor inhibitor, as an adjunct for the treatment of active severe AAV, based on its favourable safety profile and non-inferiority to glucocorticoids (GCs) (at Week 26). The novel monoclonal anti-C5a antibody vilobelimab has also been studied in a phase II trial in patients with AAV (IXchange; ClinicalTrials.gov Identifier: NCT03895801). The results appear promising; in addition to decreased GC toxicity index, a smaller number of treatment-emergent adverse events have been observed in vilobelimab-treated patients. However, the study was not powered statistically to compare the efficacy of vilobelimab to standard GC treatment. This editorial summarizes the study findings and outlines potential future directions. [ABSTRACT FROM AUTHOR]

BackgroundAntibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling.MethodsPrimary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003).ResultsTreatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement.ConclusionsDespite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR.

The efficient recruitment of immune cells is a vital cornerstone of our defense against infections and a key challenge of immunotherapeutic applications. It relies on the ability of chemotaxing cells to prioritize their responses to different stimuli. For example, immune cells are known to abandon gradients of host-cell-produced cytokines in favor of complement-derived anaphylatoxins, which then guide the cells toward nearby pathogen surfaces. The aptitude to triage stimuli depends on the cells' specific sensitivities to different chemoattractants. We here use human neutrophils as uniquely capable biodetectors to map out the anaphylatoxic cloud that surrounds microbes in the presence of host serum. We quantify the neutrophil sensitivity in terms of the ratio between the chemoattractant concentration c and the production rate j0 of the chemoattractant at the source surface. An integrative experimental/theoretical approach allows us to estimate the c/j0-threshold at which human neutrophils first detect nearby β-glucan surfaces as c/j0 ≈ 0.0044 s/μm.

C5aR1, the proinflammatory receptor for C5a, is expressed in the central nervous system on microglia, endothelial cells, and neurons. Previous work demonstrated that the C5aR1 antagonist, PMX205, decreased amyloid pathology and suppressed cognitive deficits in two Alzheimer's Disease (AD) mouse models. However, the cellular mechanisms of this protection have not been definitively demonstrated. Here, primary cultured mouse neurons treated with exogenous C5a show reproducible loss of MAP-2 staining in a dose-dependent manner within 24 hr of treatment, indicative of injury to neurons. This injury is prevented by the C5aR1 antagonist PMX53, a close analog of PMX205. Furthermore, primary neurons derived from C5aR1 null mice exhibited no MAP-2 loss after exposure to the highest concentration of C5a tested. Primary mouse neurons treated with both 100 nM C5a and 5 µM fibrillar amyloid beta (fAβ), to model what occurs in the AD brain, showed increased MAP-2 loss relative to either C5a or fAβ alone. Blocking C5aR1 with PMX53 (100 nM) blocked the loss of MAP2 in these primary neurons to the level seen with fAβ alone. Similar experiments with primary neurons derived from C5aR1 null mice showed a loss of MAP-2 due to fAβ treatment. However, the addition of C5a to the cultures did not enhance the loss of MAP-2 and the addition of PMX53 to the cultures did not change the MAP-2 loss in response to fAβ. Thus, at least part of the beneficial effects of C5aR1 antagonist in AD mouse models may be due to protection of neurons from the toxic effects of C5a.

Alzheimer's disease (AD) is characterized by abnormal inflammatory responses, and complement C5a (C5a) is known to initiate inflammation. This study aimed to investigate the associations between serum C5a, inflammatory responses, and cognitive function in AD patients. A total of 242 AD patients and 132 age-matched controls were included. Enzyme-linked immunosorbent assay revealed increased levels of C5a, interleukin (IL)-4, IL-6, IL-10, IL-1β, and tumor necrosis factor (TNF)-α with advancing stages of AD. Pearson correlation coefficient and receiver operating characteristic curve revealed positive correlations between serum C5a levels, inflammatory cytokine levels, Neuropsychiatric Inventory (NPI) and Activities of Daily Living (ADL) scores, and negative correlations with Mini-mental State Examination (MMSE) and Montreal cognitive assessment (MoCA) scores. Serum C5a above 68.68 pg/mL could aid in the diagnosis of AD. Multivariable logistic analysis revealed that serum C5a was an independent risk factor for IL-1β/IL-6/IL-10/TNF-α and an independent protective factor for IL-4. Higher serum C5a levels were associated with lower MMSE and MoCA scores. In conclusion, elevated serum C5a levels were beneficial for AD diagnosis and predictive of inflammation and cognitive dysfunction. [ABSTRACT FROM AUTHOR]

Complement activation and Rab GTPase trafficking are commonly observed in inflammatory responses. Recruitment of innate immune cells to sites of infection or injury and secretion of inflammatory chemokines are promoted by complement component 5a (C5a) that activates the cell surface protein C5a receptor1 (C5aR1). Persistent activation can lead to a myriad of inflammatory and autoimmune diseases. Here, we demonstrate that the mechanism of C5a induced chemotaxis of human monocyte-derived macrophages (HMDMs) and their secretion of inflammatory chemokines are controlled by Rab5a. We find that C5a activation of the G protein coupled receptor C5aR1 expressed on the surface of HMDMs, recruits β-arrestin2 via Rab5a trafficking, then activates downstream phosphatidylinositol 3-kinase (PI3K)/Akt signaling that culminates in chemotaxis and secretion of pro-inflammatory chemokines from HMDMs. High-resolution lattice light-sheet microscopy on live cells showed that C5a activates C5aR1-GFP internalization and colocalization with Rab5a-tdTomato but not with dominant negative mutant Rab5a-S34N-tdTomato in HEK293 cells. We found that Rab5a is significantly upregulated in differentiated HMDMs and internalization of C5aR1 is dependent on Rab5a. Interestingly, while knockdown of Rab5a inhibited C5aR1-mediated Akt phosphorylation, it did not affect C5aR1-mediated ERK1/2 phosphorylation or intracellular calcium mobilization in HMDMs. Functional analysis using transwell migration and µ-slide chemotaxis assays indicated that Rab5a regulates C5a-induced chemotaxis of HMDMs. Further, C5aR1 was found to mediate interaction of Rab5a with β-arrestin2 but not with G proteins in HMDMs. Furthermore, C5a-induced secretion of pro-inflammatory chemokines (CCL2, CCL3) from HMDMs was attenuated by Rab5a or β-arrestin2 knockdown or by pharmacological inhibition with a C5aR1 antagonist or a PI3K inhibitor. These findings reveal a C5a-C5aR1-β-arrestin2-Rab5a-PI3K signaling pathway that regulates chemotaxis and pro-inflammatory chemokine secretion in HMDMs and suggests new ways of selectively modulating C5a-induced inflammatory outputs.

Background: Thrombotic events cannot be completely prevented by antithrombotics, implicating a therapeutic gap due to inflammation, a not yet sufficiently addressed mechanism. Neutrophil extracellular traps (NETs) are an essential interface between inflammation and thrombosis, but exactly how the NETotic process is initiated and maintained during arterial thrombosis remains incompletely understood. Methods and results: We found that the plasma concentrations of C5a were higher in patients with ST-elevation myocardial infarction (STEMI) than in patients with angina and higher in mice with left common carotid artery (LCCA) thrombosis induced by FeCl3 than in control mice. We observed that the thrombus area and weight were decreased and that NET formation in the thrombi was reduced in the group treated with the selective C5aR1 receptor inhibitor PMX53 compared with the NaCl group. In vitro, NETosis was observed when C5a was added to neutrophil cultures, and this effect was reversed by PMX53. In addition, our data showed that C5a increased the production of mitochondrial reactive oxygen species (ROS) and that the promotion of NET formation by C5a was mitochondrial ROS (Mito-ROS) dependent. Furthermore, we found that C5a induced the production of Mito-ROS by inhibiting mitochondrial STAT3 activity. Conclusions: By inhibiting mitochondrial STAT3 to elicit Mito-ROS generation, C5a triggers the generation of NETs to promote the development of arterial thrombosis. Hence, our study identifies complement C5a as a potential new target for the treatment and prevention of thrombosis. [ABSTRACT FROM AUTHOR]

The loss of retinal pericytes (RPCs) is a hallmark of early stage diabetic retinopathy (DR), but the mechanism underlying RPC death is unclear. Although it was postulated in previous studies using bovine RPCs that autoantibodies against RPCs might develop and induce RPC death, it is unknown whether autoantibodies against cell-surface antigens on human RPCs exist in DR patients, whether such autoantibodies contribute to RPC damage/loss, and if they do, through which mechanism. We screened serum samples from DR patients and controls using primary human RPCs and found that that levels of IgGs reactive to RPCs were significantly higher in the DR group than the control group. Serum samples with higher RPC-reactive IgG levels induced more severe complement-mediated RPC damage than those with lower RPC-reactive IgG levels. We also assessed levels of the complement-activation products C3a, C4a and C5a in these serum samples, and found that serum levels of C3a and C5a, but not C4a, were higher in the DR group than control group. These data provide evidence the first time showing that autoantibodies against RPCs can develop in DR patients, and that these autoantibodies could contribute to pericyte damage through complement activation.

The role of the complement system in schizophrenia (Sz) is inconclusive due to heterogeneity of the disease and study designs. Here, we assessed the levels of complement activation products and functionality of the classical pathway in acutely ill unmedicated Sz patients at baseline and after 6 weeks of treatment versus matched controls. The study included analyses of the terminal complement complex (sTCC) and C5a in plasma from 96 patients and 96 controls by enzyme-linked immunosorbent assay. Sub-group analysis of serum was conducted for measurement of C4 component and activity of the classical pathway (28 and 24 cases per cohort, respectively). We found no differences in levels of C5a, C4 and classical pathway function in patients versus controls. Plasma sTCC was significantly higher in patients [486 (392-659) ng/mL, n = 96] compared to controls [389 (304-612) ng/mL, n = 96] (p = 0.027, δ = 0.185), but not associated with clinical symptom ratings or treatment. The differences in sTCC between Sz and controls were confirmed using an Aligned Rank Transformation model considering the covariates age and sex (p = 0.040). Additional analysis showed that sTCC was significantly associated with C-reactive protein (CRP; p = 0.006). These findings suggest that sTCC plays a role in Sz as a trait marker of non-specific chronic immune activation, as previously described for CRP. Future longitudinal analyses with more sampling time points from early recognition centres for psychoses may be helpful to better understand the temporal dynamics of innate immune system changes during psychosis development., (© 2024. The Author(s).)

The complement component C5a contributes to the recruitment of immune cells to inflamed tissues and local inflammation. The proinflammatory cytokine interleukin (IL)-1β is also related to inflammatory disorders through inflammasome activation. However, the association between inflammasome activation and C5a is unclear. Human peripheral blood mononuclear cells (PBMCs) were stimulated with C5a and measured for IL-1β secretion by enzyme-linked immunosorbent assay (ELISA). The pro-IL-1β expression in cell lysates was also examined by Western blot analysis. Similarly, magnetic bead-isolated CD14 + monocyte-depleted and lymphocyte-depleted PBMCs were stimulated with C5a, and immunoblot analysis was performed using an anti-cleaved-IL-1β (p17) antibody. FACS was performed to detect caspase-1-activated cells. C5a-stimulated PBMCs produced IL-1β in C5a concentration-dependent manner. The protein levels of pro-IL-1β in the cell lysates were significantly increased. Furthermore, the cleaved-IL-1β (p17) was faintly detected in the same lysates. Active caspase-1 was demonstrated in C5a-simulated CD14 + monocytes by FACS. Cleaved-IL-1β (p17) was demonstrated in the supernatant of C5a-stimulated PBMCs. Lymphocyte-depleted PBMCs stimulated with C5a but monocyte-depleted PBMCs produced cleaved-IL-1β (p17). C5a induced the production of mature IL-1β in PBMCs. The IL-1β production is mediated mainly by caspase-1 activation in CD14 + monocytes. These results suggest that C5a alone potentiates mature IL-1β production mainly in monocytes.

Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a disseminated infection, while the closely related pathogen S. enterica serovar Typhimurium (S. Typhimurium) is associated with a localized gastroenteritis in humans. Here we investigated whether both pathogens differ in the chemotactic response they induce in neutrophils using a single-cell experimental approach. Surprisingly, neutrophils extended chemotactic pseudopodia toward Escherichia coli and S. Typhimurium, but not toward S. Typhi. Bacterial-guided chemotaxis was dependent on the presence of complement component 5a (C5a) and C5a receptor (C5aR). Deletion of S. Typhi capsule biosynthesis genes markedly enhanced the chemotactic response of neutrophils in vitro. Furthermore, deletion of capsule biosynthesis genes heightened the association of S. Typhi with neutrophils in vivo through a C5aR-dependent mechanism. Collectively, these data suggest that expression of the virulence-associated (Vi) capsular polysaccharide of S. Typhi obstructs bacterial-guided neutrophil chemotaxis.

Clinical trials and observational studies have established cyclophosphamide (CY) or rituximab plus glucocorticoid (GC) as standard remission induction therapies in patients with microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA). However, because these regimens are associated with serious adverse drug reactions, the development of drugs with novel mechanisms of actions are needed. Progress in basic and clinical research has identified novel candidate targeting molecules, including B-cell activating factor (BAF), C5a receptor, and interleukin-6. The combination of rituximab and BAF blockade in patients with MPA and GPA is under investigation in an effort to strike a better benefit-risk balance. Phase II clinical trials of avacopan (CCX168), an orally administered C5a receptor antagonist, have suggested a reduction in the dosage of concomitant GC or the replacement of GC in patients with MPA and GPA. The results from a currently ongoing phase III trial are awaited. Anecdotal case reports and an open-label pilot study have indicated the effectiveness of tocilizumab in patients with MPA and GPA. A randomized clinical trial comparing tocilizumab and intravenous CY in combination with GC is currently in progress. Molecular targeted therapy is expected to transform the treatment strategy for MPA and GPA to allow GC-free or at least less GC-dependent forms of therapy.

Summary: The complement fragment C5a is closely associated with adaptive immune induction in the mucosa. However, the mechanisms that control CD8+ T cell responses by C5a have not been extensively explored. This study reveals that C5/C5a in the Peyer’s patch (PP) subepithelial dome increases upon oral Listeria infection. We hypothesize that C5aR+ PP cells play an important role in the induction of antigen-specific T cell immunity. Using single-cell RNA sequencing, we identify C5aR- and lysozyme-expressing dendritic cells (C5aR+ LysoDCs) in PP and examine their role in CD8+ T cell immune induction. Stimulation of C5aR+ LysoDCs by C5a increases reactive oxygen species levels, leading to efficient antigen cross-presentation, which elicits an antigen-specific CD8+ T cell response. In C5-deficient mice, oral co-administration of C5a and Listeria enhances Listeria-specific cytotoxic T cell levels. Collectively, these findings suggest a role of the complement system in intestinal T cell immunity.

Both, aberrant mast cell responses and complement activation contribute to allergic diseases. Since mast cells are highly responsive to C3a and C5a, while Interleukin-33 (IL-33) is a potent mast cell activator, we hypothesized that IL-33 critically regulates mast cell responses to complement anaphylatoxins. We sought to understand whether C3a and C5a differentially activate primary human mast cells, and probe whether IL-33 regulates C3a/C5a-induced mast cell activities. Primary human mast cells were generated from peripheral blood precursors or isolated from healthy human lung tissue, and mast cell complement receptor expression, degranulation, mediator release, phosphorylation patterns, and calcium flux were assessed. Human mast cells of distinct origin express constitutively higher levels of C3aR1 than C5aR1, and both receptors are downregulated by anaphylatoxins. While C3a is a potent mast cell degranulation inducer, C5a is a weaker secretagogue with more delayed effects. Importantly, IL-33 potently enhances the human mast cell reactivity to C3a and C5a (degranulation, cytokine and chemokine release), independent of changes in C3a or C5a receptor expression or the level of Ca2+ influx. Instead, this reflects differential dynamics of intracellular signaling such as ERK1/2 phosphorylation. Since primary human mast cells respond differentially to anaphylatoxin stimulation, and that IL-33 is a key regulator of mast cell responses to complement anaphylatoxins, this is likely to aggravate Th2 immune responses. This newly identified cross-regulation may be important for controlling exacerbated complement- and mast cell-dependent Th2 responses and thus provides an additional rationale for targeting anti-IL33 therapeutically in allergic diseases.

Both, aberrant mast cell responses and complement activation contribute to allergic diseases. Since mast cells are highly responsive to C3a and C5a, while Interleukin-33 (IL-33) is a potent mast cell activator, we hypothesized that IL-33 critically regulates mast cell responses to complement anaphylatoxins. We sought to understand whether C3a and C5a differentially activate primary human mast cells, and probe whether IL-33 regulates C3a/C5a-induced mast cell activities. Primary human mast cells were generated from peripheral blood precursors or isolated from healthy human lung tissue, and mast cell complement receptor expression, degranulation, mediator release, phosphorylation patterns, and calcium flux were assessed. Human mast cells of distinct origin express constitutively higher levels of C3aR1 than C5aR1, and both receptors are downregulated by anaphylatoxins. While C3a is a potent mast cell degranulation inducer, C5a is a weaker secretagogue with more delayed effects. Importantly, IL-33 potently enhances the human mast cell reactivity to C3a and C5a (degranulation, cytokine and chemokine release), independent of changes in C3a or C5a receptor expression or the level of Ca2+ influx. Instead, this reflects differential dynamics of intracellular signaling such as ERK1/2 phosphorylation. Since primary human mast cells respond differentially to anaphylatoxin stimulation, and that IL-33 is a key regulator of mast cell responses to complement anaphylatoxins, this is likely to aggravate Th2 immune responses. This newly identified cross-regulation may be important for controlling exacerbated complement- and mast cell-dependent Th2 responses and thus provides an additional rationale for targeting anti-IL33 therapeutically in allergic diseases. [ABSTRACT FROM AUTHOR]

Objective To observe the changes of serum complement C5a and nucleotide binding oligomerization domain-like receptor protein 3（NLRP3) levels in patients with Alzheimer's disease（AD) and to explore whether they are involved in inflammatory response and cognitive impairment. Methods Totally 198 patients with AD（the observation group）and 82 healthy persons who took physical examination during the same period（the control group）were selected. Serum levels of complement C5a, NLRP3, tumor necrosis factor-α（TNF-α), interleukin-6（IL-6), and IL-1β were measured by enzyme-linked immunosorbent assay, and serum high-sensitivity C-reactive protein（hs-CRP) was measured by immunoturbidimetry. The Mini Mental State Scale（MMSE）was used to evaluate cognitive function and the levels of serum indexes and MMSE scores between the two groups were compared. Results Compared with the control group，the serum levels of complement C5a，NLRP3, TNF- α, IL-6, IL-1β and hs-CRP in the observation group were higher，and the MMSE score was lower in the observation group (all P<0. 05). Pearson correlation analysis was used to analyze the correla⁃ tion between serum complement C5a，NLRP levels and TNF-α, IL-6, IL-1β, hs-CRP levels and MMSE score. The lev⁃ els of serum complement C5a，NLRP3，TNF-α, IL-6，IL-1β and hs-CRP were negatively correlated with MMSE score (r=-0. 420, -0. 418, -0. 506, -0. 444, -0. 435, -0. 524，all P<0. 05）. Serum complement C5a and NLRP levels were positively correlated with TNF- α，IL-6, IL-1β and hs-CRP levels（r=0. 538，0. 640, 0. 601, 0. 563, 0. 512, 0. 587, 0. 573, 0. 452, all P<0. 05). Conclusion The levels of serum complement C5a and NLRP3 increase in AD patients, and both of them may be involved in inflammation and cognitive impairment. [ABSTRACT FROM AUTHOR]

Complement plays a critical role in the immune response toward nanomaterials. The complement attack on a foreign surface results in the deposition of C3, assembly of C3 convertases, the release of anaphylatoxins C3a and C5a, and finally, the formation of membrane attack complex C5b-9. Various technologies can measure complement activation markers in the fluid phase, but measurements of surface C3 deposition are less common. Previously, we developed an ultracentrifugation-based dot blot immunoassay (DBI) to measure the deposition of C3 and other protein corona components on nanoparticles. Here, we validate the repeatability of the DBI and its correlation with pathway-specific and common fluid phase markers. Moreover, we discuss the advantages of DBI, such as cost-effectiveness and versatility, while addressing potential limitations. This study provides insights into complement activation at the nanosurface level, offering a valuable tool for nanomedicine researchers in the field., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Solid organ transplantation is a cost-effective treatment for end-stage organ failure. Organ donation after brain death is an important source of transplanted organs. Data are limited on the effects of brain injury or donor management on grafts. The consensus view has been that brain death creates a progressively proinflammatory environment. We aimed to investigate time-course changes across a range of cytokines in a donation after brain death cohort of donors who died of intracranial hemorrhage without any other systemic source of inflammation., Methods: A donor cohort was defined using the UK Quality in Organ Donation biobank. Serum levels of proteins involved in proinflammatory and brain injury pathways (tumor necrosis factor-alpha, interleukin-6, complement C5a, neuron-specific enolase, and glial fibrillary acidic protein) were measured from admission to organ recovery. Moving median analysis was used to combine donor trajectories and delineate a time-course., Results: A cohort of 27 donors with brain death duration between 10 and 30 h was created, with 24 donors contributing to the time-course analysis. We observed no increase in tumor necrosis factor-alpha or interleukin-6 throughout the donor management period. Neuronal injury marker and complement C5a remain high from admission to organ recovery, whereas glial fibrillary acidic protein rises around the confirmation of brain death., Conclusions: We found no evidence of a progressive rise of proinflammatory mediators with prolonged duration of brain death, questioning the hypothesis of a progressively proinflammatory environment. Furthermore, the proposed approach allows us to study chronological changes and identify biomarkers or target pathways when logistical or ethical considerations limit sample availability., Competing Interests: R.J.P. is the Principal Investigator for the Quality in Organ Donation programme. K.D.B. was supported by the National Institute for Health Research with an National Institute for Health Research Academic Clinical Fellowship. K.D.B. received a pump priming research grant from the Oxford Transplant Foundation to support this project (R56956/AA001). The other authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

In Huntington disease (HD), immune cells are activated before symptoms arise; however, it is unclear how the expression of mutant huntingtin (htt) compromises the normal functions of immune cells. Here we report that primary microglia from early postnatal HD mice were profoundly impaired in their migration to chemotactic stimuli, and expression of a mutant htt fragment in microglial cell lines was sufficient to reproduce these deficits. Microglia expressing mutant htt had a retarded response to a laser-induced brain injury in vivo. Leukocyte recruitment was defective upon induction of peritonitis in HD mice at early disease stages and was normalized upon genetic deletion of mutant htt in immune cells. Migration was also strongly impaired in peripheral immune cells from pre-manifest human HD patients. Defective actin remodeling in immune cells expressing mutant htt likely contributed to their migration deficit. Our results suggest that these functional changes may contribute to immune dysfunction and neurodegeneration in HD, and may have implications for other polyglutamine expansion diseases in which mutant proteins are ubiquitously expressed.

Complement factor C5a is an integral constituent of the complement cascade critically involved in the innate immune response, and it exerts its functions via two distinct receptors, C5aR1 and C5aR2. While C5aR1 is a prototypical G-protein-coupled receptor (GPCR), C5aR2 lacks functional coupling to heterotrimeric G proteins, although both receptors efficiently recruit β arrestins (βarrs). Here, we discuss the recent studies providing direct structural details of ligand–receptor interactions, and a framework of functional bias in this system, including the differences in terms of structural motifs and transducer coupling. We also discuss the functional analogy of C5aR2 with the atypical chemokine receptors (ACKRs), and highlight the future directions to elucidate the mechanistic basis of the functional divergence of these receptors activated by a common natural agonist. The complement cascade is a critical part of our innate immune system that plays a crucial role in combating pathogenic infections. Complement C5a, a potent anaphylatoxin in the complement cascade, activates two different seven transmembrane receptors, namely C5aR1 and C5aR2. C5aR1 is a prototypical GPCR, while C5aR2, which exhibits functional analogy to ACKRs, does not exhibit any functional coupling to G proteins but engages βarrs upon activation. Crystal structures of C5aR1 in complex with antagonists have provided important insights into ligand–receptor interaction and a potential template for novel ligand discovery. Ligand-specific functional bias at C5aR1 has been discovered recently that offers a potential framework for better therapeutic intervention. [ABSTRACT FROM AUTHOR]

Objectives: When bone filling materials are applied onto the periodontal tissues in vivo, they interact with the injured periodontal ligament (PDL) tissue and modulate its activity. This may lead to mesenchymal stem cells (MSCs) recruitment from bone marrow and initiate bone regeneration. Our hypothesis is that the filling materials affect PDL cells and MSCs functional activities by modulating PDL C5a secretion and subsequent MSCs proliferation and recruitment. Materials and methods: Materials' extracts were prepared from 3 bone-grafting materials: Gen-Os® of equine and porcine origins and bovine Bio-Oss®. Expression and secretion of C5a protein by injured PDL cells were investigated by RT-PCR and ELISA. MSCs proliferation was analyzed by MTT assay. C5a binding to MSCs C5aR and its phosphorylation was studied by ELISA. C5a implication in MSCs recruitment toward injured PDL cells was investigated using Boyden chambers. Results: MSCs proliferation significantly increased with Gen-Os® materials but significantly decreased with Bio-Oss®. C5a secretion slightly increased with Bio-Oss® while its level doubled with Gen-Os® materials. C5a fixation on MSCs C5aR and its phosphorylation significantly increased with Gen-Os® materials but not with Bio-Oss®. MSCs recruitment toward injured PDL cells increased with the three materials but was significantly higher with Gen-Os® materials than with Bio-Oss®. Adding C5a antagonist inhibited MSCs recruitment demonstrating a C5a-mediated migration. Conclusions: Injured PDL cells secrete C5a leading MSCs proliferation and recruitment to the PDL injured cells. Gen-Os® materials enhanced both C5a secretion by injured PDL cells and MSCs recruitment. Bio-Oss® inhibited MSCs and was less efficient than Gen-Os® materials in inducing MSCs recruitment. Clinical relevance: Within the limits of this study in vitro, Gen-Os® filling materials have a higher potential than Bio-Oss® on MSCs proliferation and C5a-dependent recruitment to the PDL injury site and the subsequent bone regeneration. [ABSTRACT FROM AUTHOR]

Macrophage cells that are stimulated by two different ligands that bind to G-protein-coupled receptors (GPCRs) usually respond as if the stimulus effects are additive, but for a minority of ligand combinations the response is synergistic. The G-protein-coupled receptor system integrates signaling cues from the environment to actuate cell morphology, gene expression, ion homeostasis, and other physiological states. We analyze the effects of the two signaling molecules complement factors 5a (C5a) and uridine diphosphate (UDP) on the intracellular second messenger calcium to elucidate the principles that govern the processing of multiple signals by GPCRs. We have developed a formal hypothesis, in the form of a kinetic model, for the mechanism of action of this GPCR signal transduction system using data obtained from RAW264.7 macrophage cells. Bayesian statistical methods are employed to represent uncertainty in both data and model parameters and formally tie the model to experimental data. When the model is also used as a tool in the design of experiments, it predicts a synergistic region in the calcium peak height dose response that results when cells are simultaneously stimulated by C5a and UDP. An analysis of the model reveals a potential mechanism for crosstalk between the Galphai-coupled C5a receptor and the Galphaq-coupled UDP receptor signaling systems that results in synergistic calcium release.

Immune responses rely on efficient and coordinated migration of immune cells to the site of infection or injury. To reach the site of immunological threat often requires long-range navigation of immune cells through complex tissue and vascular networks. Chemotaxis, cell migration steered by gradients of cell-attractive chemicals that bind sensory receptors, is central to this response. Chemoattractant receptors mostly belong to the G-protein-coupled receptor (GPCR) family, but the way attractant-receptor signaling directs cell migration is not fully understood. Direct-viewing chemotaxis chambers combined with time-lapse microscopy give a powerful tool to study the dynamic details of cells' responses to different attractant landscapes. Here, we describe the application of one such chamber (the Dunn chamber) to study bone marrow-derived macrophage chemotaxis to gradients of complement C5a., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Bacterial meningitis is most commonly caused by Streptococcus pneumoniae and Neisseria meningitidis and continues to pose a major public health threat. Morbidity and mortality of meningitis are driven by an uncontrolled host inflammatory response. This comprehensive update evaluates the role of the complement system in upregulating and maintaining the inflammatory response in bacterial meningitis. Genetic variation studies, complement level measurements in blood and CSF, and experimental work have together led to the identification of anaphylatoxin C5a as a promising treatment target in bacterial meningitis. In animals and patients with pneumococcal meningitis, the accumulation of neutrophils in the CSF was mainly driven by C5-derived chemotactic activity and correlated positively with disease severity and outcome. In murine pneumococcal meningitis, adjunctive treatment with C5 antibodies prevented brain damage and death. Several recently developed therapeutics target C5 conversion, C5a, or its receptor C5aR. Caution is warranted because treatment with C5 antibodies such as eculizumab also inhibits the formation of the membrane attack complex, which may result in decreased meningococcal killing and increased meningococcal disease susceptibility. The use of C5a or C5aR antagonists to specifically target the harmful anaphylatoxins-induced effects, therefore, are most promising and present opportunities for a phase 2 clinical trial. [ABSTRACT FROM AUTHOR]

The complement system significantly contributes to the development of inflammatory and neuropathic pain, but the underlying mechanisms are poorly understood. Recently, we identified the signaling pathway responsible for thermal hypersensitivity induced by the complement system component C5a. Here, we examine the mechanisms of another important action of C5a, induction of mechanical hypersensitivity. We found that intraplantar injection of C5a produced a dose-dependent mechanical sensitization and that this effect was blocked by chemogenetic ablation of macrophages in both male and female mice. Knockout of TRPV1 or pretreatment with the TRPV1 antagonists, AMG9810 or 5'-iodoresiniferatoxin (5'-IRTX), significantly reduced C5a-induced mechanical sensitization. Notably, local administration of 5'-IRTX 90 minutes after C5a injection resulted in a slow, but complete, reversal of mechanical sensitization, indicating that TRPV1 activity was required for maintaining C5a-induced mechanical hypersensitivity. This slow reversal suggests that neurogenic inflammation and neuropeptide release may be involved. Indeed, pretreatment with a calcitonin gene-related peptide (CGRP) receptor antagonist (but not an antagonist of the neurokinin 1 receptor) prevented C5a-induced mechanical sensitization. Furthermore, intraplantar injection of CGRP produced significant mechanical sensitization in both wild-type and TRPV1 knockout mice. Taken together, these findings suggest that C5a produces mechanical sensitization by initiating macrophage-to-sensory-neuron signaling cascade that involves activation of TRPV1 and CGRP receptor as critical steps in this process. [ABSTRACT FROM AUTHOR]

The complement system has demonstrated roles in regulating tumor growth, although these may differ between tumor types. The current study used two murine breast cancer models (EMT6 and 4T1) to investigate whether pharmacological targeting of receptors for complement proteins C3a (C3aR) and C5a (C5aR1) is protective in murine breast cancer models. In contrast to prior studies in other tumor models, treatment with the selective C5aR1 antagonist PMX53 had no effect on tumor growth. However, treatment of mice with a dual C3aR/C5aR1 agonist (YSFKPMPLaR) significantly slowed mammary tumor development and progression. Examination of receptor expression by quantitative polymerase chain reaction (qPCR) analysis showed very low levels of mRNA expression for either C3aR or C5aR1 by EMT6 or 4T1 mammary carcinoma cell lines compared with the J774 macrophage line or bone marrow-derived macrophages. Moreover, flow cytometric analysis found no evidence of C3aR or C5aR1 protein expression by either EMT6 or 4T1 cells, leading us to hypothesize that the tumor inhibitory effects of the dual agonist are indirect, possibly via regulation of the anti-tumor immune response. This hypothesis was supported by flow cytometric analysis of tumor infiltrating leukocyte populations, which demonstrated a significant increase in T lymphocytes in mice treated with the C3aR/C5aR1 agonist. These results support an immunoregulatory role for complement receptors in primary murine mammary carcinoma models. They also suggest that complement activation peptides can influence the anti-tumor response in different ways depending on the cancer type, the host immune response to the tumor and levels of endogenous complement activation within the tumor microenvironment.

Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Drs. Lim, Prof. Dr. Vlaar, Prof. Dr. Brouwer and Prof. Dr. Van de Beek are study investigators of the PANAMO 2 and/or 3 study. Prof. Dr. Vlaar reports consulting fees from InflaRx paid to Amsterdam UMC, outside the submitted work. All other authors declare no competing interests.

Endometriosis (EM) is a gynecological disorder that presents significant immune dysregulation in its pathophysiology. Recent studies indicate that the Complement System may play a significant role in the immune processes involved in peritoneal clearance and inflammation in EM patients. C5a is a potent anaphylatoxin molecule of complement associated with the development of inflammatory disorders, however its possible impact on EM development requires further investigation. The aim of this study was to determine the concentration of serum C5a in women with EM and to investigate its possible association with severity, symptoms, age and the timing of infertility. Ninety-four patients with EM (from stage I to IV) and 50 healthy controls were assessed for C5a serum levels. Clinical and demographic data were included in the analysis. C5a serum levels were higher in patients with EM than in controls (39.5 ng/mL vs. 26.0 ng/mL; p  < .0001), but not different between the EM stages. No association was observed between C5a serum concentration and the presence of symptoms, age, symptom time or infertility time. The C5a serum levels were higher in patients with EM than in controls but not associated with the severity or clinical findings.

Hepatocellular carcinoma (HCC) is highly refractory. β-Sitosterol has been reported to suppress proliferation and migration as well as interfere with cell metabolism in tumors. However, there is limited information on the effects of β-sitosterol on HCC. Herein, we used a xenograft mouse model to investigate the effects of β-sitosterol on HCC tumor growth. The molecular mechanism was elucidated using quantitative real-time PCR, western blotting, lentiviral transfection, CCK8, scratch, Transwell, and Ad-mCherry-GFP-LC3B assays. The results showed that HepG2 cells highly expressed complement C5a receptor 1. β-Sitosterol antagonized complement component 5a and exerted inhibitory effects on the proliferation and migration of HepG2 cells. The inhibitory effect of β-sitosterol was reversed by the knockdown of complement C5a receptor 1. Bioinformatics analysis suggested alpha fetoprotein (AFP) as a downstream factor of complement C5a receptor 1. β-Sitosterol inhibited AFP expression, which was reversed by complement C5a receptor 1 knockdown. The inhibitory effects of β-sitosterol on cell proliferation and migration were weakened by AFP overexpression. Furthermore, β-sitosterol induced autophagy in HepG2 cells, which was reversed by complement C5a receptor 1 knockdown and AFP overexpression. Blockade of autophagy by 3-MA attenuated β-sitosterol inhibition of proliferation and migration in HepG2 cells. Moreover, β-sitosterol inhibited HCC progression in vivo. Our findings demonstrate that β-sitosterol inhibits HCC advancement by activating autophagy through the complement C5a receptor 1/AFP axis. These findings recommend β-sitosterol as a promising therapy for HCC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Allergic conditions are associated with canonical and noncanonical activation of the complement system leading to the release of several bioactive mediators with inflammatory and immunoregulatory properties that regulate the immune response in response to allergens during the sensitization and/or the effector phase of allergic diseases. Further, immune sensors of complement and regulator proteins of the cascade impact on the development of allergies. These bioactive mediators comprise the small and large cleavage fragments of C3 and C5. Here, we provide an update on the multiple roles of immune sensors, regulators, and bioactive mediators of complement in allergic airway diseases, food allergies, and anaphylaxis. A particular emphasis is on the anaphylatoxins C3a and C5a and their receptors, which are expressed on many of the effector cells in allergy such as mast cells, eosinophils, basophils, macrophages, and neutrophils. Also, we will discuss the multiple pathways, by which the anaphylatoxins initiate and control the development of maladaptive type 2 immunity including their impact on innate lymphoid cell recruitment and activation. Finally, we briefly comment on the potential to therapeutically target the complement system in different allergic conditions., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

In recent years, there has been increased accessibility to cannabis for recreational and medicinal use. Incidentally, there has been an increase in reports describing allergic reactions to cannabis including exacerbation of underlying asthma. Recently, multiple protein allergens were discovered in cannabis, yet these fail to explain allergic sensitization in many patients, particularly urticaria and angioedema. Cannabis has a rich chemical profile including cannabinoids and terpenes that possess immunomodulatory potential. We examined whether major cannabinoids of cannabis such as cannabidiol (CBD) and the bicyclic sesquiterpene beta-caryophyllene (β-CP) act as contact sensitizers. The repeated topical application of mice skin with β-CP at 10 mg/mL (50 µL) induced an itch response and dermatitis at 2 weeks in mice, which were sustained for the period of study. Histopathological analysis of skin tissues revealed significant edema and desquamation for β-CP at 10 mg/mL. For CBD and β-CP, we observed a dose-dependent increase in epidermal thickening with profound thickening observed for β-CP at 10 mg/mL. Significant trafficking of CD11b cells was observed in various compartments of the skin in response to treatment with β-CP in a concentration-dependent manner. Mast cell trafficking was restricted to β-CP (10 mg/mL). Mouse proteome profiler cytokine/chemokine array revealed upregulation of complement C5/5a (anaphylatoxin), soluble intracellular adhesion molecule-1 (sICAM-1) and IL-1 receptor antagonist (IL-1RA) in animals dosed with β-CP (10 mg/mL). Moreover, we observed a dose-dependent increase in serum IgE in animals dosed with β-CP. Treatment with β-CP (10 mg/mL) significantly reduced filaggrin expression, an indicator of barrier disruption. In contrast, treatment with CBD at all concentrations failed to evoke scratching and dermatitis in mice and did not result in increased serum IgE. Further, skin tissues were devoid of any remarkable features, although at 10 mg/mL CBD we did observe the accumulation of dermal CD11b cells in skin tissue sections. We also observed increased filaggrin staining in mice repeatedly dosed with CBD (10 mg/mL). Collectively, our studies indicate that repeated exposure to high concentrations of β-CP can induce dermatitis-like pathological outcomes in mice.