Contributed equally
High mobility group protein B1 (HMGB1) has been reported to serve important roles in various pathological conditions. Toll-like receptor 4 (TLR4), as one of the HMGB1 receptors, has been reported to be involved in the development of certain inflammatory diseases by activating nuclear factor NF-κ-B (NF-κB). However, there are few studies investigating the effects of HMGB1, TLR4 and NF-κB on human inflammatory dermatoses. In the present study, the distribution and characteristics of HMGB1, TLR4 and NF-κB p65 expression in psoriasis and atopic eczema (AE) were investigated. In addition, immunohistochemical analysis was performed to evaluate their expression and distribution in normal skin, and in patients with AE or psoriasis. Spearman's correlation analysis was used to predicate their relevancy. The present study identified that the p65 level in epithelial nuclei in AE skin was increased compared with normal and psoriasis skin (P<0.01). The level of extracellular HMGB1 in AE skin was also increased compared with normal and psoriasis skin (P<0.01). Meanwhile, TLR4 expression on the epithelial membranes of AE skin was increased compared with psoriasis skin (P<0.01). Furthermore, the level of extracellular HMGB1 was positively correlated with epithelial membrane TLR4 (r=0.3856; P<0.05) and epithelial nuclear p65 (r=0.5894; P<0.01) in AE skin. These results indicated that the HMGB1-TLR4-NF-κB signaling pathway is activated in AE and may account for its pathogenesis, but not in psoriasis. Therefore, HMGB1, TLR4 and NF-κB p65 have the potential to be targets for the treatment of human inflammatory dermatoses, including AE.
As two of the most common inflammatory dermatoses, atopic eczema (AE) and psoriasis threaten the quality of life and health in humans. AE is an inflammation of the skin characterized by pruritic, papulovesicular and occasional weeping dermatitis (
HMGB1, a 30-kDa nuclear protein referred to one of the most important chromatin proteins, serves as a proinflammatory cytokine mediator, following release by macrophages or monocytes, or during various pathological necrotic conditions (
It has been recognized that HMGB1 serves significant roles in autoimmunity diseases, epidermal tumors, toxic epidermal necrolysis and Stevens-Johnson syndrome (
The present study was performed in accordance with The Declaration of Helsinki 1964 and its later amendments, and was approved by the Ethics Board of Tongji Medical College (Wuhan, China). Diagnosis of AE and psoriasis was based on the clinically apparent symptoms and histopathological criteria. The severity of AE and psoriasis were assessed by two well-trained and experienced dermatologists, according to the scoring AD (SCORAD) index and Psoriasis Area and Severity Index (PASI), respectively (
The antibodies and reagents used in the present study include anti-HMGB1 (cat. no. 2600-1; Epitomics; Abcam, Cambridge, UK), anti-TLR4 (cat. no. ab22048; Abcam), anti-NF-κB p65 (cat. no. SC-7151; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and REAL™ EnVision Detection kit (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA).
The tissue specimen preparation and immunohistochemical procedures were as previously described (
Data are expressed as the mean ± standard error of the mean. Differences between groups were analyzed using one-way analysis of variance followed by the Bonferroni correction for normally distributed datasets, or by Kruskal-Wallis one-way analysis of variance followed by Nemenyi test for skewed datasets. Spearman's rank correlation coefficient was used for correlation analysis. P<0.05 was considered to indicate a statistically significant difference. Statistical analysis was performed using R software (version 3.0, GNU Project, Boston, MA, USA).
In psoriasis, moderate to strong positive HMGB1 diffuse expression was observed in the nuclei, with weak positive focal expression in the cytoplasm of the squamous epithelium. Extracellular HMGB1 was occasionally present in the intercellular spaces of the epithelium. In the nuclei and cytoplasm of associated inflammatory cells and vascular endothelial cells, moderate positive HMGB1 diffuse expression was observed (
In AE, there was strong positive HMGB1 diffuse expression in the nuclei, focal weak expression in the cytoplasm and weak positive expression in the intercellular spaces of the squamous epithelium. The nuclei and cytoplasm exhibited diffusive moderate positive HMGB1 expression in the associated inflammatory cells, while exhibiting moderate to strong expression in the vascular endothelial cells (
In healthy skin, moderate to strong positive HMGB1 expression was observed in the nuclei, with occasional focal expression in the cytoplasm of the squamous epithelium. Little HMGB1 expression was present in the epithelial intercellular spaces. As for inflammatory cells, little HMGB1 expression was observed in the nuclei and cytoplasm, and for vascular endothelial cells, a moderate positive HMGB1 expression was observed (
Analysis of variance demonstrated that the HMGB1 expression in epithelial intercellular spaces in AE was significantly increased compared with psoriasis (P=0.0023) and normal skin (P=0.0001), and that the HMGB1 expression in epithelial intercellular spaces in psoriasis was markedly increased compared with normal skin (
On the squamous epithelium membranes, weak positive TLR4 expression was observed in psoriasis (
In psoriasis, sporadic weak positive p65 expression was observed in the nuclei, with weak positive expression in the cytoplasm of the epithelium. Extracellular p65 was occasionally present in the epithelial intercellular spaces. In the associated inflammatory cells, there was focal expression of p65 in the cytoplasm and little expression of p65 in the nuclei. As for vascular endothelial cells, there was a weak positive p65 expression in the cytoplasm; however, no p65 expression in the nuclei (
In AE skin, there was a relatively high p65 expression in the nuclei and weak positive expression in the cytoplasm of epithelium. There was also a relatively high p65 expression in the nuclei; however, weak positive focal expression in the cytoplasm of associated inflammatory cells. However, in the vascular endothelial cells, p65 was not observed in the nuclei and exhibited weak positive staining in the cytoplasm (
In healthy skin, minimal p65 expression was observed in the nuclei, with focal positive expression in the cytoplasm of the epithelium. In the associated inflammatory cells there was little p65 expression in the nuclei. As for vascular endothelial cells, there was sporadic weak positive p65 expression in the cytoplasm (
Analysis of variance showed that the p65 expression of epithelial nuclei in AE was significantly increased compared with healthy skin (P=0.0066) and psoriasis (P=0.0082), and p65 expression of epithelial nuclei in psoriasis was markedly increased compared with healthy skin (
In healthy skin and psoriasis tissues, Spearman's rank correlation coefficient analysis demonstrated that there was no significant correlation between the p65 level in epithelial cell nuclei and the HMGB1 level in epithelial intercellular spaces (
The pathogenesis of AE and psoriasis remains to be completely elucidated. Despite the clinical distinctions, the two diseases involve the activation of local proinflammatory mediators. The purpose of the present study is to gain an improved understanding of the interactions between HMGB1, TLR4 and NF-κB p65 in the two diseases. The results of the present study support possible roles for these local proinflammatory mediators in the pathogenesis of AE; however, not in psoriasis.
As important inflammatory cytokines and transcription factors, the NF-κB family primarily consists of transcription factor p65, NF-κB1, NF-κB2, proto-oncogene c-Rel and transcription factor RelB (
To evaluate the effects of HMGB1 and TLR4 on NF-κB p65-regulated inflammation, the significance of the HMGB1-TLR4-NF-κB signaling pathway in the inflammatory dermatoses was investigated. The data demonstrated that there were variant levels of HMGB1 expression in the epithelial intercellular spaces of the tissues, with major expression of extracellular HMGB1 in AE, lesser expression of extracellular HMGB1 in psoriasis and little expression of extracellular HMGB1 in normal skin. These results implied that HMGB1 is released into the extracellular environment in the inflammatory skin diseases, serving as a proinflammatory mediator of the local inflammation. Analysis of variance demonstrated that the expression of extracellular HMGB1 in AE was increased compared with healthy skin, as well as in psoriasis, and extracellular HMGB1 in psoriasis was markedly increased compared with healthy skin, indicating that HMGB1 may be a significant mediator in promoting local inflammation in AE; however, not in psoriasis. These results are inconsistent with the study by Chen
TLRs are a type of pattern recognition receptor that recognize structurally conserved molecules derived from microbes and serve a key role in the innate immune system (
In addition, the present results demonstrated that there was almost the same level of HMGB1 expression in the epithelial nuclei of psoriasis, AE and healthy skin, indicating that the nuclear HMGB1 contributes to the stabilization of DNA and chromosomes, which is consistent with the authors' previous study into the significance of nuclear HMGB1 in certain epidermal tumors (
In conclusion, the NF-κB p65-regulated inflammation was intensified in AE; however, not in psoriasis in the present study. Therefore, the HMGB1-TLR4-NF-κB signaling pathway may be involved in the pathogenesis of AE and these molecules may be promising targets for attenuating the inflammation in AE. However, there are certain limitations to the present study, as the inflammation intensity is correlated with the stage and severity of skin diseases (
The present study was supported by the National Natural Science Foundation of China (grant no. 81460304) and the Guangxi Natural Science Foundation (grant nos. 2015GXNSFAA139197 and 2015GXNSFDA139020).
Immunohistochemical analysis of HMGB1 expression in healthy skin and inflammatory dermatoses. HMGB1 expression is located in the nuclei, cytoplasm and epithelial intercellular spaces in (A and B) psoriasis, (C and D) AE and (E) healthy skin. The black arrows represent HMGB1 expression in the ec n; the red arrows represent HMGB1 expression in the ics; the blue arrows represent HMGB1 expression in inflam cells; and the green arrows represent HMGB1 expression in vascular endothelial cells. Magnification, ×400. (F) Quantification of HMGB1 in the ics. (G) Quantification of HMGB1 in the ec n. (H) Quantification of HMGB1 in the inflam cells. Data are expressed as the mean ± standard error of the mean. **P<0.01. HMGB1, high mobility group protein B1; ec n, epithelial cell nuclei; ics, intercellular space; inflam, inflammatory; AE, atopic eczema.
Immunohistochemical analysis of TLR4 expression in (A) psoriasis, (B) AE and (C) healthy skin. The purple arrows represent ec m TLR4 expression. Magnification, ×400. (D) Quantification of ec m TLR4 expression. Data are expressed as the mean ± standard error of the mean. **P<0.01. TLR4, Toll-like receptor 4; AE, atopic eczema; ec m, epithelial cell membrane.
Immunohistochemical analysis of p65 expression in normal skin and inflammatory dermatoses. p65 expression is located in the nuclei and cytoplasm in (A and B) psoriasis, (C and D) AE and (E) healthy skin. The black arrows represent p65 expression in the ec n; the yellow arrows represent p65 expression in the epithelial cytoplasm; the blue arrows represent p65 expression in inflam n; and the green arrows represent p65 expression in vec. Magnification, ×400. (F) Quantification of p65 in the ec n. (G) Quantification of p65 in the inflam n. (H) Quantification of p65 in the vec. Data are expressed as the mean ± standard error of the mean. **P<0.01. ec n, epithelial cell nuclei; inflam n, inflammatory cell nuclei; vec, vascular endothelial cells; N.D, not detected; AE, atopic eczema.
Spearman's rank correlation coefficient analysis of: (A) p65 level in ec n vs. HMGB1 level in ics in normal and psoriasis skin (r=0.1758); (B) p65 level in ec n vs. ec m TLR4 level in normal and psoriasis skin (r=−0.1122); (C) ec m TLR4 level vs. HMGB1 level in ics in normal and psoriasis skin (r=−0.3745); (D) p65 level in ec n vs. HMGB1 level in ics in normal and eczema skin (r=0.5894); (E) p65 level in ec n vs. ec m TLR4 level in normal and eczema skin (r=0.1381); and (F) ec m TLR4 level vs. HMGB1 level in ics in normal and eczema skin (r=0.3856,). *P<0.05, **P<0.01. ec n, epithelial cell nuclei; HMGB1, high mobility group protein B1; ics, intercellular space; ec m, epithelial cell membrane; TLR4, Toll-like receptor 4.