Aliskiren inhibits renin-mediated complement activation.

Certain kidney diseases are associated with complement activation although a renal triggering factor has not been identified. Here we demonstrated that renin, a kidney-specific enzyme, cleaves C3 into C3b and C3a, in a manner identical to the C3 convertase. Cleavage was specifically blocked by the renin inhibitor aliskiren. Renin-mediated C3 cleavage and its inhibition by aliskiren also occurred in serum. Generation of C3 cleavage products was demonstrated by immunoblotting, detecting the cleavage product C3b, by N-terminal sequencing of the cleavage product, and by ELISA for C3a release. Functional assays showed mast cell chemotaxis towards the cleavage product C3a and release of factor Ba when the cleavage product C3b was combined with factor B and factor D. The renin-mediated C3 cleavage product bound to factor B. In the presence of aliskiren this did not occur, and less C3 deposited on renin-producing cells. The effect of aliskiren was studied in three patients with dense deposit disease and this demonstrated decreased systemic and renal complement activation (increased C3, decreased C3a and C5a, decreased renal C3 and C5b-9 deposition and/or decreased glomerular basement membrane thickness) over a follow-up period of four to seven years. Thus, renin can trigger complement activation, an effect inhibited by aliskiren. Since renin concentrations are higher in renal tissue than systemically, this may explain the renal propensity of complement-mediated disease in the presence of complement mutations or auto-antibodies.

The combined role of galactose-deficient IgA1 and streptococcal IgA-binding M Protein in inducing IL-6 and C3 secretion from human mesangial cells: implications for IgA nephropathy.

IgA nephropathy (IgAN) is characterized by mesangial cell proliferation and extracellular matrix expansion associated with immune deposits consisting of galactose-deficient polymeric IgA1 and C3. We have previously shown that IgA-binding regions of streptococcal M proteins colocalize with IgA in mesangial immune deposits in patients with IgAN. In the present study, the IgA-binding M4 protein from group A Streptococcus was found to bind to galactose-deficient polymeric IgA1 with higher affinity than to other forms of IgA1, as shown by surface plasmon resonance and solid-phase immunoassay. The M4 protein was demonstrated to bind to mesangial cells not via the IgA-binding region but rather via the C-terminal region, as demonstrated by flow cytometry. IgA1 enhanced binding of M4 to mesangial cells, but not vice versa. Costimulation of human mesangial cells with M4 and galactose-deficient polymeric IgA1 resulted in a significant increase in IL-6 secretion compared with each stimulant alone. Galactose-deficient polymeric IgA1 alone, but not M4, induced C3 secretion from the cells, and costimulation enhanced this effect. Additionally, costimulation enhanced mesangial cell proliferation compared with each stimulant alone. These results indicate that IgA-binding M4 protein binds preferentially to galactose-deficient polymeric IgA1 and that these proteins together induce excessive proinflammatory responses and proliferation of human mesangial cells. Thus, tissue deposition of streptococcal IgA-binding M proteins may contribute to the pathogenesis of IgAN.

Identification of molecular mechanisms used by Finegoldia magna to penetrate and colonize human skin.

Finegoldia magna is a Gram-positive anaerobic commensal of the human skin microbiota, but also known to act as an opportunistic pathogen. Two primary virulence factors of F. magna are the subtilisin-like extracellular serine protease SufA and the adhesive protein FAF. This study examines the molecular mechanisms F. magna uses when colonizing or establishing an infection in the skin. FAF was found to be essential in the initial adherence of F. magna to human skin biopsies. In the upper layers of the epidermis FAF mediates adhesion through binding to galectin-7 - a keratinocyte cell marker. Once the bacteria moved deeper into the skin to the basement membrane layer, SufA was found to degrade collagen IV which forms the backbone structure of the basement membrane. It also degraded collagen V, whereby F. magna could reach deeper dermal tissue sites. In the dermis, FAF interacts with collagen V and fibrillin, which presumably helps the bacteria to establish infection in this area. The findings of this study paint a clear picture of how F. magna interacts with human skin and explain how it is such a successful opportunistic pathogen in chronic wounds and ulcers.

A novel role for pro-coagulant microvesicles in the early host defense against streptococcus pyogenes.

Previous studies have shown that stimulation of whole blood or peripheral blood mononuclear cells with bacterial virulence factors results in the sequestration of pro-coagulant microvesicles (MVs). These particles explore their clotting activity via the extrinsic and intrinsic pathway of coagulation; however, their pathophysiological role in infectious diseases remains enigmatic. Here we describe that the interaction of pro-coagulant MVs with bacteria of the species Streptococcus pyogenes is part of the early immune response to the invading pathogen. As shown by negative staining electron microscopy and clotting assays, pro-coagulant MVs bind in the presence of plasma to the bacterial surface. Fibrinogen was identified as a linker that, through binding to the M1 protein of S. pyogenes, allows the opsonization of the bacteria by MVs. Surface plasmon resonance analysis revealed a strong interaction between pro-coagulant MVs and fibrinogen with a KD value in the nanomolar range. When performing a mass-spectrometry-based strategy to determine the protein quantity, a significant up-regulation of the fibrinogen-binding integrins CD18 and CD11b on pro-coagulant MVs was recorded. Finally we show that plasma clots induced by pro-coagulant MVs are able to prevent bacterial dissemination and possess antimicrobial activity. These findings were confirmed by in vivo experiments, as local treatment with pro-coagulant MVs dampens bacterial spreading to other organs and improved survival in an invasive streptococcal mouse model of infection. Taken together, our data implicate that pro-coagulant MVs play an important role in the early response of the innate immune system in infectious diseases.

p33 (gC1q receptor) prevents cell damage by blocking the cytolytic activity of antimicrobial peptides.

The innate immune system is the first line of defense against invading microbes. Its specificity relies a great deal on host pattern recognition molecules that sense pathogen-associated molecular patterns of the invading pathogen. However, full protection is not always guaranteed, and some early defense mechanisms involved in bacterial killing, such as the complement system, can also exert cytolytic activity against host cells. Although these cascades are tightly regulated, the host has to take additional precautions to prevent its cell destruction. In this study, we describe that p33, a negatively charged surface protein found on endothelial cells also known as gC1q receptor, protects host cells from a cytolytic attack by antimicrobial peptides (AMPs), such as LL37 and ß-defensin 3. To this end, we characterized the interaction of p33 with AMPs by biochemical and functional means. Our data show that p33 forms a doughnut-shaped trimer that can bind up to three AMPs, and we identified a segment in p33 forming a ß-sheet that mediates the binding to all AMPs. Moreover, our results show that p33 abolishes the lytic activity of AMPs at an equimolar ratio, and it protects endothelial cells and erythrocytes from AMP-induced lysis. Taken together, our data suggest a novel protective mechanism of p33 in modulating innate immune response by neutralizing cytotoxic AMPs at the host cell surface.

A novel C3 mutation causing increased formation of the C3 convertase in familial atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome has been associated with dysregulation of the alternative complement pathway. In this study, a novel heterozygous C3 mutation was identified in a factor B-binding region in exon 41, V1636A (4973 T > C). The mutation was found in three family members affected with late-onset atypical hemolytic uremic syndrome and symptoms of glomerulonephritis. All three patients exhibited increased complement activation detected by decreased C3 levels and glomerular C3 deposits. Platelets from two of the patients had C3 and C9 deposits on the cell surface. Patient sera exhibited more C3 cleavage and higher levels of C3a. The C3 mutation resulted in increased C3 binding to factor B and increased net formation of the C3 convertase, even after decay induced by decay-accelerating factor and factor H, as assayed by surface plasmon resonance. Patient sera incubated with washed human platelets induced more C3 and C9 deposition on the cell surface in comparison with normal sera. More C3a was released into serum over time when washed platelets were exposed to patient sera. Results regarding C3 and C9 deposition on washed platelets were confirmed using purified patient C3 in C3-depleted serum. The results indicated enhanced convertase formation leading to increased complement activation on cell surfaces. Previously described C3 mutations showed loss of function with regard to C3 binding to complement regulators. To our knowledge, this study presents the first known C3 mutation inducing increased formation of the C3 convertase, thus explaining enhanced activation of the alternative pathway of complement.

High expression of midkine in the airways of patients with cystic fibrosis.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene result in impaired host defense during cystic fibrosis (CF), where Pseudomonas aeruginosa becomes a key pathogen. We investigated the expression pattern of the antibacterial growth factor midkine (MK) in CF and the possible interference with its activity by the altered airway microenvironment. High MK expression was found in CF lung tissue compared with control samples, involving epithelia of the large and small airways, alveoli, and cells of the submucosa (i.e., neutrophils and mast cells). In CF sputum, MK was present at 100-fold higher levels, but was also subject to increased degradation, compared with MK in sputum from healthy control subjects. MK exerted a bactericidal effect on P. aeruginosa, but increasing salt concentrations and low pH impaired this activity. Molecular modeling suggested that the effects of salt and pH were attributable to electrostatic screening and a charge-neutralization of the membrane, respectively. Both the neutrophil elastase and elastase of P. aeruginosa cleaved MK to smaller fragments, resulting in impaired bactericidal activity. Thus, MK is highly expressed in CF, but its bactericidal properties may be impaired by the altered microenvironment, as reflected by the in vitro conditions used in this study.

Streptococcal surface proteins activate the contact system and control its antibacterial activity.

Group G streptococci (GGS) are important bacterial pathogens in humans. Here, we investigated the interactions between GGS and the contact system, a procoagulant and proinflammatory proteolytic cascade that, upon activation, also generates antibacterial peptides. Two surface proteins of GGS, protein FOG and protein G (PG), were found to bind contact system proteins. Experiments utilizing contact protein-deficient human plasma and isogenic GGS mutant strains lacking FOG or PG showed that FOG and PG both activate the procoagulant branch of the contact system. In contrast, only FOG induced cleavage of high molecular weight kininogen, generating the proinflammatory bradykinin peptide and additional high molecular weight kininogen fragments containing the antimicrobial peptide NAT-26. On the other hand, PG protected the bacteria against the antibacterial effect of NAT-26. These findings underline the significance of the contact system in innate immunity and demonstrate that GGS have evolved surface proteins to exploit and modulate its effects.

Midkine is expressed and differentially processed during chronic obstructive pulmonary disease exacerbations and ventilator-associated pneumonia associated with Staphylococcus aureus infection.

Staphylococcus aureus is sometimes isolated from the airways during acute exacerbations of chronic obstructive pulmonary disease (COPD) but more commonly recognized as a cause of ventilator-associated pneumonia (VAP). Antimicrobial proteins, among them midkine (MK), are an important part of innate immunity in the airways. In this study, the levels and possible processing of MK in relation to S. aureus infection of the airways were investigated, comparing COPD and VAP, thus comparing a state of disease with preceding chronic inflammation and remodeling (COPD) with acute inflammation (that is, VAP). MK was detected in the small airways and alveoli of COPD lung tissue but less so in normal lung tissue. MK at below micromolar concentrations killed S. aureus in vitro. Proteolytic processing of MK by the staphylococcal metalloprotease aureolysin (AL), but not cysteine protease staphopain A (SA), resulted in impaired bactericidal activity. Degradation was seen foremost in the COOH-terminal portion of the molecule that harbors high bactericidal activity. In addition, MK was detected in sputum from patients suffering from VAP caused by S. aureus but less so in sputum from COPD exacerbations associated with the same bacterium. Recombinant MK was degraded more rapidly in sputum from the COPD patients than from the VAP patients and a greater proteolytic activity in COPD sputum was confirmed by zymography. Taken together, proteases of both bacteria and the host contribute to degradation of the antibacterial protein MK, resulting in an impaired defense of the airways, in particular, in COPD where the state of chronic inflammation could be of importance.

Coagulation, an ancestral serine protease cascade, exerts a novel function in early immune defense.

Phylogenetically conserved serine protease cascades play an important role in invertebrate and vertebrate immunity. The mammalian coagulation system can be traced back some 400 million years and shares homology with ancestral serine proteinase cascades that are involved in, for example, Toll receptor signaling in insects and release of antimicrobial peptides during hemolymph clotting. In the present study, we show that the induction of coagulation by bacteria leads to immobilization and killing of Streptococcus pyogenes bacteria inside the clot. The entrapment is mediated via cross-linking of bacteria to fibrin fibers by the action of coagulation factor XIII (fXIII), an evolutionarily conserved transglutaminase. In a streptococcal skin infection model, fXIII(-/-) mice developed severe signs of pathologic inflammation at the local site of infection, and fXIII treatment of wild-type animals dampened bacterial dissemination during early infection. Bacterial killing and cross-linking to fibrin networks was also detected in tissue biopsies from patients with streptococcal necrotizing fasciitis, supporting the concept that coagulation is part of the early innate immune system.

IgG glycan hydrolysis by endoglycosidase S diminishes the proinflammatory properties of immune complexes from patients with systemic lupus erythematosus: a possible new treatment?

OBJECTIVE: Systemic lupus erythematosus (SLE) is an autoimmune disease with chronic or episodic inflammation in several organ systems, related to the presence of circulating and tissue-deposited immune complexes (ICs) that stimulate leukocytes through Fcγ receptors (FcγR) with subsequent inflammation. Treatment with endoglycosidase S (EndoS), an IgG glycan-hydrolyzing bacterial enzyme from Streptococcus pyogenes, has shown beneficial effects in several experimental animal models of chronic inflammatory disease. This study was undertaken to investigate whether EndoS affects the proinflammatory properties of ICs and has the potential to be developed as a therapy for SLE. METHODS: ICs purified from SLE patients or RNA-containing ICs formed in vitro were treated with EndoS and used in several assays reflecting different important features of SLE pathogenesis, such as phagocytosis by polymorphonuclear cells (PMNs) and plasmacytoid dendritic cells (PDCs), complement activation, and interferon-α (IFNα) production by PDCs. RESULTS: EndoS treatment abolished all proinflammatory properties of the ICs investigated. This included FcγR-mediated phagocytosis by PDCs (P = 0.001) and subsequent production of IFNα (P = 0.002), IC-induced classical pathway of complement activation (P = 0.008), chemotaxis, and oxidative burst activity of PMNs (P = 0.002). EndoS treatment also had a direct effect on the molecular structure of ICs, causing decreased IC size and glycosylation. CONCLUSION: Our findings indicate that EndoS treatment has prominent effects on several pathogenetically important IC-mediated events, and suggest that EndoS has the potential to be developed as a novel therapy for SLE.

Constitutive and inflammation-dependent antimicrobial peptides produced by epithelium are differentially processed and inactivated by the commensal Finegoldia magna and the pathogen Streptococcus pyogenes.

Epithelial linings serve as physical barriers and produce antimicrobial peptides (AMPs) to maintain host integrity. Examples are the bactericidal proteins midkine (MK) and BRAK/CXCL14 that are constitutively produced in the skin epidermal layer, where the anaerobic Gram-positive coccoid commensal Finegoldia magna resides. Consequently, this bacterium is likely to encounter both MK and BRAK/CXCL14, making these molecules possible threats to its habitat. In this study, we show that MK expression is upregulated during inflammation, concomitant with a strong downregulation of BRAK/CXCL14, resulting in changed antibacterial conditions. MK, BRAK/CXCL14, and the inflammation-dependent antimicrobial ß-defensins human ß-defensin (hBD)-2 and hBD-3 all showed bactericidal activity against both F. magna and the virulent pathogen Streptococcus pyogenes at similar concentrations. SufA, a released protease of F. magna, degraded MK and BRAK/CXCL14 but not hBD-2 nor hBD-3. Cleavage was seen at lysine and arginine residues, amino acids characteristic of AMPs. Intermediate SufA-degraded fragments of MK and BRAK/CXCL14 showed stronger bactericidal activity against S. pyogenes than F. magna, thus promoting survival of the latter. In contrast, the cysteine-protease SpeB of S. pyogenes rapidly degraded all AMPs investigated. The proteins FAF and SIC, released by F. magna and S. pyogenes, respectively, neutralized the antibacterial activity of MK and BRAK/CXCL14, protein FAF being the most efficient. Quantitation and colocalization by immunoelectron microscopy demonstrated significant levels and interactions of the molecules in in vivo and ex vivo samples. The findings reflect strategies used by a permanently residing commensal and a virulent pathogen, the latter operating during the limited time course of invasive disease.

Peptidylarginine deiminases present in the airways during tobacco smoking and inflammation can citrullinate the host defense peptide LL-37, resulting in altered activities.

Bacterial colonization of the lower respiratory tract is frequently seen in chronic obstructive pulmonary disease (COPD), and may cause exacerbations leading to disease progression. Antimicrobial peptides comprise an important part of innate lung immunity, and not least the cathelicidin human cationic antimicrobial protein-18/LL-37. Peptidylarginine deiminases (PADIs) post-translationally modify proteins by converting cationic peptidylarginine residues to neutral peptidylcitrulline. An increased presence of PADI2 and citrullinated proteins was demonstrated in the lungs of smokers. In this study, preformed PADI4, stored in granulocytes and extracellularly in the lumina of bronchi, was found in lung tissue of individuals suffering from COPD. In vitro, recombinant human PADI2 and PADI4 both caused a time- and dose-dependent citrullination of LL-37. The citrullination resulted in impaired antibacterial activity against Staphylococcus aureus, Streptococcus pneumoniae, and nontypable Haemophilus influenzae, but less so against Pseudomonas aeruginosa. Using artificial lipid bilayers, we observed discrete differences when comparing the disrupting activity of native and citrullinated LL-37, suggesting that differences in cell wall composition are important during interactions with whole bacteria. Furthermore, citrullinated LL-37 showed higher chemotactic activity against mononuclear leukocytes than did native LL-37, but was less efficient at neutralizing lipolysaccharide, and also in converting apoptotic neutrophils into a state of secondary necrosis. In addition, citrullinated LL-37 was more prone to degradation by proteases, whereas the V8 endopetidase of S. aureus cleaved the modified peptide at additional sites, compared with native LL-37. Together, these findings demonstrate novel mechanisms whereby the inflammation-dependent deiminases PADI2 and PADI4 can alter the activites of antibacterial polypeptides, affecting the course of inflammatory disorders such as COPD.

Binding of albumin promotes bacterial survival at the epithelial surface.

Human serum albumin (HSA) is the dominating protein in human plasma. Many bacterial species, especially streptococci, express surface proteins that bind HSA with high specificity and affinity, but the biological consequences of these protein-protein interactions are poorly understood. Group G streptococci (GGS), carrying the HSA-binding protein G, colonize the skin and the mucosa of the upper respiratory tract, mostly without causing disease. In the case of bacterial invasion, pro-inflammatory cytokines are released that activate the epithelium to produce antibacterial peptides, in particular the chemokine MIG/CXCL9. In addition, the inflammation causes capillary leakage and extravasation of HSA and other plasma proteins, environmental changes at the epithelial surface to which the bacteria need to respond. In this study, we found that GGS adsorbed HSA from both saliva and plasma via binding to protein G and that HSA bound to protein G bound and inactivated the antibacterial MIG/CXCL9 peptide. Another surface protein of GGS, FOG, was found to mediate adherence of the bacteria to pharyngeal epithelial cells through interaction with glycosaminoglycans. This adherence was not affected by activation of the epithelium with a combination of IFN-γ and TNF-α, leading to the production of MIG/CXCL9. However, at the activated epithelial surface, adherent GGS were protected against killing by MIG/CXCL9 through protein G-dependent HSA coating. The findings identify a previously unknown bacterial survival strategy that helps to explain the evolution of HSA-binding proteins among bacterial species of the normal human microbiota.

Human Siglec-5 inhibitory receptor and immunoglobulin A (IgA) have separate binding sites in streptococcal beta protein.

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are receptors believed to be important for regulation of cellular activation and inflammation. Several pathogenic microbes bind specific Siglecs via sialic acid-containing structures at the microbial surface, interactions that may result in modulation of host responses. Recently, it was shown that the group B Streptococcus (GBS) binds to human Siglec-5 (hSiglec-5), an inhibitory receptor expressed on macrophages and neutrophils, via the IgA-binding surface ß protein, providing the first example of a protein/protein interaction between a pathogenic microbe and a Siglec. Here we show that the hSiglec-5-binding part of ß resides in the N-terminal half of the protein, which also harbors the previously determined IgA-binding region. We constructed bacterial mutants expressing variants of the ß protein with non-overlapping deletions in the N-terminal half of the protein. Using these mutants and recombinant ß fragments, we showed that the hSiglec-5-binding site is located in the most N-terminal part of ß (B6N region; amino acids 1-152) and that the hSiglec-5- and IgA-binding domains in ß are completely separate. We showed with BIAcore(TM) analysis that tandem variants of the hSiglec-5- and IgA-binding domains bind to their respective ligands with high affinity. Finally, we showed that the B6N region, but not the IgA-binding region of ß, triggers recruitment of the tyrosine phosphatase SHP-2 to hSiglec-5 in U937 monocytes. Taken together, we have identified and isolated the first microbial non-sialic acid Siglec-binding region that can be used as a tool in studies of the ß/hSiglec-5 interaction.

Midkine and pleiotrophin have bactericidal properties: preserved antibacterial activity in a family of heparin-binding growth factors during evolution.

Antibacterial peptides of the innate immune system combat pathogenic microbes, but often have additional roles in promoting inflammation and as growth factors during tissue repair. Midkine (MK) and pleiotrophin (PTN) are the only two members of a family of heparin-binding growth factors. They show restricted expression during embryogenesis and are up-regulated in neoplasia. In addition, MK shows constitutive and inflammation-dependent expression in some non-transformed tissues of the adult. In the present study, we show that both MK and PTN display strong antibacterial activity, present at physiological salt concentrations. Electron microscopy of bacteria and experiments using artificial lipid bilayers suggest that MK and PTN exert their antibacterial action via a membrane disruption mechanism. The predicted structure of PTN, employing the previously solved MK structure as a template, indicates that both molecules consist of two domains, each containing three antiparallel beta-sheets. The antibacterial activity was mapped to the unordered C-terminal tails of both molecules and the last beta-sheets of the N-terminals. Analysis of the highly conserved MK and PTN orthologues from the amphibian Xenopus laevis and the fish Danio rerio suggests that they also harbor antibacterial activity in the corresponding domains. In support of an evolutionary conserved function it was found that the more distant orthologue, insect Miple2 from Drosophila melanogaster, also displays strong antibacterial activity. Taken together, the findings suggest that MK and PTN, in addition to their earlier described activities, may have previously unrealized important roles as innate antibiotics.

Treatment of invasive streptococcal infection with a peptide derived from human high-molecular weight kininogen.

Sepsis and septic shock remain an important medical problem, emphasizing the need to identify novel therapeutic opportunities. Hypovolemic hypotension, coagulation dysfunction, disturbed microcirculation, and multiorgan failure resulting from vascular leakage are often observed in these severe conditions. In the present study, we find that HKH20, a peptide derived from human high molecular weight kininogen (HK), down-regulates inflammatory reactions caused by Streptococcus pyogenes in a mouse model of sepsis. HK is a component of the pro-inflammatory and pro-coagulant contact system. Activation of the contact system in the bloodstream by S pyogenes leads to massive tissue damage in the lungs of the infected mice, which eventually results in the death of the animals. HKH20 inhibits activation of the contact system and protects mice with invasive S pyogenes infection from lung damage. In combination with clindamycin treatment, the peptide also significantly prolongs the survival of infected mice.

Thymic stromal lymphopoietin exerts antimicrobial activities.

Thymic stromal lymphopoietin (TSLP) is an interleukin-7-like cytokine expressed by epithelial cells and reported to be involved in allergic diseases and atopic eczema. The presence of several predicted α-helical regions in TSPL, a structure characterizing many classical antimicrobial peptides (AMPs), prompted us to investigate whether TSLP exerts antimicrobial activities. Recombinant human TSLP exerted antimicrobial activity, particularly against Gram-negative bacteria. Using synthetic overlapping peptide 20-mers of TSLP, it was demonstrated that the antimicrobial effect is primarily mediated by the C-terminal region of the protein. MKK34 (MKKRRKRKVTTNKCLEQVSQLQGLWRRFNRPLLK), a peptide spanning a C-terminal α-helical region in TSLP, showed potent antimicrobial activities, in physiological salt conditions and in the presence of human plasma. Fluorescent studies of peptide-treated bacteria, electron microscopy and liposome leakage models showed that MKK34 exerted membrane-disrupting effects comparable to those of the classical AMP LL-37. Moreover, TSLP was degraded into multiple fragments by staphylococcal V8 proteinase. One major antimicrobial degradation fragment was found to encompass the C-terminal antimicrobial region defined by the MKK34 peptide. We here describe a novel antimicrobial role for TSLP. The antimicrobial activity is primarily mediated by the C-terminal part of the protein. In combination with the previously known cytokine function of TSLP, our result indicates dual functions of the molecule and a previously unknown role in host defense.

SufA of the opportunistic pathogen finegoldia magna modulates actions of the antibacterial chemokine MIG/CXCL9, promoting bacterial survival during epithelial inflammation.

The anaerobic bacterium Finegoldia magna is part of the human commensal microbiota, but is also an important opportunistic pathogen. This bacterium expresses a subtilisin-like serine proteinase, SufA, which partially degrade the antibacterial chemokine MIG/CXCL9. Here, we show that MIG/CXCL9 is produced by human keratinocytes in response to inflammatory stimuli. In contrast to the virulent human pathogen Streptococcus pyogenes, the presence of F. magna had no enhancing effect on the MIG/CXCL9 expression by keratinocytes, suggesting poor detection of the latter by pathogen-recognition receptors. When MIG/CXCL9 was exposed to SufA-expressing F. magna, the molecule was processed into several smaller fragments. Analysis by mass spectrometry showed that SufA cleaves MIG/CXCL9 at several sites in the COOH-terminal region of the molecule. At equimolar concentrations, SufA-generated MIG/CXCL9 fragments were not bactericidal against F. magna, but retained their ability to kill S. pyogenes. Moreover, the SufA-generated MIG/CXCL9 fragments were capable of activating the angiostasis-mediating CXCR3 receptor, which is expressed on endothelial cells, in an order of magnitude similar to that of intact MIG/CXCL9. F. magna expresses a surface protein called FAF that is released from the bacterial surface by SufA. Soluble FAF was found to bind and inactivate the antibacterial activity of MIG/CXCL9, thereby further potentially promoting the survival of F. magna. The findings suggest that SufA modulation of the inflammatory response could be a mechanism playing an important role in creating an ecologic niche for F. magna, decreasing antibacterial activity and suppressing angiogenesis, thus providing advantage in survival for this anaerobic opportunist compared with competing pathogens during inflammation.

Antibodies against a surface protein of Streptococcus pyogenes promote a pathological inflammatory response.

Streptococcal toxic shock syndrome (STSS) caused by Streptococcus pyogenes is a clinical condition with a high mortality rate despite modern intensive care. A key feature of STSS is excessive plasma leakage leading to hypovolemic hypotension, disturbed microcirculation and multiorgan failure. Previous work has identified a virulence mechanism in STSS where M1 protein of S. pyogenes forms complexes with fibrinogen that activate neutrophils to release heparin-binding protein (HBP), an inducer of vascular leakage. Here, we report a marked inter-individual difference in the response to M1 protein-induced HBP release, a difference found to be related to IgG antibodies directed against the central region of the M1 protein. To elicit massive HBP release, such antibodies need to be part of the M1 protein-fibrinogen complexes. The data add a novel aspect to bacterial pathogenesis where antibodies contribute to the severity of disease by promoting a pathologic inflammatory response.