In Rheumatoid Arthritis Patients, HLA-DRB1*04:01 and Rheumatoid Nodules Are Associated With ACPA to a Particular Fibrin Epitope.

Objectives: Rheumatoid arthritis (RA) is associated with HLA-DRB1 genes encoding the shared epitope (SE), a 5-amino acid motive. RA is usually preceded by the emergence of anti-citrullinated protein/peptide antibodies (ACPAs). Citrulline is a neutral amino acid resulting from post-translational modification of arginine involved in peptidic bounds (arginyl residue) by PeptidylArginine Deiminases (PADs). ACPAs recognize epitopes from citrullinated human fibrin(ogen) (hFib) and can be specifically detected by the AhFibA assay. Five citrullinated peptides derived from hFib together represent almost all of the epitopes recognized by patients with ACPA-positive RA, namely: α36-50cit, α171-185cit, α501-515cit, α621-635cit, and ß60-74cit. The use of antibody fine specificities as markers of clinical phenotypes has become a major challenge. Our objective was to study whether RA clinical characteristics and HLA-DRB1 genetic background were associated with a specific reactivity against the epitopes borne by the five peptides. Methods: 184 ACPA-positive RA patients fulfilling the 2010 ACR/EULAR criteria were studied. Patient characteristics including HLA-DRB1 genotype, were collected from their medical files. Anti-CCP2 antibodies, AhFibA, and antibodies against the five citrullinated hFib (hFib-cit) peptides were analyzed by ELISA. Results: Anti-α505-515cit antibodies were associated with HLA-DRB1*04:01 (OR = 5.52 [2.00 - 13.64]; p = 0.0003). High level anti-α505-515cit antibodies were associated with rheumatoid nodules (OR = 2.71 [1.00 - 7.16], p= 0.044). Conclusion: Immune complexes containing anti-α501-515cit antibodies and rheumatoid factors might be involved in the development of rheumatoid nodules on the HLA-DRB1*04:01 background. Apheresis of these epitope-specific antibodies might be a new therapeutic opportunity for patients with rheumatoid nodules.

IgM and IgA rheumatoid factors purified from rheumatoid arthritis sera boost the Fc receptor- and complement-dependent effector functions of the disease-specific anti-citrullinated protein autoantibodies.

Rheumatoid factors (RF) and the disease-specific anti-citrullinated protein autoantibodies (ACPA) coexist in the joints of rheumatoid arthritis (RA) patients where they probably contribute to synovitis. We investigated the influence of IgM and IgA RF on the FcR- and complement-dependent effects of ACPA immune complexes (ACPA-IC). When stimulated by ACPA-IC formed in the presence of IgM RF or IgA RF fractions purified from RA serum pools, M-CSF-generated macrophages skewed their cytokine response toward inflammation, with increases in the TNF-α/IL-10 ratio and in IL-6 and IL-8 secretion, and decreases in the IL-1Ra/IL-1ß ratio. In the IgM RF-mediated amplification of the inflammatory response of macrophages, the participation of an IgM receptor was excluded, notably by showing that they did not express any established receptor for IgM. Rather, this amplification depended on the IgM RF-mediated recruitment of more IgG into the ACPA-IC. However, the macrophages expressed FcαRI and blocking its interaction with IgA inhibited the IgA RF-mediated amplification of TNF-α secretion induced by ACPA-IC, showing its major implication in the effects of RF of the IgA class. LPS further amplified the TNF-α response of macrophages to RF-containing ACPA-IC. Lastly, the presence of IgM or IgA RF increased the capacity of ACPA-IC to activate the complement cascade. Therefore, specifically using autoantibodies from RA patients, the strong FcR-mediated or complement-dependent pathogenic potential of IC including both ACPA and IgM or IgA RF was established. Simultaneous FcR triggering by these RF-containing ACPA-IC and TLR4 ligation possibly makes a major contribution to RA synovitis.

IgM rheumatoid factor amplifies the inflammatory response of macrophages induced by the rheumatoid arthritis-specific immune complexes containing anticitrullinated protein antibodies.

OBJECTIVES: Anticitrullinated protein antibodies (ACPA) are specifically associated with rheumatoid arthritis (RA) and produced in inflamed synovial membranes where citrullinated fibrin, their antigenic target, is abundant. We showed that immune complexes containing IgG ACPA (ACPA-IC) induce FcγR-mediated tumour necrosis factor (TNF)-α secretion in macrophages. Since IgM rheumatoid factor (RF), an autoantibody directed to the Fc fragment of IgG, is also produced and concentrated in the rheumatoid synovial tissue, we evaluated its influence on macrophage stimulation by ACPA-IC. METHODS: With monocyte-derived macrophages from more than 40 healthy individuals and different human IgM cryoglobulins with RF activity, using a previously developed human in vitro model, we evaluated the effect of the incorporation of IgM RF into ACPA-IC. RESULTS: IgM RF induced an important amplification of the TNF-α secretion. This effect was not observed in monocytes and depended on an increase in the number of IgG-engaged FcγR. It extended to the secretion of interleukin (IL)-1ß and IL-6, was paralleled by IL-8 secretion and was not associated with overwhelming secretion of IL-10 or IL-1Ra. Moreover, the RF-induced increased proinflammatory bioactivity of the cytokine response to ACPA-IC was confirmed by an enhanced, not entirely TNF-dependent, capacity of the secreted cytokine cocktail to prompt IL-6 secretion by RA synoviocytes. CONCLUSIONS: By showing that it can greatly enhance the proinflammatory cytokine response induced in macrophages by the RA-specific ACPA-IC, these results highlight a previously undescribed, FcγR-dependent strong proinflammatory potential of IgM RF. They clarify the pathophysiological link between the presence of ACPA and IgM RF, and RA severity.

Importance of mitochondrial dynamin-related protein 1 in hypothalamic glucose sensitivity in rats.

AIMS: Hypothalamic mitochondrial reactive oxygen species (mROS)-mediated signaling has been recently shown to be involved in the regulation of energy homeostasis. However, the upstream signals that control this mechanism have not yet been determined. Here, we hypothesize that glucose-induced mitochondrial fission plays a significant role in mROS-dependent hypothalamic glucose sensing. RESULTS: Glucose-triggered translocation of the fission protein dynamin-related protein 1 (DRP1) to mitochondria was first investigated in vivo in hypothalamus. Thus, we show that intracarotid glucose injection induces the recruitment of DRP1 to VMH mitochondria in vivo. Then, expression was transiently knocked down by intra-ventromedial hypothalamus (VMH) DRP1 siRNA (siDRP1) injection. 72 h post siRNA injection, brain intracarotid glucose induced insulin secretion, and VMH glucose infusion-induced refeeding decrease were measured, as well as mROS production. The SiDRP1 rats decreased mROS and impaired intracarotid glucose injection-induced insulin secretion. In addition, the VMH glucose infusion-induced refeeding decrease was lost in siDRP1 rats. Finally, mitochondrial function was evaluated by oxygen consumption measurements after DRP1 knock down. Although hypothalamic mitochondrial respiration was not modified in the resting state, substrate-driven respiration was impaired in siDRP1 rats and associated with an alteration of the coupling mechanism. INNOVATION AND CONCLUSION: Collectively, our results suggest that glucose-induced DRP1-dependent mitochondrial fission is an upstream regulator for mROS signaling, and consequently, a key mechanism in hypothalamic glucose sensing. Thus, for the first time, we demonstrate the involvement of DRP1 in physiological regulation of brain glucose-induced insulin secretion and food intake inhibition. Such involvement implies DRP1-dependent mROS production.

Enhanced hypothalamic glucose sensing in obesity: alteration of redox signaling.

OBJECTIVE: Recent data demonstrated that glucose sensing in different tissues is initiated by an intracellular redox signaling pathway in physiological conditions. However, the relevance of such a mechanism in metabolic disease is not known. The aim of the present study was to determine whether brain glucose hypersensitivity present in obese Zücker rats is related to an alteration in redox signaling. RESEARCH DESIGN AND METHODS: Brain glucose sensing alteration was investigated in vivo through the evaluation of electrical activity in arcuate nucleus, changes in reactive oxygen species levels, and hypothalamic glucose-induced insulin secretion. In basal conditions, modifications of redox state and mitochondrial functions were assessed through oxidized glutathione, glutathione peroxidase, manganese superoxide dismutase, aconitase activities, and mitochondrial respiration. RESULTS: Hypothalamic hypersensitivity to glucose was characterized by enhanced electrical activity of the arcuate nucleus and increased insulin secretion at a low glucose concentration, which does not produce such an effect in normal rats. It was associated with 1) increased reactive oxygen species levels in response to this low glucose load, 2) constitutive oxidized environment coupled with lower antioxidant enzyme activity at both the cellular and mitochondrial level, and 3) overexpression of several mitochondrial subunits of the respiratory chain coupled with a global dysfunction in mitochondrial activity. Moreover, pharmacological restoration of the glutathione hypothalamic redox state by reduced glutathione infusion in the third ventricle fully reversed the cerebral hypersensitivity to glucose. CONCLUSIONS: The data demonstrated that obese Zücker rats' impaired hypothalamic regulation in terms of glucose sensing is linked to an abnormal redox signaling, which originates from mitochondria dysfunction.

Mitochondrial reactive oxygen species are required for hypothalamic glucose sensing.

The physiological signaling mechanisms that link glucose sensing to the electrical activity in metabolism-regulating hypothalamus are still controversial. Although ATP production was considered the main metabolic signal, recent studies show that the glucose-stimulated signaling in neurons is not totally dependent on this production. Here, we examined whether mitochondrial reactive oxygen species (mROS), which are physiologically generated depending on glucose metabolism, may act as physiological sensors to monitor the glucose-sensing response. Transient increase from 5 to 20 mmol/l glucose stimulates reactive oxygen species (ROS) generation on hypothalamic slices ex vivo, which is reversed by adding antioxidants, suggesting that hypothalamic cells generate ROS to rapidly increase glucose level. Furthermore, in vivo, data demonstrate that both the glucose-induced increased neuronal activity in arcuate nucleus and the subsequent nervous-mediated insulin release might be mimicked by the mitochondrial complex blockers antimycin and rotenone, which generate mROS. Adding antioxidants such as trolox and catalase or the uncoupler carbonyl cyanide m-chlorophenylhydrazone in order to lower mROS during glucose stimulation completely reverses both parameters. In conclusion, the results presented here clearly show that the brain glucose-sensing mechanism involved mROS signaling. We propose that this mROS production plays a key role in brain metabolic signaling.

Intrauterine hyperglycemia increases insulin binding sites but not glucose transporter expression in discrete brain areas in term rat fetuses.

Diabetic pregnancy results in several metabolic and hormonal disorders, both in the embryo and the fetus of different species, including humans. Insulin is a potent modulator of brain development and is suggested to promote the differentiation and maturation of hypothalamic or related extrahypothalamic structures, which are directly involved in neural inputs to the pancreas. Because these structures are known to be specifically responsive both to insulin and glucose, we examined the effects of 48-h hyperglycemic clamps in unrestrained pregnant rats on insulin binding and glucose transporter expression in hypothalamic and extrahypothalamic-related areas of their fetal offspring. The main result was an increase in insulin binding in the ventromedial hypothalamic nucleus (VMH), the arcuate nucleus (AN), and the lateral hypothalamus (LH), and in the nucleus of the tractus solitarius (NTS) for extrahypothalamic areas (+30% in the VMH, +37% in the AN, +25.8% in the LH, and +37.3% in the NTS). The deleterious effect of brain hyperinsulinism during the late gestational stage does not seem to act through glucose transporter (GLUT) expression, inasmuch as no relationship between GLUT level and hyperinsulinism in brain areas could be observed. The specific increase in insulin binding in areas involved in the nervous control of metabolism could be a factor in the increased glucose intolerance and impairment of insulin secretion that was previously observed in the adult rats from hyperglycemic mothers.