Faculty Bibliography

Ubiquitination was recently identified as a central process in the pathogenesis and development of numerous inflammatory diseases, such as obesity, atherosclerosis, and asthma. Treatment with proteasomal inhibitors led to severe side effects because ubiquitination is heavily involved in a plethora of cellular functions. Thus, new players regulating ubiquitination processes must be identified to improve therapies for inflammatory diseases. In addition to their role in adaptive immunity, endosomal MHC class II (MHCII) molecules were shown to modulate innate immune responses by fine tuning the TLR4 signaling pathway. However, the role of MHCII ubiquitination by membrane associated ring-CH-type finger 1 (MARCH1) E3 ubiquitin ligase in this process remains to be assessed. In this article, we demonstrate that MARCH1 is a key inhibitor of innate inflammation in response to bacterial endotoxins. The higher mortality of March1^-/- mice challenged with a lethal dose of LPS was associated with significantly stronger systemic production of proinflammatory cytokines and splenic NK cell activation; however, we did not find evidence that MARCH1 modulates LPS or IL-10 signaling pathways. Instead, the mechanism by which MARCH1 protects against endotoxic shock rests on its capacity to promote the transition of monocytes from Ly6C^Hi to Ly6C^+/- Moreover, in competitive bone marrow chimeras, March1^-/- monocytes and polymorphonuclear neutrophils outcompeted wild-type cells with regard to bone marrow egress and homing to peripheral organs. We conclude that MARCH1 exerts MHCII-independent effects that regulate the innate arm of immunity. Thus, MARCH1 might represent a potential new target for emerging therapies based on ubiquitination reactions in inflammatory diseases.

Dendritic cells (DCs) are central to initiate antigen-specific immunity and tolerance. The in vivo development and distribution of DCs are now better understood even in nonlymphoid tissues [1]. Atherosclerosis is a chronic inflammatory disease of blood vessels and DCs are highly enriched in the intimal area of the aorta, which is predisposed to develop atherosclerosis. Previously, we were the first to show antigen presenting DCs and their subsets in the aorta [2, 3]. Here, we discuss several useful methods to characterize not only DCs but also other immune cells in steady state and atherosclerotic aorta. These comprise multiparameter flow cytometry strategies including intracellular staining and cell sorting, en face immunohistochemistry of DCs and regulatory T cells (Tregs), and Oil Red O staining of atherosclerotic lesions in the aorta.

Plasmacytoid dendritic cells (pDCs) are unique bone-marrow-derived cells that produce large amounts of type I interferon in response to microbial stimulation. Furthermore, pDCs also promote T cell tolerance in sterile-inflammation conditions. However, the immunomodulatory role of aortic pDCs in atherosclerosis has been poorly understood. Here, we identified functional mouse and human pDCs in the aortic intima and showed that selective, inducible pDC depletion in mice exacerbates atherosclerosis. Aortic pDCs expressed CCR9 and indoleamine 2,3-dioxygenase 1 (IDO-1), an enzyme involved in driving the generation of regulatory T cells (Tregs). As a consequence, loss of pDCs resulted in decreased numbers of Tregs and reduced IL-10 levels in the aorta. Moreover, antigen presentation by pDCs expanded antigen-specific Tregs in the atherosclerotic aorta. Notably, Tregs ablation affected pDC homeostasis in diseased aorta. Accordingly, pDCs in human atherosclerotic aortas colocalized with Tregs. Collectively, we identified a mechanism of atheroprotection mediated by tolerogenic aortic pDCs.

Resident macrophages densely populate the normal arterial wall, yet their origins and the mechanisms that sustain them are poorly understood. Here we use gene-expression profiling to show that arterial macrophages constitute a distinct population among macrophages. Using multiple fate-mapping approaches, we show that arterial macrophages arise embryonically from CX3CR1(+) precursors and postnatally from bone marrow-derived monocytes that colonize the tissue immediately after birth. In adulthood, proliferation (rather than monocyte recruitment) sustains arterial macrophages in the steady state and after severe depletion following sepsis. After infection, arterial macrophages return rapidly to functional homeostasis. Finally, survival of resident arterial macrophages depends on a CX3CR1-CX3CL1 axis within the vascular niche.

BACKGROUND:Endocrine functions of the heart have been well established. We investigated the hypothesis that cardiac secretion of a unique phospholipase A2 recently identified by our laboratory (cardiac secreted phospholipase A2 [sPLA2]) establishes a heart-liver endocrine axis that is negatively regulated by matrix metalloproteinase-2 (MMP-2). METHODS AND RESULTS/RESULTS:In Mmp2(-/-) mice, cardiac (but not hepatic) sPLA2 was elevated, leading to hepatic inflammation, immune cell infiltration, dysregulation of the sterol regulatory element binding protein-2 and liver X receptor-α pathways, abnormal transcriptional responses to dietary cholesterol, and elevated triglycerides in very low-density lipoprotein and in the liver. Expression of monocyte chemoattractant protein-3, a known MMP-2 substrate, was elevated at both mRNA and protein levels in the heart. Functional studies including in vivo antibody neutralization identified cardiac monocyte chemoattractant protein 3 as a possible agonist of cardiac sPLA2 secretion. Conversely, systemic sPLA2 inhibition almost fully normalized the cardiohepatic phenotype without affecting monocyte chemoattractant protein-3. Finally, wild-type mice that received high-performance liquid chromatography-isolated cardiac sPLA2 from Mmp2(-/-) donors developed a cardiohepatic gene expression profile similar to that of Mmp2(-/-) mice. CONCLUSIONS:These findings identified the novel MMP-2/cardiac sPLA2 pathway that endows the heart with important endocrine functions, including regulation of inflammation and lipid metabolism in the liver. Our findings could also help explain how MMP2 deficiency leads to cardiac problems, inflammation, and metabolic dysregulation in patients.

Tissue effector cells of the monocyte lineage can differentiate into different cell types with specific cell function depending on their environment. The phenotype, developmental requirements, and functional mechanisms of immune protective macrophages that mediate the induction of transplantation tolerance remain elusive. Here, we demonstrate that costimulatory blockade favored accumulation of DC-SIGN-expressing macrophages that inhibited CD8(+) T cell immunity and promoted CD4(+)Foxp3(+) Treg cell expansion in numbers. Mechanistically, that simultaneous DC-SIGN engagement by fucosylated ligands and TLR4 signaling was required for production of immunoregulatory IL-10 associated with prolonged allograft survival. Deletion of DC-SIGN-expressing macrophages in vivo, interfering with their CSF1-dependent development, or preventing the DC-SIGN signaling pathway abrogated tolerance. Together, the results provide new insights into the tolerogenic effects of costimulatory blockade and identify DC-SIGN(+) suppressive macrophages as crucial mediators of immunological tolerance with the concomitant therapeutic implications in the clinic.

A key feature in the induction of pathological angiogenesis is that inflammation precedes and accompanies the formation of neovessels as evidenced by increased vascular permeability and the recruitment of inflammatory cells. Previously, we and other groups have shown that selected growth factors, namely vascular endothelial growth factor (VEGF) and angiopoietins (Ang1 and Ang2) do not only promote angiogenesis, but can also induce inflammatory response. Herein, given a pro-inflammatory environment, we addressed the individual capacity of VEGF and angiopoietins to promote the formation of mature neovessels and to identify the different types of inflammatory cells accompanying the angiogenic process over time. Sterilized polyvinyl alcohol (PVA) sponges soaked in growth factor-depleted Matrigel mixed with PBS, VEGF, Ang1, or Ang2 (200 ng/200 µl) were subcutaneously inserted into anesthetized mice. Sponges were removed at day 4, 7, 14, or 21 post-procedure for histological, immunohistological (IHC), and flow cytometry analyses. As compared to PBS-treated sponges, the three growth factors promoted the recruitment of inflammatory cells, mainly neutrophils and macrophages, and to a lesser extent, T- and B-cells. In addition, they were more potent and more rapid in the recruitment of endothelial cells (ECs) and in the formation and maturation (ensheating of smooth muscle cells around ECs) of neovessels. Thus, the autocrine/paracrine interaction among the different inflammatory cells in combination with VEGF, Ang1, or Ang2 provides a suitable microenvironment for the formation and maturation of blood vessels.