CD169+ macrophages in lymph node and spleen critically depend on dual RANK and LTbetaR signaling

CD169+ macrophages reside in lymph node (LN) and spleen and play an important role in the immune defense against pathogens. As resident macrophages, they are responsive to environmental cues to shape their tissue-specific identity. We have previously shown that LN CD169+ macrophages require RANKL for formation of their niche and their differentiation. Here, we demonstrate that they are also dependent on direct lymphotoxin beta (LTβ) receptor (R) signaling. In the absence or the reduced expression of either RANK or LTβR, their differentiation is perturbed, generating myeloid cells expressing SIGN-R1 in LNs. Conditions of combined haploinsufficiencies of RANK and LTβR revealed that both receptors contribute equally to LN CD169+ macrophage differentiation. In the spleen, the Cd169-directed ablation of either receptor results in a selective loss of marginal metallophilic macrophages (MMMs). Using a RANKL reporter mouse, we identify splenic marginal zone stromal cells as a source of RANKL and demonstrate that it participates in MMM differentiation. The loss of MMMs had no effect on the splenic B cell compartments but compromised viral capture and the expansion of virus-specific CD8+ T cells. Taken together, the data provide evidence that CD169+ macrophage differentiation in LN and spleen requires dual signals from LTβR and RANK with implications for the immune response.

Genotype and Th2 cells control monocyte to tissue resident macrophage differentiation during nematode infection of the pleural cavity

The recent revolution in tissue-resident macrophage biology has resulted largely from murine studies performed in the C57BL/6 strain. Here, we provide a comprehensive analysis of immune cells in the pleural cavity using both C57BL/6 and BALB/c mice. Unlike C57BL/6 mice, naive tissue-resident Large Cavity Macrophages (LCM) of BALB/c mice failed to fully implement the tissue residency program. Following infection with a pleural-dwelling nematode these pre-existing differences were accentuated with LCM expansion occurring in C57BL/6 but not BALB/c mice. While infection drove monocyte recruitment in both strains, only in C57BL/6 mice were monocytes able to efficiently integrate into the resident pool. Monocyte to macrophage conversion required both T cells and IL-4Rα signalling. Host genetics are therefore a key influence on tissue resident macrophage biology, and during nematode infection Th2 cells control the differentiation pathway of tissue resident macrophages.

Carbon Monoxide Regulates Macrophage Differentiation and Polarization toward the M2 Phenotype through Upregulation of Heme Oxygenase 1

Carbon monoxide (CO) is generated by heme oxygenase (HO), and HO-1 is highly induced in monocytes and macrophages upon stimulation. Monocytes differentiate into macrophages, including pro-inflammatory (M1) and anti-inflammatory (M2) cells, in response to environmental signals. The present study investigated whether CO modulates macrophage differentiation and polarization, by applying the CO-releasing molecule-3 (CORM-3). Results showed that murine bone marrow cells are differentiated into macrophages by CORM-3 in the presence of macrophage colony-stimulating factor. CORM-3 increases expressions of macrophage markers, including F4/80 and CD11b, and alters the cell morphology into elongated spindle-shaped cells, which is a typical morphology of M2 cells. CORM-3 upregulates the expressions of genes and molecules involved in M2 polarization and M2 phenotype markers, such as STAT6, PPARγ, Ym1, Fizz1, arginase-1, and IL-10. However, exposure to CORM-3 inhibits the iNOS expression, suggesting that CO enhances macrophage differentiation and polarization toward M2. Increased HO-1 expression is observed in differentiated macrophages, and CORM-3 further increases this expression. Hemin, an HO-1 inducer, results in increased macrophage differentiation, whereas the HO-1 inhibitor zinc protoporphyrin IX inhibits differentiation. In addition, CORM-3 increases the proportion of macrophages in peritoneal exudate cells and enhances the expression of HO-1 and arginase-1 but inhibits iNOS. Taken together, these results suggest that the abundantly produced CO in activated macrophages enhances proliferation, differentiation, and polarization toward M2. It will probably help clear apoptotic cells, resolve inflammation, and promote wound healing and tissue remodeling.

The Role of TGFβ1 and Macrophage Differentiation in MSU Crystal-Induced Inflammation

<p>Gout is a painful form of inflammatory arthritis that is caused by the deposition of monosodium urate (MSU) crystals in the joints. MSU crystals trigger a local inflammatory response initiated by resident macrophages followed by a large infiltration of leukocytes. The spontaneous resolution of acute gout is associated with the production of transforming growth factor β1 (TGFβ1). The overall objectives of this thesis were to investigate mechanisms that lead to TGFβ1 production and contribute to the resolution of acute gout, the effect of TGFβ1 on the functional phenotype of differentiated macrophages, and possible changes in surface marker expression by macrophages in response to MSU crystals. To determine macrophage-independent sources of TGFβ1 during the resolution of acute gout and how TGFβ1 production altered MSU crystal-recruited neutrophil functions, neutrophils were purified from MSU crystal-treated mice when levels of TGFβ1 were high. MSU crystal-recruited neutrophils and circulating blood neutrophils were identified as TGFβ1⁺ cells. The mechanism for TGFβ1 production by neutrophils was associated with their ability to phagocytose apoptotic neutrophils. TGFβ1 produced by canibalising neutrophils inhibited both respiratory burst and interleukin-1β (IL-1β) production by MSU crystal-activated neutrophils ex vivo. Importantly, neutrophils from MSU crystal-challenged mice treated with TGFβ1 neutralising antibody in vivo produced elevated levels of superoxide but neutrophil IL-1β production was unaffected. These results show that TGFβ1 produced by canibalising neutrophils can actively suppress neutrophil inflammatory functions and therefore make a significant contribution towards the resolution of gouty inflammation. To investigate the effect of TGFβ1 on macrophage differentiation in vitro, granulocyte macrophage colony-stimulating factor (GM-CSF) bone marrow macrophages (GM-BMMs) and macrophage colony-stimulating factor (M-CSF) bone marrow macrophages (M-BMMs) were generated in the presence of TGFβ1. TGFβ1 was found to drive a hyper-inflammatory GM-BMM phenotype, while contributing to the differentiation of a hypo-inflammatory M-BMM phenotype specifically in response to MSU crystals. Increased IL-1β production by TGFβ1-differentiated GM-BMMs was associated with enhanced NOD like receptor family, pyrin domain-containing 3 (NLRP3) in ammasome activation and caspase 1/caspase 8 interaction, and a down-regulation of receptor-interacting serine/threonine-protein kinase 3 (RIP3) triggered by MSU crystals. At the same, TGFβ1 inhibited antigen-specific T cell proliferation by GM-BMMs. In contrast, TGFβ1-treated M-BMMs down-regulated the expression of active IL-1β that correlated with decreased IL-1β production, and upregulated RIP3 expression in response to MSU crystals. These data indicate that TGFβ1-treated GM-BMMs exhibited a hyper-inflammatory response to MSU crystal stimulation, whereas M-BMMs were found to be hypo-responsive. Macrophages were found to upregulate the surface marker NK1.1, which is primarily expressed on natural killer (NK) cells, and occured as a consequence of phagocytosis. Following phagocytosis of MSU crystals, activated macrophages produced IL-1β and tumour necrosis factor ⍺ (TNF⍺), which triggered the upregulation of NK1.1 expression. Macrophage NK1.1 expression is an activation-driven event specifc to MSU crystals. However, phagocytosis of apoptotic neutrophils also triggered the upregulation of NK1.1 by macrophages, a non-inflammatory event that is characteristic for the resolution of acute inflammation. These findings suggest that macrophages may develop NK cell-like properties initiated by an activation-driven or apoptotic cell clearance mechanism. Taken together, the results of this thesis indicate that canibalising neutrophils self-regulate their inflammatory functions via TGFβ1 and that TGFβ1 drives a hyper-inflammatory GM-BMM phenotype, while shutting down inflammatory functions of M-BMMs. These data highlight a regulatory role for TGFβ1 during acute gouty inflammation.</p>

The Role of TGFβ1 and Macrophage Differentiation in MSU Crystal-Induced Inflammation

<p>Gout is a painful form of inflammatory arthritis that is caused by the deposition of monosodium urate (MSU) crystals in the joints. MSU crystals trigger a local inflammatory response initiated by resident macrophages followed by a large infiltration of leukocytes. The spontaneous resolution of acute gout is associated with the production of transforming growth factor β1 (TGFβ1). The overall objectives of this thesis were to investigate mechanisms that lead to TGFβ1 production and contribute to the resolution of acute gout, the effect of TGFβ1 on the functional phenotype of differentiated macrophages, and possible changes in surface marker expression by macrophages in response to MSU crystals. To determine macrophage-independent sources of TGFβ1 during the resolution of acute gout and how TGFβ1 production altered MSU crystal-recruited neutrophil functions, neutrophils were purified from MSU crystal-treated mice when levels of TGFβ1 were high. MSU crystal-recruited neutrophils and circulating blood neutrophils were identified as TGFβ1⁺ cells. The mechanism for TGFβ1 production by neutrophils was associated with their ability to phagocytose apoptotic neutrophils. TGFβ1 produced by canibalising neutrophils inhibited both respiratory burst and interleukin-1β (IL-1β) production by MSU crystal-activated neutrophils ex vivo. Importantly, neutrophils from MSU crystal-challenged mice treated with TGFβ1 neutralising antibody in vivo produced elevated levels of superoxide but neutrophil IL-1β production was unaffected. These results show that TGFβ1 produced by canibalising neutrophils can actively suppress neutrophil inflammatory functions and therefore make a significant contribution towards the resolution of gouty inflammation. To investigate the effect of TGFβ1 on macrophage differentiation in vitro, granulocyte macrophage colony-stimulating factor (GM-CSF) bone marrow macrophages (GM-BMMs) and macrophage colony-stimulating factor (M-CSF) bone marrow macrophages (M-BMMs) were generated in the presence of TGFβ1. TGFβ1 was found to drive a hyper-inflammatory GM-BMM phenotype, while contributing to the differentiation of a hypo-inflammatory M-BMM phenotype specifically in response to MSU crystals. Increased IL-1β production by TGFβ1-differentiated GM-BMMs was associated with enhanced NOD like receptor family, pyrin domain-containing 3 (NLRP3) in ammasome activation and caspase 1/caspase 8 interaction, and a down-regulation of receptor-interacting serine/threonine-protein kinase 3 (RIP3) triggered by MSU crystals. At the same, TGFβ1 inhibited antigen-specific T cell proliferation by GM-BMMs. In contrast, TGFβ1-treated M-BMMs down-regulated the expression of active IL-1β that correlated with decreased IL-1β production, and upregulated RIP3 expression in response to MSU crystals. These data indicate that TGFβ1-treated GM-BMMs exhibited a hyper-inflammatory response to MSU crystal stimulation, whereas M-BMMs were found to be hypo-responsive. Macrophages were found to upregulate the surface marker NK1.1, which is primarily expressed on natural killer (NK) cells, and occured as a consequence of phagocytosis. Following phagocytosis of MSU crystals, activated macrophages produced IL-1β and tumour necrosis factor ⍺ (TNF⍺), which triggered the upregulation of NK1.1 expression. Macrophage NK1.1 expression is an activation-driven event specifc to MSU crystals. However, phagocytosis of apoptotic neutrophils also triggered the upregulation of NK1.1 by macrophages, a non-inflammatory event that is characteristic for the resolution of acute inflammation. These findings suggest that macrophages may develop NK cell-like properties initiated by an activation-driven or apoptotic cell clearance mechanism. Taken together, the results of this thesis indicate that canibalising neutrophils self-regulate their inflammatory functions via TGFβ1 and that TGFβ1 drives a hyper-inflammatory GM-BMM phenotype, while shutting down inflammatory functions of M-BMMs. These data highlight a regulatory role for TGFβ1 during acute gouty inflammation.</p>

Calcium Dobesilate Modulates PKCδ-NADPH Oxidase- MAPK-NF-κB Signaling Pathway to Reduce CD14, TLR4, and MMP9 Expression during Monocyte-to-Macrophage Differentiation: Potential Therapeutic Implications for Atherosclerosis

Monocyte-to-macrophage differentiation results in the secretion of various inflammatory mediators and oxidative stress molecules necessary for atherosclerosis pathogenesis. Consequently, this differentiation represents a potential clinical target in atherosclerosis. Calcium dobesilate (CaD), an established vasoactive and angioprotective drug in experimental models of diabetic microvascular complications reduces oxidative stress and inhibits inflammation via diverse molecular targets; however, its effect on monocytes/macrophages is poorly understood. In this study, we investigated the anti-inflammatory mechanism of CaD during phorbol 12-myristate 13-acetate (PMA)-induced monocyte-to-macrophage differentiation in in vitro models of sepsis (LPS) and hyperglycemia, using THP-1 monocytic cell line. CaD significantly suppressed CD14, TLR4, and MMP9 expression and activity, lowering pro-inflammatory mediators, such as IL1β, TNFα, and MCP-1. The effects of CaD translated through to studies on primary human macrophages. CaD inhibited reactive oxygen species (ROS) generation, PKCδ, MAPK (ERK1/2 and p38) phosphorylation, NOX2/p47phox expression, and membrane translocation. We used hydrogen peroxide (H2O2) to mimic oxidative stress, demonstrating that CaD suppressed PKCδ activation via its ROS-scavenging properties. Taken together, we demonstrate for the first time that CaD suppresses CD14, TLR4, MMP9, and signature pro-inflammatory cytokines, in human macrophages, via the downregulation of PKCδ/NADPH oxidase/ROS/MAPK/NF-κB-dependent signaling pathways. Our data present novel mechanisms of how CaD alleviates metabolic and infectious inflammation.