The CIS association of CD47 with integrin Mac-1 regulates macrophage responses by stabilizing the extended integrin conformation.

CD47 is a ubiquitously expressed cell surface integrin-associated protein. Recently, we have demonstrated that integrin Mac-1 (αMß2, CD11b/CD18, CR3), the major adhesion receptor on the surface of myeloid cells, can be coprecipitated with CD47. However, the molecular basis for the CD47-Mac-1 interaction and its functional consequences remain unclear. Here, we demonstrated that CD47 regulates macrophage functions directly interacting with Mac-1. In particular, adhesion, spreading, migration, phagocytosis, and fusion of CD47-deficient macrophages were significantly decreased. We validated the functional link between CD47 and Mac-1 by coimmunoprecipitation analysis using various Mac-1-expressing cells. In HEK293 cells expressing individual αM and ß2 integrin subunits, CD47 was found to bind both subunits. Interestingly, a higher amount of CD47 was recovered with the free ß2 subunit than in the complex with the whole integrin. Furthermore, activating Mac-1-expressing HEK293 cells with phorbol 12-myristate 13-acetate (PMA), Mn2+, and activating antibody MEM48 increased the amount of CD47 in complex with Mac-1, suggesting CD47 has a greater affinity for the extended integrin conformation. Notably, on the surface of cells lacking CD47, fewer Mac-1 molecules could convert into an extended conformation in response to activation. Additionally, we identified the binding site in CD47 for Mac-1 in its constituent IgV domain. The complementary binding sites for CD47 in Mac-1 were localized in integrin epidermal growth factor-like domains 3 and 4 of the ß2 and calf-1 and calf-2 domains of the αM subunits. These results indicate that Mac-1 forms a lateral complex with CD47, which regulates essential macrophage functions by stabilizing the extended integrin conformation.

The essential liaison of two copper proteins: the Cu-sensing transcription factor Mac1 and the Cu/Zn superoxide dismutase Sod1 in Saccharomyces cerevisiae.

Although copper is an essential trace element for cell function and viability, its excess can lead to protein oxidation, DNA cleavage, and ultimate cell damage. Cells have established a variety of regulatory mechanisms to ensure copper ion homeostasis. In Saccharomyces cerevisiae, copper sensing and response to copper deficiency are regulated by the transcription factor Mac1. Our group has previously reported that in addition to copper, several chromatin proteins modulate Mac1 functionality. In this study, based on a synthetic growth deficiency phenotype, we showed that the Cu/Zn superoxide dismutase Sod1 plays an important role in Mac1 transcriptional activity, in unchallenged nutrient-rich growth conditions. Sod1 is a multipotent cytoplasmic and mitochondrial enzyme, whose main known function is to detoxify the cell from superoxide ions. It has been previously reported that Sod1 also enters the nucleus and affects the transcription of several genes, some of which are involved in copper homeostasis under Cu-depleted (Wood and Thiele in J Biol Chem 284:404-413, 2009) or only under specific oxidative stress conditions (Dong et al. Mol Cell Biol 33:4041-4050, 2013; Tsang et al. Nar Commun 8:3446, 2014). We have shown that Sod1 physically interacts with Mac1 transcription factor and is important for the transactivation as well as its DNA-binding activities. On the other hand, a constitutively active mutant of Mac1 is not affected functionally by the Sod1 ablation, pointing out that Sod1 contributes to the maintenance of the copper-unchelated state of Mac1. In conclusion, we showed that Sod1-Mac1 interaction is vital for Mac1 functionality, regardless of copper medium deficiency, in unchallenged growth conditions, and we suggest that Sod1 enzymatic activity may modify the redox state of the cysteine-rich motifs in the Mac1 DNA-binding and transactivation domains.

Monocyte-Derived miRNA-1914-5p Attenuates IL-1ß-Induced Monocyte Adhesion and Transmigration.

Atherosclerosis can lead to cardiovascular and cerebrovascular diseases. Atherosclerotic plaque formation is promoted by the accumulation of inflammatory cells. Therefore, modulating monocyte recruitment represents a potential therapeutic strategy. In an inflammatory state, the expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) is upregulated in endothelial cells. We previously reported that miR-1914-5p in endothelial cells suppresses interleukin (IL)-1ß-induced ICAM-1 expression and monocyte adhesion to endothelial cells. However, whether monocyte miR-1914-5p affects monocyte recruitment is unclear. In this study, IL-1ß decreased miR-1914-5p expression in a human monocyte cell line. Moreover, miR-1914-5p inhibition enhanced adhesion to endothelial cells with the upregulation of macrophage-1 antigen (Mac-1), a counter-ligand to ICAM-1. Transmigration through the endothelial layer was also promoted with the upregulation of monocyte chemotactic protein-1 (MCP-1). Furthermore, a miR-1914-5p mimic suppressed IL-1ß-induced monocyte adhesion and transmigration in monocytes with Mac-1 and MCP-1 downregulation. Further investigation of miR-1914-5p in monocytes could lead to the development of novel diagnostic markers and therapeutic strategies for atherosclerosis.

GPR55 Antagonist CID16020046 Protects against Atherosclerosis Development in Mice by Inhibiting Monocyte Adhesion and Mac-1 Expression.

GPR55 recognizes several lipid molecules such as lysophosphatidylinositol. GPR55 expression was reported in human monocytes. However, its role in monocyte adhesion and atherosclerosis development has not been studied. The role of GPR55 in monocyte adhesion and atherosclerosis development was investigated in human THP-1 monocytes and ApoE-/- mice using O-1602 (a potent agonist of GPR55) and CID16020046 (a specific GPR55 antagonist). O-1602 treatment significantly increased monocyte adhesion to human umbilical vein endothelial cells, and the O-1602-induced adhesion was inhibited by treatment with CID16020046. O-1602 induced the expression of Mac-1 adhesion molecules, whereas CID16020046 inhibited this induction. Analysis of the promoter region of Mac-1 elucidated the binding sites of AP-1 and NF-κB between nucleotides -750 and -503 as GPR55 responsive elements. O-1602 induction of Mac-1 was found to be dependent on the signaling components of GPR55, that is, Gq protein, Ca2+, CaMKK, and PI3K. In Apo-/- mice, administration of CID16020046 ameliorated high-fat diet-induced atherosclerosis development. These results suggest that high-fat diet-induced GPR55 activation leads to the adhesion of monocytes to endothelial cells via induction of Mac-1, and CID16020046 blockage of GPR55 could suppress monocyte adhesion to vascular endothelial cells through suppression of Mac-1 expression, leading to protection against the development of atherosclerosis.

Interaction between the integrin Mac-1 and signal regulatory protein α (SIRPα) mediates fusion in heterologous cells.

Macrophage fusion leading to the formation of multinucleated giant cells is a hallmark of chronic inflammation. Several membrane proteins have been implicated in mediating cell-cell attachment during fusion, but their binding partners remain unknown. Recently, we demonstrated that interleukin-4 (IL-4)-induced fusion of mouse macrophages depends on the integrin macrophage antigen 1 (Mac-1). Surprisingly, the genetic deficiency of intercellular adhesion molecule 1 (ICAM-1), an established ligand of Mac-1, did not impair macrophage fusion, suggesting the involvement of other counter-receptors. Here, using various approaches, including signal regulatory protein α (SIRPα) knockdown, recombinant proteins, adhesion and fusion assays, biolayer interferometry, and peptide libraries, we show that SIRPα, which, similar to ICAM-1, belongs to the Ig superfamily and has previously been implicated in cell fusion, interacts with Mac-1. The following results support the conclusion that SIRPα is a ligand of Mac-1: (a) recombinant ectodomain of SIRPα supports adhesion of Mac-1-expressing cells; (b) Mac-1-SIRPα interaction is mediated through the ligand-binding αMI-domain of Mac-1; (c) recognition of SIRPα by the αMI-domain conforms to general principles governing binding of Mac-1 to many of its ligands; (d) SIRPα reportedly binds CD47; however, anti-CD47 function-blocking mAb produced only a limited inhibition of macrophage adhesion to SIRPα; and (e) co-culturing of SIRPα- and Mac-1-expressing HEK293 cells resulted in the formation of multinucleated cells. Taken together, these results identify SIRPα as a counter-receptor for Mac-1 and suggest that the Mac-1-SIRPα interaction may be involved in macrophage fusion.

Double-stranded RNA-induced dopaminergic neuronal loss in the substantia nigra in the presence of Mac1 receptor.

BACKGROUND: The underlying mechanism of viral infection as a risk factor for Parkinson's disease (PD), the second most common neurodegenerative disease, remains unclear. OBJECTIVE: We used Mac-1-/- and gp91phox-/- transgene animal models to investigate the mechanisms by which poly I:C, a mimic of virus double-stranded RNA, induces PD neurodegeneration. METHOD: Poly I:C was stereotaxically injected into the substantia nigra (SN) of wild-type (WT), Mac-1-knockout (Mac-1-/-) and gp91 phox-knockout (gp91 phox-/-) mice (10 µg/µl), and nigral dopaminergic neurodegeneration, α-synuclein accumulation and neuroinflammation were evaluated. RESULT: Dopaminergic neurons in the nigra and striatum were markedly reduced in WT mice after administration of poly I:C together with abundant microglial activation in the SN, and the expression of α-synuclein was also elevated. However, these pathological changes were greatly dampened in Mac-1-/- and gp91 phox-/- mice. CONCLUSIONS: Our findings demonstrated that viral infection could result in the activation of microglia as well as NADPH oxidase, which may lead to neuron loss and the development of Parkinson's-like symptoms. Mac-1 is a key receptor during this process.

Distinct associations of the Saccharomyces cerevisiae Rad9 protein link Mac1-regulated transcription to DNA repair.

While it is known that ScRad9 DNA damage checkpoint protein is recruited to damaged DNA by recognizing specific histone modifications, here we report a different way of Rad9 recruitment on chromatin under non DNA damaging conditions. We found Rad9 to bind directly with the copper-modulated transcriptional activator Mac1, suppressing both its DNA binding and transactivation functions. Rad9 was recruited to active Mac1-target promoters (CTR1, FRE1) and along CTR1 coding region following the association pattern of RNA polymerase (Pol) II. Hir1 histone chaperone also interacted directly with Rad9 and was partly required for its localization throughout CTR1 gene. Moreover, Mac1-dependent transcriptional initiation was necessary and sufficient for Rad9 recruitment to the heterologous ACT1 coding region. In addition to Rad9, Rad53 kinase also localized to CTR1 coding region in a Rad9-dependent manner. Our data provide an example of a yeast DNA-binding transcriptional activator that interacts directly with a DNA damage checkpoint protein in vivo and is functionally restrained by this protein, suggesting a new role for Rad9 in connecting factors of the transcription machinery with the DNA repair pathway under unchallenged conditions.

ß2 Integrins-Multi-Functional Leukocyte Receptors in Health and Disease.

ß2 integrins are heterodimeric surface receptors composed of a variable α (CD11a-CD11d) and a constant ß (CD18) subunit and are specifically expressed by leukocytes. The α subunit defines the individual functional properties of the corresponding ß2 integrin, but all ß2 integrins show functional overlap. They mediate adhesion to other cells and to components of the extracellular matrix (ECM), orchestrate uptake of extracellular material like complement-opsonized pathogens, control cytoskeletal organization, and modulate cell signaling. This review aims to delineate the tremendous role of ß2 integrins for immune functions as exemplified by the phenotype of LAD-I (leukocyte adhesion deficiency 1) patients that suffer from strong recurrent infections. These immune defects have been largely attributed to impaired migratory and phagocytic properties of polymorphonuclear granulocytes. The molecular base for this inherited disease is a functional impairment of ß2 integrins due to mutations within the CD18 gene. LAD-I patients are also predisposed for autoimmune diseases. In agreement, polymorphisms within the CD11b gene have been associated with autoimmunity. Consequently, ß2 integrins have received growing interest as targets in the treatment of autoimmune diseases. Moreover, ß2 integrin activity on leukocytes has been implicated in tumor development.

The many faces of Mac-1 in autoimmune disease.

Mac-1 (CD11b/CD18) is a ß2 integrin classically regarded as a pro-inflammatory molecule because of its ability to promote phagocyte cytotoxic functions and enhance the function of several effector molecules such as FcγR, uPAR, and CD14. Nevertheless, recent reports have revealed that Mac-1 also plays significant immunoregulatory roles, and genetic variants in ITGAM, the gene that encodes CD11b, confer risk for the autoimmune disease systemic lupus erythematosus (SLE). This has renewed interest in the physiological roles of this integrin and raised new questions on how its seemingly opposing biological functions may be regulated. Here, we provide an overview of the CD18 integrins and how their activation may be regulated as this may shed light on how the opposing roles of Mac-1 may be elicited. We then discuss studies that exemplify Mac-1's pro-inflammatory versus regulatory roles particularly in the context of IgG immune complex-mediated inflammation. This includes a detailed examination of molecular mechanisms that could explain the risk-conferring effect of rs1143679, a single nucleotide non-synonymous Mac-1 polymorphism associated with SLE.

The pattern-recognition molecule mindin binds integrin Mac-1 to promote macrophage phagocytosis via Syk activation and NF-κB p65 translocation.

Mindin has a broad spectrum of roles in the innate immune system, including in macrophage migration, antigen phagocytosis and cytokine production. Mindin functions as a pattern-recognition molecule for microbial pathogens. However, the underlying mechanisms of mindin-mediated phagocytosis and its exact membrane receptors are not well established. Herein, we generated mindin-deficient mice using the CRISPR-Cas9 system and show that peritoneal macrophages from mindin-deficient mice were severely defective in their ability to phagocytize E  coli. Phagocytosis was enhanced when E  coli or fluorescent particles were pre-incubated with mindin, indicating that mindin binds directly to bacteria or non-pathogen particles and promotes phagocytosis. We defined that 131 I-labelled mindin binds with integrin Mac-1 (CD11b/CD18), the F-spondin (FS)-fragment of mindin binds with the αM -I domain of Mac-1 and that mindin serves as a novel ligand of Mac-1. Blockade of the αM -I domain of Mac-1 using either a neutralizing antibody or si-Mac-1 efficiently blocked mindin-induced phagocytosis. Furthermore, mindin activated the Syk and MAPK signalling pathways and promoted NF-κB entry into the nucleus. Our data indicate that mindin binds with the integrin Mac-1 to promote macrophage phagocytosis through Syk activation and NF-κB p65 translocation, suggesting that the mindin/Mac-1 axis plays a critical role during innate immune responses.

Leukocyte integrin Mac-1 (CD11b/CD18, αMß2, CR3) acts as a functional receptor for platelet factor 4.

Platelet factor 4 (PF4) is one of the most abundant cationic proteins secreted from α-granules of activated platelets. Based on its structure, PF4 was assigned to the CXC family of chemokines and has been shown to have numerous effects on myeloid leukocytes. However, the receptor for PF4 remains unknown. Here, we demonstrate that PF4 induces leukocyte responses through the integrin Mac-1 (αMß2, CD11b/CD18). Human neutrophils, monocytes, U937 monocytic and HEK293 cells expressing Mac-1 strongly adhered to immobilized PF4 in a concentration-dependent manner. The cell adhesion was partially blocked by anti-Mac-1 mAb and inhibition was enhanced when anti-Mac-1 antibodies were combined with glycosaminoglycans, suggesting that cell-surface proteoglycans act cooperatively with Mac-1. PF4 also induced Mac-1-dependent migration of human neutrophils and murine WT, but not Mac-1-deficient macrophages. Coating of Escherichia coli bacteria or latex beads with PF4 enhanced their phagocytosis by macrophages by ∼4-fold, and this process was blocked by different Mac-1 antagonists. Furthermore, PF4 potentiated phagocytosis by WT, but not Mac-1-deficient macrophages. As determined by biolayer interferometry, PF4 directly bound the αMI-domain, the major ligand-binding region of Mac-1, and this interaction was governed by a Kd of 1.3 ± 0.2 µm Using the PF4-derived peptide library, synthetic peptides duplicating the αMI-domain recognition sequences and recombinant mutant PF4 fragments, the binding sites for αMI-domain were identified in the PF4 segments Cys12-Ser26 and Ala57-Ser70 These results identify PF4 as a ligand for the integrin Mac-1 and suggest that many immune-modulating effects previously ascribed to PF4 are mediated through its interaction with Mac-1.

Pattern recognition receptors mediate pro-inflammatory effects of extracellular mitochondrial transcription factor A (TFAM).

Neuroinflammation is a common pathogenic mechanism for a number of neurodegenerative disorders including Alzheimer's and Parkinson's diseases. Microglia, the immune cells of the brain, contribute to the onset and progression of the neuroinflammation observed in these diseases. Microglia become activated and initiate an inflammatory response by interacting with a diverse set of molecules, including the group of endogenous proteins released upon cell damage, termed damage-associated molecular patterns (DAMPs). One of these molecules, mitochondrial transcription factor A (TFAM), has been shown to induce pro-inflammatory and cytotoxic responses of microglia in vitro. Here, we demonstrate that TFAM injected into the cisterna magna of male Sprague-Dawley rats upregulates (i) the expression of monocyte chemotactic protein (MCP)-1, interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α and nuclear factor-kappa B inhibitor alpha (NF-κBIA) in the hippocampus; (ii) the expression of MCP-1, IL-1ß and TNF-α in the frontal cortex; and (iii) IL-1ß protein concentration in both these brain regions. These same inflammatory mediators are upregulated in isolated rat microglia following their in vitro exposure to extracellular TFAM. Blocking the receptor for advanced glycation endproducts (RAGE) and the macrophage antigen complex (Mac)-1 by specific antibodies inhibited the TFAM-induced secretion of MCP-1 by THP-1 monocytic cells, which were used to model human microglia. Our data support the hypothesis that extracellular TFAM can interact with RAGE and Mac-1 to function as a DAMP that causes pro-inflammatory microglial activation. Blocking this interaction may represent a potential target for attenuating the neuroinflammation observed in neurodegenerative diseases.

Effects of sinomenine in LPS-associated diseases are related to inhibition of LBP, Mac-1, and L-selectin levels.

The aim of the research was to investigate the anti-endotoxin and anti-inflammatory effects of Sinomenine, an agent commonly found in Chinese herbal medicines. Endotoxin (i.e., 1 mg lipopolysaccharide (LPS)/kg)) was administered via intraperitoneal (IP) injection to piglets in high-, middle-, and low-dose sinomenine groups. Piglets were then treated with 1, 5 or 10 mg/kg sinomenine, intramuscularly (i.m.), 3 hr after LPS. Vehicle was administered, as above, to drug control group piglets followed 3 hr later by 10 mg/kg sinomenine i.m.. LPS control group piglets were challenged with 1 mg/kg LPS IP, followed by vehicle i.m., and naïve control piglets were treated with normal saline IP, followed by normal saline i.m., as above. Temperatures were measured, and blood samples were collected from the precaval veins of piglets at 12, 24, and 48 hr post-LPS or vehicle injection. Clinical signs were recorded, and index levels were analyzed via ELISA. Sinomenine was found to reduce the incidence and severity of LPS-induced toxicities, including body temperature elevation, cell adhesion, and systemic inflammation. These data suggest that sinomenine may be effective for regulating inflammatory responses and has the potential for use as an anti-endotoxin therapy.

Pleiotrophin, a multifunctional cytokine and growth factor, induces leukocyte responses through the integrin Mac-1.

Pleiotrophin (PTN) is a multifunctional, cationic, glycosaminoglycan-binding cytokine and growth factor involved in numerous physiological and pathological processes, including tissue repair and inflammation-related diseases. PTN has been shown to promote leukocyte responses by inducing their migration and expression of inflammatory cytokines. However, the mechanisms through which PTN mediates these responses remain unclear. Here, we identified the integrin Mac-1 (αMß2, CD11b/CD18) as the receptor mediating macrophage adhesion and migration to PTN. We also found that expression of Mac-1 on the surface of human embryonic kidney (HEK) 293 cells induced their adhesion and migration to PTN. Accordingly, PTN promoted Mac-1-dependent cell spreading and initiated intracellular signaling manifested in phosphorylation of Erk1/2. While binding to PTN, Mac-1 on Mac-1-expressing HEK293 cells appears to cooperate with cell-surface proteoglycans because both anti-Mac-1 function-blocking mAb and heparin were required to block adhesion. Moreover, biolayer interferometry and NMR indicated a direct interaction between the αMI domain, the major ligand-binding region of Mac-1, and PTN. Using peptide libraries, we found that in PTN the αMI domain bound sequences enriched in basic and hydrophobic residues, indicating that PTN conforms to the general principle of ligand-recognition specificity of the αMI domain toward cationic proteins/peptides. Finally, using recombinant PTN-derived fragments, we show that PTN contains two distinct Mac-1-binding sites in each of its constitutive domains. Collectively, these results identify PTN as a ligand for the integrin Mac-1 on the surface of leukocytes and suggest that this interaction may play a role in inflammatory responses.

Activated protein C inhibits neutrophil extracellular trap formation in vitro and activation in vivo.

Activated protein C (APC) is a multifunctional serine protease with anticoagulant, cytoprotective, and anti-inflammatory activities. In addition to the cytoprotective effects of APC on endothelial cells, podocytes, and neurons, APC cleaves and detoxifies extracellular histones, a major component of neutrophil extracellular traps (NETs). NETs promote pathogen clearance but also can lead to thrombosis; the pathways that negatively regulate NETosis are largely unknown. Thus, we studied whether APC is capable of directly inhibiting NETosis via receptor-mediated cell signaling mechanisms. Here, by quantifying extracellular DNA or myeloperoxidase, we demonstrate that APC binds human leukocytes and prevents activated platelet supernatant or phorbol 12-myristate 13-acetate (PMA) from inducing NETosis. Of note, APC proteolytic activity was required for inhibiting NETosis. Moreover, antibodies against the neutrophil receptors endothelial protein C receptor (EPCR), protease-activated receptor 3 (PAR3), and macrophage-1 antigen (Mac-1) blocked APC inhibition of NETosis. Select mutations in the Gla and protease domains of recombinant APC caused a loss of NETosis. Interestingly, pretreatment of neutrophils with APC prior to induction of NETosis inhibited platelet adhesion to NETs. Lastly, in a nonhuman primate model of Escherichia coli-induced sepsis, pretreatment of animals with APC abrogated release of myeloperoxidase from neutrophils, a marker of neutrophil activation. These findings suggest that the anti-inflammatory function of APC at therapeutic concentrations may include the inhibition of NETosis in an EPCR-, PAR3-, and Mac-1-dependent manner, providing additional mechanistic insight into the diverse functions of neutrophils and APC in disease states including sepsis.

The balance between Treg and TH17 cells: CD11b and interleukin-6.

One of the gold standards for animal models of rheumatoid arthritis is the murine collagen-induced arthritis model. Native type II collagen together with CFA is injected into susceptible mouse strains. Unfortunately, only mice with H-2q or H-2r MHC haplotypes are susceptible, making the widely used C57BL/6 mouse strain, which carries the H-2b haplotype, resistant against the disease. In this issue of the European Journal of Immunology, Stevanin et al. [Eur. J. Immunol. 2017. 47: 637-645] now convincingly show that although WT C57BL/6 mice are resistant to collagen-induced arthritis, mice with a homozygous deletion of CD11b on the same genetic background are fully susceptible in this important animal model of rheumatoid arthritis. They clearly demonstrate that the injection of type II collagen together with CFA leads to early onset of the disease with high incidence and with sustained severity. The authors further characterize this disease with an increase of leukocyte infiltration and enhanced TH17 differentiation.

Functional characterization of the copper transcription factor AfMac1 from Aspergillus fumigatus.

Although copper functions as a cofactor in many physiological processes, copper overload leads to harmful effects in living cells. Thus, copper homeostasis is tightly regulated. However, detailed copper metabolic pathways have not yet been identified in filamentous fungi. In this report, we investigated the copper transcription factor AfMac1 ( Aspergillus fumigatusMac1 homolog) and identified its regulatory mechanism in A. fumigatus AfMac1 has domains homologous to the DNA-binding and copper-binding domains of Mac1 from Saccharomyces cerevisiae, and AfMac1 efficiently complemented Mac1 in S. cerevisiae Expression of Afmac1 resulted in CTR1 up-regulation, and mutation of the DNA-binding domain of Afmac1 failed to activate CTR1 expression in S. cerevisiae The Afmac1 deletion strain of A. fumigatus failed to grow in copper-limited media, and its growth was restored by introducing ctrC We found that AfMac1 specifically bound to the promoter region of ctrC based on EMSA. The AfMac1-binding motif 5'-TGTGCTCA-3' was identified from the promoter region of ctrC, and the addition of mutant ctrC lacking the AfMac1-binding motif failed to up-regulate ctrC in A. fumigatus Furthermore, deletion of Afmac1 significantly reduced strain virulence and activated conidial killing activity by neutrophils and macrophages. Taken together, these results suggest that AfMac1 is a copper transcription factor that regulates cellular copper homeostasis in A. fumigatus.

Cu-sensing transcription factor Mac1 coordinates with the Ctr transporter family to regulate Cu acquisition and virulence in Aspergillus fumigatus.

Copper (Cu) is an essential trace element and is regarded as an important virulence factor in fungal pathogens. Previous studies suggest that a putative Cu-sensing transcription factor Mac1 and the Cu transporter Ctr family play important roles during fungal development and virulence. However, how Cu importers of the Ctr family are involved in the Cu acquisition and what is the functional relationship between them have not been fully investigated yet. Here, we demonstrate that the yeast Mac1 homolog in the opportunistic human pathogen Aspergillus fumigatus is required during colony development under low Cu conditions. Transcriptional profiling combined with LacZ reporter analyses indicate that Cu transporters ctrA2 and ctrC are expressed in an Afmac1-dependent manner upon Cu starvation, and over-expression of ctrA2 or ctrC transporters almost completely rescue the Afmac1-deletion defects, suggesting a redundancy of both transporters in Afmac1-mediated Cu uptake. Genetic analysis showed that ctrC may play a dominant role against Cu starvation relative to ctrA2 and elevated expression of ctrA2 can compensate for ctrC deletion under Cu starvation. Interestingly, both ctrA2 and ctrC deletions can suppress ctrB deletion colony defects. Our findings suggest that Ctr family proteins might coordinately regulate their functions to adapt to different Cu environments. Compared to yeast homologs, Cu family proteins in A. fumigatus may have their own working styles. Most importantly, the Afmac1 deletion strain shows a significantly attenuated pathogenicity in the neutropenic immunocompromised (a combination of cyclophosphamide and hydrocortisone) mice model, demonstrating that Afmac1 is required for pathogenesis in vivo.

Microglial-mediated PDGF-CC activation increases cerebrovascular permeability during ischemic stroke.

Treatment of acute ischemic stroke with the thrombolytic tissue plasminogen activator (tPA) can significantly improve neurological outcomes; however, thrombolytic therapy is associated with an increased risk of intra-cerebral hemorrhage (ICH). Previously, we demonstrated that during stroke tPA acting on the parenchymal side of the neurovascular unit (NVU) can increase blood-brain barrier (BBB) permeability and ICH through activation of latent platelet-derived growth factor-CC (PDGF-CC) and signaling by the PDGF receptor-α (PDGFRα). However, in vitro, activation of PDGF-CC by tPA is very inefficient and the mechanism of PDGF-CC activation in the NVU is not known. Here, we show that the integrin Mac-1, expressed on brain microglia/macrophages (denoted microglia throughout), acts together with the endocytic receptor LRP1 in the NVU to promote tPA-mediated activation of PDGF-CC. Mac-1-deficient mice (Mac-1-/-) are protected from tPA-induced BBB permeability but not from permeability induced by intracerebroventricular injection of active PDGF-CC. Immunofluorescence analysis demonstrates that Mac-1, LRP1, and the PDGFRα all localize to the NVU of arterioles, and following middle cerebral artery occlusion (MCAO) Mac-1-/- mice show significantly less PDGFRα phosphorylation, BBB permeability, and infarct volume compared to wild-type mice. Bone-marrow transplantation studies indicate that resident CD11b+ cells, but not bone-marrow-derived leukocytes, mediate the early activation of PDGF-CC by tPA after MCAO. Finally, using a model of thrombotic stroke with late thrombolysis, we show that wild-type mice have an increased incidence of spontaneous ICH following thrombolysis with tPA 5 h after MCAO, whereas Mac-1-/- mice are resistant to the development of ICH even with late tPA treatment. Together, these results indicate that Mac-1 and LRP1 act as co-factors for the activation of PDGF-CC by tPA in the NVU, and suggest a novel mechanism for tightly regulating PDGFRα signaling in the NVU and controlling BBB permeability.

Staphylococcal enterotoxin A regulates bone marrow granulocyte trafficking during pulmonary inflammatory disease in mice.

Pulmonary neutrophil infiltration produced by Staphylococcal enterotoxin A (SEA) airway exposure is accompanied by marked granulocyte accumulation in bone marrow (BM). Therefore, the aim of this study was to investigate the mechanisms of BM cell accumulation, and trafficking to circulating blood and lung tissue after SEA airway exposure. Male BALB/C mice were intranasally exposed to SEA (1µg), and at 4, 12 and 24h thereafter, BM, circulating blood, bronchoalveolar lavage (BAL) fluid and lung tissue were collected. Adhesion of BM granulocytes and flow cytometry for MAC-1, LFA1-α and VLA-4 and cytokine and/or chemokine levels were assayed after SEA-airway exposure. Prior exposure to SEA promoted a marked PMN influx to BAL and lung tissue, which was accompanied by increased counts of immature and/or mature neutrophils and eosinophils in BM, along with blood neutrophilia. Airway exposure to SEA enhanced BM neutrophil MAC-1 expression, and adhesion to VCAM-1 and/or ICAM-1-coated plates. Elevated levels of GM-CSF, G-CSF, INF-γ, TNF-α, KC/CXCL-1 and SDF-1α were detected in BM after SEA exposure. SEA exposure increased production of eosinopoietic cytokines (eotaxin and IL-5) and BM eosinophil VLA-4 expression, but it failed to affect eosinophil adhesion to VCAM-1 and ICAM-1. In conclusion, BM neutrophil accumulation after SEA exposure takes place by integrated action of cytokines and/or chemokines, enhancing the adhesive responses of BM neutrophils and its trafficking to lung tissues, leading to acute lung injury. BM eosinophil accumulation in SEA-induced acute lung injury may occur via increased eosinopoietic cytokines and VLA-4 expression.