Hidradenitis suppurativa/acne inversa (HS) is a potentially severe, chronic, inflammatory, recurrent skin disease of the hair follicle. Some of its variants belong to the rare diseases (Orphanet codes 387, 289478) (1). A meta-analysis by Jfri et al. estimated the prevalence of HS at 0.40% (2). Although the precise pathogenesis of HS remains uncertain, current perspectives are shifting from the traditional view of HS as primarily a disorder of follicular occlusion to a model emphasizing autoinflammatory keratinization (3). Evidence indicates that HS may involve inflammatory, autoimmune, or a combination of both mechanisms, with hyperkeratotic plugging of the follicular infundibulum identified as a central pathogenic event (4). Clear evidence suggests the involvement of proinflammatory cytokines in the immune dysregulation of HS (4). Notably, the interleukin-1 (IL-1) family of cytokines (IL-1α and IL-1β) (5) as well as multiple IL-36 transcripts (6) are elevated in HS lesions as demonstrated by ribonucleic acid (RNA) sequencing and real-time reverse transcription polymerase chain reaction (qRT-PCR). IL-23A (encoding IL-23p19) transcripts are elevated in HS-affected tissues, and expression of IL-23A mRNA has been found in activated macrophages in HS lesions using qRT-PCR and IL-23 subunits by immunohistochemistry and immunofluorescence confocal microscopy (7). Multiple IL-17 family transcripts are elevated in HS lesions, including IL-17A and the resultant protein IL-17A as well as IL-17C and IL-17F, the latter forming heterodimers with IL-17A (8).

The most favorable anti-inflammatory treatment for HS is biologics that directly target cytokines. At present, the only European Medicines Agency (EMA) and US Food and Drug Administration approved medications for the treatment of HS are the tumor necrosis factor α (TNF-α) inhibitor adalimumab, the IL-17A inhibitor secukinumab, and the IL-17A/F inhibitor bimekizumab (9). Preclinical in vivo or ex vivo studies were published for adalimumab and bimekizumab (10, 11). In general, the optimal target for successful HS treatment remains undiscovered.

Previous studies have suggested that mesenchymal stem/stromal cells (MSCs) possess intrinsic immunosuppressive capabilities that can alleviate inflammation and immune responses (12). MSCs obtained from different tissues inhibit the production of IFN-γ, TNF-α, and IL-1β (13, 14) and promote the synthesis of TGF-β and IL-10 cytokines (13). They can also suppress inflammation by means of cell-to-cell contact, extracellular vesicles, and various other secreted factors such as IDO, iNOS, HGF, TSG6, and CCL2 (15). These findings suggest MSCs as a potential multitarget treatment approach for HS, addressing both innate and adaptive inflammation.

The safety of envisioned cell therapy methods was proven at both preclinical as well as clinical phases, concluding safe clinical administration of allogeneic MSCs (16). The low expression of class I and II human leukocyte antigen (HLA) antibodies on MSCs reduces the risk of rejection in allogeneic applications in normal immunocompetent hosts, making them safe for universal application and cryopreserved, off-the-shelf product storage (17). This approach streamlines treatment processes and enhances accessibility for critical interventions.

In our previous pilot study, we investigated the immunomodulatory effects of MSCs on peripheral blood mononuclear cells (PBMCs) from 3 healthy controls and 3 HS patients. Our results showed that MSCs can effectively suppress PBMC proliferation and inhibit the production of inflammatory cytokines. Furthermore, pre-activation of MSCs with IFN-γ and TNF-α before use may enhance their therapeutic efficacy (18).

In the current study, we developed an experiment using in vitro PBMCs and ex vivo HS explant models to comprehensively evaluate the therapeutic potential of MSCs and alleviate the systemic (PBMCs) and localized (explant) immune aberrant responses that are pathogenic in HS. To our knowledge, no prior study investigating MSCs’ immunomodulatory effects on both PBMCs in vitro and HS explants ex vivo has been published before.

Previous studies have established that MSCs possess the ability to prevent immune system overstimulation through the modulation of various inflammatory pathways (12). Although the mechanisms of MSC-mediated regulation on inflammation are clear, MSCs are reported to suppress T cell proliferation and differentiation, as well as inhibit Th1 and Th17 activity, and promote Treg induction and IL-10 production (21). Thus, MSCs are prone to sense multifactorial signals and modulate various proinflammatory cytokines, such as TNF-α, IL-1β and IFN-γ (22, 23). Two clinical studies have demonstrated that intralesional injection of allogeneic MSCs in combination with adalimumab-maintained therapy (24) or surgery (25) can induce clinical remission of persistent HS fistulas. Their authors hypothesized that intralesional MSCs may have regulatory effects on inflammatory mediators, including Th1 and Th17 cells, macrophages, and dendritic cells, as well as their regenerative properties that promote tissue healing (24). Other studies highlighted the immunomodulatory potential of adipose tissue-derived MSCs (26, 27), suggesting their ability to upregulate IL-10 secretion by dendritic cells, leading to a less potent immune response or even T cell anergy (26). Interestingly, MSCs derived from patients with skin inflammatory diseases often demonstrate compromised inflammation-regulating abilities (28). For example, MSCs derived from HS patients are characterized by elevated levels of several helper T-cell cytokines (IL-6, IL-10, IL-12, IL-17A, IFN-γ) (29), leading to the hypothesis that MSCs are involved in HS pathogenesis at certain inflammation stages.

In our study, n-MSCs showed a consistent trend of reducing IL-1β, IL-17A, and TNF-α in both lesional and perilesional HS samples, while a-MSCs significantly decreased IL-17A and IFN-γ in PBMC samples. Although adalimumab also reduced inflammation, it had a more limited effect, targeting fewer pro-inflammatory cytokines (10). Our experimental results suggest that MSCs may have broader immunomodulatory potential for the treatment of HS compared to adalimumab. Another key observation is that MSCs inhibited the overall lymphocyte proliferation and reduced the number of CD8 lymphocytes, shifting the population towards a less cytotoxic profile. For instance, research has demonstrated that MSCs are able to downregulate CD8 expression on CD8 cells, therefore promoting a less aggressive immune response (30), which could be beneficial in such conditions as HS, as it is characterized by excessive autoinflammation.

It is worth mentioning that the HS cytokine pathway is also associated with the anti-inflammatory mediator IL-10, which suppresses T-lymphocyte activation and inflammatory cytokine production (31, 32). The elevated levels of IL-10 may indicate T-reg cell recruitment to reduce inflammation as a response to early macrophage activation (33). Consistent with our findings, IL-10 has been found to have a marked expression in the lesional and perilesional skin of HS patients (34, 35). Other anti-inflammatory cytokines in HS, such as IL-4 and IL-13, inhibit IL-1β synthesis while stimulating IL-1 receptor antagonist production (32). Moreover, IL-13 is reported to correlate inversely with the presence of Th1/Th17-associated cytokines (36). Based on our findings, IL-10 levels were significantly elevated in lesional/perilesional skin and PBMC samples of HS patients when compared to healthy controls. However, the effect of MSCs on IL-10 expression proved to be inconsistent. In the PBMC model, MSCs reduced IL-10 levels, while the skin biopsy co-culture model with MSCs had higher IL-10 concentrations. We hypothesize that this could be explained by inherent differences between the models. In the PBMC co-culture model, MSCs reduced lymphocyte proliferation, leading to a smaller overall lymphocyte count in each culture well, which reduced the overall cytokine production. On the other hand, in the skin biopsy model, during the 24-h MSC co-culture period, the cell count is expected to remain stable, as the effect on cytokine production is likely due to changes in transcription and translation. Interestingly, this discrepancy also reflects the inherent biological differences between systemic and local immune environments. The IL-10 is secreted by a wide range of immune and non-immune cells, including regulatory T cells, monocytes, dendritic cells, macrophages, and keratinocytes. The abundance, activation status, and microenvironmental context of these cells differ significantly between peripheral blood and chronically inflamed skin. In HS, the local skin environment is characterized by sustained inflammation and tissue stress, which may induce IL-10 as a compensatory, though often insufficient, anti-inflammatory response.

Moreover, we determined significantly elevated IL-1β and IL-17A cytokines in HS lesional skin when compared with healthy controls, with a marked increase in perilesional skin and PBMC samples as well. IL-17 is known to promote neutrophil activation and upregulate the expression of proinflammatory molecules (e.g. S100A7, S100A8, S100A9) (31). Data from several previous studies report an upregulation of this cytokine in HS-affected skin (10, 34, 37–39). Moreover, HS-affected skin is characterized by a Th17-skewed cytokine profile, with cells expressing CD161 and IL-17, thus creating a dysregulation of the Th17: Treg cell axis (40). IL-17 levels are not only found to be elevated in HS patients’ serum but also tend to be higher with more advanced disease stages (41).

The methodological choices in this study were carefully made to ensure experimental reliability, biological relevance, and reproducibility. While we acknowledge that in vivo experiments could offer additional insights, our primary aim was to evaluate the immunomodulatory potential of MSCs using well-established, human-based models. To date, only one in vivo model of HS has been published, which is limited in scope and does not adequately reflect the complexity of the human disease (42). Moreover, in adherence to the 3R principles and considering the interspecies challenges associated with using human-derived MSCs in animal models, we chose to rely on validated in vitro and ex vivo systems. As shown in our previous studies (18), the PBMC model employed in this study has consistently provided reliable and interpretable results. We also utilized an ex vivo skin explant culture system that closely reflects the local immune microenvironment in HS lesions. Our decision to use allogeneic, placenta-derived MSCs was based on the results of previous studies (17). They demonstrated several superior characteristics of MSCs - primarily in terms of manufacturing feasibility and therapeutic potency. Placenta-derived MSCs were obtained from placental tissue, a readily available and ethically acceptable source following caesarean section. These MSCs are known to possess superior proliferative capacity compared to MSCs from other sources, likely due to their early life origin (43), making them particularly suitable for future clinical applications. For MSC activation, we selected IFN-γ and TNF-α based on robust evidence supporting their ability to prime MSCs (44). Although stimulating MSCs with patient-derived serum or lesion fluid might provide a more disease-specific inflammatory milieu, such an approach would introduce significant variability and potential unwanted effects, also due to immunological incompatibility. In contrast, the administration of defined concentrations of IFN-γ and TNF-α ensured standardized and reproducible activation. In future studies, we plan to integrate more advanced human skin models, such as the 3D-SeboSkin (10), to enhance the translational relevance and complexity of our experimental platform.

The limitations of this study include several aspects. Firstly, the size of the samples was small, which may limit the generalizability of the findings to the entire HS population. HS is a clinically heterogeneous disease. We chose a focused approach in order to detect at least minimal effects in any HS subpopulation. Consequently, we successfully included a relatively small HS patient cohort by employing strict inclusion criteria (e.g., biologic-naïve patients, no antibiotic treatment for at least six weeks). The statistically compelling results we obtained allowed us to draw conclusions regarding preliminary efficacy in the selected population. These results cannot be generalized to the entire HS population but should be viewed as a starting point for further confirmatory studies, both in the same patient group and in other HS subpopulations. Future studies are needed to include a broader representation of the HS population, with a larger number of patients and controls. Secondly, only adalimumab was used as a positive control, meaning that no direct comparison was made with the gold standard antibiotics. At this stage of MSCs development we did not intend to compare all potential alternatives including other biologics (secukinumab and bimekizumab), that target additional inflammatory axes relevant to HS pathogenesis (e.g., IL-17A and IL-17A/F). The absence of these comparators in this study is a limitation, and future investigations will aim to include these agents. Additionally, there is a growing body of evidence supporting the use of more advanced HS skin models, such as the 3D-SeboSkin model (10), which could be utilized for the ex vivo part of the study. This study provides a preliminary but important assessment of the immunomodulatory effects of MSCs in HS. The results demonstrate consistent changes in key cytokines such as IFN-γ and IL-17A and reveal novel findings on compartment-specific modulation of IL-10. Together, these observations offer insight into potential immunological targets of MSCs and lay a critical foundation for future in-depth mechanistic investigations.

To conclude, our findings further confirm that HS is driven by complex proinflammatory cytokine pathway dysregulation, as stated in previous studies of HS immunopathogenesis. Accordingly, MSC-based therapies should come into consideration and be deemed as a promising therapeutic intervention on account of their ability to modulate several immune pathways, thereby decreasing inflammation driven by HS. Our approach in applying in vitro PBMCs and ex vivo HS explant models allow for evaluation of MSCs’ therapeutic potential in alleviating systemic (PBMCs) and localized (explant) immune aberrant responses, which are pathogenic in HS as described previously. This approach can be applied for therapeutic intervention, screening and prototyping.