This study investigated the role of the alternative receptor for complement activation fragment C5a, C5aR2, in secondary inflammatory pathology after contusive spinal cord injury (SCI) in mice. C5ar2-/- mice exhibited decreased intraparenchymal tumor necrosis factor alpha and interleukin-6 acutely post-injury, but these reductions did not translate into improved outcomes. We show that loss of C5aR2 leads to increased lesion volumes, reduced myelin sparing, and significantly worsened recovery from SCI in C5ar2-/- animals compared to wild-type (WT) controls. Loss of C5aR2 did not alter leukocyte mobilization from the bone marrow in response to SCI, and neutrophil recruitment/presence at the lesion site was also not different between genotypes. Acute treatment of SCI mice with the selective C5aR1 antagonist, PMX205, improved SCI outcomes, compared to vehicle controls, and, importantly, fully alleviated the worsened recovery of C5ar2-/- mice compared to their WT counterparts. Collectively, these findings indicate that C5aR2 is neuroprotective and a novel target to restrain injurious C5a signaling after a major neurotraumatic event.

Clinical studies have identified a cytokine storm in the third stage of disease progression in critical ill patients with coronavirus disease 2019 (COVID-19). Hence, effectively suppressing the uncontrolled immune response of the host towards the invaded viruses in a cytokine storm is a critical step to prevent the deterioration of patient conditions and decrease the rate of mortality. Therapeutic monoclonal antibodies (mAbs) are found to be effective for the management of acute respiratory distress syndrome in patients with COVID-19. In this review, we compiled all therapeutic mAbs targeting cytokine storm, which are in clinical trials for its repurposing in the management of COVID-19. Compilation of clinical trial data indicated that therapeutic monoclonal antibodies targeting interleukins (IL-6, IL-1ra, IL-8, IL-1β, IL-17A, IL-33), interferon-gamma, tumor necrosis factor-alpha, P-selectin, connective tissue growth factor<b>,</b> plasma kallikrein, tumor necrosis factor superfamily 14, granulocyte macrophage colony stimulating factor, colony stimulating factor 1 receptor, C-C chemokine receptor type 5, cluster of differentiation 14 and 147, vascular endothelial growth factor, programmed cell death protein-1, Angiopoietin - 2, human factor XIIa, <em>complement</em>ary protein 5, natural killer cell receptor G2A, human epidermal growth factor receptor 2<b>,</b> immunoglobulin-like transcript 7 receptor, <em>complement</em> <em>component</em> fragment <em>5a</em> receptor and viral attachment to the human cell were under investigation for management of severely ill patients with COVID-19. Among these, about 65 clinical trials are targeting IL-6 inhibition as the most promising one and Tocilizumab, an IL-6 inhibitor is considered to be the potential candidate to treat cytokine storm associated with the COVID-19.

Keywords: ARDS; COVID-19; Cytokine Storm; SARS-CoV-2; therapeutic monoclonal antibody.

Background: During atherogenesis, cholesterol precipitates into cholesterol crystals (CC) in the vessel wall, which trigger plaque inflammation by activating the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome. We investigated the relationship between CC, complement and NLRP3 in patients with cardiovascular disease.

Methods: We analysed plasma, peripheral blood mononuclear cells (PBMC) and carotid plaques from patients with advanced atherosclerosis applying ELISAs, multiplex cytokine assay, qPCR, immunohistochemistry, and gene profiling.

Findings: Transcripts of interleukin (IL)-1beta(β) and NLRP3 were increased and correlated in PBMC from patients with acute coronary syndrome (ACS). Priming of these cells with complement factor 5a (C5a) and tumour necrosis factor (TNF) before incubation with CC resulted in increased IL-1β protein when compared to healthy controls. As opposed to healthy controls, systemic complement was significantly increased in patients with stable angina pectoris or ACS. In carotid plaques, complement C1q and C5b-9 complex accumulated around CC-clefts, and complement receptors C5aR1, C5aR2 and C3aR1 were higher in carotid plaques compared to control arteries. Priming human carotid plaques with C5a followed by CC incubation resulted in pronounced release of IL-1β, IL-18 and IL-1α. Additionally, mRNA profiling demonstrated that C5a and TNF priming followed by CC incubation upregulated plaque expression of NLRP3 inflammasome components.

Interpretation: We demonstrate that CC are important local- and systemic complement activators, and we reveal that the interaction between CC and complement could exert its effect by activating the NLRP3 inflammasome, thus promoting the progression of atherosclerosis.

Keywords: Atherosclerosis; Carotid atherosclerosis; Cholesterol crystals; Complement system; Coronary artery disease; Inflammation; NLRP3 inflammasome.

Background: Mortality rates are high among hospitalized patients with COVID-19, especially in those intubated on the ICU. Insight in pathways associated with unfavourable outcome may lead to new treatment strategies.

Methods: We performed a prospective cohort study of patients with COVID-19 admitted to general ward or ICU who underwent serial blood sampling. To provide insight in the pathways involved in disease progression, associations were estimated between outcome risk and serial measurements of 64 biomarkers in potential important pathways of COVID-19 infection (inflammation, tissue damage, complement system, coagulation and fibrinolysis) using joint models combining Cox regression and linear mixed-effects models. For patients admitted to the general ward, the primary outcome was admission to the ICU or mortality (unfavourable outcome). For patients admitted to the ICU, the primary outcome was 12-week mortality.

<strong class="sub-title"> Findings: </strong> A total of 219 patients were included: 136 (62%) on the ward and 119 patients (54%) on the ICU; 36 patients (26%) were included in both cohorts because they were transferred from general ward to ICU. On the general ward, 54 of 136 patients (40%) had an unfavourable outcome and 31 (23%) patients died. On the ICU, 54 out of 119 patients (45%) died. Unfavourable outcome on the general ward was associated with changes in concentrations of IL-6, IL-8, IL-10, soluble Receptor for Advanced Glycation End Products (sRAGE), vascular cell adhesion molecule 1 (VCAM-1) and Pentraxin-3. Death on the ICU was associated with changes in IL-6, IL-8, IL-10, sRAGE, VCAM-1, Pentraxin-3, urokinase-type plasminogen activator receptor, IL-1-receptor antagonist, CD14, procalcitonin, tumor necrosis factor alfa, tissue factor, <em>complement</em> <em>component</em> <em>5a</em>, Growth arrest-specific 6, angiopoietin 2, and lactoferrin. Pathway analysis showed that unfavourable outcome on the ward was mainly driven by chemotaxis and interleukin production, whereas death on ICU was associated with a variety of pathways including chemotaxis, cell-cell adhesion, innate host response mechanisms, including the <em>complement</em> system, viral life cycle regulation, angiogenesis, wound healing and response to corticosteroids.

Interpretation: Clinical deterioration in patients with severe COVID-19 involves multiple pathways, including chemotaxis and interleukin production, but also endothelial dysfunction, the complement system, and immunothrombosis. Prognostic markers showed considerable overlap between general ward and ICU patients, but we identified distinct differences between groups that should be considered in the development and timing of interventional therapies in COVID-19.

Funding: Amsterdam UMC, Amsterdam UMC Corona Fund, and Dr. C.J. Vaillant Fonds.

Keywords: Biomarker; COVID-19; Clinical features; Pathway analysis; Prognosis.

Differentiated human leukemia (HL 60) cells contain high numbers of receptors for the chemotactic factors, N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) and <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>), both coupled to pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins (G proteins). Agonist activation of either receptor stimulated binding of the GTP analog, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), to membrane G proteins and by a similar extent in a non-additive manner. The possible interaction of the two receptors was studied by measuring agonist binding to one receptor in the presence of the other receptor agonist. fMet-Leu-Phe and C<em>5a</em> had no effects on [125I]C<em>5a</em> and fMet-Leu-[3H]Phe receptor binding, respectively, when studied in the absence of regulatory ligands. Similarly, the inhibitory effects of NaCl and GDP on agonist receptor binding were not altered in the presence of the other receptor agonist. In contrast, in the presence of the GTP analogs, GTP[S] and guanosine 5'-[beta,gamma-imino] triphosphate, fMet-Leu-Phe and C<em>5a</em> reduced the binding of [125I]C<em>5a</em> and fMet-Leu-[3H]Phe, respectively, in a concentration-dependent manner. The potencies of the GTP analogs to inhibit binding of [125I]C<em>5a</em> and fMet-Leu-[3H]Phe was increased about 3-fold by fMet-Leu-Phe and C<em>5a</em>, respectively. The data presented suggest that fMet-Leu-Phe and C<em>5a</em> receptors share the same G protein pool in membranes of HL 60 cells and that activation of these G proteins by one of the two receptors decreases the availability of G proteins for the other receptor.

OBJECTIVE

Adenosine (ADO) can enhance and inhibit mast cell degranulation. Potentiation of degranulation occurs at relatively low concentrations of ADO (10−6–10−5 M) through triggering of A3AR, whereas, inhibition occurs at higher concentrations of ADO reportedly through triggering of A2aAR. However, the discrepancy in the concentration of ADO that inhibits degranulation and that required to trigger ADORs suggests a different mechanism. The purpose of this study is to determine the mechanism by which ADO inhibits human mast cell degranulation.

METHODS

We compare the effectiveness of A2aAR specific antagonist ZM241385 and equilibrative nucleoside transporter inhibitors Dipyridamole and NBMPR in preventing ADO-mediated inhibition of FcεRI-induced degranulation of human skin mast cells (hSMCs). Western blotting is done to analyze the effect of ADO on FcεRI-induced Syk phosphorylation.

RESULTS

Dipyridamole and NBMPR completely and dose-dependently prevented ADO from inhibiting FcεRI-induced degranulation in all hSMC preparations. In contrast, ZM241385 at 10−5 M was effective in only 3 of 10 hSMC preparations. Moreover, NBMPR was effective even in those hSMC preparations not responsive to ZM241385. ADO inhibited degranulation induced by FcεRI crosslinking, but not that induced by <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>), Substance P or calcium ionophore. Accordingly, ADO significantly attenuated FcεRI-induced phosphorylation of Syk at the critical activating tyrosine (Y525).

CONCLUSIONS

Blocking the influx of ADO, but not A2aAR signals, is necessary and sufficient to prevent ADO from inhibiting FcεRI-induced mast cell degranulation. Thus, ADO specifically inhibits FcεRI-induced degranulation of hSMCs primarily by an intracellular mechanism that requires its influx via equilibrative nucleoside transporter 1 (ENT1).

Protein kinase (PK) C comprises a family of isoenzymes that play key roles in downstream signalling and cell functions. We studied PKC zeta participation in the effector functions of human eosinophils stimulated with platelet-activating factor (PAF) or <em>complement</em> (C) <em>5a</em>. After pretreating eosinophils with a myristoylated specific PKC zeta inhibitor; bisindlolylmaleimide I (BisI), an inhibitor of conventional and novel PKCs; or rottlerin, a PKC delta inhibitor, we examined PAF- and C<em>5a</em>-evoked functions. Induced PKC translocation was characterized by confocal laser scanning microscopy. The PKC zeta inhibitor blocked PAF- or C<em>5a</em>-induced eosinophil superoxide anion generation as effectively as BisI or rottlerin. The PKC zeta inhibitor also attenuated PAF- or C<em>5a</em>-induced eosinophil degranulation and adhesion. In contrast, the PKC zeta inhibitor did not affect PAF- or C<em>5a</em>-induced CD11b expression. Finally, both eosinophil shape changes and the translocation of PKC zeta and p47phox, a <em>component</em> of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, to the plasma membrane induced by PAF or C<em>5a</em> were completely inhibited by the PKC inhibitor. Thus, the atypical PKC zeta regulates human eosinophil adhesion and effector functions.

Effective therapy for protecting dying neurons against cerebral ischemia-reperfusion injury (IRI) represents a substantial challenge in the treatment of ischemic strokes. Oxidative stress coupled with excessive inflammation is the main culprit for brain IRI that results in neuronal damage and disability. Specifically, <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>) exacerbates the vicious cycle between oxidative stress and inflammatory responses. Herein, we propose that a framework nucleic acid (FNA) conjugated with anti-C<em>5a</em> aptamers (aC<em>5a</em>) can selectively reduce C<em>5a</em>-mediated neurotoxicity and effectively alleviate oxidative stress in the brain. Intrathecal injection of the aC<em>5a</em>-conjugated FNA (aC<em>5a</em>-FNA) was applied for the treatment of rats with ischemic strokes. Positron emission tomography (PET) imaging was performed to investigate the accumulation of aC<em>5a</em>-FNA in the penumbra and its therapeutic efficacy. Results demonstrated that aC<em>5a</em>-FNA could rapidly penetrate different brain regions after brain IRI. Furthermore, aC<em>5a</em>-FNA effectively protected neurons from brain IRI, as verified by serum tests, tissue staining, biomarker detection, and functional assessment. The protective effect of aC<em>5a</em>-FNA against cerebral IRI in living animals may pave the way for the translation of FNA from bench to bedside and broaden the horizon of FNA in the field of biomedicine.

An effective immune response relies on efficient activation of polymorphonuclear neutrophilic leukocytes (PMNL). The PMNL release cellular ATP in response to inflammatory mediators. Although extracellular ATP is rapidly degraded to adenosine, both compounds can readily bind to either the purinergic receptor P1 (adenosine) or P2 (ATP). The P1 and P2 receptors are members of the G-protein-coupled receptor family. The peripartal period is characterized by marked changes in metabolic and inflammatory status that are functionally related with immune responses in the cow. We evaluated the mRNA expression of genes associated with purinergic signaling in PMNL during the peripartal period. Seven multiparous Holstein cows were dried off at d -50 relative to expected parturition and fed a controlled-energy diet (net energy for lactation=1.24 Mcal/kg of dry matter) for ad libitum intake during the entire dry period. After calving, all cows were fed a common lactation diet (net energy for lactation=1.65 Mcal/kg of dry matter) until 30 d in milk. Blood PMNL collected at -10, 3, and 21 d in milk were used to study the expression of 22 genes associated with adhesion to endothelium, chemoattractant binding at the plasma membrane, and purinergic signaling. Other blood samples around calving were used to analyze concentrations of insulin, metabolites, and whole-blood phagocytosis. The expression of purinergic receptor P2Y, G-protein coupled, 2 (P2RY2) increased on d 3 and then decreased on d 21. This response suggested that ATP could play a role in the amplification of chemotactic signals. In contrast, the expression of genes encoding cell adhesion [selectin L (SELL) and selectin P ligand (SELPLG)], chemoattractant receptors [<em>complement</em> <em>component</em> <em>5a</em> receptor 1 (C5AR1), IL-8 receptor α (CXCR1), IL-8 receptor β (CXCR2), and platelet-activating factor receptor (PTAFR)], and adenosine receptors [adenosine A1 receptor (ADORA1) and adenosine A3 receptor (ADORA3)] decreased between -10 and 3 d. The decrease coincided with a marked increase in blood nonesterified fatty acids and hydroxybutyrate concentrations, and a decrease in glucose and insulin concentrations. The increase in metabolites also was associated with greater expression of leukotriene B4 receptor (LTB4R) on d 3 and 21 compared with d -10, which is involved in inflammatory prostaglandin synthesis. Most chemoattractant receptors increased by 21 d, but cell adhesion genes and blood leukocyte phagocytosis was lower. The expression of adenosine A2a receptor (ADORA2A), which is associated with immunosuppression of PMNL and that of adenosine uptake channels [solute carrier family 29 (nucleoside transporters), member 1 (SLC29A1) and member 2 (SLC29A2)] and the nucleotidase adenosine deaminase (ADA) was greater at 3 and 21 d compared with -10d. The reduction in key immune responses, such as cell adhesion and chemotaxis, by bovine PMNL could partly be a function of changes in mRNA expression of genes associated with purinergic signaling.

Skeletal muscle injury presents a challenging traumatological dilemma, and current therapeutic options remain mediocre. This study was designed to delineate if engraftment of mesenchymal stem cells derived from umbilical cord Wharton's jelly (uMSCs) could aid in skeletal muscle healing and persuasive molecular mechanisms. We established a skeletal muscle injury model by injection of myotoxin bupivacaine (BPVC) into quadriceps muscles of C57BL/6 mice. Post BPVC injection, neutrophils, the first host defensive line, rapidly invaded injured muscle and induced acute inflammation. Engrafted uMSCs effectively abolished neutrophil infiltration and activation, and diminished neutrophil chemotaxis, including <em>Complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>), Keratinocyte chemoattractant (KC), Macrophage inflammatory protein (MIP)-2, LPS-induced CXC chemokine (LIX), Fractalkine, Leukotriene B4 (LTB4), and Interferon-γ, as determined using a Quantibody Mouse Cytokine Array assay. Subsequently, uMSCs noticeably prevented BPVC-accelerated collagen deposition and fibrosis, measured by Masson's trichrome staining. Remarkably, uMSCs attenuated BPVC-induced Transforming growth factor (TGF)-β1 expression, a master regulator of fibrosis. Engrafted uMSCs attenuated TGF-β1 transmitting through interrupting the canonical Sma- And Mad-Related Protein (Smad)2/3 dependent pathway and noncanonical Smad-independent Transforming growth factor beta-activated kinase (TAK)-1/p38 mitogen-activated protein kinases signaling. The uMSCs abrogated TGF-β1-induced fibrosis by reducing extracellular matrix <em>components</em> including <i>fibronectin-1</i>, <i>collagen (COL) 1A1</i>, and <i>COL10A1</i>. Most importantly, uMSCs modestly extricated BPVC-impaired gait functions, determined using CatWalk™ XT gait analysis. This work provides several innovative insights into and molecular bases for employing uMSCs to execute therapeutic potential through the elimination of neutrophil-mediated acute inflammation toward protecting against fibrosis, thereby rescuing functional impairments post injury.

BACKGROUND

<em>Complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>) might be involved in the formation of wheals in patients with chronic urticaria (CU). We sought to compare the in vitro responsiveness of basophils to C<em>5a</em> in patients with CU and in a control group.

METHODS

Basophil surface expression of activation marker CD63 induced by C<em>5a</em>, anti-FcepsilonRI mAb or anti-IgE pAb was measured using flow cytometry in 17 patients with CU and in 10 healthy controls.

RESULTS

Patients with CU showed significantly greater basophil CD63 surface expression induced by C<em>5a</em> (median [interquartile range]; 16.4% [13-25.1]; P = 0.011) than the group of healthy controls (10.7% [7.2-16.8]). In contrast, basophil CD63 response to anti-IgE and anti-FcepsilonRI was lower in the CU group (12.3% [6-36.3]; 25.9% [12.5-60.5]) than in the control group (51.7% [6.7-84.3]; 62.1% [9.7-89.2]), although not statistically significant.

CONCLUSIONS

Results of this pilot study suggest that patients with CU might have an enhanced basophil response to stimulation with C<em>5a</em>, indicating that further studies in CU basophil responsiveness are needed.

In neutrophils, toll-like receptor and <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>) signaling are critical pathways regulating innate immunity. In cows, not much is known about the second C<em>5a</em> receptor, <em>complement</em> <em>component</em> <em>5a</em> receptor 2 (C5AR2). It is an interesting player in sepsis treatment because it is considered to have an anti-inflammatory effect during normal inflammation. Periparturient cows are prone to severe infections, and the objectives of this study were to investigate the expression and functionality of C5AR2 during peripartum. We investigated the effect of 2 major inflammatory stimuli, C<em>5a</em> and lipopolysaccharide (LPS), on the expression of a selected number of genes (C5AR1, C5AR2, TLR4, ITGAM, COX2, and CXCL8) and functions linked to these receptors. Overall, TLR4, ITGAM, and C5AR2, all of which are involved in early inflammation, showed a lower expression in periparturient cows. However, an overall lower expression seems not to be the only explanation for the increased risk of sepsis in periparturient cows. Normally, in response to inflammation and as seen in the mid-lactation group, the expression of these genes increases after stimulation with LPS. However, in periparturient cows, stimulation with LPS led to a decrease in expression of these receptors, indicating a different response of neutrophils in response to LPS during this period. A decrease in ITGAM (coding for CD11b) expression complicates correct neutrophil localization and phagocytosis. Its downregulation upon stimulation might be detrimental for adequate eradication of the pathogen and might increase the risk of an imbalanced inflammation; C5AR2 seems to play a central role in this altered response. In addition, myeloperoxidase (MPO) activity in periparturient cows is lower in response to C<em>5a</em> stimulation. It has been suggested that MPO plays an important role in neutrophil shutdown and, thereby, timely resolution of inflammation. A decreased MPO activity might thus prolong the inflammatory reaction of the neutrophils. This finding was supported by the increased viability of the neutrophils obtained from periparturient cows. Even after stimulation, we found a lower caspase-3 activity in this group, indicating that they might be activated for a longer time compared with the neutrophils from mid-lactation cows. Accordingly, these alterations might contribute to a temporal mismatch in inflammatory responses, as often seen in severe periparturient infections.

Immune function was assessed in 93 adult hemophiliacs in the 2 years prior to HTLV-III antibody testing of blood donors and routine heat treatment of coagulation factor concentrates. Parameters of humoral and cellular immunity studied included serum <em>complements</em>, immunoglobulins, immune complexes, blood cell counts, lymphocyte subsets, lymphocyte transformation, 2-<em>5A</em> synthetase, serology for HTLV-III and hepatitis B, skin tests, and clinical status. HTLV-III seropositivity was significantly more prevalent in patients treated with factor VIII concentrate; seropositive patients had a higher proportion of abnormal test results and were more symptomatic. Although many subjects had abnormal test results, specific test results generally did not correlate with type of blood <em>component</em> received or time of preceding treatment. The proportion of tests abnormal in individual patients was correlated with the intensity of factor replacement therapy when the time interval from the last treatment was a covariate. While T8+ lymphocytes were increased and T4/T8 ratios were decreased in many patients, significantly reduced T4+ lymphocytes were seen only in seropositive patients and in those treated with factor VIII concentrate. Neither seropositivity nor presence of symptoms correlated with abnormal lymphocyte mitogenic response or 2-<em>5A</em> synthetase levels, but synthetase levels were increased in 65% of patients tested. Over the 2-year study period, no significant deterioration of clinical or laboratory variables was observed in the patients. Abnormalities of immune function were found to be common in hemophiliacs regardless of type of treatment or of evidence of HTLV-III infection.

This study was undertaken to assess the effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), a known activator of soluble guanylyl cyclase, on formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>)-induced homotypic human neutrophil aggregation. YC-1 as well as the phosphodiesterase (PDE)4 inhibitors rolipram and Ro 20-1724, but not the PDE3 inhibitor milrinone, inhibited the aggregation responses stimulated by FMLP and C<em>5a</em>. In contrast, sodium nitroprusside (SNP) had no effect on FMLP- or C<em>5a</em>-induced neutrophil aggregation. Moreover, SNP together with YC-1 failed to modify the YC-1-induced responses. In addition, YC-1 and rolipram, but not milrinone, induced substantial increases in cAMP levels, which occurred through the inhibition of PDE activity but not an increase in adenylate cyclase function. Interestingly, adenosine deaminase abolished the inhibitory effects and cAMP levels of YC-1, rolipram, and Ro 20-1724. In conclusion, these results indicate that the inhibitory effect of YC-1 on homotypic neutrophil aggregation is attributed to an elevation in the cAMP concentration through inhibition of the activity of PDE, which may potentiate the autocrine functions of endogenous adenosine.

Crosstalk between <em>complement</em> <em>component</em> <em>5a</em> receptors (C<em>5a</em>Rs) and TLRs in dendritic cells (DCs) occurs upon pathogen invasion; however, studies on C<em>5a</em>R and TLR crosstalk mainly focused on the modulating effect of C<em>5a</em> on TLR-induced cytokine production. To elucidate the breadth of C<em>5a</em>R and TLR4 crosstalk, the effect of simultaneous treatment with C<em>5a</em> and LPS was investigated in human monocyte-derived DCs (moDCs) 2 h after stimulation using whole transcriptome sequencing analysis. Although the effect of C<em>5a</em> on hallmark genes defining TLR4-induced DC maturation was limited at this time point, RNA sequencing analysis revealed a great variety of novel C<em>5a</em> targets, of which many interfere with TLR4-mediated immune activation. Analysis of functional relationships among these genes uncovered induction of a central immune regulatory network upon C<em>5a</em>R and TLR4 crosstalk, involving the transcription factors forkhead box (FOX)O1 and FOXO3 and the signaling molecules serum- and glucocorticoid-inducible kinase (SGK1), ribosomal S6 kinase 2 (RSK2), and PI3Kβ. C<em>5a</em>R and TLR crosstalk, furthermore, yielded down-regulation of mainly proinflammatory network branches, including IL-12B, IL-2Rα (IL-2RA), and jagged 1 (JAG1) and cooperative induction of predominantly anti-inflammatory network branches, including sphingosine kinase 1 (SPHK1), β2 adrenergic receptor (ADRB2), gastric inhibitory polypeptide receptor (GIPR), and four-and-a-half Lin11, Isl-1, and Mec-3 domains protein 2 (FHL2). Together, these data point toward induction of generalized immune regulation of DC function. Motif enrichment analysis indicate a prominent role for basic leucine zipper (bZIP) and IFN regulatory factor 4 (IRF4) transcription factors upon C<em>5a</em>R and TLR4 crosstalk. Additionally, differences were observed in the modulating capacity of C<em>5a</em> on DCs in the absence or presence of a pathogen (TLR stimulus). Our findings shed new light on the depth and complexity of C<em>5a</em>R and TLR4 crosstalk and provide new foci of research for future studies.

Neutrophils sense and respond to diverse chemotactic cues through G-protein-coupled receptors (GPCRs). However, the precise trafficking dynamics of chemoattractant GPCRs during neutrophil activation and chemotaxis remain unclear. Here, by using small-molecule inhibitors and CRISPR-based knockouts, we establish that two primary chemoattractant GPCRs - formyl peptide receptor 1 (FPR1) and <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>) receptor 1 (C<em>5a</em>R1) - internalize in a CDC42-actin-dependent manner. Through live-cell imaging, we demonstrate that, upon stimulation, FPR1 rapidly clusters and re-distributes along the plasma membrane to the trailing edge, where it internalizes and is directionally trafficked towards the front of migrating primary human neutrophils. In contrast to FPR1 and C<em>5a</em>R1, the leukotriene B4 (LTB4) receptor (BLT1, also known as LTB4R), which relays LTB4 signals in response to primary chemoattractants during neutrophil chemotaxis, fails to internalize upon physiological stimulation with LTB4, N-formyl-Met-Leu-Phe (fMLF) or C<em>5a</em>. Importantly, we report that blocking the LTB4-BLT1 axis or downstream myosin activation enhances the internalization of FPR1 and C<em>5a</em>R1, thus reducing downstream signaling and impairing chemotaxis to primary chemoattractants. The polarized trafficking of chemoattractant GPCRs and its regulation by the BLT1-mediated myosin activation therefore drives persistent chemotactic signaling in neutrophils.This article has an associated First Person interview with the first author of the paper.

Primary ciliary dyskinesia (PCD), cystic fibrosis (CF), and chronic obstructive airway disease are characterized by neutrophilic inflammation in the lungs. In CF and chronic obstructive airway disease, improper functioning of neutrophils has been demonstrated. We hypothesized that the pulmonary damage in PCD might be aggravated by abnormal functioning neutrophils either as a primary consequence of the PCD mutation or secondary to chronic inflammation. We analyzed chemotactic responses and chemoattractant receptor expression profiles of peripheral blood neutrophils from 36 patients with PCD, 21 healthy children and 19 healthy adults. We stimulated peripheral blood monocytes from patients and healthy controls and measured CXCL8 and IL-1β production with ELISA. PCD neutrophils displayed reduced migration toward CXCR2 ligands (CXCL5 and CXCL8) in the shape change, microchamber and microslide chemotaxis assays, whereas leukotriene B4 and <em>complement</em> <em>component</em> <em>5a</em> chemotactic responses were not significantly different. The reduced response to CXCL8 was observed in all subgroups of patients with PCD (displaying either normal ultrastructure, dynein abnormalities or central pair deficiencies) and correlated with lung function. CXCR2 was downregulated in about 65% of the PCD patients, suggestive for additional mechanisms causing CXCR2 impairment. After treatment with the TLR ligands lipopolysaccharide and peptidoglycan, PCD monocytes produced more CXCL8 and IL-1β compared to controls. Moreover, PCD monocytes also responded stronger to IL-1β stimulation in terms of CXCL8 production. In conclusion, we revealed a potential link between CXCR2 and its ligand CXCL8 and the pathogenesis of PCD.

Multiple sclerosis (MS) is an autoimmune, demyelinating disease of the central nervous system. Patients with MS typically present with visual, motor, and sensory deficits. However, an additional complication of MS in large subset of patients is neuropathic pain. To study the underlying immune-mediated pathophysiology of pain in MS we employed the myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalitis (EAE) model in mice. Since sensory neurons are crucial for nociceptive transduction, we investigated the effect of this disease on sensory neurons of the lumbar dorsal root ganglia (DRG). Here, we report the disease was associated with activation of the <em>complement</em> system and the NLRP3 inflammasome in the DRG. We further observe a transient increase in the number of <em>complement</em> <em>component</em> <em>5a</em> receptor 1-positive (C<em>5a</em>R1+) immune cells, CD4+ T-cells, and Iba1+ macrophages in the DRG. The absence of any significant change in the levels of mRNA for myelin proteins in the DRG and the sciatic nerve suggests that demyelination in the PNS is not a trigger for the immune response in the DRG. However, we did observe an induction of activating transcription factor 3 (ATF3) at disease onset and chronic disruption of cytoskeletal proteins in the DRG demonstrating neuronal injury in the PNS in response to the disease. Electrophysiological analysis revealed the emergence of hyperexcitability in medium-to-large (≥26 µm) diameter neurons, especially at the onset of MOG-EAE signs. These results provide conclusive evidence of immune activation, neuronal injury, and peripheral sensitization in MOG-EAE, a model classically considered to be centrally mediated.

Exosomes are 30-150 nm extracellular vesicles mediating intercellular communication. Disease states can alter exosome composition affecting the message carried and thereby, its functional impact. The objective of this study was to identify proteins present in these vesicles in a mouse model of neuropathic pain induced by spared nerve injury (SNI). Small extracellular vesicles (sEVs) were purified from serum four weeks after SNI surgery and the protein composition was determined using tandem mass spectrometry and cytokine array. Proteomic analysis detected 274 gene products within sEVs. Of these, 24 were unique to SNI model, 100 to sham surgery control and five to naïve control samples. In addition to commonly expressed sEVs proteins, multiple members of serpin and <em>complement</em> family were detected in sEVs. Cytokine profiling using a membrane-based antibody array showed significant upregulation of <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>) and Intercellular Adhesion Molecule 1 (ICAM-1) in sEVs from SNI model compared to sham control. We observed a differential distribution of C<em>5a</em> and ICAM-1 within sEVs and serum between sham and SNI, indicating changes from local or paracrine to long distance signaling under neuropathic pain. Our studies suggest critical roles for cargo sorting of vesicular proteins in mediating signaling mechanisms underlying neuropathic pain. SIGNIFICANCE: Approximately 100 million U.S. adults are burdened by chronic pain. Neuropathic pain resulting from injury or dysfunction of the nervous system is challenging to treat. Unlike acute pain that resolves over time, chronic pain persists resulting in changes in the peripheral and central nervous system. The transport of biomolecular cargo comprised of proteins and RNAs by small extracellular vesicles (sEVs) including exosomes has been proposed to be a fundamental mode of intercellular communication. To obtain insights on the role of exosome-mediated information transfer in the context of neuropathic pain, we investigated alterations in protein composition of sEVs in a mouse model of neuropathic pain induced by spared nerve injury (SNI). Our studies using mass spectrometry and cytokine array show that sEVs from SNI model harbor unique proteins. We observed an upregulation of C<em>5a</em> and ICAM-1 in exosomes from SNI model compared to control. There was a differential distribution of C<em>5a</em> and ICAM-1 within exosomes and serum, between control and SNI suggesting a switch from local to long distance signaling. Our studies suggest critical roles for cargo sorting of vesicular proteins in mediating signaling under neuropathic pain.

The interaction of hC5a with C5aR, previously hypothesized to involve a "two-site" binding, (i) recognition of the bulk of hC5a by the N-terminus (NT) of C5aR ("site1"), and (ii) recognition of C-terminus (CT) of hC5a by the extra cellular surface (ECS) of the C5aR ("site2"). However, the pharmacological landscapes of such recognition sites are yet to be illuminated at atomistic resolution. In the context, unique model complexes of C5aR, harboring pharmacophores of diverse functionality at the "site2" has recently been described. The current study provides a rational illustration of the "two-site" binding paradigm in C5aR, by recruiting the native agonist hC5a and engineered antagonist hC5a(A8). The hC5a-C5aR and hC5a(A8)-C5aR complexes studied over 250 ns of molecular dynamics (MD) each in POPC bilayer illuminate the hallmark of activation mechanism in C5aR. The intermolecular interactions in the model complexes are well supported by the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) based binding free energy calculation, strongly correlating with the reported mutational studies. Exemplified in two unique and contrasting molecular complexes, the study provides an exceptional understanding of the pharmacological divergence observed in C5aR, which will certainly be useful for search and optimization of new generation "neutraligands" targeting the hC5a-C5aR interaction.

<AbstractText>Chronic spontaneous urticaria presents as a heterogeneous syndrome characterised by wheals, angioedema, or both for greater than 6 weeks. Spleen tyrosine kinase mediates allergen-induced mast cell degranulation via the IgE signalling pathway, a central <em>component</em> of wheal formation and inflammation. In this study, we investigated the effects of perfused or topically administered GSK2646264 on IgE-mediated histamine release from mast cells in an ex vivo human skin model.</AbstractText><AbstractText>Using a novel SkiP device, ex vivo human skin from mastectomy surgeries was challenged with anti-IgE, <em>complement</em> <em>5a</em> (C<em>5a</em>), and buffer to induce histamine release from skin mast cells. Histamine was collected via microdialysis fibres and measured fluorometrically. GSK2646264 was delivered via perfusion either using microdialysis fibres or topically in a cream. Drug concentrations in the skin were measured by LC-MS, and a pharmacokinetic/ pharmacodynamic (PK/PD) relationship developed.</AbstractText><p><div><b>KEY RESULTS</b></div>Perfused GSK2646264 significantly inhibited anti-IgE (but not C<em>5a</em>)-induced histamine release in a concentration-dependent manner. The 0.5, 1, and 3% cream delivered GSK2646264 to the dermis above the IC₉₀ and dose-dependently attenuated anti-IgE-induced histamine release.</p><AbstractText>GSK2646264 administered topically or direct to the dermis blocked histamine release from in situ skin mast cells. A PK/PD relationship curve suggests that dermal concentrations above 6.8 μM should lead to approximately 90% inhibition of histamine release from skin mast cells following activation of the Fc fragment of IgE receptor 1a, implicating a potential use for the compound in skin mast cell diseases such as urticaria.</AbstractText>

BACKGROUND

Hidradenitis suppurativa (HS) is a chronic, inflammatory, and debilitating skin disease, which usually occurs after puberty with painful, deep-seated, inflammatory lesions in the apocrine gland-bearing areas of the body. Several pharmacologic agents have been described to reduce lesion activity and inflammation in HS. However, conventional treatment may not always get the desired results. Therefore, unconventional therapies must be taken into account. Areas covered: Recently, the better understanding of HS pathogenesis has been used to improve treatment strategies with many emerging conventional and unconventional therapeutics options. Adalimumab is the only FDA-approved biologic available for therapy of moderate-to-severe HS. Nevertheless, novel therapeutic approach, including both topical and systemic as well as novel laser device, showed good clinical outcome. Several molecules, such as tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), IL-17, IL-12, IL-23, phosphodiesterase 4 (PDE4), lymphocyte function-associated antigen 1 (LFA-1), and <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>), are modulated by such new biologic agents in HS. Expert commentary: In the next years, many therapeutic options for HS will be available. Clinical trials showed the efficacy of several biologic drugs, antibiotics, laser light device. Novel therapeutic options seem to be promising, but dermatologists will have to evaluate their effectiveness and safety in daily clinical practice.

In infants, the major <em>components</em> of the innate immune system appear weakened, and it has been shown that both polymorphonuclear neutrophil (PMN) production and function are immature. This study was conducted to assess the expression of a number of receptors important to normal PMN function and the integrity of PMN degranulation in cord blood and in uninfected children of varying ages born to human immunodeficiency virus type 1 (HIV-1) seropositive mothers. Although the expression of l-selectin (CD62L) on PMN did not differ between the infants aged 12, 15 and 18 months, the expression of the interleukin-8 (IL-8) receptors CXCR1 and CXCR2, and the <em>complement</em> <em>5a</em> (C<em>5a</em>) receptor CD88 displayed a similar pattern, with the highest levels expressed on PMN from infants in the 12 month old age group, and declining with age. It was also observed that PMN from a substantial proportion of the younger infants were unresponsive to a variety of stimuli including IL-8, C<em>5a</em>, stromal cell-derived factor (SDF)-1alpha, SDF-1beta, and phorbol 12-myristate 13-acetate (PMA), with the proportions of children showing positive (adult-like) PMN degranulation responses increasing with age. Exposure to HIV-1 did not appear to be the cause of impaired degranulation responses, since a similar proportion of cord blood PMN from uninfected infants born to HIV-1 infected and HIV-1 uninfected mothers were unresponsive. The altered expression of these important receptors and inefficient agonist-induced degranulation in early life may contribute to the increased susceptibility of infants to secondary microbial infections.

Gingipains secreted by Porphyromonas gingivalis (P. gingivalis, Pg) play an important role in maintaining macrophage infiltrating. And, this study is to evaluate effects of gingipain on M1 macrophage polarization after exposure to Porphyromonas gingivalis (P. gingivalis, Pg) and if these effects are through <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>) pathway. Mouse RAW264.7 macrophages were exposed to gingipain extracts, Escherichia coli lipopolysaccharides (Ec-LPS), Pg-LPS with or without the C<em>5a</em>R antagonist: PMX-53 for 24 h. Then, gene expressions and protein of IL-12, IL-23, iNOS, IL-10, TNF-α, IL-1β, and IL-6 were determined by qRT-PCR and ELISA assays. Surface markers CD86 for M1 and CD206 for M2 were also evaluated by flow cytometry. The results show that gingipain extracts alone increased expressions of IL-12, IL-23, iNOS, TNF-α, IL-1β, and IL-6, but not IL-10. Gingipain extracts plus Ec-LPS decreased expressions of IL-12, IL-23, iNOS, TNF-α, IL-1β, and IL-6 in which Ec-LPS induced increase. For gingipain extracts plus Pg-LPS-treated RAW264.7, macrophages, gingipain extracts enhanced expressions of IL-12 and IL-23 in which Pg-LPS induced increase, but not iNOS and IL-10 while gingipain extracts decreased expressions of TNF-α, IL-1β, and IL-6 in which Pg-LPS induced increase. Interestingly, PMX-53 increased expressions of IL-12, IL-23, and iNOS when RAW264.7 macrophages were treated with gingipain extracts plus Ec-LPS or Pg-LPS and PMX-53, while PMX-53 decreased expressions of TNF-α, IL-1β, and IL-6. Changes of CD86-positive macrophages were consistent with cytokine changes. Our data indicate that gingipain is a critical regulator, more like a promoter to manipulate M1 macrophage polarization in order to benefit P. gingivalis infection through the C<em>5a</em> pathway.