Retinoic Acid Signaling in Thymic Epithelial Cells Regulates Thymopoiesis.

Despite the essential role of thymic epithelial cells (TEC) in T cell development, the signals regulating TEC differentiation and homeostasis remain incompletely understood. In this study, we show a key in vivo role for the vitamin A metabolite, retinoic acid (RA), in TEC homeostasis. In the absence of RA signaling in TEC, cortical TEC (cTEC) and CD80loMHC class IIlo medullary TEC displayed subset-specific alterations in gene expression, which in cTEC included genes involved in epithelial proliferation, development, and differentiation. Mice whose TEC were unable to respond to RA showed increased cTEC proliferation, an accumulation of stem cell Ag-1hi cTEC, and, in early life, a decrease in medullary TEC numbers. These alterations resulted in reduced thymic cellularity in early life, a reduction in CD4 single-positive and CD8 single-positive numbers in both young and adult mice, and enhanced peripheral CD8+ T cell survival upon TCR stimulation. Collectively, our results identify RA as a regulator of TEC homeostasis that is essential for TEC function and normal thymopoiesis.

CX3CR1-dependent renal macrophage survival promotes Candida control and host survival.

Systemic Candida albicans infection causes high morbidity and mortality and is associated with neutropenia; however, the roles of other innate immune cells in pathogenesis are poorly defined. Here, using a mouse model of systemic candidiasis, we found that resident macrophages accumulated in the kidney, the main target organ of infection, and formed direct contacts with the fungus in vivo mainly within the first few hours after infection. Macrophage accumulation and contact with Candida were both markedly reduced in mice lacking chemokine receptor CX3CR1, which was found almost exclusively on resident macrophages in uninfected kidneys. Infected Cx3cr1-/- mice uniformly succumbed to Candida-induced renal failure, but exhibited clearance of the fungus in all other organs tested. Renal macrophage deficiency in infected Cx3cr1-/- mice was due to reduced macrophage survival, not impaired proliferation, trafficking, or differentiation. In humans, the dysfunctional CX3CR1 allele CX3CR1-M280 was associated with increased risk of systemic candidiasis. Together, these data indicate that CX3CR1-mediated renal resident macrophage survival is a critical innate mechanism of early fungal control that influences host survival in systemic candidiasis.

Type I IFNs regulate effector and regulatory T cell accumulation and anti-inflammatory cytokine production during T cell-mediated colitis.

We explored the function of endogenous type I IFNs (IFN-1) in the colon using the T cell adoptive transfer model of colitis. Colon mononuclear phagocytes (MPs) constitutively produced IFN-1 in a Toll/IL-1R domain-containing adapter-inducing IFN-ß-dependent manner. Transfer of CD4(+)CD45RB(hi) T cells from wild-type (WT) or IFN-α/ß receptor subunit 1 knockout (IFNAR1(-/-)) mice into RAG(-/-) hosts resulted in similar onset and severity of colitis. In contrast, RAG(-/-) × IFNAR1(-/-) double knockout (DKO) mice developed accelerated severe colitis compared with RAG(-/-) hosts when transferred with WT CD4(+)CD45RB(hi) T cells. IFNAR signaling on host hematopoietic cells was required to delay colitis development. MPs isolated from the colon lamina propria of IFNAR1(-/-) mice produced less IL-10, IL-1R antagonist, and IL-27 compared with WT MPs. Accelerated colitis development in DKO mice was characterized by early T cell proliferation and accumulation of CD11b(+)CD103(-) dendritic cells in the mesenteric lymph nodes, both of which could be reversed by systemic administration of IL-1R antagonist (anakinra). Cotransfer of CD4(+)CD25(+) regulatory T cells (Tregs) from WT or IFNAR1(-/-) mice prevented disease caused by CD4(+)CD45RB(hi) T cells. However, WT CD4(+)CD25(+)Foxp3(GFP+) Tregs cotransferred with CD4(+)CD45RB(hi) T cells into DKO hosts failed to expand or maintain Foxp3 expression and gained effector functions in the colon. To our knowledge, these data are the first to demonstrate an essential role for IFN-1 in the production of anti-inflammatory cytokines by gut MPs and the indirect maintenance of intestinal T cell homeostasis by both limiting effector T cell expansion and promoting Treg stability.

NK cell-derived interferon-γ orchestrates cellular dynamics and the differentiation of monocytes into dendritic cells at the site of infection.

Dendritic cells (DCs), monocytes, and/or macrophages initiate host-protective immune responses to intracellular pathogens in part through interleukin-12 (IL-12) production, although the relative contribution of tissue resident versus recruited cells has been unclear. Here, we showed that after intraperitoneal infection with Toxoplasma gondii cysts, resident mononuclear phagocytes are replaced by circulating monocytes that differentiate in situ into inflammatory DCs (moDCs) and F4/80(+) macrophages. Importantly, NK cell-derived interferon-γ (IFN-γ) was required for both the loss of resident mononuclear phagocytes and the local differentiation of monocytes into macrophages and moDCs. This newly generated moDC population and not the resident DCs (or macrophages) served as the major source of IL-12 at the site of infection. Thus, NK cell-derived IFN-γ is important in both regulating inflammatory cell dynamics and in driving the local differentiation of monocytes into the cells required for initiating the immune response to an important intracellular pathogen.

Inflammation switches the differentiation program of Ly6Chi monocytes from antiinflammatory macrophages to inflammatory dendritic cells in the colon.

Dendritic cells (DCs) and macrophages (MPs) are important for immunological homeostasis in the colon. We found that F4/80(hi)CX3CR1(hi) (CD11b(+)CD103(-)) cells account for 80% of mouse colonic lamina propria MHC-II(hi) cells. Both CD11c(+) and CD11c(-) cells within this population were identified as MPs based on multiple criteria, including an MP transcriptome revealed by microarray analysis. These MPs constitutively released high levels of IL-10 at least partially in response to the microbiota via an MyD88-independent mechanism. In contrast, cells expressing low to intermediate levels of F4/80 and CX3CR1 were identified as DCs based on phenotypic and functional analysis and comprise three separate CD11c(hi) cell populations: CD103(+)CX3CR1(-)CD11b(-) DCs, CD103(+)CX3CR1(-)CD11b(+) DCs, and CD103(-)CX3CR1(int)CD11b(+) DCs. In noninflammatory conditions, Ly6C(hi) monocytes (MOs) differentiated primarily into CD11c(+) but not CD11c(-) MPs. In contrast, during colitis, Ly6C(hi) MOs massively invaded the colon and differentiated into proinflammatory CD103(-)CX3CR1(int)CD11b(+) DCs, which produced high levels of IL-12, IL-23, iNOS, and TNF. These findings demonstrate the dual capacity of Ly6C(hi) blood MOs to differentiate into either regulatory MPs or inflammatory DCs in the colon and that the balance of these immunologically antagonistic cell types is dictated by microenvironmental conditions.

Genetic deletion of chemokine receptor Ccr6 decreases atherogenesis in ApoE-deficient mice.

RATIONALE: The chemokine receptor Ccr6 is a G-protein-coupled receptor expressed on various types of leukocytes identified in mouse atherosclerotic lesions. Recent evidence suggests that both CCR6 and its ligand CCL20 are also present in human atheroma; however, their functional roles in atherogenesis remain undefined. OBJECTIVE: Our objective was to delineate the role of Ccr6 in atherogenesis in the apolipoprotein E-deficient (ApoE(-/-)) mouse model of atherosclerosis. METHODS AND RESULTS: Both Ccr6 and Ccl20 are expressed in atherosclerotic aorta from ApoE(-/-) mice. Aortic lesion area in Ccr6(-/-)ApoE(-/-) mice was ∼40% and ∼30% smaller than in Ccr6(+/+)ApoE(-/-) mice at 16 and 24 weeks of age, respectively. Transplantation of bone marrow from Ccr6(-/-) mice into ApoE(-/-) mice resulted in ∼40% less atherosclerotic lesion area than for bone marrow from Ccr6(+/+) mice; lesions in Ccr6(-/-)ApoE(-/-) mice had 44% less macrophage content than lesions in Ccr6(+/+)ApoE(-/-) mice. Ccr6 was expressed on a subset of primary mouse monocytes. Accordingly, Ccl20 induced chemotaxis of primary monocytes from wild-type but not Ccr6(-/-) mice; moreover, Ccl20 induced monocytosis in ApoE(-/-) mice in vivo. Consistent with this, we observed 30% fewer monocytes in circulating blood of Ccr6(-/-)ApoE(-/-) mice, mainly because of fewer CD11b(+)Ly6C(high) inflammatory monocytes. CONCLUSIONS: Ccr6 promotes atherosclerosis in ApoE-deficient mice, which may be due in part to Ccr6 support of normal monocyte levels in blood, as well as direct Ccr6-dependent monocyte migration.

Chemokine receptor Ccr2 is critical for monocyte accumulation and survival in West Nile virus encephalitis.

West Nile virus (WNV) is a re-emerging pathogen responsible for outbreaks of fatal meningoencephalitis in humans. Previous studies have suggested a protective role for monocytes in a mouse model of WNV infection, but the molecular mechanisms have remained unclear. In this study, we show that genetic deficiency in Ccr2, a chemokine receptor on Ly6c(hi) inflammatory monocytes and other leukocyte subtypes, markedly increases mortality due to WNV encephalitis in C57BL/6 mice; this was associated with a large and selective reduction of Ly6c(hi) monocyte accumulation in the brain. WNV infection in Ccr2(+/+) mice induced a strong and highly selective monocytosis in peripheral blood that was absent in Ccr2(-/-) mice, which in contrast showed sustained monocytopenia. When a 1:1 mixture of Ccr2(+/+) and Ccr2(-/-) donor monocytes was transferred by vein into WNV-infected Ccr2(-/-) recipient mice, monocyte accumulation in the CNS was not skewed toward either component of the mixture, indicating that Ccr2 is not required for trafficking of monocytes from blood to brain. We conclude that Ccr2 mediates highly selective peripheral blood monocytosis during WNV infection of mice and that this is critical for accumulation of monocytes in the brain.

Langerhans cell histiocytosis reveals a new IL-17A-dependent pathway of dendritic cell fusion.

IL-17A is a T cell-specific cytokine that is involved in chronic inflammations, such as Mycobacterium infection, Crohn's disease, rheumatoid arthritis and multiple sclerosis. Mouse models have explained the molecular basis of IL-17A production and have shown that IL-17A has a positive effect not only on granuloma formation and neurodegeneration through unknown mechanisms, but also on bone resorption through Receptor activator of NF-kappaB ligand (RANKL) induction in osteoblasts. Langerhans cell histiocytosis (LCH) is a rare disease of unknown etiology, lacking an animal model, that cumulates symptoms that are found separately in various IL-17A-related diseases, such as aggressive chronic granuloma formation, bone resorption and soft tissue lesions with occasional neurodegeneration. We examined IL-17A in the context of LCH and found that there were high serum levels of IL-17A during active LCH and unexpected IL-17A synthesis by dendritic cells (DCs), the major cell type in LCH lesions. We also found an IL-17A-dependent pathway for DC fusion, which was highly potentiated by IFN-gamma and led to giant cells expressing three major tissue-destructive enzymes: tartrate resistant acidic phosphatase and matrix metalloproteinases 9 and 12. IFN-gamma expression has been previously documented in LCH and observed in IL-17A-related diseases. Notably, serum IL-17A-dependent fusion activity correlates with LCH activity. Thus, IL-17A and IL-17A-stimulated DCs represent targets that may have clinical value in the treatment of LCH and other IL-17A-related inflammatory disorders.

High expression of antioxidant proteins in dendritic cells: possible implications in atherosclerosis.

Dendritic cells (DCs) display the unique ability to activate naive T cells and to initiate primary T cell responses revealed in DC-T cell alloreactions. DCs frequently operate under stress conditions. Oxidative stress enhances the production of inflammatory cytokines by DCs. We performed a proteomic analysis to see which major changes occur, at the protein expression level, during DC differentiation and maturation. Comparative two-dimensional gel analysis of the monocyte, immature DC, and mature DC stages was performed. Manganese superoxide dismutase (Mn-SOD) reached 0.7% of the gel-displayed proteins at the mature DC stage. This important amount of Mn-SOD is a primary antioxidant defense system against superoxide radicals, but its product, H(2)O(2), is also deleterious for cells. Peroxiredoxin (Prx) enzymes play an important role in eliminating such peroxide. Prx1 expression level continuously increased during DC differentiation and maturation, whereas Prx6 continuously decreased, and Prx2 peaked at the immature DC stage. As a consequence, DCs were more resistant than monocytes to apoptosis induced by high amounts of oxidized low density lipoproteins containing toxic organic peroxides and hydrogen peroxide. Furthermore DC-stimulated T cells produced high levels of receptor activator of nuclear factor kappaB ligand, a chemotactic and survival factor for monocytes and DCs. This study provides insights into the original ability of DCs to express very high levels of antioxidant enzymes such as Mn-SOD and Prx1, to detoxify oxidized low density lipoproteins, and to induce high levels of receptor activator of nuclear factor kappaB ligand by the T cells they activate and further emphasizes the role that DCs might play in atherosclerosis, a pathology recognized as a chronic inflammatory disorder.

Immature dendritic cell transdifferentiation into osteoclasts: a novel pathway sustained by the rheumatoid arthritis microenvironment.

Dendritic cells (DCs), the mononuclear cells that initiate immune response, and osteoclasts, the multinucleated bone-resorbing cells, are derived from monocyte/macrophage precursor cells. Granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor (M-CSF) reciprocally regulate the differentiation of both lineages in mice. Using human monocyte-derived DCs generated in vitro, we show that immature DCs transdifferentiate into functional osteoclasts (OCs) in the presence of M-CSF and receptor activator of nuclear factor-kappaB ligand (RANKL). Transdifferentiation operates through fusion of intermediate adherent bipolar fusiform mononuclear cells expressing CD14, CD1a, and RANKL and able to induce RANKL(+) T-cell proliferation. Surprisingly, DC fusion in vitro is faster and more efficient than monocyte fusion to form multinucleated giant cells. The transdifferentiation process reported here supports the existence of a high cellular plasticity within differentiated myeloid phagocytes. Importantly, this process is greatly enhanced by rheumatoid arthritis synovial fluid and involves proinflammatory cytokines such as interleukin 1 or tumor necrosis factor alpha, as well as components of the extracellular matrix such as hyaluronic acid. Our data therefore suggest that DC-derived OCs may be directly involved in the osteolytic lesions observed in human inflammatory bone diseases such as rheumatoid arthritis or in particular forms of Langerhans cell histiocytosis, characterized by accumulation of immature skin DCs and chronic lytic bone lesions.

Flt3+ macrophage precursors commit sequentially to osteoclasts, dendritic cells and microglia.

BACKGROUND: Macrophages, osteoclasts, dendritic cells, and microglia are highly specialized cells that belong to the mononuclear phagocyte system. Functional and phenotypic heterogeneity within the mononuclear phagocyte system may reveal differentiation plasticity of a common progenitor, but developmental pathways leading to such diversity are still unclear. RESULTS: Mouse bone marrow cells were expanded in vitro in the presence of Flt3-ligand (FL), yielding high numbers of non-adherent cells exhibiting immature monocyte characteristics. Cells expanded for 6 days, 8 days, or 11 days (day 6-FL, day 8-FL, and day 11-FL cells, respectively) exhibited constitutive potential towards macrophage differentiation. In contrast, they showed time-dependent potential towards osteoclast, dendritic, and microglia differentiation that was detected in day 6-, day 8-, and day 11-FL cells, in response to M-CSF and receptor activator of NFkappaB ligand (RANKL), granulocyte-macrophage colony stimulating-factor (GM-CSF) and tumor necrosis factor-alpha (TNFalpha), and glial cell-conditioned medium (GCCM), respectively. Analysis of cell proliferation using the vital dye CFSE revealed homogenous growth in FL-stimulated cultures of bone marrow cells, demonstrating that changes in differential potential did not result from sequential outgrowth of specific precursors. CONCLUSIONS: We propose that macrophages, osteoclasts, dendritic cells, and microglia may arise from expansion of common progenitors undergoing sequential differentiation commitment. This study also emphasizes differentiation plasticity within the mononuclear phagocyte system. Furthermore, selective massive cell production, as shown here, would greatly facilitate investigation of the clinical potential of dendritic cells and microglia.