Embryonic vitamin D deficiency programs hematopoietic stem cells to induce type 2 diabetes.

Environmental factors may alter the fetal genome to cause metabolic diseases. It is unknown whether embryonic immune cell programming impacts the risk of type 2 diabetes in later life. We demonstrate that transplantation of fetal hematopoietic stem cells (HSCs) made vitamin D deficient in utero induce diabetes in vitamin D-sufficient mice. Vitamin D deficiency epigenetically suppresses Jarid2 expression and activates the Mef2/PGC1a pathway in HSCs, which persists in recipient bone marrow, resulting in adipose macrophage infiltration. These macrophages secrete miR106-5p, which promotes adipose insulin resistance by repressing PIK3 catalytic and regulatory subunits and down-regulating AKT signaling. Vitamin D-deficient monocytes from human cord blood have comparable Jarid2/Mef2/PGC1a expression changes and secrete miR-106b-5p, causing adipocyte insulin resistance. These findings suggest that vitamin D deficiency during development has epigenetic consequences impacting the systemic metabolic milieu.

Novel SIRPα Antibodies That Induce Single-Agent Phagocytosis of Tumor Cells while Preserving T Cells.

The signal regulatory protein α (SIRPα)/CD47 axis has emerged as an important innate immune checkpoint that enables cancer cell escape from macrophage phagocytosis. SIRPα expression is limited to macrophages, dendritic cells, and neutrophils-cells enriched in the tumor microenvironment. In this study, we present novel anti-SIRP Abs, SIRP-1 and SIRP-2, as an approach to targeting the SIRPα/CD47 axis. Both SIRP-1 and SIRP-2 bind human macrophage SIRPα variants 1 and 2, the most common variants in the human population. SIRP-1 and SIRP-2 are differentiated among reported anti-SIRP Abs in that they induce phagocytosis of solid and hematologic tumor cell lines by human monocyte-derived macrophages as single agents. We demonstrate that SIRP-1 and SIRP-2 disrupt SIRPα/CD47 interaction by two distinct mechanisms: SIRP-1 directly blocks SIRPα/CD47 and induces internalization of SIRPα/Ab complexes that reduce macrophage SIRPα surface levels and SIRP-2 acts via disruption of higher-order SIRPα structures on macrophages. Both SIRP-1 and SIRP-2 engage FcγRII, which is required for single-agent phagocytic activity. Although SIRP-1 and SIRP-2 bind SIRPγ with varying affinity, they show no adverse effects on T cell proliferation. Finally, both Abs also enhance phagocytosis when combined with tumor-opsonizing Abs, including a highly differentiated anti-CD47 Ab, AO-176, currently being evaluated in phase 1 clinical trials, NCT03834948 and NCT04445701 SIRP-1 and SIRP-2 are novel, differentiated SIRP Abs that induce in vitro single-agent and combination phagocytosis and show no adverse effects on T cell functionality. These data support their future development, both as single agents and in combination with other anticancer drugs.

Deletion of JNK2 prevents vitamin-D-deficiency-induced hypertension and atherosclerosis in mice.

The c-Jun N-terminal kinase 2 (JNK2) signaling pathway contributes to inflammation and plays a key role in the development of obesity-induced insulin resistance and cardiovascular disease. Macrophages are key cells implicated in these metabolic abnormalities. Active vitamin D downregulates macrophage JNK activation, suppressing oxidized LDL cholesterol uptake and foam cell formation and promoting an anti-inflammatory phenotype. To determine whether deletion of JNK2 prevents high blood pressure and atherosclerosis known to be induced by vitamin D deficiency in mice, we generated mice with knockout of JNK2 in a background susceptible to diet-induced atherosclerosis (LDLR-/-). JNK2-/- LDLR-/- and LDLR-/- control mice were fed vitamin D-deficient chow for 8 weeks followed by vitamin D-deficient high fat diet (HFD) for 10 weeks and assessed before and after HFD. There was no difference in fasting glucose, cholesterol, triglycerides, or free fatty acid levels. However, JNK2-/- mice, despite vitamin D-deficient diet, had 20-30mmHg lower systolic (SBP) and diastolic (DBP) blood pressure before HFD compared to control mice fed vitamin D-deficient diets, with persistent SBP differences after HFD. Moreover, deletion of JNK2 reduced HFD-induced atherosclerosis by 30% in the proximal aorta when compared to control mice fed vitamin D-deficient diets. We have previously shown that peritoneal macrophages obtained from LDLR-/- mice fed vitamin D-deficient HFD diets have higher foam cell formation compared to those from mice on vitamin D-sufficient HFD. The increased total cellular cholesterol and modified cholesterol uptake in macrophages from mice on vitamin D-deficient HFD were blunted by deletion of JNK2. These data suggest that JNK2 signaling activation is necessary for the atherosclerosis and hypertension induced by vitamin D deficiency.

Vitamin D3 supplementation decreases a unique circulating monocyte cholesterol pool in patients with type 2 diabetes.

Cross-sectional studies indicate consistent associations between low 25(OH)D concentration and increased risk of cardiovascular disease (CVD), but results of randomized control trials (RCTs) are mixed. However, the majority of the RCTs do not focus on type 2 diabetics, potentially obscuring the effects of vitamin D in this population. In vitro 1,25(OH)2D3 downregulates macrophage cholesterol deposition, but the in vivo effects are unknown. To explore potential mechanisms of the effects of vitamin D on CVD risk in patients with type 2 diabetes, we isolated monocytes in a subset of 26 patients from our RCT of diabetics with baseline serum 25(OH)D <25ng/mL randomized to vitamin D3 4000 IU/day or placebo for 4 months. Upon enrollment, the mean 25(OH)D level was 17ng/mL, which increased to 36ng/mL after vitamin D and remained unchanged in the placebo group. Before randomization, groups demonstrated similar mean hemoglobin A1c and plasma lipids levels, none of which was significantly altered by vitamin D supplementation. Moreover, assessment of oxidized LDL uptake in monocytes cultured in the patient's own serum before vs. after treatment resulted in >50% reduction in the vitamin D group with no change in the placebo group. This was mediated through suppression of endoplasmic reticulum stress and scavenger receptor CD36 protein expression. The reduction in monocyte cholesterol uptake was reflected in a 19% decrease in total monocyte cholesterol content. Interestingly, cross-sectional analysis of circulating monocytes from vitamin D-deficient vs. sufficient diabetic patients revealed 8-fold higher cholesteryl ester content, confirming the capacity of these monocytes to uptake and carry cholesterol in the circulation. This study identifies a unique circulating cholesterol pool within monocytes that is modulated by vitamin D and has the potential to contribute to CVD in type 2 diabetes.

Deletion of macrophage Vitamin D receptor promotes insulin resistance and monocyte cholesterol transport to accelerate atherosclerosis in mice.

Intense effort has been devoted to understanding predisposition to chronic systemic inflammation because it contributes to cardiometabolic disease. We demonstrate that deletion of the macrophage vitamin D receptor (VDR) in mice (KODMAC) is sufficient to induce insulin resistance by promoting M2 macrophage accumulation in the liver as well as increasing cytokine secretion and hepatic glucose production. Moreover, VDR deletion increases atherosclerosis by enabling lipid-laden M2 monocytes to adhere, migrate, and carry cholesterol into the atherosclerotic plaque and by increasing macrophage cholesterol uptake and esterification. Increased foam cell formation results from lack of VDR-SERCA2b interaction, causing SERCA dysfunction, activation of ER stress-CaMKII-JNKp-PPARγ signaling, and induction of the scavenger receptors CD36 and SR-A1. Bone marrow transplant of VDR-expressing cells into KODMAC mice improved insulin sensitivity, suppressed atherosclerosis, and decreased foam cell formation. The immunomodulatory effects of vitamin D in macrophages are thus critical in diet-induced insulin resistance and atherosclerosis in mice.

Epimorphin(-/-) mice are protected, in part, from acute colitis via decreased interleukin 6 signaling.

Epimorphin (Epim), a member of the syntaxin family of membrane-bound, intracellular vesicle-docking proteins, is expressed in intestinal myofibroblasts and macrophages. We demonstrated previously that Epimorphin(-/-)(Epim(-/-)) mice are protected, in part, from dextran sodium sulfate (DSS)-induced colitis. Although interleukin (IL)-6/p-Stat3 signaling has been implicated in the pathogenesis of colitis, the myofibroblast contribution to IL-6 signaling in colitis remains unexplored. Our aim was to investigate the IL-6 pathway in Epim(-/-) mice in the DSS colitis model. Whole colonic tissue, epithelium, and stroma of WT and congenic Epim(-/-) mice treated with 5% DSS for 7 days were analyzed for IL-6 and a downstream effector, p-Stat3, by immunostaining and immunoblot. Colonic myofibroblast and peritoneal macrophage IL-6 secretion were evaluated by enzyme-linked immunosorbent assay. IL-6 and p-Stat3 expression were decreased in Epim(-/-) vs WT colon. A relative increase in stromal vs epithelial p-Stat3 expression was observed in WT mice but not in Epim(-/-) mice. Epim deletion abrogates IL-6 secretion from colonic myofibroblasts treated with IL-1ß and decreases IL-6 secretion from peritoneal macrophages in a subset of DSS-treated mice. Epim deletion inhibits IL-6 secretion most profoundly from colonic myofibroblasts. Distribution of Stat3 activation is altered in DSS-treated Epim(-/-) mice. Our findings support the notion that myofibroblasts modulate IL-6/p-Stat3 signaling in DSS-treated Epim(-/-) mice.

25(OH) vitamin D suppresses macrophage adhesion and migration by downregulation of ER stress and scavenger receptor A1 in type 2 diabetes.

Cardiovascular disease (CVD) is the leading cause of mortality in patients with type 2 diabetes mellitus (T2DM). Vitamin D deficiency is not only more prevalent in diabetics but also doubles the risk of developing CVD. However, it is unknown whether 25-hydroxy vitamin D [25(OH)D3] replacement slows monocyte adhesion and migration, critical mechanisms involved in atherosclerosis progression. In this study, monocytes from vitamin D-deficient diabetic patients were cultured either in the patient's serum or in vitamin D-deficient media with or without 25(OH)D3 treatment. Adding 25(OH)D3 to monocytes cultured in vitamin D-deficient serum or media decreased monocyte adhesion to fibronectin and migration stimulated by monocyte chemotactic protein 1 (MCP-1). Accordingly, 25(OH)D3 decreased adhesion marker ß1- and ß2-integrin expression and migration receptor chemokine (C-C motif) receptor 2 (CCR2) expression. 25(OH)D3 treatment downregulated monocyte endoplasmic reticulum (ER) stress and scavenger receptor class A, type 1 (SR-A1) expression. The absence of SR-A1 prevented the increased macrophage adhesion and migration induced by vitamin D deficiency. Moreover, the absence of SR-A1 prevented the induction of adhesion and migration and expression of their associated membrane receptors by Thapsigargin, an ER stress inducer. These results identify cellular activation of monocyte/macrophage vitamin D signaling through 25(OH)D3 as a potential mechanism that could modulate adhesion and migration in diabetic subjects. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

Stromal cells participate in the murine esophageal mucosal injury response.

We identified α-smooth muscle actin (α-SMA)- and vimentin-expressing spindle-shaped esophageal mesenchymal cells in the adult and neonate murine esophageal lamina propria. We hypothesized that these esophageal mesenchymal cells express and secrete signaling and inflammatory mediators in response to injury. We established primary cultures of esophageal mesenchymal cells using mechanical and enzymatic digestion. We demonstrate that these primary cultures are nonhematopoietic, nonendothelial, stromal cells with myofibroblast-like features. These cells increase secretion of IL-6 in response to treatment with acidified media and IL-1ß. They also increase bone morphogenetic protein (Bmp)-4 secretion in response to sonic hedgehog. The location of these cells and their biological functions demonstrate their potential role in regulating esophageal epithelial responses to injury and repair.

Tyrosine phosphorylation is required for IkappaB kinase-beta (IKKbeta) activation and function in osteoclastogenesis.

The transcription factor NF-kappaB is crucial for numerous cellular functions such as survival, differentiation, immunity, and inflammation. A key function of this family of transcription factors is regulation of osteoclast differentiation and function, which in turn controls skeletal homeostasis. The IkappaB kinase (IKK) complex, which contains IKKalpha, IKKbeta, and IKKgamma, is required for activation of NF-kappaB, and deletion of either IKKalpha or IKKbeta resulted with defective osteoclast differentiation and survival. We have recently investigated the details of the mechanisms governing the role of IKKbeta in osteoclastogenesis and found that constitutively active IKKbeta in which serine residues 177/181 were mutated into negatively charged glutamic acids instigates spontaneous bona fide receptor activator of NF-kappaB ligand (RANKL)-independent osteoclastogenesis. To better understand and define the functional role of IKKbeta domains capable of regulating the osteoclastogenic activity of IKK, we investigated key motifs in the activation T loop of IKKbeta, which are potentially capable of modulating its osteoclastogenic activity. We discovered that dual serine (traditional serine residues 177/181) and tyrosine (188/199) phosphorylation events are crucial for IKKbeta activation. Mutation of the latter tyrosine residues blunted the NF-kappaB activity of wild type and constitutively active IKKbeta, and tyrosine 188/199-deficient IKKbeta inhibited osteoclastogenesis. Thus, tyrosines 188/199 are a novel target for regulating IKKbeta activity, at least in osteoclasts.

IKKbeta activation is sufficient for RANK-independent osteoclast differentiation and osteolysis.

Monocytes differentiate into osteoclasts through stimulation of receptor activator of NF-kappaB (RANK). Many downstream effectors of RANK play a positive role in osteoclastogenesis, but their relative importance in osteoclast differentiation is unclear. We report the discovery that activation of a single pathway downstream of RANK is sufficient for osteoclast differentiation. In this regard, introduction of constitutively activated IKKbeta (IKKbeta(SSEE)) but not wild-type IKKbeta into monocytes stimulates differentiation of bona fide osteoclasts in the absence of RANK ligand (RANKL). This phenomenon is independent of upstream signals because IKKbeta(SSEE) induced the development of bone-resorbing osteoclasts from RANK and IKKalpha knockout monocytes and in conditions in which NEMO-IKKbeta association was inhibited. NF-kappaB p100 and p105, but not RelB, were critical mediators of this effect. Inflammatory autocrine signaling by tumor necrosis factor alpha (TNF-alpha) and interleukin 1 (IL-1) were dispensable for the spontaneous osteoclastogenesis driven by IKKbeta(SSEE). More important, adenoviral gene transfer of IKKbeta(SSEE) induced osteoclasts and osteolysis in calvariae and knees of mice. Our data establish the sufficiency of IKKbeta activation for osteolysis and suggest that IKKbeta hyperactivation may play a role in conditions of pathologic bone destruction refractory to RANK/RANKL proximal therapeutic interventions.

Impediment of NEMO oligomerization inhibits osteoclastogenesis and osteolysis.

The transcription factor NF-kappaB is essential for osteoclastogenesis and is considered an immune-modulator of rheumatoid arthritis and inflammatory osteolysis. Activation of NF-kappaB subunits is regulated by the upstream IkappaB kinase (IKK) complex which contains IKKalpha, IKKbeta, and IKKgamma; the latter also known as NF-kappaB essential modulator (NEMO). The role of IKKalpha and IKKbeta in the skeletal development and inflammatory osteolysis has been described, whereas little is known regarding the role of NEMO in this setting. Typically, signals induced by RANK ligand (RANKL) or TNF prompt oligomerization of NEMO monomers through the coiled-coil-2 (CC2) and leucine zipper (LZ) motifs. This step facilitates binding to IKKs and further relaying signal transduction. Given the central role of NF-kappaB in osteoclastogenesis, we asked whether NEMO is essential for osteoclastogenesis and whether interruption of NEMO oligomerization impedes osteoclast differentiation in vitro and in vivo. Using cell-permeable short peptides overlapping the CC2 and LZ motifs we show that these peptides specifically bind to NEMO monomers, prevent trimer formation, and render NEMO monomers susceptible for ubiquitin-mediated degradation. Further, CC2 and LZ peptides attenuate RANKL- and TNF-induced NF-kappaB signaling in bone marrow-derived osteoclast precursors (OCPs). More importantly, these peptides potently inhibit osteoclastogenesis, in vitro, and arrest RANKL-induced osteolysis, in mice. To further ascertain its role in osteoclastogenesis, we were able to block osteoclastogenesis using NEMO siRNA knockdown approach. Collectively, our data establish that obstruction of NEMO oligomerization destabilizes NEMO monomers, inhibits NF-kappaB activation, impedes osteoclastogenesis and arrests inflammatory osteolysis. Thus, NEMO presents itself as a promising target for anti-osteolytic intervention.

Role of the NF-kappaB axis in immune modulation of osteoclasts and bone loss.

NF-kappaB is a vital component of the molecular programs for immune cell development and activation, inflammatory responses, and osteoclast differentiation. This transcriptional regulatory family is activated by diverse immunological and inflammatory stimuli and contributes to both positive feedback of the immune and osteolytic responses as well as their resolution. The ubiquilous expression of NF-kappaB components in osteoclasts and other immune cells creates an opportunity to gain a better understanding of the complex interplay between the immune and skeletal systems in physiological and pathological conditions and also makes NF-kappaB an important target in the treatment of autoimmune, inflammatory, and osteolytic diseases. Indeed, many genetic murine models have recently been developed which highlight the importance of NF-kappaB in basic processes including lymphocyte development, macrophage activation, and osteoclast differentiation. Furthermore, inhibition of NF-kappaB signaling has been demonstrated to ameliorate tissue inflammation and osteolysis in mouse models of inflammatory disease. A more complete understanding of the immunological factors that regulate NF-kappaB and the role that NF-kappaB plays in the immune and skeletal systems will elucidate potential avenues for intervening therapeutically in the pathological conditions of inflammation and osteolysis.

Aseptic loosening of total joint replacements: mechanisms underlying osteolysis and potential therapies.

Total joint replacement, although considered an excellent surgical procedure, can be complicated by osteolysis induced by implant particles and subsequent aseptic loosening of the implant. The pathogenesis of implant-associated osteolysis includes inflammatory and osteolytic processes. The sustained chronic inflammatory response initiated by particulate debris at the implant-bone interface is manifested by recruitment of a wide array of cell types. These cells include macrophages, fibroblasts, giant cells, neutrophils, lymphocytes, and--most importantly--osteoclasts, which are the principal bone resorbing cells. The 'cellular response' entails secretion of osteoclastogenic and inflammatory cytokines that favor exacerbated osteoclast activity and enhanced osteolysis. An appreciation of the complex network that leads to these cellular and inflammatory responses will form a foundation on which to develop therapeutic interventions to combat inflammatory periprosthetic bone loss.