Mer-mediated eosinophil efferocytosis regulates resolution of allergic airway inflammation.

BACKGROUND: Eosinophils play a central role in propagation of allergic diseases, including asthma. Both recruitment and retention of eosinophils regulate pulmonary eosinophilia, but the question of whether alterations in apoptotic cell clearance by phagocytes contributes directly to resolution of allergic airway inflammation remains unexplored. OBJECTIVES: In this study we investigated the role of the receptor tyrosine kinase Mer in mediating apoptotic eosinophil clearance and allergic airway inflammation resolution in vivo to establish whether apoptotic cell clearance directly affects the resolution of allergic airway inflammation. METHODS: Alveolar and bone marrow macrophages were used to study Mer-mediated phagocytosis of apoptotic eosinophils. Allergic airway inflammation resolution was modeled in mice by using ovalbumin. Fluorescently labeled apoptotic cells were administered intratracheally or eosinophil apoptosis was driven by administration of dexamethasone to determine apoptotic cell clearance in vivo. RESULTS: Inhibition or absence of Mer impaired phagocytosis of apoptotic human and mouse eosinophils by macrophages. Mer-deficient mice showed delayed resolution of ovalbumin-induced allergic airway inflammation, together with increased airway responsiveness to aerosolized methacholine, increased bronchoalveolar lavage fluid protein levels, altered cytokine production, and an excess of uncleared dying eosinophils after dexamethasone treatment. Alveolar macrophage phagocytosis was significantly Mer dependent, with the absence of Mer attenuating apoptotic cell clearance in vivo to enhance inflammation in response to apoptotic cells. CONCLUSIONS: We demonstrate that Mer-mediated apoptotic cell clearance by phagocytes contributes to resolution of allergic airway inflammation, suggesting that augmenting apoptotic cell clearance is a potential therapeutic strategy for treating allergic airway inflammation.

11ß-hydroxysteroid dehydrogenase-1 deficiency alters brain energy metabolism in acute systemic inflammation.

Chronically elevated glucocorticoid levels impair cognition and are pro-inflammatory in the brain. Deficiency or inhibition of 11ß-hydroxysteroid dehydrogenase type-1 (11ß-HSD1), which converts inactive into active glucocorticoids, protects against glucocorticoid-associated chronic stress- or age-related cognitive impairment. Here, we hypothesised that 11ß-HSD1 deficiency attenuates the brain cytokine response to inflammation. Because inflammation is associated with altered energy metabolism, we also examined the effects of 11ß-HSD1 deficiency upon hippocampal energy metabolism. Inflammation was induced in 11ß-HSD1 deficient (Hsd11b1Del/Del) and C57BL/6 control mice by intraperitoneal injection of lipopolysaccharide (LPS). LPS reduced circulating neutrophil and monocyte numbers and increased plasma corticosterone levels equally in C57BL/6 and Hsd11b1Del/Del mice, suggesting a similar peripheral inflammatory response. However, the induction of pro-inflammatory cytokine mRNAs in the hippocampus was attenuated in Hsd11b1Del/Del mice. Principal component analysis of mRNA expression revealed a distinct metabolic response to LPS in hippocampus of Hsd11b1Del/Del mice. Expression of Pfkfb3 and Ldha, key contributors to the Warburg effect, showed greater induction in Hsd11b1Del/Del mice. Consistent with increased glycolytic flux, levels of 3-phosphoglyceraldehyde and dihydroxyacetone phosphate were reduced in hippocampus of LPS injected Hsd11b1Del/Del mice. Expression of Sdha and Sdhb, encoding subunits of succinate dehydrogenase/complex II that determines mitochondrial reserve respiratory capacity, was induced specifically in hippocampus of LPS injected Hsd11b1Del/Del mice, together with increased levels of its product, fumarate. These data suggest 11ß-HSD1 deficiency attenuates the hippocampal pro-inflammatory response to LPS, associated with increased capacity for aerobic glycolysis and mitochondrial ATP generation. This may provide better metabolic support and be neuroprotective during systemic inflammation or aging.

The cyclin-dependent kinase inhibitor AT7519 accelerates neutrophil apoptosis in sepsis-related acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a neutrophil-dominant disorder with no effective pharmacological therapies. While the cyclin-dependent kinase inhibitor AT7519 induces neutrophil apoptosis to promote inflammation resolution in preclinical models of lung inflammation, its potential efficacy in ARDS has not been examined. Untreated peripheral blood sepsis-related ARDS neutrophils demonstrated prolonged survival after 20 hours in vitro culture. AT7519 was able to override this phenotype to induce apoptosis in ARDS neutrophils with reduced expression of the pro-survival protein Mcl-1. We demonstrate the first pharmacological compound to induce neutrophil apoptosis in sepsis-related ARDS, highlighting cyclin-dependent kinase inhibitors as potential novel therapeutic agents.

Novel role for endogenous mitochondrial formylated peptide-driven formyl peptide receptor 1 signalling in acute respiratory distress syndrome.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is an often fatal neutrophil-dominant lung disease. Although influenced by multiple proinflammatory mediators, identification of suitable therapeutic candidates remains elusive. We aimed to delineate the presence of mitochondrial formylated peptides in ARDS and characterise the functional importance of formyl peptide receptor 1 (FPR1) signalling in sterile lung inflammation. METHODS: Mitochondrial formylated peptides were identified in bronchoalveolar lavage fluid (BALF) and serum of patients with ARDS by liquid chromatography-tandem mass spectrometry. In vitro, human neutrophils were stimulated with mitochondrial formylated peptides and their effects assessed by flow cytometry and chemotaxis assay. Mouse lung injury was induced by mitochondrial formylated peptides or hydrochloric acid. Bone marrow chimeras determined the contribution of myeloid and parenchymal FPR1 to sterile lung inflammation. RESULTS: Mitochondrial formylated peptides were elevated in BALF and serum from patients with ARDS. These peptides drove neutrophil activation and chemotaxis through FPR1-dependent mechanisms in vitro and in vivo. In mouse lung injury, inflammation was attenuated in Fpr1-/- mice, effects recapitulated by a pharmacological FPR1 antagonist even when administered after the onset of injury. FPR1 expression was present in alveolar epithelium and chimeric mice demonstrated that both myeloid and parenchymal FPR1 contributed to lung inflammation. CONCLUSIONS: We provide the first definitive evidence of mitochondrial formylated peptides in human disease and demonstrate them to be elevated in ARDS and important in a mouse model of lung injury. This work reveals mitochondrial formylated peptide FPR1 signalling as a key driver of sterile acute lung injury and a potential therapeutic target in ARDS.

11ß-hydroxysteroid dehydrogenase type 1 deficiency in bone marrow-derived cells reduces atherosclerosis.

11ß-Hydroxysteroid dehydrogenase type-1 (11ß-HSD1) converts inert cortisone into active cortisol, amplifying intracellular glucocorticoid action. 11ß-HSD1 deficiency improves cardiovascular risk factors in obesity but exacerbates acute inflammation. To determine the effects of 11ß-HSD1 deficiency on atherosclerosis and its inflammation, atherosclerosis-prone apolipoprotein E-knockout (ApoE-KO) mice were treated with a selective 11ß-HSD1 inhibitor or crossed with 11ß-HSD1-KO mice to generate double knockouts (DKOs) and challenged with an atherogenic Western diet. 11ß-HSD1 inhibition or deficiency attenuated atherosclerosis (74-76%) without deleterious effects on plaque structure. This occurred without affecting plasma lipids or glucose, suggesting independence from classical metabolic risk factors. KO plaques were not more inflamed and indeed had 36% less T-cell infiltration, associated with 38% reduced circulating monocyte chemoattractant protein-1 (MCP-1) and 36% lower lesional vascular cell adhesion molecule-1 (VCAM-1). Bone marrow (BM) cells are key to the atheroprotection, since transplantation of DKO BM to irradiated ApoE-KO mice reduced atherosclerosis by 51%. 11ß-HSD1-null macrophages show 76% enhanced cholesterol ester export. Thus, 11ß-HSD1 deficiency reduces atherosclerosis without exaggerated lesional inflammation independent of metabolic risk factors. Selective 11ß-HSD1 inhibitors promise novel antiatherosclerosis effects over and above their benefits for metabolic risk factors via effects on BM cells, plausibly macrophages.

Modulation of 11ß-hydroxysteroid dehydrogenase as a strategy to reduce vascular inflammation.

Atherosclerosis is a chronic inflammatory disease in which initial vascular damage leads to extensive macrophage and lymphocyte infiltration. Although acutely glucocorticoids suppress inflammation, chronic glucocorticoid excess worsens atherosclerosis, possibly by exacerbating systemic cardiovascular risk factors. However, glucocorticoid action within the lesion may reduce neointimal proliferation and inflammation. Glucocorticoid levels within cells do not necessarily reflect circulating levels due to pre-receptor metabolism by 11ß-hydroxysteroid dehydrogenases (11ß-HSDs). 11ß-HSD2 converts active glucocorticoids into inert 11-keto forms. 11ß-HSD1 catalyses the reverse reaction, regenerating active glucocorticoids. 11ß-HSD2-deficiency/inhibition causes hypertension, whereas deficiency/inhibition of 11ß-HSD1 generates a cardioprotective lipid profile and improves glycemic control. Importantly, 11ß-HSD1-deficiency/inhibition is atheroprotective, whereas 11ß-HSD2-deficiency accelerates atherosclerosis. These effects are largely independent of systemic risk factors, reflecting modulation of glucocorticoid action and inflammation within the vasculature. Here, we consider whether evidence linking the 11ß-HSDs to vascular inflammation suggests these isozymes are potential therapeutic targets in vascular injury and atherosclerosis.

Macrophage/monocyte depletion by clodronate, but not diphtheria toxin, improves renal ischemia/reperfusion injury in mice.

The role of resident renal mononuclear phagocytes in acute kidney injury is controversial with experimental data suggesting both deleterious and protective functions. To help resolve this, we used mice transgenic for the human diphtheria toxin receptor under the control of the CD11b promoter and treated them with diphtheria toxin, or liposomal clodronate, or both to deplete monocyte/mononuclear phagocytes prior to renal ischemia/reperfusion injury. Although either system effectively depleted circulating monocytes and resident mononuclear phagocytes, depletion was most marked in diphtheria toxin-treated mice. Despite this, diphtheria toxin treatment did not protect from renal ischemia. In contrast, mice treated with clodronate exhibited reduced renal failure and acute tubular necrosis, suggesting key differences between these depletion strategies. Clodronate did not deplete CD206-positive renal macrophages and, unlike diphtheria toxin, left resident CD11c-positive cells unscathed while inducing dramatic apoptosis in hepatic and splenic mononuclear phagocyte populations. Abolition of the protected phenotype by administration of diphtheria toxin to clodronate-treated mice suggested that the protective effect of clodronate resulted from the presence of a cytoprotective intrarenal population of mononuclear phagocytes sensitive to diphtheria toxin-mediated ablation.

Inhibition of Cyclin-Dependent Kinase 9 Downregulates Cytokine Production Without Detrimentally Affecting Human Monocyte-Derived Macrophage Viability.

Cyclin-dependent kinase (CDK) inhibitor drugs (CDKi), such as R-roscovitine and AT7519, induce neutrophil apoptosis in vitro and enhance the resolution of inflammation in a number of in vivo models. This class of compounds are potential novel therapeutic agents that could promote the resolution of acute and chronic inflammatory conditions where neutrophil activation contributes to tissue damage and aberrant tissue repair. In this study we investigated CDKi effects on macrophage pro-inflammatory mediator production and viability. Treatment of human monocyte-derived macrophages (MDMs) with the CDKi AT7519 and R-roscovitine at concentrations that induce neutrophil apoptosis had no significant effect on control or LPS-activated MDM apoptosis and viability, and did not detrimentally affect MDM efferocytosis of apoptotic cells. In addition, enhanced efferocytosis, induced by the glucocorticoid dexamethasone, was also unaffected after a short time treatment with R-roscovitine. Macrophage cytokine responses to inflammatory stimuli are also of importance during inflammation and resolution. As a key target of CDKi, CDK9, is involved in protein transcription via the RNA polymerase II complex, we investigated the effect of CDKi drugs on cytokine production. Our data show that treatment with AT7519 significantly downregulated expression and release of key MDM cytokines IL-6, TNF, IL-10 and IL-1ß, as well as markers of pro-inflammatory macrophage polarisation. R-Roscovitine was also able to downregulate inflammatory cytokine protein secretion from MDMs. Using siRNA transfection, we demonstrate that genetic knock-down of CDK9 replicates these findings, reducing expression and release of pro-inflammatory cytokines. Furthermore, overexpression of CDK9 in THP-1 cells can promote a pro-inflammatory phenotype in these cells, suggesting that CDK9 plays an important role in the inflammatory phenotype of macrophages. Overall, this study demonstrates that pharmacological and genetic targeting of CDK9 inhibits an inflammatory phenotype in human MDMs. As such these data indicate that CDK9 may be key to therapeutically targeting pro-inflammatory macrophage functions during chronic inflammation.

Conditional ablation of macrophages disrupts ovarian vasculature.

Macrophages are the most abundant immune cell within the ovary. Their dynamic distribution throughout the ovarian cycle and heterogenic array of functions suggest the involvement in various ovarian processes, but their functional role has yet to be fully established. The aim was to induce conditional macrophage ablation to elucidate the putative role of macrophages in maintaining the integrity of ovarian vasculature. Using the CD11b-diphtheria toxin receptor (DTR) mouse, in which expression of human DTR is under the control of the macrophage-specific promoter sequence CD11b, ovarian macrophages were specifically ablated in adult females by injections of diphtheria toxin (DT). CD11b-DTR mice were given DT treatment or vehicle and ovaries collected at 2, 8, 16, 24 and 48  h. Histochemical stains were employed to characterise morphological changes, immunohistochemistry for F4/80 to identify macrophages and the endothelial cell marker CD31 used to quantify vascular changes. In normal ovaries, macrophages were detected in corpora lutea and in the theca layer of healthy and atretic follicles. As macrophage ablation progressed, increasing amounts of ovarian haemorrhage were observed affecting both luteal and thecal tissue associated with significant endothelial cell depletion, increased erythrocyte accumulation and increased follicular atresia by 16  h. These events were followed by necrosis and profound structural damage. Changes were limited to the ovary, as DT treatment does not disrupt the vasculature of other tissues likely reflecting the unique cyclical nature of the ovarian vasculature and heterogeneity between macrophages within different tissues. These results show that macrophages play a critical role in maintaining ovarian vascular integrity.

Peritubular capillary rarefaction and lymphangiogenesis in chronic allograft failure.

BACKGROUND: Chronic renal allograft failure is a common and multifactorial but incompletely understood process with no effective treatment strategy. METHODS: We used immunohistochemistry to evaluate changes in density and turnover (proliferation) of the microvasculature and lymphatic vessels in endstage human transplant nephrectomies and control tissue derived from macroscopically normal areas of native nephrectomy specimens removed for renal carcinoma. We also examined the expression of angiogenic and lymphangiogenic growth factors in the associated inflammatory infiltrate. RESULTS: Endstage allografts showed reduced microvascular density in cortex and medulla compared with controls (P<0.0001), despite the presence of endothelial cell proliferation. However, the grafts also showed new lymphatic vessels in the tubulointerstitium, not evident in controls, and which appeared to be functional with luminal macrophages. Double labeling studies showed a subpopulation of the graft-infiltrating macrophages to be immunopositive for inducible nitric oxide synthase or vascular endothelial growth factor-C (a lymphatic-specific growth factor). B cells also strongly expressed the inflammatory and angiogenic cytokine vascular endothelial growth factor A. CONCLUSIONS: The present results identify contrasting changes in the microanatomy of vascular and lymphatic beds in endstage renal allografts associated with subpopulations of infiltrating macrophages and B cells that potentially regulate some of these changes. These cells and processes could become a new therapeutic target in chronic allograft failure.

Macrophage 11ß-HSD-1 deficiency promotes inflammatory angiogenesis.

11ß-Hydroxysteroid dehydrogenase-1 (11ß-HSD1) predominantly converts inert glucocorticoids into active forms, thereby contributing to intracellular glucocorticoid levels. 11ß-HSD1 is dynamically regulated during inflammation, including in macrophages where it regulates phagocytic capacity. The resolution of inflammation in some disease models including inflammatory arthritis is impaired by 11ß-HSD1 deficiency or inhibition. However, 11ß-HSD1 deficiency/inhibition also promotes angiogenesis, which is beneficial in mouse models of surgical wound healing, myocardial infarction or obesity. The cell types responsible for the anti-inflammatory and anti-angiogenic roles of 11ß-HSD1 have not been characterised. Here, we generated Hsd11b1MKO mice with LysM-Cre mediated deletion of Hsd11b1 to investigate whether 11ß-HSD1 deficiency in myeloid phagocytes is pro-angiogenic and/or affects the resolution of inflammation. Resolution of inflammatory K/BxN-induced arthritis was impaired in Hsd11b1MKO mice to a similar extent as in mice globally deficient in 11ß-HSD1. This was associated with >2-fold elevation in levels of the endothelial marker Cdh5 mRNA, suggesting increased angiogenesis in joints of Hsd11b1MKO mice following arthritis. A pro-angiogenic phenotype was confirmed by measuring angiogenesis in subcutaneously implanted polyurethane sponges, in which Hsd11b1MKO mice showed 20% greater vessel density than their littermate controls, associated with higher expression of Cdh5 Thus, 11ß-HSD1 deficiency in myeloid phagocytes promotes angiogenesis. Targeting 11ß-HSD1 in macrophages may be beneficial in tissue repair.

11ß-Hydroxysteroid Dehydrogenase Type 1 Is Expressed in Neutrophils and Restrains an Inflammatory Response in Male Mice.

Endogenous glucocorticoid action within cells is enhanced by prereceptor metabolism by 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), which converts intrinsically inert cortisone and 11-dehydrocorticosterone into active cortisol and corticosterone, respectively. 11ß-HSD1 is highly expressed in immune cells elicited to the mouse peritoneum during thioglycollate-induced peritonitis and is down-regulated as the inflammation resolves. During inflammation, 11ß-HSD1-deficient mice show enhanced recruitment of inflammatory cells and delayed acquisition of macrophage phagocytic capacity. However, the key cells in which 11ß-HSD1 exerts these effects remain unknown. Here we have identified neutrophils (CD11b(+),Ly6G(+),7/4(+) cells) as the thioglycollate-recruited cells that most highly express 11ß-HSD1 and show dynamic regulation of 11ß-HSD1 in these cells during an inflammatory response. Flow cytometry showed high expression of 11ß-HSD1 in peritoneal neutrophils early during inflammation, declining at later states. In contrast, expression in blood neutrophils continued to increase during inflammation. Ablation of monocytes/macrophages by treatment of CD11b-diphtheria-toxin receptor transgenic mice with diphtheria toxin prior to thioglycollate injection had no significant effect on 11ß-HSD1 activity in peritoneal cells, consistent with neutrophils being the predominant 11ß-HSD1 expressing cell type at this time. Similar to genetic deficiency in 11ß-HSD1, acute inhibition of 11ß-HSD1 activity during thioglycollate-induced peritonitis augmented inflammatory cell recruitment to the peritoneum. These data suggest that neutrophil 11ß-HSD1 increases during inflammation to contribute to the restraining effect of glucocorticoids upon neutrophil-mediated inflammation. In human neutrophils, lipopolysaccharide activation increased 11ß-HSD1 expression, suggesting the antiinflammatory effects of 11ß-HSD1 in neutrophils may be conserved in humans.

Pregnancy in obese mice protects selectively against visceral adiposity and is associated with increased adipocyte estrogen signalling.

Maternal obesity is linked with increased adverse pregnancy outcomes for both mother and child. The metabolic impact of excessive fat within the context of pregnancy is not fully understood. We used a mouse model of high fat (HF) feeding to induce maternal obesity to identify adipose tissue-mediated mechanisms driving metabolic dysfunction in pregnant and non-pregnant obese mice. As expected, chronic HF-feeding for 12 weeks preceding pregnancy increased peripheral (subcutaneous) and visceral (mesenteric) fat mass. However, unexpectedly at late gestation (E18.5) HF-fed mice exhibited a remarkable normalization of visceral but not peripheral adiposity, with a 53% reduction in non-pregnant visceral fat mass expressed as a proportion of body weight (P<0.001). In contrast, in control animals, pregnancy had no effect on visceral fat mass proportion. Obesity exaggerated glucose intolerance at mid-pregnancy (E14.5). However by E18.5, there were no differences, in glucose tolerance between obese and control mice. Transcriptomic analysis of visceral fat from HF-fed dams at E18.5 revealed reduced expression of genes involved in de novo lipogenesis (diacylglycerol O-acyltransferase 2--Dgat2) and inflammation (chemokine C-C motif ligand 20--Ccl2) and upregulation of estrogen receptor α (ERα) compared to HF non pregnant. Attenuation of adipose inflammation was functionally confirmed by a 45% reduction of CD11b+CD11c+ adipose tissue macrophages (expressed as a proportion of all stromal vascular fraction cells) in HF pregnant compared to HF non pregnant animals (P<0.001). An ERα selective agonist suppressed both de novo lipogenesis and expression of lipogenic genes in adipocytes in vitro. These data show that, in a HF model of maternal obesity, late gestation is associated with amelioration of visceral fat hypertrophy, inflammation and glucose intolerance, and suggest that these effects are mediated in part by elevated visceral adipocyte ERα signaling.

Changing glucocorticoid action: 11ß-hydroxysteroid dehydrogenase type 1 in acute and chronic inflammation.

Since the discovery of cortisone in the 1940s and its early success in treatment of rheumatoid arthritis, glucocorticoids have remained the mainstay of anti-inflammatory therapies. However, cortisone itself is intrinsically inert. To be effective, it requires conversion to cortisol, the active glucocorticoid, by the enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1). Despite the identification of 11ß-HSD in liver in 1953 (which we now know to be 11ß-HSD1), its physiological role has been little explored until recently. Over the past decade, however, it has become apparent that 11ß-HSD1 plays an important role in shaping endogenous glucocorticoid action. Acute inflammation is more severe with 11ß-HSD1-deficiency or inhibition, yet in some inflammatory settings such as obesity or diabetes, 11ß-HSD1-deficiency/inhibition is beneficial, reducing inflammation. Current evidence suggests both beneficial and detrimental effects may result from 11ß-HSD1 inhibition in chronic inflammatory disease. Here we review recent evidence pertaining to the role of 11ß-HSD1 in inflammation. This article is part of a Special Issue entitled 'CSR 2013'.

Novel fat depot-specific mechanisms underlie resistance to visceral obesity and inflammation in 11 ß-hydroxysteroid dehydrogenase type 1-deficient mice.

OBJECTIVE: The study objective was to determine the key early mechanisms underlying the beneficial redistribution, function, and inflammatory profile of adipose tissue in 11ß-hydroxysteroid dehydrogenase type 1 knockout (11ß-HSD1(-/-)) mice fed a high-fat (HF) diet. RESEARCH DESIGN AND METHODS: By focusing on the earliest divergence in visceral adiposity, subcutaneous and visceral fat depots from 11ß-HSD1(-/-) and C57Bl/6J control mice fed an HF diet for 4 weeks were used for comparative microarray analysis of gene expression, and differences were validated with real-time PCR. Key changes in metabolic signaling pathways were confirmed using Western blotting/immunoprecipitation, and fat cell size was compared with the respective chow-fed control groups. Altered adipose inflammatory cell content and function after 4 weeks (early) and 18 weeks (chronic) of HF feeding was investigated using fluorescence (and magnetic)-activated cell sorting analysis, immunohistochemistry, and in situ hybridization. RESULTS: In subcutaneous fat, HF-fed 11ß-HSD1(-/-) mice showed evidence of enhanced insulin and ß-adrenergic signaling associated with accretion of smaller metabolically active adipocytes. In contrast, reduced 11ß-HSD1(-/-) visceral fat accumulation was characterized by maintained AMP kinase activation, not insulin sensitization, and higher adipocyte interleukin-6 release. Intracellular glucocorticoid deficiency was unexpectedly associated with suppressed inflammatory signaling and lower adipocyte monocyte chemoattractant protein-1 secretion with strikingly reduced cytotoxic T-cell and macrophage infiltration, predominantly in visceral fat. CONCLUSIONS: Our data define for the first time the novel and distinct depot-specific mechanisms driving healthier fat patterning and function as a result of reduced intra-adipose glucocorticoid levels.

Galectin-3 expression and secretion links macrophages to the promotion of renal fibrosis.

Macrophages have been proposed as a key cell type in the pathogenesis of renal fibrosis; however, the mechanism by which macrophages drive fibrosis is still unclear. We show that expression of galectin-3, a beta-galactoside-binding lectin, is up-regulated in a mouse model of progressive renal fibrosis (unilateral ureteric obstruction, UUO), and absence of galectin-3 protects against renal myofibroblast accumulation/activation and fibrosis. Furthermore, specific depletion of macrophages using CD11b-DTR mice reduces fibrosis severity after UUO demonstrating that macrophages are key cells in the pathogenesis of renal fibrosis. Disruption of the galectin-3 gene does not affect macrophage recruitment after UUO, or macrophage proinflammatory cytokine profiles in response to interferon-gamma/lipopolysaccharide. In addition, absence of galectin-3 does not affect transforming growth factor-beta expression or Smad 2/3 phosphorylation in obstructed kidneys. Adoptive transfer of wild-type but not galectin-3(-/-) macrophages did, however, restore the fibrotic phenotype in galectin-3(-/-) mice. Cross-over experiments using wild-type and galectin-3(-/-) macrophage supernatants and renal fibroblasts confirmed that secretion of galectin-3 by macrophages is critical in the activation of renal fibroblasts to a profibrotic phenotype. Therefore, we demonstrate for the first time that galectin-3 expression and secretion by macrophages is a major mechanism linking macrophages to the promotion of renal fibrosis.

Nitric oxide is an important mediator of renal tubular epithelial cell death in vitro and in murine experimental hydronephrosis.

Macrophages play a pivotal role in tissue injury and fibrosis during renal inflammation. Although macrophages may induce apoptosis of renal tubular epithelial cells, the mechanisms involved are unclear. We used a microscopically quantifiable co-culture assay to dissect the cytotoxic interaction between murine bone marrow-derived macrophages and Madin-Darby canine kidney cells and primary murine renal tubular epithelial cells. The induction of tubular cell apoptosis by cytokine-activated macrophages was reduced by inhibitors of nitric oxide synthase whereas tubular cell proliferation was unaffected. Furthermore, cytokine-activated macrophages derived from mice targeted for the deletion of inducible nitric oxide synthase were noncytotoxic. We then examined the role of nitric oxide in vivo by inhibiting inducible nitric oxide synthase in the model of murine experimental hydronephrosis. l-N(6)-(1-iminoethyl)-lysine was administered in the drinking water between days 5 and 7 after ureteric obstruction. Macrophage infiltration was comparable between groups, but treatment significantly inhibited tubular cell apoptosis at day 7. Tubular cell proliferation was unaffected. Inducible nitric oxide synthase blockade also reduced interstitial cell apoptosis and increased collagen III deposition. These data indicate that nitric oxide is a key mediator of macrophage-directed tubular cell apoptosis in vitro and in vivo and also modulates tubulointerstitial fibrosis.

Resident pleural macrophages are key orchestrators of neutrophil recruitment in pleural inflammation.

RATIONALE: The role played by resident pleural macrophages in the initiation of pleural inflammation is currently unclear. OBJECTIVE: To evaluate the role of resident pleural macrophages in the initiation of inflammation. METHODS: We have used a conditional macrophage ablation strategy to determine the role of resident pleural macrophages in the regulation of neutrophil recruitment in a murine model of experimental pleurisy induced by the administration of carrageenan and formalin- fixed Staphylococcus aureus. MEASUREMENTS AND MAIN RESULTS: Conditional macrophage ablation mice express the human diphtheria toxin receptor under the control of the CD11b promoter such that the administration of diphtheria toxin induces ablation of nearly 97% of resident macrophages. Ablation of resident pleural macrophages before the administration of carrageenan or S. aureus dramatically reduced neutrophil influx into the pleural cavity. In the carrageenan model, the reduction in neutrophil infiltration was associated with marked early reduction in the level of macrophage inflammatory protein 2 as well as reduced levels of various cytokines, including tumor necrosis factor alpha, interleukin 6, and interleukin 10. Adoptive transfer of nontransgenic macrophages partially restored neutrophil infiltration. We also stimulated macrophage-depleted and nondepleted pleural cell populations with carrageenan in vitro and determined the production of chemokines and cytokines. Chemokine and cytokine production was markedly reduced by macrophage depletion, reinforcing the role of resident pleural macrophages in the generation of mediators that initiate acute inflammation. CONCLUSION: These studies indicate a critical role for resident pleural macrophages in sensing perturbation to the local microenvironment and orchestrating subsequent neutrophil infiltration.

Conditional ablation of macrophages halts progression of crescentic glomerulonephritis.

The presence of macrophages in inflamed glomeruli of rat kidney correlates with proliferation and apoptosis of resident glomerular mesangial cells. We assessed the contribution of inflammatory macrophages to progressive renal injury in murine crescentic glomerulonephritis (GN). Using a novel transgenic mouse (CD11b-DTR) in which tissue macrophages can be specifically and selectively ablated by minute injections of diphtheria toxin, we depleted renal inflammatory macrophages through days 15 and 20 of progressive crescentic GN. Macrophage depletion reduced the number of glomerular crescents, improved renal function, and reduced proteinuria. Morphometric analysis of renal tubules and interstitium revealed a marked attenuation of tubular injury that was associated with reduced proliferation and apoptosis of tubular cells. The population of interstitial myofibroblasts decreased after macrophage depletion and interstitial fibrosis also decreased. In the presence of macrophages, interstitial myofibroblasts exhibited increased levels of both proliferation and apoptosis, suggesting that macrophages act to support a population of renal myofibroblasts in a high turnover state and in matrix deposition. Finally, deletion of macrophages reduced CD4 T cells in the diseased kidney. This study demonstrates that macrophages are key effectors of disease progression in crescentic GN, acting to regulate parenchymal cell populations by modulating both cell proliferation and apoptosis.