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1.  Protection against inhaled oxidants through scavenging of oxidized lipids by macrophage receptors MARCO and SR-AI/II 
Journal of Clinical Investigation  2007;117(3):757-764.
Alveolar macrophages (AMs) express the class A scavenger receptors (SRAs) macrophage receptor with collagenous structure (MARCO) and scavenger receptor AI/II (SRA-I/II), which recognize oxidized lipids and provide innate defense against inhaled pathogens and particles. Increased MARCO expression in lungs of ozone-resistant mice suggested an additional role protecting against inhaled oxidants. After ozone exposure, MARCO–/– mice showed greater lung injury than did MARCO+/+ mice. Ozone is known to generate oxidized, proinflammatory lipids in lung lining fluid, such as 5β,6β-epoxycholesterol (β-epoxide) and 1-palmitoyl-2-(9′-oxo-nonanoyl)-glycerophosphocholine (PON-GPC). Intratracheal instillation of either lipid caused substantial neutrophil influx in MARCO–/– mice, but had no effect in MARCO+/+ mice. Normal AMs showed greater uptake in vitro of β-epoxide compared with MARCO–/– AMs, consistent with SRA function in binding oxidized lipids. SR-AI/II–/– mice showed similar enhanced acute lung inflammation after β-epoxide or another inhaled oxidant (aerosolized leachate of residual oil fly ash). In contrast, subacute ozone exposure did not enhance inflammation in SR-AI/II–/– versus SR-AI/II+/+ mice, reflecting increased AM expression of MARCO. These data identify what we believe to be a novel function for AM SRAs in decreasing pulmonary inflammation after oxidant inhalation by scavenging proinflammatory oxidized lipids from lung lining fluids.
PMCID: PMC1804372  PMID: 17332894
2.  Critical Role of MARCO in Crystalline Silica–Induced Pulmonary Inflammation 
Toxicological Sciences  2009;108(2):462-471.
Chronic exposure to crystalline silica can lead to the development of silicosis, an irreversible, inflammatory and fibrotic pulmonary disease. Although, previous studies established the macrophage receptor with collagenous structure (MARCO) as an important receptor for binding and uptake of crystalline silica particles in vitro, the role of MARCO in regulating the inflammatory response following silica exposure in vivo remains unknown. Therefore, we determined the role of MARCO in crystalline silica–induced pulmonary pathology using C57Bl/6 wild-type (WT) and MARCO−/− mice. Increased numbers of MARCO+ pulmonary macrophages were observed following crystalline silica, but not phosphate-buffered saline and titanium dioxide (TiO2), instillation in WT mice, highlighting a specific role of MARCO in silica-induced pathology. We hypothesized that MARCO−/− mice will exhibit diminished clearance of silica leading to enhanced pulmonary inflammation and exacerbation of silicosis. Alveolar macrophages isolated from crystalline silica–exposed mice showed diminished particle uptake in vivo as compared with WT mice, indicating abnormalities in clearance mechanisms. Furthermore, MARCO−/− mice exposed to crystalline silica showed enhanced acute inflammation and lung injury marked by increases in early response cytokines and inflammatory cells compared with WT mice. Similarly, histological examination of MARCO−/− lungs at 3 months post–crystalline silica exposure showed increased chronic inflammation compared with WT; however, only a small difference was observed with respect to development of fibrosis as measured by hydroxyproline content. Altogether, these results demonstrate that MARCO is important for clearance of crystalline silica in vivo and that the absence of MARCO results in exacerbations in innate pulmonary immune responses.
PMCID: PMC2664690  PMID: 19151164
fibrosis; silicosis; particle clearance; macrophages; scavenger receptors
3.  The Scavenger Receptor MARCO Is Required for Lung Defense against Pneumococcal Pneumonia and Inhaled Particles 
Alveolar macrophages (AMs) express the class A scavenger receptor macrophage receptor with collagenous structure (MARCO), but its role in vivo in lung defense against bacteria and environmental particles has not been studied. We used MARCO-deficient mice to directly test the in vivo role of AM MARCO in innate defense against pneumococcal infection and environmental particles. In a murine model of pneumococcal pneumonia, MARCO−/− mice displayed an impaired ability to clear bacteria from the lungs, increased pulmonary inflammation and cytokine release, and diminished survival. In vitro binding of Streptococcus pneumoniae and in vivo uptake of unopsonized particles by MARCO−/− AMs were dramatically impaired. MARCO−/− mice treated with the “inert” environmental particle TiO2 showed enhanced inflammation and chemokine expression, indicating that MARCO-mediated clearance of inert particles by AMs prevents inflammatory responses otherwise initiated by other lung cells. Our findings point to an important role of MARCO in mounting an efficient and appropriately regulated innate immune response against inhaled particles and airborne pathogens.
PMCID: PMC2212010  PMID: 15263032
macrophage; phagocytosis; environment; innate immunity
4.  MARCO, TLR2, and CD14 Are Required for Macrophage Cytokine Responses to Mycobacterial Trehalose Dimycolate and Mycobacterium tuberculosis 
PLoS Pathogens  2009;5(6):e1000474.
Virtually all of the elements of Mycobacterium tuberculosis (Mtb) pathogenesis, including pro-inflammatory cytokine production, granuloma formation, cachexia, and mortality, can be induced by its predominant cell wall glycolipid, trehalose 6,6′-dimycolate (TDM/cord factor). TDM mediates these potent inflammatory responses via interactions with macrophages both in vitro and in vivo in a myeloid differentiation factor 88 (MyD88)-dependent manner via phosphorylation of the mitogen activated protein kinases (MAPKs), implying involvement of toll-like receptors (TLRs). However, specific TLRs or binding receptors for TDM have yet to be identified. Herein, we demonstrate that the macrophage receptor with collagenous structure (MARCO), a class A scavenger receptor, is utilized preferentially to “tether” TDM to the macrophage and to activate the TLR2 signaling pathway. TDM-induced signaling, as measured by a nuclear factor-kappa B (NF-κB)-luciferase reporter assay, required MARCO in addition to TLR2 and CD14. MARCO was used preferentially over the highly homologous scavenger receptor class A (SRA), which required TLR2 and TLR4, as well as their respective accessory molecules, in order for a slight increase in NF-κB signaling to occur. Consistent with these observations, macrophages from MARCO−/− or MARCO−/−SRA−/− mice are defective in activation of extracellular signal-related kinase 1/2 (ERK1/2) and subsequent pro-inflammatory cytokine production in response to TDM. These results show that MARCO-expressing macrophages secrete pro-inflammatory cytokines in response to TDM by cooperation between MARCO and TLR2/CD14, whereas other macrophage subtypes (e.g. bone marrow–derived) may rely somewhat less effectively on SRA, TLR2/CD14, and TLR4/MD2. Macrophages from MARCO−/− mice also produce markedly lower levels of pro-inflammatory cytokines in response to infection with virulent Mtb. These observations identify the scavenger receptors as essential binding receptors for TDM, explain the differential response to TDM of various macrophage populations, which differ in their expression of the scavenger receptors, and identify MARCO as a novel component required for TLR signaling.
Author Summary
The causative agent of tuberculosis, Mycobacterium tuberculosis, has a lipid-rich cell wall that contains a high percentage of mycolic acids. These mycolic acids contribute to both the impermeable nature of the cell wall and to the immunostimulatory properties of the bacterium. Indeed, it has been known for over 50 years that trehalose 6,6′-dimycolate (TDM/cord factor) is the major immunogenic lipid of M. tuberculosis, which induces potent pro-inflammatory responses from macrophages, although the receptor has not been identified. We have demonstrated that the toll-like receptor (TLR) pathway is required for pro-inflammatory cytokine production in response to TDM; however, the TLRs alone, or in conjunction with known co-receptors, are not sufficient to induce a response. We demonstrate that the macrophage receptor MARCO, a scavenger receptor, is utilized preferentially to “tether” TDM to the macrophage and activate the TLR2 signaling pathway, and is used preferentially over the related SRA. Macrophages from MARCO−/− mice are defective in activation of TDM-induced signaling and subsequent pro-inflammatory cytokine production in response to both TDM-coated beads and virulent M. tuberculosis. By identifying the macrophage receptors involved in initial recognition we can now explain variable responses to TDM between different macrophage populations (which differ in scavenger receptor expression), and have identified a novel co-receptor that may be involved in lipid presentation to TLRs.
PMCID: PMC2688075  PMID: 19521507
5.  Role of Lysosomes in Silica-Induced Inflammasome Activation and Inflammation in Absence of MARCO 
Journal of Immunology Research  2014;2014:304180.
MARCO is the predominant scavenger receptor for recognition and binding of silica particles by alveolar macrophages (AM). Previously, it was shown that mice null for MARCO have a greater inflammatory response to silica, but the mechanism was not described. The aim of this study was to determine the relationship between MARCO and NLRP3 inflammasome activity. Silica increased NLRP3 inflammasome activation and release of the proinflammatory cytokine, IL-1β, to a greater extent in MARCO−/− AM compared to wild type (WT) AM. Furthermore, in MARCO−/− AM there was greater cathepsin B release from phagolysosomes, Caspase-1 activation, and acid sphingomyelinase activity compared to WT AM, supporting the critical role played by lysosomal membrane permeabilization (LMP) in triggering silica-induced inflammation. The difference in sensitivity to LMP appears to be in cholesterol recycling since increasing cholesterol in AM by treatment with U18666A decreased silica-induced NLRP3 inflammasome activation, and cells lacking MARCO were less able to sequester cholesterol following silica treatment. Taken together, these results demonstrate that MARCO contributes to normal cholesterol uptake in macrophages; therefore, in the absence of MARCO, macrophages are more susceptible to a greater inflammatory response by particulates known to cause NLRP3 inflammasome activation and the effect is due to increased LMP.
PMCID: PMC4099041  PMID: 25054161
6.  Role of the Scavenger Receptor MARCO in Alveolar Macrophage Binding of Unopsonized Environmental Particles  
The Journal of Experimental Medicine  1999;189(9):1497-1506.
Alveolar macrophages (AMs) avidly bind and ingest unopsonized environmental particles and bacteria through scavenger-type receptors (SRs). AMs from mice with a genetic deletion of the major macrophage SR (types AI and AII; SR−/−) showed no decrease in particle binding compared with SR+/+ mice, suggesting that other SRs are involved. To identify these receptors, we generated a monoclonal antibody (mAb), PAL-1, that inhibits hamster AM binding of unopsonized particles (TiO2, Fe2O3, and latex beads; 66 ± 5, 77 ± 2, and 85 ± 2% inhibition, respectively, measured by flow cytometry). This antibody identifies a protein of ∼70 kD on the AM surface (immunoprecipitation) that is expressed by AMs and other macrophages in situ. A cDNA clone encoding the mAb PAL-1–reactive protein isolated by means of COS cell expression was found to be 84 and 77% homologous to mouse and human scavenger receptor MARCO mRNA, respectively. Transfection of COS cells with MARCO cDNA conferred mAb-inhibitable TiO2 binding. Hamster MARCO also mediates AM binding of unopsonized bacteria (67 ± 5 and 47 ± 4% inhibition of Escherichia coli and Staphylococcus aureus binding by mAb PAL-1). A polyclonal antibody to human MARCO identified the expected ∼70-kD band on Western blots of lysates of normal bronchoalveolar lavage (BAL) cells (>90% AMs) and showed strong immunolabeling of human AMs in BAL cytocentrifuge preparations and within lung tissue specimens. In normal mouse AMs, the anti-MARCO mAb ED31 also showed immunoreactivity and inhibited binding of unopsonized particles (e.g., TiO2 ∼40%) and bacteria. The novel function of binding unopsonized environmental dusts and pathogens suggests an important role for MARCO in the lungs' response to inhaled particles.
PMCID: PMC2193067  PMID: 10224290
MARCO; alveolar; macrophage; unopsonized; environmental particle
7.  The Scavenger Receptor MARCO Modulates TLR-Induced Responses in Dendritic Cells 
PLoS ONE  2014;9(8):e104148.
The scavenger receptor MARCO mediates macrophage recognition and clearance of pathogens and their polyanionic ligands. However, recent studies demonstrate MARCO expression and function in dendritic cells, suggesting MARCO might serve to bridge innate and adaptive immunity. To gain additional insight into the role of MARCO in dendritic cell activation and function, we profiled transcriptomes of mouse splenic dendritic cells obtained from MARCO deficient mice and their wild type counterparts under resting and activating conditions. In silico analysis uncovered major alterations in gene expression in MARCO deficient dendritic cells resulting in dramatic alterations in key dendritic cell-specific pathways and functions. Specifically, changes in CD209, FCGR4 and Complement factors can have major consequences on DC-mediated innate responses. Notably, these perturbations were magnified following activation with the TLR-4 agonist lipopolysaccharide. To validate our in silico data, we challenged DC‘s with various agonists that recognize all mouse TLRs and assessed expression of a set of immune and inflammatory marker genes. This approach identified a differential contribution of MARCO to TLR activation and validated a major role for MARCO in mounting an inflammatory response. Together, our data demonstrate that MARCO differentially affects TLR-induced DC activation and suggest targeting of MARCO could lead to different outcomes that depend on the inflammatory context encountered by DC.
PMCID: PMC4121322  PMID: 25089703
8.  MARCO is required for TLR2- and NOD2-mediated responses to Streptococcus pneumoniae and clearance of pneumococcal colonization in the murine nasopharynx 
Streptococcus pneumoniae is a common human pathogen that accounts for over a million deaths every year. Colonization of the nasopharynx by S. pneumoniae precedes pulmonary and other invasive diseases, and is therefore a promising target for intervention. Since the receptors Scavenger Receptor A (SRA), Macrophage Receptor with Collagenous Structure (MARCO), and Mannose Receptor (MR) have previously been identified as non-opsonic receptors for S. pneumoniae in the lung, we utilized scavenger receptor knock out mice to study the roles of these receptors in the clearance of S. pneumoniae from the nasopharynx. MARCO−/−, but not SRA−/− or MR−/−, mice had significantly impaired clearance of S. pneumoniae from the nasopharynx. In addition to impairment in bacterial clearance, MARCO−/− mice had abrogated cytokine production and cellular recruitment to the nasopharynx following colonization. Furthermore, macrophages from MARCO−/− mice were deficient in cytokine and chemokine production, including type I interferons, in response to S. pneumoniae. MARCO was required for maximal TLR2- and NOD2-dependent NF-κB activation and signaling that ultimately resulted in clearance. Thus, MARCO is an important component of anti-S. pneumoniae responses in the murine nasopharynx during colonization.
PMCID: PMC3529821  PMID: 23197261
Innate Immunity; Macrophage; Scavenger Receptor; MARCO; Streptococcus pneumoniae; Colonization; Nasopharynx
9.  Protective or Deleterious Role of Scavenger Receptors SR-A and CD36 on Host Resistance to Staphylococcus aureus Depends on the Site of Infection 
PLoS ONE  2014;9(1):e87927.
Staphylococcus aureus is a major human opportunistic pathogen responsible for a broad spectrum of infections ranging from benign skin infection to more severe life threatening disorders (e.g. pneumonia, sepsis), particularly in intensive care patients. Scavenger receptors (SR-A and CD36) are known to be involved in S. aureus recognition by immune cells in addition to MARCO, TLR2, NOD2 and α5β1 integrin. In the present study, we further deciphered the contribution of SR-A and CD36 scavenger receptors in the control of infection of mice by S. aureus. Using double SR-A/CD36 knockout mice (S/C-KO) and S. aureus strain HG001, a clinically relevant non-mutagenized strain, we showed that the absence of these two scavenger receptors was protective in peritoneal infection. In contrast, the deletion of these two receptors was detrimental in pulmonary infection following intranasal instillation. For pulmonary infection, susceptible mice (S/C-KO) had more colony-forming units (CFU) in their broncho-alveolar lavages fluids, associated with increased recruitment of macrophages and neutrophils. For peritoneal infection, susceptible mice (wild-type) had more CFU in their blood, but recruited less macrophages and neutrophils in the peritoneal cavity than resistant mice. Exacerbated cytokine levels were often observed in the susceptible mice in the infected compartment as well as in the plasma. The exception was the enhanced compartmentalized expression of IL-1β for the resistant mice (S/C-KO) after peritoneal infection. A similar mirrored susceptibility to S. aureus infection was also observed for MARCO and TLR2. Marco and tlr2 -/- mice were more resistant to peritoneal infection but more susceptible to pulmonary infection than wild type mice. In conclusion, our results show that innate immune receptors can play distinct and opposite roles depending on the site of infection. Their presence is protective for local pulmonary infection, whereas it becomes detrimental in the peritoneal infection.
PMCID: PMC3909292  PMID: 24498223
10.  Genetic variants of MARCO are associated with susceptibility to pulmonary tuberculosis in a Gambian population 
BMC Medical Genetics  2013;14:47.
The two major class A scavenger receptors are scavenger receptor A (SRA), which is constitutively expressed on most macrophage populations, and macrophage receptor with collagenous structure (MARCO), which is constitutively expressed on a more restricted subset of macrophages, (e.g. alveolar macrophages) but whose expression increases on most macrophages during the course of infection. Although the primary role of SRA appears to be clearance of modified host proteins and lipids, mice defective in expression of either MARCO or SRA are immunocompromised in multiple models of infection and in vitro assays, the scavenger receptors have been demonstrated to bind bacteria and to enhance pro-inflammatory signalling to many bacterial lung pathogens; however their importance in Mycobacterium tuberculosis infection, is less clear.
To determine whether polymorphisms in either SRA or MARCO were associated with tuberculosis, a case–control study of was performed. DNA samples from newly-detected, smear-positive, pulmonary tuberculosis cases were collected from The Gambia. Controls for this study consisted of DNA from cord bloods obtained from routine births at local Gambian health clinics. Informed written consent was obtained from patients or their parents or guardians. Ethical approval was provided by the joint The Gambian Government/MRC Joint Ethics Committee.
We studied the frequencies of 25 polymorphisms of MSR1 (SRA) and 22 in MARCO in individuals with tuberculosis (n=1284) and matched controls (n=1349). No SNPs within the gene encoding or within 1 kb of the promoter sequence of MSR1 were associated with either susceptibility or resistance to tuberculosis. Three SNPs in MARCO (rs4491733, Mantel-Haenszel 2x2 χ2 = 6.5, p = 0.001, rs12998782, Mantel-Haenszel 2x2 χ2 = 6.59, p = 0.001, rs13389814 Mantel-Haenszel 2x2 χ2 = 6.9, p = 0.0009) were associated with susceptibility to tuberculosis and one (rs7559955, Mantel-Haenszel 2x2 χ2 = 6.9, p = 0.0009) was associated with resistance to tuberculosis.
These findings identify MARCO as a potentially important receptor in the host response to tuberculosis.
PMCID: PMC3652798  PMID: 23617307
Scavenger receptors; Mycobacterium tuberculosis; Single nucleotide polymorphisms; Case control study; MARCO
11.  Differential Binding of Inorganic Particles to MARCO 
Toxicological Sciences  2008;107(1):238-246.
Alveolar macrophages (AM) in the lung have been documented to play pivotal roles in inflammation and fibrosis (silicosis) following inhalation of crystalline silica (CSiO2). In contrast, exposure to either titanium dioxide (TiO2) or amorphous silica (ASiO2) is considered relatively benign. The scavenger receptor macrophage receptor with collagenous structure (MARCO), expressed on AM, binds and internalizes environmental particles such as silica and TiO2. Only CSiO2 is toxic to AM, while ASiO2 and TiO2 are not. We hypothesize that differences in induction of pathology between toxic CSiO2 and nontoxic particles ASiO2 and TiO2 may be related to their differential binding to MARCO. In vitro studies with Chinese hamster ovary (CHO) cells transfected with human MARCO and mutants were conducted to better characterize MARCO-particulate (ASiO2, CSiO2, and TiO2) interactions. Results with MARCO-transfected CHO cells and MARCO-specific antibody demonstrated that the scavenger receptor cysteine-rich (SRCR) domain of MARCO was required for particle binding for all the tested particles. Only TiO2 required divalent cations (viz., Ca+2 and/or Mg+2) for binding to MARCO, and results from competitive binding studies supported the notion that TiO2 and both the silica particles bound to different motifs in SRCR domain of MARCO. The results also suggest that particle shape and/or crystal structure may be the determinants linking particle binding to MARCO and cytotoxicity. Taken together, these results demonstrate that the SRCR domain of MARCO is required for particle binding and that involvement of different regions of SRCR domain may distinguish downstream events following particle binding.
PMCID: PMC2735421  PMID: 18836211
MARCO; crystalline silica; amorphous silica; TiO2; binding; apoptosis
12.  The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis 
Recent studies have suggested that the scavenger receptor MARCO (macrophage receptor with collagenous structure) mediates activation of the immune response in bacterial infection of the central nervous system (CNS). The chemotactic G-protein-coupled receptor (GPCR) formyl-peptide-receptor like-1 (FPRL1) plays an essential role in the inflammatory responses of host defence mechanisms and neurodegenerative disorders such as Alzheimer's disease (AD). Expression of the antimicrobial peptide cathelicidin CRAMP/LL-37 is up-regulated in bacterial meningitis, but the mechanisms underlying CRAMP expression are far from clear.
Using a rat meningitis model, we investigated the influence of MARCO and FPRL1 on rCRAMP (rat cathelin-related antimicrobial peptide) expression after infection with bacterial supernatants of Streptococcus pneumoniae (SP) and Neisseria meningitides (NM). Expression of FPRL1 and MARCO was analyzed by immunofluorescence and real-time RT-PCR in a rat meningitis model. Furthermore, we examined the receptor involvement by real-time RT-PCR, extracellular-signal regulated kinases 1/2 (ERK1/2) phosphorylation and cAMP level measurement in glial cells (astrocytes and microglia) and transfected HEK293 cells using receptor deactivation by antagonists. Receptors were inhibited by small interference RNA and the consequences in NM- and SP-induced Camp (rCRAMP gene) expression and signal transduction were determined.
We show an NM-induced increase of MARCO expression by immunofluorescence and real-time RT-PCR in glial and meningeal cells. Receptor deactivation by antagonists and small interfering RNA (siRNA) verified the importance of FPRL1 and MARCO for NM- and SP-induced Camp and interleukin-1β expression in glial cells. Furthermore, we demonstrated a functional interaction between FPRL1 and MARCO in NM-induced signalling by real-time RT-PCR, ERK1/2 phosphorylation and cAMP level measurement and show differences between NM- or SP-induced signal transduction.
We propose that NM and SP induce glial cell activation and rCRAMP expression also via FPRL1 and MARCO. Thus the receptors contribute an important part to the host defence against infection.
PMCID: PMC3040686  PMID: 21299846
13.  The Role of Macrophage Class A Scavenger Receptors in a Laser-Induced Murine Choroidal Neovascularization Model 
Laser-induced choroidal neovascularization (CNV) is a widely used model to mimic many features of CNV resulting from wet AMD. Macrophages have been implicated in the pathogenesis of AMD. Class A scavenger receptors, scavenger receptor-A (SR-A) and macrophage receptor with collagenous domain (MARCO), are expressed on macrophages and are associated with macrophage function. The goal of this study is to examine the role of macrophage scavenger receptors in immune cell recruitment and the formation of CNV.
Laser photocoagulation was performed in wild-type and knockout mice with deletion of SR-A (SR-A−/−), MARCO (MARCO−/−), or both SR-A and MARCO double knockout (DKO). Immune cell recruitment at different time points and CNV lesions at 14 days after laser treatment were evaluated through immunostaining and confocal microscopy. Microarray analysis was performed in eyes 1 day after laser injury.
Wild-type eyes showed higher chemokine/receptor expression compared with knockout eyes after laser injury. Scavenger receptor deficiency markedly impaired the recruitment of neutrophils and macrophages to CNV lesions at 1- and 3-days post laser injury, respectively. Significantly reduced CNV volumes were found in the eyes from scavenger receptor knockout mice compared with wild-type mice.
The deficiency of scavenger receptors impairs the formation of CNV and immune cell recruitment. Our findings suggest a potential role for scavenger receptors in contributing to CNV formation and inflammation in AMD.
Class A scavenger receptor deficiency impairs immune cell recruitment and the formation of choroidal neovascularization (CNV). This suggests that scavenger receptors may contribute to CNV formation and inflammation seen in AMD.
PMCID: PMC3771553  PMID: 23927892
AMD; CNV; macrophages; scavenger receptors
14.  A20 (Tnfaip3) Deficiency in Myeloid Cells Protects against Influenza A Virus Infection 
PLoS Pathogens  2012;8(3):e1002570.
The innate immune response provides the first line of defense against viruses and other pathogens by responding to specific microbial molecules. Influenza A virus (IAV) produces double-stranded RNA as an intermediate during the replication life cycle, which activates the intracellular pathogen recognition receptor RIG-I and induces the production of proinflammatory cytokines and antiviral interferon. Understanding the mechanisms that regulate innate immune responses to IAV and other viruses is of key importance to develop novel therapeutic strategies. Here we used myeloid cell specific A20 knockout mice to examine the role of the ubiquitin-editing protein A20 in the response of myeloid cells to IAV infection. A20 deficient macrophages were hyperresponsive to double stranded RNA and IAV infection, as illustrated by enhanced NF-κB and IRF3 activation, concomitant with increased production of proinflammatory cytokines, chemokines and type I interferon. In vivo this was associated with an increased number of alveolar macrophages and neutrophils in the lungs of IAV infected mice. Surprisingly, myeloid cell specific A20 knockout mice are protected against lethal IAV infection. These results challenge the general belief that an excessive host proinflammatory response is associated with IAV-induced lethality, and suggest that under certain conditions inhibition of A20 might be of interest in the management of IAV infections.
Author Summary
Influenza virus or flu epidemics represent a recurrent threat to the public health, especially for individuals which are part of a high-risk group such as children, elderly or immune-compromised people. Sporadic pandemic flu outbreaks, such as the Spanish flu of 1918, may cause high grades of mortality among healthy persons. A better understanding of how the immune system deals with these pathogens is of key importance. The protein A20 is an important negative regulator of both innate and adaptive immune responses. We show that the specific deletion of A20 in myeloid cells, such as macrophages and neutrophils, improves the resistance against otherwise lethal influenza infections. This protective effect is mediated by an enhanced innate immune response following respiratory challenge with influenza virus. Although exaggerated pulmonary immune responses are believed to be the primary cause of often life threatening influenza virus induced pneumonia, we demonstrate that boosting the innate immune response by selectively targeting the functionality of A20 in myeloid cells is beneficial for the host survival. This finding provides us with a novel valuable approach for treating influenza and potentially other respiratory viral infections.
PMCID: PMC3291650  PMID: 22396652
15.  Detrimental Contribution of the Toll-Like Receptor (TLR)3 to Influenza A Virus–Induced Acute Pneumonia 
PLoS Pathogens  2006;2(6):e53.
Influenza A virus (IAV) is the etiological agent of a highly contagious acute respiratory disease that causes epidemics and considerable mortality annually. Recently, we demonstrated, using an in vitro approach, that the pattern recognition Toll-like receptor (TLR)3 plays a key role in the immune response of lung epithelial cells to IAV. In view of these data and the fact that the functional role of TLR3 in vivo is still debated, we designed an investigation to better understand the role of TLR3 in the mechanisms of IAV pathogenesis and host immune response using an experimental murine model. The time-course of several dynamic parameters, including animal survival, respiratory suffering, viral clearance, leukocyte recruitment into the airspaces and secretion of critical inflammatory mediators, was compared in infected wild-type and TLR3−/− mice. First, we found that the pulmonary expression of TLR3 is constitutive and markedly upregulated following influenza infection in control mice. Notably, when compared to wild-type mice, infected TLR3−/− animals displayed significantly reduced inflammatory mediators, including RANTES (regulated upon activation, normal T cell expressed and secreted), interleukin-6, and interleukin-12p40/p70 as well as a lower number of CD8+ T lymphocytes in the bronchoalveolar airspace. More important, despite a higher viral production in the lungs, mice deficient in TLR3 had an unexpected survival advantage. Hence, to our knowledge, our findings show for the first time that TLR3-IAV interaction critically contributes to the debilitating effects of a detrimental host inflammatory response.
Influenza A virus (IAV) is responsible for highly contagious acute respiratory disease. Recent concerns have risen concerning a possible influenza pandemic in the near future. Thus, a better understanding of the molecular mechanisms of IAV pathogenesis and host immune responses is required for the development of more efficient means of prevention and treatment of influenza. The Toll-like receptor (TLR)3 is a member of a family of receptors that detects microbes and triggers host defenses. We previously demonstrated using an in vitro approach, that the TLR3 plays a key role in the response of lung epithelial cells to IAV. Here, we used a mouse model to dissect the in vivo importance of TLR3-dependent responses during influenza. The time-course of several parameters, including animal survival, respiratory distress, viral clearance, and inflammation, was compared in infected control wild-type and TLR3-deficient mice. Our findings reveal that TLR3−/− mice have an unexpected advantage against IAV challenge as we show for the first time that a reduction of TLR3-mediated inflammatory response reduces the clinical manifestations of IAV-induced pneumonia.
PMCID: PMC1475659  PMID: 16789835
16.  Interleukin-22 Reduces Lung Inflammation during Influenza A Virus Infection and Protects against Secondary Bacterial Infection 
Journal of Virology  2013;87(12):6911-6924.
Interleukin-22 (IL-22) has redundant, protective, or pathogenic functions during autoimmune, inflammatory, and infectious diseases. Here, we addressed the potential role of IL-22 in host defense and pathogenesis during lethal and sublethal respiratory H3N2 influenza A virus (IAV) infection. We show that IL-22, as well as factors associated with its production, are expressed in the lung tissue during the early phases of IAV infection. Our data indicate that retinoic acid receptor-related orphan receptor-γt (RORγt)-positive αβ and γδ T cells, as well as innate lymphoid cells, expressed enhanced Il22 transcripts as early as 2 days postinfection. During lethal or sublethal IAV infections, endogenous IL-22 played no role in the control of IAV replication and in the development of the IAV-specific CD8+ T cell response. During lethal infection, where wild-type (WT) mice succumbed to severe pneumonia, the lack of IL-22 did not accelerate or delay IAV-associated pathogenesis and animal death. In stark contrast, during sublethal IAV infection, IL-22-deficient animals had enhanced lung injuries and showed a lower airway epithelial integrity relative to WT littermates. Of importance, the protective effect of endogenous IL-22 in pulmonary damages was associated with a more controlled secondary bacterial infection. Indeed, after challenge with Streptococcus pneumoniae, IAV-experienced Il22−/− animals were more susceptible than WT controls in terms of survival rate and bacterial burden in the lungs. Together, IL-22 plays no major role during lethal influenza but is beneficial during sublethal H3N2 IAV infection, where it limits lung inflammation and subsequent bacterial superinfections.
PMCID: PMC3676141  PMID: 23596287
17.  Complement C5 Activation during Influenza A Infection in Mice Contributes to Neutrophil Recruitment and Lung Injury 
PLoS ONE  2013;8(5):e64443.
Influenza virus A (IAV) causes annual epidemics and intermittent pandemics that affect millions of people worldwide. Potent inflammatory responses are commonly associated with severe cases of IAV infection. The complement system, an important mechanism of innate and humoral immune responses to infections, is activated during primary IAV infection and mediates, in association with natural IgM, viral neutralization by virion aggregation and coating of viral hemmagglutinin. Increased levels of the anaphylatoxin C5a were found in patients fatally infected with the most recent H1N1 pandemic virus. In this study, our aim was to evaluate whether targeting C5 activation alters inflammatory lung injury and viral load in a murine model of IAV infection. To address this question C57Bl/6j mice were infected intranasally with 104 PFU of the mouse adapted Influenza A virus A/WSN/33 (H1N1) or inoculated with PBS (Mock). We demonstrated that C5a is increased in bronchoalveolar lavage fluid (BALF) upon experimental IAV infection. To evaluate the role of C5, we used OmCI, a potent arthropod-derived inhibitor of C5 activation that binds to C5 and prevents release of C5a by complement. OmCI was given daily by intraperitoneal injection from the day of IAV infection until day 5. Treatment with OmCI only partially reduced C5a levels in BALF. However, there was significant inhibition of neutrophil and macrophage infiltration in the airways, Neutrophil Extracellular Traps (NETs) formation, death of leukocytes, lung epithelial injury and overall lung damage induced by the infection. There was no effect on viral load. Taken together, these data suggest that targeting C5 activation with OmCI during IAV infection could be a promising approach to reduce excessive inflammatory reactions associated with the severe forms of IAV infections.
PMCID: PMC3655967  PMID: 23696894
18.  HSV-1 exploits the innate immune scavenger receptor MARCO to enhance epithelial adsorption and infection 
Nature communications  2013;4:1963.
HSV-1 is an important epithelial pathogen and has the potential for significant morbidity in humans. Here we demonstrate that a cell surface scavenger receptor, macrophage receptor with collagenous structure (MARCO), previously thought to enhance antiviral defense by enabling nucleic acid recognition, is usurped by HSV-1 and functions together with heparan sulfate proteoglycans to mediate adsorption to epithelial cells. Ligands of MARCO dramatically inhibit HSV-1 adsorption and infection of human keratinocytes and protect mice against infection. HSV-1 glycoprotein C (gC) closely co-localizes with MARCO at the cell surface, and gC binds directly to purified MARCO with high affinity. Increasing MARCO expression enhances HSV-1 infection while MARCO-/- mice have reduced susceptibility to infection by HSV-1. These findings demonstrate that HSV-1 binds to MARCO to enhance its capacity for disease, and suggests a new therapeutic target to alter pathogenicity of HSV-1 in skin infection.
PMCID: PMC3681428  PMID: 23739639
19.  Influenza A Virus Inhibits Alveolar Fluid Clearance in BALB/c Mice 
Rationale: Pulmonary infections can impair alveolar fluid clearance (AFC), contributing to formation of lung edema. Effects of influenza A virus (IAV) on AFC are unknown.
Objectives: To determine effects of IAV infection on AFC, and to identify intercellular signaling mechanisms underlying influenza-mediated inhibition of AFC.
Methods: BALB/c mice were infected intranasally with influenza A/WSN/33 (10,000 or 2,500 focus-forming units per mouse). AFC was measured in anesthetized, ventilated mice by instilling 5% bovine serum albumin into the dependent lung.
Measurements and Main Results: Infection with high-dose IAV resulted in a steady decline in arterial oxygen saturation and increased lung water content. AFC was significantly inhibited starting 1 hour after infection, and remained suppressed through Day 6. AFC inhibition at early time points (1–4 h after infection) did not require viral replication, whereas AFC inhibition later in infection was replication-dependent. Low-dose IAV infection impaired AFC for 10 days, but induced only mild hypoxemia. High-dose IAV infection increased bronchoalveolar lavage fluid ATP and UTP levels. Impaired AFC at Day 2 resulted primarily from reduced amiloride-sensitive AFC, mediated by increased activation of the pyrimidine-P2Y purinergic receptor axis. However, an additional component of AFC impairment was due to activation of A1 adenosine receptors and stimulation of increased cystic fibrosis transmembrane regulator–mediated anion secretion. Finally, IAV-mediated inhibition of AFC at Day 2 could be reversed by addition of β-adrenergic agonists to the AFC instillate.
Conclusions: AFC inhibition may be an important feature of early IAV infection. Its blockade may reduce the severity of pulmonary edema and hypoxemia associated with influenza pneumonia.
PMCID: PMC2577730  PMID: 18689466
orthomyxovirus infections; pneumonia, viral; pulmonary edema; ion transport; adenosine
20.  IL-1α and Complement Cooperate in Triggering Local Neutrophilic Inflammation in Response to Adenovirus and Eliminating Virus-Containing Cells 
PLoS Pathogens  2014;10(3):e1004035.
Inflammation is a highly coordinated host response to infection, injury, or cell stress. In most instances, the inflammatory response is pro-survival and is aimed at restoring physiological tissue homeostasis and eliminating invading pathogens, although exuberant inflammation can lead to tissue damage and death. Intravascular injection of adenovirus (Ad) results in virus accumulation in resident tissue macrophages that trigger activation of CXCL1 and CXCL2 chemokines via the IL-1α-IL-1RI signaling pathway. However, the mechanistic role and functional significance of this pathway in orchestrating cellular inflammatory responses to the virus in vivo remain unclear. Resident metallophilic macrophages expressing macrophage receptor with collagenous structure (MARCO+) in the splenic marginal zone (MZ) play the principal role in trapping Ad from the blood. Here we show that intravascular Ad administration leads to the rapid recruitment of Ly-6G+7/4+ polymorphonuclear leukocytes (PMNs) in the splenic MZ, the anatomical compartment that remains free of PMNs when these cells are purged from the bone marrow via a non-inflammatory stimulus. Furthermore, PMN recruitment in the splenic MZ resulted in elimination of virus-containing cells. IL-1α-IL-1RI signaling is only partially responsible for PMN recruitment in the MZ and requires CXCR2, but not CXCR1 signaling. We further found reduced recruitment of PMNs in the splenic MZ in complement C3-deficient mice, and that pre-treatment of IL-1α-deficient, but not wild-type mice, with complement inhibitor CR2-Crry (inhibits all complement pathways at C3 activation) or CR2-fH (inhibits only the alternative complement activation pathway) prior to Ad infection, abrogates PMN recruitment to the MZ and prevents elimination of MARCO+ macrophages from the spleen. Collectively, our study reveals a non-redundant role of the molecular factors of innate immunity – the chemokine-activating IL-1α-IL-1RI-CXCR2 axis and complement – in orchestrating local inflammation and functional cooperation of PMNs and resident macrophages in the splenic MZ, which collectively contribute to limiting disseminated pathogen spread via elimination of virus-containing cells.
Author Summary
Adenovirus (Ad) induces a potent activation of pro-inflammatory cytokines and chemokines upon interaction with tissue macrophages in vivo. However, critical factors affecting cellular inflammatory responses to Ad and their functional significance remain unclear. Here we show that in the model of disseminated infection, intravenous Ad administration leads to a rapid release of pro-inflammatory Ly-6G+7/4+ leukocytes (PMNs) from the bone marrow into the blood. PMNs enter into peripheral tissues and, in the case of spleen, are accumulated in proximity to the virus-containing MARCO+ macrophages within the splenic marginal zone (MZ). Mechanistic dissection of molecular queues that guide PMN migration reveals that CXCL1 and CXCL2 chemokines are only partially responsible for CXCR2-dependent PMN recruitment into the splenic MZ. We further found that complement cooperates with IL-1α-IL-1RI-CXCR2 signaling pathways in recruitment of PMNs to the splenic MZ, which results in elimination of virus-containing MARCO+ macrophages from the spleen. Administration of complement-blocking CR2-Crry or CR2-fH proteins into IL-1α-deficient, but not wild-type, mice prevents PMN accumulation in the splenic MZ and elimination of virus-containing macrophages from the spleen. Our study defines the functional significance of molecular and cellular host defense mechanisms that cooperate in eliminating virus-containing cells in the model of acute disseminated Ad infection.
PMCID: PMC3961377  PMID: 24651866
21.  17β-Estradiol Protects Females against Influenza by Recruiting Neutrophils and Increasing Virus-Specific CD8 T Cell Responses in the Lungs 
Journal of Virology  2014;88(9):4711-4720.
17β-Estradiol (E2) treatment limits the pathology associated with pulmonary diseases caused by pathogens, allergens, and asthma, partly by reducing the production of proinflammatory cytokines and chemokines. To test the hypothesis that E2 protects against influenza A virus (IAV) infection by altering the recruitment and activity of innate immune cells and T cells, chemokine concentrations were measured and innate and adaptive immune cells were enumerated from the lungs of E2- and placebo-treated ovariectomized female C57BL/6 mice following infection. Females treated with E2 experienced less morbidity but had similar lung virus titers to placebo-treated females. Females treated with E2 had lower induction of CCL2 but higher CCL3 and CXCL1 responses in their lungs than placebo-treated females. Pulmonary recruitment of neutrophils, NK cells, macrophages, and dendritic cells was increased following infection, but only neutrophil numbers were greater in E2-treated than placebo-treated females. Neutrophils enhance the responses of influenza virus-specific CD8 T cells to promote virus clearance and improve the outcome of infection. Total numbers of virus-specific CD8 T cells were not altered by treatment with E2, but the proportion of gamma interferon (IFN-γ)- and tumor necrosis factor alpha (TNF-α)-producing, virus-specific CD8 T cells was increased. Neutrophil depletion in E2-treated females increased morbidity, reduced pulmonary production of chemoattractants for neutrophils, and reduced IFN-γ production by virus-specific CD8 T cells. Neutrophils mediate both inflammation and tissue repair during IAV infection and are regulated by E2 to improve the outcome of influenza in females.
IMPORTANCE Severe influenza is associated with excessive inflammation that leads to tissue damage. We demonstrate that estradiol (E2) is a potent anti-inflammatory hormone that reduces the severity of influenza A virus infection in females. Treatment of female C57BL/6 mice with E2 does not affect virus replication but rather alters the production of chemokines, pulmonary recruitment of neutrophils, and the cytokine responses of virus-specific CD8 T cells to protect females against severe influenza.
PMCID: PMC3993800  PMID: 24522912
22.  Late Engagement of CD86 after Influenza Virus Clearance Promotes Recovery in a FoxP3+ Regulatory T Cell Dependent Manner 
PLoS Pathogens  2014;10(8):e1004315.
Influenza A virus (IAV) infection in the respiratory tract triggers robust innate and adaptive immune responses, resulting in both virus clearance and lung inflammation and injury. After virus clearance, resolution of ongoing inflammation and tissue repair occur during a distinct recovery period. B7 family co-stimulatory molecules such as CD80 and CD86 have important roles in modulating T cell activity during the initiation and effector stages of the host response to IAV infection, but their potential role during recovery and resolution of inflammation is unknown. We found that antibody-mediated CD86 blockade in vivo after virus clearance led to a delay in recovery, characterized by increased numbers of lung neutrophils and inflammatory cytokines in airways and lung interstitium, but no change in conventional IAV-specific T cell responses. However, CD86 blockade led to decreased numbers of FoxP3+ regulatory T cells (Tregs), and adoptive transfer of Tregs into αCD86 treated mice rescued the effect of the blockade, supporting a role for Tregs in promoting recovery after virus clearance. Specific depletion of Tregs late after infection mimicked the CD86 blockade phenotype, confirming a role for Tregs during recovery after virus clearance. Furthermore, we identified neutrophils as a target of Treg suppression since neutrophil depletion in Treg-depleted mice reduced excess inflammatory cytokines in the airways. These results demonstrate that Tregs, in a CD86 dependent mechanism, contribute to the resolution of disease after IAV infection, in part by suppressing neutrophil-driven cytokine release into the airways.
Author Summary
Influenza A virus (IAV) infection can cause severe inflammation and injury in the respiratory tract, which must be resolved and repaired for the host to fully recover after virus clearance. Evidence is emerging that host immune responses may regulate tissue repair and resolution of inflammation after IAV infection. Early in IAV infection, the co-stimulatory molecules CD80 and CD86 promote inflammation through triggering IAV-specific T cell responses, but no role for CD80/86 in recovery after virus clearance has been previously established. By in vivo antibody-mediated blockade of CD80 or CD86 after virus clearance, we found that engagement of CD86 (but not CD80) was required for optimal recovery after influenza infection. Furthermore, we determined that CD86 was essential for maintaining the FoxP3+regulatory T cell (Treg) population in the respiratory tract, and CD86-dependent Tregs promoted recovery by suppressing pulmonary inflammation and supporting regain of weight after virus clearance. In addition, we demonstrated that Tregs suppress neutrophils late after infection, preventing neutrophils from driving excess inflammatory cytokine release into the airways. Taken together, we propose a novel role for CD86 engagement late after IAV infection to promote resolution of inflammation and host recovery through a Treg-dependent mechanism.
PMCID: PMC4140856  PMID: 25144228
23.  Cigarette smoke worsens lung inflammation and impairs resolution of influenza infection in mice 
Respiratory Research  2008;9(1):53.
Cigarette smoke has both pro-inflammatory and immunosuppressive effects. Both active and passive cigarette smoke exposure are linked to an increased incidence and severity of respiratory virus infections, but underlying mechanisms are not well defined. We hypothesized, based on prior gene expression profiling studies, that upregulation of pro-inflammatory mediators by short term smoke exposure would be protective against a subsequent influenza infection.
BALB/c mice were subjected to whole body smoke exposure with 9 cigarettes/day for 4 days. Mice were then infected with influenza A (H3N1, Mem71 strain), and analyzed 3 and 10 days later (d3, d10). These time points are the peak and resolution (respectively) of influenza infection.
Inflammatory cell influx into the bronchoalveolar lavage (BALF), inflammatory mediators, proteases, histopathology, viral titres and T lymphocyte profiles were analyzed. Compared to smoke or influenza alone, mice exposed to smoke and then influenza had more macrophages, neutrophils and total lymphocytes in BALF at d3, more macrophages in BALF at d10, lower net gelatinase activity and increased activity of tissue inhibitor of metalloprotease-1 in BALF at d3, altered profiles of key cytokines and CD4+ and CD8+ T lymphocytes, worse lung pathology and more virus-specific, activated CD8+ T lymphocytes in BALF. Mice smoke exposed before influenza infection had close to 10-fold higher lung virus titres at d3 than influenza alone mice, although all mice had cleared virus by d10, regardless of smoke exposure. Smoke exposure caused temporary weight loss and when smoking ceased after viral infection, smoke and influenza mice regained significantly less weight than smoke alone mice.
Smoke induced inflammation does not protect against influenza infection.
In most respects, smoke exposure worsened the host response to influenza. This animal model may be useful in studying how smoke worsens respiratory viral infections.
PMCID: PMC2483272  PMID: 18627612
24.  Role of MARCO in Innate Immune Tolerance 
Macrophages play a key role in host defense against microbes, in part, through phagocytosis. MARCO (macrophage receptor with collagenous structure) is a scavenger receptor on the cell surface of macrophages that mediates opsonin-independent phagocytosis. The goal of our study is to investigate the role of MARCO in lipopolysaccharide (LPS) or lipotechoic acid (LTA)-induced macrophage tolerance. While it has been established that expression of MARCO and phagocytosis is increased in tolerant macrophages, the transcriptional regulation and biological role of MARCO in tolerant macrophages has not been investigated. Here, we confirm that tolerized mouse bone marrow derived macrophages (BMDM) selectively increase expression of MARCO (both transcript and cell surface receptor) and increase phagocytosis. We found that H3K4me3 dynamic modification of a promoter site of MARCO was increased in tolerized BMDM. Blocking methylation by treatment with 5-Aza-2′-deoxycytidine (5-AZA) resulted in reduced H3K4me3 binding in the promoter of MARCO, decreased expression of MARCO, and impaired phagocytosis in tolerized BMDM. However, 5-AZA had no effect on the inflammatory component of innate immune tolerance. In aggregate, we found that histone methylation was critical to MARCO expression and phagocytosis in tolerized macrophages but did not affect the inflammatory component of innate immune tolerance.
PMCID: PMC3679202  PMID: 23667110
Phagocytosis; MARCO; Macrophage tolerance; Chromatin modification
25.  Pulmonary Collectins Modulate Strain-Specific Influenza A Virus Infection and Host Responses 
Journal of Virology  2004;78(16):8565-8572.
Collectins are secreted collagen-like lectins that bind, agglutinate, and neutralize influenza A virus (IAV) in vitro. Surfactant proteins A and D (SP-A and SP-D) are collectins expressed in the airway and alveolar epithelium and could have a role in the regulation of IAV infection in vivo. Previous studies have shown that binding of SP-D to IAV is dependent on the glycosylation of specific sites on the HA1 domain of hemagglutinin on the surface of IAV, while the binding of SP-A to the HA1 domain is dependent on the glycosylation of the carbohydrate recognition domain of SP-A. Here, using SP-A and SP-D gene-targeted mice on a common C57BL6 background, we report that viral replication and the host response as measured by weight loss, neutrophil influx into the lung, and local cytokine release are regulated by SP-D but not SP-A when the IAV is glycosylated at a specific site (N165) on the HA1 domain. SP-D does not protect against IAV infection with a strain lacking glycosylation at N165. With the exception of a small difference on day 2 after infection with X-79, we did not find any significant difference in viral load in SP-A−/− mice with either IAV strain, although small differences in the cytokine responses to IAV were detected in SP-A−/− mice. Mice deficient in both SP-A and SP-D responded to IAV similarly to mice deficient in SP-D alone. Since most strains of IAV currently circulating are glycosylated at N165, SP-D may play a role in protection from IAV infection.
PMCID: PMC479098  PMID: 15280465

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