Variable region in streptococcal M-proteins provides stable binding with host fibrinogen for plasminogen-mediated bacterial invasion.

Dimeric M-proteins (M-Prt) in group A Streptococcus pyogenes (GAS) are surface-expressed virulence factors implicated in processes that contribute to the pathogenicity of infection. Sequence analyses of various GAS M-Prts have shown that they contain a highly conserved sortase A-dependent cell wall-anchored C terminus, whereas the surface-exposed N terminus is highly variable, a feature used for identification and serotyping of various GAS strains. This variability also allows for strain-specific responses that suppress host defenses. Previous studies have indeed identified the N-terminal M-Prt B-domain as the site interacting with antiphagocytotic human-host fibrinogen (hFg). Herein, we show that hFg strongly interacts with M-Prts containing highly variable B-domains. We further demonstrate that specific GAS clinical isolates display high affinity for the D-domain of hFg, and this interaction allowed for subsequent surface binding of human-host plasminogen (hPg) to the E-domain of hFg. This GAS surface-bound hPg is then activated by GAS-secreted streptokinase, leading to the generation of an invasive proteolytic bacterial surface. Our results underscore the importance of the human fibrinolytic system in host-pathogen interactions in invasive GAS infections.

Streptococcus pyogenes Employs Strain-dependent Mechanisms of C3b Inactivation to Inhibit Phagocytosis and Killing of Bacteria.

Evasion of complement-mediated opsonophagocytosis enables group A Streptococcus pyogenes (GAS) to establish infection. Different strain-dependent mechanisms are employed by the host to accomplish this goal. In general, GAS inhibits the amplification of the complement cascade on its cell surface by facilitating the degradation of C3b, an opsonin, to an inactive product, inactivated C3b (iC3b), in a step catalyzed by factor I (FI) and its cofactor, factor H (FH), with or without the participation of human host plasmin (hPm). GAS recruits FH to its cell surface via FH receptors, which are transcriptionally controlled by the two-component cluster of virulence responder-sensor system. The manner in which FI-FH and hPm function together on GAS cells is unknown. Using GAS strain AP53, which strongly binds host human plasminogen/plasmin (hPg/hPm) directly via an hPg/hPm surface receptor (PAM), we show that both FI-FH and hPm sequentially cleave C3b. Whereas FI-FH proteolytically cleaves C3b into iC3b, PAM-bound hPm catalyzes cleavage of iC3b into multiple smaller peptides. Unlike AP53, GAS strain M23ND weakly binds FH and recruits hPg/hPm to its cell surface indirectly via fibrinogen bound to M-protein, M23. In this case, FH-FI cleaves C3b into iC3b, with negligible degradation of iC3b by hPm that is bound to fibrinogen on the cells. AP53 and M23ND display similar resistance to human neutrophil-mediated phagocytosis, which results in a corresponding high lethality in mice after injection of these cells. These results suggest that GAS utilizes diverse mechanisms to degrade C3b and thus to protect bacterial cells from the complement response of the host.

Direct Host Plasminogen Binding to Bacterial Surface M-protein in Pattern D Strains of Streptococcus pyogenes Is Required for Activation by Its Natural Coinherited SK2b Protein.

Streptokinase (SK), secreted by Group A Streptococcus (GAS), is a single-chain ∼47-kDa protein containing three consecutive primary sequence regions that comprise its α, ß, and γ modules. Phylogenetic analyses of the variable ß-domain sequences from different GAS strains suggest that SKs can be arranged into two clusters, SK1 and SK2, with a subdivision of SK2 into SK2a and SK2b. SK2b is secreted by skin-tropic Pattern D M-protein strains that also express plasminogen (human Pg (hPg)) binding Group A streptococcal M-protein (PAM) as its major cell surface M-protein. SK2a-expressing strains are associated with nasopharynx tropicity, and many of these strains express human fibrinogen (hFg) binding Pattern A-C M-proteins, e.g. M1. PAM interacts with hPg directly, whereas M1 binds to hPg indirectly via M1-bound hFg. Subsequently, SK is secreted by GAS and activates hPg to plasmin (hPm), thus generating a proteolytic surface on GAS that enhances its dissemination. Due to these different modes of hPg/hPm recognition by GAS, full characterizations of the mechanisms of activation of hPg by SK2a and SK2b and their roles in GAS virulence are important topics. To more fully examine these subjects, isogenic chimeric SK- and M-protein-containing GAS strains were generated, and the virulence of these chimeric strains were analyzed in mice. We show that SK and M-protein alterations influenced the virulence of GAS and were associated with the different natures of hPg activation and hPm binding. These studies demonstrate that GAS virulence can be explained by disparate hPg activation by SK2a and SK2b coupled with the coinherited M-proteins of these strains.

A natural inactivating mutation in the CovS component of the CovRS regulatory operon in a pattern D Streptococcal pyogenes strain influences virulence-associated genes.

A skin-tropic invasive group A Streptococcus pyogenes (GAS) strain, AP53, contains a natural inactivating mutation in the covS gene (covS(M)) of the two-component responder (CovR)/sensor (CovS) gene regulatory system. The effects of this mutation on specific GAS virulence determinants have been assessed, with emphasis on expression of the extracellular protease, streptococcal pyrogenic exotoxin B (SpeB), capsular hyaluronic acid, and proteins that allow host plasmin assembly on the bacterial surface, viz. a high affinity plasminogen (Pg)/plasmin receptor, Pg-binding group A streptococcal M protein (PAM), and the human Pg activator streptokinase. To further illuminate mechanisms of the functioning of CovRS in the virulence of AP53, two AP53 isogenic strains were generated, one in which the natural covS(M) gene was mutated to WT-covS (AP53/covS(WT)) and a strain that contained an inactivated covR gene (AP53/ΔcovR). Two additional strains that do not contain PAM, viz. WT-NS931 and NS931/covS(M), were also employed. SpeB was not measurably expressed in strains containing covR(WT)/covS(M), whereas in strains with natural or engineered covR(WT)/covS(WT), SpeB expression was highly up-regulated. Alternatively, capsule synthesis via the hasABC operon was enhanced in strain AP53/covS(M), whereas streptokinase expression was only slightly affected by the covS inactivation. PAM expression was not substantially influenced by the covS mutation, suggesting that covRS had minimal effects on the mga regulon that controls PAM expression. These results demonstrate that a covS inactivation results in virulence gene alterations and also suggest that the CovR phosphorylation needed for gene up- or down-regulation can occur by alternative pathways to CovS kinase.

HIF-1α is Required to Differentiate the Neonatal Macrophage Secretome from Adults.

The immune response to stress diverges with age, with neonatal macrophages implicated in tissue regeneration versus tissue scarring and maladaptive inflammation in adults. Integral to the macrophage stress response is the recognition of hypoxia and pathogen-associated molecular patterns (PAMPs), which are often coupled. The age-specific, cell-intrinsic nature of this stress response remains vague. To uncover age-defined divergences in macrophage crosstalk potential after exposure to hypoxia and PAMPs, we interrogated the secreted proteomes of neonatal versus adult macrophages via non-biased mass spectrometry. Through this approach, we newly identified age-specific signatures in the secretomes of neonatal versus adult macrophages in response to hypoxia and the prototypical PAMP, lipopolysaccharide (LPS). Neonatal macrophages polarized to an anti-inflammatory, regenerative phenotype protective against apoptosis and oxidative stress, dependent on hypoxia inducible transcription factor-1α ( HIF-1α). In contrast, adult macrophages adopted a pro-inflammatory, glycolytic phenotypic signature consistent with pathogen killing. Taken together, these data uncover fundamental age and HIF-1α dependent macrophage programs that may be targeted to calibrate the innate immune response during stress and inflammation.

Single-cell profiling reveals inflammatory polarization of human carotid versus femoral plaque leukocytes.

Femoral atherosclerotic plaques are less inflammatory than carotid plaques histologically, but limited cell-level data exist regarding comparative immune landscapes and polarization at these sites. We investigated intraplaque leukocyte phenotypes and transcriptional polarization in 49 patients undergoing femoral (n = 23) or carotid (n = 26) endarterectomy using single-cell RNA-Seq (scRNA-Seq; n = 13), flow cytometry (n = 24), and IHC (n = 12). Comparative scRNA-Seq of CD45+-selected leukocytes from femoral (n = 9; 35,265 cells) and carotid (n = 4; 30,655 cells) plaque revealed distinct transcriptional profiles. Inflammatory foam cell-like macrophages and monocytes comprised higher proportions of myeloid cells in carotid plaques, whereas noninflammatory foam cell-like macrophages and LYVE1-overexpressing macrophages comprised higher proportions of myeloid cells in femoral plaque (P < 0.001 for all). A significant comparative excess of CCR2+ macrophages in carotid versus plaque was observed by flow cytometry in a separate validation cohort. B cells were more prevalent and exhibited a comparatively antiinflammatory profile in femoral plaque, whereas cytotoxic CD8+ T cells were more prevalent in carotid plaque. In conclusion, human femoral plaques exhibit distinct macrophage phenotypic and transcriptional profiles as well as diminished CD8+ T cell populations compared with human carotid plaques.

Macrophage-produced VEGFC is induced by efferocytosis to ameliorate cardiac injury and inflammation.

Clearance of dying cells by efferocytosis is necessary for cardiac repair after myocardial infarction (MI). Recent reports have suggested a protective role for vascular endothelial growth factor C (VEGFC) during acute cardiac lymphangiogenesis after MI. Here, we report that defective efferocytosis by macrophages after experimental MI led to a reduction in cardiac lymphangiogenesis and Vegfc expression. Cell-intrinsic evidence for efferocytic induction of Vegfc was revealed after adding apoptotic cells to cultured primary macrophages, which subsequently triggered Vegfc transcription and VEGFC secretion. Similarly, cardiac macrophages elevated Vegfc expression levels after MI, and mice deficient for myeloid Vegfc exhibited impaired ventricular contractility, adverse tissue remodeling, and reduced lymphangiogenesis. These results were observed in mouse models of permanent coronary occlusion and clinically relevant ischemia and reperfusion. Interestingly, myeloid Vegfc deficiency also led to increases in acute infarct size, prior to the amplitude of the acute cardiac lymphangiogenesis response. RNA-Seq and cardiac flow cytometry revealed that myeloid Vegfc deficiency was also characterized by a defective inflammatory response, and macrophage-produced VEGFC was directly effective at suppressing proinflammatory macrophage activation. Taken together, our findings indicate that cardiac macrophages promote healing through the promotion of myocardial lymphangiogenesis and the suppression of inflammatory cytokines.

Macrophage AXL receptor tyrosine kinase inflames the heart after reperfused myocardial infarction.

Tyro3, AXL, and MerTK (TAM) receptors are activated in macrophages in response to tissue injury and as such have been proposed as therapeutic targets to promote inflammation resolution during sterile wound healing, including myocardial infarction. Although the role of MerTK in cardioprotection is well characterized, the unique role of the other structurally similar TAMs, and particularly AXL, in clinically relevant models of myocardial ischemia/reperfusion infarction (IRI) is comparatively unknown. Utilizing complementary approaches, validated by flow cytometric analysis of human and murine macrophage subsets and conditional genetic loss and gain of function, we uncover a maladaptive role for myeloid AXL during IRI in the heart. Cross signaling between AXL and TLR4 in cardiac macrophages directed a switch to glycolytic metabolism and secretion of proinflammatory IL-1ß, leading to increased intramyocardial inflammation, adverse ventricular remodeling, and impaired contractile function. AXL functioned independently of cardioprotective MerTK to reduce the efficacy of cardiac repair, but like MerTK, was proteolytically cleaved. Administration of a selective small molecule AXL inhibitor alone improved cardiac healing, which was further enhanced in combination with blockade of MerTK cleavage. These data support further exploration of macrophage TAM receptors as therapeutic targets for myocardial infarction.

Bone marrow-derived AXL tyrosine kinase promotes mitogenic crosstalk and cardiac allograft vasculopathy.

Cardiac Allograft Vasculopathy (CAV) is a leading contributor to late transplant rejection. Although implicated, the mechanisms by which bone marrow-derived cells promote CAV remain unclear. Emerging evidence implicates the cell surface receptor tyrosine kinase AXL to be elevated in rejecting human allografts. AXL protein is found on multiple cell types, including bone marrow-derived myeloid cells. The causal role of AXL from this compartment and during transplant is largely unknown. This is important because AXL is a key regulator of myeloid inflammation. Utilizing experimental chimeras deficient in the bone marrow-derived Axl gene, we report that Axl antagonizes cardiac allograft survival and promotes CAV. Flow cytometric and histologic analyses of Axl-deficient transplant recipients revealed reductions in both allograft immune cell accumulation and vascular intimal thickness. Co-culture experiments designed to identify cell-intrinsic functions of Axl uncovered complementary cell-proliferative pathways by which Axl promotes CAV-associated inflammation. Specifically, Axl-deficient myeloid cells were less efficient at increasing the replication of both antigen-specific T cells and vascular smooth muscle cells (VSMCs), the latter a key hallmark of CAV. For the latter, we discovered that Axl-was required to amass the VSMC mitogen Platelet-Derived Growth Factor. Taken together, our studies reveal a new role for myeloid Axl in the progression of CAV and mitogenic crosstalk. Inhibition of AXL-protein, in combination with current standards of care, is a candidate strategy to prolong cardiac allograft survival.

Innate Functions of Dendritic Cell Subsets in Cardiac Allograft Tolerance.

Survival rates after heart transplant have significantly improved over the last decade. Nevertheless, long-term allograft viability after 10 years remains poor and the sequelae of transplant-associated immunosuppression increases morbidity. Although several studies have implicated roles for lymphocyte-mediated rejection, less is understood with respect to non-major histocompatibility, and innate immune reactivity, which influence graft viability. As immature and mature dendritic cells (DCs) engage in both Major Histocompatibility Complex (MHC)-dependent and MHC-independent immune responses, these cells are at the crossroads of therapeutic strategies that seek to achieve both allograft tolerance and suppression of innate immunity to the allograft. Here we review emerging roles of DC subsets and their molecular protagonists during allograft tolerance and allograft rejection, with a focus on cardiac transplant. New insight into emerging DC subsets in transplant will inform novel strategies for operational tolerance and amelioration of cardiac vasculopathy.

Acute and chronic phagocyte determinants of cardiac allograft vasculopathy.

Post-transplant immunosuppression has reduced the incidence of T cell-mediated acute rejection, yet long-term cardiac graft survival rates remain a challenge. An important determinant of chronic solid organ allograft complication is accelerated vascular disease of the transplanted graft. In the case of cardiac allograft vasculopathy (CAV), the precise cellular etiology remains inadequately understood; however, histologic evidence hints at the accumulation and activation of innate phagocytes as a causal contributing factor. This includes monocytes, macrophages, and immature dendritic cell subsets. In addition to crosstalk with adaptive T and B immune cells, myeloid phagocytes secrete paracrine signals that directly activate fibroblasts and vascular smooth muscle cells, both of which contribute to fibrous intimal thickening. Though maladaptive phagocyte functions may promote CAV, directed modulation of myeloid cell function, at the molecular level, holds promise for tolerance and prolonged cardiac graft function.

Efferocytosis and Outside-In Signaling by Cardiac Phagocytes. Links to Repair, Cellular Programming, and Intercellular Crosstalk in Heart.

Phagocytic sensing and engulfment of dying cells and extracellular bodies initiate an intracellular signaling cascade within the phagocyte that can polarize cellular function and promote communication with neighboring non-phagocytes. Accumulating evidence links phagocytic signaling in the heart to cardiac development, adult myocardial homeostasis, and the resolution of cardiac inflammation of infectious, ischemic, and aging-associated etiology. Phagocytic clearance in the heart may be carried out by professional phagocytes, such as macrophages, and non-professional cells, including myofibrolasts and potentially epithelial cells. During cardiac development, phagocytosis initiates growth cues for early cardiac morphogenesis. In diseases of aging, including myocardial infarction, heightened levels of cell death require efficient phagocytic debridement to salvage further loss of terminally differentiated adult cardiomyocytes. Additional risk factors, including insulin resistance and other systemic risk factors, contribute to inefficient phagocytosis, altered phagocytic signaling, and delayed cardiac inflammation resolution. Under such conditions, inflammatory presentation of myocardial antigen may lead to autoimmunity and even possible rejection of transplanted heart allografts. Increased understanding of these basic mechanisms offers therapeutic opportunities.

Functional differences between Streptococcus pyogenes cluster 1 and cluster 2b streptokinases are determined by their ß-domains.

Cluster 1 streptokinases (SK1) from Streptococcus pyogenes (GAS) show substantially higher human plasminogen (hPg) activation activities and tighter hPg binding affinities than cluster 2b streptokinases (SK2b) in solution. The extent to which the different domains of SK are responsible for these differences is unknown. We exchanged each of the three known SK domains (α, ß, and γ) between SK1 and SK2b and assessed the function of the resulting variants. Our results show that primary structural differences in the ß-domains dictate these functional differences. This first report on the primary structure-functional relationship between naturally occurring SK1 and SK2b sheds new light on the mechanism of hPg activation by SK, a critical virulence determinant in this species of human pathogenic bacteria.