αMI-domain of integrin Mac-1 binds the cytokine pleiotrophin using multiple mechanisms.

The integrin Mac-1 (αMß2, CD11b/CD18, CR3) is an adhesion receptor expressed on macrophages and neutrophils. Mac-1 is also a promiscuous integrin that binds a diverse set of ligands through its αMI-domain. However, the binding mechanism of most ligands remains unclear. We have characterized the interaction of αMI-domain with the cytokine pleiotrophin (PTN), a protein known to bind αMI-domain and induce Mac-1-mediated cell adhesion and migration. Our data show that PTN's N-terminal domain binds a unique site near the N- and C-termini of the αMI-domain using a metal-independent mechanism. However, a stronger interaction is achieved when an acidic amino acid in a zwitterionic motif in PTN's C-terminal domain chelates the divalent cation in the metal ion-dependent adhesion site of active αMI-domain. These results indicate that αMI-domain can bind ligands using multiple mechanisms and that the active αMI-domain has a preference for motifs containing both positively and negatively charged amino acids.

Ablation of the integrin CD11b mac-1 limits deleterious responses to traumatic spinal cord injury and improves functional recovery in mice.

Background: Spinal cord injury (SCI) causes long-term sensorimotor deficits and posttraumatic neuropathic pain, with no effective treatment. In part, this reflects an incomplete understanding of the complex secondary pathobiological mechanisms involved. SCI triggers microglial/macrophage activation with distinct pro-inflammatory or inflammation-resolving phenotypes, which potentiate tissue damage or facilitate functional repair, respectively. The major integrin Mac-1 (CD11b/CD18, αMß2 or CR3), a heterodimer consisting of αM (CD11b) and ß2 (CD18) chains, is generally regarded as a pro-inflammatory receptor in neurotrauma. Multiple immune cells of the myeloid lineage express CD11b, including microglia, macrophages, and neutrophils. In the present study, we examined the effects of CD11b gene ablation on posttraumatic neuroinflammation and functional outcomes after SCI. Methods: Young adult age-matched female CD11b knockout (KO) mice and their wildtype (WT) littermates were subjected to moderate thoracic spinal cord contusion. Neuroinflammation in the injured spinal cord was assessed with qPCR, flow cytometry, NanoString, and RNAseq. Neurological function was evaluated with the Basso Mouse Scale (BMS), gait analysis, thermal hyperesthesia, and mechanical allodynia. Lesion volume was evaluated by GFAP-DAB immunohistochemistry, followed by analysis with unbiased stereology. Results: qPCR analysis showed a rapid and persistent upregulation of CD11b mRNA starting from 1d after injury, which persisted up to 28 days. At 1d post-injury, increased expression levels of genes that regulate inflammation-resolving processes were observed in CD11b KO mice. Flow cytometry analysis of CD45intLy6C-CX3CR1+ microglia, CD45hiLy6C+Ly6G- monocytes, and CD45hiLy6C+Ly6G+ neutrophils revealed significantly reduced cell counts as well as reactive oxygen production in CD11b KO mice at d3 post-injury. Further examination of the injured spinal cord with NanoString Mouse Neuroinflammation Panel and RNAseq showed upregulated expression of pro-inflammatory genes, but downregulated expression of the reactive oxygen species pathway. Importantly, CD11b KO mice exhibited significantly improved locomotor function, reduced cutaneous mechanical/thermal hypersensitivity, and limited tissue damage at 8 weeks post-injury. Conclusion: Collectively, our data suggest an important role for CD11b in regulating tissue inflammation and functional outcome following SCI. Thus, the integrin CD11b represents a potential target that may lead to novel therapeutic strategies for SCI.

αMI-domain of Integrin Mac-1 Binds the Cytokine Pleiotrophin Using Multiple Mechanisms.

The integrin Mac-1 (αMß2, CD11b/CD18, CR3) is an important adhesion receptor expressed on macrophages and neutrophils. Mac-1 is also the most promiscuous member of the integrin family that binds a diverse set of ligands through its αMI-domain. However, the binding mechanism of most ligands is not clear. We have determined the interaction of αMI-domain with the cytokine pleiotrophin (PTN), a cationic protein known to bind αMI-domain and induce Mac-1-mediated cell adhesion and migration. Our data show that PTN's N-terminal domain binds a unique site near the N- and C-termini of the αMI-domain using a metal-independent mechanism. However, stronger interaction is achieved when an acidic amino acid in a zwitterionic motif in PTN's C-terminal domain chelates the divalent cation in the metal ion-dependent adhesion site of the active αMI-domain. These results indicate that αMI-domain can bind ligands using multiple mechanisms, and suggest that active αMI-domain prefers acidic amino acids in zwitterionic motifs.

Integrins in Health and Disease-Suitable Targets for Treatment?

Integrin receptors are heterodimeric surface receptors that play multiple roles regarding cell-cell communication, signaling, and migration. The four members of the ß2 integrin subfamily are composed of an alternative α (CD11a-d) subunit, which determines the specific receptor properties, and a constant ß (CD18) subunit. This review aims to present insight into the multiple immunological roles of integrin receptors, with a focus on ß2 integrins that are specifically expressed by leukocytes. The pathophysiological role of ß2 integrins is confirmed by the drastic phenotype of patients suffering from leukocyte adhesion deficiencies, most often resulting in severe recurrent infections and, at the same time, a predisposition for autoimmune diseases. So far, studies on the role of ß2 integrins in vivo employed mice with a constitutive knockout of all ß2 integrins or either family member, respectively, which complicated the differentiation between the direct and indirect effects of ß2 integrin deficiency for distinct cell types. The recent generation and characterization of transgenic mice with a cell-type-specific knockdown of ß2 integrins by our group has enabled the dissection of cell-specific roles of ß2 integrins. Further, integrin receptors have been recognized as target receptors for the treatment of inflammatory diseases as well as tumor therapy. However, whereas both agonistic and antagonistic agents yielded beneficial effects in animal models, the success of clinical trials was limited in most cases and was associated with unwanted side effects. This unfavorable outcome is most probably related to the systemic effects of the used compounds on all leukocytes, thereby emphasizing the need to develop formulations that target distinct types of leukocytes to modulate ß2 integrin activity for therapeutic applications.

Mac-1 deficiency ameliorates pressure overloaded heart failure through inhibiting macrophage polarization and activation.

Persistent pressure overload commonly leads to pathological cardiac hypertrophy and remodeling, ultimately leading to heart failure (HF). Cardiac remodeling is associated with the involvement of immune cells and the inflammatory response in pathogenesis. The macrophage-1 antigen (Mac-1) is specifically expressed on leukocytes and regulates their migration and polarization. Nonetheless, the involvement of Mac-1 in cardiac remodeling and HF caused by pressure overload has not been determined. The Mac-1-knockout (KO) and wild-type (WT) mice were subjected to transverse aortic constriction (TAC) for 6 weeks. Echocardiography and pressure-volume loop assessments were used to evaluate cardiac function, and cardiac remodeling and macrophage infiltration and polarization were estimated by histopathology and molecular techniques. The findings of our study demonstrated that Mac-1 expression was markedly increased in hearts subjected to TAC treatment. Moreover, compared with WT mice, Mac-1-KO mice exhibited dramatically ameliorated TAC-induced cardiac dysfunction, hypertrophy, fibrosis, oxidative stress and apoptosis. The potential positive impacts may be linked to the inhibition of macrophage infiltration and M1 polarization via reductions in NF-kB and STAT1 expression and upregulation of STAT6. In conclusion, this research reveals a new function of Mac-1 deficiency in reducing pathological cardiac remodeling and HF caused by pressure overload. Additionally, inhibiting Mac-1 could be a potential treatment option for patients with HF in a clinical setting.

Rutin prevents pyroptosis and M1 microglia via Nrf2/Mac-1/caspase-1-mediated inflammasome axis to improve POCD.

BACKGROUND: Neuroinflammation following peripheral surgery plays a key role in postoperative cognitive dysfunction (POCD) development and there is no effective therapy to inflammation-mediated cognitive impairment. Recent studies showed that rutin, a natural flavonoid compound, conferred neuroprotection. However, the effects and mechanisms of rutin on cognition of surgical and aged mice and LPS-induced BV2 need deeper exploration. METHODS: The effect of rutin in vivo and vitro were evaluated by Morris water maze test, HE stainin, Golgi-Cox staining, IF, IHC, RT-PCR, Flow Cytometer and Western blotting. In vivo, aged mice were treated with rutin and surgery. In vitro, rutin, Nrf2 knockdown, MAC-1 overexpression and VX765, a caspase-1 inhibitor, were administration on BV2 microglial cells. RESULTS: Surgery led to compensatory increase in nuclear Nrf2 and rutin could further increase it. Neural damage was accompanied with high level in MAC-1, caspase-1-mediated pyroptosis and M1 microglia, while rutin recovered the process. Nrf2 inhibition abolished the effect of rutin with the increase of MAC-1, caspase-1-mediated pyroptosis and M1 microglia. Activation of MAC-1 abrogated protection of rutin by increase in pyroptosis and M1 microglia. Finally, we found that treatment with VX765 improved injury and increased M2 microglia against overexpression of MAC-1. CONCLUSIONS: Our study indicated that rutin may be a potential therapy in POCD and exerted neural protection via Nrf2/ Mac-1/ caspase-1-mediated inflammasome axis to regulate pyroptosis and microglial polarization.

Structure-Based High-Throughput Virtual Screening and Molecular Dynamics Simulation for the Discovery of Novel SARS-CoV-2 NSP3 Mac1 Domain Inhibitors.

Since the emergence of SARS-CoV-2, many genetic variations within its genome have been identified, but only a few mutations have been found in nonstructural proteins (NSPs). Among this class of viral proteins, NSP3 is a multidomain protein with 16 different domains, and its largest domain is known as the macrodomain or Mac1 domain. In this study, we present a virtual screening campaign in which we computationally evaluated the NCI anticancer library against the NSP3 Mac1 domain, using Molegro Virtual Docker. The top hits with the best MolDock and Re-Rank scores were selected. The physicochemical analysis and drug-like potential of the top hits were analyzed using the SwissADME data server. The binding stability and affinity of the top NSC compounds against the NSP3 Mac1 domain were analyzed using molecular dynamics (MD) simulation, using Desmond software, and their interaction energies were analyzed using the MM/GBSA method. In particular, by applying subsequent computational filters, we identified 10 compounds as possible NSP3 Mac1 domain inhibitors. Among them, after the assessment of binding energies (ΔGbind) on the whole MD trajectories, we identified the four most interesting compounds that acted as strong binders of the NSP3 Mac1 domain (NSC-358078, NSC-287067, NSC-123472, and NSC-142843), and, remarkably, it could be further characterized for developing innovative antivirals against SARS-CoV-2.

Platelet factor 4 improves survival in a murine model of antibiotic-susceptible and methicillin-resistant Staphylococcus aureus peritonitis.

The complement receptor CR3, also known as integrin Mac-1 (CD11b/CD18), is one of the major phagocytic receptors on the surface of neutrophils and macrophages. We previously demonstrated that in its protein ligands, Mac-1 binds sequences enriched in basic and hydrophobic residues and strongly disfavors negatively charged sequences. The avoidance by Mac-1 of negatively charged surfaces suggests that the bacterial wall and bacterial capsule possessing net negative electrostatic charge may repel Mac-1 and that the cationic Mac-1 ligands can overcome this evasion by acting as opsonins. Indeed, we previously showed that opsonization of Gram-negative Escherichia coli with several cationic peptides, including PF4 (Platelet Factor 4), strongly augmented phagocytosis by macrophages. Here, we investigated the effect of recombinant PF4 (rPF4) on phagocytosis of Gram-positive Staphylococcus aureus in vitro and examined its impact in a mouse model of S. aureus peritonitis. Characterization of the interaction of rPF4 with nonencapsulated and encapsulated S. aureus showed that rPF4 localizes on the bacterial surface, thus making it available for Mac-1. Furthermore, rPF4 did not have direct bactericidal and bacteriostatic activity and was not toxic to host cells. rPF4 enhanced phagocytosis of S. aureus bioparticles by various primary and cultured Mac-1-expressing leukocytes by several folds. It also increased phagocytosis of live nonencapsulated and encapsulated bacteria. Notably, the augmentation of phagocytosis by rPF4 did not compromise the intracellular killing of S. aureus by macrophages. Using a murine S. aureus peritonitis model, we showed that treatment of infected mice with rPF4 caused a significant increase in the clearance of antibiotic-susceptible S. aureus and its methicillin-resistant (MRSA) variant and markedly improved survival. These findings indicate that rPF4 binding to the bacterial surface circumvents its antiphagocytic properties, improving host defense against antibiotic-susceptible and antibiotic-resistant bacteria.

An Update on the Current State of SARS-CoV-2 Mac1 Inhibitors.

Non-structural protein 3 (nsp3) from all coronaviruses (CoVs) contains a conserved macrodomain, known as Mac1, that has been proposed as a potential therapeutic target for CoVs due to its critical role in viral pathogenesis. Mac1 is an ADP-ribose binding protein and ADP-ribosylhydrolase that promotes replication and blocks IFN responses, though the precise mechanisms it uses to carry out these functions remain unknown. Over the past 3 years following the onset of COVID-19, several groups have used high-throughput screening with multiple assays and chemical modifications to create unique chemical inhibitors of the SARS-CoV-2 Mac1 protein. Here, we summarize the current efforts to identify selective and potent inhibitors of SARS-CoV-2 Mac1.

PLATELET FACTOR 4 (PF4) IMPROVES SURVIVAL IN A MURINE MODEL OF ANTIBIOTIC-SUSCEPTIBLE AND METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS PERITONITIS.

The complement receptor CR3, also known as integrin Mac-1 (CD11b/CD18), is one of the major phagocytic receptors on the surface of neutrophils and macrophages. We previously demonstrated that in its protein ligands, Mac-1 binds sequences enriched in basic and hydrophobic residues and strongly disfavors negatively charged sequences. The avoidance by Mac-1 of negatively charged surfaces suggests that the bacterial wall and bacterial capsule possessing net negative electrostatic charge may repel Mac-1 and that the cationic Mac-1 ligands can overcome this evasion by acting as opsonins. Indeed, we previously showed that opsonization of Gram-negative Escherichia coli with several cationic peptides, including PF4 (Platelet Factor 4), strongly augmented phagocytosis by macrophages. Here, we investigated the effect of recombinant PF4 (rPF4) on phagocytosis of Gram-positive Staphylococcus aureus in vitro and examined its impact in a mouse model of S. aureus peritonitis. Characterization of the interaction of rPF4 with nonencapsulated and encapsulated S. aureus showed that rPF4 localizes on the bacterial surface, thus making it available for Mac-1. Furthermore, rPF4 did not have direct bactericidal and bacteriostatic activity and was not toxic to host cells. rPF4 enhanced phagocytosis of S. aureus bioparticles by various primary and cultured Mac-1-expressing leukocytes by several folds. It also increased phagocytosis of live nonencapsulated and encapsulated bacteria. Notably, the augmentation of phagocytosis by rPF4 did not compromise the intracellular killing of S. aureus by macrophages. Using a murine S. aureus peritonitis model, we showed that treatment of infected mice with rPF4 caused a significant increase in the clearance of antibiotic-susceptible S. aureus and its methicillin-resistant (MRSA) variant and markedly improved survival. These findings indicate that rPF4 binding to the bacterial surface circumvents its antiphagocytic properties, improving host defense against antibiotic-susceptible and antibiotic-resistant bacteria.

An Activation-Specific Anti-Mac-1 Designed-Ankyrin-Repeat-Protein Attenuates Colitis in Mice.

(1) Background: Inflammatory bowel diseases are complex and multifactorial disorders of unknown etiology. The extravasation of activated leukocytes is a critical step in the pathogenesis of these diseases. Leukocyte integrin Mac-1 (αMß2; CD11b/CD18) is crucial for the extravasation of myeloid cells, and a novel activation-specific anti-Mac-1 Designed Ankyrin Repeat protein (DARPin F7) is a promising therapeutic agent for inflammatory diseases. In its activated conformation, Mac-1 expresses the high-affinity binding site I-domain, which the DARPin F7 selectively targets. In our study, we aimed to explore the therapeutic potential of anti-Mac-1 DARPin F7 in murine dextrane sodium sulfate (DSS)-induced colitis. (2) Methods: C57BL/6J mice received 3% DSS drinking water for five days, followed by normal drinking water for one week. The mice were treated with DARPin F7 or a control substance daily via intraperitoneal injections. Disease activity index (DAI), colon length, myeloperoxidase (MPO) activity measurements, H&E staining, and qRT-PCR were conducted after euthanizing the mice on day 12. (3) Results: Treatment with DARPin F7 resulted in less pronounced colon shortening and significantly lower histological scores. The DARPin F7-treated animals experienced substantially less disease and myeloperoxidase (MPO) activity. Animals that received DARPin F7 treatment suffered less weight loss and recovered from the weight loss more efficiently. Treatment with DARPin F7 also led to significantly reduced mRNA expression of inflammatory cytokines. (4) Conclusion: Anti-Mac-1 treatment markedly reduced disease activity and inflammatory reaction accompanying DSS-induced colitis in mice.

Dual wave of neutrophil recruitment determines the outcome of C. albicans infection.

Candida albicans is a ubiquitous fungus that can cause superficial and systemic infections in humans. Neutrophils play a crucial role in controlling C. albicans infections. When C. albicans enters the bloodstream, it tends to get trapped in capillary vessels. However, the behavior of neutrophils in combating capillary-residing fungi has not been fully characterized. In this study, we used transgenic mice and whole mount imaging to investigate the growth of C. albicans and its interaction with innate immune cells in different organs. We observed that C. albicans rapidly grows hyphae within hours of infection. Following intravenous infection, we observed two waves of neutrophil recruitment, both of which significantly contributed to the elimination of the fungi. The first wave of neutrophils was induced by complement activation and could be prevented by C5aR blockade. Interestingly, we discovered that the fungicidal effect in the lungs was independent of adhesion molecules such as Mac-1, LFA-1, and ICAM-1. However, these molecules played a more significant role in the optimal killing of C. albicans in the kidney. Importantly, the initial difference in killing efficiency resulted in significantly reduced survival in knockout mice lacking these adhesion molecules. We identified a second wave of neutrophil recruitment associated with hyphal growth and tissue damage, which was independent of the aforementioned adhesion molecules. Overall, this study elucidates the dual wave of neutrophil recruitment during C. albicans infection and highlights the importance of early fungal clearance for favorable disease outcomes.

The complement receptor C5aR2 regulates neutrophil activation and function contributing to neutrophil-driven epidermolysis bullosa acquisita.

Introduction: The function of the second receptor for the complement cleavage product C5a, C5aR2, is poorly understood and often neglected in the immunological context. Using mice with a global deficiency of C5aR2, we have previously reported an important role of this receptor in the pathogenesis of the neutrophil-driven autoimmune disease epidermolysis bullosa acquisita (EBA). Based on in vitro analyses, we hypothesized that the absence of C5aR2 specifically on neutrophils is the cause of the observed differences. Here, we report the generation of a new mouse line with a LysM-specific deficiency of C5aR2. Methods: LysM-specific deletion of C5aR2 was achieved by crossing LysMcre mice with tdTomato-C5ar2fl/fl mice in which the tdTomato-C5ar2 gene is flanked by loxP sites. Passive EBA was induced by subcutaneous injection of rabbit anti-mouse collagen type VII IgG. The effects of targeted deletion of C5ar2 on C5a-induced effector functions of neutrophils were examined in in vitro assays. Results: We confirm the successful deletion of C5aR2 at both the genetic and protein levels in neutrophils. The mice appeared healthy and the expression of C5aR1 in bone marrow and blood neutrophils was not negatively affected by LysM-specific deletion of C5aR2. Using the antibody transfer mouse model of EBA, we found that the absence of C5aR2 in LysM-positive cells resulted in an overall amelioration of disease progression, similar to what we had previously found in mice with global deficiency of C5aR2. Neutrophils lacking C5aR2 showed decreased activation after C5a stimulation and increased expression of the inhibitory Fcγ receptor FcγRIIb. Discussion: Overall, with the data presented here, we confirm and extend our previous findings and show that C5aR2 in neutrophils regulates their activation and function in response to C5a by potentially affecting the expression of Fcγ receptors and CD11b. Thus, C5aR2 regulates the finely tuned interaction network between immune complexes, Fcγ receptors, CD11b, and C5aR1 that is important for neutrophil recruitment and sustained activation. This underscores the importance of C5aR2 in the pathogenesis of neutrophil-mediated autoimmune diseases.

Complement C3 Enhances LPS-Elicited Neuroinflammation and Neurodegeneration Via the Mac1/NOX2 Pathway.

Recent studies showed increased expression of complements in various neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. However, the mechanism regulating the expression of complements and their roles in the pathogenesis of neurodegeneration are unclear. We hypothesized that acute neuroinflammation increases the expression and activation of brain complements, which, in turn, participate in chronic neuroinflammation and progressive neurodegeneration. We initially focused on the complement component C3, because C3 can activate microglia by binding to C3 receptors and attaching to damaged neurons destined to be phagocytosed by microglia. We found that complement C3 is upregulated in lipopolysaccharide (LPS)-stimulated neuron/glial cultures. Mechanistic studies revealed that microglia-released proinflammatory factors initiated the enhanced expression of C3 in astroglia during acute neuroinflammation. On the other hand, the sustained C3 expression during chronic neuroinflammation requires releasing damage-associated molecule patterns (DAMPs) from damaged/degenerating brain cells. Our results suggested that DAMPs might act on microglial integrin receptor Mac1 to trigger the activation of NADPH oxidase (NOX2). Activated microglial NOX2 increases the production of extracellular reactive oxygen species (ROS), elevating the levels of intracellular ROS of astroglia and sustaining the astroglial C3 expression. This was supported by the findings showing reduced C3 expression and attenuated neurodegeneration in LPS-treated neuron/glial cultures prepared from mice deficient in Mac1 or NOX2. LPS-induced neurodegeneration and oxidative stress are significantly reduced in C3 KO neuron/glial cultures and mouse brains. Together, this study provides the first evidence demonstrating the role of C3 in regulating chronic neuroinflammation and in driving progressive neurodegeneration.

MAC-1 marks a quiescent and functionally superior HSC subset during regeneration.

Mouse hematopoietic stem cells (HSCs) have been extensively defined both molecularly and functionally at steady state, while regenerative stress induces immunophenotypical changes that limit high purity isolation and analysis. It is therefore important to identify markers that specifically label activated HSCs to gain further knowledge about their molecular and functional properties. Here, we assessed the expression of macrophage-1 antigen (MAC-1) on HSCs during regeneration following transplantation and observed a transient increase in MAC-1 expression during the early reconstitution phase. Serial transplantation experiments demonstrated that reconstitution potential was highly enriched in the MAC-1+ portion of the HSC pool. Moreover, in contrast to previous reports, we found that MAC-1 expression inversely correlates with cell cycling, and global transcriptome analysis showed that regenerating MAC-1+ HSCs share molecular features with stem cells with low mitotic history. Taken together, our results suggest that MAC-1 expression marks predominantly quiescent and functionally superior HSCs during early regeneration.

Cathelicidin promotes liver repair after acetaminophen-induced liver injury in mice.

Background & Aims: Acetaminophen (APAP)-induced acute liver injury (AILI) is a leading cause of acute liver failure (ALF). N-acetylcysteine (NAC) is only effective within 24 h after APAP intoxication, raising an urgent need for alternative approaches to treat this disease. This study aimed to test whether cathelicidin (Camp), which is a protective factor in chronic liver diseases, protects mice against APAP-induced liver injury and ALF. Methods: A clinically relevant AILI model and an APAP-induced ALF model were generated in mice. Genetic and pharmacological approaches were used to interfere with the levels of cathelicidin in vivo. Results: An increase in hepatic pro-CRAMP/CRAMP (the precursor and mature forms of mouse cathelicidin) was observed in APAP-intoxicated mice. Upregulated cathelicidin was derived from liver-infiltrating neutrophils. Compared with wild-type littermates, Camp knockout had no effect on hepatic injury but dampened hepatic repair in AILI and reduced survival in APAP-induced ALF. CRAMP administration reversed impaired liver recovery observed in APAP-challenged Camp knockout mice. Delayed CRAMP, CRAMP(1-39) (the extended form of CRAMP), or LL-37 (the mature form of human cathelicidin) treatment exhibited a therapeutic benefit for AILI. Co-treatment of cathelicidin and NAC in AILI displayed a stronger hepatoprotective effect than NAC alone. A similar additive effect of CRAMP(1-39)/LL-37 and NAC was observed in APAP-induced ALF. The pro-reparative role of cathelicidin in the APAP-damaged liver was attributed to an accelerated resolution of inflammation at the onset of liver repair, possibly through enhanced neutrophil phagocytosis of necrotic cell debris in an autocrine manner. Conclusions: Cathelicidin reduces APAP-induced liver injury and ALF in mice by promoting liver recovery via facilitating inflammation resolution, suggesting a therapeutic potential for late-presenting patients with AILI with or without ALF. Impact and implications: Acetaminophen-induced acute liver injury is a leading cause of acute liver failure. The efficacy of N-acetylcysteine, the only clinically approved drug against acetaminophen-induced acute liver injury, is significantly reduced for late-presenting patients. We found that cathelicidin exhibits a great therapeutic potential in mice with acetaminophen-induced liver injury or acute liver failure, which makes up for the limitation of N-acetylcysteine therapy by specifically promoting liver repair after acetaminophen intoxication. The pro-reparative role of cathelicidin, as a key effector molecule of neutrophils, in the APAP-injured liver is attributed to an accelerated resolution of inflammation at the onset of liver repair, possibly through enhanced phagocytic function of neutrophils in an autocrine manner.

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.

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.

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.

Spatiotemporal characteristics of P-selectin-induced ß2 integrin activation of human neutrophils under flow.

Activation of integrins is crucial for recruitment of flowing leukocytes to inflammatory or injured vascular sites, but their spatiotemporal characteristics are incompletely understood. We discovered that ß2-integrin activation over the entire surface of neutrophils on immobilized P-selectin occurred via mitogen-activated protein kinase (MAPK) or non-MAPK signaling with a minute-level timescale in a force-dependent manner. In flow, MAPK signaling required intracellular Ca2+ release to activate integrin within 2 min. Integrin activation via non-MAPK signaling occurred first locally in the vicinity of ligated P-selectin glycoprotein ligand-1 (PSGL-1) within sub-seconds, and then over the entire cell surface within 1 min in an extracellular Ca2+ influx-dependent manner. The transition from a local (but rapid) to global (but slow) activation mode was triggered by ligating the freshly activated integrin. Lipid rafts, moesin, actin, and talin were involved in non-MAPK signaling. Fluid loads had a slight effect on local integrin activation with a second-level timescale, but served as enhancers of global integrin activation.