Lev Becker

Gender Bias in Autoimmunity Is Influenced by Microbiota

Gender bias and the role of sex hormones in autoimmune diseases are well established. In specific pathogen-free nonobese diabetic (NOD) mice, females have 1.3-4.4 times higher incidence of type 1 diabetes (T1D). Germ-free (GF) mice lost the gender bias (female-to-male ratio 1.1-1.2). Gut microbiota differed in males and females, a trend reversed by male castration, confirming that androgens influence gut microbiota. Colonization of GF NOD mice with defined microbiota revealed that some, but not all, lineages overrepresented in male mice supported a gender bias in T1D. Although protection of males did not correlate with blood androgen concentration, hormone-supported expansion of selected microbial lineages may work as a positive-feedback mechanism contributing to the sexual dimorphism of autoimmune diseases. Gene-expression analysis suggested pathways involved in protection of males from T1D by microbiota. Our results favor a two-signal model of gender bias, in which hormones and microbes together trigger protective pathways.

Diabetes promotes an inflammatory macrophage phenotype and atherosclerosis through acyl-CoA synthetase 1

The mechanisms that promote an inflammatory environment and accelerated atherosclerosis in diabetes are poorly understood. We show that macrophages isolated from two different mouse models of type 1 diabetes exhibit an inflammatory phenotype. This inflammatory phenotype associates with increased expression of long-chain acyl-CoA synthetase 1 (ACSL1), an enzyme that catalyzes the thioesterification of fatty acids. Monocytes from humans and mice with type 1 diabetes also exhibit increased ACSL1. Furthermore, myeloid-selective deletion of ACSL1 protects monocytes and macrophages from the inflammatory effects of diabetes. Strikingly, myeloid-selective deletion of ACSL1 also prevents accelerated atherosclerosis in diabetic mice without affecting lesions in nondiabetic mice. Our observations indicate that ACSL1 plays a critical role by promoting the inflammatory phenotype of macrophages associated with type 1 diabetes; they also raise the possibilities that diabetic atherosclerosis has an etiology that is, at least in part, distinct from the etiology of nondiabetic vascular disease and that this difference is because of increased monocyte and macrophage ACSL1 expression.

Intermittent Hypoxia-induced Changes in Tumor-associated Macrophages and Tumor Malignancy in a Mouse Model of Sleep Apnea

RATIONALE An increased cancer aggressiveness and mortality have been recently reported among patients with obstructive sleep apnea (OSA). Intermittent hypoxia (IH), a hallmark of OSA, enhances melanoma growth and metastasis in mice. OBJECTIVES To assess whether OSA-related adverse cancer outcomes occur via IH-induced changes in host immune responses, namely tumor-associated macrophages (TAMs). MEASUREMENTS AND MAIN RESULTS Lung epithelial TC1 cell tumors were 84% greater in mice subjected to IH for 28 days compared with room air (RA). In addition, TAMs in IH-exposed tumors exhibited reductions in M1 polarity with a shift toward M2 protumoral phenotype. Although TAMs from tumors harvested from RA-exposed mice increased TC1 migration and extravasation, TAMs from IH-exposed mice markedly enhanced such effects and also promoted proliferative rates and invasiveness of TC1 cells. Proliferative rates of melanoma (B16F10) and TC1 cells exposed to IH either in single culture or in coculture with macrophages (RAW 264.7) increased only when RAW 264.7 macrophages were concurrently present. CONCLUSIONS Our findings support the notion that IH-induced alterations in TAMs participate in the adverse cancer outcomes reported in OSA.

S100A9 Differentially Modifies Phenotypic States of Neutrophils, Macrophages, and Dendritic Cells Implications for Atherosclerosis and Adipose Tissue Inflammation

Background— S100A9 is constitutively expressed in neutrophils, dendritic cells, and monocytes; is associated with acute and chronic inflammatory conditions; and is implicated in obesity and cardiovascular disease in humans. Most of the constitutively secreted S100A9 is derived from myeloid cells. A recent report demonstrated that mice deficient in S100A9 exhibit reduced atherosclerosis compared with controls and suggested that this effect was due in large part to loss of S100A9 in bone marrow–derived cells. Methods and Results— To directly investigate the role of bone marrow–derived S100A9 in atherosclerosis and insulin resistance in mice, low-density lipoprotein receptor–deficient, S100A9-deficient bone marrow chimeras were generated. Neither atherosclerosis nor insulin resistance was reduced in S100A9-deficient chimeras fed a diet rich in fat and carbohydrates. To investigate the reason for this lack of effect, myeloid cells were isolated from the peritoneal cavity or bone marrow. S100A9-deficient neutrophils exhibited a reduced secretion of cytokines in response to toll-like receptor-4 stimulation. In striking contrast, S100A9-deficient dendritic cells showed an exacerbated release of cytokines after toll-like receptor stimulation. Macrophages rapidly lost S100A9 expression during maturation; hence, S100A9 deficiency did not affect the inflammatory status of macrophages. Conclusions— S100A9 differentially modifies phenotypic states of neutrophils, macrophages, and dendritic cells. The effect of S100A9 deficiency on atherosclerosis and other inflammatory diseases is therefore predicted to depend on the relative contribution of these cell types at different stages of disease progression. Furthermore, S100A9 expression in nonmyeloid cells is likely to contribute to atherosclerosis. # Clinical Perspective {#article-title-38}Background— S100A9 is constitutively expressed in neutrophils, dendritic cells, and monocytes; is associated with acute and chronic inflammatory conditions; and is implicated in obesity and cardiovascular disease in humans. Most of the constitutively secreted S100A9 is derived from myeloid cells. A recent report demonstrated that mice deficient in S100A9 exhibit reduced atherosclerosis compared with controls and suggested that this effect was due in large part to loss of S100A9 in bone marrow–derived cells. Methods and Results— To directly investigate the role of bone marrow–derived S100A9 in atherosclerosis and insulin resistance in mice, low-density lipoprotein receptor–deficient, S100A9-deficient bone marrow chimeras were generated. Neither atherosclerosis nor insulin resistance was reduced in S100A9-deficient chimeras fed a diet rich in fat and carbohydrates. To investigate the reason for this lack of effect, myeloid cells were isolated from the peritoneal cavity or bone marrow. S100A9-deficient neutrophils exhibited a reduced secretion of cytokines in response to toll-like receptor-4 stimulation. In striking contrast, S100A9-deficient dendritic cells showed an exacerbated release of cytokines after toll-like receptor stimulation. Macrophages rapidly lost S100A9 expression during maturation; hence, S100A9 deficiency did not affect the inflammatory status of macrophages. Conclusions— S100A9 differentially modifies phenotypic states of neutrophils, macrophages, and dendritic cells. The effect of S100A9 deficiency on atherosclerosis and other inflammatory diseases is therefore predicted to depend on the relative contribution of these cell types at different stages of disease progression. Furthermore, S100A9 expression in nonmyeloid cells is likely to contribute to atherosclerosis.

High-Density Lipoprotein Suppresses the Type I Interferon Response, a Family of Potent Antiviral Immunoregulators, in Macrophages Challenged With Lipopolysaccharide

Background— High-density lipoprotein (HDL) protects the artery wall by removing cholesterol from lipid-laden macrophages. However, recent evidence suggests that HDL might also inhibit atherogenesis by combating inflammation. Methods and Results— To identify potential antiinflammatory mechanisms, we challenged macrophages with lipopolysaccharide, an inflammatory microbial ligand for Toll-like receptor 4. HDL inhibited the expression of 30% (277 of 911) of the genes normally induced by lipopolysaccharide, microarray analysis revealed. One of its major targets was the type I interferon response pathway, a family of potent viral immunoregulators controlled by Toll-like receptor 4 and the TRAM/TRIF signaling pathway. Unexpectedly, the ability of HDL to inhibit gene expression was independent of macrophage cholesterol stores. Immunofluorescent studies suggested that HDL promoted TRAM translocation to intracellular compartments, which impaired subsequent signaling by Toll-like receptor 4 and TRIF. To examine the potential in vivo relevance of the pathway, we used mice deficient in apolipoprotein A-I, the major protein of HDL. After infection with Salmonella typhimurium, a Gram-negative bacterium that expresses lipopolysaccharide, apolipoprotein A-I–deficient mice had 6-fold higher plasma levels of interferon-&bgr;, a key regulator of the type I interferon response, than did wild-type mice. Conclusions— HDL inhibits a subset of lipopolysaccharide-stimulated macrophage genes that regulate the type I interferon response, and its action is independent of sterol metabolism. These findings raise the possibility that regulation of macrophage genes by HDL might link innate immunity and cardioprotection.

Unique Proteomic Signatures Distinguish Macrophages and Dendritic Cells

Monocytes differentiate into heterogeneous populations of tissue macrophages and dendritic cells (DCs) that regulate inflammation and immunity. Identifying specific populations of myeloid cells in vivo is problematic, however, because only a limited number of proteins have been used to assign cellular phenotype. Using mass spectrometry and bone marrow-derived cells, we provided a global view of the proteomes of M-CSF-derived macrophages, classically and alternatively activated macrophages, and GM-CSF-derived DCs. Remarkably, the expression levels of half the plasma membrane proteins differed significantly in the various populations of cells derived in vitro. Moreover, the membrane proteomes of macrophages and DCs were more distinct than those of classically and alternatively activated macrophages. Hierarchical cluster and dual statistical analyses demonstrated that each cell type exhibited a robust proteomic signature that was unique. To interrogate the phenotype of myeloid cells in vivo, we subjected elicited peritoneal macrophages harvested from wild-type and GM-CSF-deficient mice to mass spectrometric and functional analysis. Unexpectedly, we found that peritoneal macrophages exhibited many features of the DCs generated in vitro. These findings demonstrate that global analysis of the membrane proteome can help define immune cell phenotypes in vivo.

A Macrophage Sterol-Responsive Network Linked to Atherogenesis

Cholesteryl ester accumulation by macrophages is a critical early event in atherogenesis. To test the hypothesis that sterol loading promotes foam cell formation and vascular disease by perturbing a network of interacting proteins, we used a global approach to identify proteins that are differentially expressed when macrophages are loaded with cholesterol in vivo. Our analysis revealed a sterol-responsive network that is highly enriched in proteins with known physical interactions, established roles in vesicular transport, and demonstrated atherosclerotic phenotypes in mice. Pharmacologic intervention with a statin or rosiglitazone and use of mice deficient in LDL receptor or apolipoprotein E implicated the network in atherosclerosis. Biochemical fractionation revealed that most of the sterol-responsive proteins resided in microvesicles, providing a physical basis for the networks functional and biochemical properties. These observations identify a highly integrated network of proteins whose expression is influenced by environmental, genetic, and pharmacological factors implicated in atherogenesis.

Metastasis Suppressors Regulate the Tumor Microenvironment by Blocking Recruitment of Prometastatic Tumor-Associated Macrophages

Triple-negative breast cancer (TNBC) patients have the highest risk of recurrence and metastasis. Because they cannot be treated with targeted therapies, and many do not respond to chemotherapy, they represent a clinically underserved group. TNBC is characterized by reduced expression of metastasis suppressors such as Raf kinase inhibitory protein (RKIP), which inhibits tumor invasiveness. Mechanisms by which metastasis suppressors alter tumor cells are well characterized; however, their ability to regulate the tumor microenvironment and the importance of such regulation to metastasis suppression are incompletely understood. Here, we use species-specific RNA sequencing to show that RKIP expression in tumors markedly reduces the number and metastatic potential of infiltrating tumor-associated macrophages (TAM). TAMs isolated from nonmetastatic RKIP(+) tumors, relative to metastatic RKIP(-) tumors, exhibit a reduced ability to drive tumor cell invasion and decreased secretion of prometastatic factors, including PRGN, and shed TNFR2. RKIP regulates TAM recruitment by blocking HMGA2, resulting in reduced expression of numerous macrophage chemotactic factors, including CCL5. CCL5 overexpression in RKIP(+) tumors restores recruitment of prometastatic TAMs and intravasation, whereas treatment with the CCL5 receptor antagonist Maraviroc reduces TAM infiltration. These results highlight the importance of RKIP as a regulator of TAM recruitment through chemokines such as CCL5. The clinical significance of these interactions is underscored by our demonstration that a signature comprised of RKIP signaling and prometastatic TAM factors strikingly separates TNBC patients based on survival outcome. Collectively, our findings identify TAMs as a previously unsuspected mechanism by which the metastasis-suppressor RKIP regulates tumor invasiveness, and further suggest that TNBC patients with decreased RKIP activity and increased TAM infiltration may respond to macrophage-based therapeutics.

Macrophage Metalloelastase (MMP12) Regulates Adipose Tissue Expansion, Insulin Sensitivity, and Expression of Inducible Nitric Oxide Synthase

Macrophage metalloelastase, a matrix metallopeptidase (MMP12) predominantly expressed by mature tissue macrophages, is implicated in pathological processes. However, physiological functions for MMP12 have not been described. Because mRNA levels for the enzyme increase markedly in adipose tissue of obese mice, we investigated the role of MMP12 in adipose tissue expansion and insulin resistance. In humans, MMP12 expression correlated positively and significantly with insulin resistance, TNF-α expression, and the number of CD14(+)CD206(+) macrophages in adipose tissue. MMP12 was the most abundant matrix metallopeptidase detected by proteomic analysis of conditioned medium of M2 macrophages and dendritic cells. In contrast, it was detected only at low levels in bone marrow derived macrophages and M1 macrophages. When mice received a high-fat diet, adipose tissue mass increased and CD11b(+)F4/80(+)CD11c(-) macrophages accumulated to a greater extent in MMP12-deficient (Mmp12(-/-)) mice than in wild-type mice (Mmp12(+/+)). Despite being markedly more obese, fat-fed Mmp12(-/-) mice were more insulin sensitive than fat-fed Mmp12(+/+) mice. Expression of inducible nitric oxide synthase (Nos2) by Mmp12(-/-) macrophages was significantly impaired both in vivo and in vitro, suggesting that MMP12 might mediate nitric oxide production during inflammation. We propose that MMP12 acts as a double-edged sword by promoting insulin resistance while combatting adipose tissue expansion.

Contextualised urinary biomarker analysis facilitates diagnosis of paediatric obstructive sleep apnoea

BACKGROUND Intrinsic variance of the urine proteome limits the discriminative power of proteomic analysis and complicates potential biomarker detection in the context of paediatric sleep disorders. METHODS AND RESULTS Using a rigorous workflow for proteomic analysis of urine, we demonstrate that gender and diurnal effects constitute two important sources of variability in healthy children. In the context of disease, complex pathophysiological perturbations magnify these proteomic differences and therefore require contextualised biomarker analysis. Indeed, by performing biomarker discovery in a gender- and diurnal-dependent manner, we identified ∼30-fold more candidate biomarkers of paediatric obstructive sleep apnoea (OSA), a highly prevalent condition in children characterised by repetitive episodes of intermittent hypoxia and hypercapnia, and sleep fragmentation in the context of recurrent upper airway obstructive events during sleep. Remarkably, biomarkers were highly specific for gender and sampling time as poor overlap (∼3%) was observed in the proteins identified in boys and girls across morning and bedtime samples. CONCLUSIONS As no clinical basis to explain gender-specific effects in OSA or healthy children is apparent, we propose that implementation of contextualised biomarker strategies will be applicable to a broad range of human diseases, and may be specifically applicable to paediatric OSA.