Cellular Metabolism and Macrophage Functional Polarization

Macrophages are a functionally heterogeneous cell population that is mainly shaped by a variety of microenvironmental stimuli. Interferon γ (IFN-γ), interleukin-1β (IL-1β), and lipopolysaccharide (LPS) induce a classical activation of macrophages (M1), whereas IL-4 and IL-13 induce an alternative activation program in macrophages (M2). Reprogramming of intracellular metabolisms is required for the proper polarization and functions of activated macrophages. Similar to the Warburg effect observed in tumor cells, M1 macrophages increase glucose consumption and lactate release and decreased oxygen consumption rate. In comparison, M2 macrophages mainly employ oxidative glucose metabolism pathways. In addition, fatty acids, vitamins, and iron metabolisms are also related to macrophage polarization. However, detailed metabolic pathways involved in macrophages have remained elusive. Understanding the bidirectional interactions between cellular metabolism and macrophage functions in physiological and pathological situations and the regulatory pathways involved may offer novel therapies for macrophage-associated diseases.     

M1和M2型巨噬细胞表型的比较分析

通过对M1和M2型巨噬细胞表型相关指标的比较分析,评价各鉴定巨噬细胞类型的表型指标及其意义。按常规方法以IFN-γ及LPS将骨髓来源巨噬细胞诱导成M1型巨噬细胞,以IL-4诱导出M2型巨噬细胞。分别以RT-PCR和酶活性定量方法检测精氨酸代谢相关酶的表达和活性;以ELISA检测IL-12和IL-10的分泌;以FACS检测巨噬细胞膜分子的表达。结果显示:M1型巨噬细胞诱导性一氧化氮合酶(inducible nitric oxide synthase,iNOS)表达和活性水平较未刺激组明显升高,IL-12产生显著增加,CD16/32表达上调;而M2型巨噬细胞I型精氨酸酶(arginase 1,Arg-1)的表达水平和酶活性较未刺激巨噬细胞显著提高,IL-10分泌轻度增加,并且表达高水平的CD206和DECTIN-1。表型比较分析结果表明,iN-OS表达和活性、IL-12的分泌和膜蛋白CD16/32可用于鉴定M1型巨噬细胞,而Arg-1、CD206和DECTIN-1是鉴定M2型巨噬细胞较为理想的表型指标。     

Advanced age impairs macrophage polarization

Aging affects many aspects of the cellular function of macrophages. Macrophages play a critical role in innate immunity, acting as sentinels to fight pathogens, promoting wound healing, and orchestrating the development of the specific acquired immune response. However, little is known about how age influences the ability of macrophage to change phenotypes in response to environmental factors. This study examined the age-associated defects on macrophage polarization toward a pro-inflammatory (M1) or an anti-inflammatory (M2) phenotype. Adherent splenocytes enriched for macrophages were cultured with or without lipopolysaccharide (LPS), a combination of interferon (IFN)-γ and tumor necrosis factor (TNF)-α or interleukin (IL)-4. A panel of M1 markers, inducible nitric oxide synthase (iNOS), IL-6, IL-1β, and TNF-α, and M2 markers, including arginase-1 (Arg1), Ym1, and Found In Inflammatory Zone 1 (FIZZ1), were analyzed. IL-6 mRNA in cells from aged mice was decreased by 78% and 58% compared with young after stimulation with LPS or IFN-γ and TNF-α (P<0.05), respectively. Also, there was a marked reduction in the induced levels of iNOS, IL-1β, and TNF-α in cells from aged mice relative to young controls. Similarly, IL-4 exposure resulted in a reduction of M2 markers in adherent splenocytes from aged mice compared with younger animals. This was consistent with a 28% decrease in splenic F4/80(+)IL-4R(+) cells in aged mice relative to controls, although IL-4R expression on these cells did not vary between age groups. In contrast, levels of M1 and most M2 markers, save for FIZZ1, in bone marrow-derived macrophages were similar between the age groups, irrespective of stimuli. These data imply that impaired macrophage polarization in the elderly may dysregulate the development of the host response, making them more susceptible to infectious diseases and that the aging microenvironment may be a key modulator of these macrophage-elicited responses.     

TLR-mediated distinct IFN-γ/IL-10 pattern induces protective immunity against murine visceral leishmaniasis

Resistance to murine visceral leishmaniasis (VL) correlates with the development of an IFN-γ predominant immune response. Beta1,4-galactose terminal glycans are potent inducers of IFN-γ. Here, we demonstrate the efficacy of a 29 kDa β1,4-galactose terminal glycoprotein (GP29) of Leishmania donovani (LD) in an in vitro macrophage model and an in vivo mouse model of VL. GP29 induced splenic macrophages to release NO and ROS in appreciable amounts that resulted in effective parasite clearance from macrophages. This was associated with the toll-like receptor (TLR)-4 mediated IL-12 induction and inhibition of TLR2-mediated IL-10 production. Two subcutaneous injections of GP29 at fortnightly intervals resulted in dominant IL-12-mediated IFN-γ production and 100% animals were protected against a subsequent challenge with virulent LD parasites. Vaccinated mice showed a reversal of T-cell anergy, significantly elevated expression of iNOS and a type-1 IgG subclass response. Moreover, vaccinated mice downregulated arginase1 and IL-10 expression but did not alter IL-4 expression. The IFN-γ/IL-10 ratio regulated the intensity of the protective immune response. Experiments with IFN-γ and IL-10 knockout mice reiterated the role IL-10 and IFN-γ play in disease progression or resolution in the murine model of VL.     

Liposomes of phosphatidylcholine and cholesterol induce an M2-like macrophage phenotype reprogrammable to M1 pattern with the involvement of B-1 cells

Macrophages respond to endogenous and non-self stimuli acquiring the M1 or M2 phenotypes, corresponding to classical or alternative activation, respectively. The role of B-1 cells in the regulation of macrophage polarization through the secretion of interleukin (IL)-10 has been demonstrated. However, the influence of B-1 cells on macrophage phenotype induction by an immunogen that suppress their ability to secrete IL-10 has not been explored. Here, we studied the peritoneal macrophage pattern induced by liposomes comprised of dipalmitoylphosphatidylcholine (DPPC) and cholesterol (Chol) carrying ovalbumin (OVA) (Lp DPPC/OVA), and the involvement of B-1 cells in macrophage polarization. Peritoneal cells from BALB/c, B-1 cells-deficient BALB/xid and C57BL/6 mice immunized with Lp DPPC/OVA and OVA in soluble form (PBS/OVA) were analyzed and stimulated or not in vitro with lipopolysaccharide (LPS). Peritoneal macrophages from BALB/c and C57BL/6 mice immunized with Lp DPPC/OVA showed an M2-like phenotype as evidenced by their high arginase activity without LPS stimulation. Upon stimulation, these macrophages were reprogrammable toward the M1 phenotype with the upregulation of nitric oxide (NO) and a decrease in IL-10 secretion. In addition, high IFN-γ levels were detected in the culture supernatant of peritoneal cells from BALB/c and C57BL/6 mice immunized with Lp DPPC/OVA. Nevertheless, still high levels of arginase activity and undetectable levels of IL-12 were found, indicating that the switch to a classical activation state was not complete. In the peritoneal cells from liposomes-immunized BALB/xid mice, levels of arginase activity, NO, and IL-6 were below those from wild type animals, but the last two products were restored upon adoptive transfer of B-1 cells, together with an increase in IFN-γ secretion. Summarizing, we have demonstrated that Lp DPPC/OVA induce an M2-like pattern in peritoneal macrophages reprogrammable to M1 phenotype after LPS stimulation, with the involvement of B-1 cells.     

Modulation of the inflammatory response to chitosan through M2 macrophage polarization using pro-resolution mediators

Tissue engineering and regenerative medicine have created a demand for biomaterials with specific functions such as the ability to modify the host immune response. The objective of this study was to evaluate the effect of two different pro-resolution lipid mediators, lipoxin A4 (LxA4) and resolvin D1 (RvD1), in the modulation of the inflammatory response to biomaterials through M2 macrophage polarization. This was investigated in vivo using a mouse air-pouch model of inflammation. Our results demonstrated that both LxA4 and RvD1 are able to shift the macrophage response to implanted Ch scaffolds to an M2 reparative response. The injection of these pro-resolution mediators caused a decrease in inflammatory cells recruited to the implant site together with higher numbers of F4/80⁺/CD206⁺ cells (M2 macrophages) and lower numbers of F4/80⁺/CCR7⁺ cells (M1 macrophages); it also induced a general decrease in several pro-inflammatory cytokines; and caused a significant decrease in the thickness and area of the fibrous capsule formed around the implanted scaffolds. In conclusion, the use of either LxA4 or RvD1 allowed the in vivo control of macrophage phenotypic profile and thus may play a significant role in regenerative medicine applications, namely through modulation of the inflammatory response.     

Aire 对巨噬细胞极化影响的研究

目的:研究自身免疫调节因子(Aire)对巨噬细胞极化的影响。方法:分别用LPS、IL-4 以及LPS 联合免疫复合物刺激小鼠单核巨噬细胞系RAW264郾7 细胞、稳定表达GFP-Aire 的RAW264.7 细胞(A33-3) 细胞和稳定表达GFP 的RAW264.7 细胞(C1-6),使其向M1(LPS)、M2a(IL-4)和M2b(LPS 联合免疫复合物)型巨噬细胞极化。通过Real-time PCR 检测各组细胞中M1 型巨噬细胞特征分子IL-1、iNOS 和IL-6,M2a 型特征分子Arg-1 和M2b 型特征分子IL-10 的表达水平,研究Aire 对各种类型巨噬细胞极化的影响。结果:LPS 在0.5 g/ ml 浓度时,RAW264.7 细胞中M1 型巨噬细胞产物IL-1 、iNOS和IL-6 基因表达量最高;而IL-4 以及LPS 联合免疫复合物的刺激作用有显著的剂量依赖性,都在浓度最高时RAW264.7 细胞中Arg1(M2a)和IL-10(M2b)基因表达量最高。LPS 刺激后,A33-3 细胞中IL-1 和iNOS 表达水平明显高于C1-6 细胞,IL-6 则相反;IL-4 及LPS 联合免疫复合物刺激后,A33-3 细胞中Arg1 和IL-10 的表达水平明显低于C1-6 细胞。结论:Aire 可能促进巨噬细胞向M1 极化,同时抑制其向M2a 和M2b 极化。     

Attenuated expression of interferon-β and interferon-λ1 by human alternatively activated macrophages

Macrophages can be polarized into classically (CAM) or alternatively (AAM) activated macrophages with IFN-γ or IL-4, respectively. CAM are associated with type 1 immune responses and are implicated in autoimmunity; AAM are associated with type 2 responses and are implicated in allergic diseases. An impediment in investigating macrophage biology using primary human monocyte derived macrophages is the wide inter-donor heterogeneity and the limited quantity of cells that survive in vitro polarization. To overcome this impediment, we established a protocol to generate CAM and AAM cultures derived from the THP-1 human promonocytic cell line. In this report, we demonstrate that THP-CAM and -AAM express gene and protein markers that define their primary human monocyte derived counterparts, such as IL-1β, CXCL10, and CXCL11 for CAM, and MRC1, IL-4 and CCL22 for AAM. In addition, we demonstrate that STAT6 is selectively activated in THP-AAM which, upon LPS stimulation, have an attenuated or delayed expression of IFN-β, IFN-λ1, and IFN α/β pathway genes compared to their CAM counterparts. Taken together, these findings may help further investigate human diseases associated with the alternatively activated macrophage phenotype using this reproducible in vitro macrophage model.     

Differential potentiation of anti-mycobacterial activity and reactive nitrogen intermediate-producing ability of murine peritoneal macrophages activated by interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α)

The anti-mycobacterial activities of IFN-γ and TNF-α-treated murine peritoneal macrophages were determined. Resident macrophages pretreated with IFN-γ or TNF-α for 2 days were infected with test organisms and subsequently cultured for up to 7 days. First, the early-phase growth of Mycobacterium tuberculosis (days 0–3) was strongly suppressed in IFN-γ-treated macrophages, and progressive bacterial elimination was subsequently observed. Although TNF-α treatment of macrophages did not affect the early phase growth of organisms, bacterial killing was observed in the later phase of cultivation. Second, although IFN-γ-treated macrophages killed M. avium during the first 3 days of culture, regrowth of the intracellular organisms was subsequently observed. TNF-α treatment of macrophages did not influence the mode of intracellular growth of M. avium. Third, IFN-γ but not TNF-α enhanced production of reactive nitrogen intermediates (RNI) by macrophages infected with M. tuberculosis or M. avium, whereas both cytokines increased macrophage release of reactive oxygen intermediates (ROI). The present findings therefore show that IFN-γ and TNF-α potentiated the anti-mycobacterial activity of murine peritoneal macrophages in different fashions. They also suggest that RNI played more important roles than did ROI in the expression of macrophage anti-mycobacterial, particularly anti-M. avium, activity.     

小鼠骨髓源性巨噬细胞极化的体外诱导方法探究

目的:探究小鼠骨髓前体细胞体外诱导成为不同极化状态(M1和M2)巨噬细胞的优化方法。方法:健康C57BL/6小鼠麻醉处死,收集其股骨和胫骨腔内容物,经筛网过滤、红细胞裂解后,在RPMI-1640完全培养基中培养16h,收集未贴壁的骨髓前体细胞重新接种于6孔板。根据培养基中所加刺激剂的种类、剂量不同进行实验分组,于不同时点收集细胞,光镜下观察各组细胞形态学变化,流式细胞术及RT-qPCR检测不同极化状态巨噬细胞的相应标志物。结果:(1)小鼠骨髓前体细胞经50μg/L巨噬细胞集落刺激因子(M-CSF)刺激72 h后,CD11b阳染率达到90%以上;刺激96 h后,F4/80的阳染率达到95%以上。40μg/L的粒-巨噬细胞集落刺激因子(GM-CSF)刺激96 h后,CD11b阳染率也达到了90%以上,F4/80阳染率至144 h达到峰值(58.2%);(2)在M-CSF刺激所得单核-巨噬细胞的基础上,给予M1型巨噬细胞诱导剂(25μg/L LPS和10μg/L IFN-γ)刺激24 h,可见CD86的阳染率大于90%;给予M2型巨噬细胞诱导剂(20μg/L IL-4和IL-13)刺激后CD206的阳染率始终处于较低水平(10%左右);(3)在GM-CSF刺激的基础上,给予M1型巨噬细胞诱导剂刺激24 h,可见CD86的阳染率大于90%;而当细胞接受M2型巨噬细胞诱导剂刺激96 h,CD206的阳染率达68.98%;(4)RT-qPCR结果显示在给予相应极化诱导剂刺激后,M1型巨噬细胞标志物诱导型一氧化氮合酶(iNOS)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)、IL-12,以及M2型巨噬细胞标志物:几丁质酶3样蛋白3(Chi3l3/Ym1)、甘露糖受体(MR)和精氨酸酶1(Arg-1)的mRNA表达均明显高于对照组(P<0.01)。结论:(1)C57BL/6小鼠骨髓前体细胞受到M-CSF或GM-CSF诱导后90%以上细胞均可向单核细胞分化,M-CSF可诱导90%以上的细胞为成熟巨噬细胞,GM-CSF可诱导58%的细胞为成熟巨噬细胞;(2)在M-CSF前期诱导的基础上,联合LPS和IFN-γ易于诱导出M1型巨噬细胞,但联合IL-4和IL-13难以获得M2型巨噬细胞;(3)在GM-CSF前期诱导的基础上,联合LPS和IFN-γ易于诱导出M1型巨噬细胞,联合IL-4和IL-13也可将大部分细胞诱导成为M2型巨噬细胞。     

小鼠骨髓源性巨噬细胞极化的体外诱导方法探究

目的:探究小鼠骨髓前体细胞体外诱导成为不同极化状态(M1和M2)巨噬细胞的优化方法。方法:健康C57BL/6小鼠麻醉处死,收集其股骨和胫骨腔内容物,经筛网过滤、红细胞裂解后,在RPMI-1640完全培养基中培养16h,收集未贴壁的骨髓前体细胞重新接种于6孔板。根据培养基中所加刺激剂的种类、剂量不同进行实验分组,于不同时点收集细胞,光镜下观察各组细胞形态学变化,流式细胞术及RT-qPCR检测不同极化状态巨噬细胞的相应标志物。结果:(1)小鼠骨髓前体细胞经50μg/L巨噬细胞集落刺激因子(M-CSF)刺激72 h后,CD11b阳染率达到90%以上;刺激96 h后,F4/80的阳染率达到95%以上。40μg/L的粒-巨噬细胞集落刺激因子(GM-CSF)刺激96 h后,CD11b阳染率也达到了90%以上,F4/80阳染率至144 h达到峰值(58.2%);(2)在M-CSF刺激所得单核-巨噬细胞的基础上,给予M1型巨噬细胞诱导剂(25μg/L LPS和10μg/L IFN-γ)刺激24 h,可见CD86的阳染率大于90%;给予M2型巨噬细胞诱导剂(20μg/L IL-4和IL-13)刺激后CD206的阳染率始终处于较低水平(10%左右);(3)在GM-CSF刺激的基础上,给予M1型巨噬细胞诱导剂刺激24 h,可见CD86的阳染率大于90%;而当细胞接受M2型巨噬细胞诱导剂刺激96 h,CD206的阳染率达68.98%;(4)RT-qPCR结果显示在给予相应极化诱导剂刺激后,M1型巨噬细胞标志物诱导型一氧化氮合酶(iNOS)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)、IL-12,以及M2型巨噬细胞标志物:几丁质酶3样蛋白3(Chi3l3/Ym1)、甘露糖受体(MR)和精氨酸酶1(Arg-1)的mRNA表达均明显高于对照组(P<0.01)。结论:(1)C57BL/6小鼠骨髓前体细胞受到M-CSF或GM-CSF诱导后90%以上细胞均可向单核细胞分化,M-CSF可诱导90%以上的细胞为成熟巨噬细胞,GM-CSF可诱导58%的细胞为成熟巨噬细胞;(2)在M-CSF前期诱导的基础上,联合LPS和IFN-γ易于诱导出M1型巨噬细胞,但联合IL-4和IL-13难以获得M2型巨噬细胞;(3)在GM-CSF前期诱导的基础上,联合LPS和IFN-γ易于诱导出M1型巨噬细胞,联合IL-4和IL-13也可将大部分细胞诱导成为M2型巨噬细胞。     

Macrophage polarization in response to ECM coated polypropylene mesh

The host response to implanted biomaterials is a highly regulated process that influences device functionality and clinical outcome. Non-degradable biomaterials, such as knitted polypropylene mesh, frequently elicit a chronic foreign body reaction with resultant fibrosis. Previous studies have shown that an extracellular matrix (ECM) hydrogel coating of polypropylene mesh reduces the intensity of the foreign body reaction, though the mode of action is unknown. Macrophage participation plays a key role in the development of the foreign body reaction to biomaterials, and therefore the present study investigated macrophage polarization following mesh implantation. Spatiotemporal analysis of macrophage polarization was conducted in response to uncoated polypropylene mesh and mesh coated with hydrated and dry forms of ECM hydrogels derived from either dermis or urinary bladder. Pro-inflammatory M1 macrophages (CD86+/CD68+), alternatively activated M2 macrophages (CD206+/CD68+), and foreign body giant cells were quantified between 3 and 35 days. Uncoated polypropylene mesh elicited a dominant M1 response at the mesh fiber surface, which was decreased by each ECM coating type beginning at 7 days. The diminished M1 response was accompanied by a reduction in the number of foreign body giant cells at 14 and 35 days, though there was a minimal effect upon the number of M2 macrophages at any time. These results show that ECM coatings attenuate the M1 macrophage response and increase the M2/M1 ratio to polypropylene mesh in vivo.     

Effect of interferon-gamma,tumor necrosis factor,interleukin-1 and interleukin-6 on the modulation of anti-tumor responses of bone marrow derived macrophages

Non-adherent bone marrow cells (NABMC) obtained from BALB/c mice were incubated in medium alone or containing granulocyte — macrophage-colony stimulating factor(GM-CSF) or macrophage-colony stimulating factor (M-CSF) for 4 days to obtain bone marrow derived macrophages. Treatment of GM-CSF or M-CSF derived macrophages with interferon-gamma (IFN-γ) (50 U/ml), tumor necrosis factor (TNF) (500 U/ml), interleukin-1 (IL-1) (200 U/ml) or interleukin-6 (IL-6) (100 U/ml) for 24 h rendered them significantly cytotoxic to different tumor cells. These macrophages also produced enhanced amounts of soluble or membrane associated TNF. Medium derived macrophages showed little cytotoxicity against tumor cells and production of TNF on treatment with TNF, IFN-γ, IL-1 or IL-6. M-CSF or GM-CSF derived macrophages on treatment with IFN-γ showed enhanced release of nitrite as compared to medium derived macrophages. TNF, IL-1 or IL-6 did not induce nitrite production in bone marrow derived macrophages. Out of the different combinations tested, only IFN-γ plus TNF-treated macrophages showed enhancement in nitrite production as compared to that of IFN-γ alone.     

IL-15 and GM-CSF stimulated macrophages enhances phagocytic activity in ENU induced leukemic mice

Murine splenic macrophage plays a decisive role in host immunity through phagocytosis against pathogens. It was reported that, macrophages also involves in phagocytosis of some tumour cells upon its activation initiated by certain cytokines produced by other immune cell or by indigenously treated. In this study, we have investigated the killing of leukemic blast cells by macrophages upon stimulated with IL-15 and GM-CSF alone or in combination in ENU challenged leukemic murine model. Along with, the release of TNF-α, IL-12 and IFN-γ by macrophages were assayed by ELISA. NO production by macrophages was also investigated. The molecular expressions like GM-CSF and TLRs were investigated for better understand of macrophage-leukemic cell interaction. Result shows that in disease condition macrophages have poor phagocytic activities which may be due to less release of TNF-α, IL-12 and IFN-γ by macrophages. This impaired phagocytic activity in leukemic mice was increase upon stimulation with IL-15 and GM-CSF.     

IFN-α cannot substitute lack of IFN-γ responsiveness in cells of an IFN-γR1 deficient patient

Patients with complete IFN-γR deficiency are unable to respond to IFN-γ and have impaired Th1-immunity and recurrent, severe infections with weakly virulent Mycobacteria. Since IFN-α and IFN-γ share signalling pathways, treatment with IFN-α has been proposed in complete IFN-γR deficiency. We stimulated cells from healthy controls and from a patient lacking IFN-γR1 with IFN-α and IFN-γ, to establish whether IFN-α would substitute for IFN-γ effects. IFN-α induced STAT1 phosphorylation in monocytes of the IFN-γR1(-/-) patient, but did not prime for LPS-induced IL-12p70, IL-12p40, IL-23 or TNF production. In control cells, IFN-α inhibited the priming effect of IFN-γ on LPS-induced pro-inflammatory cytokine release. Finally, IFN-γ but not IFN-α induced killing of M. smegmatis in cultured macrophages. In conclusion, no evidence was found to support the use of IFN-α in IFN-γR-deficient patients as intervention against mycobacterial infection; on the contrary, treatment of individuals with IFN-α may even adversely affect host defence against Mycobacteria.     

Transient depletion of CD4+ CD25+ regulatory T cells results in multiple autoimmune diseases in wild‐type and B‐cell‐deficient NOD mice

Plasticity is a hallmark of macrophages, and in response to environmental signals these cells undergo different forms of polarized activation, the extremes of which are called classic (M1) and alternative (M2). Rapamycin (RAPA) is crucial for survival and functions of myeloid phagocytes, but its effects on macrophage polarization are not yet studied. To address this issue, human macrophages obtained from six normal blood donors were polarized to M1 or M2 in vitro by lipopolysaccharide plus interferon-γ or interleukin-4 (IL-4), respectively. The presence of RAPA (10 ng/ml) induced macrophage apoptosis in M2 but not in M1. Beyond the impact on survival in M2, RAPA reduced CXCR4, CD206 and CD209 expression and stem cell growth factor-β, CCL18 and CCL13 release. In contrast, in M1 RAPA increased CD86 and CCR7 expression and IL-6, tumour necrosis factor-α and IL-1β release but reduced CD206 and CD209 expression and IL-10, vascular endothelial growth factor and CCL18 release. In view of the in vitro data, we examined the in vivo effect of RAPA monotherapy (0·1 mg/kg/day) in 12 patients who were treated for at least 1 month before islet transplant. Cytokine release by Toll-like receptor 4-stimulated peripheral blood mononuclear cells showed a clear shift to an M1-like profile. Moreover, macrophage polarization 21 days after treatment showed a significant quantitative shift to M1. These results suggest a role of mammalian target of rapamycin (mTOR) into the molecular mechanisms of macrophage polarization and propose new therapeutic strategies for human M2-related diseases through mTOR inhibitor treatment.     

Heme Oxygenase-1 expression in M-CSF-polarized M2 macrophages contributes to LPS-induced IL-10 release

The shift between pro-inflammatory (M1) and anti-inflammatory (M2) states of macrophage polarization allows the resolution of inflammatory processes as well as the maintenance of a basal anti-inflammatory environment in tissues continuously exposed to harmless antigens (e.g., lung and gut). To identify markers for the anti-inflammatory state of macrophages, expression profiling was performed on human macrophages polarized by either GM-CSF or M-CSF, which lead to the generation of TNF-α and IL-12p40-producing pro-inflammatory macrophages [M1 (GM-CSF)] or IL-10-producing anti-inflammatory macrophages [M2 (M-CSF)] upon exposure to LPS, respectively. A different iron metabolism gene signature was detected in both macrophage types, with the heme regulatory molecules CD163 and Heme Oxygenase-1 (HO-1) being preferentially expressed by M2 (M-CSF) macrophages. M1-polarizing cytokines (GM-CSF, IFNγ) inhibited, while IL-4 enhanced, the M-CSF-driven HO-1 expression. In agreement with this in vitro data, HO-1 expression in metastatic melanoma was primarily detected in CD163⁺ tumor-associated macrophages, which are known to exhibit an M2-skewed polarization phenotype. In contrast to the HO-1 inhibitor tin protoporphyrin (SnPP), the administration of cobalt protoporphyrin (CoPP), a potent inducer of HO-1 resulted in increased LPS-triggered IL-10 release from M2 (M-CSF) macrophages. The data suggests that HO-1 is important for the anti-inflammatory activities of M-CSF-polarized M2 macrophages. Moreover, since M2 (M-CSF) macrophages also express higher levels of the CD163 scavenger receptor, the CD163/HO-1/IL-10 axis appears to contribute to the generation of an immunosuppressive environment within the tumor stroma.     

Macrophage polarization in response to wear particles in vitro

Total joint replacement is a highly successful surgical procedure for treatment of patients with disabling arthritis and joint dysfunction. However, over time, with high levels of activity and usage of the joint, implant wear particles are generated from the articulating surfaces. These wear particles can lead to activation of an inflammatory reaction, and subsequent bone resorption around the implant （periprosthetic osteolysis）. Cells of the monocyte/macrophage lineage orchestrate this chronic inflammatory response, which is dominated by a pro-inflammatory （M 1） macrophage phenotype rather than an anti-inflammatory pro-tissue healing （M2） macrophage phenotype. While it has been shown that interleukin-4 （IL-4） selectively polarizes macrophages towards an M2 anti-inflammatory phenotype which promotes bone healing, rather than inflammation, little is known about the time course in which this occurs or conditions in which repolarization through I L-4 is most effective. The goal of this work was to study the time course of murine macrophage polarization and cytokine release in response to challenge with combinations of polymethyl methacrylate （PMMA） particles, lipopolysaccharide （LPS） and IL-4 in vitro. Treatment of particle-challenged monocyte/macrophages with IL-4 led to an initial suppression of pro-inflammatory cytokines and inducible nitric oxide synthase （iNOS） production and subsequent polarization into an M2 anti-inflammatory phenotype. This result was optimized when IL-4 was delivered before PMMA particle challenge, to an M 1 phenotype rather than to uncommitted （MO） macrophages. The effects of this polarization were sustained over a 5-day time course. Polarization of M1 macrophages into an M2 phenotype may be a strategy to mitigate wear particle associated periprosthetic osteolysis.