Markers Of Alternative Macrophage Activation Research Articles

Abstract MP04: Interleukin-33 Drives Cardiac Remodeling And Alters Macrophage Polarization In Doca-salt Hypertension

Interleukin-33 (IL-33) is an alarmin critical for alternative macrophage activation and has been linked to hypertension in humans. The goal of this study was to evaluate whether IL-33 contributes to (deoxycorticosterone acetate) DOCA-salt induced cardiac remodeling and macrophage polarization. C57Bl/6J (WT, n=8) and IL33-deficient (IL-33 KO, n=8) mice underwent uninephrectomy, implantation of a DOCA pellet, and 1% NaCl supplementation of drinking water for three weeks. A control group of WT mice (n=10) underwent a sham procedure and were maintained on normal water. Systolic blood pressure measured by tail cuff showed a significant increase in WT DOCA-salt mice (128±9 mmHg; mean±SD) and IL-33 KO DOCA-salt mice (129±11) compared to WT sham (103±11). Hypertension was accompanied by significantly increased heart to body weight ratios in WT DOCA-salt (7.0±1.0 mg/g) and IL-33 KO DOCA-salt (6.8±0.6) compared to WT sham (5.3±0.3) mice. Glomerular injury was assessed by urine albumin-to-creatinine ratios, which were similarly increased in WT DOCA-salt (0.92±0.4) and IL-33 KO DOCA-salt (0.42±0.09) mice compared to WT controls (0.03±0.003). Echocardiography was performed to assess left ventricle (LV) dimensions and systolic function. IL-33 KO DOCA-salt mice exhibited a significantly higher cardiac output (22±2 mL/min) compared to WT sham (16±3) and WT DOCA-salt (15±3) mice, driven by increased stroke volume. No significant changes in heart rate or ejection fraction were observed between groups. Additionally, IL33-KO DOCA-salt mice had an increased diastolic LV internal dimension (3.2±0.2 mm) compared to WT sham (2.7±0.3) and WT DOCA-salt (2.7±0.3). DOCA-salt induced increased mRNA expression of alternative macrophage activation markers in both LV ( Chil3, Il10) and isolated peritoneal macrophages ( Arg1, Retnla, Tgfb1 ). Peritoneal macrophages deficient in IL-33 expressed less Il10 compared to WT. Cardiac fibrosis evaluated by Masson’s trichrome showed a significant increase in LV collagen deposition in WT DOCA-salt mice (1.09±0.8% trichrome positive) compared to WT sham (0.43±0.3) but no increase in IL-33 KO DOCA-salt mice (0.54±0.3). In conclusion, IL-33 deficiency alters macrophage and cardiac function in the DOCA-salt model of hypertension.

Fli1 Downregulation in Scleroderma Myeloid Cells Has Profibrotic and Proinflammatory Effects.

Scleroderma (SSc) is an autoimmune connective tissue disease characterized by immune dysregulation, vasculopathy, and fibrosis. We have previously demonstrated that low Fli1 expression in SSc fibroblasts and endothelial cells plays an important role in SSc pathogenesis. Cells of myeloid and lymphoid origin also express Fli1 and are dysregulated in patients with SSc, playing key roles in disease pathogenesis. However, the role for immune Fli1 in SSc is not yet clear. Our aim was to elucidate whether Fli1 contributes to the immune dysregulation seen in SSc. Comparison of the expression of Fli1 in monocytes, B- and T-cell fractions of PBMCs isolated from SSc patients and healthy controls (HC), showed an increase in Fli1 levels in monocytes. We used siRNA transfected human myeloid cells and mouse peritoneal macrophages obtained from Fli1flox/floxLysMCre+/+ mice, and found that markers of alternative macrophage activation were increased with Fli1 deletion. Coculture of Fli1-deficient myeloid cells and primary human or mouse fibroblasts resulted in a potent induction of collagen type I, independent of TGFβ upregulation. We next analyzed global gene expression profile in response to Fli1 downregulation, to gain further insight into the molecular mechanisms of this process and to identify differentially expressed genes in myeloid cells. Of relevance to SSc, the top most upregulated pathways were hallmark IFN-γ and IFN-α response. Additionally, several genes previously linked to SSc pathogenesis and fibrosis in general were also induced, including CCL2, CCL7, MMP12, and CXCL10. ANKRD1, a profibrotic transcription co-regulator was the top upregulated gene in our array. Our results show that Fli1-deficient myeloid cells share key features with cells from SSc patients, with higher expression of profibrotic markers and activation of interferon responsive genes, thus suggesting that dysregulation of Fli1 in myeloid cells may contribute to SSc pathogenesis.

Azithromycin Polarizes Macrophages to an M2 Phenotype via Inhibition of the STAT1 and NF-κB Signaling Pathways

Azithromycin is effective at controlling exaggerated inflammation and slowing the long-term decline of lung function in patients with cystic fibrosis. We previously demonstrated that the drug shifts macrophage polarization toward an alternative, anti-inflammatory phenotype. In this study we investigated the immunomodulatory mechanism of azithromycin through its alteration of signaling via the NF-κB and STAT1 pathways. J774 murine macrophages were plated, polarized (with IFN-γ, IL-4/-13, or with azithromycin plus IFN-γ) and stimulated with LPS. The effect of azithromycin on NF-κB and STAT1 signaling mediators was assessed by Western blot, homogeneous time-resolved fluorescence assay, nuclear translocation assay, and immunofluorescence. The drug's effect on gene and protein expression of arginase was evaluated as a marker of alternative macrophage activation. Azithromycin blocked NF-κB activation by decreasing p65 nuclear translocation, although blunting the degradation of IκBα was due, at least in part, to a decrease in IKKβ kinase activity. A direct correlation was observed between increasing azithromycin concentrations and increased IKKβ protein expression. Moreover, incubation with the IKKβ inhibitor IKK16 decreased arginase expression and activity in azithromycin-treated cells but not in cells treated with IL-4 and IL-13. Importantly, azithromycin treatment also decreased STAT1 phosphorylation in a concentration-dependent manner, an effect that was reversed with IKK16 treatment. We conclude that azithromycin anti-inflammatory mechanisms involve inhibition of the STAT1 and NF-κB signaling pathways through the drug's effect on p65 nuclear translocation and IKKβ.

Rac2 is required for alternative macrophage activation and bleomycin induced pulmonary fibrosis; a macrophage autonomous phenotype.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by cellular phenotype alterations and deposition of extracellular matrix proteins. The alternative activation of macrophages in the lungs has been associated as a major factor promoting pulmonary fibrosis, however the mechanisms underlying this phenomenon are poorly understood. In the present study, we have defined a molecular mechanism by which signals transmitted from the extracellular matrix via the α4β1 integrin lead to the activation of Rac2 which regulates alternative macrophage differentiation, a signaling axis within the pulmonary macrophage compartment required for bleomycin induced pulmonary fibrosis. Mice deficient in Rac2 were protected against bleomycin-induced fibrosis and displayed diminished collagen deposition in association with lower expression of alternatively activated profibrotic macrophage markers. We have demonstrated a macrophage autonomous process by which the injection of M2 and not M1 macrophages restored the bleomycin induced pulmonary fibrosis susceptibility in Rac2-/- mice, establishing a critical role for a macrophage Rac2 signaling axis in the regulation of macrophage differentiation and lung fibrosis in vivo. We also demonstrate that markers of alternative macrophage activation are increased in patients with IPF. Taken together, these studies define an important role for an integrin-driven Rac2 signaling axis in macrophages, and reveal that Rac2 activation is required for polarization of macrophages towards a profibrotic phenotype and progression of pulmonary fibrosis in vivo.

Adverse local tissue reaction (ALTR) associated with corrosion products in metal-on-metal and dual modular neck total hip replacements is associated with upregulation of interferon gamma-mediated chemokine signaling.

Adverse local tissue reactions (ALTR) associated with tribocorrosion following total hip arthroplasty (THA) have become a significant clinical concern in recent years. In particular, implants featuring metal-on-metal bearing surfaces and modular femoral stems have been reported to result in elevated rates of ALTR. These tribocorrosion-related tissue reactions are characterized by marked necrosis and lymphocytic infiltration, which contrasts sharply with the macrophagic and foreign body giant cell inflammation associated with polyethylene wear particle induced peri-implant osteolysis. In this study, we characterize tribocorrosion-associated ALTR at a molecular level. Gene expression profiling of peri-implant tissue around failing implants identifies upregulation of numerous inflammatory mediators in ALTR, including several interferon gamma inducible factors, most notably the chemokines MIG/CXCL9 and IP-10/CXCL10. This expression profile is distinct from that associated with polyethylene wear induced osteolysis, which is characterized by induction of markers of alternative macrophage activation, such as chitotriosidase (CHIT-1). Importantly, MIG/CXCL9 and IP-10/CXCL10 are also elevated at the protein level in the synovial fluid and, albeit more moderately, the serum, of ALTR patients, raising the possibility that these factors may serve as circulating biomarkers for the early detection of ALTR in at-risk patients.

IRAK-M Promotes Alternative Macrophage Activation and Fibroproliferation in Bleomycin-Induced Lung Injury

Idiopathic pulmonary fibrosis is a devastating lung disease characterized by inflammation and the development of excessive extracellular matrix deposition. Currently, there are only limited therapeutic intervenes to offer patients diagnosed with pulmonary fibrosis. Although previous studies focused on structural cells in promoting fibrosis, our study assessed the contribution of macrophages. Recently, TLR signaling has been identified as a regulator of pulmonary fibrosis. IL-1R-associated kinase-M (IRAK-M), a MyD88-dependent inhibitor of TLR signaling, suppresses deleterious inflammation, but may paradoxically promote fibrogenesis. Mice deficient in IRAK-M (IRAK-M(-/-)) were protected against bleomycin-induced fibrosis and displayed diminished collagen deposition in association with reduced production of IL-13 compared with wild-type (WT) control mice. Bone marrow chimera experiments indicated that IRAK-M expression by bone marrow-derived cells, rather than structural cells, promoted fibrosis. After bleomycin, WT macrophages displayed an alternatively activated phenotype, whereas IRAK-M(-/-) macrophages displayed higher expression of classically activated macrophage markers. Using an in vitro coculture system, macrophages isolated from in vivo bleomycin-challenged WT, but not IRAK-M(-/-), mice promoted increased collagen and α-smooth muscle actin expression from lung fibroblasts in an IL-13-dependent fashion. Finally, IRAK-M expression is upregulated in peripheral blood cells from idiopathic pulmonary fibrosis patients and correlated with markers of alternative macrophage activation. These data indicate expression of IRAK-M skews lung macrophages toward an alternatively activated profibrotic phenotype, which promotes collagen production, leading to the progression of experimental pulmonary fibrosis.

150: Haematopoietic cell kinase activation promotes IL-11 dependent gastric cancer progression and invasion in mice

Gastric cancer has a high mortality rate associated with its late stage diagnosis. Although Gp130 knock-in mutant mice (gp130 F / F ) develop benign gastric tumours dependent upon IL-11-induced Stat3/mTORC activation with hallmarks of early stage intestinal-type gastric cancer in humans [1] , [2] , there is an urgent need to develop mouse models that mimic the progression from adenoma to carcinoma and metastasis. Macrophages are a major component of the tumour micro-environment, and their increased numbers and alternative activation is commonly associated with tumour progression and metastasis. Haematopoietic Cell Kinase (Hck) is a myeloid-specific Src family kinase that is involved in polarization of alternatively activated macrophages, which correlate with a poor prognosis for human gastric cancer. To establish whether aberrant Hck activation would induce tumourigenesis, Hck mutant mice that exhibit constitutive kinase activation (Hck Up / Up ) were crossed with gp130 F /+ mice that are predisposed to gastric tumour development. Surprisingly, the resulting gp130 F /+ ;Hck Up / Up compound mutants developed tumours similar to gp130[F/F] animals, while gp130 F /+ mice remained tumour-free. Furthermore, tumours in 9 month old gp130 F /+ ;Hck Up / Up mutants showed sub-mucosal invasion, which was not observed in gp130 F / F mice. Western blotting and immunohistochemical analysis revealed a significant increase in tumour cell proliferation in gp130 F /+ ;Hck Up / Up mutants compared to gp130 F / F animals, consistent with an increased abundance of the activated forms of Stat3, Erk, Akt and S6 cytoplasmic signalling proteins. While the total number of macrophages remained unaffected, qPCR analysis of purified tumour-associated macrophages from gp130 F /+ ; Hck Up / Up mice showed a significant increase in markers of alternative macrophage activation, including Il4 , Il13 , Arg1 and Ym1 when compared to cells from gp130 F / F animals. Furthermore, reciprocal adoptive transfer of Hck Up / Up bone-marrow into gp130 mutant recipients was sufficient to increase tumour burden and promote alternative macrophage polarisation. Collectively, our results demonstrate that aberrant Hck activation promotes polarisation towards alternative macrophage activation and confers an invasive gastric cancer phenotype.

Acute chlorine gas exposure produces transient inflammation and a progressive alteration in surfactant composition with accompanying mechanical dysfunction

Acute Cl2 exposure following industrial accidents or military/terrorist activity causes pulmonary injury and severe acute respiratory distress. Prior studies suggest that antioxidant depletion is important in producing dysfunction, however a pathophysiologic mechanism has not been elucidated. We propose that acute Cl2 inhalation leads to oxidative modification of lung lining fluid, producing surfactant inactivation, inflammation and mechanical respiratory dysfunction at the organ level. C57BL/6J mice underwent whole-body exposure to an effective 60ppm-hour Cl2 dose, and were euthanized 3, 24 and 48h later. Whereas pulmonary architecture and endothelial barrier function were preserved, transient neutrophilia, peaking at 24h, was noted. Increased expression of ARG1, CCL2, RETLNA, IL-1b, and PTGS2 genes was observed in bronchoalveolar lavage (BAL) cells with peak change in all genes at 24h. Cl2 exposure had no effect on NOS2 mRNA or iNOS protein expression, nor on BAL NO3− or NO2−. Expression of the alternative macrophage activation markers, Relm-α and mannose receptor was increased in alveolar macrophages and pulmonary epithelium. Capillary surfactometry demonstrated impaired surfactant function, and altered BAL phospholipid and surfactant protein content following exposure. Organ level respiratory function was assessed by forced oscillation technique at 5 end expiratory pressures. Cl2 exposure had no significant effect on either airway or tissue resistance. Pulmonary elastance was elevated with time following exposure and demonstrated PEEP refractory derecruitment at 48h, despite waning inflammation. These data support a role for surfactant inactivation as a physiologic mechanism underlying respiratory dysfunction following Cl2 inhalation.

Human Female Genital Tract Infection by the Obligate Intracellular Bacterium Chlamydia trachomatis Elicits Robust Type 2 Immunity

While Chlamydia trachomatis infections are frequently asymptomatic, mechanisms that regulate host response to this intracellular Gram-negative bacterium remain undefined. This investigation thus used peripheral blood mononuclear cells and endometrial tissue from women with or without Chlamydia genital tract infection to better define this response. Initial genome-wide microarray analysis revealed highly elevated expression of matrix metalloproteinase 10 and other molecules characteristic of Type 2 immunity (e.g., fibrosis and wound repair) in Chlamydia-infected tissue. This result was corroborated in flow cytometry and immunohistochemistry studies that showed extant upper genital tract Chlamydia infection was associated with increased co-expression of CD200 receptor and CD206 (markers of alternative macrophage activation) by endometrial macrophages as well as increased expression of GATA-3 (the transcription factor regulating TH2 differentiation) by endometrial CD4+ T cells. Also among women with genital tract Chlamydia infection, peripheral CD3+ CD4+ and CD3+ CD4- cells that proliferated in response to ex vivo stimulation with inactivated chlamydial antigen secreted significantly more interleukin (IL)-4 than tumor necrosis factor, interferon-γ, or IL-17; findings that repeated in T cells isolated from these same women 1 and 4 months after infection had been eradicated. Our results thus newly reveal that genital infection by an obligate intracellular bacterium induces polarization towards Type 2 immunity, including Chlamydia-specific TH2 development. Based on these findings, we now speculate that Type 2 immunity was selected by evolution as the host response to C. trachomatis in the human female genital tract to control infection and minimize immunopathological damage to vital reproductive structures.

Role of Galectin-3 in Classical and Alternative Macrophage Activation in the Liver following Acetaminophen Intoxication

Inflammatory macrophages have been implicated in hepatotoxicity induced by the analgesic acetaminophen (APAP). In these studies, we characterized the phenotype of macrophages accumulating in the liver following APAP intoxication and evaluated the role of galectin-3 (Gal-3) in macrophage activation. Administration of APAP (300 mg/kg, i.p.) to wild-type mice resulted in the appearance of two distinct subpopulations of CD11b(+) cells in the liver, which expressed high or low levels of the monocyte/macrophage activation marker Ly6C. Whereas CD11b(+)/Ly6C(hi) macrophages exhibited a classically activated proinflammatory phenotype characterized by increased expression of TNF-α, inducible NO synthase, and CCR2, CD11b(+)/Ly6C(lo) macrophages were alternatively activated, expressing high levels of the anti-inflammatory cytokine IL-10. APAP intoxication was also associated with an accumulation of Gal-3(+) macrophages in the liver; the majority of these cells were Ly6C(hi). APAP-induced increases in CD11b(+)/Ly6C(hi) macrophages were significantly reduced in Gal-3(-/-) mice. This reduction was evident 72 h post APAP and was correlated with decreased expression of the classical macrophage activation markers, inducible NO synthase, IL-12, and TNF-α, as well as the proinflammatory chemokines CCL2 and CCL3, and chemokine receptors CCR1 and CCR2. Conversely, numbers of CD11b(+)/Ly6C(lo) macrophages increased in livers of APAP-treated Gal-3(-/-) mice; this was associated with increased expression of the alternative macrophage activation markers Ym1 and Fizz1, increased liver repair, and reduced hepatotoxicity. These data demonstrate that both classically and alternatively activated macrophages accumulate in the liver following APAP intoxication; moreover, Gal-3 plays a role in promoting a persistent proinflammatory macrophage phenotype.

Schistosoma mansoni Hemozoin Modulates Alternative Activation of Macrophages via Specific Suppression of Retnla Expression and Secretion

The trematode Schistosoma mansoni is one of the etiological agents of schistosomiasis, a key neglected tropical disease responsible for an estimated annual loss of 70 million disability-adjusted life years. Hematophagy represents the primary nutrient acquisition pathway of this parasite, but digestion of hemoglobin also liberates toxic heme. Schistosomes detoxify heme via crystallization into hemozoin, which is subsequently regurgitated into the host's circulation. Here we demonstrate that during experimental schistosomiasis, hemozoin accumulating in the mouse liver is taken up by phagocytes at a time coincident with the development of the egg-induced T-helper 2 (Th2) granulomatous immune response. Furthermore, the uptake of hemozoin also coincides with the hepatic expression of markers of alternative macrophage activation. Alternatively activated macrophages are a key effector cell population associated with protection against schistosomiasis, making hemozoin well placed to play an important immunomodulatory role in this disease. To systematically explore this hypothesis, S. mansoni hemozoin was purified and added to in vitro bone marrow-derived macrophage cultures concurrently exposed to cytokines chosen to reflect the shifting state of macrophage activation in vivo. Macrophages undergoing interleukin-4 (IL-4)-induced alternative activation in the presence of hemozoin developed a phenotype specifically lacking in Retnla, a characteristic alternatively activated macrophage product associated with regulation of Th2 inflammatory responses. As such, in addition to its important detoxification role during hematophagy, we propose that schistosome hemozoin also provides a potent immunomodulatory function in the coevolved network of host-parasite relationships during schistosomiasis.

Human Amniotic Epithelial Cell Transplantation Induces Markers of Alternative Macrophage Activation and Reduces Established Hepatic Fibrosis

Chronic hepatic inflammation from multiple etiologies leads to a fibrogenic response that can progress to cirrhosis and liver failure. Transplantation of human amniotic epithelial cells (hAEC) from term delivered placenta has been shown to decrease mild to moderate hepatic fibrosis in a murine model. To model advanced human liver disease and assess the efficacy of hAEC therapy, we transplanted hAEC in mice with advanced hepatic fibrosis. Immunocompetent C57BL/6 mice were administered carbon tetrachloride (CCl4) twice weekly resulting in bridging fibrosis by 12 weeks. hAEC (2×106) were infused via the tail vein at week 8 or weeks 8 and 10 (single and double dose, respectively). Human cells were detected in mouse liver four weeks after transplantation showing hAEC engraftment. CCl4 treated mice receiving single or double hAEC doses showed a significant but similar decrease in liver fibrosis area associated with decreased activation of collagen-producing hepatic stellate cells and decreased hepatic protein levels of the pro-fibrogenic cytokine, transforming growth factor-beta1. CCl4 administration caused hepatic T cell infiltration that decreased significantly following hAEC transplantation. Hepatic macrophages play a crucial role in both fibrogenesis and fibrosis resolution. Mice exposed to CCl4 demonstrated increased numbers of hepatic macrophages compared to normal mice; the number of macrophages decreased significantly in CCl4 treated mice given hAEC. These mice had significantly lower hepatic protein levels of the chemokine monocyte chemoattractant protein-1 than mice given CCl4 alone. Alternatively activated M2 macrophages are associated with fibrosis resolution. CCl4 treated mice given hAEC showed increased expression of genes associated with M2 macrophages including YM-1, IL-10 and CD206. We provide novel data showing that hAEC transplantation induces a wound healing M2 macrophage phenotype associated with reduction of established hepatic fibrosis that justifies further investigation of this potential cell-based therapy for advanced hepatic fibrosis.

Tubular Atrophy and Interstitial Fibrosis After Renal Transplantation Is Dependent on Galectin-3

Chronic allograft injury (CAI), characterized by interstitial fibrosis and tubular atrophy, leads to a progressive decline in graft function, resulting in the loss of 5% of renal transplants per annum, and eludes specific therapies. Galectin-3 (gal-3) is a β-galactoside-binding lectin expressed in diverse fibrotic tissue, and mice deficient in gal-3 have reduced fibrosis in kidney, liver, and lung models. The role of gal-3 in CAI is examined in this study. We adopted a murine model of CAI, characterized by a single class II mismatch between BM12 donor and C57BL/6 recipient strains. Syngeneic transplants served as controls (C57BL/6). Transplants were then performed between BM12 donors and gal-3 null recipients on a C57BL/6 background. Transplantation of BM12 kidneys into C57BL6 mice was associated with interstitial fibrosis (P<0.0001), tubular atrophy (P<0.0001), and upregulation in gal-3 expression (P=0.002), compared with syngeneic controls. Transplanting BM12 kidneys into gal-3 null mice resulted in significant preservation of tubules (P=0.008) and reduced interstitial fibrosis (P=0.01), with decreased myofibroblast activation (P=0.01) and collagen I expression (P=0.04), compared with wild type controls. The number of infiltrating leukocytes was unaltered by abrogation of gal-3, but reduced expression of YM1 (P=0.0001), a marker of alternative macrophage activation, along with a reduction in the number of circulating CD4-positive T cells (P=0.01), and reduced expression of interleukin-4 (P=0.02) in gal-3 null mice suggest possible mechanisms by which gal-3 may promote renal transplant fibrosis. Our results suggest a potential role for gal-3 in CAI, and this represents a potentially exciting therapeutic target.

S125 Ly6Chi circulating monocytes direct alternatively activated, pro-fibrotic, lung macrophage regulation of pulmonary fibrosis

Introduction and objectivesIdiopathic pulmonary fibrosis (IPF) remains one of the few respiratory conditions for which there are no effective therapies. The role of monocytes and macrophages in IPF has been...

Src-family tyrosine kinases regulate alveolar macrophage alternative activation during Pneumocystis murina lung infection (37.28)

Abstract We have previously shown that mice triple-deficient in the Src-family tyrosine kinases (SFKs) Hck, Fgr and Lyn (SrcTKO mice) have augmented innate lung clearance of P.murina as a result of elevated proinflammatory cytokine (e.g. IL-1β) and chemokine (e.g. CCL2/MCP-1) levels and increased inflammatory cell recruitment. Augmented clearance in SrcTKO mice correlates with a higher ability of alveolar macrophages (AMs) from these mice to kill P.murina in vitro, though this enhancement is not associated with elevated AM inflammatory responses. Since SrcTKO AMs are not hyper-inflammatory yet kill more efficiently, we hypothesized that they have a different activation pattern compared to WT. We show that AMs and whole lungs from P.murina-infected SrcTKO mice express higher mRNA and protein levels of the alternative macrophage activation markers RELM-α and Arg-1 compared to WT. There is surprisingly no difference in the levels of the pro-alternative activation cytokines IL-4 and IL-13 or other cytokines known to modulate AM activation, e.g. IFN-γ or GM-CSF. However, infected SrcTKO mice have higher lung levels of M-CSF, CCL17/TARC and the IL-4/IL-13 receptor, IL4ra. Treatment of WT AMs with IL-4 or IL-13 enhances their ability to kill P.murina, while a blockade of IL4ra or inhibition of phagolysosome maturation inhibits killing. These results indicate that SFKs regulate AM activation during P.murina infection, and that alternatively activated AMs are better equipped to kill P.murina.

Azithromycin Alters Macrophage Phenotype and Pulmonary Compartmentalization during Lung Infection with Pseudomonas

Infection with mucoid strains of Pseudomonas aeruginosa in chronic inflammatory diseases of the airway is difficult to eradicate and can cause excessive inflammation. The roles of alternatively activated and regulatory subsets of macrophages in this pathophysiological process are not well characterized. We previously demonstrated that azithromycin induces an alternatively activated macrophage-like phenotype in vitro. In the present study, we tested whether azithromycin affects the macrophage activation status and migration in the lungs of P. aeruginosa-infected mice. C57BL/6 mice received daily doses of oral azithromycin and were infected intratracheally with a mucoid strain of P. aeruginosa. The properties of macrophage activation, immune cell infiltration, and markers of pulmonary inflammation in the lung interstitial and alveolar compartments were evaluated postinfection. Markers of alternative macrophage activation were induced by azithromycin treatment, including the surface expression of the mannose receptor, the upregulation of arginase 1, and a decrease in the production of proinflammatory cytokines. Additionally, azithromycin increased the number of CD11b(+) monocytes and CD4(+) T cells that infiltrated the alveolar compartment. A predominant subset of CD11b(+) cells was Gr-1 positive (Gr-1(+)), indicative of a subset of cells that has been shown to be immunoregulatory. These differences corresponded to decreases in neutrophil influx into the lung parenchyma and alteration of the characteristics of peribronchiolar inflammation without any change in the clearance of the organism. These results suggest that the immunomodulatory effects of azithromycin are associated with the induction of alternative and regulatory macrophage activation characteristics and alteration of cellular compartmentalization during infection.

Regulation of Alternative Macrophage Activation by Galectin-3

Alternative macrophage activation is implicated in diverse disease pathologies such as asthma, organ fibrosis, and granulomatous diseases, but the mechanisms underlying macrophage programming are not fully understood. Galectin-3 is a carbohydrate-binding lectin present on macrophages. We show that disruption of the galectin-3 gene in 129sv mice specifically restrains IL-4/IL-13-induced alternative macrophage activation in bone marrow-derived macrophages in vitro and in resident lung and recruited peritoneal macrophages in vivo without affecting IFN-gamma/LPS-induced classical activation or IL-10-induced deactivation. IL-4-mediated alternative macrophage activation is inhibited by siRNA-targeted deletion of galectin-3 or its membrane receptor CD98 and by inhibition of PI3K. Increased galectin-3 expression and secretion is a feature of alternative macrophage activation. IL-4 stimulates galectin-3 expression and release in parallel with other phenotypic markers of alternative macrophage activation. By contrast, classical macrophage activation with LPS inhibits galectin-3 expression and release. Galectin-3 binds to CD98, and exogenous galectin-3 or cross-linking CD98 with the mAb 4F2 stimulates PI3K activation and alternative activation. IL-4-induced alternative activation is blocked by bis-(3-deoxy-3-(3-methoxybenzamido)-beta-D-galactopyranosyl) sulfane, a specific inhibitor of extracellular galectin-3 carbohydrate binding. These results demonstrate that a galectin-3 feedback loop drives alternative macrophage activation. Pharmacological modulation of galectin-3 function represents a novel therapeutic strategy in pathologies associated with alternatively activated macrophages.

Expression profiling reveals alternative macrophage activation and impaired osteogenesis in periprosthetic osteolysis

Interactions between periprosthetic cells and prosthetic wear debris have been recognized as an important event in the development of osteolysis and aseptic loosening. Although the ability of wear debris to activate pro-inflammatory macrophage signaling has been documented, the full repertoire of macrophage responses to wear particles has not been established. Here, we examined the involvement of alternative macrophage activation and defective osteogenic signaling in osteolysis. Using real-time RT-PCR analysis of periprosthetic soft tissue from osteolysis patients, we detected elevated levels of expression of alternative macrophage activation markers (CHIT1, CCL18), chemokines (IL8, MIP1 alpha) and markers of osteoclast precursor cell differentiation and multinucleation (Cathepsin K, TRAP, DC-STAMP) relative to osteoarthritis controls. The presence of cathepsin K positive multinuclear cells was confirmed by immunohistochemistry. Reduced expression levels of the osteogenic signaling components BMP4 and FGF18 were detected. Expression levels of TNF-alpha, IL-6, and RANKL were unchanged, while the anti-osteoclastogenic cytokine OPG was reduced in osteolysis patients, resulting in elevated RANKL:OPG ratios. In vitro studies confirmed the role of particulate debris in alternative macrophage activation and inhibition of osteogenic signaling. Taken together, these results suggest involvement in osteolysis of alternative macrophage activation, accompanied by elevated levels of various chemokines. Increased recruitment and maturation of osteoclast precursors is also observed, as is reduced osteogenesis. These findings provide new insights into the molecular pathogenesis of osteolysis, and identify new potential candidate markers for disease progression and therapeutic targeting.

Identification of factor XIII-A as a marker of alternative macrophage activation.

Factor XIII subunit A of blood coagulation (FXIII-A) is known to be synthesized but not secreted by the monocyte/macrophage cell line. On the basis of its intracellular localization and substrate profile, FXIII-A is thought to be involved in certain intracellular processes. Our present study was designed to monitor the changes in FXIII-A gene expression and protein production in long-term culture of human monocytes during their differentiation into macrophages in the presence of activating agents (interleukin-4, interferon-gamma, Mycobacterium bovis BCG) inducing classical and alternative activation pathways. By using quantitative RT-PCR and fluorescent image analysis at the single-cell level we demonstrated that the expression of FXIII-A both at the mRNA as well as at the protein level is inversely regulated during the two activation programmes. Here we conclude that FXIII-A expression is an intracellular marker for alternatively activated macrophages, while its absence in monocyte-derived macrophages indicates their classically activated state.