The nuclear receptor LXRα controls the functional specialization of splenic macrophages.

Macrophages are professional phagocytic cells that orchestrate innate immune responses and have considerable phenotypic diversity at different anatomical locations. However, the mechanisms that control the heterogeneity of tissue macrophages are not well characterized. Here we found that the nuclear receptor LXRα was essential for the differentiation of macrophages in the marginal zone (MZ) of the spleen. LXR-deficient mice were defective in the generation of MZ and metallophilic macrophages, which resulted in abnormal responses to blood-borne antigens. Myeloid-specific expression of LXRα or adoptive transfer of wild-type monocytes restored the MZ microenvironment in LXRα-deficient mice. Our results demonstrate that signaling via LXRα in myeloid cells is crucial for the generation of splenic MZ macrophages and identify an unprecedented role for a nuclear receptor in the generation of specialized macrophage subsets.

Apoptotic cells promote their own clearance and immune tolerance through activation of the nuclear receptor LXR.

Effective clearance of apoptotic cells by macrophages is essential for immune homeostasis. The transcriptional pathways that allow macrophages to sense and respond to apoptotic cells are poorly defined. We found that liver X receptor (LXR) signaling was important for both apoptotic cell clearance and the maintenance of immune tolerance. Apoptotic cell engulfment activated LXR and thereby induced the expression of Mer, a receptor tyrosine kinase critical for phagocytosis. LXR-deficient macrophages exhibited a selective defect in phagocytosis of apoptotic cells and an aberrant proinflammatory response to them. As a consequence of these defects, mice lacking LXRs manifested a breakdown in self-tolerance and developed autoantibodies and autoimmune glomerulonephritis. Treatment with an LXR agonist ameliorated disease progression in a mouse model of lupus-like autoimmunity. Thus, activation of LXR by apoptotic cells engages a virtuous cycle that promotes their own clearance and couples engulfment to the suppression of inflammatory pathways.

Ribonucleases 6 and 7 have antimicrobial function in the human and murine urinary tract.

Recent evidence suggests antimicrobial peptides protect the urinary tract from infection. Ribonuclease 7 (RNase 7), a member of the RNase A superfamily, is a potent epithelial-derived protein that maintains human urinary tract sterility. RNase 7 expression is restricted to primates, limiting evaluation of its antimicrobial activity in vivo. Here we identified ribonuclease 6 (RNase 6) as the RNase A superfamily member present in humans and mice that is most conserved at the amino acid level relative to RNase 7. Like RNase 7, recombinant human and murine RNase 6 has potent antimicrobial activity against uropathogens. Quantitative real-time PCR and immunoblot analysis indicate that RNase 6 mRNA and protein are upregulated in the human and murine urinary tract during infection. Immunostaining located RNase 6 to resident and infiltrating monocytes, macrophages, and neutrophils. Uropathogenic E. coli induces RNase 6 peptide expression in human CD14(+) monocytes and murine bone marrow-derived macrophages. Thus, RNase 6 is an inducible, myeloid-derived protein with markedly different expression from the epithelial-derived RNase 7 but with equally potent antimicrobial activity. Our studies suggest RNase 6 serves as an evolutionarily conserved antimicrobial peptide that participates in the maintenance of urinary tract sterility.

Inhibition of Notch1 signaling reduces abdominal aortic aneurysm in mice by attenuating macrophage-mediated inflammation.

OBJECTIVE: Activation of inflammatory pathways plays a critical role in the development of abdominal aortic aneurysms (AAA). Notch1 signaling is a significant regulator of the inflammatory response; however, its role in AAA is unknown. METHODS AND RESULTS: In an angiotensin II-induced mouse model of AAA, activation of Notch1 signaling was observed in the aortic aneurysmal tissue of Apoe(-/-) mice, and a similar activation of Notch1 was observed in aneurysms of humans undergoing AAA repair. Notch1 haploinsufficiency significantly reduced the incidence of AAA in Apoe(-/-) mice in response to angiotensin II. Reconstitution of bone marrow-derived cells from Notch1(+/-);Apoe(-/-) mice (donor) in lethally irradiated Apoe(-/-) mice (recipient) decreased the occurrence of aneurysm. Flow cytometry and immunohistochemistry demonstrated that Notch1 haploinsufficiency prevented the influx of inflammatory macrophages at the aneurysmal site by causing defects in macrophage migration and proliferation. In addition, there was an overall reduction in the inflammatory burden in the aorta of the Notch1(+/-);Apoe(-/-) mice compared with the Apoe(-/-) mice. Last, pharmacological inhibition of Notch1 signaling also prevented AAA formation and progression in Apoe(-/-) mice. CONCLUSIONS: Our data suggest that decreased levels of Notch1 protect against the formation of AAA by preventing macrophage recruitment and attenuating the inflammatory response in the aorta.

Involvement of adenosine A2A receptors in engulfment-dependent apoptotic cell suppression of inflammation.

Efficient execution of apoptotic cell death followed by efficient clearance mediated by professional macrophages is a key mechanism in maintaining tissue homeostasis. Removal of apoptotic cells usually involves three central elements: 1) attraction of phagocytes via soluble "find me" signals, 2) recognition and phagocytosis via cell surface-presenting "eat me" signals, and 3) suppression or initiation of inflammatory responses depending on additional innate immune stimuli. Suppression of inflammation involves both direct inhibition of proinflammatory cytokine production and release of anti-inflammatory factors, which all contribute to the resolution of inflammation. In the current study, using wild-type and adenosine A(2A) receptor (A2AR) null mice, we investigated whether A2ARs, known to mediate anti-inflammatory signals in macrophages, participate in the apoptotic cell-mediated immunosuppression. We found that macrophages engulfing apoptotic cells release adenosine in sufficient amount to trigger A2ARs, and simultaneously increase the expression of A2ARs, as a result of possible activation of liver X receptor and peroxisome proliferators activated receptor δ. In macrophages engulfing apoptotic cells, stimulation of A2ARs suppresses the NO-dependent formation of neutrophil migration factors, such as macrophage inflammatory protein-2, using the adenylate cyclase/protein kinase A pathway. As a result, loss of A2ARs results in elevated chemoattractant secretion. This was evident as pronounced neutrophil migration upon exposure of macrophages to apoptotic cells in an in vivo peritonitis model. Altogether, our data indicate that adenosine is one of the soluble mediators released by macrophages that mediate engulfment-dependent apoptotic cell suppression of inflammation.

Immunomodulatory effects of plasma products on monocyte function in vitro.

BACKGROUND: Restoration of a balanced innate immune response is paramount to recovery from critical injury. Plasma transfusion may modulate innate immune responses; however, little is known about the immunomodulatory potential of various plasma products. We conducted in vitro experiments to determine the effects of fresh frozen plasma, thawed plasma, solvent/detergent plasma, and an investigational spray-dried solvent/detergent plasma product on monocyte function. METHODS: Monocytes were isolated from healthy adult volunteers and cocultured with aliquots of autologous plasma (control), fresh frozen plasma, thawed plasma, solvent/detergent treated plasma, or spray-dried solvent/detergent plasma. Monocyte function was assessed by cytokine production with and without lipopolysaccharide (LPS) stimulation, and flow cytometric assessment of HLA-DR cell surface expression. RESULTS: Monocyte cytokine production was not significantly altered after exposure to fresh frozen plasma or thawed plasma. In the absence of LPS, spray-dried solvent/detergent plasma exposure resulted in markedly increased IL-8 production compared to other plasma groups and controls (p = 0.01, analysis of variance [ANOVA]). Likewise, spray-dried SD plasma exposure resulted in higher LPS-induced IL-8, TNFα, and IL-1ß production compared with autologous plasma controls (p < 0.0001; p < 0.0001, p = 0.002, respectively; ANOVA). LPS-induced IL-8 and TNFα production was lowest after exposure to solvent/detergent plasma (p < 0.0001, ANOVA). CONCLUSION: Exposure to spray-dried solvent/detergent plasma resulted in marked augmentation of monocyte inflammatory cytokine production. Solvent/detergent plasma exposure resulted in the lowest cytokine production, suggesting lower immunomodulatory potential. Further work is needed to determine how these in vitro findings may translate to the bedside.

Liver X Receptor Nuclear Receptors Are Transcriptional Regulators of Dendritic Cell Chemotaxis.

The liver X receptors (LXRs) are ligand-activated nuclear receptors with established roles in the maintenance of lipid homeostasis in multiple tissues. LXRs exert additional biological functions as negative regulators of inflammation, particularly in macrophages. However, the transcriptional responses controlled by LXRs in other myeloid cells, such as dendritic cells (DCs), are still poorly understood. Here we used gain- and loss-of-function models to characterize the impact of LXR deficiency on DC activation programs. Our results identified an LXR-dependent pathway that is important for DC chemotaxis. LXR-deficient mature DCs are defective in stimulus-induced migration in vitro and in vivo Mechanistically, we show that LXRs facilitate DC chemotactic signaling by regulating the expression of CD38, an ectoenzyme important for leukocyte trafficking. Pharmacological or genetic inactivation of CD38 activity abolished the LXR-dependent induction of DC chemotaxis. Using the low-density lipoprotein receptor-deficient (LDLR-/-) LDLR-/- mouse model of atherosclerosis, we also demonstrated that hematopoietic CD38 expression is important for the accumulation of lipid-laden myeloid cells in lesions, suggesting that CD38 is a key factor in leukocyte migration during atherogenesis. Collectively, our results demonstrate that LXRs are required for the efficient emigration of DCs in response to chemotactic signals during inflammation.

The Nuclear Receptor LXR Limits Bacterial Infection of Host Macrophages through a Mechanism that Impacts Cellular NAD Metabolism.

Macrophages exert potent effector functions against invading microorganisms but constitute, paradoxically, a preferential niche for many bacterial strains to replicate. Using a model of infection by Salmonella Typhimurium, we have identified a molecular mechanism regulated by the nuclear receptor LXR that limits infection of host macrophages through transcriptional activation of the multifunctional enzyme CD38. LXR agonists reduced the intracellular levels of NAD+ in a CD38-dependent manner, counteracting pathogen-induced changes in macrophage morphology and the distribution of the F-actin cytoskeleton and reducing the capability of non-opsonized Salmonella to infect macrophages. Remarkably, pharmacological treatment with an LXR agonist ameliorated clinical signs associated with Salmonella infection in vivo, and these effects were dependent on CD38 expression in bone-marrow-derived cells. Altogether, this work reveals an unappreciated role for CD38 in bacterial-host cell interaction that can be pharmacologically exploited by activation of the LXR pathway.