Multifacted Role of TNF-α During the Pathogenesis of Rheumatoid Arthritis

Tumor necrosis factor alpha (TNF-α) a cytokine has been shown to be the key player during the pathogenesis of several autoimmune inflammatory disorders (presumably sterile inflammation) including rheumatoid arthritis (RA). Several studies have shown that TNF-α is mainly involved in the proinflammatory responses. However recent studies have reported multifunctional role of TNF-α during the development of RA. Therefore, in this article we have highlighted the distinct functions of TNF-α during pathogenesis of R

Novel Anti-Obesity Effects of Beer Hops Compound Xanthohumol: Role of AMPK Signaling Pathway

Background: Obesity alters adipose tissue metabolic and endocrine functioning, leading to an increased adiposity and release of pro-inflammatory cytokines. Various phytochemicals have been reported to contribute to the beiging of white adipose tissue in order to ameliorate obesity by increasing thermogenesis. Here, we show that the prenylated chalcone, xanthohumol (XN), induces beiging of white adipocytes, stimulates lipolysis, and inhibits adipogenesis of murine 3T3-L1 adipocytes and primary human subcutaneous preadipocytes and these effects are partly mediated by the activation of the AMP-activated protein kinase (AMPK) signaling pathway. Methods: 3T3-L1 adipocytes and primary human subcutaneous preadipocytes were differentiated using a standard protocol and were treated with various concentrations of XN, dorsomorphin, an AMPK inhibitor, or AICAR, an AMPK activator, to investigate the effects on adipogenesis, beiging and lipolysis. Results: XN induced beiging of white adipocytes as witnessed by the increased expression of beige markers CIDE-A and TBX-1. XN increased mitochondrial biogenesis, as evidenced by increased mitochondrial content, enhanced expression of PGC-1α, and the thermogenic protein UCP1. Following 24 h of treatment, XN also increased oxygen consumption rate. XN stimulated lipolysis of mature 3T3-L1 and primary human subcutaneous adipocytes and inhibited adipogenesis of maturing adipocytes. XN activated AMPK and in turn, XN-induced upregulation of UCP1, p-ACC, HSL, and ATGL was downregulated in the presence of dorsomorphin. Likewise, an XN-induced decrease in adipogenesis was reversed in the presence of dorsomorphin. Conclusions: Taken together, XN demonstrates anti-obesity effects by not only inducing beiging but also decreasing adipogenesis and inducing lipolysis. The anti-obesity effects of XN are partly mediated by AMPK signaling pathway suggesting that XN may have potential therapeutic implications for obesity

Xanthohumol Stimulates the Secretion of Catecholamines and Induces M2 Polarization in Raw 264.7 Macrophages

Anti-inflammatory, anti-oxidant, and anti-cancer effects of xanthohumol (XN), a prenylated chalcone extracted from common hop plants, are gaining attention and research has been expanding on the beneficial effects of this compound. In this study, we have investigated the anti-inflammatory effects of XN using a mouse monocytic cell line, RAW 264.7. We hypothesized that the anti-inflammatory effects of XN are due to M2 polarization of macrophages which, in turn, is mediated partly through the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway. RAW 264.7 cells were treated with either 0.1% DMSO or XN at varying concentrations for 24hrs. Cell culture supernatant was collected for ELISA and whole cell lysates were collected for Western blotting experiments. Our results suggest that XN upregulated the secretion of interleukin 10 (IL10), a signature cytokine for M2 polarization, in RAW 264.7 cells after 24 hours. We further demonstrated that XN increased arginase expression, a marker for M2 polarization, and failed to increase inducible nitric oxide synthase (iNOS) expression, a marker for M1 polarization. XN decreased interferon-γ (IFNγ) induced elevation of nitrite release, indicating the inhibitory effects of XN against M1 polarization. Additionally, XN at 25µM increased the secretion of catecholamines from macrophages comparable to interleukin 4 (IL4), an inducer of the M2 phenotype. Finally, XN upregulated the expression of phospho-AMPK in RAW 264.7 cells, indicating the role of AMPK signaling pathway in XN-induced effects. These results provide evidence for the anti-inflammatory properties of XN–mediated induction of M2 polarization. The M2 macrophage mediated anti-inflammatory effects, coupled with catecholamine secretion, and previously anti-adipogenic effects, makes XN an attractive molecule to study its beneficial effects on metabolic disease, like obesity and diabetes, that are associated with underlying chronic, low-grade inflammation

Direct and Indirect Effects of Guggulsterone on the Induction of Beiging in Mature 3T3-L1 Adipocytes

Phytochemicals have long demonstrated anti-obesity properties in adipocytes. Their ability, however, to induce browning in white adipose tissue is only beginning to emerge. We have recently established that the white adipocyte cell line, 3T3-L1, is capable of beiging under beta-adrenergic conditions. Using this information, we sought to investigate if the plant steroid guggulsterone (GS) can induce beiging in 3T3-L1s. 3T3-L1 preadipocytes were differentiated using established protocols supplemented with rosiglitazone and thyroid hormone. Direct effects of GS were measured by treating mature 3T3-L1s for 24 hours. Indirect effects were measured by treating mature 3T3-L1s with conditioned media from GS-treated RAW 264.7 macrophages. Direct treatment of 3T3-L1s with GS resulted in increased lipolysis, increased mitochondrial activity (11%), and increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) levels by 250% more than control. 3T3-L1s also demonstrated an increase in uncoupling protein 1 (UCP1) expression by 200% and beige marker, T-box protein 1 (TBX1) expression by 80% more than control. Furthermore, this was accompanied by increased levels of G protein-coupled bile acid receptor (TGR5) and its downstream target iodothyronine deiodinase 2 (DIO2). Treatment of RAW 264.7 macrophages with GS induced a 60% increase in catecholamine release into the media compared to control. Using this conditioned media from macrophages, 3T3-L1 adipocytes increased the expression of DIO2 and UCP1 following 24 hours of incubation. Results from this study demonstrate that GS can potentially induce beiging in white adipose tissue through two distinct mechanisms: (1) direct signaling through the TGR5-cAMP-DIO2 pathway and (2) indirectly through stimulating catecholamine release in macrophages. Thus, it is reasonable to conclude that GS may improve the metabolic capacity of adipose tissue thereby counteracting the effects of obesity

Guggulsterone Activates Adipocyte Beiging through Direct Effects on 3T3-L1 Adipocytes and Indirect Effects Mediated through RAW264.7 Macrophages.

Background: Plant-derived phytochemicals have been of emerging interest as anti-obesity compounds due to their apparent effects on promoting reduced lipid accumulation in adipocytes. Despite such promising evidence, little is known about the potential mechanisms behind their anti-obesity effects. The aim of this study is to establish potential anti-obesity effects of the phytochemical guggulsterone (GS). Methods: Mature 3T3-L1 adipocytes were treated with GS, derived from the guggul plant native in northern India, to investigate its effects on mitochondrial biogenesis and adipocyte beiging. Further, to explore the relationship between macrophages and adipocytes, 3T3-L1s were treated with conditioned media from GS-treated RAW264.7 macrophages. Markers of mitochondrial biogenesis and beiging were measured by western blot. Results: GS treatment in adipocytes resulted in increased mitochondrial density, biogenesis (PGC1α and PPARγ), and increased markers of a beige adipocyte phenotype (UCP1, TBX1, and β-3AR). This upregulation in mitochondrial expression was accompanied by increases oxygen consumption. In GS-treated macrophages, markers of M2 polarization were elevated (e.g., arginase and IL-10), along with increased catecholamine release into the media. Lastly, 3T3-L1 adipocytes treated with conditioned media from macrophages induced a 167.8% increase in UCP1 expression, suggestive of a role of macrophages in eliciting an anti-adipogenic response to GS. Conclusions: Results from this study provide the first mechanistic understanding of the anti-obesity effects of GS and suggests a role for both direct GS-signaling and indirect stimulation of M2 macrophage polarization in this model

Effects of Anchor Structure and Glycosylation of Fcγ Receptor III on Ligand Binding Affinity

Isoforms of the Fcγ receptor III (FcγRIII or CD16) are cell surface receptors for the Fc portion of IgG and important regulators of humoral immune responses. Different ligand binding kinetics of FcγRIII isoforms are obtained in three dimensions by surface plasmon resonance and in two dimensions by a micropipette adhesion frequency assay. We show that the anchor structure of CD16 isoforms isolated from the cell membrane affects their binding affinities in a ligand-specific manner. Changing the receptor anchor structure from full to partial to none decreases the ligand binding affinity for human IgG1 (hIgG1) but increases it for murine IgG2a (mIgG2a). Removing N-glycosylation from the CD16 protein core by tunicamycin also increases the ligand binding affinity. Molecular dynamics simulations indicate that deglycosylation at Asn-163 of CD16 removes the steric hindrance for the CD16-hIgG1 Fc binding and thus increases the binding affinity. These results highlight an unexpected sensitivity of ligand binding to the receptor anchor structure and glycosylation and suggest their respective roles in controlling allosterically the conformation of the ligand binding pocket of CD16

Immune Complex-Induced, Nitric Oxide-Mediated Vascular Endothelial Cell Death by Phagocytes Is Prevented with Decoy FcyReceptors

Autoimmune vasculitis is an endothelial inflammatory disease that results from the deposition of immune-complexes (ICs) in blood vessels. The interaction between Fcgamma receptors (FcyRs) expressed on inflammatory cells with ICs is known to cause blood vessel damage. Hence, blocking the interaction of ICs and inflammatory cells is essential to prevent the IC-mediated blood vessel damage. Thus we tested if uncoupling the interaction of FcyRs and ICs prevents endothelium damage. Herein, we demonstrate that dimeric FcyR-Igs prevented nitric oxide (NO) mediated apoptosis of human umbilical vein endothelial cells (HUVECs) in an in vitro vasculitis model. Dimeric FcyR-Igs significantly inhibited the IC-induced upregulation of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) release by murine monocytic cell line. However, FcyR-Igs did not affect the exogenously added NO-induced upregulation of pro-apoptotic genes such as Bax (15 fold), Bak (35 fold), cytochrome-C (11 fold) and caspase-3 (30 fold) in HUVECs. In conclusion, these data suggest that IC-induced NO could be one of the major inflammatory mediator promoting blood vessel inflammation and endothelial cell death during IC-mediated vasculitis which can be effectively blocked by dimeric decoy FcyRs

Effect of Tunicamycin Treatment on the Cell Surface Expression and Function of Low Affinity Fc Gamma Receptor CD16

Only title is posted. Authors have restricted access to abstract and poster

Recombinant Dimeric FcgR-Ig Molecules

Autoimmune vasculitis is an endothelial inflammatory disease that results from deposition of immune-complexes (ICs) in blood vessels. Interaction of FcγRs expressed on inflammatory cells with ICs is known to cause blood vessel damage. Hence, blocking the interaction of ICs and inflammatory cells is prerequisite to prevent the blood vessel damage. Herein, we have shown that dimeric FcγR-Ig (CD16A-Ig and CD32A-Ig) molecules are able to block these interactions using in vitro and in vivo vasculitis models. FcγR-Igs could block 70% of RAW 267.4 cells binding to antibody-coated Human umbilical vein endothelial cells (HUVEC). FcγR-Igs significantly inhibited the IC-mediated expression of inducible nitric oxide synthase (iNOS) and Nitric oxide (NO) release in RAW 264.7 cells. We observed that exogenous NO induced the upregulation of pro-apoptotic genes such as Bax, Bak, caspase-3 and caspase-8 in HUVEC cells. Further, in vivo studies revealed that circulating ICs deposits in the capillaries of various vital organs but not in large arteries. Interestingly, dimeric FcγR-Igs are distributed in the areas where ICs are deposited. The co-localization of ICs and FcγR-Igs revealed that dimeric FcγR-Ig molecules bind specifically to ICs and thus prevent the vascular damage. Taken together, these results suggest that IC-induced NO might be a major factor promoting the blood vessel damage, which can effectively be blocked using recombinant dimeric FcγRs molecules during IC mediated vasculitis

Lipocalin2 Mediates Anti-Inflammatory Functions Through the Inhibition of STAT3 and Activation of STAT5 Signaling in Bone Marrow Derived Macrophages

Lipocalin2 (Lcn2) is significantly up-regulated during various inflammatory disorders including autoimmune arthritis, however, its mechanisms are still unknown. We have shown that Lcn2-knockout (Lcn2KO) mice developed severe serum-induced arthritis (STA) compared to wild-type (WT) mice with reduced neutrophil infiltration but more macrophage migration. Therefore, in this study we have investigated the role of Lcn2 in regulating macrophages. Bone marrow derived macrophages were prepared from Lcn2KO and WT mice and polarized to inflammatory (M1) and anti-inflammatory (M2) macrophages. The polarization of M1 (iNOS) and M2 (arginase-1) phenotypes was confirmed by Western blot analysis. We did not observe a difference in polarization of Lcn2KO and WT macrophages. However, anti-inflammatory cytokines such as TGF-β1 and IL-10 were significantly reduced in IC-stimulated M2 phenotype from Lcn2KO mice than WT mice. In agreement, we observed systemically elevated pro-inflammatory cytokines in Lcn2KO (TNF-α, IL-1β and IL-6) than WT mice during STA. Further, Lcn2 deficient M1 macrophages displayed increased STAT3 activation than WT cells. In addition, WT M2 phenotype exhibited elevated STAT5 activation than Lcn2KO cells. These results suggest that Lcn2 may promote the down-regulation of pro-inflammatory but up-regulate anti-inflammatory cytokines possibly through STAT3/STAT5 signaling pathways as a negative feedback loop, limiting inflammation during autoimmune arthritis conditions