IgA autoantibodies promote inflammation, Th17 polarization and fibrotic responses in hidradenitis suppurativa

Domain: biology
Published: 2026-03-24
Editorial outcome: minor_revision

Abstract
Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease with complex immune dysregulation. While previous studies have reported tertiary lymphoid structures (TLS) and IgA-producing B cells in HS skin lesions, the pathogenic role of IgA autoantibodies remains insufficiently characterized. Here, we demonstrate that lesional skin from patients with HS exhibits elevated expression of IGHA1, IGHA2, and J chain, as confirmed by qPCR, immunofluorescence, and Western blot analyses. Single-cell RNA sequencing identified local plasma cells and B cells as the primary source of IgA. Autoantigen profiling revealed a diverse repertoire of IgA autoantibodies targeting nuclear, cytoplasmic, and membrane antigens, with levels correlating with disease severity and other clinical manifestations. We identified IgA autoantibodies binding to CD68⁺ macrophages, which induced secretion of TNF, IL-6, and IL-1β and upregulated inflammasome and profibrotic pathways. Anti-neutrophil extracellular trap (NET) IgA was elevated in HS and promoted NET formation, establishing a pathogenic feedback loop. NET–IgA immune complexes induced macrophages to secrete CCL18, driving collagen production by fibroblasts and promoting a type I interferon (IFN) gene signature. IgA immune complexes presented by myeloid dendritic cells activated CD4⁺ T cells, triggering IFN-γ production and further amplifying local inflammation. Notably, supernatants from macrophages exposed to IgA were able to polarize naïve CD4⁺ T cells toward a Th17 phenotype, linking innate immune activation to the expansion of pathogenic adaptive immune responses. Direct exposure of fibroblasts to NET–IgA complexes triggered expression of adhesion molecules, chemokines, and regulators of adaptive immunity. Together, these findings uncover a role for IgA autoantibodies in HS, implicating them as central drivers of chronic inflammation, fibrosis, and immune crosstalk across neutrophils, macrophages, fibroblasts, and T cells.

Introduction
Hidradenitis suppurativa (HS) is a chronic, relapsing inflammatory skin disorder characterized by painful nodules, abscesses, and draining tunnels, most commonly affecting intertriginous regions such as the axillae, groin, and buttocks(1-3). Despite increasing recognition of its multifactorial pathogenesis including genetic susceptibility, follicular occlusion, and immune dysregulation, the underlying immunologic mechanisms driving HS remain incompletely understood(4-11). While early studies emphasized the role of neutrophil-mediated inflammation and complement activation, recent transcriptomic and histopathologic analyses have also identified robust infiltration of B cells and plasma cells within HS lesions, suggesting a significant contribution of humoral immunity to disease pathophysiology(5, 7, 9, 12-15).

One particularly intriguing finding has been the presence of tertiary lymphoid structures (TLS) (9, 15, 16). These organized lymphoid aggregates, composed of T cells, B cells, and follicular dendritic cells (FDCs), are known to serve as local sites of antigen presentation, B cell activation, and antibody production in chronic inflammatory diseases(9, 15, 17, 18). In HS, their presence raises the possibility that in situ antibody generation may play a more central role than previously appreciated. Indeed, autoantibodies including IgG reactive to citrullinated proteins have been detected in patient sera and lesional tissue, and B cell/plasma cell signatures are increasingly recognized in HS transcriptomic profiles(5, 7, 13, 14).

However, while IgG autoantibodies have received some attention, the potential role of immunoglobulin A (IgA), a mucosal isotype with both immunoregulatory and pro-inflammatory properties, remains poorly characterized in HS. Emerging evidence indicates local class switching to IgA in HS lesions(7, 9, 15). However, it remains unclear whether IgA autoantibodies are produced in HS skin lesion, what antigens they target, and what functional effects they exert within the inflamed skin microenvironment. Given the ability of IgA immune complexes to activate myeloid(19) and stromal cells via Fcα receptors(20), and the established presence of TLSs as niches supporting local B cell maturation, the possibility that IgA contributes to sustained inflammation and tissue remodeling in HS warrants focused investigation.

This study aims to address this critical gap by investigating the cellular sources, antigenic targets, and downstream immunologic effects of IgA in HS. By characterizing the expression of IgA-related genes and proteins, profiling the specificity of IgA autoantibodies, and examining their impact on key immune and resident skin cells, we seek to clarify how IgA contributes to the chronic inflammation, fibrosis, and immune amplification that define advanced HS.

Results
IgA antibodies are present in HS skin lesions

Histological analysis of skin lesions from patients with HS revealed organized lymphoid aggregates consistent with TLSs (9, 15, 17). To investigate local IgA production, we performed quantitative PCR on skin biopsies from healthy controls and HS patients. Expression of JCHAIN, and IGHA1was significantly elevated in HS lesions compared to control skin (Fig. 1A, B, Sup Fig s1 A-B; p = 0.0368). Similarly, ELISA analysis demonstrated significant increased IgA protein levels in HS lesional tissue (p = 0.0013) but not in serum (Fig. 1C, D, Suppl Fig s1C, D). To determine whether IgA elevation extended beyond lesional areas, we analyzed paired lesional and non-lesional skin samples from publicly available RNA sequencing data obtained from the same patients (21). Lesional tissue exhibited significant upregulation of JCHAIN (p = 0.0004) and IGHA1(p < 0.0001), whereas non-lesional sites showed minimal or no expression (Fig. 1E, F). Immunofluorescence staining confirmed IgA protein localization throughout lesional tissue, with prominent (Fig. 1G). Western blot analysis of lesional extracts further validated elevated levels of J chain, IgA1, and IgA2 proteins in HS skin compared to healthy controls (Fig. 1H). IgA2 (heavy chain [Hc] and light chain [Lc]) appears to be more prevalent in HS skin lesions than the IgA1 subtype (Fig. 1H). To identify the cellular source of IgA in skin, we analyzed publicly available single-cell RNA sequencing (scRNA-seq) data from lesional HS skin (9). This analysis revealed that B cells and plasma cells expressing IGHA1, IGHA2, and JCHAIN, consistent with local class-switch recombination and in situ IgA production (Fig. 1I). To further substantiate that B cells contribute to the IgA detected in HS skin lesions, we performed confocal microscopy analysis. Immunofluorescence revealed IgA-producing cells clustered within CD20+/CD21+ B cell-rich areas, consistent with TLS-like aggregates (Figure 1J). Notably, IgA-producing cells were also detected outside these aggregates and distributed throughout the tissue. Collectively, these findings demonstrate the presence of IgA-producing plasma cells within HS lesions, frequently in proximity to B cell clusters, supporting a localized, tissue-specific humoral immune response in HS.

IgA autoantibodies are present in HS and correlate with clinical manifestations

Given the evidence for local IgA production in HS lesions, we next investigated whether these antibodies were directed against self-antigens. Using comprehensive autoantigen profiling across two independent HS patient cohorts, we identified a broad and diverse repertoire of IgA autoantibodies targeting nuclear (e.g., dsDNA, nucleolin, genomic DNA, PCNA), cytoplasmic (e.g., vimentin, MDA5, complement components), and extracellular antigens (e.g., collagen, fibronectin, IFN-γ) (Fig. 2A, B). Many of these autoantibodies were more prevalent in HS patients with chronic or severe disease. The demographic and clinical characteristics of the cohorts are summarized in Table 1.

Notably, IgA autoantibodies against melanoma differentiation-associated protein 5 (MDA5; p = 0.002), thyroglobulin (p = 0.0022), complement proteins C3 (p = 0.0023) and C4 (p = 0.0079), NLR family pyrin domain containing 1 (NLRP1; p = 0.0182), and angiotensin-converting enzyme (ACE; p = 0.002) were significantly elevated in patients with advanced disease (higher Hurley stage), suggesting an association between these IgA autoantibodies and disease progression and tissue injury. Conversely, IgA autoantibodies targeting myeloperoxidase (MPO; p = 0.0042), IL-17A (p = 0.0122), and nucleolin (p = 0.0198) were more frequently observed in patients with milder disease or in healthy controls, indicating potential context-dependent regulatory functions (Figure 2C). Correlation analyses further revealed associations between specific IgA autoantibodies and clinical manifestations. For example, IgA antibodies to aggrecan (r = 0.4505, p = 0.0231), calprotectin (r = 0.4560, p = 0.0226), and collagen II (r = 0.4719, p = 0.0178) were significantly associated with acne (Figure 2D). Diabetes showed significant correlations with IgA against nuclear antigens, including Ro/SSA (52 kDa) (r = 0.7299, p = 0.0001), Sm/RNP (r = 0.4994, p = 0.0125), and small nuclear ribonucleoprotein SmD3 (r = 0.6332, p = 0.0013).

Disease severity, as measured by Hurley stage, was positively associated with IgA autoantibodies to MDA5 (r = 0.4997, p = 0.0146), genomic DNA (r = 0.4800, p = 0.0187), and dsDNA (r = 0.4372, p = 0.0306). The number of draining sinuses correlated with anti-calprotectin (r = 0.4416, p = 0.0256) and proteinase-3 (PR3) IgA (r = 0.4749, p = 0.0171), while anti-alpha fodrin IgA was associated with nodule count (r = 0.4011, p = 0.0398), local IHS4 score (r = 0.4713, p = 0.0179), and number of draining sinuses (r = 0.4994, p = 0.0125).

Smoking, an established HS risk factor, was significantly associated with IgA responses to laminin (r = 0.6104, p = 0.0021), complement C1q (r = 0.6103, p = 0.0021), and histone H2B (r = 0.4590, p = 0.0209). The presence of comedones was linked to IgA targeting Sm (r = 0.6380, p = 0.0012), glomerular basement membrane (GBM; r = 0.4135, p = 0.0349), IFN-γ (r = 0.5377, p = 0.0072), lysozyme C (r = 0.4315, p = 0.0287), and nucleosomes (r = 0.4031, p = 0.0390).

Of note, several IgA autoantibodies were inversely associated with the use of anti-TNF therapy, including those against BAFF (r = –0.4125, p = 0.0353), CD40 (r = –0.3936, p = 0.0429), collagen IV (r = –0.4379, p = 0.0267), La/SSB (r = –0.3884, p = 0.0452), MDA5 (r = –0.4560, p = 0.0216), myosin (r = –0.4088, p = 0.0367), and PR3 (r = –0.5194, p = 0.0094) (Fig. 2D).

Because IgA derived from mucosal and tissue compartments can exhibit a degree of polyreactivity (22), we next assessed whether the IgA detected in HS lesions recognized bacterial antigens and might therefore confound our interpretation. ELISA analyses of IgA reactivity against E. coli, Lactobacillus, and P. gingivalis revealed no significant differences between HS tissue samples and controls (Figure 2E, Suppl Fig S2A-C). In addition, correlation analyses showed no positive association between anti-bacterial IgA levels and the clinical parameters examined (Figure 2F). Notably, anti–E. coli IgA levels in HS samples positively correlated with draining sinus count, mirroring the only autoantibody that correlated with this parameter, anti-CRP IgA. This observation suggests possible cross-reactivity or a shared inflammatory context rather than a direct microbe-driven response. Although total IgG levels were significantly elevated in HS samples (Supplementary Figure S1E, F), IgG levels correlated with male sex but not with other clinical parameters (Figure 2F), further supporting a distinct functional role for IgA in HS pathogenesis.

These findings suggest that IgA autoantibodies in HS display a dynamic and heterogeneous repertoire, with both potentially pathogenic and regulatory roles. Their distribution appears to be shaped by disease severity, clinical phenotype, and therapeutic exposure.

HS-IgA facilitates keratinocyte autoantigen presentation through FcαRI and co-stimulatory molecules

To further characterize the IgA antibody repertoire in HS, we performed Western blot analysis using lysates from control neutrophils, skin fibroblasts, and keratinocytes (HaCaT cells). Notably, IgA2 autoantibodies purified from HS skin samples robustly recognized antigens from all three cell types, whereas minimal reactivity was observed in control samples (Fig. 3A, upper panel), suggesting the presence of IgA2 antibodies recognizing autoantigens in neutrophils, skin fibroblasts and keratinocytes in HS skin lesions. In contrast, IgA1 antibodies from healthy control and HS skin showed no difference in their ability to recognize autoantigens in these 3 cell types (Fig. 3A, lower panel), indicating that IgA2 is the predominant subtype targeting skin and immune cells in HS. To investigate the functional consequences of IgA autoreactivity, IgA was purified from HS lesional tissue using protein M. We hypothesized that IgA bound to keratinocyte antigens could form immune complexes capable of being presented to CD4⁺ T cells via myeloid DCs (mDCs), contributing to local immune activation. To test this, we isolated autologous CD4⁺ T cells and mDCs from healthy donors and co-cultured them in the presence or absence of keratinocyte protein–IgA immune complexes for 4–5 days. IFN-γ and TNF-α secretion was used as a readout for T cell activation. Co-cultures exposed to keratinocyte–IgA complexes exhibited significantly elevated levels of both cytokines (IFN-γ, p =0.0003, TNF-α, p =0.0157), indicating effective T cell activation (Fig. 3B, C). To determine the mechanism of autoantigen uptake and presentation, we used neutralizing antibodies against the IgA receptor FcαRI (CD89) and co-stimulatory molecules CD80/CD86. Blocking these pathways significantly reduced IFN-γ production (p =0.0347) (Figure 3D), suggesting that keratinocyte–IgA immune complexes are internalized via CD89 and require co-stimulation for T cell activation. These findings support a model in which IgA autoantibodies facilitate antigen uptake and presentation by mDCs, leading to activation of autoreactive CD4⁺ T cells and promoting IFN-γ/TNF-α–driven skin inflammation. This mechanism highlights a potential bridge between innate and adaptive immunity in HS.

IgA autoantibodies target macrophages and promote a pro-inflammatory and pro-fibrotic response in HS

Building on the discovery of a broad IgA autoantibody repertoire in HS, we identified a specific subset of IgA autoantibodies recognizing macrophage proteins that were significantly elevated in HS skin (p < 0.0001) compared to control skin (Fig. 4A) and positively associated with disease severity (r= 0.3845, p = 0.0238; Figure 4B). Immunofluorescence staining of HS lesions revealed co-localization of IgA with CD68⁺ cells, supporting the presence of IgA-bound resident or infiltrating macrophages in lesional tissue (Fig. 4C). To determine whether these antibodies specifically recognize macrophage antigens, we performed ELISA using recombinant CD68. HS samples exhibited significantly higher levels of anti-CD68 IgA (p =0.0026) compared to controls (Fig. 4D), with a strong correlation between antibody levels and disease severity (R = 0.4671, p = 0.0189; Fig. 4E). To assess the functional consequences of IgA binding, we exposed primary human M2-like macrophages to IgA purified from HS lesions, control IgA, or vehicle for 24 hours. RNA sequencing of macrophages treated with HS IgA revealed substantial upregulation of genes involved in inflammation, leukocyte chemotaxis, fibroblast activation, and the NLRP3 inflammasome pathway (Fig. 4F, G). ELISA of culture supernatants confirmed significantly increased secretion of TNF-α (p =0.0308), IL-6 (p =0.0002), and IL-1β (p = 0.0169) by macrophages treated with HS IgA compared to control IgA or untreated macrophages (Figure 4H–J). Given our previous observation of IgG deposition in HS skin lesions(13), we next assessed whether IgG purified from the same HS tissues elicited proinflammatory responses comparable to those induced by IgA. IgG isolated from HS samples induced significant release of TNF-α (p = 0.0047), and IL-6 (p = 0.0016), but did not stimulate significant IL-1β production (Figure 4H–J), highlighting a functional divergence between IgG- and IgA-mediated macrophage activation.

Given the cytokine profile, we hypothesized that HS IgA–activated macrophages could promote Th17 polarization. To test this hypothesis, naïve CD4⁺ T cells from healthy donors were purified and cultured with 10% supernatant from macrophages treated with either HS-IgA or control IgA. After 4 days, qPCR revealed significant upregulation of RORC (p =0.0055), encoding the Th17 lineage defining transcription factor RORγt, in T cells exposed to HS IgA-conditioned media (Fig. 4K). Additionally, IL-17A (p =0.0005) levels were significantly elevated in these cultures (Fig. 4L), indicating that macrophage-derived factors driven by HS IgA promote Th17 differentiation. Consistent with these findings, supernatants from macrophages treated with HS-derived IgG failed to promote polarization of naïve T cells toward a Th17 phenotype, as evidenced by significantly lower IL-17A production (p = 0.0025) compared with naïve T cells exposed to supernatants from macrophages stimulated with HS-derived IgA (Figure 4L).Together, these results indicate that IgA-but not IgG- autoantibodies in HS preferentially activate macrophages to produce Th17-polarizing cytokines, thereby linking humoral autoimmunity to pathogenic adaptive immune responses in the skin.

IgA autoantibodies recognize NETs and amplify inflammation and fibrosis via macrophage and fibroblast crosstalk

Further expanding the spectrum of IgA autoreactivity in HS, we identified significantly elevated levels of IgA antibodies directed against neutrophil extracellular traps (NETs, p = 0.002) in lesional HS skin compared to controls (Fig. 5A). This was relevant as NETs have been implicated in HS pathogenesis (5, 14). These anti-NET antibodies were significantly elevated in Hurley stage III when compared to controls (p = 0.0025) (Fig. 5B). Immunofluorescence staining of HS tissue revealed co-localization of IgA and neutrophil elastase, a hallmark component of NETs, supporting the presence of IgA autoantibodies bound to NET structures in vivo (Fig. 5C). To assess whether IgA can modulate NET formation, control neutrophils were incubated with purified IgA from HS or control skin. Indeed, HS IgA induced significant NET release (p = 0.0047) when compared to control IgA (Fig. 5D). These findings suggest a vicious cycle, in which IgA antibodies recognize and stimulate NET formation, potentially perpetuating local inflammation.

To explore the impact of NET–IgA immune complexes on other cell types involved in HS pathogenesis, we incubated M2 polarized macrophages with NETs alone or with NET–IgA complexes. Supernatant analysis revealed that NET–IgA complexes induced significant release of CCL18 (p = 0.0022) (Fig. 5E), a chemokine known to stimulate collagen synthesis in dermal fibroblasts and proposed to play a pathogenic role in HS. Of note, IgA alone did not induce significant CCL18 release (Suppl Fig S3). To determine the downstream impact of this macrophage-derived signal, primary skin fibroblasts were incubated with supernatants from M2 macrophages treated with NETs or NET–IgA complexes and RNA sequencing was performed. Notably, skin fibroblasts treated with NET–IgA supernatant exhibited an upregulated type I interferon (IFN) gene signature, as well as significant upregulation of pro-inflammatory genes including IL6, CXCL3, CCL2, and IL1B (Fig. 5F, G). ELISA analysis showed that fibroblasts exposed to NET–IgA-conditioned M2 supernatants produced significantly elevated levels of collagen when compared to those exposed to NETs alone (p =0.0317), suggesting that NET–IgA immune complexes may drive fibroblast-mediated fibrosis via macrophage signaling. These results suggest that NET–IgA immune complexes not only promote fibrosis but also amplify innate immune activation in fibroblasts.

To assess whether NET–IgA complexes can act directly on fibroblasts, these cells were also incubated with purified NETs or NET–IgA complexes. RNA-seq analysis revealed that NET–IgA-treated fibroblasts upregulated genes involved in leukocyte recruitment and adhesion, such as CD9, ITGA2, VCAM1, ICAM1, and those encoding for several CXCL chemokines, consistent with a pro-inflammatory and pro-chemotactic phenotype (Fig. 5I). Additionally, genes associated with adaptive immune modulation including TNFSF13B (BAFF), TNFAIP3, and NFKBIZ were significantly upregulated (Fig. 5J), suggesting that NET–IgA complexes may also prime fibroblasts to act as immune-competent stromal cells that influence local adaptive responses. These findings support a model in which NET–IgA immune complexes serve as pleiotropic and potent inflammatory amplifiers, capable of activating macrophages and fibroblasts in a way that bridges innate immunity, tissue remodeling, and adaptive immune regulation, key processes that underlie the chronic and fibrotic nature of HS lesions.

Methods
Collection of tissue samples

Healthy controls were recruited through advertisements, and written informed consent was obtained from all healthy volunteers. HS samples were obtained under protocols NA_00013177 and NA_00031269, approved by the Johns Hopkins University Institutional Review Board (IRB), IRB-19-MED-50 approved by the Howard University Office of Regulatory Research Compliance, and IRB-17-1506 approved by the University of North Carolina (UNC) Institutional Review Board. Lesional skin samples from HS patients were collected through the UNC Department of Dermatology’s HS Program for Clinical and Research Excellence (ProCARE), following written informed consent. Skin samples were obtained during standard-of-care surgical procedures using a 6 mm punch biopsy. Clinical assessments were conducted by the surgeon or investigator at the time of surgery, documenting lesion type, regional and global lesion counts, current medications, and other relevant clinical and demographic information from electronic medical records or clinical registries. Additional lesional and non-HS samples were obtained from the Johns Hopkins tissue bank, sourced from patients undergoing HS lesion excision. All diagnoses were confirmed through clinical, visual, and histopathological evaluation. Collected tissues were stored at –80 °C until further analysis. All participants provided written informed.

Isolation of protein from tissue and cells

Protein extraction from tissues or cells was performed as previously described(48). Briefly, pulverized tissue samples were resuspended in RIPA buffer supplemented with a protease inhibitor cocktail (Roche). Following a 1-hour incubation at 4°C, the samples were centrifuged at 16,000 × g for 10 minutes. The resulting supernatants were collected and transferred to fresh Eppendorf tubes. Protein concentrations were determined using the BCA Protein Assay Kit (Thermo Fisher), following the manufacturer’s instructions.

Antibodies

The following monoclonal antibodies were used: mouse anti-human IgA2-biotin (1:10,000, SouthernBiotech, cat no. 9140-08), mouse anti-human IgA1 (1:10,000, SouthernBiotech, cat no. 29130-08), mouse anti-human CD89 (1:100, Bio-Rad, cat no.MCA1824), mouse anti-human CD80 (1:100, Bio-Rad, cat no. MCA2071GA), mouse anti-human CD86 (1:100, Bio-Rad, cat no. MCA1118), and mouse anti-human GAPDH (clone 6C5; 1:1,000, Thermo Fisher Scientific, cat no. AM4300). Antibodies were used for Western blotting according to manufacturer-recommended dilutions.

NET isolation

NETs were isolated as previously described(48). In brief, neutrophils from HS patients were resuspended in RPMI medium without phenol red and seeded into 24-well tissue culture plates. The cells were incubated at 37°C for 3 hours. Following incubation, supernatants were removed, and the adherent NETs were digested using micrococcal nuclease (10 U/mL; Thermo Fisher Scientific) in RPMI for 15 minutes at 37°C. The resulting supernatants were collected and centrifuged at 2,800 × g for 5 minutes at 4°C. NET-containing supernatants were then transferred to eppendorf tubes and stored at −80°C until further use.

Anti-NET, macrophage and CD68 IgA detection

A 96-well plate was coated overnight at 4°C with one of the following antigens diluted in PBS(14): 10 μg/mL of spontaneously generated neutrophil extracellular traps (NETs), 2.5 μg/mL recombinant CD68 (antibodies-online, cat no. ABIN7092757), or 10 μg/mL macrophage lysate. Following coating, wells were blocked with 1% BSA for 1 hour at room temperature. One microgram of total protein extracted from control or HS skin samples was added to each well and incubated overnight at 4°C. After washing, HRP-conjugated goat anti-human IgA secondary antibody (1:5,000; Life technologies, cat no. a18781) was incubated for 1 hour at room temperature. ELISA was developed using TMB substrate, and absorbance was measured at 450 nm using a Synergy HT microplate reader (Bio-Tek, Rockville, MD). Results are expressed as the optical density (OD) index, calculated as the ratio of the sample OD to the mean OD of healthy control.

NET quantification plate assay

To quantify NET formation, neutrophils were resuspended in RPMI without phenol red medium containing 0.2 µM Sytox Green (Invitrogen)(14). Cells (1 × 10⁴) were seeded into wells of a plate previously coated with either control or HS IgA. Cells were incubated for 2 hours at 37°C. Extracellular DNA was quantified by measuring fluorescence using a BioTek Synergy H1 Hybrid Reader (excitation: 485 nm; emission: 520 nm). Results are expressed as relative fluorescence units (RFU).

Quantitative PCR analysis

RNA was isolated from tissue sections and cells as previously describes(5). Briefly, RNA was isolated using the Direct-zol RNA MiniPrep Kit (Zymo Research) according to the manufacturer’s instructions. Total RNA (300-500 ng) was reverse- transcribed using iScript RT single-strand complementary DNA (cDNA) (Bio-Rad). qPCR was performed using the TaqMan Gene Expression Master Mix (Thermo Fisher Scientific), human GAPDH primers (Hs99999905_m1), and sequence-specific primers for IGHA1(Hs00733892_m1), JCHAIN (H00376160_m1), RORC (Hs01076112_m1) were used. Fold difference was calculated using the DCt equation.

RNA sequencing and analysis

Between 50 and 500 ng of total RNA were used for cDNA synthesis, with RNA quantity normalized across all samples per experiment. RNA integrity was evaluated by analyzing an aliquot of each sample on an Agilent TapeStation using the High Sensitivity RNA ScreenTape. cDNA libraries were prepared using either the NEBNext Poly(A) mRNA Magnetic Isolation Kit or the NEBNext rRNA Depletion Kit (New England Biolabs), followed by library construction with the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs). Library fragment size and quality were assessed using the Agilent TapeStation with D1000 ScreenTape. Library concentrations were quantified using the PicoGreen dsDNA assay (Thermo Fisher Scientific). Libraries were diluted to 3 nM, pooled, and sequenced on the Illumina Novaseq X platform. Raw sequencing data were converted to FASTQ format using bcl2fastq (v2.20.0). Adapter trimming was performed using Trim Galore (v0.64.7). Pair-end 50 bp reads were mapped to the human genome (hg38) using STAR2.7.8a as implemented in Partek Flow (version 11.0.24.0604) with default setting. Gene expression levels were quantified as FPKM (Fragments Per Kilobase of exon per Million mapped reads) using the “quantify to annotation model” (Partek E/M) based on ensemble transcript release 109. Differential gene expression was conducted with ANOVA with Partek Flow.

Western blot

Proteins isolated from tissue, macrophages, keratinocytes (HaCaT), and skin fibroblasts were quantified using the BCA Protein Assay Kit (Thermo Fisher) following the manufacturer’s protocol. Equal amounts of total protein were separated on 4–12% Bis-Tris gradient gels (Invitrogen), then transferred onto nitrocellulose membranes. Membranes were blocked with 10% BSA for 30 minutes at room temperature, followed by overnight incubation with primary antibodies at 4°C. After washing three times with PBS containing 0.1% Tween-20 (PBS-T), membranes were incubated with (1:10,000) secondary antibodies conjugated to IRDye 680 or 800CW. Blots were visualized using the LI-COR Odyssey CLx imaging system.

IgA autoantibody detection assay

The assay was performed as previously described (13, 49). Briefly, skin lysates from 45 HS patients and 10 controls were pretreated with DNase I and diluted 1:50. Samples were then hybridized to nitrocellulose membrane-coated slides printed in duplicate with a panel of autoantigens related to various autoimmune conditions, including systemic lupus erythematosus (SLE), systemic sclerosis, Sjogren's syndrome, idiopathic inflammatory myositis, and rheumatoid arthritis (RA) (available at: https://utsw.corefacilities.org/service_center/show_external/5596?name=utsw-microarray-immune-phenotyping-core-facility). Following hybridization and washing steps, bound antibodies were detected using Cy3-labeled anti-human IgA (1:2,000; Jackson ImmunoResearch Laboratories, cat no. 109-165-011). Arrays were scanned using a GenePix 4400A Microarray Scanner, and fluorescence intensities were analyzed with GenePix 7.0 software (Molecular Devices). Background signal (from PBS) was subtracted from each sample’s average signal intensity. A signal-to-noise ratio (SNR) was calculated, and antibody scores were determined using the formula log₂ (SNR × NFI + 1), as previously described(50).

Frozen Sections and Immunofluorescence Staining

Frozen tissue sections (25 µm) were methanol fixed and permeabilized for 10 minutes at 4°C, followed by blocking with 10% bovine serum albumin (BSA) for 1 hour at room temperature. Where indicated, sections were incubated overnight at 4°C with primary antibodies, including anti-CD68 (1:200, Abcam, cat no. ab201340), elastase (1:200, Abcam, cat no. ab68672), and FITC-conjugated IgA antibodies (1:200, Sigma, cat no. F5259), all diluted in 5% BSA. Following incubation, slides were washed three times with PBS. Sections were then incubated with secondary antibodies Alexa Fluor 555-conjugated anti-rabbit IgG or Alexa Fluor 488-conjugated donkey anti-mouse IgG (1:400; Invitrogen, cat no. A-21428, A-21202) for 1 hour at room temperature. Nuclei were counterstained with Hoechst 33342 (1:1,000, Invitrogen, cat no. H3570) for 10 minutes. After five PBS washes, sections were mounted with ProLong Gold Antifade Mounting (Invitrogen, cat no. P36930) and coverslip. Images were acquired using a Zeiss LSM780 confocal laser scanning microscope.

IgA and IgG Isolation from Skin Lysates

IgA and IgG antibodies were purified from 100 µg of Chronic stage (II-III) HS or control skin lysates using Protein M and G beads (InvivoGen), respectively. Samples were incubated with Protein M or G beads overnight at 4°C. After incubation, the beads were washed three times with binding buffer (Cytiva, Sweden). IgA and IgG were then eluted using elution buffer and immediately neutralized with neutralization solution (Cytiva, Sweden). Buffer exchange was performed using Microcon-10 centrifugal filters (Millipore) according to the manufacturer’s instructions.

IgA or IgG Stimulation and Macrophage Activation

Human CD14⁺ monocytes were isolated from control PBMCs using positive selection (Miltenyi Biotec). CD14⁺ monocytes were cultured with M-CSF (50 ng/mL; PeproTech) for 5–7 days. For stimulation, 96-well plates were coated overnight at 4°C with purified IgA or IgG (isolated from control or HS skin lysates) diluted in PBS. Plates were then washed twice with phosphate-buffered saline before seeding with macrophages. Differentiated macrophages were detached using Dissociation solution (StemCells) and 50,000 cells were seeded per well in 200 uL. Cells were incubated for 24 or 72 hours, after which supernatants were collected and analyzed for TNF-α, IL-6, and IL-1β using commercial ELISA kits (Invitrogen).

Skin fibroblast treatment

Primary human skin fibroblasts were obtained from Coriell and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% L-glutamine at 37°C in a humidified incubator with 5% CO₂. For stimulation experiments, fibroblasts were seeded into 12-well plates that had been pre-coated for 24 hours with 50 µg of NETs alone or NETs + HS-IgA. After 24 hours of incubation, total RNA was extracted for RNA sequencing analysis. In a parallel experiment, fibroblasts were also treated for 24 hours with 10% supernatant from macrophages previously stimulated with either NETs alone or NETs + HS-IgA. RNA was isolated for transcriptomic profiling by RNA sequencing, and culture supernatants were collected and analyzed for collagen type I alpha 1 (COL1A1) levels using ELISA.

mDC and CD4 T cell co-culture

Myeloid dendritic cells (mDCs) and CD4⁺ T cells were isolated from peripheral blood mononuclear cells (PBMCs) obtained from healthy control donors using the Myeloid Dendritic Cell Isolation Kit (Miltenyi Biotec, cat no. 130-094-487) and the CD4⁺ T Cell Isolation Kit (Miltenyi Biotec, cat no. 130-096-533), respectively, following the manufacturer's instructions. To generate immune complexes, 96-well plates were pre-coated overnight at 4°C with IgA purified from HS skin lesion. Keratinocyte lysates were prepared and denatured by heating at 95°C for 5 minutes, then incubated with the IgA-coated wells for 2 hours at room temperature. Plates were washed twice with PBS to remove unbound proteins. Isolated mDCs and CD4⁺ T in a ratio 1:2 cells were then co-cultured in the presence or absence of keratinocyte-IgA immune complexes, with or without the addition of blocking antibodies against CD89 (1:100, Bio-Rad, cat no. MCA1824), CD80 (1:100, Bio-Rad, cat no. MCA2071GA), or CD86 (1:100, Bio-Rad, cat no. MCA1118) for 4–5 days. Culture supernatants were collected and analyzed for IFN-γ and/or TNF-α production.

Th17 polarization

Naïve CD4⁺ T cells were isolated from peripheral blood mononuclear cells (PBMCs) using the Naïve CD4⁺ T Cell Isolation Kit II, human (Miltenyi Biotec, cat no. 130-094-131), according to the manufacturer’s protocol. Isolated naïve T cells were activated with CD3/CD28 ImmunoCult antibodies (1:40, Stem Cell, cat no.10971) and cultured in the presence of 10% of supernatant from macrophages that had been previously exposed to either control or HS purified IgA or IgG. Cells were incubated at 37°C in a humidified incubator with 5% CO₂ for 4 days. Following incubation, total RNA was extracted for qPCR analysis of the Th17 lineage-defining transcription factor RORC. Culture supernatants were analyzed for IL-17A levels using ELISA to assess Th17 polarization.

Statistical Analysis

All statistical analyses were performed using GraphPad Prism version 9.5.0 (GraphPad Software, La Jolla, CA). For comparisons involving non-normally distributed data, the Mann–Whitney U test was applied. Group comparisons were conducted using Kruskal–Wallis test followed by Dunn’s multiple comparison test. Pearson correlation was used to assess associations between continuous variables. A p-value of < 0.05 was considered statistically significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Hidradenitis suppurativa (HS) is a relatively common inflammatory skin disease with potentially devastating impacts on\npatient quality of life. Mechanisms of disease pathogenesis remain poorly defined. In their manuscript Carmona-Rivera et al.,\nbuild on recent findings that B cells and supporting tertiary lymphoid structures are highly active in HS to comprehensively\ndetail the functional role of lesional auto-IgA. The current work identifies high enrichment of auto-IgA in lesional HS skin\nsamples and maps which specific antigens are targeted including correlations with clinical parameters. A number of strong\nex vivo experiments with IgA isolated from lesional skin are performed to demonstrate that these antibodies are functionally\nrelevant for regulating antigen presentation to T cells, macrophage activation, and fibroblast activity. Altogether the findings\npresent a strong new line of evidence that local autoimmunity contribute to the inflammatory pathogenesis of HS. The\nmanuscript is well written and robust experiments/analyses are presented. Findings have important implications for HS but\nalso may inform our thinking about the link between autoimmunity and inflammatory disease more generally. Therefore, I\nbelieve the manuscript is well suited for the broad interest readership at Nature Communications. I have minor comments\nlisted below:\n-In Figure 1, total IgA gene expression and protein levels present in lesional skin are quantified. As done with most other\nfigures it would be helpful to correlate this by Hurley stage given this in of itself could be a biomarker.\n-At the end of the first results section “IgAs are present in HS skin lesions” it is stated that the “findings indicate TLS support\nlocal differentiation of IgA-producing cells…” while this is logical, the presented data in of themselves do not test this\nhypothesis. I would be more comfortable if the sentence was deleted or amended to remove reference to the role of TLS.\n-The image quality of Fig 2A,B,D is low and I had trouble reading the individual antigens/parameters.\n-The correlation analysis in Fig 2D is important but as a comparator it would be helpful to also include total IgA.\n-In Figure 4c the CD4/mDCs are from healthy donors but the figure legend wording could be misconstrued as indicating the\ncells are isolated from HS patients.\n-Fig 5f is slightly cut off.", "reviewer_2": "Overall Assessment\nThis manuscript investigates the role of IgA autoantibodies in hidradenitis suppurativa, demonstrating their ability to activate\nmacrophages, promote Th17 polarization, and contribute to fibrotic responses. The topic is timely and important, linking\nhumoral immunity to key inflammatory and tissue-remodeling processes in HS. The study is original and clinically relevant,\nbut several methodological and interpretative weaknesses limit the strength of the conclusions.\nStrengths\n1. Originality: The potential role of IgA autoantibodies in HS has been scarcely studied, making this manuscript highly\ninnovative.\n2. Integrated approach: The authors connect autoantibodies, macrophage activation, Th17 polarization, and fibrosis within a\nunified mechanistic framework.\nWeaknesses\nIgA specificity\n- IgA autoantibodies are often polyreactive and prone to nonspecific binding. The broad reactivity observed in Figures 2A\nand 2B might reflect nonspecific interactions rather than true antigen-specific responses. This issue should be discussed.\n- If IgA are indeed polyreactive, they may target microbial as well as self-antigens. This possibility should be considered,\nespecially given the suspected role of dysbiosis in HS.\n- Since many HS patients receive antibiotics, it would be valuable to explore whether IgA responses are modulated in this\ncontext. This could help disentangle whether the observed activity is linked to microbial exposure or autoreactive.\nVariability in IgA immunostaining\nThe in situ IgA staining shown in Figures 1G, 4C, and 5C appears inconsistent. Quantification or more standardized\nrepresentative images would strengthen confidence in these findings.\nOrigin of IgA production within TLS\nIt remains unclear whether TLS are the main sites of IgA production. Demonstrating or discussing this point would\nsignificantly enhance the mechanistic interpretation.\nComparison with IgG responses is missing\nIn chronic inflammation, isotype switching typically favors IgG rather than IgA. It would be highly informative to quantify IgG\nresponses in parallel, to assess whether IgG autoantibodies might contribute even more significantly to macrophage\nactivation, Th17 polarization, or NET recognition. Without this comparison, the specific contribution of IgA remains difficult to\nestablish.\nRequirement for immune complexes\nIn figure 3, it is unclear whether IgA alone can activate myeloid dendritic cells (mDCs), or whether immune complex\nformation is required. Clarification is essential, since FcαR signaling usually depends on multivalent engagement.\nImpact of IgA on M2 macrophages\nIt would be highly relevant to test whether HS-derived IgA alters the phenotype of M2 macrophages. For example, do they\nproduce fibrotic mediators such as CCL18, or undergo transcriptomic changes consistent with a pro-fibrotic program?"}, "author_response": "We thank the reviewer for this thoughtful and comprehensive evaluation of our work. We\nappreciate the positive assessment of the novelty, experimental rigor, and broader\nimplications of our findings in HS and other chronic inflammatory diseases.\n-In Figure 1, total IgA gene expression and protein levels present in lesional skin are\nquantified. As done with most other figures it would be helpful to correlate this by Hurley\nstage given this in of itself could be a biomarker.\nWe thank the reviewer for this helpful suggestion. We have now incorporated the distribution\nof total IgA gene expression and protein levels stratified by Hurley stage. These results are\nnow presented in Supplementary Figure S1.\n-At the end of the first results section “IgAs are present in HS skin lesions” it is stated that\nthe “findings indicate TLS support local differentiation of IgA-producing cells…” while this is\nlogical, the presented data in of themselves do not test this hypothesis. I would be more\ncomfortable if the sentence was deleted or amended to remove reference to the role of\nTLS.\nWe thank the reviewer for this important point and agree that the original wording could be\ninterpreted as overextending beyond the data initially presented. To address this concern,\nwe have revised the text to more accurately reflect the experimental evidence.\nIn the revised manuscript, we have added immunostaining for IgA producing cells in\nconjunction with B-cell markers in HS skin, demonstrating the presence of IgA-expressing B\ncells within cellular aggregates resembling tertiary lymphoid structures (Figure 1J). These\ndata, together with our qPCR findings showing increased IgA transcripts in lesional\ncompared with non-lesional skin, and the detection of IgA in HS skin lesions but not in the\nmatched serum samples, support the presence of locally differentiated IgA-producing cells\nin the skin.\nWe have revised the Results text to avoid implying a definitive mechanistic role of TLS\nstructures in IgA class switching and instead describe the findings as evidence consistent\nwith local IgA production. We believe these changes appropriately align our conclusions\nwith the scope of the experimental data.\n-The image quality of Fig 2A,B,D is low and I had trouble reading the individual\nantigens/parameters.\nWe apologize for the lack of clarity and agree that the heatmaps in Fig. 2A, B, and D contain\na large number of antigens, which made them difficult to read at the original resolution. To\naddress this, we have increased the figure size and optimized the image resolution. When\nviewed at higher magnification, individual antigens and expression patterns are now clearly\ndiscernible.\n-The correlation analysis in Fig 2D is important but as a comparator it would be helpful to\nalso include total IgA.\nWe thank the reviewer for this request. In response, we have performed an additional\ncorrelation analysis using total IgA and IgG levels measured in HS skin lesions. These\nresults are now included in Figure 2F.\n-In Figure 4c the CD4/mDCs are from healthy donors but the figure legend wording could be\nmisconstrued as indicating the cells are isolated from HS patients.\nTo clarify, the mDC–CD4 T cell experiments shown in Figure 3 were performed using mDCs\nand CD4 T cells isolated from healthy donors and cultured in the presence of IgA purified\n⁺\n⁺\nfrom HS patients. The corresponding figure legend reads: “IgA facilitates keratinocyte\nantigen uptake by mDCs via FcαR and promotes CD4 T cell activation.”\n⁺\n-Fig 5f is slightly cut off.\nThanks for noticing this; we have now corrected this.\nReviewer #2 (Remarks to the Author):\nOverall Assessment\nThis manuscript investigates the role of IgA autoantibodies in hidradenitis suppurativa,\ndemonstrating their ability to activate macrophages, promote Th17 polarization, and\ncontribute to fibrotic responses. The topic is timely and important, linking humoral\nimmunity to key inflammatory and tissue-remodeling processes in HS. The study is original\nand clinically relevant, but several methodological and interpretative weaknesses limit the\nstrength of the conclusions.\nWe thank the reviewer for recognizing the novelty, clinical relevance, and potential impact\nof our study linking IgA autoantibodies to key inflammatory and tissue-remodeling\nprocesses in HS. We also appreciate the constructive feedback regarding methodological\nand interpretative aspects and have carefully addressed each of the points raised to\nstrengthen the rigor and clarity of our conclusions.\nStrengths\n1. Originality: The potential role of IgA autoantibodies in HS has been scarcely studied,\nmaking this manuscript highly innovative.\nWe thank the reviewer for highlighting the originality and integrative nature of our study.\n2. Integrated approach: The authors connect autoantibodies, macrophage activation, Th17\npolarization, and fibrosis within a unified mechanistic framework.\nWe are pleased that the innovative aspect of investigating IgA autoantibodies in HS and our\napproach linking humoral immunity to macrophage activation, Th17 polarization, and\nfibrotic responses are recognized. We consider that these insights provide a valuable\nframework for understanding the multifaceted mechanisms driving HS pathogenesis.\nWeaknesses\nIgA specificity\n- IgA autoantibodies are often polyreactive and prone to nonspecific binding. The broad\nreactivity observed in Figures 2A and 2B might reflect nonspecific interactions rather than\ntrue antigen-specific responses. This issue should be discussed.\nWe appreciate the reviewer’s comment regarding IgA polyreactivity. While IgA antibodies,\nparticularly those arising in mucosal and tissue compartments, are known to exhibit a\ndegree of polyreactivity, this property does not necessarily imply nonspecific or artifactual\nbinding. Indeed, accumulating evidence indicates that IgA polyreactivity can be biologically\nmeaningful and functionally relevant ( Bunker et al., Science 2018; Lindner, J Exp Med 2012;\nBarone et al., Gastro 2011; Kabbert et al., J Exp Med 2020). In our study, the reproducible and\nselective downstream responses following IgA engagement—including induction of IL-1β\nproduction by macrophages and preferential Th17 polarization—argue against nonspecific\ninteractions as the primary explanation for the broad reactivity patterns observed in Figures\n2A-B. We therefore interpret these data as reflecting biologically relevant IgA-antigen\ninteractions, while acknowledging that IgA polyreactivity may contribute to the breadth of\nantigen recognition. We have added text to the Discussion to clarify this point and to\nappropriately contextualize the findings.\n- If IgA are indeed polyreactive, they may target microbial as well as self-antigens. This\npossibility should be considered, especially given the suspected role of dysbiosis in HS.\nWe appreciate the reviewer’s comment regarding IgA polyreactivity and the possibility that\nIgA antibodies in HS may target both microbial and self-antigens, particularly in the context\nof dysbiosis. To address this, we assessed IgA reactivity against several microbes implicated\nin HS or commonly present on the skin, including E. coli, Lactobacillus casei, and P.\ngingivalis. We did not detect significant differences in IgA reactivity to these microbial\nantigens between healthy skin and HS lesions (Figure 2E, F; Supplementary Figure S2).\nThese findings suggest that, for the microbial antigens examined, IgA responses do not differ\nbetween disease and control tissue and are unlikely to account for the increased self-\nreactive IgA observed in HS lesions. Moreover, IgA reactivity to these microbial targets did\nnot confound the associations observed between IgA self-reactivity and clinical parameters.\nWhile IgA polyreactivity remains a theoretical consideration, our data indicate that IgA\ntargeting of these microbial antigens is unlikely to be the primary driver the self-reactive IgA\nsignatures identified in HS skin.\n- Since many HS patients receive antibiotics, it would be valuable to explore whether IgA\nresponses are modulated in this context. This could help disentangle whether the observed\nactivity is linked to microbial exposure or autoreactive.\nWe agree that antibiotic use is an important variable to consider when interpreting IgA\nresponses in HS. To address this, we performed correlation analyses stratifying patients\nbased on treatment with topical clindamycin or doxycycline. We did not observe significant\ncorrelations between antibiotic exposure IgA levels in HS skin (clindamycin r = 0.2271, p =\n0.1678; doxycycline r = 0.3780, p = 0.1004) nor with IgG levels (clindamycin r = 0.1325, p =\n0.2889; doxycycline r = 0.3382, p = 0.1147). If local IgA or IgG accumulation were primarily\ndriven by microbial exposure, reduced antibody levels would be expected in patients\nreceiving antibiotics. The absence of such associations suggests that the observed antibody\nresponses are more likely autoreactive rather than directly linked to microbial burden. We\nacknowledge that the relatively small cohort size and imbalance between treated and\nuntreated patients limit definitive conclusions. Accordingly, indirect or context-dependent\nmicrobial contributions cannot be entirely excluded and warrant further investigation in\nlarger, more balanced cohorts.\nVariability in IgA immunostaining\nThe in situ IgA staining shown in Figures 1G, 4C, and 5C appears inconsistent.\nQuantification or more standardized representative images would strengthen confidence in\nthese findings.\nThank you for the comment. In response, we repeated immunofluorescence for IgA, NETs,\nand macrophages in both control and HS skin samples. We have replaced Figures 1G, 4C,\nand 5C with more standardized and representative images that better illustrate the\ndistribution and co-localization of these components across samples. These updated\nimages more accurately reflect the reproducibility of our observations.\nOrigin of IgA production within TLS\nIt remains unclear whether TLS are the main sites of IgA production. Demonstrating or\ndiscussing this point would significantly enhance the mechanistic interpretation.\nTo address this, we have added confocal images of HS skin lesions demonstrating B\ncell aggregates consistent with tertiary lymphoid structures (TLS) and the presence of IgA-\npositive cells within or adjacent to these structures in Figure 1J. While these data support a\nspatial association between TLS and IgA-producing cells, we acknowledge that they do not\ndefinitively establish TLS as the sole or primary sites of IgA production. We have therefore\ntempered our interpretation accordingly and discussed this limitation in the revised\nmanuscript.\nComparison with IgG responses is missing\nIn chronic inflammation, isotype switching typically favors IgG rather than IgA. It would be\nhighly informative to quantify IgG responses in parallel, to assess whether IgG\nautoantibodies might contribute even more significantly to macrophage activation, Th17\npolarization, or NET recognition. Without this comparison, the specific contribution of IgA\nremains difficult to establish.\nWe thank the reviewer for this insightful comment. In response, we have now quantified\ntotal IgG levels in the same HS skin samples analyzed for IgA (Supplementary Figure S1).\nNotably, IgG and IgA levels were comparable, indicating that IgA accumulation in HS\nlesions is not simply secondary to a dominant IgG response.\nTo further delineate functional differences, we purified IgA and IgG from the same HS tissue\nsamples and compared their effects on macrophage activation. Both isotypes induced IL-6\nand TNF production at similar or greater levels following IgG stimulation. However, IL-1β\nproduction differed significantly between IgA- and IgG-stimulated macrophages (Figures\n4H–J). Supernatants from these macrophage cultures were then used in Th17 polarization\nassays. While supernatants from IgG-stimulated macrophages failed to promote Th17\npolarization, those from IgA-stimulated macrophages robustly induced Th17 polarization\n(Figure 4L). Together, these findings suggest that although IgG contributes to inflammatory\ncytokine production, IgA uniquely promotes a macrophage-TH17 axis, supporting a\nnonredundant role for IgA in shaping pathogenic immune responses in HS.\nRequirement for immune complexes\nIn figure 3, it is unclear whether IgA alone can activate myeloid dendritic cells (mDCs), or\nwhether immune complex formation is required. Clarification is essential, since FcαR\nsignaling usually depends on multivalent engagement.\nWe have now included experiments in which mDCs were incubated with purified IgA alone\nas a control condition. These data are included in the revised Figure 3\nImpact of IgA on M2 macrophages\nIt would be highly relevant to test whether HS-derived IgA alters the phenotype of M2\nmacrophages. For example, do they produce fibrotic mediators such as CCL18, or undergo\ntranscriptomic changes consistent with a pro-fibrotic program?\nWe thank the reviewer for this insightful suggestion. To address this, we incubated M2\nmacrophages with purified IgA derived from either healthy control skin or HS lesional skin.\nTreatment with control IgA reduced CCL18 production, whereas HS-derived IgA did not\nsignificantly alter CCL18 levels compared with untreated M2 macrophages\n(Supplementary Figure S3). These findings indicate that HS IgA alone is insufficient to\ndirectly induce a pro-fibrotic macrophage phenotype.\nImportantly, these results suggest that additional contextual signals, such as NET priming\nor IgA-NET immune complex formation, as demonstrated in Figure 5, may be required to\ndrive fibrotic or tissue-remodeling programs. While our data does not support a direct pro-\nfibrotic effect of HS IgA in isolation, they highlight a multi-step mechanism in which IgA\ncontributes to inflammation and tissue remodeling in concert with other immune factors.\nWe believe this adds important mechanistic nuance to the role of IgA in HS pathogenesis."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have thoughtfully and carefully addressed my initial comments/concerns. My strong enthusiasm for this\nmanuscript remains as I believe it applies a rigorous approach to dissecting a the potential pathogenic role of lesional\nantibody (especially IgA) in HS. Conclusions are well supported by the data presented and these data are highly important\nfor the field.\nA few small errors should be addressed:\n-In figure S1A the split axis cuts off some of the data points\n-Line 62: it's misleading to shorten follicular dendritic cells to \"DCs\" as they are mesenchymal in origin. Use FDC\n-Line 315: Is this a typo?\n-Line 326: typo \"BAFF ,\"", "reviewer_2": "The revised manuscript has been substantially improved following the initial review. The authors have addressed all of my\ncomments carefully and comprehensively, providing additional data, analyses, and clarifications that significantly strengthen\nboth the rigor and the mechanistic interpretation of the study.\nIn particular, the added analyses examining IgA reactivity against microbial antigens, as well as the direct functional\ncomparison between IgA and IgG derived from the same lesional samples, meaningfully reinforce the central conclusions.\nThe clarification regarding immune complex requirements and the tempered interpretation of TLS involvement further\nenhance the scientific precision of the manuscript.\nOverall, the revised version presents a coherent and well-supported framework linking\nlesional IgA autoantibodies to macrophage activation, Th17 polarization, and fibrotic responses in hidradenitis suppurativa.\nThe manuscript is now considerably strengthened."}, "author_response": "We sincerely thank the reviewer for the thoughtful and encouraging feedback. We greatly\nappreciate your recognition of the rigor of our approach and the significance of our\nfindings.\nA few small errors should be addressed:\n-In figure S1A the split axis cuts off some of the data points\nWe thank the reviewer for highlighting this issue. We have revised Figure S1A and ensured\nthat all data points are fully visible.\n-Line 62: it's misleading to shorten follicular dendritic cells to \"DCs\" as they are\nmesenchymal in origin. Use FDC\nThanks for the clarification, we have updated the revised version accordingly.\n-Line 315: Is this a typo?\nWe have corrected accordingly.\n-Line 326: typo \"BAFF ,\"\nWe have corrected accordingly.\nReviewer #2 (Remarks to the Author):\nThe revised manuscript has been substantially improved following the initial review. The\nauthors have addressed all of my comments carefully and comprehensively, providing\nadditional data, analyses, and clarifications that significantly strengthen both the rigor and\nthe mechanistic interpretation of the study.\nIn particular, the added analyses examining IgA reactivity against microbial antigens, as\nwell as the direct functional comparison between IgA and IgG derived from the same\nlesional samples, meaningfully reinforce the central conclusions. The clarification\nregarding immune complex requirements and the tempered interpretation of TLS\ninvolvement further enhance the scientific precision of the manuscript.\nOverall, the revised version presents a coherent and well-supported framework linking\nlesional IgA autoantibodies to macrophage activation, Th17 polarization, and fibrotic\nresponses in hidradenitis suppurativa. The manuscript is now considerably strengthened.\nWe sincerely thank the reviewer for this thoughtful and highly encouraging assessment. We\nare pleased that these revisions have strengthened the rigor, mechanistic clarity, and\noverall framework of the manuscript."}]

Decision letter