BACKGROUND

Defining conserved molecular pathways in animal models of successful cardiac regeneration could yield insight into why adult mammals have inadequate cardiac regeneration after injury. Insight into the transcriptomic landscape of early cardiac regeneration from model organisms will shed light on evolutionarily conserved pathways in successful cardiac regeneration.

METHODS

Here we describe a cross-species transcriptomic screen in 3 model organisms for cardiac regeneration: axolotl, neonatal mice, and zebrafish. Apical resection to remove ≈10% to 20% of ventricular mass was carried out in these model organisms. RNA-sequencing analysis was performed on the hearts harvested at 3 time points: 12, 24, and 48 hours after resection. Sham surgery was used as internal control.

RESULTS

Genes associated with inflammatory processes were found to be upregulated in a conserved manner. <em>Complement</em> receptors (activated by <em>complement</em> <em>components</em>, part of the innate immune system) were found to be highly upregulated in all 3 species. This approach revealed induction of gene expression for <em>complement</em> <em>5a</em> receptor 1 in the regenerating hearts of zebrafish, axolotls, and mice. Inhibition of <em>complement</em> <em>5a</em> receptor 1 significantly attenuated the cardiomyocyte proliferative response to heart injury in all 3 species. Furthermore, after left ventricular apical resection, the cardiomyocyte proliferative response was diminished in mice with genetic deletion of <em>complement</em> <em>5a</em> receptor 1.

CONCLUSIONS

These data reveal that activation of <em>complement</em> <em>5a</em> receptor 1 mediates an evolutionarily conserved response that promotes cardiomyocyte proliferation after cardiac injury and identify <em>complement</em> pathway activation as a common pathway of successful heart regeneration.

<AbstractText>Anti-neutrophil cytoplasmic antibody-associated vasculitis (ANCA-associated vasculitis) is a serious, often life-threatening disease. In new-onset disease or relapses, the standard treatment is immunosuppressive therapy with glucocorticoids; these therapies are associated with substantial short- and long-term toxicity. <em>Complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>) binding to C<em>5a</em> receptor (C<em>5a</em>R) may play a central role in the pathogenesis of ANCA-associated vasculitis. Avacopan is a novel, orally bioavailable, and highly selective antagonist of human C<em>5a</em>R. Avacopan does not interfere with the production of C5b or the membrane attack complex (i.e., terminal <em>complement</em> complex), and does not block C<em>5a</em> binding to a second receptor, C5L2 (also called C<em>5a</em>R2), shown to be protective in anti-MPO glomerulonephritis. This trial will evaluate if avacopan replaces the need for chronic glucocorticoids in the treatment of ANCA-associated vasculitis.</AbstractText><AbstractText>To determine the proportions of patients in remission at week 26 and with sustained remission at week 52, defined as Birmingham Vasculitis Activity Score = 0 and not taking glucocorticoids within the 4 weeks prior to week 26 and 52, respectively.</AbstractText><AbstractText>The "Avacopan Development in Vasculitis to Obtain Corticosteroid elimination And Therapeutic Efficacy study (ADVOCATE)" is a randomized, double-blind, active-comparator (prednisone), 2-arm study evaluating the safety and efficacy of avacopan versus prednisone, administered in combination with other immunosuppressive therapy. Eligible subjects will have active disease requiring induction of remission. Subjects are stratified based on type of immunosuppressive therapy, ANCA subtype, and new or relapsing disease. Target sample size is 300 patients, enrolled at over 200 sites globally. All authors and local ethics committees approved the study design. All patients will provide informed consent.</AbstractText><AbstractText>Patient enrollment was completed in Q4 2018. Topline results are anticipated by Q1 2020.</AbstractText><AbstractText>Results will be released irrespective of whether the findings are positive or negative.</AbstractText><AbstractText>ClinicalTrials.gov NCT02994927.</AbstractText>

Most G-protein-coupled receptors (GPCRs) form di(oligo)-meric structures that constitute signaling and trafficking units and might be essential for receptor functions. Cell responses to complement C5a receptor (C5aR) are tightly controlled by receptor desensitization and internalization. To examine the implication of dimerization in C5aR regulation, we generated an NH(2)-terminally modified C5aR mutant, unable to bind C5a, and a phosphorylation-deficient mutant. Neither an intact NH(2) terminus nor the presence of COOH-terminal phosphorylation sites appeared to be required for the formation of C5aR dimers. Upon C5a stimulation, mutant receptors did not internalize when individually expressed. C5a stimulation of cells that co-expressed wild type C5aR together with either unliganded or phosphorylation-deficient mutant resulted in co-internalization of mutant receptors with C5aR. Unliganded GPCRs can be cross-phosphorylated within a heterologous receptor dimer or by second messenger-activated kinases. C5a stimulation of (32)P-labeled cells that co-expressed the unliganded mutant with either C5aR or the phosphorylation-deficient mutant did not induce phosphorylation of the unliganded mutant. We can thus postulate that, in the case of C5aR, the stimulation and phosphorylation of one monomer is enough to lead to dimer internalization. The existence and functional implication of di(oligo)mer formation may be important for an accurate C5aR down-regulation in pathological conditions.

Leukotriene B4 (LTB4), a naturally occurring pro-inflammatory product of arachidonic acid metabolism, has been associated with human inflammatory disease. This study compares the abilities of two LTB4 receptor antagonists, 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]- propoxy]phenoxy]benzoic acid (LY293111) and 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]- 3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid (SC-41930), to displace LTB4 binding and their functional blockade of human neutrophil activation. LY293111 inhibited the binding of [3H]LTB4 with a Ki of 25 nM; SC-41930 displayed a similar potency (Ki = 17 nM). In contrast, LY293111 prevented LTB4-induced calcium mobilization with an IC50 = 20 nM, or 40 times more effectively than SC-41930 (IC50 = 808 nM). LY293111 was 300 times more potent than SC-41930 in blocking LTB4-induced CD11b up-regulation on isolated neutrophils. LY293111 also arrested LTB4-induced up-regulation of CD11b on neutrophils in whole human blood. LY293111 was not effective in blocking human neutrophil activation responses induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP), platelet-activating factor (PAF), human recombinant endothelial interleukin-8 (IL-8) or human recombinant <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>).

Inherited and age-related macular degenerations (AMDs) are important causes of vision loss. An early hallmark of these disorders is the formation of sub-retinal pigment epithelium (RPE) basal deposits. A role for the <em>complement</em> system in MDs was suggested by genetic association studies, but direct functional connections between alterations in the <em>complement</em> system and the pathogenesis of MD remain to be defined. We used primary RPE cells from a mouse model of inherited MD due to a p.R345W mutation in EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) to investigate the role of the RPE in early MD pathogenesis. Efemp1(R345W) RPE cells recapitulate the basal deposit formation observed in vivo by producing sub-RPE deposits in vitro. The deposits share features with basal deposits, and their formation was mediated by EFEMP1(R345W) or <em>complement</em> <em>component</em> 3a (C3a), but not by <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>). Increased activation of <em>complement</em> appears to occur in response to an abnormal extracellular matrix (ECM), generated by the mutant EFEMP1(R345W) protein and reduced ECM turnover due to inhibition of matrix metalloproteinase 2 by EFEMP1(R345W) and C3a. Increased production of C3a also stimulated the release of cytokines such as interleukin (IL)-6 and IL-1B, which appear to have a role in deposit formation, albeit downstream of C3a. These studies provide the first direct indication that <em>complement</em> <em>components</em> produced locally by the RPE are involved in the formation of basal deposits. Furthermore, these results suggest that C3a generated by RPE is a potential therapeutic target for the treatment of EFEMP1-associated MD as well as AMD.

BACKGROUND

Eosinophils play an important role in the pathogenesis of allergic diseases. Chemoattractants, including platelet-activating factor (PAF) and <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>), induce eosinophil infiltration and promote eosinophil effector functions.

OBJECTIVE

To compare eosinophil degranulation and superoxide anion (O2-) generation induced by various chemoattractants, and to elucidate the role of cellular adhesion on these effector functions.

METHODS

Human eosinophils were stimulated with PAF, C<em>5a</em>, eotaxin, or leukotriene B4 (LTB4). O2- generation was assayed by a chemiluminescence method using a Cypridina luciferin analog as the amplifier. Degranulation and adhesion were measured by quantitating eosinophil protein X by radioimmunoassay. Expression of CD11b on eosinophils was measured by flow cytometry.

RESULTS

PAF and C<em>5a</em> induced significant degranulation and O2- generation from eosinophils. In contrast, the potency of eotaxin or LTB4 for these functions was much less. PAF and C<em>5a</em> also significantly enhanced eosinophil adhesion, whereas eotaxin and LTB4 did not. CD11b expression on eosinophils was enhanced by all four stimulants, and the order of potency to induce CD11b expression was C<em>5a</em>>> PAF>> eotaxin>> LTB4.

CONCLUSIONS

The potency of PAF and C<em>5a</em> for inducing effector function in eosinophils was greater than that of eotaxin or LTB4. The magnitude of the effector function was consistent with the degree of eosinophil adherence induced by each stimulant. These results suggest that effector functions of eosinophils which are mediated through G-protein coupled receptors are dependent on cellular adhesion.

It is well known that signaling in neutrophils through both the <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>) and C<em>5a</em> receptor (C<em>5a</em>R) and the toll-like receptor 4 (TLR4) pathways plays an essential role in innate defense. Neutrophil dysfunction, as seen during sepsis in severe mastitis during the periparturient period, is correlated with elevated concentrations of anaphylatoxin C<em>5a</em>. The aim of the current study was to elucidate the effect of C<em>5a</em> on TLR4 signaling in bovine neutrophils. Neutrophils were incubated with a high (but physiological) dose of purified C<em>5a</em>, and mRNA was extracted from neutrophils at different time points postincubation (PI). The incubation with C<em>5a</em> resulted in a biphasic C<em>5a</em>R expression profile, a phenomenon that might be explained by internalization (at 10 min PI) with subsequent reconstitution (starting at 40 min PI) of this receptor. The expression of TLR4, as well as its coreceptor, CD14, showed a similar biphasic change as observed with C<em>5a</em>R. In addition, changes in the mRNA expression levels of several genes belonging to the TLR4 pathway, such as TICAM-1, IKKα, and MAP3K7 were noted. The maximal expression of TLR4, CD14, and C<em>5a</em>R mRNA at 80 min PI was accompanied by a peak in IL8 mRNA, indicating that C<em>5a</em> is able to induce IL-8 production in neutrophils in vitro without the need of a costimulatory factor such as lipopolysaccharide. Moreover, a relatively constant expression of RELA was accompanied by increased expression of ATF3, an endogenous inhibitor of nuclear factor-κB mediated transcription, implying that C<em>5a</em> regulates TLR4 signaling and IL-8 synthesis independently. A significant time-dependent correlation was found between C<em>5a</em>R and TLR4, with the majority of the selected TLR4-dependent genes showing a significant correlation with C<em>5a</em>R at 80 min PI, when C<em>5a</em>R and TLR4 mRNA expression reached its maximum, suggesting crosstalk between both receptors. Taken together, this study showed that C<em>5a</em> is able to (1) alter the expression of genes belonging to the TLR4 pathway and (2) induce IL8 gene expression in bovine neutrophils. In addition, indications for cross-talk between <em>complement</em> activation and TLR4 signaling were found in the present study.

The anaphylatoxins <em>complement</em> <em>component</em> 3a and <em>5a</em> (C3a and C<em>5a</em>, respectively) are classically seen as proinflammatory mediators of allergic asthma that recruit inflammatory cells, induce edema, and cause bronchoconstriction. A few years ago, controversy arose when it was shown that C5-deficient mice were more susceptible to experimental asthma compared with C5-sufficient mice. In a study by Köhl et al. in this issue of the JCI, it is shown in a series of truly "<em>complement</em>ary" experiments that C<em>5a</em> receptor (C<em>5a</em>R) blockade promotes Th2 sensitization upon first exposure to inhaled allergen, whereas C<em>5a</em>R blockade during established inflammation suppresses the cardinal features of asthma (see the related article beginning on page 783). Blockade of C<em>5a</em>R alters the function of airway DCs, crucial for inducing and maintaining Th2 responses in the lung. Targeting C<em>5a</em>R as a treatment for established asthma could be beneficial, but might be accompanied by sensitization to novel antigens.

BACKGROUND

The t(9;22)(q34;q11) generating the BCR/ABL1 fusion gene represents the cytogenetic hallmark of chronic myeloid leukemia (CML). About 5-10% of CML cases show variant translocations with the involvement of other chromosomes in addition to chromosomes 9 and 22. The molecular bases of biological differences between CML patients with classic and variant t(9;22) have never been clarified.

RESULTS

In this study, we performed gene expression microarray analysis to compare CML patients bearing variant rearrangements and those with classic t(9;22)(q34;q11). We identified 59 differentially expressed genes significantly associated with the two analyzed groups. The role of specific candidate genes such as TRIB1 (tribbles homolog 1), PTK2B (protein tyrosine kinase 2 beta), and C5AR1 (<em>complement</em> <em>component</em> <em>5a</em> receptor 1) is discussed.

CONCLUSIONS

Our results reveal that in CML cases with variant t(9;22) there is an enhancement of the MAPK pathway deregulation and show that kinases are a common target of molecular alterations in hematological disorders.

The antiphospholipid syndrome is characterized clinically by fetal loss and thrombosis and serologically by the presence of autoantibodies to lipid-binding proteins. In a model of this procoagulant condition in which these antibodies are injected into pregnant mice, fetal loss was prevented by blocking of <em>complement</em> activation. Specifically, interaction of <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>) with its receptor is necessary for thrombosis of placental vasculature. Inhibition of <em>complement</em> activation may have a therapeutic role in this disease.

BACKGROUND

Inflammation and immunity play crucial roles in the development of hypertension. Complement activation-mediated innate immune response is involved in the regulation of hypertension and target-organ damage. However, whether complement-mediated T-cell functions could regulate blood pressure elevation in hypertension is still unclear.

OBJECTIVE

We aim to determine whether C3aR (<em>complement</em> <em>component</em> 3a receptor) and C<em>5a</em>R (<em>complement</em> <em>component</em> <em>5a</em> receptor) could regulate blood pressure via modulating regulatory T cells (Tregs).

RESULTS

We showed that angiotensin II (Ang II)-induced hypertension resulted in an elevated expression of C3aR and C<em>5a</em>R in Foxp3 (forkhead box P3)+ Tregs. By using C3aR and C<em>5a</em>R DKO (double knockout) mice, we showed that C3aR and C<em>5a</em>R deficiency together strikingly decreased both systolic and diastolic blood pressure in response to Ang II compared with WT (wild type), single C3aR-deficient (C3aR-/-), or C<em>5a</em>R-deficient (C<em>5a</em>R-/-) mice. Flow cytometric analysis showed that Ang II-induced Treg reduction in the kidney and blood was also blocked in DKO mice. Histological analysis indicated that renal and vascular structure remodeling and damage after Ang II treatment were attenuated in DKO mice compared with WT mice. In vitro, Ang II was able to stimulate C3aR and C<em>5a</em>R expression in cultured CD4+CD25+ natural Tregs. CD3 and CD28 antibody stimuli downregulated Foxp3 expression in WT but not DKO Tregs. More important, depletion of Tregs with CD25 antibody abolished the protective effects against Ang II-induced hypertension and target-organ damage in DKO mice. Adoptive transfer of DKO Tregs showed much more profound protective effects against Ang II-induced hypertension than WT Treg transfer. Furthermore, we demonstrated that C<em>5a</em>R expression in Foxp3+ Tregs was higher in hypertensive patients compared with normotensive individuals.

CONCLUSIONS

C3aR and C<em>5a</em>R DKO-mediated Treg function prevents Ang II-induced hypertension and target-organ damage. Targeting C3aR and C<em>5a</em>R in Tregs specifically may be an alternative novel approach for hypertension treatment.

Polymicrobial sepsis in mice causes myocardial dysfunction after generation of the <em>complement</em> anaphylatoxin, <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>). C<em>5a</em> interacts with its receptors on cardiomyocytes (CMs), resulting in redox imbalance and cardiac dysfunction that can be functionally measured and quantitated using Doppler echocardiography. In this report we have evaluated activation of MAPKs and Akt in CMs exposed to C<em>5a</em> in vitro and after cecal ligation and puncture (CLP) in vivo In both cases, C<em>5a</em> in vitro caused activation (phosphorylation) of MAPKs and Akt in CMs, which required availability of both C<em>5a</em> receptors. Using immunofluorescence technology, activation of MAPKs and Akt occurred in left ventricular (LV) CMs, requiring both C<em>5a</em> receptors, C<em>5a</em>R1 and -2. Use of a water-soluble p38 inhibitor curtailed activation in vivo of MAPKs and Akt in LV CMs as well as the appearance of cytokines and histones in plasma from CLP mice. When mouse macrophages were exposed in vitro to LPS, activation of MAPKs and Akt also occurred. The copresence of the p38 inhibitor blocked these activation responses. Finally, the presence of the p38 inhibitor in CLP mice reduced the development of cardiac dysfunction. These data suggest that polymicrobial sepsis causes cardiac dysfunction that appears to be linked to activation of MAPKs and Akt in heart.-Fattahi, F., Kalbitz, M., Malan, E. A., Abe, E., Jajou, L., Huber-Lang, M. S., Bosmann, M., Russell, M. W., Zetoune, F. S., Ward, P. A. <em>Complement</em>-induced activation of MAPKs and Akt during sepsis: role in cardiac dysfunction.

Rheumatoid arthritis (RA) is a systemic autoimmune disease and collagen-induced arthritis (CIA) is an animal model for RA. Micheliolide (MCL) is a novel compound with strong anti-inflammatory effects. The present study was conducted to evaluate the therapeutic effects of MCL on RA. Mice were randomly divided into four groups and the CIA model mice were treated with methotrexate (MTX), MCL and dimethyl sulfoxide. A score associated with the severity of arthritis was assigned on alternate days from the 22nd day for 60 days. Histopathological changes and the serum levels of cytokines were measured on day 85. The results demonstrated that the MCL treatment group had arthritis scores lower than the CIA group and higher than the MTX group; compared with the CIA group, MCL and MTX significantly reduced the swelling of the paws and suppressed the degeneration of articular cartilage. Expression levels of macrophage colony-stimulating factor (M-CSF), tissue inhibitors of metalloproteinases-1 (TIMP-1) and <em>complement</em> <em>component</em> <em>5a</em> (C5/C<em>5a</em>) were lower in the mice with arthritis compared with normal mice, however, following treatment with MCL and MTX, all the mice exhibited significant recovery to differing degrees. Unlike the MTX group, the MCL group failed to recover the level of soluble intercellular adhesion molecule-1. In addition, the cytokine of B-lymphocyte chemoattractant (BLC) solely presented in the MCL group. These results suggest that MCL may be considered for use as a novel therapeutic treatment against RA and that changes in the expression of cytokines C5/C<em>5a</em>, TIMP-1, M-CSF and BLC may underlie the mechanism by which MCL effects changes in this disease.

OBJECTIVE

Targeted, immune-modulating drugs are at the forefront of therapy for HS, and a comprehensive clinical trial registry is needed to facilitate data pooling and clinical efficacy comparison.

METHODS

A systematic review of the ClinicalTrials.gov database was searched for planned, in-progress, completed, or terminated trials investigating the effect of targeted biologic therapies for hidradenitis suppurativa (HS). When results of RCTs were not available, case reports or series were included.

RESULTS

Inflammatory mediators that are targeted by biologic agents include 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>). Clinical efficacy was measured by reduction in Sartorius score, Hidradenitis Suppurativa Clinical Response (HiSCR), Dermatology Life Quality Index (DLQI), or pain Visual Analog Scale (VAS). TNF inhibitors (adalimumab, etanercept, and infliximab), IL-1 receptor antagonist (Anakinra), IL-17A inhibitor (secukinumab), IL-12/23 inhibitor (ustekinumab), and PDE4 inhibitor (apremilast) show promise due to statistically significant improvements in disease severity.

CONCLUSIONS

Currently, adalimumab is the only FDA-approved biologic available for the treatment of HS. However, results from trials of other biologic agents targeting downstream mediators are promising. Large-scale, randomized, placebo-controlled trials in patients with skin of color, as well as weight-based dosing trials, are needed.

BACKGROUND

Main indications for liver transplantation in the pediatric population include biliary atresia and inherited metabolic diseases. The present study evaluated whether there are differences between pediatric patients undergoing living-related liver transplantation due to the two diseases in terms of their oxidative and immunological status during their regular outpatient follow-up visits.

METHODS

A clinical outpatient study measuring serum oxidative stress index (calculated as serum oxidant/antioxidant ratio, in the form of serum total hydroperoxide/serum biological antioxidative potential), serum terminal <em>complement</em> <em>component</em> <em>5a</em>, as an indicator of <em>complement</em> activity and immunological status, and transforming growth factor-ß1, as a marker of liver fibrosis, in 16 patients (6 males and 10 females, 2.5-15 years old) who received living-related liver transplantation due to inherited metabolic diseases (n=6; in the form of propionic acidemia [n=1], methylmalonic acidemia [n=1], arginase deficiency [n=1], tyrosinemia [n=2], and glycogen storage disease type 1b [n=1], with an age range of 2.4-14.6 years old) and due to biliary atresia ([n=10], with an age range of 2.9-14.5 years old).

RESULTS

Serum oxidative stress index, <em>complement</em> <em>component</em>-<em>5a</em>, and transforming growth factor-ß1 were significantly higher in the inherited metabolic diseases group than in the biliary atresia group. In all patients, serum oxidative stress index correlated positively with <em>complement</em> <em>component</em>-<em>5a</em> and transforming growth factor-ß1.

CONCLUSIONS

Patients who receive living-related liver transplantation due to inherited metabolic diseases are prone to higher oxidative stress, complement activity, and serum transforming growth factor-ß1.

The interaction of blood with artificial surfaces is of particular interest during hemodialysis treatments with extracorporeal blood circuits. <em>Components</em> of the extracorporeal blood circuit are known to have only a moderate, sometimes even an unfavorable hemocompatibility, and thus may provoke adverse biochemical or clinical sequelae. This article describes a newly established hemocompatibility assessment score. This score is based on on a standardized series of in vitro tests and is applied to commercially available hemodialysis membranes. It relates to a variety of membrane polymers, such as regenerated cellulose, diethylaminoethyl-modified cellulose, polyethersulfone/polyarylate blends and polysulfone. In order to compare different polymers used in the manufacturing of dialysis membranes, a set of the following hemocompatibility parameters was assessed and assembled to an overall score: generation of <em>complement</em> factor <em>5a</em>, thrombin-antithrombin III-complex, release of platelet factor 4, generation and release of elastase from polymorphonuclear granulocytes, and platelet count. With respect to these parameters, the results reveal major differences between the selected dialysis membranes. This new score model proves to be an efficient tool to derive objective results, and it may, thus, be used in the future to facilitate the selection of membrane polymers with an appropriate hemocompatibility pattern for dialysis therapy.

The canonical <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>) receptor (C<em>5a</em>R) 1 has well-described roles in tumorigenesis but the contribution of the second receptor, C<em>5a</em>R2, is unclear. The present study demonstrates that B16.F0 melanoma cells express mRNA for both <i>C<em>5a</em>R1</i> and <i>C<em>5a</em>R2</i> and signal through ERK and p38 MAPKs in response to C<em>5a</em>. Despite this, C<em>5a</em> had no impact on melanoma cell proliferation or migration <i>in vitro</i>. <i>In vivo</i> studies demonstrated that the growth of B16.F0 melanoma tumors was increased in C<em>5a</em>R2^-/- mice but reduced in C<em>5a</em>R1^-/- mice and wild-type mice treated with a C<em>5a</em>R1 antagonist. Analysis of tumor-infiltrating leukocyte populations showed no significant differences between wild-type and C<em>5a</em>R2^-/- mice. Conversely, percentages of myeloid-derived suppressor cells, macrophages, and regulatory T lymphocytes were lower in tumors from C<em>5a</em>R1^-/- mice, whereas total (CD3⁺) T lymphocytes and CD4⁺ subsets were higher. Analysis of cytokine and chemokine levels also showed plasma IFN-γ was higher and tumor C-C motif chemokine ligand 2 was lower in the absence of C<em>5a</em>R1. The results suggest that C<em>5a</em>R1 signaling supports melanoma growth by promoting infiltration of immunosuppressive leukocyte populations into the tumor microenvironment, whereas C<em>5a</em>R2 has a more restricted but beneficial role in limiting tumor growth. Overall, these data support the potential of C<em>5a</em>R1-inhibitory therapies for melanoma.-Nabizadeh, J. A., Manthey, H. D., Panagides, N., Steyn, F. J., Lee, J. D., Li, X. X., Akhir, F. N. M., Chen, W., Boyle, G. M., Taylor, S. M., Woodruff, T. M., Rolfe, B. E. C<em>5a</em> receptors C<em>5a</em>R1 and C<em>5a</em>R2 mediate opposing pathologies in a mouse model of melanoma.

<em>Complement</em> activation, an integral arm of innate immunity, may be the critical link to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Whereas we have previously reported elevated anaphylatoxins-<em>complement</em> <em>component</em> 3a (C3a) and <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>)-in IPF, which interact with TGF-β and augment epithelial injury in vitro, their role in IPF pathogenesis remains unclear. The objective of the current study is to determine the mechanistic role of the binding of C3a/C<em>5a</em> to their respective receptors (C3aR and C<em>5a</em>R) in the progression of lung fibrosis. In normal primary human fetal lung fibroblasts, C3a and C<em>5a</em> induces mesenchymal activation, matrix synthesis, and the expression of their respective receptors. We investigated the role of C3aR and C<em>5a</em>R in lung fibrosis by using bleomycin-injured mice with fibrotic lungs, elevated local C3a and C<em>5a</em>, and overexpression of their receptors via pharmacologic and RNA interference interventions. Histopathologic examination revealed an arrest in disease progression and attenuated lung collagen deposition (Masson's trichrome, hydroxyproline, collagen type I α 1 chain, and collagen type I α 2 chain). Pharmacologic or RNA interference-specific interventions suppressed <em>complement</em> activation (C3a and C<em>5a</em>) and soluble terminal <em>complement</em> complex formation (C5b-9) locally and active TGF-β1 systemically. C3aR/C<em>5a</em>R antagonists suppressed local mRNA expressions of tgfb2, tgfbr1/2, ltbp1/2, serpine1, tsp1, bmp1/4, pdgfbb, igf1, but restored the proteoglycan, dcn Clinically, compared with pathologically normal human subjects, patients with IPF presented local induction of C<em>5a</em>R, local and systemic induction of soluble C5b-9, and amplified expression of C3aR/C<em>5a</em>R in lesions. The blockade of C3aR and C<em>5a</em>R arrested the progression of fibrosis by attenuating local <em>complement</em> activation and TGF-β/bone morphologic protein signaling as well as restoring decorin, which suggests a promising therapeutic strategy for patients with IPF.-Gu, H., Fisher, A. J., Mickler, E. A., Duerson, F., III, Cummings, O. W., Peters-Golden, M., Twigg, H. L., III, Woodruff, T. M., Wilkes, D. S., Vittal, R. Contribution of the anaphylatoxin receptors, C3aR and C<em>5a</em>R, to the pathogenesis of pulmonary fibrosis.

<AbstractText>Macrophages can polarize to M2 phenotype to decrease inflammation and encourage tissue repair. Nonetheless, its role in sepsis-induced acute lung injury and its effect on endothelial cells (ECs) regeneration remains unknown. The aim of the current study was to explore the impact of M2 macrophages on pulmonary ECs proliferation in sepsis-induced acute lung injury.</AbstractText><AbstractText>We co-cultured mouse lung microvascular endothelial cells (MLMVECs) with M2 macrophages following LPS challenge. M2 macrophages were intratracheally transplanted into mice subjected to cecal ligation and puncture (CLP). We further performed cytokine array for the supernatant from M2 macrophages and serum from mice subjected with CLP.</AbstractText><p><div><b>Results</b></div>We found both co-culture with M2 macrophages and treating with supernatant from M2 macrophages increased ECs viability following LPS challenge. Intratracheal transplantation of M2 macrophages markedly promoted pulmonary ECs proliferation, manifesting as attenuation of lung microvascular permeability and lung tissue edema, as well as improvement of survival rate. We further found that CXCL12, IL-1ra, TIMP-1, IL-4, and CXCL1 were increased in the supernatant of M2 macrophages <i>in vitro</i>. G-CSF and <em>Complement</em> <em>Component</em> <em>5a</em> (C5/C<em>5a</em>) were increased in the serum of the M2-transplanted mice.</p><AbstractText>The present study suggested M2 macrophages could promote ECs proliferation in sepsis-induced ALI through secretion of anti-inflammatory cytokines and growth factors.</AbstractText>

<em>Complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>) is a 74 amino acid glycoprotein and an important proinflammatory mediator that is cleaved enzymatically from its precursor, C5, on activation of the <em>complement</em> cascade. C<em>5a</em> is quickly metabolised by carboxypeptidases, forming the less-potent C<em>5a</em> desArg. C<em>5a</em> and C<em>5a</em> desArg interact with their receptors (C<em>5a</em>R and C5L2), which results in a number of effects which are essential to the immune response. C<em>5a</em> has a broad range of biological effects throughout the human body because the widespread expression of C<em>5a</em> receptors throughout the human organs enables C<em>5a</em> and C<em>5a</em> desArg to elicit a broad range of biological effects. Recently, accumulating evidence in humans and experimental animal models shows that the C<em>5a</em>-C<em>5a</em>R axis is involved in the development of atherosclerosis lesions. The absence or blockade of C<em>5a</em>Rs greatly reduces the formation of atherosclerotic lesions or wire-injury-induced neointima formation in atherosclerosis-prone mice. Serum C<em>5a</em> level was related to the major adverse cardiovascular events in patients with advanced atherosclerosis and those with drug-eluting stent implantation. Thus, the C<em>5a</em>-C<em>5a</em>R axis may be a significant pathogenic driver of arteriosclerotic vascular disease, making C<em>5a</em>-C<em>5a</em>R inhibition an attractive therapeutic strategy.

The efficacy of antitumoral responses can be increased using combinatorial vaccine strategies. We recently showed that vaccination could be optimized by local administration of diverse molecular or bacterial agents to target and augment antitumoral CD8 T cells in the genital mucosa (GM) and increase regression of cervical cancer in an animal model. Non muscle-invasive bladder cancer is another disease that is easily amenable to local therapies. In contrast to data obtained in the GM, in this study we show that intravesical (IVES) instillation of synthetic toll-like receptor (TLR) agonists only modestly induced recruitment of CD8 T cells to the bladder. However, IVES administration of Ty21a, a live bacterial vaccine against typhoid fever, was much more effective and increased the number of total and vaccine-specific CD8 T cells in the bladder approximately 10 fold. Comparison of chemokines induced in the bladder by either CpG (a TLR-9 agonist) or Ty21a highlighted the preferential increase in <em>complement</em> <em>component</em> <em>5a</em>, CXCL5, CXCL2, CCL8, and CCL5 by Ty21a, suggesting their involvement in the attraction of T cells to the bladder. IVES treatment with Ty21a after vaccination also significantly increased tumor regression compared to vaccination alone, resulting in 90% survival in an orthotopic murine model of bladder cancer expressing a prototype tumor antigen. Our data demonstrate that combining vaccination with local immunostimulation may be an effective treatment strategy for different types of cancer and also highlight the great potential of the Ty21a vaccine, which is routinely used worldwide, in such combinatorial therapies.

OBJECTIVE

The macrophage scavenger receptor 1 (Msr1, also called SRA) is a pattern recognition receptor primarily expressed on myeloid cells, which plays an important role in the maintenance of immune homeostasis. Since MSR1 expression was upregulated in the livers of patients with fulminant hepatitis (FH), we investigated the functional mechanism of Msr1 in FH pathogenesis.

METHODS

Msr1-deficient (Msr1-/-) mice and their wild-type (WT) littermates were infected with mouse hepatitis virus strain-A59 (MHV-A59) to induce FH, and the levels of tissue damage, serum alanine aminotransferase, inflammatory cytokines and <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>) were measured and compared. Liver injury was studied after MHV infection with or without neutrophil depletion.

RESULTS

Our results showed that Msr1-/- mice were resistant to MHV-induced hepatitis. Treatment with the C<em>5a</em> receptor antagonist (C<em>5a</em>Ra) diminished the differences in inflammatory responses and liver injury between MHV-infected wild-type and Msr1-/- mice, suggesting that C<em>5a</em>-induced pro-inflammatory response plays a critical role in the Msr1-mediated regulation of FH pathogenesis. We demonstrated that Msr1 efficiently enhanced transforming growth factor-activated kinase-1 phosphorylation in neutrophils upon MHV-A59 stimulation, thereby promoting the activation of the extracellular signal-regulated kinase pathway and subsequent NETosis formation. Moreover, we provided evidence that blockage of Msr1 attenuated the liver damage caused by MHV-A59 infection.

CONCLUSIONS

Msr1 promotes the pathogenesis of virus-induced FH by enhancing induction of neutrophil NETosis and subsequent complement activation. Targeting Msr1 may be employed as a new immunotherapeutic strategy for FH.

UNASSIGNED

Virus-induced fulminant hepatitis (FH) is a disease with a high mortality worldwide. Enhanced levels of macrophage scavenger receptor 1 (Msr1) in the liver of patients with FH and of murine experimental FH indicated Msr1 plays a role in the pathogenesis of FH. Herein, we demonstrate that mice deficient in Msr1 are resistant to FH induced by MHV-A59, and the Msr1 inhibitor fucoidan suppresses the progression of FH in mice. Our study suggests that use of drugs inhibiting MSR1 function could be beneficial to patients with FH.

Neutrophil granulocytes constitute the main <em>component</em> of innate immunity in the clearance of bacterial infections. However, during systemic inflammation, immunoparalysis may occur resulting in neutrophil dysfunction. This study presents a new in vitro model for analyzing the dysfunction of human peripheral blood neutrophils resulting from the interaction with Staphylococcus aureus <em>components</em> in whole blood. After induction of a massive <em>complement</em> activation by S. aureus supernatant, the neutrophils exhibit a reduced phagocytic capacity resulting in a dramatic reduction of the antibacterial activity similar to that of neutrophils isolated from septic patients. The number of phagocytozing neutrophils is drastically reduced as well as the phagocytic capacity designated by a significantly lower number of ingested microbes. This dysfunction correlates with the loss of <em>complement</em> <em>component</em> <em>5a</em> receptor 1 from the neutrophil cell surface and can be further characterized by a C<em>5a</em>-induced CD66b overexpression. The presented in vitro model offers a new platform for preclinical testing of immunosuppressive drugs and delivers new information for the understanding of neutrophil dysfunctions under the conditions described.

A large body of evidence supports a central role for <em>complement</em> activation in the pathobiology of age-related macular degeneration (AMD), including plasma <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>). Interestingly, C<em>5a</em> is a chemotactic agent for monocytes, a cell type also shown to contribute to AMD. However, the role monocytes play in the pathogenesis of "dry" AMD and the pharmacologic potential of targeting C<em>5a</em> to regulate these cells are unclear. We addressed these questions via C<em>5a</em> blockade in a unique model of early/intermediate dry AMD and large panel flow cytometry to immunophenotype monocytic involvement.

Heterozygous <em>complement</em> factor H (Cfh+/-) mice aged to 90 weeks were fed a high-fat, cholesterol-enriched diet (Cfh+/-∼HFC) for 8 weeks and were given weekly intraperitoneal injections of 30 mg/kg anti-C<em>5a</em> (4C9, Pfizer). Flow cytometry, retinal pigmented epithelium (RPE) flat mounts, and electroretinograms were used to characterize anti-C<em>5a</em> treatment.

Aged Cfh+/- mice developed RPE damage, sub-RPE basal laminar deposits, and attenuation of visual function and immune cell recruitment to the choroid that was accompanied by expression of inflammatory and extracellular matrix remodeling genes following 8 weeks of HFC diet. Concomitant systemic administration of an anti-C<em>5a</em> antibody successfully inhibited local recruitment of mononuclear phagocytes to the choroid-RPE interface but did not ameliorate these AMD-like pathologies in this mouse model.

These results show that immunotherapy targeting C<em>5a</em> is not sufficient to block the development of the AMD-like pathologies observed in Cfh+/-∼HFC mice and suggest that other <em>complement</em> <em>component</em>s or molecules/mechanisms may be driving "early" and "intermediate" AMD pathologies.