Autoantibodies Present in Hidradenitis Suppurativa Correlate with Disease Severity and Promote the Release of Proinflammatory Cytokines in Macrophages

Abstract

Hidradenitis suppurativa (HS), also known as acne inversa, is a debilitating inflammatory skin disorder that is characterized by nodules that lead to the development of connected tunnels and scars as it progresses from Hurley stages I to III. HS has been associated with several autoimmune diseases, including inflammatory bowel disease and spondyloarthritis. We previously reported dysregulation of humoral immune responses in HS, characterized by elevated serum total IgG, B-cell activation, and antibodies recognizing citrullinated proteins. In this study, we characterized IgG autoreactivity in HS sera and lesional skin compared with those in normal healthy controls using an array-based high-throughput autoantibody screening. The Cy3-labeled anti-human assay showed the presence of autoantibodies against nuclear antigens, cytokines, cytoplasmic proteins, extracellular matrix proteins, neutrophil proteins, and citrullinated antigens. Most of these autoantibodies were significantly elevated in stages II‒III in HS sera and stage III in HS skin lesions compared with those of healthy controls. Furthermore, immune complexes containing both native and citrullinated versions of antigens can activate M1 and M2 macrophages to release proinflammatory cytokines such as TNF-α, IL-8, IL-6, and IL-12. Taken together, the identification of specific IgG autoantibodies that recognize circulating and tissue antigens in HS suggests an autoimmune mechanism and uncovers putative therapeutic targets.

Figure 1.. Autoantibodies recognizing nuclear and citrullinated antigens are present in HS serum.

Ctrl (n = 9) and HS (stage I, n = 6; stage II, n = 7; stage III, n = 8) sera were tested for the presence of autoantibodies recognizing (a) dsDNA, (b) nucleolin, (c) La/SSB, (d) citrullinated fibrinogen, and (e) MDA5. Results are the mean SEM, *P < 0.05, *P < 0.01; Mann–Whitney U test analysis was used. (f–j) Autoantibodies present in HS were stratified by Hurley stage (stage I, n = 6; stage II, n = 7; stage III, n = 8). Results are the mean SEM, *P < 0.05, **P < 0.01; one-way ANOVA was used. (k) ANA assay Hep-2 assay was used to detect ANAs in HS sera. Original magnification ×20. (l) Heat map of unsupervised clustering of autoantibodies detected in HS serum stratified by Hurley stage. Ab, antibody; ANA, antinuclear antibody; Ctrl, control; dsDNA, double-stranded DNA; HS, hidradenitis suppurativa; RFU, relative fluorescence units.

Figure 2.. Autoantibodies targeting multiple antigens detected in serum are higher in Hurley stages II and III.

Antibodies present in HS serum were classified on the basis of the role of the antigen and localization in the cells as (a) cytokine, (b) chemotaxis, (c) membrane, (d) extracellular, (e) nuclear, or (f) cytoplasmic. The average of the patients per Hurley stage was used to generate a score and perform a supervised clustering for each category. Ctrl, control; HS, hidradenitis suppurativa.

Figure 3.. Autoantibodies recognizing nuclear and citrullinated antigens are present in HS skin lesions.

Ctrl (n = 5) and HS (stage I, n = 3; stage II, n = 11; stage III, n = 11) skin tissue lysates were tested for the presence of autoantibodies against (a) dsDNA, (c) histone H2A, (e) total citrullinated histones, (g) histone H4, (i) IL-17A, and (k) LL37. Results are the mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001; Mann–Whitney U test analysis was used. (b, d, f, h, j, l) Autoantibodies present in the lysate from HS skin lesions were stratified by Hurley stage (stage I, n = 3; stage II, n = 11; stage III, n = 11). Results are the mean± SEM, *P < 0.05; one-way ANOVA was used. (m) Heat map of unsupervised clustering of all autoantibodies detected in HS tissue stratified by Hurley stage. Ab, antibody; Ctrl, control; dsDNA, double-stranded DNA; HS, hidradenitis suppurativa; RFU, relative fluorescence units.

Figure 4.. Autoantibodies detected in HS tissue are increased in Hurley stage III.

Antibodies present in HS skin samples were classified on the basis of the role of the antigen and localization in the cells as (a) cytokine, (b) chemotaxis, (c) membrane, (d) extracellular, (e) nuclear, or (f) cytoplasmic. The average of the patients per stage was used to generate a score and perform a supervised clustering for each category. Ctrl, control; HS, hidradenitis suppurativa

Figure 5.. Native and citrullinated antigen–IgG immune complexes activate M1-like macrophages to release proinflammatory cytokines.

(a) Heat map of unsupervised clustering of all autoantibodies detected in HS serum compared with those detected in tissue. M1-like macrophages were incubated with native or citrullinated histone H2B, H4, or FN and immune complexes for 24–72 h. Supernatants were analyzed for (b) TNF-α, (c) IL-8, (d) IL-6, and (e) IL-12/IL-23p40. LPS was used as a positive control. Results are the mean SEM of four independent experiments. *P < 0.05; Mann–Whitney U test was used. FN, fibronectin; h, hour; HS, hidradenitis suppurativa; LPS, lipopolysaccharide.

Figure 6.. Native and citrullinated antigen–IgG immune complexes activate M2-like macrophages to release proinflammatory cytokines.

M2-like macrophages were incubated with native or citrullinated histone H2B, H4, or FN and immune complexes for 24–72 h. Supernatants were analyzed for (a) TNF-α, (b) IL-8, (c) IL-6, and (d) IL-12/IL-23p40. LPS was used as a positive control. Results are the mean ± SEM of four independent experiments. Mann–Whitney U test was used. *P < 0.05, **P < 0.01. FN, fibronectin; h, hour; LPS, lipopolysaccharide.