2023 Volume 46 Issue 10 Pages 1484-1489
Psoriasis is classically regarded as a T-helper 1 (Th1) response-dominant disease believed to be antagonized by the Th2 response, which is responsible for allergic diseases, such as atopic dermatitis. The roles of these responses in psoriasis and the relationship between psoriasis and atopic dermatitis have received increasing attention because it is estimated that more than one million patients are concomitantly affected by psoriasis and atopic dermatitis. To address this, we attempted to determine the characteristics of imiquimod-induced psoriasiform lesions in mice with a concomitant allergic response after co-application of the unrelated allergen ovalbumin onto the skin. Imiquimod cream containing ovalbumin was successively applied to the right back skin of hairless HR female mice. Psoriasiform scores were determined for 11 d, and then, the resected skin thickness, spleen weight, and serum antibody levels were examined. In some experiments, mice were allowed free access to ovalbumin-containing water for 10 d before skin application to induce oral tolerance. Imiquimod cream induced psoriasis, and its severity increased upon simultaneous ovalbumin treatment. Increases in anti-ovalbumin immunoglobulin G2a (IgG2a) levels, a Th1 response indicator, and IgG1 and IgE levels, Th2 response indicators, were mediated by ovalbumin addition. Oral tolerance against ovalbumin effectively decreased ovalbumin-exacerbated imiquimod-induced psoriasis, in parallel with a decrease in levels of anti-ovalbumin antibodies. These results suggest that the concomitant allergic response induced by ovalbumin application exacerbates imiquimod-induced psoriasis. This implies that allergic responses to unrelated allergens might exacerbate psoriasis in humans and that modulating such responses could be an effective new approach to treat psoriasis.
Psoriasis causes skin manifestations, such as erythema, thickening, and scales,1) and 125 million patients worldwide are estimated to have psoriasis.1) Many drugs that control the characteristics of autoimmune and inflammatory disorders are beneficial for patients with psoriasis.1) For example, anti-interleukin (IL)-12 and anti-IL-17 antibodies have been used, and the effectiveness of these treatments implies the importance of T-helper 1 (Th1) and Th17 responses in psoriasis. However, resistance to these established treatments is occasionally observed; thus, the development of new treatments for psoriasis is warranted.
Classically, it is believed that Th1-dependent psoriasis1) and Th2-dependent atopic dermatitis2) are rarely induced simultaneously according to the Th1 versus Th2 balance.3) However, currently, there is a substantial population (more than one million) of patients presenting with co-induction of psoriasis and atopic dermatitis.4) Additionally, the Th2 response is expected to be implicated in psoriasis.5) However, the relationship between the two diseases remains controversial. Dai et al.6) revealed that the presence of each disease increases the risk of the other, but Kirsten et al.7) found that the prevalence of atopic diseases was lower in patients with psoriasis than in those without. To understand this discrepancy in humans, studies using mice with psoriasis and allergic conditions could provide important information; however, such studies have not been conducted. Moreover, such information might be useful for establishing new therapeutic methods for patients with psoriasis and allergic conditions that control Th1, Th2, and Th17 responses.
Here, we analyzed the characteristics of imiquimod-induced psoriasis and allergic responses to the unrelated allergen ovalbumin (OVA), concomitantly applied to the skin. These experiments revealed an exacerbating effect of co-induced allergic responses to psoriasis, as relationships between psoriasis and atopic dermatitis are predicted but not identified in humans.6) Additionally, to propose alternative therapeutic options for patients with psoriasis and allergic conditions, we examined whether the suppression of allergic responses to a co-administered allergen mediated by oral tolerance exerts therapeutic effects on psoriasiform lesions in mice.
Female hairless HR mice (6–9-weeks-old; Laboratory Animal Resource Bank at NIBIOHN, Japan) were used. All animal procedures were approved by the Animal Ethics Committee of Kobe Pharmaceutical University.
Imiquimod-Induced Psoriasiform Lesions with the Topical Administration of an Unrelated AllergenBESELNA cream (62.5 mg; imiquimod cream; Mochida Pharmaceutical Co., Ltd., Tokyo, Japan), with or without 1% OVA (Sigma-Aldrich Japan K.K., Tokyo, Japan), was applied onto the right side of the backs of the mice, as shown in Fig. 1A.8) Until day 11, the severity of psoriasis was scored based on the Psoriasis Area and Severity Index (PASI). The mice were then euthanized, and their blood, skin, and spleens were obtained. For oral tolerance testing, 8 mg/mL OVA-containing water was placed in the housing cage from day−10 to day−19) (Fig. 2A). To induce mild psoriasis, 15.625 mg of imiquimod cream and 46.875 mg of a hydrophilic ointment mixture were used.
Mice were treated with imiquimod (Imi) cream with or without OVA (A). Total psoriasiform (B), erythema (C), thickness (D), and scaling (E) scores were determined optically. On day 11, the resected skin thickness (F) and spleen weight (G) were measured. Anti-OVA antibody (H) and total antibody levels (I) in sera obtained on day 11 were measured using ELISA. The symbols represent individual data points. The horizontal lines in the plots indicate the mean + standard error of the mean (S.E.M.) (B–I). Data were collected from two independent experiments (B) or representatives of two independent experiments (C–I). * p < 0.05.
Mice were housed with or without OVA-containing water for 10 d to induce oral tolerance (A). The mice were then treated with imiquimod (Imi) cream with OVA (A). Total scores for psoriasis (B) were determined optically. On day 11, resected skin thickness (C), spleen weight (D), anti-OVA antibody levels (E), and total antibody levels (F) were measured. The symbols represent individual data. The horizontal lines in the plots indicate the mean + S.E.M. (B–F). Data were collected from three independent experiments (B) or representatives of two or three independent experiments (C–F). * p < 0.05.
Anti-OVA antibody levels in sera obtained on day 11 (Figs. 1A, 2A) were measured as previously described.10) The procedure for total immunoglobulin G1 (IgG1) and IgG2a ELISA was the same, except that ELISA plates were coated with anti-IgG1 or anti-IgG2a antibodies (Bethyl Laboratories Inc., Montgomery, TX, U.S.A.). For determining total IgE, plates were coated with an anti-mouse IgE antibody (Southern Biotech, Birmingham, AL, U.S.A.). After serum application, bound IgE was detected using an alkaline phosphatase-labeled anti-mouse IgE antibody (Novus Biologicals, LLC., Centennial, CO, U.S.A.).
IL-17A Production from OVA-Restimulated SplenocytesOn day 11, spleens were obtained and dispersed splenocytes were cultured in RPMI1640 (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) containing 10% fetal bovine serum (Serana Europe GmbH, Brandenburg, Germany) and antibiotics in the presence of 200 µg/mL OVA for 3 d. IL-17A levels in the cultured supernatants were determined using an IL-17A (homodimer) Mouse Uncoated ELISA Kit (Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.).
Statistical AnalysisThe Mann–Whitney U test was used to statistically analyze the data.
Five applications of imiquimod cream (62.5 mg) gradually increased the total score (Fig. 1B), the sum of the erythema (Fig. 1C), thickness (Fig. 1D), and scaling (Fig. 1E) scores, until day 4. Cream application between days 7 and 10 induced more severe skin manifestations than that during the initial days of the study period (Figs. 1B–E). When cream mixed with OVA was applied to the skin, the scores were higher than those for mice treated with cream without OVA (Figs. 1B–E). The differences in the total and scaling scores between the two groups on day 9 were significant (Figs. 1B, E).
The skin thickness of mice treated with OVA and imiquimod cream tended to increase compared to that of mice treated with cream without OVA on day 11 (Fig. 1F). Spleen weights, an indicator of the total immune and inflammatory response, were similar between the groups (Fig. 1G). Both Th1-dependent anti-OVA IgG2a and Th2-dependent anti-OVA IgG1 and IgE antibody levels were increased in mice upon OVA addition to imiquimod cream (Fig. 1H), but total IgG2a, IgG1, and IgE levels were not significantly affected (Fig. 1I).
IL-17A, a Th17 cytokine, is important for the induction of imiquimod-induced psoriasis,8) and thus, this cytokine has been regarded as a target for the development of anti-psoriatic drugs.11) To measure the OVA-dependent Th17 response, the splenocytes of mice obtained on day 11 were cultured with OVA for 3 d. Here, the IL-17A concentration increased in the splenocyte culture supernatants of imiquimod and OVA-treated mice compared with the concentration in those of imiquimod-treated mice (Supplementary Fig. 1A).
Suppression of Allergic Responses to the Unrelated Allergen OVA Mediated by Oral Tolerance Attenuates Co-existing Imiquimod-Induced PsoriasisOral allergen administration suppresses antigen-specific immune responses, known as oral tolerance.12) Hence, we determined whether inhibiting the allergic response to co-existing allergens could ameliorate the exacerbation of imiquimod-induced psoriasis. Psoriasiform lesions induced by 62.5 mg imiquimod cream were believed to be too severe for demonstrating this effect, and 15.625–62.5 mg of imiquimod cream was found to induce psoriasiform lesions in a dose-dependent manner (data not shown). Thus, 15.625 mg of imiquimod cream was used to examine the inhibitory effects of oral tolerance on psoriasis. Oral tolerance to OVA significantly diminished total psoriasiform scores on days 8–10 (Fig. 2B) and the resected skin thickness on day 11 (Fig. 2C). Anti-OVA IgG2a, IgG1, and IgE antibody levels also decreased upon the oral administration of OVA (Fig. 2E), suggesting that oral tolerance was successfully induced. In addition, oral tolerance induction successively reduced IL-17A production in splenocyte cultured supernatants in two of three experiments (Supplementary Fig. 1B). However, total immune response indicators, spleen weight (Fig. 2D), and total antibody levels (Fig. 2F), were not significantly affected.
In this study, we revealed that the application of the allergen OVA exacerbates imiquimod-induced psoriasis (Figs. 1B–E). This clearly shows, for the first time, that an allergic response to co-existing allergens aggravates psoriasis. Psoriasis is classically regarded as a Th1-dependent disease that is antagonized by the Th2 response.3) However, Th1 and Th2 responses have recently been proposed to cooperatively exacerbate immune diseases. The roles of Th2 responses in systemic lupus erythematosus,13) a representative Th1-dominant disease,14) and Th1 responses in asthma,15) a representative Th2-dominant disease, have been reported.15) In our study, mixing OVA with imiquimod cream induced the production of anti-OVA IgG1 and IgE antibodies, indicators of the Th2 immune response, and anti-OVA IgG2a antibodies, an indicator of the Th1 response (Fig. 1H). Increases in both Th1 and Th2 responses to unrelated allergens could be induced in parallel with increases in psoriasis scores in mice, thereby suggesting that psoriasis in humans can be exacerbated by Th1 and Th2 responses to unrelated allergens. In addition to Th1 cells playing an important role in psoriasis, Th2 cells in psoriatic skin,16) the increased production of IgE in some patients with psoriasis,17) and a correlation between IgE levels and PASI score increases support this proposal.17) Although reports that directly indicate a role of IgE directly in the mouse model are lacking, mast cells in the psoriatic skin regions increase in imiquimod-induced psoriasis in mice,18) similarly to the psoriatic skin regions19) of patients. Moreover, mast cell activation mediated by IL-3318) and through MRGPRX220) is important for imiquimod-induced psoriasis. These results support the possible participation of mast cell activation by IgE, including anti-OVA IgE in this study, in the development of imiquimod-induced psoriasis. Whereas the relationship between total IgG levels and severity of psoriasis in humans is not clarified, the amounts of IgG against heat shock protein,21) apolipoprotein,22) and advanced glycation endproducts (AGE)23) in serum correlate with disease severity. Moreover, since genetic deletion of the inhibitory IgG receptor FcγRIIb exacerbates imiquimod-induced psoriasis in mice,24) IgG and the activating IgG receptors FcγRI and FcγRIII may be responsible for disease induction. The anti-OVA IgG detected here might co-operate with IgG against unidentified important antigens (possibly heat shock protein, apolipoprotein, and AGE) in the development of imiquimod-induced psoriasis through IgG receptors. From a different perspective, the revealed enhancing effect on antibody production in the psoriasis model is important, regardless of the underlying mechanisms. This may be because the enhancement of antibody production leading allergic responses in our study supports the observation in humans that psoriasis increases the risk for atopic dermatitis,6) an allergic disease.
Concurrent inhibition of Th1 and Th2 responses is considered relatively effective for treating psoriasiform lesions associated with both responses. The calculated daily consumption of OVA per mouse in our study was approximately 40 mg, which has been reported to inhibit both Th1 and Th2 responses.12) As predicted, tolerance successfully suppressed anti-OVA IgG2a (Th1 response) and anti-OVA IgE/IgG1 (Th2 response) (Fig. 2E) levels in parallel with a considerable decrease in psoriasiform lesions (Fig. 2B). Hence, the regulation of allergic responses to a co-existing allergen exerts beneficial effects on concomitant psoriasis. Oral tolerance induces mainly fed antigen-specific immune suppression, although bystander suppression to non-specific antigens through inhibitory cytokine productions can occur in various degrees. Thus, the bystander suppressive mechanisms induced by OVA feeding may contribute to the attenuation of imiquimod-induced and OVA-exacerbated psoriasis in our study (Fig. 2B). The suppressive cytokine IL-10 is a candidate factor responsible for psoriasis attenuation through bystander suppression, since oral tolerance induces IL-10 production,25) genetic deletion of IL-10 exacerbates imiquimod-induced psoriasis,26) and blockade of IL-10 function increases the production of antigen-specific IgE27) and IgG2a28) in mice. Although we did not clarify whether oral tolerance induced production of the anti-inflammatory IL-35, IL-35 administration ameliorates imiquimod-induced psoriasis29) and decreases antigen-specific IgE and IgG1 production.30) The effect of oral tolerance on IL-35 production should be elucidated in the future. In our experimental conditions, the contribution of bystander suppression did not to appear sufficient to ameliorate disease symptoms and OVA-specific antibody production, since our oral tolerance induction did not affect imiquimod-induced increase in spleen weights (Fig. 2D). IL-10, a main cytokine responsible for bystander suppression, inhibits imiquimod-induced increase in spleen weight.26)
Suppression of the Th17 response against allergens has also been suggested in ex vivo experiments. Production of IL-17A, an emerging important inflammatory cytokine in psoriasis, was induced in OVA-stimulated cultured splenocytes of imiquimod-treated mice (Supplementary Fig. 1A). Moreover, enhancement of IL-17A production by imiquimod was attenuated after induction of oral tolerance (Supplementary Fig. 1B). Thus, OVA application to the skin induced immune responses against OVA in the whole body including the spleen and IL-17A production, driven by allergen exposure, played a considerable role in psoriasis exacerbation. The detected Th17 response in the spleen might distribute to psoriatic region and contribute to disease development. Inhibition of the Th17 response by oral tolerance also contributed to psoriasis suppression in this study. Ueyama et al.31) reported that the expression of IL-23, another important cytokine in psoriasis, in addition to that of IL-17, is upregulated in psoriatic skin. The importance and roles of IL-23 should be clarified in more detail to understand the disease mechanism.
There are two limitations in our study. One is the moderate suppressive effect of oral tolerance on imiquimod-induced and allergen-exacerbated psoriasis. Establishment of sensitization, namely detectable antigen-specific antibody production, requires approximately one to two weeks from antigen exposure.32) We thus expected that the production of OVA-specific antibodies and related exacerbation of imiquimod-induced psoriasis would be achieved in about 7 d after initiating application of OVA with imiquimod cream to the skin. Consistent with the expectation, an increasing tendency of the psoriasis score due to OVA addition was relatively clearer in latter days (days 7–11) and the increase was significant on day 10 (Fig. 1B). We speculate that the limited enhancing effect of OVA on imiquimod-induced psoriasis caused the significant effect only on day 10. At this time, the balance between imiquimod-induced psoriasis and OVA-dependent exacerbating effect is likely the best detector of the enhancing effect of OVA. Improved experimental settings may provide more obvious evidence of allergen participation on concomitant psoriasis.
A second limitation regards the successful demonstration of a correlation between suppression of the Th2 response and attenuation of psoriasis. We attempted to reveal the effect of signal transducer and activator of transcription 6 (STAT6) inhibitor AS151749933) on imiquimod-induced and OVA-exacerbated psoriasis. STAT6 is indispensable for IL-4/IL-13 signaling.33) AS1517499 (10 mg/kg; Selleck Chemicals, Houston, TX, U.S.A.) was intraperitoneally administered 1 h before imiquimod and OVA application to the skin. AS1517499 treatment tended to, but not significantly, decrease the total score of psoriasis, without affecting other parameters, including resected skin thickness, spleen weight, and antibody production (data not shown). Thus, our results with the STAT6 inhibitor were insufficient to show an indispensable role for Th2 responses, including IL-4/IL-13 and IgE/IgG1 in imiquimod-induced and OVA-exacerbated psoriasis, although Th2 responses are thought to participate in psoriasis development.5) The beneficial potential of the STAT6 inhibitor on imiquimod-induced psoriasis should be examined in more detail, and, in turn, the importance of the role of Th2 in human psoriasis may become an attractive research focus.
In conclusion, our results suggest the following: first, imiquimod-induced psoriasis is exacerbated by a co-existing allergic response to the unrelated allergen OVA. This reinforces the fact that psoriasis in humans is exacerbated by concomitant allergic reactions, such as atopic dermatitis. Additionally, oral tolerance induction to the unrelated allergen OVA attenuates concomitant psoriasis, in addition to allergic responses to the allergen. Immunomodulatory methods, including oral tolerance to allergens, might thus be an efficacious psoriasis treatment for patients affected by both psoriasis and allergic diseases, such as atopic dermatitis.
The authors declare no conflict of interest.
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