The genetic difference between C57Bl/6J and C57Bl/6N mice significantly impacts Aldara™‐induced psoriasiform dermatitis

Since its first description in 2009,1 the Aldara™ (MEDA Pharma GmbH, Bad Homburg, Germany)-induced psoriasiform dermatitis (AIPD) model has become a most commonly used mouse model of plaque psoriasis. It is mostly conducted in the C57Bl/6 mouse strain. Two substrains of this mouse strain, C57Bl/6J and C57Bl/6N, are in parallel widely used for research purposes. The two substrains are phenotypically identical but genetically differ by 34 SNPs, two indels and 15 gene variants that overlap a gene.2-4 Notably, gene knockout and transgenic mice on the C57Bl/6 background are often held on an intermediate genotype between the two substrains due to incomplete backcrossing after genetic manipulation. This issue has become even more common in recent years with the International Knockout Mouse Consortium's (IKMC) decision to preferentially generate genetic knockouts in C57Bl/6N embryonic stem cells,4 while, in contrast, most investigators still preferably use C57Bl/6J mice. We have addressed the question whether the genetic difference between the C57Bl/6J and C57Bl/6N substrains translates into relevant differences in the clinical phenotype of these two substrains in the AIPD mouse model. C57Bl/6J (C57Bl/6JRj) and C57Bl/6N (C57Bl/6NRj) mice were purchased from JANVIER LABS (Le Genest Saint Isle, France). The code ‘Rj’ in the elaborate strain nomenclature indicates that these stocks of the C57Bl/6J and the C57Bl/6N substrain, respectively, were bred at JANVIER LABS. JANVIER regularly reconfirms that the genotype of these stocks remains correspondent to the origin substrains bred at the Jackson Laboratory (Bar Harbor, ME, USA) and at the National Institute of Health (Bethesda, MD, USA), respectively. C57Bl/6J and C57Bl/6N mice were housed in the animal facility of the University of Lübeck for at least 4 weeks prior to use in experiments. The local government had approved all animal experiments. Aldara™-induced psoriasiform dermatitis (AIPD), a model in current literature also often designated as ‘imiquimod-induced psoriasiform dermatitis’, was induced in parallel in C57Bl/6J and C57Bl/6N wild-type mice by application of 50 mg of Aldara™ cream (MEDA) on the shaved back of the mice for five consecutive days, as previously described.5 The severity of resulting dermatitis was scored daily using a modification of the Psoriasis Activity and Severity Index (PASI), which factors in erythema, infiltration, and desquamation on a scale from 0 to 4, as previously described.1, 5 Methods for qPCR and histopathologies are described in Supplement 1. Statistical differences in the clinical course of disease were assessed by two-way ANOVA with Sidak's post hoc test. Statistical differences in cell infiltration, proliferation or mRNA expression were assessed by t-test. P<.05 was regarded as statistically significant. Data were analysed GraphPad Prism 6.0 (GraphPad, San Diego, Ca, USA). Comparing AIPD in C57Bl/6J vs C57Bl/6N mice, we have found most significant differences in the course of skin inflammation in all its single aspects, that is erythema, infiltration and desquamation, between the two closely related mouse substrains (Fig. 1a, b). Although, in general, both substrains were susceptible to AIPD, the course of skin inflammation was significantly more severe in C57Bl/6J than in C57Bl/6N mice. Thus, erythema and desquamation were reduced in C57Bl/6N mice by approximately 30% and 50%, respectively, at the end of the experiment on day 6. The difference in skin infiltration was even more pronounced with C57Bl/6N mice showing a delayed onset of detectable skin infiltration and a reduction by 60% in comparison with C57Bl/6J mice on day 6. These differences in erythema, infiltration and desquamation accumulated to a difference of 50% in the cumulative score for AIPD diseases activity. In line with this clinical difference, C57Bl/6J and C57Bl/6N mice also differed in histopathological signs of psoriasiform dermatitis (Fig. 1c). Thus, at the end of the experiment on day 6, C57Bl/6N mice exhibited significantly less epidermal proliferation, as reflected by Ki-67 expression in the epidermis (Fig. 1d), and consequently less epidermal thickness than C57Bl/6J mice (Fig. 1e). In addition, while there was no difference in the overall number of T cells infiltrating the skin (Fig. 1f), C57Bl/6N mice showed reduced skin macrophage infiltration (Fig. 1g). With the IL-23/IL-17 axis and IL-22 playing a critical role in the pathogenesis of AIPD,6, 7 we determined the mRNA expression of these three cytokines in inflamed skin on day 6. This revealed that IL-23p19 mRNA expression levels equalled in C57Bl/6J and C57Bl/6N mice, whereas IL-22 expression was significantly lower C57Bl/6N mice than in C57Bl/6J mice (Fig. 1h). Likewise, IL-17A expression was lower in C57Bl/6N mice than in C57Bl/6J mice (Fig. 1h). This difference, however, did not reach statistical significance. Notably, in another set of experiments, we excluded a difference in disease activity in C57Bl/6J mice from different commercial sources, precisely Charles River Laboratories Germany (Sulzfeld, Germany) and JANVIER LABS (results not shown). Furthermore, subjecting C57Bl/6J and C57Bl/6N mice to mock treatment with softcream for 6 days, we excluded that pharmacologically inactive cream induces signs of psoriasiform dermatitis in the skin of C57Bl/6J or C57Bl/6N mice on the macroscopic or microscopic level (Fig. S1). Our results reveal that the course of AIPD significantly differs in C57Bl/6J vs C57Bl/6N mice. The magnitude of this difference is remarkable regarding the fact that induction of AIPD relies on several parallel, redundant molecular pathways, which can often compensate deficiencies in single contributors.8, 9 Consequently, only comparatively few single gene deficiencies were found to reduce dermatitis in this model by 50%. As a corollary, the genetic differences between the C57Bl/6J and C57Bl/6N substrains must include genes, which significantly modulate some of the core processes driving AIPD and, possibly, also human plaque psoriasis. Our results hint at IL-22, one of the major drivers of AIPD, to be more tightly regulated and, consequently, less aggravating psoriasiform dermatitis in the C57Bl/6N substrain than in the C57Bl/6J substrain. Furthermore, our results suggest that macrophages may play a significant role in the establishment of dermatitis in the AIPD model. The genetic differences between the C57Bl/6J and C57Bl/6N substrains have been comprehensively described in recent years;2-4 however, among those genes, none has been described to contribute to psoriasis. We are therefore in the process of identifying those genes responsible for the difference between C57Bl/6J and C57Bl/6N mice in AIPD. Most importantly, our study warrants great caution when matching up groups of C57Bl/6 mice for studies in the AIPD mouse model. If the genetic background between these two groups is not exactly the same, there is a high risk for false results and, consequently, false conclusions. The use of littermates in both groups is therefore strongly recommended. This work was supported by the Deutsche Forschungsgemeinschaft (KFO 303; Sa1960/5-1). C.D.S. designed the research study, analysed and wrote the manuscript; S.B. performed the research and analysed results; A.H., T.S. and S.M. performed the research; D.Z., S.I. and R.J.L analysed data and wrote the manuscript. All author approved the final version of the article and consented to its publication. Figure S1 Mock cream does not induce signs of psoriasiform dermatitis or elicits macroscopic or microscopic differences between C57Bl/6N and C57Bl/6J mice Data S1 Supplementary Methods and Materials Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.