Inflammation in the hippocampus affects IGF1 receptor signaling and contributes to neurological sequelae in rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA) is an inflammatory joint disease with a neurological component including depression, cognitive deficits, and pain, which substantially affect patients' quality of daily life. Insulin-like growth factor 1 receptor (IGF1R) signaling is one of the factors in RA pathogenesis as well as a known regulator of adult neurogenesis. The purpose of this study was to investigate the association between IGF1R signaling and the neurological symptoms in RA. In experimental RA, we demonstrated that arthritis induced enrichment of IBA1⁺ microglia in the hippocampus. This coincided with inhibitory phosphorylation of insulin receptor substrate 1 (IRS1) and up-regulation of IGF1R in the pyramidal cell layer of the cornus ammoni and in the dentate gyrus, reproducing the molecular features of the IGF1/insulin resistance. The aberrant IGF1R signaling was associated with reduced hippocampal neurogenesis, smaller hippocampus, and increased immobility of RA mice. Inhibition of IGF1R in experimental RA led to a reduction of IRS1 inhibition and partial improvement of neurogenesis. Evaluation of physical functioning and brain imaging in RA patients revealed that enhanced functional disability is linked with smaller hippocampus volume and aberrant IGF1R/IRS1 signaling. These results point to abnormal IGF1R signaling in the brain as a mediator of neurological sequelae in RA and provide support for the potentially reversible nature of hippocampal changes.

Keywords: IGF1 receptor; MRI; arthritis; hippocampus; pain.

Fig. 1.

Collagen-induced arthritis causes behavior changes in mice. (A) Subcutaneous immunization of DBA/1 mice with chicken CII resulted in spontaneous arthritis, which progressed in frequency and severity. Insets show clinical arthritis in the digital and metatarsal areas, and skeletal damage in a 3D micro-computed tomography image of the same areas. The red boxes indicate the areas of analysis. (B) The changes in body weight and (C) serum IL-6 levels in RA mice compared with naive siblings. (D) Spearman’s correlation shows a gradual reduction in proportion of bone volume within tissue volume (BV/TV) with arthritis severity. (E) Behavioral pattern of RA mice was registered by filming and presented as time occupied in each activity (locomotion, rearing, minor movements, grooming, and immobility). The dotted boxes present median and interquartile range of the naive siblings filmed on the same occasion. (F) Spearman’s correlations (rho values) of arthritis severity, body weight loss, and serum IL-6 levels with locomotion and rearing is shown. Boxplots present median, interquartile range, and full range. P values are calculated with the Mann–Whitney U test between RA mice (filled boxes) vs. naive, nonimmunized mice (open boxes). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2.

Enrichment of IBA1⁺ cells in the HC during arthritis. (A) The localization pattern of IBA1⁺ cells (red, arrowheads) in DG and CA area of RA mice and naive controls. The area of the HC was determined from nuclei staining (blue). (B) The density of IBA1⁺ cells within CA areas and layers of DG. (C) Gene transcription analysis in the HC tissue of naive and arthritic mice done with RT-PCR. (D) Spider chart of Spearman’s correlations (rho values) between the density of IBA1⁺ cells within DG_mo (black line) and CA_sp (gray line). Boxplots indicate median, interquartile range, and full range. P values are calculated with the Mann–Whitney U test between RA mice (filled boxes) vs. naive, nonimmunized mice (open boxes). *P < 0.05; **P < 0.01; ***P < 0.001. CA, cornu ammonis; DG, dentate gyrus; mo, molecular layer; po, polymorph layer; sg, subgranular layer; slm, stratum lacunosum-moleculare; slu, stratum lucidum; so, stratum oriencs; sp, pyramidal layer; sr, stratum radiatum.

Fig. 3.

Resistance to IGF1R signaling inhibits neurogenesis in HC during arthritis. Morphometric analysis of the HC of arthritic and naive mice (A–K). (A) The area of DG, CA, and total HC was measured from nuclei staining (blue) of 4-μm tissue sections. (B) The change in average thickness of DG_sg. (C) Density of DCX⁺ cells in DG_sg layer as normalized to the length. (D) Spearman’s correlation indicates a reduction in the thickness of DG_sg with lower number of DCX⁺ cells. (E) The density of IGF1R⁺ cells and (F) pS⁶¹²IRS1⁺ cells in DG. (G) The Spearman correlation between the density of DCX⁺ and pS⁶¹²IRS1⁺ cells in DG. (H) Representative images of the HC staining show distribution of DCX⁺ (indicated by arrowheads), IGF1R⁺, and pS⁶¹²IRS1⁺ cells in DG of naive (Upper row) and arthritis mice (Middle row). Bottom shows colocalization (yellow staining) of pS⁶¹²IRS1 and DCX in DG. (I) Representative image of the CA_sp subfield shows enrichment of IGF1R⁺ and pS⁶¹²IRS1⁺ cells in arthritis compared with naive mice. (J and K) The density of IGF1R⁺ cells (J) and pS⁶¹²IRS1⁺ cells (K) within CA_sp layer. (L) Heatmap shows transcriptional analysis of genes involved in neurogenesis and IGF1 signaling in the HC tissue of naive and arthritis mice, done by RT-PCR. Box plots present median, interquartile range, and full range. P values are calculated with the Mann–Whitney U test between RA mice (filled boxes) vs. naive, nonimmunized mice (open boxes). CA, cornu ammonis; DCX, doublecortin; DG, dentate gyrus; sg, subgranular layer; sp, pyramidal layer.

Fig. 4.

Inhibition of IGF1R alleviates collagen-induced arthritis and supports early neurogenesis. (A) IGF1R was inhibited with shRNA (shIGF1R, empty symbols) provided intraperitoneally on days 19, 26, and 33 (indicated by arrows). The control group received nontargeting scrambled RNA (shNT) (filled symbols). Inhibition of IGF1R alleviated severity of arthritis and weight loss (Inset) in mice. Inhibition of IGF1R (B) reduced IRS1 mRNA in spleen, (C) increased serum levels of IGF1, and reduced the cell density of IBA1⁺ cells in DG (D) and CA (E). (F) Change in density of pS⁶¹²IRS1⁺ cells in DG. (G and H) Change in density of GFAP⁺ cells in total DG and in DG_sg. (I) Reduction in behavioral activity of shIGF1R-treated mice. (J) The brain sections show costaining for GFAP and DCX in DG. The dotted line indicates the borders of DG_sg. The arrowheads indicate positive cells. (K) Different morphology of GFAP⁺ cells in DG_sg, distinct from vertical-oriented neuronal stem cells from astrocytes. (L) Heatmap shows transcriptional analysis of genes involved in neurogenesis and IGF1R signaling in the hippocampal tissue of shIGF1R-treated and shNT-treated RA mice, done by RT-PCR. Boxplots present median, interquartile range, and full range. P values are calculated with the Mann–Whitney U test, between shIGF1R-treated mice (open boxes) vs. control, shNT (filled boxes). *P < 0.05. CA, cornu ammonis; DG, dentate gyrus; GFAP, glial fibrillary acidic protein; mo, molecular layer; po, polymorph layer; sg, subgranular layer; slm, stratum lacunosum-moleculare; slu, stratum lucidum; so, stratum oriencs; sp, pyramidal layer; sr, stratum radiatum.

Fig. 5.

The volume of HC in patients with RA correlates with IGF1R signaling and functional disability. (A, 1) A 3D surface rendering of the HC in the postero-anterior view is shown. Anatomical segmentation of MR images in 15 female RA patients separated the group with the HC volume above the median (bigger HC, n = 8) and below the median (smaller HC, n = 7). Clinical characteristics of the patients are shown in the table. Boxplots show (A, 2) gray matter volumes in the left and right HC gyrus, in absolute numbers normalized by intracranial volume; (A, 3) difference in functional disability, (A, 4) pain perception, and (A, 5) serum levels of IGF1 in RA patients with different HC volume. (A, 6) Change in pain perception recorded with replication of pressure applied on metacarpophalangeal joint II. (A, 7) Heatmap shows transcriptional analysis of genes involved in IGF1R signaling in WBC, done by RT-PCR. (B) Expression of IGF1R in WBC was measured in 84 randomly selected female RA patients by RT-PCR, and the groups with IGF1R above and below the lower tertile for the group were compared. Boxplot shows (B, 1) functional disability assessed by the Fibromyalgia Impact Questionnaire (FIQ); perception of (B, 2) depression and (B, 3) anxiety; (B, 4) pain threshold measured by algometry; and (B, 5) levels of IRS1 mRNA and (B, 6) IRS2 mRNA in WBCs measured by RT-PCR. (C) Functional disability (C, 1) was assessed by FIQ and compared in 230 RA patients (female, 171; male, 59) within the groups of (C, 2) nondepressed (group A), nondepressed with severe pain (group B; VAS > 45 mm, HADS ≤ 7) and with depression (group C; HADS ≥ 8). Box plots demonstrate median, interquartile range, and full range. P values are estimated with the Mann–Whitney U test or the Kruskal–Wallis nonparametric ANOVA. B, bigger; FIQ, Fibromyalgia Impact Questionnaire; HADS, Hospital Anxiety and Depression Scale; HAQ, Health Assessment Questionnaire; HC, hippocampus; MR, magnetic resonance; RA, rheumatoid arthritis; S, smaller; VAS, visual analog scale; WBC, white blood cell.