HMGB1 Inhibitor Effectively Alleviates Psoriasis-Like Lesions and Inflammatory Cytokines in K14-VEGF Transgenic Mice
Anti-high-mobility group box 1 (HMGB1) is involved in the pathogenesis of many inflammatory and autoimmune diseases, including psoriasis. The present study aimed to investigate the therapeutic effects of HMGB1 monoclonal antibody (mAb) in keratin 14 (K14)-vascular endothelial growth factor (VEGF) transgenic homozygous mice.
Twelve VEGF transgenic mice were randomly divided into two groups of six mice each: the anti-HMGB1 mAb group and the immune complex (IC) mAb group. The mice underwent intraperitoneal injection of anti-HMGB1 mAb or IC mAb once every 2 days for a total of three treatments. Compare the lesions on the ears of the mice and evaluate the severity of the lesions using the baseline and clinical scores on the last day of treatment. The changes in psoriasis-like lesions, cellular infiltration of T cells, dendritic cells, and neutrophils were detected by hematoxylin-eosin staining and immunohistochemistry. The mRNA expression of the inflammatory cytokines, including interleukin (IL)-6, tumor necrosis factor-α, interferon-γ, and IL-17 in the lesions were assessed by real-time quantitative polymerase chain reaction. The number of γδ T cells in the lesions of two groups were detected by flow cytometry. The t test was used to compare their differences.
The anti-HMGB1 mAb effectively ameliorated the clinical skin lesions. The clinical scores in the anti-HMGB1 mAb group were lower than those in the IC mAb group (6.00±0.52 vs. 10.83±0.48, P < 0.001). Histopathologic changes and improvements in the K14-VEGF transgenic homozygous mice were evident after three treatments. The scores of mice in the anti-HMGB1 mAb group were significantly lower than those in the IC mAb group (3.25±0.71 vs. 6.95±0.83, P = 0.0033). The average epidermal thickness in the anti-HMGB1 mAb group was reduced by about 45% when compared with that in the IC mAb group (32.15±7.08 vs. 64.69±7.93, P = 0.0054). Moreover, anti-HMGB1 mAb also decreased the number of infiltrating CD3+ T cells, myeloperoxidase-positive neutrophils, and CD11c+ dendritic cells. The ratio of ear skin γδ T cells was reduced in anti-HMGB1 mAb treated group. The mRNA expression of IL-6, tumor necrosis factor-α, interferon-γ, and IL-17 in the anti-HMGB1 mAb group were significantly reduced when compared with IC mAb group (0.36±0.070 vs.1.98±0.62, P = 0.0148; 6.43±1.37 vs. 13.80±1.33, P = 0.0006; 2.62±0.83 vs. 7.77±1.32, P = 0.0026; 4.69±1.13 vs. 11.41±1.92, P = 0.0054).
HMGB1 blockade (anti-HMGB1 mAb) reduced leukocyte infiltration and suppressed inflammatory cytokine expression in this K14-VEGF transgenic mouse model, markedly reducing the severity of the psoriasis-like lesions. HMGB1 blockade might serve as a potential target for the treatment of psoriasis.
Psoriasis is a common immune-mediated chronic inflammatory skin disorder that is histopathologically characterized by keratinocyte and dermal vascular proliferation1 as well as cellular infiltration of neutrophils, T cells, macrophages, and dendritic cells (DCs).2,3,4,5,6 However, the molecular mechanisms involved in the development of this disease remain unclear.
High-mobility group box 1 (HMGB1) was initially identified as a non-histone protein located in the nucleus of almost all cells.7 HMGB1 can be secreted extracellularly when exposed to inflammatory mediators and in the course of cellular necrosis.8 Recent evidence has revealed that extracellular HMGB1 is also a late mediator in septic shock9,10 and a proinflammatory cytokine in the pathogenesis of many inflammatory and autoimmune diseases, such as rheumatoid arthritis, ankylosing spondylitis, and systemic lupus erythematosus.11,12,13
In our previous study, we explored the role of HMGB1 in psoriasis vulgaris (PV) and found that patients with PV had increased serum levels of HMGB1 and an altered distribution of HMGB1 in lesional skin.14 To further understand the roles of HMGB1 in psoriasis and other inflammatory skin diseases, we investigated the therapeutic effects of anti-HMGB1 monoclonal antibody (mAb) in keratin 14 (K14)-vascular endothelial growth factor (VEGF) transgenic homozygous mice, an animal model of psoriasis that displays many clinical and histopathologic characteristics of human psoriasis.15 We then observed the changes in psoriatic lesions; the cellular infiltration of CD3+ T cells, myeloperoxidase (MPO)-positive neutro-phils, and CD11c+ DCs16,17; and the expression of inflammatory cytokine mRNA levels [including interleu-kin (IL)-6, tumor necrosis factor a (TNF-α), interferon γ (IFN-γ), and IL-17] of the psoriatic lesioned skin.18,19,20,21
The K14-VEGF transgenic homozygous mice were donated by the State Key Laboratory of Biotherapy, Sichuan University. Twelve 3-month-old mice with psoriasis-like lesions were selected for the experiment and maintained in specific pathogen-free conditions.
The mice were randomly divided into two groups of six mice each: the anti-HMGB1 mAb group and the immune complex (IC) mAb group. The mice in the anti-HMGB1 mAb group underwent intraperitoneal injection of anti-HMGB1 mAb (2 mg/kg; Shino-Test Corporation, Sagamihara-shi, Kanagawa). The mice in the IC mAb group underwent intraperitoneal injection of matching IC mAb (2 mg/kg; Sigma-Aldrich, St. Louis, MO, USA). The two groups were treated once every 2 days for three treatments. We observed the lesions on the ears and evaluated the lesional severity using a clinical score at baseline and the last day of treatment. The scoring system was built on the Psoriasis Area and Severity Index and was scored from 0 to 4, where 0 is none, 1 is slight, 2 is moderate, 3 is marked, and 4 is very marked, independently from the commonly used scale of thickening, erythema and scaling. The cumulative score served as a measure of the severity of inflammation (total score of 0-12).22
After 6 days, all mice in both groups were euthanized. Their ears were cut off, and one ear was used for hematoxylin-eosin (HE) staining and immunohistochemistry whereas the other ear was used for real-time quantitative polymerase chain reaction (RT-qPCR). The tissues were embedded in paraffin, sectioned (approximately 5 µm), and stained by HE. Immunohistochemical examination was performed using antibodies to CD3 (Abcam, Cambridge, UK), CD11c (Beijing Biosynthesis, Beijing, China), and MPO (Beijing Biosynthesis). The pathological severity of psoriasis, based on the Baker scoring system,23 wasobservedin10random fields (× 100) of each HE-stained section. The Baker score23 was based on the following psoriasiform histological features: keratin (Munro abscess = 2.0, hyperkeratosis = 0.5, parakeratosis = 1.0), epidermis (thinning above papillae = 0.5, lengthening of rete ridges = 1.0, clubbing of rete ridges = 0.5, 1 mitosis/3 rete ridges = 0.5, lack of granular layer = 1.0), and dermis (lymphocytic infiltration: mild = 0.5, moderate = 1.0, marked = 2.0; papillary congestion = 0.5). The epidermal thickness in tissue samples was also measured by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
The ear tissue was cut up with tissue scissors, and then treated with collagenase I at 37 °C for 30 minutes, filtered through a cell sieve, washed twice with phosphate-buffered saline (PBS), and finally resuspended in 1 mL of PBS to prepare a single-cell suspension. This process provided the ear cells for further analysis. The ear cells from the two groups were isolated individually, and then washed twice with PBS, and 1×106 cells were then thoroughly mixed with 2 µL of anti-CD3e-PE and anti-γδ-FITC mAbs and incubated in the dark at 4 °C for 30 minutes. Next, the cells from both groups were analyzed with a flow cytometer (FACSCa-libur; BD Biosciences, San Jose, CA, USA) and the data were processed using the CellQuest Pro (BD Biosciences).
The mRNA expression of IL-6, TNF-α, IFN-γ, and IL-17 was measured by RT-qPCR. Total RNA of the ear samples was extracted by Trizol reagent according to the manufacturer’s instructions. The complementary DNA (cDNA) was synthesized from the total RNA by reverse transcription using a PrimeScript® RT reagent kit with genomic DNA Eraser (Takara, Dalian, China). The expression of GAPDH and other genes of interest was amplified from cDNA in a 20-µL reaction volume including 2 µL cDNA, 10 µL SYBR qPCR Mix (Takara), and 0.25 µmol/L qPCR primers each of IL-6 (Catalog no. MQP036632; GeneCopoeia, Rockville, MD, USA), TNF-α (Catalog no. MQP031019; GeneCopoeia), INF-γ (Catalog no. MQP027401; GeneCopoeia), and IL-17 (Catalog no. MQP029457; GeneCopoeia). The qPCR conditions were 95 °C for 5 minutes, 95 °C for 15 seconds, and 60 °C for 30 seconds for 40 cycles with a final extension at 72 °C for 5 minutes. The mRNA levels of IL-6, TNF-α, IFN-γ, and IL-17 were expressed as relative mRNA levels compared with control and determined by the 2-ΔΔCt method.
All experiments were carried out at least three times. Data were analyzed with GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA) and expressed as mean ± standard deviation. The t-test was used to compare the data. A P-value of < 0.05 was considered statistically significant.
Mice treated by anti-HMGB1 mAb tolerated the treatment well and showed no signs of illness or weight loss. The psoriasis-like lesions in the anti-HMGB1 mAb group were moderately alleviated. However, the skin lesions in the IC mAb group were significantly worse (Fig. 1A). Furthermore, the clinical scores in the anti-HMGB1 mAb group were lower than those in the IC mAb group (Fig. 1B).
Histologic examination of the IC mAb group showed epidermal thickening, epidermal rete elongation, hyper-keratosis, and infiltration of many lymphocytes (Fig. 2A). Anti-HMGB1 mAb effectively inhibited the proliferation of keratinocytes and caused significant improvement of the psoriasis-like histological appearance compared with the IC mAb group (Fig. 2A), as evaluated by the Baker scoring system.23 The scores of mice in the anti-HMGB1 mAb group were significantly lower than those in the IC mAb group (Fig. 2B). Moreover, the average epidermal thickness in the anti-HMGB1 mAb group was reduced by about 45% when compared with that in the IC mAb group (Fig. 2C).
Immunohistochemical examination showed a huge number of CD3+ T cells, MPO+ neutrophils, and CD11c+ DCs infiltrating the dermis of the mice in the IC mAb group. However, the mice treated by anti-HMGB1 mAb showed much less cellular infiltration of CD3+ T cells, MPO+ neutrophils, and CD11c+ DCs than the mice in the IC mAb group (Fig. 3).
The ratio of ear skin γδ T cells was lower in the anti-HMGB1 mAb group than in the IC mAb group (Fig. 4).
As shown in Figure 5, the mRNA expression of IL-6, TNF-α, IFN-γ, and IL-17 were significantly lower in the anti-HMGB1 mAb group than in the IC mAb group. This finding suggests that anti-HMGB1 mAb moderately alleviates psoriasis-like lesions in K14-VEGF transgenic mice via decreased expression of IL-6, TNF-α, IFN-γ, and IL-17.
Several studies have established that extracellular HMGB1 is a proinflammatory cytokine and is relevant in the pathogenesis of many autoimmune or inflammatory diseases. Although psoriasis is a common chronic immune-mediated inflammatory disease, the underlying molecular mechanisms are still incompletely elucidated.24,25,26 Whether HMGB1 is extracellularly released in patients with psoriasis and whether targeting HMGB1 with anti-HMGB1 mAb can be therapeutically useful for psoriasis remain unclear. In our previous study, we explored the relationship between HMGB1 and PV. We found that the serum levels of HMGB1 were increased and the distribution of HMGB1 was altered in the lesional skin of patients with PV.14 This suggests that HMGB1 may play a significant role in the pathogenesis of psoriasis.
To further study the potential effects of HMGB1 in the pathogenesis of psoriasis, an in vivo experiment was performed using a K14-VEGF transgenic homozygous mouse model of psoriasis. In this study, we found that anti-HMGB1 mAb clinically alleviated the psoriasis-like lesions and effectively improved the histopathologic changes in K14-VEGF transgenic mice. Moreover, anti-HMGB1 mAb reduced the number of infiltrated CD3+ T cells, MPO+ neutrophils, and CD11C+ DCs in psoriasis-like lesions and obviously decreased the expression of IL-6, TNF-α, IFN-γ, and IL-17 in psoriasis-like lesions. These data suggest that HMGB1 may be a promising molecular target in the treatment of psoriasis.
Many studies have shown that Th1/Th17 cells play crucial roles in the pathogenesis of psoriasis.27,28,29 Psoriatic lesions can secrete inflammatory cytokines that contribute to the development of psoriasis, such as IFN-γ, IL-6, IL-1β, IL-8, IL-17, and TNF-α.30 As described above, HMGB1 can be released extracellularly by inflammatory mediators, such as TNF-α, IFN-γ, and lipopolysaccharide. Our unpublished work also showed that HMGB1 can promote the release of chemokines and proinflammatory cytokines in keratinocytes. Hence, a positive feedback mechanism may be present in patients with psoriasis, constituting a source of extracellular cytokines and contributing to the development of psoriasis. In the present study, anti-HMGB1 mAb treatment inhibited the expression and function of HMGB1 on the epidermal cells, thereby down-regulating the production of IL-6, TNF-α, IFN-γ, and IL-17. This may also hinder the differentiation of Th1 and Th17 cells by suppressing the polarization and differentiation of T cells, DCs, and neutrophils. The inhibition of Th1/Th17 may ameliorate inflammation and the immune response, leading to subsequent suppression of T-cell cascade reactions. All of these effects may have contributed to the obvious therapeutic effect of anti-HMGB1 mAb in the mouse model. Moreover, the mRNA expression of IL-10 and IL-4 in the psoriatic lesioned skin showed no difference between the two groups in our preliminary study (data not shown). Together, our findings show that HMGB1 is therefore a promising molecular target for psoriasis therapy through interference with Th1/Th17 cells. However, further study is required because the specific signal pathway remains unclear.
The study has following limitation: The results of this study are from mice models, and the evidence for its role in human is insufficient, and further research in human is needed in the future.
In summary, HMGB1 blockade (anti-HMGB1 mAb) reduced leukocyte infiltration and suppressed the expression of inflammatory cytokines such as IL-6, TNF-α, IFN-γ, and IL-17, helping to ameliorate the disease severity in this K14-VEGF transgenic mouse model of psoriasis. These findings suggest that HMGB1 might be involved in the pathogenesis of psoriasis in K14-VEGF transgenic mice and that targeting HMGB1 by anti-HMGB1 mAb might provide a solid foundation and novel therapeutic endeavor in the treatment of psoriasis. However, the results of this study are only available in mouse models, and the evidence for its role in human psoriasis is still insufficient, and further research in this area is needed in the future.
All the data related to this manuscript will be shared upon request to the corresponding authors. No additional data are available.
The authors reported no conflicts of interest.