Chemokine receptor 5 blockade modulates macrophage trafficking in renal ischaemic-reperfusion injury

Chemokine receptor 5 (CCR5) is a pivotal regulator of macrophage trafficking in the kidneys in response to an inflammatory cascade. We investigated the role of CCR5 in experimental ischaemic-reperfusion injury (IRI) pathogenesis. To establish IRI, we clamped the bilateral renal artery pedicle for 30 min and then reperfused the kidney. We performed adoptive transfer of lipopolysaccharide (LPS)-treated RAW 264.7 macrophages following macrophage depletion in mice. B6.CCR5^−/− mice showed less severe IRI based on tubular epithelial cell apoptosis than did wild-type mice. CXCR3 expression in CD11b⁺ cells and inducible nitric oxide synthase levels were more attenuated in B6.CCR5^−/− mice. B6.CCR5^−/− mice showed increased arginase-1 and CD206 expression. Macrophage-depleted wild-type mice showed more injury than B6.CCR5^−/− mice after M1 macrophage transfer. Adoptive transfer of LPS-treated RAW 264.7 macrophages reversed the protection against IRI in wild-type, but not B6.CCR5^−/− mice. Upon knocking out CCR5 in macrophages, migration of bone marrow-derived macrophages from wild-type mice towards primary tubular epithelial cells with recombinant CCR5 increased. Phospho-CCR5 expression in renal tissues of patients with acute tubular necrosis was increased, showing a positive correlation with tubular inflammation. In conclusion, CCR5 deficiency favours M2 macrophage activation, and blocking CCR5 might aid in treating acute kidney injury.     

TREM2 expression promotes liver and peritoneal M2 macrophage polarization in mice infected with Schistosoma japonicum

Schistosomiasis is a tropical parasitic disease that damages the liver and poses a serious threat to human health. Macrophages play a key role in the development of liver granulomas and fibrosis by undergoing polarization from M1 to M2 type during schistosomiasis. Therefore, regulating macrophage polarization is important for controlling pathological changes that occur during this disease. Triggering receptor expressed on myeloid cells 2 (TREM2) expressed on the surface of macrophages, dendritic cells and other immune cells has been shown to play a role in inhibiting inflammatory responses and regulating M2 macrophage polarization, however its role in macrophage polarization in schistosomiasis has not been investigated. In this study, we confirmed that TREM2 expression was upregulated in the livers and peritoneal macrophages of mice infected with Schistosoma japonicum. Moreover, the TREM2 expression trend correlated with the expression of M2 macrophage polarization-related molecules in the liver tissues of S. japonicum-infected mice. Using Trem2^−/− mice, we also showed that Trem2 deletion inhibited Arg1 and Ym1 expression in liver tissues. Trem2 deletion also increased the number of F4/80 + CD86+ cells in peritoneal macrophages of infected mice. In summary, our study suggests that TREM2 may be involved in M2 macrophage polarization during schistosomiasis.     

Protective role of G-CSF in dextran sulfate sodium-induced acute colitis through generating gut-homing macrophages

Granulocyte colony-stimulating factor (G-CSF) is a pleiotropic cytokine best known for its role in promoting the generation and function of neutrophils. G-CSF is also found to be involved in macrophage generation and immune regulation; however, its in vivo role in immune homeostasis is largely unknown. Here, we examined the role of G-CSF in dextran sulfate sodium (DSS)-induced acute colitis using G-CSF receptor-deficient (G-CSFR^−/−) mice. Mice were administered with 1.5% DSS in drinking water for 5 days, and the severity of colitis was measured for the next 5 days. GCSFR^−/− mice were more susceptible to DSS-induced colitis than G-CSFR^+/+ or G-CSFR^−/+ mice. G-CSFR^−/− mice harbored less F4/80⁺ macrophages, but a similar number of neutrophils, in the intestine. In vitro, bone marrow-derived macrophages prepared in the presence of both G-CSF and macrophage colony-stimulating factor (M-CSF) (G-BMDM) expressed higher levels of regulatory macrophage markers such as programmed death ligand 2 (PDL2), CD71 and CD206, but not in arginase I, transforming growth factor (TGF)-β, Ym1 (chitinase-like 3) and FIZZ1 (found in inflammatory zone 1), and lower levels of inducible nitric oxide synthase (iNOS), CD80 and CD86 than bone marrow-derived macrophages prepared in the presence of M-CSF alone (BMDM), in response to interleukin (IL)-4/IL-13 and lipopolysaccharide (LPS)/interferon (IFN)-γ, respectively. Adoptive transfer of G-BMDM, but not BMDM, protected G-CSFR^−/− mice from DSS-induced colitis, and suppressed expression of tumor necrosis factor (TNF)-α, IL-1β and iNOS in the intestine. These results suggest that G-CSF plays an important role in preventing colitis, likely through populating immune regulatory macrophages in the intestine.     

PPARγ Ligands Switched High Fat Diet-Induced Macrophage M2b Polarization toward M2a Thereby Improving Intestinal Candida Elimination

Obesity is associated with a chronic low-grade inflammation that predisposes to insulin resistance and the development of type 2 diabetes. In this metabolic context, gastrointestinal (GI) candidiasis is common. We recently demonstrated that the PPARγ ligand rosiglitazone promotes the clearance of Candida albicans through the activation of alternative M2 macrophage polarization. Here, we evaluated the impact of high fat diet (HFD)-induced obesity and the effect of rosiglitazone (PPARγ ligand) or WY14643 (PPARα ligand) both on the phenotypic M1/M2 polarization of peritoneal and cecal tissue macrophages and on the outcome of GI candidiasis. We demonstrated that the peritoneal macrophages and the cell types present in the cecal tissue from HF fed mice present a M2b polarization (TNF-α^high, IL-10^high, MR, Dectin-1). Interestingly, rosiglitazone induces a phenotypic M2b-to-M2a (TNF-α^low, IL-10^low, MR^high, Dectin-1^high) switch of peritoneal macrophages and of the cells present in the cecal tissue. The incapacity of WY14643 to switch this polarization toward M2a state, strongly suggests the specific involvement of PPARγ in this mechanism. We showed that in insulin resistant mice, M2b polarization of macrophages present on the site of infection is associated with an increased susceptibility to GI candidiasis, whereas M2a polarization after rosiglitazone treatment favours the GI fungal elimination independently of reduced blood glucose. In conclusion, our data demonstrate a dual benefit of PPARγ ligands because they promote mucosal defence mechanisms against GI candidiasis through M2a macrophage polarization while regulating blood glucose level.     

Type I Interferon Induction Is Detrimental during Infection with the Whipple's Disease Bacterium,Tropheryma whipplei

Macrophages are the first line of defense against pathogens. Upon infection macrophages usually produce high levels of proinflammatory mediators. However, macrophages can undergo an alternate polarization leading to a permissive state. In assessing global macrophage responses to the bacterial agent of Whipple''s disease, Tropheryma whipplei, we found that T. whipplei induced M2 macrophage polarization which was compatible with bacterial replication. Surprisingly, this M2 polarization of infected macrophages was associated with apoptosis induction and a functional type I interferon (IFN) response, through IRF3 activation and STAT1 phosphorylation. Using macrophages from mice deficient for the type I IFN receptor, we found that this type I IFN response was required for T. whipplei-induced macrophage apoptosis in a JNK-dependent manner and was associated with the intracellular replication of T. whipplei independently of JNK. This study underscores the role of macrophage polarization in host responses and highlights the detrimental role of type I IFN during T. whipplei infection.     

Effective-components combination improves airway remodeling in COPD rats by suppressing M2 macrophage polarization via the inhibition of mTORC2 activity

BackgroundIn chronic obstructive pulmonary disease (COPD), M2 macrophages release multiple tissue repair-related factors, leading to airway remodeling, a significant pathological characteristic. Meanwhile, effective-components combination (ECC), derived from Bufei Yishen formula (BYF), is an effective treatment for COPD.PurposeTo determine the potential mechanisms of ECC in airway remodeling in COPD by suppressing M2 macrophage polarization.MethodsWe established a rat COPD Model using exposure to cigarette smoke and bacterial infection to investigate the efficacy of ECC. We also treated macrophages with IL-4 for 12 h to explore the in vivo effect of ECC on M2 macrophage polarization and mTORC2 signals.ResultsThe disease severity of COPD rats could be alleviated by ECC treatment, which improved pulmonary function and alleviated pathological injuries in lung tissue and the inflammatory cytokine levels. Meanwhile, ECC could ameliorate airway remodeling by reducing collagen deposition, hindering airway mucus hypersecretion and smooth muscle cell proliferation, and reducing the number of M2 macrophages in the lung tissues of COPD rats. Furthermore, with IL-4-induced macrophages, we found that ECC could suppress M2 macrophage polarization by decreasing the levels of M2 macrophage markers. Finally, we discovered that ECC inhibited mTORC2 activity by examining p-mTOR²⁴⁸¹ and its downstream protein p-Akt⁴⁷³.ConclusionsECC exerts beneficial effects on airway remodeling in COPD rats, likely by suppressing M2 macrophage polarization via the inhibition of mTORC2 activity.     

Enhanced M1 macrophage polarization in human helicobacter pylori-associated atrophic gastritis and in vaccinated mice

Background

Infection with Helicobacter pylori triggers a chronic gastric inflammation that can progress to atrophy and gastric adenocarcinoma. Polarization of macrophages is a characteristic of both cancer and infection, and may promote progression or resolution of disease. However, the role of macrophages and their polarization during H. pylori infection has not been well defined.

Methodology/Principal Findings

By using a mouse model of infection and gastric biopsies from 29 individuals, we have analyzed macrophage recruitment and polarization during H. pylori infection by flow cytometry and real-time PCR. We found a sequential recruitment of neutrophils, eosinophils and macrophages to the gastric mucosa of infected mice. Gene expression analysis of stomach tissue and sorted macrophages revealed that gastric macrophages were polarized to M1 after H. pylori infection, and this process was substantially accelerated by prior vaccination. Human H. pylori infection was characterized by a mixed M1/M2 polarization of macrophages. However, in H. pylori-associated atrophic gastritis, the expression of inducible nitric oxide synthase was markedly increased compared to uncomplicated gastritis, indicative of an enhanced M1 macrophage polarization in this pre-malignant lesion.

Conclusions/Significance

These results show that vaccination of mice against H. pylori amplifies M1 polarization of gastric macrophages, and that a similar enhanced M1 polarization is present in human H. pylori-induced atrophic gastritis.     

M2-like macrophages polarized by Foxp3− Treg-of-B cells ameliorate imiquimod-induced psoriasis

Our group have demonstrated that splenic B cells contributed to the CD4⁺CD25⁻ naive T cells conversion into CD4⁺CD25⁺Foxp3⁻ regulatory T cells without adding appended cytokines, named Treg-of-B cells which were potent suppressors of adaptive immunity. We like to investigate whether Treg-of-B cells could promote alternatively activated macrophage (M2 macrophages) polarization and alleviate inflammatory disease, psoriasis. In this study, we co-cultured the bone marrow-derived macrophages (BMDMs) with Treg-of-B cells under LPS/IFN-γ stimulation and analyzed the M2-associated gene and protein using qPCR, western blotting, and immunofluorescence staining. We also examined the therapeutic effect of Treg-of-B cell-induced M2 macrophage for skin inflammation using imiquimod (IMQ)-induced psoriatic mouse model. Our results showed that BMDMs co-cultured with Treg-of-B cells upregulated typical M2-associated molecules, including Arg-1, IL-10, Pdcd1lg2, MGL-1, IL-4, YM1/2 and CD206. In an inflammatory environment, TNF-α and IL-6 production by macrophages co-cultured with Treg-of-B cells was decreased significantly. The molecular mechanism revealed that Treg-of-B cells promoted M2 macrophage polarization via STAT6 activation in a cell contact-dependent manner. Moreover, the treatment with Treg-of-B cell-induced M2 macrophages attenuated the clinical manifestations of psoriasis, such as scaling, erythema and thickening in the IMQ-induced psoriatic mouse model. T cell activation in draining lymph nodes was decreased in the Treg-of-B cell-induced M2 macrophage group after IMQ application. In conclusion, our findings suggested that Foxp3⁻ Treg-of-B cells could induce alternatively activated M2 macrophages through STAT6 activation, providing a cell-based therapeutic strategy for psoriasis.     

Transgenic CGI-58 expression in macrophages alleviates the atherosclerotic lesion development in ApoE knockout mice

Comparative Gene Identification-58 (CGI-58), as an adipose triglyceride lipase (ATGL) activator, strongly increases ATGL-mediated triglyceride (TG) catabolism. Previous studies have shown that CGI-58 affects intestinal cholesterol homeostasis independently of ATGL activity. Therefore, we hypothesized that CGI-58 was involved in macrophage cholesterol metabolism and consequently atherosclerotic lesion formation. Here, we generated macrophage-specific CGI-58 transgenic mice (Mac-CGI-58 Tg) using an SRA promoter, which was further mated with ApoE^−/− mice to create litters of CGI-58 Tg/ApoE^−/− mice. These CGI-58 Tg/ApoE^−/− mice exhibited an anti-atherosclerosis phenotype compared with wild type (WT) controls (CGI-58 WT/ApoE^−/−), illustrated by less plaque area in aortic roots. Moreover, macrophage-specific CGI-58 overexpression in mice resulted in up-regulated levels of plasma total cholesterol and HDL-cholesterol. Consequently, higher expression levels of PPARa, PPARγ, LXRα, ABCA1, and ABCG1 were detected in macrophages from CGI-58 Tg/ApoE^−/− mice compared to CGI-58 WT/ApoE^−/− counterparts, which were accompanied by elevated macrophage cholesterol efflux toward HDL and Apo A1. Nevertheless, serum levels of TNF-α and IL-6 were reduced by macrophage-specific CGI-58 overexpression. Finally, bone marrow (BM) transplantation experiments further revealed that ApoE^−/− mice reconstituted with Mac-CGI-58 Tg BM cells (ApoE^−/−/Tg-BM chimera) displayed a significant reduction of atherosclerosis lesions compared with control mice reconstituted with Mac-CGI-58 WT BM cells (ApoE^−/−/WT-BM chimera). Collectively, these data strongly suggest that CGI-58 overexpression in macrophages may protect against atherosclerosis development in mice.     

TLR3 regulates mycobacterial RNA-induced IL-10 production through the PI3K/AKT signaling pathway

Cytokine induction in response to Mycobacterium tuberculosis (Mtb) infection is critical for pathogen control, by (i) mediating innate immune effector functions and (ii) instructing specific adaptive immunity. IL-10 is an important anti-inflammatory cytokine involved in pathogenesis of tuberculosis (TB). Here, we show that TLR3, a sensor of extracellular viral or host RNA with stable stem structures derived from infected or damaged cells, is essential for Mtb-induced IL-10 production. Upon Mycobacterium bovis Bacillus Calmette–Guérin (BCG) infection, TLR3^−/− macrophages expressed lower IL-10 but higher IL-12p40 production, accompanied by reduced phosphorylation of AKT at Ser473. BCG-infected TLR3^−/− mice exhibited reduced IL-10 but elevated IL-12 expression compared to controls. Moreover, higher numbers of splenic Th1 cells and reduced pulmonary bacterial burden and tissue damage were observed in BCG-infected TLR3^−/− mice. Finally, BCG RNA induced IL-10 in macrophages via TLR3-mediated activation of PI3K/AKT. Our findings demonstrate a critical role of TLR3-mediated regulation in the pathogenesis of mycobacterial infection involving mycobacterial RNA, which induces IL-10 through the PI3K/AKT signaling pathway.     

Mouse macrophage specific knockout of SIRT1 influences macrophage polarization and promotes angiotensin Ⅱ-induced abdominal aortic aneurysm formation

Abdominal aortic aneurysm (AAA) is a vascular degenerative disease. Macrophage polarization and the balance between classically activated macrophages (M1) and alternatively activated macrophages (M2) are crucial for AAA pathogenesis. The present study aims to investigate the roles of macrophage SIRT1 in AAA formation and macrophage polarization. We found that in mouse peritoneal macrophages, SIRT1 expression was decreased after M1 stimulation, but was enhanced after M2 stimulation. Results from SIRT1^flox/flox mice and macrophage specific SIRT1 knockout mice with treatment of angiotensin II (Ang II) for 4 weeks showed that macrophage specific deficiency of SIRT1 increased the incidence of AAA and exacerbated the severity, including more severe aneurysm types, enlarged diameter of the aneurysm and increased degradation of elastin. In mouse aortas, SIRT1 deficiency increased the pro-inflammatory M1 molecule inducible nitric oxide synthase (iNOS), and decreased M2 molecules such as arginase 1 (Arg1) and mannose receptor (MR). Furthermore, in peritoneal macrophages, SIRT1 deficiency increased the expression of M1 inflammatory molecules, but decreased the expression of M2 molecules. Overexpression of SIRT1 had the opposite effects. Thus, macrophage specific knockout of SIRT1 influences macrophage polarization and accelerates Ang II-induced AAA formation.     

TAp73 is required for macrophage-mediated innate immunity and the resolution of inflammatory responses

The multiple isoforms of p73, a member of the p53 family, share the ability to modulate p53 activities but also have unique properties, leading to a complex and poorly understood functional network. In vivo, p73 isoforms have been implicated in tumor suppression (TAp73^−/− mice), DNA damage (ΔNp73^−/− mice) and development (p73^−/− mice). In this study, we investigated whether TAp73 contributes to innate immunity and septic shock. In response to a lethal lipopolysaccharide (LPS) challenge, TAp73^−/− mice showed higher blood levels of proinflammatory cytokines and greater mortality than their wild-type littermates. In vitro, TAp73^−/− macrophages exhibited elevated production of tumor necrosis factor alpha , interleukin-6 and macrophage inflammatory protein-2 as well as prolonged survival, decreased phagocytosis and increased major histocompatibility complex class II expression. Mice depleted of endogenous macrophages and reconstituted with TAp73^−/− macrophages showed increased sensitivity to LPS challenge. These results suggest that macrophage polarization is altered in the absence of TAp73 such that maintenance of the M1 effector phenotype is prolonged at the expense of the M2 phenotype, thus impairing resolution of the inflammatory response. Our data indicate that TAp73 has a role in macrophage polarization and innate immunity, enhancing the action field of this important regulatory molecule.     

Defective adipose tissue development associated with hepatomegaly in cathepsin E-deficient mice fed a high-fat diet

Cathepsin E is an intracellular aspartic proteinase, which is predominantly distributed in immune-related and epithelial cells. However, the role of the enzyme in adipose tissues remains unknown. In this study, we investigated the characteristics of cathepsin E-deficient (CatE^−/−) mice fed a high-fat diet (HFD), as a mouse model of obesity. HFD-fed CatE^−/− mice displayed reduced body weight gain and defective development of white adipose tissue (WAT) and brown adipose tissue (BAT), compared with HFD-fed wild-type mice. Moreover, fat-induced CatE^−/− mice showed abnormal lipid accumulation in non-adipose tissues characterized by hepatomegaly, which is probably due to defective adipose tissue development. Detailed pathological and biochemical analyses showed that hepatomegaly was accompanied by hepatic steatosis and hypercholesterolemia in HFD-induced CatE^−/− mice. In fat-induced CatE^−/− mice, the number of macrophages infiltrating into WAT was significantly lower than in fat-induced wild-type mice. Thus, the impaired adipose tissue development in HFD-induced CatE^−/− mice was probably due to reduced infiltration of macrophages and may lead to hepatomegaly accompanied by hepatic steatosis and hypercholesterolemia.     

Local Delivery of Polarized Macrophages Improves Reperfusion Recovery in a Mouse Hind Limb Ischemia Model

Aims

Enhancement of collateral development in coronary or peripheral artery disease is a therapeutic target, but it has proven difficult to achieve. Macrophages are key players in collateral remodeling, yet the effect of different macrophage subsets on arteriogenesis has not been investigated.

Methods and Results

Murine macrophages were cultured from bone marrow and polarized into M1 (IFNγ), M2a (IL-4) or M2c (IL-10) subsets. C57BL/6 mice underwent femoral artery ligation followed by intramuscular injection of macrophage subsets. Using eGFP expressing macrophages, cells could be detected at least 6 days after ligation and were located in the perivascular space of collateral vessels. After 14 days, perfusion ratio was increased in animals treated with M1 as well as M2a and M2c macrophages compared to control. Depletion of circulating monocytes by clodronate liposome injections did not hamper reperfusion recovery, however, treatment with exogenous polarized macrophages improved perfusion ratio after 14 days again. We used IL10R^fl/fl/LysMCre⁺ mice to study the effect of inhibition of endogenous polarization towards specifically M2c macrophages on arteriogenesis. Deletion of the IL10-receptor (IL10R) in the myeloid lineage did not affect reperfusion recovery, yet the pro-arteriogenic effect of exogenously injected M2c macrophages was still present.

Conclusions

Local injection of polarized macrophages promotes reperfusion recovery after femoral artery ligation and is not influenced by depletion of circulatory monocytes. Preventing endogenous M2c polarization did not affect reperfusion recovery suggesting that M2c’s are not required for collateralization, but are sufficient to induce collateral formation upon exogenous administration. This is the first study using local injection of macrophage subsets showing the pro-arteriogenic effect of polarized macrophages.