Vascular lipid droplets formed in response to TNF,hypoxia, or OA: biochemical composition and prostacyclin generation

Biogenesis of lipid droplets (LDs) in various cells plays an important role in various physiological and pathological processes. However, the function of LDs in endothelial physiology and pathology is not well understood. In the present work, we investigated the formation of LDs and prostacyclin (PGI₂) generation in the vascular tissue of isolated murine aortas following activation by proinflammatory factors: tumor necrosis factor (TNF), lipopolysaccharides (LPS), angiotensin II (AngII), hypoxic conditions, or oleic acid (OA). The abundance, size, and biochemical composition of LDs were characterized based on Raman spectroscopy and fluorescence imaging. We found that blockade of lipolysis by the adipose triglyceride lipase (ATGL) delayed LDs degradation and simultaneously blunted PGI₂ generation in aorta treated with all tested proinflammatory stimuli. Furthermore, the analysis of Raman spectra of LDs in the isolated vessels stimulated by TNF, LPS, AngII, or hypoxia uncovered that these LDs were all rich in highly unsaturated lipids and had a negligible content of phospholipids and cholesterols. Additionally, by comparing the Raman signature of endothelial LDs under hypoxic or OA-overload conditions in the presence or absence of ATGL inhibitor, atglistatin (Atgl), we show that Atgl does not affect the biochemical composition of LDs. Altogether, independent of whether LDs were induced by pro-inflammatory stimuli, hypoxia, or OA and of whether they were composed of highly unsaturated or less unsaturated lipids, we observed LDs formation invariably associated with ATGL-dependent PGI₂ generation. In conclusion, vascular LDs formation and ATGL-dependent PGI₂ generation represent a universal response to vascular proinflammatory insult.     

Genome-wide CRISPR-Cas9 viability screen reveals genes involved in TNF-α-induced apoptosis of human umbilical vein endothelial cells

Tumor necrosis factor α (TNF-α), a pivotal cytokine in sepsis, protects the host against pathogens by promoting an inflammatory response while simultaneously inducing apoptosis of the vascular endothelium. Unfortunately, inhibitors targeting certain components of the TNF-α signaling pathway to reduce cellular apoptosis have failed to translate into clinical applications, partly due to the adverse effects of excessive immunosuppression. In an attempt to discover potential targets in the TNF-α signaling pathway to modulate moderate inflammation and apoptosis during the development of sepsis, we performed a pooled genome-wide CRISPR/Cas9 knockout screen in human umbilical vein endothelial cells (HUVECs). Tumor necrosis factor receptor superfamily member 1A (TNFRSF1A), B-cell lymphoma 2 (BCL2), Bcl2-associated death promoter (BAD), and NLR family member X1 (NLRX1) deficiencies were identified as the effective genetic suppressors of TNF-α cytotoxicity on a list of candidate regulators. CRISPR-mediated NLRX1 knockout conferred cellular resistance to challenge with TNF-α, and NLRX1 could be induced to colocalize with mitochondria following TNF-α stimulation. Thus, our work demonstrates the advantage of genome-scale screening with Cas9 and validates NLRX1 as a potential modulator of TNF-α-induced vascular endothelial apoptosis during sepsis.     

Estimation of the content of lipids composing endothelial lipid droplets based on Raman imaging

Lipid droplets (LDs) are dynamic organelles involved in intracellular lipid metabolism, and the biogenesis of LDs in endothelium is triggered by the excess of lipids in the environment. In this paper we present the methodology aimed to define the composition of endothelial LDs formed upon stimulation with oleic acid (OA) in two models: endothelial cells cultured in vitro and in isolated blood vessel ex vivo. The biochemical composition of LDs was determined using Raman imaging, followed by the lipid unsaturation calibration analysis and modelling of spectral bands based on individual spectra of selected lipids. Among LDs formed in response to OA in vitro or ex vivo conditions there were two types of LDs; those with more unsaturated (average number of CC bonds equalled 1.40) or saturated (average number of CC bonds equalled 0.95) lipids. The modelling of endothelial LDs composition revealed the OA represented a major component of LDs (80.6–91.3%) with an important content of arachidonic acid (8.7–19.4%). In conclusion, endothelial LDs consist of exogenous oleic acid uptaken from the extracellular space, and the endogenous arachidonic acid released from plasma membranes.     

Differential response of liver sinusoidal endothelial cells and hepatocytes to oleic and palmitic acid revealed by Raman and CARS imaging

Excess circulating fatty acids contribute to endothelial dysfunction that subsequently aggravates the metabolic conditions such as fatty liver diseases. However, the exact mechanism of this event is not fully understood, and the investigation on the effect of a direct exposure to fatty acids together with their subsequent fate is of interest. In this work we employed a chemically specific and label-free techniques such as Raman and CARS microscopies, to investigate the process of lipid droplets (LDs) formation in endothelial cells and hepatocytes after exposure to oleic and palmitic acid. We aimed to observe the changes in the composition of LDs associated with metabolism and degradation of lipids. We were able to characterize the diversity in the formation of LDs in endothelium as compared to hepatocytes, as well as the differences in the formation of LDs and degradation manner with respect to the used fatty acid. Thus, for the first time the spectral characteristics of LDs formed in endothelial cells after incubation with oleic and palmitic acid is presented, including the time-dependent changes in their chemical composition.     

Lipid droplets formation in human endothelial cells in response to polyunsaturated fatty acids and 1‐methyl‐nicotinamide (MNA); confocal Raman imaging and fluorescence microscopy studies

In this work the formation of lipid droplets (LDs) in human endothelial cells culture in response to the uptake of polyunsaturated fatty acids (PUFAs) was studied. Additionally, an effect of 1‐methylnicotinamide (MNA) on the process of LDs formation was investigated. LDs have been previously described structurally and to some degree biochemically, however neither the precise function of LDs nor the factors responsible for LD induction have been clarified. Lipid droplets, sometimes referred in the literature as lipid bodies are organelles known to regulate neutrophil, eosinophil, or tumor cell functions but their presence and function in the endothelium is largely unexplored. 3D linear Raman spectroscopy was used to study LDs formation in vitro in a single endothelial cell. The method provides information about distribution and size of LDs as well as their composition. The incubation of endothelial cells with various PUFAs resulted in formation of LDs. As a complementary method for LDs identification a fluorescence microscopy was applied. Fluorescence measurements confirmed the Raman results suggesting endothelial cells uptake of PUFAs and subsequent LDs formation in the cytoplasm of the endothelium. Furthermore, MNA seem to potentiate intracellular uptake of PUFAs to the endothelium that may bear physiological and pharmacological significance.

Confocal Raman imaging of HAoEC cell with LDs.     

Effects of tumour necrosis factor-alpha on activity and nitric oxide synthase of endothelial progenitor cells from peripheral blood

The aim of this investigation was to determine whether tumour necrosis factor-alpha (TNF-α) has any effect on endothelial progenitor cells (EPCs). Total mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days culture, attached cells were stimulated with tumour necrosis factor-α (final concentrations: 0, 10, 20, 50 and 100 mg/l) for 0, 6, 12, 24 and 48 h. EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding, by direct fluorescence staining. EPC proliferation and migration were assayed using the MTT assay and modified Boyden chamber assay, respectively. EPC adhesion assay was performed by re-plating those cells on fibronectin-coated dishes, and adherent cells were counted. Tube formation activity was assayed using a tube formation kit. Levels of apoptosis were revealed using an annexin V apoptosis detection kit. Vascular endothelial growth factor Receptor-1 (VEGF-R1) and stromal derived factor-1 (SDF-1) mRNA, assessed by real-time RT-PCR inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were assayed by western blot analysis. Incubation of EPCs with tumour necrosis factor-α reduced EPC proliferation, migration, adhesion, tube formation capacity, iNOS and eNOS in concentration- and time-dependent manners. Tumour necrosis factor-α reduced proliferation, migration, adhesion and tube formation capacity of EPCs. TNF-α increased EPC apoptosis level, reduced VEGF-R1 and SDF-1 mRNA expression; tumour necrosis factor-α also reduced iNOS and eNOS in the EPCs.     

Renal tubular fluid shear stress promotes endothelial cell activation

Modified urinary fluid shear stress (FSS) induced by variations of urinary fluid flow and composition is observed in early phases of most kidney diseases. In this study, we hypothesized that changes in urinary FSS represent a tubular aggression that contributes to the development of inflammation, a key event in progression of nephropathies. Human renal tubular cells (HK-2) were exposed to FSS for 30 min at 0.01 Pa. Treatment of human endothelial cells (HMEC-1) with the resulting conditioned medium (FSS-CM) increased C-C chemokine ligand 2 (CCL2) and tumor necrosis factor (TNF)-α protein secretion, increased endothelial vascular adhesion molecule-1 (VCAM-1) mRNA expression and stimulated adhesion of human (THP-1) monocytes to the endothelial monolayer. These effects were TNF-α dependent as they were abolished by neutralization of TNF-α. Interestingly, the origin of TNF-α was not epithelial, but resulted from autocrine endothelial production. However, in contrast to short term FSS, long term FSS (5 h) induced the release of the key inflammatory proteins CCL2 and TNF-α directly from tubular cells. In conclusion, these results suggest for the first time that urinary FSS can contribute to the inflammatory state involved in initiation/perpetuation of renal diseases.     

Tumour necrosis factor-α stimulates human neutrophils to release preformed activin A

Activin A, a member of the transforming growth factor-β superfamily, is a critical early mediator of acute inflammation. Activin A release coincides with the release of tumour necrosis factor-α (TNF-α) in models of lipopolysaccharide (LPS)-induced inflammation. The source of circulating activin A during acute inflammation has not been identified and the potential contribution of leukocyte subsets was examined in the following study. Human leukocytes from healthy volunteers were fractionated using Ficoll gradients and cultured under serum-free conditions. Freshly isolated human neutrophils contained 20-fold more activin A than blood mononuclear cells as measured by enzyme-linked immunosorbent assay (ELISA), and both dimeric and monomeric forms of activin A were detected in these cells by western blotting. Activin A was predominantly immunolocalized in the neutrophil cytoplasm. Purified neutrophils secreted activin A in culture when stimulated by TNF-α, but were unable to respond to LPS directly. Although TNF-α stimulated activin A release from neutrophils within 1 h, activin subunit mRNA expression did not increase until 12 h of culture, and the amount of activin A released following TNF-α stimulation did not change between 1 and 12 h. Specific inhibition of the p38 MAP kinase signalling pathway blocked TNF-α-induced activin release, and the secretion of activin A was not due to TNF-α-induced neutrophil apoptosis. These data provide the first evidence that neutrophils are a significant source of mature, stored activin A. Stimulation of the release of neutrophil activin A by TNF-α may contribute to the early peak in circulating activin A levels during acute inflammation.     

Autocrine stimulation of osteoblast activity by Wnt5a in response to TNF-α in human mesenchymal stem cells

Although anti-tumor necrosis factor (TNF)-α treatments efficiently block inflammation in ankylosing spondylitis (AS), they are inefficient to prevent excessive bone formation. In AS, ossification seems more prone to develop in sites where inflammation has resolved following anti-TNF therapy, suggesting that TNF-α indirectly stimulates ossification. In this context, our objectives were to determine and compare the involvement of Wnt proteins, which are potent growth factors of bone formation, in the effects of TNF-α on osteoblast function. In human mesenchymal stem cells (MSCs), TNF-α significantly increased the levels of Wnt10b and Wnt5a. Associated with this effect, TNF-α stimulated tissue-non specific alkaline phosphatase (TNAP) and mineralization. This effect was mimicked by activation of the canonical β-catenin pathway with either anti-Dkk1 antibodies, lithium chloride (LiCl) or SB216763. TNF-α reduced, and activation of β-catenin had little effect on expression of osteocalcin, a late marker of osteoblast differentiation. Surprisingly, TNF-α failed to stabilize β-catenin and Dkk1 did not inhibit TNF-α effects. In fact, Dkk1 expression was also enhanced in response to TNF-α, perhaps explaining why canonical signaling by Wnt10b was not activated by TNF-α. However, we found that Wnt5a also stimulated TNAP in MSCs cultured in osteogenic conditions, and increased the levels of inflammatory markers such as COX-2. Interestingly, treatment with anti-Wnt5a antibodies reduced endogenous TNAP expression and activity. Collectively, these data suggest that increased levels of Dkk1 may blunt the autocrine effects of Wnt10b, but not that of Wnt5a, acting through non-canonical signaling. Thus, Wnt5a may be potentially involved in the effects of inflammation on bone formation.     

Raman spectroscopy–based insight into lipid droplets presence and contents in liver sinusoidal endothelial cells and hepatocytes

Liver sinusoidal endothelial cells (LSECs), a type of endothelial cells with unique morphology and function, play an important role in the liver hemostasis, and LSECs dysfunction is involved in the development of nonalcoholic fatty liver disease (NAFLD). Here, we employed Raman imaging and chemometric data analysis in order to characterize the presence of lipid droplets (LDs) and their lipid content in primary murine LSECs, in comparison with hepatocytes, isolated from mice on high‐fat diet. On NAFLD development, LDs content in LSECs changed toward more unsaturated lipids, and this response was associated with an increased expression of stearylo‐CoA desaturase‐1. To the best of our knowledge, this is a first report characterizing LDs in LSECs, where their chemical composition is analyzed along the progression of NAFLD at the level of single LD using Raman imaging.      

Pterostilbene exerts an anti-inflammatory effect via regulating endoplasmic reticulum stress in endothelial cells

Pterostilbene (PT), an analog of resveratrol, exerts a potent anti-inflammatory effect. However, the protective effects of PT against inflammation in endothelial cells have not been elucidated. Previous studies have confirmed that endoplasmic reticulum stress (ERS) plays an important role in regulating the pathological process of endothelial cell inflammation. In this study, we explored the effect of PT on the tumor necrosis factor-α (TNF-α)-induced inflammatory response in human umbilical vein endothelial cells (HUVECs) and elaborated the role of ERS in this process. TNF-α treatment significantly upregulated the levels of inflammation-related molecules in cell culture media, increased the adhesion of monocytes to HUVECs, and enhanced the expression of the MMP9 and ICAM proteins in HUVECs. Additionally, TNF-α potently increased ERS-related protein levels, such as GRP78 and p-eIF2α. However, PT treatment reversed the increased production of inflammatory cytokines and the adhesion of monocytes to HUVECs, as well as reduced the TNF-α-induced effects exerted by ERS-related molecules. Furthermore, thapsigargin (THA), an ERS inducer, attenuated the protective effect of PT against TNF-α-induced inflammation and ERS in HUVECs. Additionally, the downregulation of ERS signaling using siRNA targeting eIF2α and IRE1 not only inhibited ERS-related molecules but also simulated the therapeutic effects of PT on TNF-α-induced inflammation. In summary, PT treatment potently attenuates inflammation in vascular endothelial cells, which at least partly depends on the reduction of ERS.     

Atherogenic effects of TNF-α and IL-6 via up-regulation of scavenger receptors

Patients with chronic inflammatory disorders such as rheumatoid arthritis (RA) have a high risk of developing cardiovascular disease. We evaluated the effects of TNF-α and IL-6 on foam cell formation, a pivotal process in atherogenesis. Accumulation of intracellular oxidized LDL (oxLDL) was induced when THP-1/macrophages were stimulated with TNF-α or IL-6. TNF-α induced the expressions of scavenger receptors SR-A and LOX-1, and IL-6 induced SR-A expression. Inhibition of the NF-κB signaling markedly decreased TNF-α-induced foam cell formation and SR-A expression. Serum from RA patients, but not healthy subjects, induced foam cell formation, which was partially reversed by either IL-6 or TNF-α blockade in conjunction with inhibiting the induction of scavenger receptors. The present study clearly showed that in patients with chronic inflammation mediated by TNF-α and IL-6, these cytokines are directly implicated in atherosclerotic plaque formation.     

Cannabidiol protects against hepatic ischemia/reperfusion injury by attenuating inflammatory signaling and response, oxidative/nitrative stress, and cell death

Ischemia/reperfusion (I/R) is a pivotal mechanism of liver damage after liver transplantation or hepatic surgery. We have investigated the effects of cannabidiol (CBD), the nonpsychotropic constituent of marijuana, in a mouse model of hepatic I/R injury. I/R triggered time-dependent increases/changes in markers of liver injury (serum transaminases), hepatic oxidative/nitrative stress (4-hydroxy-2-nonenal, nitrotyrosine content/staining, and gp91phox and inducible nitric oxide synthase mRNA), mitochondrial dysfunction (decreased complex I activity), inflammation (tumor necrosis factor α (TNF-α), cyclooxygenase 2, macrophage inflammatory protein-1α/2, intercellular adhesion molecule 1 mRNA levels; tissue neutrophil infiltration; nuclear factor κB (NF-κB) activation), stress signaling (p38MAPK and JNK), and cell death (DNA fragmentation, PARP activity, and TUNEL). CBD significantly reduced the extent of liver inflammation, oxidative/nitrative stress, and cell death and also attenuated the bacterial endotoxin-triggered NF-κB activation and TNF-α production in isolated Kupffer cells, likewise the adhesion molecule expression in primary human liver sinusoidal endothelial cells stimulated with TNF-α and attachment of human neutrophils to the activated endothelium. These protective effects were preserved in CB2 knockout mice and were not prevented by CB1/2 antagonists in vitro. Thus, CBD may represent a novel, protective strategy against I/R injury by attenuating key inflammatory pathways and oxidative/nitrative tissue injury, independent of classical CB1/2 receptors.     

Endothelial TNF-α induction by Hsp60 secreted from THP-1 monocytes exposed to hyperglycaemic conditions

A non-resolving inflammation of the endothelium is recognised to be an important process leading to atherosclerosis. In diabetes, this process is thought to account for a significant number of cardiovascular disease-associated death and disability. However, the molecular mechanisms by which diabetes contributes to endothelial inflammation remain to be established. Whilst there is some evidence linking hyperglycaemia-induced reactive oxygen species (ROS) formation by the mitochondrial electron-transport chain to oxidative stress, cellular injury and apoptosis in the endothelium, a clear link to endothelium inflammation has not yet been established. The mitochondrial molecular stress protein Hsp60 is known to be secreted from mammalian cells and is capable of activating pro-inflammatory mediators on target cells expressing Toll-like receptors (TLRs). Hsp60 is also known to be elevated in serum of diabetes patients and has been shown to be upregulated by hyperglycaemic growth conditions in cultured human HeLa cells. This study shows that Hsp60 induced in human acute monocyte leukaemia cell line (THP-1) cells grown under hyperglycaemic conditions (25 mM glucose) was able to be secreted into growth media. Furthermore, the secretion of Hsp60 from THP-1 cells was able to be inhibited by 5,5-(N-N-dimethyl)-amiloride hydrochloride (DMA), an exosomal inhibitor. Interestingly, the conditioned media obtained from THP-1 cells grown in the presence of 25 mM glucose was able to induce the secretion of TNF-α in human vascular endothelium cell line (HUVEC). When conditioned media was immuno-depleted of Hsp60, there was a significant reduction in the release of TNF-α from the HUVEC cells. This suggests that a potential link may exist between hyperglycaemia-induced expression of Hsp60 in monocyte cells and vascular inflammation. Circulating levels of Hsp60 due to mitochondrial stress in diabetes patients could therefore be an important modulator of inflammation in endothelial cells and thus contribute to the increased incidences of atherosclerosis in diabetes mellitus.     

Involvement of Mst1 in tumor necrosis factor-alpha-induced apoptosis of endothelial cells

Mammalian sterile 20-kinase 1 (Mst1), a member of the sterile-20 family protein kinase, plays an important role in the induction of apoptosis. However, little is know about the physiological activator of Mst1 and the role of Mst1 in endothelial cells (ECs). We examined whether Mst1 is involved in the tumor necrosis factor (TNF)-α-induced apoptosis of ECs. Western blot analysis revealed that TNF-α induced activation of caspase 3 and Mst1 in a time- and dose-dependent manner. TNF-α-induced Mst1 activation is almost completely prevented by pretreatment with Z-DEVD-FMK, a caspase 3 inhibitor. Nuclear staining with Hoechst 33258 and fluorescence-activated cell sorting of propidium iodide-stained cells showed that TNF-α induced apoptosis of EC. Diphenyleneiodonium, an inhibitor of NADPH oxidase, and N-acetylcysteine, a potent antioxidant, also inhibited TNF-α-induced activation of Mst1 and caspase 3, as well as apoptosis. Knockdown of Mst1 expression by short interfering RNA attenuated TNF-α-induced apoptosis but not cleavage of caspase 3. These results suggest that Mst1 plays an important role in the induction of TNF-α-induced apoptosis of EC. However, positive feedback mechanism between Mst1 and caspase 3, which was shown in the previous studies, was not observed. Inhibition of Mst1 function may be beneficial for maintaining the endothelial integrity and inhibition of atherogenesis.     

Endogenous HMGB1 contributes to ischemia-reperfusion-induced myocardial apoptosis by potentiating the effect of TNF-α/JNK

High-mobility group box 1 (HMGB1) is a nuclear protein that has been implicated in the myocardial inflammation and injury induced by ischemia-reperfusion (I/R). The purpose of the present study was to assess the role of HMGB1 in myocardial apoptosis induced by I/R. In vivo, myocardial I/R induced an increase in myocardial HMGB1 expression and apoptosis. Inhibition of HMGB1 (A-box) ameliorated the I/R-induced myocardial apoptosis. In vitro, isolated cardiac myocytes were challenged with anoxia-reoxygenation (A/R; in vitro correlate to I/R). A/R-challenged myocytes also generated HMGB1 and underwent apoptosis. Inhibition of HMGB1 attenuated the A/R-induced myocyte apoptosis. Exogenous HMGB1 had no effect on myocyte apoptosis. However, inhibition of HMGB1 attenuated myocyte TNF-α production after the A/R was challenged; surprisingly, HMGB1 itself did not induce myocyte TNF-α production. Exogenous TNF-α induced a moderate proapoptotic effect on the myocytes, an effect substantially potentiated by coadministration of HMGB1. It is generally accepted that apoptosis induced by TNF-α is regulated by the balance of activation of c-Jun NH(2)-terminal kinase (JNK) and NF-κB. Indeed, in the present study, TNF-α increased the phosphorylation status of JNK and p65, a subunit of NF-κB; HMGB1 greatly potentiated TNF-α-induced JNK phosphorylation. Furthermore, inhibition of JNK (SP-600125) prevented the myocyte apoptosis induced by a TNF-α/HMGB1 cocktail. Finally, A/R increased HMGB1 production in both wild-type and toll-like receptor 4-deficient myocytes; however, deficiency in toll-like receptor 4 diminished A/R-induced myocyte apoptosis, TNF-α, and JNK activation. Our results indicate that myocyte-derived HMGB1 and TNF-α work in concert to promote I/R-induced myocardial apoptosis through JNK activation.     

Tumor necrosis factor alpha-induced inflammation is increased but apoptosis is inhibited by common food additive carrageenan

Tumor necrosis factor (TNF)-α, a homotrimeric, pleiotropic cytokine, is secreted in response to inflammatory stimuli in diseases such as rheumatoid arthritis and inflammatory bowel disease. TNF-α mediates both apoptosis and inflammation, stimulating an inflammatory cascade through the non-canonical pathway of NF-κB activation, leading to increased nuclear RelB and p52. In contrast, the common food additive carrageenan (CGN) stimulates inflammation through both the canonical and non-canonical pathways of NF-κB activation and utilizes the adaptor molecule BCL10 (B-cell leukemia/lymphoma 10). In a series of experiments, colonic epithelial cells and mouse embryonic fibroblasts were treated with TNF-α and carrageenan in order to simulate the possible effects of exposure to dietary CGN in the setting of a TNF-α-mediated inflammatory disease process. A marked increase in secretion of IL-8 occurred, attributable to synergistic effects on phosphorylated NF-κB-inducing kinase (NIK) in the non-canonical pathway. TNF-α induced the ubiquitination of TRAF2 (TNF receptor-associated factor 2), which interacts with NIK, and CGN induced phosphorylation of BCL10, leading to increased NIK phosphorylation. These results suggest that TNF-α and CGN in combination act to increase NIK phosphorylation, thereby increasing activation of the non-canonical pathway of NF-κB activation. In contrast, the apoptotic effects of TNF-α, including activation of caspase-8 and PARP-1 (poly(ADP-ribose) polymerase 1) fragmentation, were markedly reduced in the presence of CGN, and CGN caused reduced expression of Fas. These findings demonstrate that exposure to CGN drives TNF-α-stimulated cells toward inflammation rather than toward apoptotic cell death and suggest that CGN exposure may compromise the effectiveness of anti-TNF-α therapy.