HMGB1 regulates Th17 cell differentiation and function in patients with psoriasis

Abstract Background Psoriasis is an immune‐mediated chronic inflammatory skin disease, in which T helper 17 (Th17) cells and its effective cytokine interleukin (IL)‐17A play a pivotal pathogenic role. High mobility group box 1 (HMGB1) is an important proinflammatory cytokine, which has been confirmed to be highly expressed in the peripheral circulation and epidermis tissues of psoriasis patients. The regulatory effect of HMGB1 on IL‐17A expression and function has been reported in some inflammatory and autoimmune diseases by the HMGB1‐Toll‐like receptor 4 (TLR4)‐interleukin (IL)‐23‐IL‐17A pathway. While, in the pathological environment of psoriasis, whether HMGB1 can exert the regulatory effect on IL‐17A is not clear. Objective We aimed to evaluate the role of HMGB1‐TLR4‐IL‐23‐IL‐17A pathway in the pathogenesis of psoriasis and explore the possible regulatory mechanism of HMGB1 on Th17 cell differentiation. Methods Serum levels of HMGB1, TLR4, IL‐23, and IL‐17A were quantified in 50 patients with moderate‐to‐severe plaque psoriasis and 30 healthy controls. Peripheral blood mononuclear cells were acquired from 10 severe psoriasis patients and administrated by different concentrations of recombinant‐HMGB1 (rHMGB1) to detect the Th17 cell percentage, mRNA and protein levels of TLR4, IL‐23, IL‐17A and retinoid‐related orphan receptor γt (RORγt). Results The serum levels of HMGB1, TLR4, IL‐23, and IL‐17A in psoriasis patients were significantly higher than healthy controls, especially in severe patients, and positively correlated with the severity index. There were also positive correlations between every two detected indicators of HMGB1, TLR4, IL‐23, and IL‐17A. In vitro study, rHMGB1 can promote the elevated expression of Th17 cell percentage as well as TLR4, IL‐23, IL‐17A, and RORγt in a dose‐dependent manner. Conclusion HMGB1 can contribute to the pathogenesis of psoriasis by regulating Th17 cell differentiation through HMGB1‐TLR4‐IL‐23‐RORγt pathway, then promotes IL‐17A production and aggravates inflammation process. Targeting HMGB1 may be a possible potential candidate for the immunotherapy of psoriasis.


| INTRODUCTION
Psoriasis is an immune-mediated chronic and relapsing inflammatory skin disease, characterized by a dysregulation of the immune system with multifactorial pathogenesis.Immune cells infiltration, such as T cells, dendritic cells, macrophages, and neutrophils, is linked to the pathological characters of psoriasis. 1Therapeutic effect on mouse psoriatic inflammation by downregulating oxidative and inflammatory mediators in neutrophils and dendritic cells has been reported by Al-Harbi et al. 2 T helper 17 (Th17) cells and its effective cytokine interleukin (IL)-17A play a pivotal pathogenic role in the development of psoriasis, and IL-17A inhibitors also present excellent therapeutic effects for moderate-to-severe plaque psoriasis. 3IL-23 is an upstream regulatory cytokine that acts early in the inflammatory cascade in psoriasis. 3The most important effect of IL-23 in psoriasis lesions is the stabilization of a cytokine-secreting pathogenic phenotype of Th17 cells 4,5 and the promotion of IL-17 production by effector and memory T cells. 6Retinoidrelated orphan receptor γt (RORγt) is the specific transcription factor necessary for Th17-cell lineage differentiation and function.][10][11] HMGB1 has been confirmed to be highly expressed in psoriasis patients' serum and epidermis tissues. 9,10Additionally, HMGB1 treatment can significantly aggravate psoriatic inflammation in imiquimod (IMQ)-induced mouse psoriasis-like inflammation, 12 which further indicates that HMGB1 participates in the pathogenesis of psoriasis.4][15] Th cells and other immune cells express TLRs and RAGE, so HMGB1 can act as an endogenous ligand for these receptors and regulate their function.7][18] In the murine asthma model study, HMGB1 has been confirmed to directly and indirectly induce Th17 immune response by TLR4-NF-κB signal pathway. 19And, in the study of renal ischemia-reperfusion injury, HMGB1 was demonstrated to activate macrophages to secrete IL-23 by binding TLR4 and form the HMGB1-TLR4-IL-23-IL-17A axis to further promote the inflammatory cascade reaction. 20However, whether HMGB1 can regulate Th17 cell differentiation within the pathological environment of psoriasis remains unclear.In the present study, we detected the serum levels of HMGB1-TLR4-IL-23-IL-17A pathway in psoriasis patients, carried out in vitro experiments to explore the regulatory effects of HMGB1 on Th17 cell differentiation and IL-17A expression, and tried to provide new ideas for the immune targeted therapy of psoriasis.

| Patients and controls
Fifty moderate-to-severe plaque psoriasis diagnosed by clinical features and/or histopathology (33 males and 17 females, aged 15-77 years old), psoriasis area and severity index (PASI) 4.4-37.8were enrolled in the study.All the patients had not undergone systemic or local therapy within 1 month or 1 week, without pregnant, breastfeeding, psoriatic arthritis (previous/current signs and symptoms of joint involvement), or other concomitant systemic inflammatory diseases, such as lupus erythematosus and rheumatoid arthritis.Meanwhile, 30 age-and sex-matched healthy volunteers (20 males and 10 females, aged 21-60 years old) were enrolled as healthy controls.This study was approved by the Ethics Committee of Binzhou Medical University Hospital (approval number: KYLL-2022-64), and written informed consents were obtained from all participants.

| Real-time quantitative RT-PCR analysis for TLR4, RORγt, IL-23 and IL-17A mRNA expression levels
Total RNA was extracted from PBMCs using Trizol (cat.no.133 B511311; Sangon Biotech Co., Ltd.) following the manufacturer's instructions.Complementary DNA was synthesized by Evo M-MLV Mix Kit with gDNA Clean (cat.no.AG11728; Hunan Accurate Biotechnology Co., Ltd.).The mRNA expression levels of TLR4, RORγt, IL-23, and IL-17A were quantified by RT-qPCR® Green Premium Pro Taq136 HS qPCR Kit (cat.no.AG11701; Hunan Accurate Biotechnology Co., Ltd.) on the CFX96137 Touch™ Real-time PCR detection system (Bio-Rad Laboratories Inc.).The primers were designed by China Hunan Precision Biotechnology Co., Ltd. and listed in Table 1.β-actin was used as the endogenous reference gene, and the expression level of the interest gene was calculated using the following formula:

| rHMGB1 promotes Th17 cell percentage and its effective cytokine IL-17A production in psoriasis patients' PBMCs
Th17 cell percentage was presented as IL-17A + CD4 + cells gating on CD4 + cells.After rHMGB1 treatment for 24 h in vitro, Th17 cell percentages of psoriasis patients' PBMCs were significantly increased in a dose-related fashion with rHMGB1 concentration (0, 10, 100, 200, and 400 ng/mL, F = 55.39,all p < .001, Figure 4).Consistent with the increased Th17 cell percentage, rHMGB1 treatment resulted in a dose-dependent promotional effect on IL-17A mRNA and protein expression levels in psoriasis patients' PBMCs (F = 83.94 and F = 36.13,both p < .001,Figures 5A and 6A).

| rHMGB1 upregulates the expression levels of TLR4 and IL-23, in psoriasis patients' PBMCs
As shown in Figures 5B,C and 6B,C, both the mRNA and protein expression levels of TLR4 and IL-23 were upgraded in a dose-related manner following the increased rHMGB1 stimulating concentration (TLR4: F = 25.58 and F = 39.58,IL-23: F = 87.08,F = 46.61,all p < .001).

| rHMGB1 promotes the expression levels of Th17 cell-specific transcription factor RORγt
To further determine whether the increased Th17 cell percentage and IL-17A expression levels in the presence of rHMGB1 were associated with Th17 cell-specific transcription factor RORγt, the mRNA and protein expression levels were measured.In accordance with the tendency of Th17 cell percentage and IL-17A levels, rHMGB1 treatment can significantly promote RORγt mRNA and protein expression levels (F = 43.43 and F = 103.37,both p < .001,Figures 5D and 6D).

| DISCUSSION
Psoriasis is a chronic immune-mediated inflammatory disease, in which IL-17A is regarded as a preponderant inflammatory cytokine mediating its pathogenesis.HMGB1, an important proinflammatory cytokine, has been reported to exert a regulatory effect on Th17 immune response, 19 but this effect has not been confirmed in the psoriatic pathological conditions.Therefore, the present study aimed to further evaluate the pathogenic role of HMGB1 in psoriasis by exploring the possible regulatory effect of HMGB1 on Th17 cell differentiation, which revealed that HMGB1 can promote Th17 cell differentiation and IL-17A production in a dose-dependent manner in the PBMCs from psoriasis patients and supplemented data for HMGB1-based therapeutic strategies.In this study, we first confirmed that HMGB1 was significantly increased in the peripheral circulation of psoriasis patients, which is consistent with previous reports. 9,10In addition, HMGB1 serum levels were obviously higher in severe patients than those in moderate patients and, more importantly, positively correlated with the index of psoriasis severity degree, which further demonstrates that HMGB1 is involved in the development and progress of psoriasis.The crucial role of IL-17A in the pathological conditions of psoriasis has been strongly supported, 3,21,22 which is produced predominantly by Th17 cells. 23,24Our previous studies have confirmed the increased percentage of Th17 cells and expression levels of IL-17A in the peripheral blood of psoriasis patients. 25HMGB1 promoting IL-17A release has been shown in myocardial ischemia-reperfusion injury. 26In this present study, significantly increased serum IL-17A levels were demonstrated again and positively correlated with HMGB1 expression levels, which indicates that HMGB1 may exert a regulatory effect on IL-17A expression.
IMQ-induced psoriasis-like inflammation is a classic animal model of psoriasis, which is mediated via the IL-23/IL-17 axis. 27Similar to the abundant cytoplasmic expression of HMGB1 in the lesional skin of psoriasis patients, IMQ-induced psoriatic mice presented high HMGB1 cytoplasmic levels. 12Wild-type mice intradermally injection with rHMGB1 resulted in epidermal thickening; moreover, administration of HMGB1 into IMQ-induced lesional skin can further worsen the severity of psoriasis-like inflammation. 120][31] IL-23 is the pro-inflammatory cytokine and essential for the differentiation of Th17 lymphocytes. 32MGB1 blockade and TLR-4 deficiency can both significantly reduce the production of IL-23 and IL-17A; furthermore, exogenous IL-23 administration can abrogate the decreased IL-17 expression induced by HMGB1 blockade, which indicates that HMGB1-TLR-4 pathway contributes to IL-17A secretion dependent on IL-23 and forms HMGB1-TLR4-IL-23-IL-17A pathway.26 However, in pathological conditions of psoriasis, it is still unclear whether the HMGB1-TLR4-IL-23-IL-17A pathway may be involved in and contribute to the IL-17A aggregation and its inflammatory reaction.
In this study, we detected the increased serum levels of HMGB1, TLR4, IL-23, and IL-17A and evaluated the positive correlation between HMGB1 and TLR4, HMGB1 and IL-23, HMGB1 and IL-17A, as well as TLR4 and IL-23 and TLR4 and IL-17A, which indicates that HMGB1-TLR4-IL-23-IL-17A pathway may produce a pathogenic effect on psoriasis.To further explore the role of HMGB1 in the pathogenesis of psoriasis, different concentrations of rHMGB1 were exerted on PBMCs from psoriasis patients in vitro.HMGB1 performs its functions via interactions with TLRs. 33,34The expression levels of TLR4 were found to be obviously increased when a higher concentration of rHMGB1 was administrated, which is similar to in vitro study of asthma. 226][37] And, in the study of asthma, HMGB1 has been demonstrated to induce Th17 cell differentiation by directly acting on naïve T cells to induce polarization and indirectly mediating the maturation and antigenpresenting ability of dendritic cells to promote Th17 cell differentiation. 19RORγt is required for the effective induction, development, and function of Th17 cells. 38,39ur present study showed that the Th17 cell percentage and RORγt expression levels were gradually elevated following the increased rHMGB1 administration concentration, which indicates that rHMGB1 can promote Th17 cell differentiation in the pathological environment of psoriasis.1][42] Studies on renal ischemia-reperfusion injury have shown that HMGB1 can stimulate the activation of macrophages and provoke the generation of IL-23 in a TLR-4-dependent manner. 20In the present study, we confirmed that both HMGB1 and TLR4 serum levels were positively correlated with IL-23 in psoriasis and rHMGB1 can dose-dependently increase its expression levels, further indicating the regulatory effect of HMGB1 on Th17 cell differentiation.In addition, Th17 cell differentiation is medicated by multiple factors, among which IL-1β and IL-6 are essential for human Th17 cell differentiation. 43HMGB1 has been reported to contribute to the secretion of IL-6 and IL-1β by macrophages and sustain the inflammatory reaction, 44 which may be another approach way for HMGB1 regulating Th17 cell differentiation.Previous research about the regulatory effect exerted on macrophages from dental pulp tissue specimens of pulpitis and healthy pulps by HMGB1 stimulation has shown that compared with HMGB1-unstimulated cells, the IL-17 levels in culture supernatants were significantly increased in the case of HMGB1 stimulation, especially in pulpitis samples, 45 which further indicates that the promotional effect of HMGB1 on Th17 cell differentiation is an important immune regulatory pathway in pathological conditions.On the contrary, IL-17A and HMGB1 have been shown to regulate mutually to exhibit a synergistic effect.The study on oxygen-glucose deprivation reported that the downregulation of HMGB1 expression caused a reduced IL-17A expression, while the downregulation of IL-17A expression resulted in a reduction of HMGB1 expression. 46Our previous study also confirmed that the increased serum levels of HMGB1 were significantly decreased after IL-17A inhibitor Secukinumab induction treatment.So, there may exist a feedback loop between HMGB1 and IL-17A, which can promote reciprocally and enhance the inflammatory effects synergistically.
Psoriatic inflammation is associated with numerous comorbidities, in which pathological parameters of psoriasis may mediate the potential mechanisms, including endoplasmic reticulum stress, pro-inflammatory cytokine releases, excess production of reactive oxygen species, alterations in adipocytokine levels, and gut microbiota dysbiosis. 47Al-Harbi et al. have reported that psoriasis can induce renal dysfunction by upregulation of NADPH oxidases and inducible nitric oxide synthase and mediate hepatic inflammation by oxidative stress and pro-inflammatory cytokines production with IL-17RC/ NF-κB signaling. 48,49So, as a systemic disease, the mechanism of psoriasis is complex, interactive, and integrated, including innate and adaptive immune systems, and multiple and interweaving pathogenic factors contribute to its development and aggravation.Inspiringly, Bruton's tyrosine kinase inhibitor was reported to suppress IMQ-induced psoriasis-like inflammation through regulation of IL-23/IL-17A in innate immune cells, 50 which enriches the recognition of psoriasis's pathogenesis and treatment idea.
In summary, there are several limitations in this study.First, we did not design to detect the regulatory effect of HMGB1 on PBMCs of healthy controls.Second, although HMGB1 has the potential to promote Th17 cell differentiation and induce IL-17 expression in PBMCs of psoriasis patients by IL-23 and TLR4 signaling pathways, more detailed mechanisms need to be explored and explained.Third, intervention experiments by drug blockade and genetic modification are required to further confirm the possible therapeutic treatment of HMGB1 both in human samples and animal models.
However, our present study provides further evidence that HMGB1 participates in the pathogenesis of psoriasis, which may exert its regulatory effect on Th17 cell differentiation and IL-17A production by IL-23 and TLR4 signaling pathways to amplify its inflammatory effects.So, inhibiting HMGB1 may be a candidate idea for the treatment of psoriasis.Indeed, to further identify the underlying mechanisms, more detailed and specific intervention experiments need to be established.

AUTHOR CONTRIBUTIONS
Lei Ma was responsible for the design of this study and reviewed and edited this article.Xiaofeng Zhu carried out the experiments, performed the statistical analysis, and wrote the first draft of the manuscript.YD, Yue Dou, Yawen Lin, and Gaoping Chu performed the experiments.JW Jing Wang collected the clinical data.All authors read and approved the final manuscript, and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of the work are appropriately investigated and resolved.

F I G U R E 1
Abbreviation: PASI, psoriasis area and severity index.

F
I G U R E 6 rHMGB1 elevated the protein expression levels of IL-17A, TLR4, IL-23, and RORγt in psoriasis patients' peripheral blood mononuclear cells (n = 10).Protein expression levels of IL-17A (A), TLR4 (B), IL-23 (C), and RORγt (D) after treatment by different concentrations of rHMGB1 (0, 10, 100, 200, and 400 ng/mL) in vitro.Representative western blot bands were listed in the top location, and the quantitative analysis results were listed in the bottom location.All the experiments were repeated three times.**p < .01.
Expression levels of serum HMGB1, TLR4, IL-23, and IL-17A in patients with psoriasis vulgaris and healthy control, which are denoted by (x ¯± s) or M (P25, P75).
T A B L E 2 bNote: Compared with healthy controls, a p < .05,comparedwithmoderate patients, b p < .05.Abbreviation: PASI, psoriasis area and severity index. in 5% skim milk for 60 min, the membranes were incubated with primary antibodies against RORγt, IL-23, and IL-17A (all 1:1000 dilution, cat.no.ab113434, ab45420, and ab13556, Abcam) and TLR4 antibody (1:500 dilution, cat.no.sc-293072;SantaCruz) at 4°C overnight and then incubated with the corresponding peroxidase-conjugated goat anti-rabbit IgG antibodies (1:10,000 dilution, cat.no.ZB-2306, ZSGB-BIO) for 1 h at room temperature.β-actinantibody(1:10,000 dilution, cat.no.ab179467,Abcam) was used to confirm equal protein loading in each lane.The protein bands were detected by an ECL kit (cat.no.MA0186, Dalian Meilun Biology Technology Co., Ltd.) and analyzed with ImageJ software v 4.1 (National Institutes of Health).2.7 | Statistical analysisWhitney U test, respectively.Pearson correlation and Spearman's test were used to conduct correlation analysis.According to Levene's test of homogeneity of variance, oneway analysis of variance was used to compare the differences among in vitro experimental data for homogeneous variance, followed by the least-significant difference LSD test performing multiple comparisons.Statistical analysis was conducted using the SPSS 27.0 (IBM Corp.) and GraphPad Prism 5 (GraphPad Software Inc.).The p-value of <.05 was considered statistically significant.