Anti-hypothalamus autoantibodies in anorexia nervosa: a possible new mechanism in neuro-physiological derangement?

Abstract

Purpose: Anorexia nervosa (AN) is a serious and complex mental disorder affecting mainly young adult women. AN patients are characterized by low body weight in combination with self-induced starvation, intense fear of gaining weight, and distortion of body image. AN is a multifactorial disease, linked by recent evidence to a dysregulation of the immune system.

Methods: In this pilot study, 22 blood serums from AN patients were tested for the presence of autoantibodies against primate hypothalamic periventricular neurons by immunofluorescence and by a home-made ELISA assay. Cellular fluorescence suggests the presence of autoantibodies which are able to recognize these neurons (both to body cell and fiber levels). By means of ELISA, these autoantibodies are quantitatively evaluated. In addition, orexigenic and anorexigenic molecules were measured by ELISA. As control, 18 blood serums from healthy age matched woman were analysed.

Results: All AN patients showed a reactivity against hypothalamic neurons both by immunofluorescence and ELISA. In addition, ghrelin, pro-opiomelanocortin (POMC), and agouti-related peptide (AGRP) were significantly higher than in control serums (p < 0.0001). In contrast, leptin was significantly lower in AN patients than controls (p < 0.0001).

Conclusions: Immunoreaction and ELISA assays on AN blood serum suggest the presence of autoantibodies AN related. However, it is not easy to determine the action of these antibodies in vivo: they could interact with specific ligands expressed by hypothalamic cells preventing their physiological role, however, it is also possible that they could induce an aspecific stimulation in the target cells leading to an increased secretion of anorexigenic molecules. Further studies are needed to fully understand the involvement of the immune system in AN pathogenesis.

Level of evidence: V, descriptive study.

Keywords: Anorexia nervosa; Anorexigenic molecules; Autoimmunity; Hypothalamic autoantibodies; Orexigenic molecules.

Fig. 1

IgG and IgM autoantibody standard curves. IgG and IgM autoantibodies ELISA standard curves used to calculate the anti-hypothalamus antibodies concentrations in human subject serum. In (A) IgG standard curve and in (B) IgM standard curve are presented. Standard deviation (STD) from three independent runs were shown. Linear regression fitting results (continuous line) and 95% confidence bands of the best-fit line (dotted line) are shown

Fig. 2

Serum from AN patients show an immune-reaction against hypothalamic cells. Immunofluorescent detection of binding of immunoglobulin G autoantibodies from 22 patients with AN to a primate hypothalamus section at the level of the arcuate nucleus (Pz 1, 2 and 8 are presented of examples of score 3 immunoreactive pattern, and Pz 10 of score 2). As control, 3 out 18 representative sera from healthy control are shown (Ctr1-3 as examples of score 0, and Ctr7 and Crt9 of score 1). The experiment was repeated four times. In the graph, the different reactivity among AN and healthy control is statistical different. The intensity of the fluorescence was determined by two different readers blind to subject characteristics and a scale from 0 to 3 was assigned (0 no reaction, 1 uncertain fluorescence, 2 nuclear fluorescence, 3 fibers and/or nuclear fluorescence). Samples scored positive if a 2 or 3 fluorescence reaction was observed

Fig. 3

Anti-hypothalamic antibody IgG levels are present in sera from AN patients. A measurable anti-hypothalamic IgG antibody level is present exclusively in AN patients (Panel A). The amount of anti-hypothalamic IgG antibody correlates positively with the intensity of fluorescence on hypothalamus section (Panel B)

Fig. 4

Orexigenic and anorexigenic molecules evaluation. A comparison of plasma ghrelin, POMC, AGRP and leptin levels in women with AN and healthy control women are shown

Fig. 5

Correlation analysis among the amount of orexigenic and anorexigenic molecules was performed

Fig. 6

Correlation analysis among the amount of orexigenic and anorexigenic molecules and anti-hypothalamic antibodies evaluated by immunofluorescence (left panels) and ELISA (right panels)

Fig. 7

Analysis of the correlation among ghrelin, POMC, ARGP, leptin, and fluorescent intensity (IFI) with BMI

Fig. 8

Orexigenic and anorexigenic molecules are able to interfere with autoantibodies binding. As orexigenic and anorexigenic molecules are thought to play prominent roles in integration of peripheral and central signals to modulate appetite and metabolism, experiments were set up to investigate whether they may interfere with the binding of reactive autoantibodies to hypothalamic tissues. All 22 AN sera were titred alone and in presence of recombinant human soluble molecules of ghrelin, POMC, AGRP, and α-MSH