lnflammatory response of macrophages in infection

Ling Zhang and Cheng-Cai Wang

Shanghai, China

lnflammatory response of macrophages in infection

Ling Zhang and Cheng-Cai Wang

Shanghai, China

BACKGROUND:Macrophages are widely-distributed innate immune cells playing diverse roles in various physiological and pathological processes. The primary function of macrophages is to phagocytize and clear invading pathogens.

DATA SOURCES:A systematic search of PubMed was performed to identify relevant studies in English language literature using the key words such as macrophage and inflammation. A total of 122 articles related to inflammatory response of macrophages in infection were systematically reviewed.

RESULTS:The inflammatory responses of macrophages triggered by infection comprise four interrelated phases: recognition of pathogen-associated molecular patterns by pattern-recognition receptors expressed on/in macrophages; enrichment of quantity of macrophages in local infected tissue by recruitment of circulating monocytes and/orin situproliferation; macrophage-mediation of microbicidal activity and conversion to anti-inflammatory phenotype to terminate anti-infectious response and to promote tissue repair. Complicated regulation of macrophage activation at molecular level recognized in the past decade is also reviewed, including intracellular multiple signaling molecules, membrane molecules, microRNAs and even epigenetic-associated molecules.

CONCLUSION:The inflammatory response of macrophages in infection is an orderly and complicated process under elaborate regulation at molecular level.

(Hepatobiliary Pancreat Dis Int 2014;13:138-152)

macrophage;

inflammation;

infection;

Toll-like receptor

Introduction

Macrophage is an important component of innate cellular immunity with versatile functions prominently involved in host defense and immunity against foreign microorganisms, including bacteria, viruses, fungi and parasites.[1-3]Macrophages possess a broad array of cell surface receptors, intracellular mediators and essential secretory molecules for recognition, engulfment and destruction of invading pathogens and also regulation of other kinds of immune cells.[4]Although more than a century has passed since Elie Metchnikoff first identified macrophages and described their phagocytosis of harmful microbes,[5]the crucial and diverse functions of macrophages and the underlying mechanisms and the regulation of their functions need to be updated. In addition to their well-known functions of immune defense against various infections, macrophages also have been found to play essential roles in diverse physiological and pathological processes, for example, tumor-associated macrophages (TAMs) facilitate angiogenesis and extracellular matrix degradation, and directly inhibit anti-tumor T cell response, thus promoting tumor cell progression and motility;[6,7]adipose-infiltrating macrophage-mediated inflammation is responsible for insulin resistance and subsequent type 2 diabetes;[8]the accumulation of cholesterol-laden macrophages (foam cells) in the artery wall is closely related to atherosclerosis by driving the imbalance of lipid metabolism and adaptive immune response;[9]CD169+bone marrow macrophages support erythropoiesis under pathological conditions;[10,11]and muscle-associated macrophages support the regeneration of skeletal muscle following injury.[12]In this review, we focus on the updating progression on the functions and underlying mechanisms of macrophages in inflammatory responses mainly induced by invading pathogens, as well as the regulation of their function at the molecular level.

Macrophage polarization and subsets

In terms of their anatomical location as well as phenotype and function, macrophages possess remarkable heterogeneity, mainly including microglial cells in the brain, boneresorbing osteoclasts in the skeletal system, Kupffer cells in the liver, alveolar macrophages in the lung, histiocytes in interstitial connective tissue and foam cells in plague of atherosclerosis, reflecting the specialization of tissueresident macrophages in different microenvironments of various organs and tissues, all of which mainly differentiate from circulating monocytes.[13]These tissuespecific macrophages can ingest foreign microbes and recruit other macrophages from circulation during an infection. Furthermore, depending on different settings, inflammatory macrophages can be polarized into two well-established functional subsets, referring to classically activated macrophages (M1) and alternatively activated macrophages (M2).[14,15]

M1 macrophages

The typical characteristic of M1 macrophages is their ability to participate in and promote type 1 immune response, accompanied with increased synthesis of proinflammatory cytokines (TNF-α, IL-1β, IL-12, IL-18, CCL15, CCL20, CXCL8-11 and CXCL13), reactive oxygen and nitrogen species, increased complementmediated phagocytosis and antigen presenting function.[14]IFN-γ and/or bacterial lipopolysaccharide (LPS) usually induce M1-type activationin vitro. The main function of M1 macrophages is to kill intracellular pathogens. In mice, M1-associated markers include IL-12, MHC class II molecules and nitric oxide synthase 2 (NOS2), while in humans, M1 macrophages do not induce NOS2.[13]

M2 macrophages

The typical characteristic of M2 macrophages is their ability to participate in and promote type 2 immune response such as parasitic infection, asthma and allergic disorders.[15]M2 macrophages can be further subdivided into M2a that induced by IL-4 or IL-13, M2b that induced by immune complexes in combination with LPS or IL-1β, and M2c that induced by IL-10, transforming growth factor-β (TGF-β) or glucocorticoids.[16,17]In addition, IL-33 and thymic stromal lymphopoietin (TSLP) have been proved to amplify the differentiation of M2 macrophages in an IL-13-dependent manner.[18,19]In mice, M2-associated markers include arginase 1, mannose receptor (MR, CD206, Mrc1), resistinlike molecule α (Relmα) and chitinase 3-like 3 (also known as Ym1), while in humans, M2 macrophages express indoleamine 2, 3-dioxygenase (IDO).[13,15]M2 macrophages are also associated with anti-inflammatory functions and homeostatic functions such as tissue repair and wound healing by expressing profibrotic factors including fibronectin, matrix metalloproteinases (MMPs), IL-1β and TGF-β. Unexpectedly, M2 macrophages also produce catecholamines to sustain adaptive thermogenesis in response to cold.[20]TAM is another polarized macrophage phenotype that has been extensively studied, and is often considered to belong to M2 macrophage. However, transcriptional profile of TAMs is quite different from that of M1 and M2 macrophages, although they share some other characteristics.[21]

The flexibility of macrophage phenotype

Unlike T helper subsets, such as Th1 and Th2, which are definite and discrete subpopulations, it is well accepted that the activated phenotype of macrophages is flexible and continually changeable, and macrophage can change from one functional phenotype to another in response to the variable microenvironment.[22]Numerous reports have supported this viewpoint. Macrophages sequentially change their functional phenotypein vitroin response to changes in cytokine stimulation.[23]M2 macrophages elicited by helminth infectionin vivocan be reprogrammedin vitroby LPS and IFN-γ stimulation to M1-like macrophages with the microbicidal ability via NO.[24]After injury, the transition of muscle-associated macrophages from proinflammatory (M1) to anti-inflammatory (M2) phenotype is critical for skeletal muscle regeneration, and AMP-activated protein kinase α1 (AMPKα1) regulates this macrophage skewing.[25,26]TAMs can be re-educated by targeting NF-κB to M1 phenotype with the ability to kill tumor cells.[27]This ability of macrophages can help the host adapt variable environments but also can be used by microbes to attack against the host.

Otherwise, as to M2 macrophages, it seems that these cells act as both proinflammatory potential, such as in helminth infection, and anti-inflammatory potential to eliminate inflammation after infection. Although it is possible that different M2 macrophage subsets are responsible for the two opposite roles of these cells, considering the flexibility of these cells, it seems more probable that the different status of M2 macrophages is the key. At the early stage of infection, M2 macrophages show the proinflammatory potential, and at the late stage, some certain characteristics of these cells change according to the changed environment, thus showing the anti-inflammatory potential. Of course, which one is correct needs further examination in the future.

The process and mechanism of macrophages in antimicrobial defense

During infection initiated by various foreign pathogens, the inflammatory response of macrophages mainly comprises four orderly stages, recognition of infection by pattern-recognition receptors (PRRs) of macrophages, recruitment of monocytes and/orin situproliferation of macrophages in local infected tissue, elimination of pathogens, and conversion to suppressive cells thus restoration of tissue homeostasis (Fig.). Macrophages participate in the whole inflammatory anti-infection process by changing their functional phenotype according to the changing microenvironment, thus indicating the importance of macrophages in infection.

Recognition of infection by macrophages and subsequent signaling pathways

Once pathogens invade the host, germlineencoded PRRs on the surface or in the cytoplasm of macrophages, including Toll-like receptors (TLRs), C-type lectin receptors (CLRs), NOD-like receptors (NLRs) and retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs) recognize microbial components and subsequently activate transcriptional programs through respective signaling pathways, leading to phagocytosis, cellular activation and the release of proinflammatory cytokines and chemokines of macrophages, thus starting anti-infection immune response.[28-31]

Fig.The inflammatory process of macrophages in infection. After foreign pathogens invade the host, the inflammatory process of macrophages has four orderly and interrelated stages. ①PRRs expressed on or in macrophages recognize PAMPs from foreign pathogens, thus initiating anti-infection inflammatory response.②Two ways including recruitment of circulating monocytes in response to CCL2 and IL-4-triggeredin situmacrophage proliferation have been identified for the enrichment of the quantity of macrophages in infected local tissue. ③Macrophages differentiate into classically activated macrophages (M1) for intracellular bacteria or virus infection, or alternatively activated macrophages (M2) for helminth infection, which can express microbicidal molecules such as ROS, NO, defensins and MMP12, and produce proinflammatory cytokines and anti-virus IFN-I to clear invading pathogens. ④After infection, macrophages convert to anti-inflammatory phenotype to eliminate inflammation and promote tissue repair.

TLRs

TLRs, as the first identified and the most well characterized PRRs, recognize components derived from a wide range of pathogens known as pathogen-associated molecular patterns (PAMPs) and subsequently initiate anti-infection innate immune response.[28,32]TLRs belong to type I transmembrane proteins containing an ectodomain for recognition of PAMPs, a transmembrane region, and cytosolic Toll-IL-1 receptor (TIR) domains for activating downstream signaling pathways. Ten and 12 functional TLRs have been identified in humans and mice, respectively. TLR1, TLR2, TLR4, TLR5 and TLR6 are located on the cell surface, while TLR3, TLR7, TLR8 and TLR9 are within intracellular vesicles. Each TLR detects distinct PAMPs, for example, TLR1, TLR2 and TLR6 recognize lipoproteins; TLR3 identifies double-stranded (ds) RNA; TLR4 recognizes LPS; TLR5 recognizes flagellin; TLR7 and TLR8 recognize singlestranded (ss) RNA; and TLR9 identifies DNA.

TLR signaling pathways that induced by recognition of various PAMPs initiate the recruitment of one single or a specific combination of TIR-domain containing adaptor proteins, including myeloid differentiation primary response gene 88 (MyD88), TIR-domaincontaining adaptor inducing interferon β (TRIF), TIR-domain containing adaptor protein (TIRAP) and TRIF-related adaptor molecule (TRAM).[28,32]All TLRs with the exception of TLR3 as well as members of the IL-1 receptor family utilize the MyD88-dependent signaling pathway; TLR3 and TLR4 can use the alternative TRIF-dependent signaling pathway; TLR2 and TLR4 use TIRAP as an additional adaptor to recruit MyD88; and TRAM bridges TLR4 and TRIF. Therefore, TLR4 is the only one that recruits all four adaptor proteins and utilizes both MyD88- and TRIF-dependent pathways. MyD88 then recruits IL-1 receptor-associated kinases (IRAKs), tumor necrosis factor-associated factor 6 (TRAF6), and the TGF-β-associated kinase 1 (TAK1) complex, leading to NF-κB and mitogen-activated protein kinase (MAPK) activation and the induction of proinflammatory cytokines; TRIF recruits TRAF3, TANK binding kinase 1 (TBK1) and IκB kinase i (IKKi), leading to the interferon regulatory factor 3 (IRF3) activation and type I IFN (IFN-I) expression; inaddition, TLR4-associated TRAM-TRIF also recruits TRAF6 and TAK1 to mediate late-phase NF-κB and MAPK activation.

The functions of TLRs and the underlying mechanisms are more complicated than what have been realized. TLR4 and TLR2 specifically activate the endoplasmic reticulum (ER) stress sensor kinase IRE1α and its downstream target, the transcription factor XBP1, which is required for optimal and sustained production of proinflammatory cytokines in macrophages.[33]A group[34]also found that TLR1, TLR2 and TLR6 engagement on macrophages results in recruitment of mitochondria to phagosomes and augments of mitochondrial reactive oxygen species (ROS) production, which has the direct ability of bacterial killing. This process involves translocation of TRAF6 to mitochondria, where it engages the protein evolutionarily conserved signaling intermediate in Toll pathway (ECSIT), leading to ECSIT ubiquitination and subsequent enrichment of ROS.

CLRs

CLRs comprise a large heterogeneous group of transmembrane proteins containing one or more C-type lectin-like domains (CTLDs), mainly including mannose receptor, DEC-205, langerin, and dectin-1.[29]Some of them effectively function as PRRs to initiate inflammatory response by recognizing bacterial and fungal PAMPs, while others dampen or alter macrophage activation and modulate inflammatory response. Some CLRs alone are not sufficient to elicit microbicidal effector functions and some are "selfsufficient" PRRs. The latter utilize spleen tyrosine kinase (Syk) as their signaling adaptor, which normally binds to proteins containing immunoreceptor tyrosine-based activation motifs (ITAMs).[29,35]ITAM binding leads to Syk activation and phosphorylation of a variety of substrates, initiating a signaling cascade.

NLRs

The main members of NLRs are NOD1 and NOD2, which are cytosolic receptors recognizing distinct building blocks of peptidoglycan (PGN), the component of gram-positive bacteria.[30]The recognition of PGN by NOD1 and NOD2 activates receptor-interacting serine-threonine kinase 2 (RIP2), subsequently leading to ubiquitination of NF-κB essential modulator (NEMO) and activation of the NF-κB pathway, and NOD2 can also activate the MAPK pathway through RIP2. Studies[36,37]also showed that there are additional agonists for NOD2 of macrophages. N-glycolyl muramyl dipeptide from mycobacteria induces the production of TNF of macrophages through NOD2 recognition and subsequent activation of RIP2 and NF-κB, which is stronger than conventional stimulator PGN. NOD2 also can recognize viral ssRNA, and then activates adaptor protein mitochondrial antiviral signaling (MAVS) and IRF3, leading to the production of IFN-β.[37]In addition, several NLR family members, such as NLRP1, NLRP3 and NLRC4, are capable of forming inflammasomes in response to their specific stimulators, which are multiprotein complexes and serve as platforms for the activation of caspase 1 that leads to the processing and secretion of the proinflammatory cytokines IL-1β and IL-18.[30]

RLRs

The RLRs, mainly including RIG-I, melanoma differentiation associated factor 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2), are a family of RNA helicases and play important roles in pathogen sensing of RNA virus infection to initiate and modulate antiviral inflammatory response mainly through the induction of IFN-I production.[31]RLRs are typically expressed at low levels in resting cells but are massively increased with IFN exposure and viral infection. RIG-I and MDA5 signal through a common adaptor MAVS (also known as IFN-β promoter stimulator 1, IPS-1), a membrane-bound, caspase activation and recruitment domain (CARD)-containing protein. Detection of RNA rival PAMPs induces RLRs activation and association with MAVS through homotypic CARD-CARD interaction, leading to relocation of RLRs to MAVS-associated membranes and forming an MAVS signalosome with other downstream signaling molecules that drives IFN production. A recent study located MAVS on peroxisomes and mitochondria, and found that peroxisomal MAVS induces the rapid IFN-independent expression of defense factors that provide short-term protection, whereas mitochondrial MAVS activates a delayed IFN-dependent pathway that amplifies and stabilizes antiviral inflammatory response.[38]

Enrichment of quantity of macrophages in infected tissue

An important and unresolved question in macrophage immunobiology of infection is whether tissue-resident macrophages themselves are sufficient for responding to infection. It seems that in many infections, tissue-resident macrophages are not sufficient to control microbial infection, thus needing enrichment of their quantity somehow. The well accepted and extensively studied way is the recruitment of inflammatory monocytes to infectedtissue. In mice, CCL2/CCR2-mediated monocyte recruitment is essential for defense againstListeria monocytogenes,Mycobacterium tuberculosis,Toxoplasma gondii,Cryptococcus neoformansand influenza and fungi infection.[39,40]Circulating monocytes can be divided into two subsets: one has a "patrolling" function in and around the vascular endothelium with the phenotype of CX3CR1highLy6Clow, which cannot respond to CCL2 for lacking CCR2; the other Ly6Chighinflammatory monocytes express CCR2 and can be rapidly recruited into infected tissue.[41]

The process of chemokine-induced monocyte recruitment is complicated. NOD2 is thought to be related to CCL2-CCR2-dependent recruitment of inflammatory monocytes.[41]In acuteCitrobacter rodentiumin the gut, NOD2 in non-hematopoietic cells is responsible for the production of CCL2 by colonic stromal cells and subsequent recruitment of monocytes differentiating into macrophages, which is essential to bacterial clearance.[42]Accordingly, in intranasalStreptococcus pneumoniaeinfection of mice, bacterial uptake, digestion by lysozyme and sensing of peptidoglycan by NOD2 of macrophages contribute to the production of CCL2 and subsequent recruitment of monocytes for the clearance of invading bacteria.[43]However, coinfection of the upper respiratory tract of mice with influenza virus andS. pneumoniae, which is a common phenomenon of human respiratory infection, leads to synergistic stimulation of IFN-I that impairs the recruitment of macrophages due to decreased production of CCL2, thus promotingS. pneumoniaecolonization.[44]Furthermore, inLeishmaniamajor infection, activated platelets produce platelet-derived growth factor (PDGF) that induces the rapid release of CCL2 from leukocytes and mesenchymal cells.[45]

However, the recruitment of circulating monocytes is not the only way to enrich the quantity of macrophages in the infected tissue, andin situproliferation of macrophages is another important way. In infection with parasiteLitomosoides sigmodontis, Jenkins et al[46]found that IL-4 produced by innate immune cells and Th2 cells is sufficient to cause local tissue-resident macrophage proliferation, thus resulting in an increased number of effector M2 macrophages to erase worms, and this process is not associated with the recruitment of monocytes and macrophages. They also found that with additional proinflammatory stimuli such as thioglycollate to induce monocyte recruitment, IL-4 drives the proliferation and alternative activation of the recruited cells. The underlying mechanism remains unclear, but the transcription factors, macrophageactivating factor (Maf) and MafB, might be involved, because MafB/Maf deficiency enables self-renewal of differentiated functional macrophages.[47,48]A study[49]identified that the phosphoinositide-3 kinase (PI3K)/ Akt pathway is essential for IL-4-driven macrophage proliferation. The phenomenon ofin situmacrophage proliferation during infection raises more important questions that should be answered, such as, is this phenomenon restricted to M2 macrophages? In other words, can M1 macrophages self-renewin situ? Which mechanism is more important for the enrichment of the quantity of macrophages, monocyte recruitment or selfrenewal? Is it correct that M1 macrophages depend on monocyte recruitment and M2 macrophages depend on self-renewalin situ?

Macrophage activity in response to infection

After infection, activated macrophages with proinflammatory phenotype produce various proinflammatory mediators, including TNF-α, IL-1, IL-6 and IFN-I, which participate in the activation of various microbicidal mechanisms and contribute to the clearance of invading pathogens.[1-3]Activated macrophages can also mediate the following adaptive immune response against severe infection by producing IL-12 and IL-23 to promote the polarization of Th1 and Th17 cells respectively, or by producing IL-4 and IL-13 to support the differentiation of Th2 cells with the infection of extracellular microorganisms. Macrophages possess several substances with which they can kill bacteria, including ROS, NO and various antimicrobial proteins like defensin. Otherwise, the NO production of macrophages is not always beneficial. NO increases the susceptibility of TLR-activated macrophages to spreadingL. monocytogenesby promoting them escaping from secondary vacuoles in recipient cells and delaying maturation of phagosomes.[50]A study[51]found that macrophage elastase, also known as MMP12, kills both gram-negative and gram-positive bacteria within macrophages. Intracellular MMP12 is mobilized to macrophage phagolysosomes after ingestion of bacteria, and then adheres to bacterial cell walls, which disrupt bacterial membranes and result in bacterial death. Furthermore, macrophages can collaborate with platelets to kill bacteria. After infection withBacillus cereusor methicillin-resistantStaphylococcus aureus, Kupffer cells rapidly catch these invading bacteria and trigger platelets to switch from "touch-and-go" adhesion under basal conditions to sustained GPIIb-mediated adhesion on the Kupffer cell surface to encase the bacteria.[52]

However, uncontrolled macrophage activation is considered to be a critical event in the pathogenesis ofchronic inflammatory diseases such as atherosclerosis, multiple sclerosis, and chronic venous leg ulcers. For example, iron overloading of macrophages, occurring in patients with chronic leg venous ulcers and related mouse model, induces unrestrained proinflammatory M1 macrophages via enhanced TNF-α and hydroxyl radical release, which perpetuate inflammation and induce a p16 (INK4a)-dependent senescence program in resident fibroblasts, thus leading to impaired tissue healing.[53]

Anti-inflammatory and tissue repair activity of macrophages after infection

Once the invading pathogens have been completely erased, the anti-infection inflammatory response must be stopped and the damaged tissue needs to be repaired, and macrophages also participate in the two processes.[54]Recent studies[22-27]have shown that M1 proinflammatory macrophages can themselves convert into M2 anti-inflammatory macrophages, with the potent anti-inflammatory activity and important roles in tissue healing. M2 macrophage-derived arginase 1 protects hosts against excessive tissue injury caused by worm eggs during acute schistosomiasis by suppressing IL-12/IL-23p40 production and maintaining the regulatory T cells (Treg)/Th17 balance within the intestinal mucosa.[55]M2 macrophages can control respiratory syncytial virus (RSV)-induced lung injury in an IL-4Rα, TLR4 and IFN-β-dependent manner.[56]Preexisting helminth infection induces inhibition of innate pulmonary anti-tuberculosis defense by IL-4-dependent M2 macrophages.[57]In a helminth infection model, nematode larvae migrating transiently through the lung resulted in hemorrhage and inflammation, indicating that IL-17 initially contributes to inflammation and lung damage, whereas subsequent IL-4 receptor signaling stimulates M2 macrophage development, which contributes to the rapid resolution of tissue damage.[58]Interestingly, human anti-inflammatory M2 macrophages induce Foxp3+GITR+CD25+Tregs that suppress immune response via membrane-bound TGF-β1.[59]

M2 macrophages not only play anti-inflammatory roles during and/or after infection, but also contribute to tissue repair after infection and diminishment of tissue fibrosis, a pathological feature of most chronic inflammatory diseases which eventually leads to organ malfunction and death.[60]Healing processes in many tissues, organs and systems including the central nervous system after injury caused by infection require the assistance of healing macrophages for clearance of dead cells and cell debris and production of growth factors such as TGF-β1 and PDGF, thus supporting the regrowth and cell renewal.[61]Moreover, uptake of apoptotic neutrophils by macrophages after infection reprograms macrophages towards a resolving M2 phenotype, which is a key event to restore tissue homeostasis.[62]In the infection of Th2-inducingSchistosoma mansoni, arginase 1 derived from M2 macrophages not only suppresses type 2 inflammation but also suppresses lethal liver fibrosis.[63]A report[64]also showed that milk fat globule epidermal growth factor 8 (Mfge8) diminishes the severity of bleomycininduced tissue fibrosis in mice by binding and targeting collagen for uptake by macrophages.

Molecular regulation of macrophage activation in infection

As described above, macrophages play diverse and essential roles in the entire anti-infection inflammatory process, which undoubtedly needs fine regulation at different levels because attenuated macrophage activation often leads to uncompleted pathogen clearance and chronic infection, whereas overactivation of macrophages usually results in immunopathological damage to self tissue and induces inflammatory diseases or autoimmune disorders. Next, we would uncover the regulation mechanisms of macrophage activation in diverse infections at the molecular level, including regulation by intracellular multiple signaling molecules, membrane molecules, microRNAs and even epigeneticassociated molecules (Table).

Regulation by intracellular signaling molecules

The suppressors of the cytokine signaling (SOCS) family of cytoplasmic proteins, including the cytokineinducible Src homology 2 (SH2) domain-containing protein (CIS) and SOCS1 through SOCS7, mainly act as feedback inhibitors to attenuate signal transduction from cytokines that act via the janus kinase/signal transducer and activator of the transcription (JAK/ STAT) pathway.[65]It is suggested that SOCS mediate macrophage development and polarization by regulating cytokine and TLR signaling. SOCS3 deficiency promotes M1 macrophage polarization and LPS-induced sepsis, indicating that SOCS3 represses M1 proinflammatory phenotype, thereby deactivating inflammatory response in macrophages.[66]SOCS2 and SOCS3 are recently proved as key diametric regulators of M1 and M2 macrophage polarization and LPS-induced endotoxic shock.[67]SOCS3 deficient mice showed striking bias toward to M2-like macrophages, whereas SOCS2deficient mice showed enriched M1-like macrophages. The controversial results of the functions of SOCS3 in the polarization of macrophages in these two studies might be due to the different deficient mice they used (LysMCre-Socs3fl/flmice vs Socs3Lyz2cremice) and/or different doses of LPS they used (2.5 vs 6 mg/kg). Obviously, further investigation is needed to clarify this controversy.

A20 (also known as TNF-α-induced protein 3, TNFAIP3) is an inducible and broadly expressed cytoplasmic ubiquitin-editing enzyme, which is required for the termination of TLR-induced activity of NF-κB and proinflammatory gene expression in macrophagesby directly removing ubiquitin moieties from the signaling molecule TRAF6 (deubiquitination).[68]The pivotal regulatory role of A20 has also been confirmed by its involvement in protecting hosts from endotoxin shock and TLR4-dependent erosive polyarthritis.[69,70]Also inducible immune responsive gene 1 (IRG1) promotes endotoxin tolerance in LPS re-challenged macrophages by increasing A20 expression through ROS.[71]

Table.Molecular regulation of macrophage activation in infection

In the NLR family, pyrin domain-containing 3 (NLRP3) inflammasome is activated by cellular infection or stress, which is responsible for the maturation of proinflammatory cytokines IL-1β and IL-18.[72]A recent report[73]showed that leucine-rich repeat Fli-I-interacting protein 2 (LRRFIP2), as an NLRP3-associated protein, inhibits NLRP3 inflammasome activation in LPS activated macrophages by recruiting the caspase-1 inhibitor Flightless-I. LRRFIP2 could bind both NLRP3 and Flightless-I by its N-terminal and coiled motif respectively, thus acting as a negative regulator for NLRP3 by bridging of Flightless-I. However, LRRFIP1 displays a different role in macrophage activation. LRRFIP1, as a cytosolic nucleic acid-binding protein, contributes to the production of IFN-β induced by vesicular stomatitis virus (VSV) and L. monocytogenes in macrophages through interacting with and promoting the activation of β-catenin, thus increasing IFN-β expression by binding to the C-terminal domain of the transcription factor IRF3 and recruiting the acetyltransferase p300 to the IFN-β enhanceosome.[74]

E3 ubiquitin ligase-mediated post-transcriptional polyubiquitination has been proved to specifically regulate the TLR signaling in macrophages.[75]K63-linked ubiquitination often leads to the activation of target proteins, such as NEMO and TRAF6, whereas K48-linked ubiquitination often results in proteasomal degradation of target molecules, thus limiting innate immune response. Several E3 ubiquitin ligase, such as PDLIM2, Cbl-b and Itch, have been proved to negatively regulate TLR signaling in macrophages. Neuregulin receptor degradation protein 1 (Nrdp1), a newly defined E3 ubiquitin ligase, is recently found to inhibit proinflammatory cytokine production but increase IFN-β production in TLR-triggered macrophages by suppressing MyD88-dependent activation of NF-κB and AP-1 while promoting the activation of kinase TBK1 and IRF3.[76]Nrdp1 directly binds and polyubiquitinates MyD88 and TBK1, leading to degradation of MyD88 but activation of TBK1, thereby acting as a switch of MyD88-and TRIF-dependent signaling pathways. In addition, Nrdp1 also promotes M2 macrophage polarization by ubiquitinating and activating transcription factor CCAAT/enhancer-binding protein β (C/EBPβ).[77]In contrast to Nrdp1, another ubiquitin ligase DTX4, recruited by NLRP4 in dsRNA and DNA-mediated activation, induces K48-linked polyubiquitination of TBK1 leading to degradation of TBK1 and subsequently suppresses IFN-I production.[78]Besides, the carboxyl terminus of constitutive heat shock cognate 70 (HSC70)-interacting protein (CHIP), as a U box-containing E3 ubiquitin ligase, promotes TLR-4 and TLR-9 driven signaling of macrophages by recruiting the tyrosine kinase Src and atypical protein kinase C ζ (PKC ζ) to the TLR complex, thereby resulting in activation of IRAK1, TBK1, IRF3 and IRF7.[79]Another E3 ubiquitin ligase, WW domain-containing protein 2 (WWP2), as a TRIF-associated protein, negatively regulates TLR3-mediated macrophage activation and inflammatory responses by targeting TRIF for ubiquitination and degradation.[80]

Intracellular protein tyrosine phosphatases, such as SH2-containing protein tyrosine phosphatase 1 (SHP-1) and SHP-2, are also involved in the regulation of macrophage activation. SHP-2 negatively regulates TLR4 and TLR3 activated IFN-β production in macrophages, but not TLR2, TLR7 and TLR9 activated proinflammatory cytokine IL-6 and TNF-α production, partially through inhibiting TBK1-activated signal transduction.[81]In contrast, SHP-1 negatively regulates TLR-mediated production of proinflammatory cytokines in macrophages by inhibiting the activation of NF- κB and MAPKs, and simultaneously increases IFN-I production mediated by TLRs and RIG-I by directly binding to and inhibiting the activation of the kinase IRAK1, thus contributing to immune homeostasis by balancing the production of proinflammatory cytokines and IFN-I in the innate immune response.[82]Protein tyrosine phosphatase with proline-glutamine-serinethreonine-rich motifs (PTP-PEST) also regulates macrophage-mediated inflammation. The up-regulated PTP-PEST feedback inhibits TNF-α, IL-6, and IFN-β production in TLR-triggered macrophages, by directly interacting with IKKβ, then inhibiting IKKβ phosphorylation, and subsequently suppressing IKKβ activation and kinase activity as well as downstream NF-κB activation.[83]Moreover, SH2-containing inositol-5'-phosphatase (SHIP) negatively regulates TLR3[84]and TLR4[85]triggered macrophages activation through the regulation of TBK1 localization and activity, and a phosphatase activity- and PI3K-independent mechanism, respectively.

Zinc finger protein (ZFP) is an important transcription factor family involved in a variety of cell functions, which mainly includes three types of C2H2, C4 and C6. Recently, ZFP has been found to participate in the regulation of immune response. ZFP64, a memberof C2H2 type ZFP, has recently been found to act as a downstream positive regulator of TLR-initiated macrophage activation by associating with the NF-κB p65 subunit, enhancing p65 recruitment to the target gene promoters and promoting p65 activation, thus leading to the promotion of proinflammatory cytokines production such as TNF-α, IL-6, and IFN-β.[86]In contrast, another ZFP Gfi1 interacts with p65 and inhibits p65-mediated transcriptional activation by interfering with p65 binding to target gene promoter DNA in LPS stimulated bone marrow-derived macrophages.[87]Moreover, another ZFP, zinc finger and BTB domain containing 20 (ZBTB20), as a transcription repressor, is needed to promote full activation of TLR signaling and TLR-triggered macrophage activation by selectively suppressing the suppressor IκBα gene transcription.[88]So, myeloid cell-specific ZBTB20 knockout mice are resistant to endotoxin shock andEscherichia coli-caused sepsis.

Small guanosine triphosphatase (GTPase) is also involved in the regulation of macrophage activation in infection. Lysosome-associated small Rab GTPase Rab7b has been determined to negatively regulate TLR4- and TLR9-initiated proinflammatory cytokine production of macrophages, by colocalizing with TLR4 and TLR9 in lysosomal-associated membrane protein 1 (LAMP1)-positive subcellular compartments and down-regulating the expression of TLR4 and TLR9 in macrophages via promoting their degradation once activated, respectively.[89,90]In contrast, another small GTPase Rab10 is found to be essential to optimal macrophage activation after LPS stimulation, by promoting the replenishment of cell surface TLR4 from intracellular compartments like Golgi and endosomes.[91]

Furthermore, other intracellular molecules are recently found to regulate macrophage-mediated inflammation. Notch signaling is involved in the regulation of TLR-triggered macrophage activation. Notch-RBP-J signaling augments TLR4-induced expression of key mediators of M1 macrophages in responses toL. monocytogenesby promoting the synthesis of IRF8 that induces downstream M1 macrophage-associated genes.[92]Notch1 and Notch2, as well as their target genes Hes1 and Hes5 expression are up-regulated in macrophages stimulated by LPS, which suppresses proinflammatory cytokine production by suppressing ERK phosphorylation. As a result, NF-κB activity is inhibited through the MyD88/TRAF6 and TRIF pathways.[93]Endosome/lysosome-localized down-regulated CMRF-35-like molecule 3 (CLM-3) can promote full activation of TLR9-triggered macrophage activation by enhancing TRAF6 ubiquitination and subsequently activating MAPKs and NF-κB.[94]The orphan nuclear receptor small heterodimer partner (SHP) negatively regulates TLR-triggered macrophage activation by repressing transactivation of the NF-κB subunit p65 and inhibiting polyubiquitination of TRAF6.[95]Grb2-associated binder 1 (Gab1), a member of scaffolding/adaptor proteins, is needed for full activation of TLR3/4- and RIG-I-triggered macrophage activation by promoting activation of the PI3K/Akt, MAPKs, and NF-κB pathways.[96]Nonpathogenic immune complex and immunoglobulin can negatively regulate TLR4-triggered inflammatory response in macrophages through FcγRIIb-dependent prostaglandin E2.[97]Calmodulin-dependent protein kinase II (CaMKII), activated by TLR ligands, in turn promotes both MyD88- and TRIF-dependent inflammatory responses in macrophages, by directly activating TAK1 and IRF3, so the calcium/CaMKII pathway is required for full activation of TLR signaling.[98]

Bacterial components also can inhibit macrophage inflammatory response for their survival. For example, the immunoreceptor tyrosine-based inhibition motifs (ITIMs)-containing protein translocated intimin receptor (Tir) from enteropathogenicE. coli, suppressesE. coli-stimulated expression of proinflammatory cytokines, by interacting with tyrosine phosphatase SHP-1 in macrophages and subsequently facilitating the recruitment of SHP-1 to the adaptor TRAF6 and inhibiting the ubiquitination of TRAF6,[99]or by recruitment of SHP-2 and subsequent deubiquitination of TRAF6 in an ITIM-dependent manner.[100]

Regulation by membrane molecules

Major histocompatibility complex (MHC) molecules have recently been found to be involved in regulation of macrophage activation. MHC class I and II molecules possess opposite functions. Constitutive membrane MHC class I molecules attenuate TLR-triggered macrophage activation via reverse signaling, which protects mice from sepsis.[101]After TLR activation, the intracellular domain of MHC class I molecules in macrophages is phosphorylated by the kinase Src, then recruits tyrosine kinase Fps via its SH2 domain, thus leading to enhanced Fps activity and the recruitment of the phosphatase SHP-2 that interferes with the TLR signaling mediated by signaling molecule TRAF6. However, intracellular but not cell surface MHC class II molecules promote TLR-triggered macrophage activation, by interacting with the tyrosine kinase Btk via the costimulatory molecule CD40 and maintaining Btk activation, which then interacts with MyD88 and TRIF and thereby promotes TLR signaling.[102]

CD11b is the αM subunit of αMβ2 (CD11b/CD18,Mac-1, CR3) integrin which is mainly expressed in dendritic cells (DCs), granulocytes, monocytes/ macrophages and NK cells. A recent study[103]found that CD11b deficiency enhances TLR-mediated responses in macrophages, rendering mice more susceptible to endotoxin shock andE. coli-caused sepsis. CD11b is activated by TLR-triggered inside-out signaling through PI3K and the effector RapL and feedback inhibits TLR signaling by activating the tyrosine kinases Src and Syk. Syk interacts with and induces tyrosine phosphorylation of MyD88 and TRIF, resulting in their degradation by the E3 ubiquitin ligase Cbl-b. Thus, TLR-triggered, active CD11b integrin cross-talks with the MyD88 and TRIF pathways and subsequently inhibits TLR-triggered macrophage inflammation.

Sialic acid-binding immunoglobulin-like lectins (Siglecs), mainly expressed in various immune cells, are mostly inhibitory receptors that regulate PAMPsmediated inflammation by binding to cis ligands (expressed in the same cells) or by responding to pathogen-derived sialoglycoconjugates.[104]A study[105]showed that one member Siglec-G, known as inhibitory receptor of B1 cells, inhibits VSV-induced macrophage activation and innate immune response by promoting RIG-I degradation. VSV infection up-regulates Siglec-G expression in macrophages by RIG-I- and NF-κB-dependent mechanisms, which recruits SHP-2 and the E3 ubiquitin ligase c-Cbl to RIG-I, thus feedback leading to RIG-I degradation via K48-linked ubiquitination at Lys813 by c-Cbl.

Regulation by microRNAs

microRNAs represent a class of highly conserved, small, noncoding RNAs that suppress gene expression by binding to the 3'-untranslational region of target mRNAs.[106]microRNAs are essential regulators of diverse physiological and pathological processes, such as development, tumorigenesis, inflammation, immune response, and metabolism. Undoubtedly, microRNAs also regulate the function of macrophages in infection.

Studies[107,108]found that miR-146a and miR-155 were up-regulated in murine peritoneal macrophages upon VSV challenge, through a TLR/MyD88-independent but RIG-I/JNK/NF-κB-dependent mechanism. More importantly, the up-regulated miR-146a directly targets TRAF6, IRAK1 and IRAK2 in macrophages and subsequently down-regulates VSV-triggered IFN-I production, thus promoting VSV replication in macrophages.[107]However, the up-regulated miR-155 suppresses SOCS1 expression in macrophages and subsequently enhances IFN-I effector gene expression and this cytokine-mediated antiviral response, thus suppressing viral infection.[108]Although miR-146a and miR-155 are both up-regulated in macrophages upon RNA virus infection, these two microRNAs display opposite functions, the former negative feedback regulating IFN-I signaling in antiviral immunity through targeting TRAF6, IRAK1 and IRAK2, and the latter positive feedback regulating IFN-I signaling through directly targeting SOCS1.

IL-10 is one of the most important immunosuppressive cytokines, whose expression needs precise regulation. A study[109]showed that miR-145 can promote IL-10 production in TLR4-triggered macrophages by directly targeting the epigenetic histone deacetylase 11 (HDAC11), the recently identified member of the HDAC family, which has been proved to function as a crucialIl10gene silencer in antigen-presenting cells (APCs).[110]And this miR-145 is markedly down-regulated in macrophages upon VSV infection as well as TLR signals, depending on IFN-I production and the subsequent IFN-I receptor/JAK1/STAT1 pathway. Thus, IFN-I could feedback inhibit IL-10 production in macrophages through HDAC11 by down-regulating miR-145. Moreover, miR-466I also up-regulates IL-10 expression in TLR-triggered macrophages, but by another mechanism, i.e., by antagonizing RNA-binding protein tristetraprolin-mediated IL-10 mRNA degradation at the IL-10 3'-UTR AU-rich elements.[111]

TLRs stimulation reduces the expression of miR-92a[112]but induces miR-147[113]expression in macrophages, and both of them have the ability to feedback negatively regulate TLR-triggered inflammatory response in macrophages, and miR-92a acts by targeting mitogenactivated protein kinase kinase 4 (MKK4). Furthermore, the protein kinase Akt1 controls macrophage response to LPS by up-regulating microRNAs let-7e and downregulating miR-155, which represses TLR4 and SOCS1, respectively.[114]However, miR-125b has an ability to promote the generation of the activated status of macrophages, at least partially by reducing IRF4 levels, and potentiating the functional role of macrophages in inducing immune responses.[115]In addition, DCs share many characteristics with macrophages in many settings including infection, and it has been shown that miR-146a, which can be up-regulated by CD11b, negatively regulates TLR9-triggered DC cross-priming by directly targeting Notch1.[116]miR-30b, significantly up-regulated via the TGF-β/Smad3-mediated epigenetic pathway in regulatory DCs, can also target Notch1 to promote IL-10 and NO production of DCs.[117]

Regulation by epigenetic modulation

Epigenetic modulation is the hereditable trans-criptional regulation of genes without altering genome DNA sequence and plays important roles in multiple physiological and pathological processes, including DNA methylation, histone modification and chromatin remolding.[118]Aberrant epigenetic modifications are tightly associated with various human diseases. Recently, the role of epigenetic modulation in macrophage activation has also been studied.

Ash1l, the mammalian homolog of Drosophila Ash, is a H3K4 methyltransferase. In the peritoneal macrophages of TLR-triggered inflammatory response, Ash1l enhances A20 expression through induction of H3K4 methylation at the Tnfaip3 promoter, thus suppressing NF-κB, MAPK and subsequent IL-6 and TNF production by facilitating A20-mediated NF- κB signal modulator NEMO and transducer TRAF6 deubiquitination.[119]Hence, Ash1l silence with siRNA increases the LPS-induced IL-6 production of macrophages, and Ash1l-silenced mice were more susceptible to autoimmune disease as a result of enhanced IL-6 production by macrophages.

Wbp7 (also known as MLL4), another H3K4 methyltransferase, is required for the expression of Pigp, an essential component of the glycosylphosphatidylinositol (GPI)-GlcNAc transferase, or the enzyme catalyzing the first step of GPI anchor synthesis. It is found that impaired Pigp expression in Wbp7-/-macrophages abolishes GPI anchor-dependent loading of proteins on the cell membrane, such as CD14, the coreceptor for LPS and other bacterial molecules, thus markedly attenuating LPS-triggered intracellular signals and gene expression changes of macrophages.[120]

Recently, a small-molecule inhibitor of H3K27me3 (trimethylated lysine 27 on histone 3) demethylation GSK-J4 is identified as the first specific inhibitor of the jumonji family of histone demethylases, based on a structure-guided approach, which opens a new door for specifically studying the roles of the jumonji family in biology.[121]GSK-J4 is selective for the H3K27 demethylases jumonji domain 3 (JMJD3) and ubiquitously transcribed tetratricopeptide repeat gene on X chromosome (UTX). In human primary macrophages, GSK-J4 inhibits LPS-induced production of cytokines including TNF, for GSK-J4 prevents LPS-induced loss of H3K27me3 associated with the TNF transcription start sites. Thus the recruitment of RNA polymerase II is blocked, indicating that JMJD3 and UTX are involved in the LPS-induced activation of macrophages. Undoubtedly, this method will help more epigenetic-associated molecules to regulate macrophage activation. In addition, JMJD3 has also been proved crucial for regulating M2 macrophage development leading to anti-helminth host response through promoting IRF4 expression.[122]

Conclusions

Macrophages are highly heterogeneous populations which widely distribute in almost all tissues and organs, displaying tissue- and organ-specific multiple functions. The primary function of macrophages is to defense against invading pathogens, including bacteria, viruses, fungui, and parasites. Macrophages participate in the whole inflammatory anti-infection process, from recognizing microbial components with particular PRRs expressed on the membrane or in the cytoplasm of macrophages, to closure of inflammation by converting to anti-inflammatory phenotype and simultaneously promoting tissue repair. Although they can polarize to M1 macrophages and M2 macrophages to take part in type 1 and 2 immune responses respectively, the activation phenotype of macrophages is so alterable as to convert into counterpart according to the changing microenvironment. On the one hand, the instable and plastic property of macrophages enables them to adapt the changeable environments, and that's why they are highly multifunctional populations; on the other hand, foreign pathogens or transforming tumor cells also can take advantage of it to re-educate macrophages to help them have a long-term survival or proliferation in the host. Therefore, deeper understanding of the inflammatory process and underlying mechanisms will undoubtedly help us develop promising procedures to defense against infection and other disorders.

Macrophages play important and indispensable roles in the anti-infection immune response. Insufficient macrophage activation may lead to uncompleted elimination of invading pathogens, but excessive macrophage activation may result in self-tissue damage and inflammatory diseases and even autoimmune disorders. Therefore, the inflammatory process of macrophages in infection needs fine regulation. Until now, multiple complicated signaling pathways have been found in regulating the activation of macrophages, including various intracellular signaling molecules, cell surface molecules, and even recently identified microRNAs and epigenetic-associated molecules, mainly through feedback negatively or positively regulate the TLR signaling pathways activated in infection. We in this review outline the representing mechanisms found in the past decade, and the research of macrophage regulation by microRNAs and epigenetic-associated molecules is only at its starting stage. Undoubtedly, the regulation mechanisms at different levels will be foundin the future.

Although the complicated inflammatory response of macrophages in infection and underlying regulatory mechanisms have been well studied in recent years, many questions remain to be answered. For example, macrophages and DCs share many characteristics under various conditions including infection, so what's the relationship between macrophages and DCs in inflammatory response in infection? Which one is more important? Can they replace the other or do they show different important parts in different phases of anti-infection response? With regard to the subsets of macrophages, only M1 and M2 macrophages have been identified. Thus, M1 and M2 as real subsets of macrophages other than different functional status, can macrophages further differentiate into various subsets just as the increasing Th subsets, and if so, do they play different roles in different phases of anti-infection response? More importantly, how significant discoveries in basic research into the inflammatory response of macrophages in infection are translated into clinical usage.

Contributors:WCC proposed the study. Both authors collected and analyzed the data, performed research and wrote the first draft. WCC is the guarantor.

Funding:This study was supported by a grant from the National Natural Science Foundation of China (81373131).

Ethical approval:Not needed.

Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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Received November 22, 2013

Accepted after revision March 10, 2014

Author Affiliations: Center for Educational Technology, Second Military Medical University, Shanghai 200433, China (Zhang L); Department of Anesthesiology, Changzhen Hospital, Second Military Medical University, Shanghai 200003, China (Wang CC)

Cheng-Cai Wang, PhD, Department of Anesthesiology, Changzhen Hospital, Second Military Medical University, Shanghai 200003, China (Tel: 86-21-81885833; Email: wangchengcai81@sina.com)

© 2014, Hepatobiliary Pancreat Dis Int. All rights reserved.

10.1016/S1499-3872(14)60024-2