Co-occurrence of non-alcoholic steatohepatitis exacerbates psoriasis associated with decreased adiponectin expression in a murine model

Abstract

Introduction: Clinical studies have suggested a bidirectional association between non-alcoholic steatohepatitis (NASH) and psoriasis, affecting each other's development and severity. Here, we explored bidirectional causal linkages between NASH and psoriasis using a murine model.

Methods: NASH was induced in mice by streptozotocin injection at 2 days of age and by high-fat diet feeding (STAM™ model). Psoriasis was induced by topical application of imiquimod (IMQ) on the ear. The severities of liver damage and psoriatic skin changes were determined using histological analysis. Gene expression in the skin tissues was evaluated using quantitative PCR analysis. Serum cytokine levels were determined using enzyme-linked immunosorbent assay. To examine the innate immune responses of normal human epidermal keratinocytes (NHEKs), the cells were treated with interleukin (IL)-17A, tumor necrosis factor (TNF)-α, and AdipoRon, an adiponectin receptor agonist.

Results and discussion: There were no differences in the degree of liver tissue damage (fat deposition, inflammation, and fibrosis) between NASH mice with and those without psoriasis. Conversely, the co-occurrence of NASH significantly augmented psoriatic skin changes, represented by epidermal hyperplasia, in psoriatic mice. Pro-inflammatory cytokines were expressed in the inflamed skin of psoriatic mice, and the expression of genes, especially Il23a, Il1b, Il36g, and Mip2, was significantly upregulated by the co-occurrence of NASH. The expression of keratinocyte activation marker genes Defb4b and Krt16 was also upregulated by the co-occurrence of NASH. The serum TNF-α and IL-17 levels were increased by the co-occurrence of NASH and psoriasis. The serum adiponectin levels decreased in NASH mice compared with that in non-NASH mice. In NHEK culture, TNF-α and IL-17A synergistically upregulated CXCL1, CXCL8, and IL1B expression. The upregulated pro-inflammatory gene expression was suppressed by AdipoRon treatment, reflecting the anti-inflammatory capacity of adiponectin.

Conclusion: The co-occurrence of NASH exacerbated psoriatic skin changes associated with increased serum inflammatory cytokine levels and decreased serum adiponectin levels. Combined with in vitro findings, increased inflammatory cytokine levels and decreased adiponectin levels likely promote innate immune responses in epidermal keratinocytes in psoriatic skin lesions. Overall, therapeutic intervention for co-occurring NASH is essential to achieve a favorable prognosis of psoriasis in clinical practice.

Keywords: adiponectin; epidermal keratinocyte; interleukin-17; non-alcoholic steatohepatitis; psoriasis; tumor necrosis factor-α.

Figure 1

Generation of a murine model with both NASH and psoriasis. (A) Study protocol. Healthy mice were fed a normal diet (ND) for 11 weeks. Two-day-old male pups assigned to the STAM groups were subcutaneously injected with streptozotocin (STZ; 200 μg/mouse) on the back and fed the ND for 4 weeks until weaning, after which they were fed a high-fat diet (HFD) for 7 weeks. Imiquimod cream (IMQ; 25 mg/mouse) or Vaseline (as control) was topically applied to the ear of the mice at the age of 10 weeks for 5 consecutive days (n = 8 per group). Blood, liver, and skin samples were collected 1 day after the last treatment. (B) Photographs of the liver. (C) Liver-to-body weight ratio. (D) Serum alanine aminotransferase (ALT) level. Each dot denotes an individual mouse. Values are presented as mean with standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. indicated group. NASH, non-alcoholic steatohepatitis. ns, not significant.

Figure 2

Histological analyses of the liver. Non-alcoholic steatohepatitis (NASH) and psoriatic skin changes were induced as described in Methods and Figure 1 . Liver tissues were collected 1 day after the last treatment. (A) Representative images of the liver stained by hematoxylin–eosin, Sirius Red, and Oil Red O. (B) Non-alcoholic fatty liver disease (NAFLD) activity score according to Kleiner’s criteria. (C, D) Percentage of Sirius Red- and Oil Red O-stained areas. Each dot denotes an individual mouse. Values are presented as mean with standard deviation. *** p < 0.001, **** p < 0.0001 vs. indicated group. ns, not significant.

Figure 3

Co-occurrence of NASH exacerbated psoriatic skin changes. (A) Changes in ear thickness. Mice were topically treated with imiquimod (IMQ) cream (25 mg/mouse) or Vaseline (control; n = 8 per group). The ear thickness of each mouse was measured using a digital thickness gauge daily. Values are presented as mean ± standard error of the mean. (B) Representative images of hematoxylin–eosin-stained ears of indicated mice. Bar = 100 μm. (C) Histological analysis of epidermal thickness. Ten representative areas of the epidermis were measured for individual mice, and their average value was calculated. Each dot denotes an individual mouse. Values are presented as mean with standard deviation. ** p < 0.01, **** p < 0.0001 vs. indicated group. NASH, non-alcoholic steatohepatitis. ns, not significant.

Figure 4

Quantitative polymerase chain reaction analysis of the ear. Alterations in the mRNA expression of pro-inflammatory cytokines associated with psoriasis. RNA samples were extracted from the ear tissues of the indicated mice. mRNA expression levels were determined using quantitative PCR analysis. The levels were calculated relative to the level of 36b4 and normalized to the expression level in the healthy group. Each dot denotes an individual mouse. Values are presented as mean with standard deviation. ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. indicated group. IMQ, imiquimod. ns, not significant.

Figure 5

Serum cytokine levels. Blood samples were collected from the indicated mice treated with imiquimod (IMQ) cream (25 mg/mouse) or Vaseline (control; n = 8 per group). (A–C) Serum levels of tumor necrosis factor (TNF)-α, interleukin (IL)-17, and adiponectin were measured using enzyme-linked immunosorbent assay (ELISA). Each dot denotes an individual mouse. Values are presented as mean with standard deviation. *** p < 0.001, **** p < 0.0001 vs. indicated group. ns, not significant.

Figure 6

AdipoRon suppressed innate immune responses of epidermal keratinocytes. Normal human epidermal keratinocytes were stimulated with interleukin (IL)-17A (30 ng/mL) and tumor necrosis factor (TNF)-α (30 ng/mL) for 24 h. AdipoRon (10, 30, 100, and 300 μM) was added 30-min before (A) or 60-min after (B) the IL-17A and/or TNF-α treatment. Gene expression levels relative to GAPDH level were calculated and normalized to the levels in the non-stimulated control. Values are presented as mean with standard deviation. * p < 0.05 vs. indicated group.