The severity of imiquimod-induced mouse skin inflammation is independent of endogenous IL-38 expression

<div><p>The IL-1 cytokine family includes eleven members, among which Il-36α, β and γ, IL-36Ra and IL-38. The IL-36 cytokines are involved in the pathogenesis of psoriasis. IL-38 is also expressed in the skin and was previously proposed to act as an IL-36 antagonist. In this study, we thus examined expression and function of Il-38 in a mouse model of imiquimod (IMQ)-induced skin inflammation. <i>Il-38</i> mRNA was detected in the epidermis and in primary mouse keratinocytes, but not in dermal fibroblasts. At the peak of IMQ-induced inflammation, skin <i>Il-38</i> mRNA levels were reduced, whereas <i>Il-36ra</i> mRNA expression increased. The severity of IMQ-induced skin inflammation, as assessed by recording ear thickness and histological changes, was similar in Il-38 KO and WT littermate control mice, while, in contrast, Il-36ra-deficient mice displayed more severe skin pathology than their WT littermates. Il-38-deficiency had no impact on IMQ-induced expression of proinflammatory mediators in the skin <i>in vivo</i>, on the basal expression of various cytokines or chemokines by cultured primary keratinocytes and dermal fibroblasts <i>in vitro</i>, or on the response of these cells to Il-36β. Finally, after cessation of topical IMQ application, the resolution of skin inflammation was also not altered in Il-38 KO mice. In conclusion, Il-38-deficiency did not impact the development or resolution of IMQ-induced skin inflammation. Our observations further suggest that endogenous Il-38 does not exert Il-36 inhibitory activity in this model, or in cultured skin cells. A potential anti-inflammatory function of Il-38 in mouse skin thus still remains to be demonstrated.</p></div

Primers used for qPCR.

<p>Primers used for qPCR.</p

Il-38-deficiency does not influence the severity of IMQ-induced skin inflammation.

<p><i>Il-38</i>^<i>-/-</i> mice (n = 5) and WT littermates (n = 5) were treated daily with a topical dose of 12.5mg of Aldara^™ cream (0.625mg IMQ), for 7 days. Ear thickness was followed daily (A) and expressed as ear thickness variation vs. day 0. Microscopic histopathology was studied on HE-stained slides of IMQ-treated ears on day 7. Scale bar = 100μm. (B); neutrophil-infiltrated areas, epidermal thickness and the number of neutrophil-filled abscess-like structures were evaluated (C). Results are from one experiment representative of two and are expressed as mean ± SEM of individual mice (n = 5 mice per group). <i>Il-36ra</i>^<i>-/-</i> mice (n = 6) and WT littermates (n = 4) were treated daily with a topical dose of 12.5mg of Aldara^™ cream (0.625mg IMQ), for 8 days. Ear thickness was followed daily (D) and expressed as ear thickness variation vs. day 0. Microscopic histopathology was studied on HE-stained slides of IMQ-treated ears on day 8. Scale bar = 100μm. (E); neutrophil-infiltrated areas, epidermal thickness and the number of neutrophil-filled abscess-like structures were evaluated (F). Results are expressed as mean ± SEM of individual mice (n = 4–6 mice per group). Statistical analysis was performed using a paired two-way ANOVA followed by a Sidak post-test for A and and D, and an unpaired Mann-Whitney comparison test in C and F. A <i>p</i>-value < 0.05 was considered significant. ** p<0.01, * p<0.05.</p

Expression of proinflammatory mediators in IMQ-treated skin and in cultured skin cells of Il-38 deficient mice.

<p><i>Il-38</i>^<i>-/-</i> mice and WT littermates were treated daily with a topical dose of 12.5mg of Aldara^™ cream (0.625mg IMQ), for 7 days. Skin mRNA levels for <i>Il-36α</i>, <i>Il-36β</i>, <i>Il-36γ</i>, <i>Il-36ra</i>, <i>Il-38</i>, <i>Cxcl-1 and Il-6</i> were quantified by real-time RT-qPCR on day 7 in the IMQ-treated ears (A). Data were expressed relative to <i>L32</i> levels. Results represent individual values and mean ± SEM of n = 5 mice per group. Statistical analysis was performed by unpaired Mann-Whitney comparison test. A <i>p</i>-value < 0.05 was considered significant. ** p<0.01. Cultured primary keratinocytes (Kera) and dermal fibroblasts (Fibro) isolated from naïve <i>Il-38</i>^<i>-/-</i> (dark symbols) or WT mice (white symbols), were stimulated with rec. mouse Il-36β at 100ng/ml for 6 h, or left unstimulated (Med). <i>Il-36α</i>, <i>Il-36β</i>, <i>Il-36γ</i>, <i>Il-36ra</i>, <i>Il-38</i>, <i>Cxcl-1</i>, <i>Il-6</i> and <i>S100a9</i> mRNA levels were quantified by real-time RT-qPCR (B). Data are expressed relative to <i>L32</i> levels. Results represent individual values and mean ± SEM of n = 6–9 biological replicates per group. Statistical analysis was performed by two-way ANOVA followed by a Holm–Sidak’s comparison test. A <i>p</i>-value < 0.05 was considered significant. *** p<0.001, ** p<0.01, * p<0.05.</p

Il-38-deficiency does not affect resolution of IMQ-induced inflammation.

<p><i>Il-38</i>^<i>-/-</i> mice (n = 5) and WT littermates (n = 5) were treated daily with a topical dose of 12.5mg of Aldara^™ cream (0.625mg IMQ), for 7 days, then left untreated until day 11. Ear thickness was followed daily (A) and expressed as ear thickness variation vs. day 0. Microscopic histopathology was studied on HE-stained slides of IMQ-treated ears at day 11. Scale bar = 100 μm. (B); neutrophil-infiltrated areas, epidermal thickness and the number of neutrophil-filled abscess-like structures were evaluated (C). Results are representative of 2 independent experiments and expressed as mean ± SEM of individual mice (n = 5 mice per group). Statistical analysis was performed using a paired two-way ANOVA followed by a Sidak’s post-test for A and an unpaired Mann-Whitney comparison test in C. A <i>p</i>-value < 0.05 was considered significant.</p

Expression of <i>Il-38</i> and <i>Il-36</i> family cytokines in total skin, epidermis, cultured primary keratinocytes and dermal fibroblasts.

<p>Basal <i>Il-38</i>, <i>Il-36ra</i>, <i>Il-36a</i>, <i>Il-36b</i> and <i>Il-36g</i> mRNA expression was quantified by real-time RT-qPCR in total skin (n = 5) and epidermis (n = 5) of naïve BALB/c WT mice (A); and in cultured primary keratinocytes (Kera, n = 4 independent cultures) and dermal fibroblasts (Fibro, n = 5 independent cultures) isolated from naïve BALB/c WT mice (B). Data were expressed relative to <i>L32</i> levels. Results are shown as individual values and mean ± SEM. Statistical analysis was performed using an unpaired Mann-Whitney comparison test. A <i>p</i>-value < 0.05 was considered significant. *** p<0.001, ** p<0.01, * p<0.05.</p

ILC2 promote M2 macrophage polarization.

<p>(A) BMDM from C57BL/6 mice were cultured in the lower chamber of a 24-transwell plate in complete medium alone (M0) or supplemented with IL-4 (M2). In some experiments ILC2, sorted from naïve WT mice pre-treated with IL-33, were added to the upper chamber. After 48 h, BMDM were collected and assayed for the expression of M2 markers by qPCR (relative to <i>Hprt1</i>). (B) ST2-deficient BMDM were co-cultured in transwell plates as above with WT ILC2 in the presence of IL-33 alone or in combination with IL-7. After 48 h, BMDM were collected and assayed for the expression of M2 markers by qPCR (relative to <i>Hprt1</i>). Type 2 cytokines in the supernatants of ILC2 cultured in the presence of IL-33 or IL-33 + IL-7 were determined by ELISA (C), or by FACS (D, E). Data are mean ± SEM (n = 3 per group), representative of two independent experiments, *P<0.05, **P<0.01, ***P<0.001 by two-tailed ANOVA. (F) ILC2 sorted from mice pre-treated with IL-33 were adoptively transferred (2×10⁶ cells, i.v., on day −1) into naïve C57BL/6 mice which were infected with PbA (10⁴ pRBCs, i.v., on day 0). The recipients, were given 2 injections of IL-33 (0.2 μg, i.p.) 30 min and 24 h after cell transfer. Expression of <i>Arg-1, Ym1</i> and <i>Fizz1</i> mRNA in the spleen was measured by qPCR (relative to <i>Hprt1</i>) on day 7. Data are mean ± SEM (n = 5 per group) *P<0.05, **P<0.01, ***P<0.001 by two-tailed ANOVA. </p

IL-33 protects mice from PbA-induced cerebral malaria.

<p>C57BL/6 mice were infected i.v. with <i>Plasmodium berghei</i> ANKA (PbA) (10⁴ pRBCs) and injected i.p. daily with PBS or IL-33 (0.2 μg) from day 0. (A) Kaplan–Meier survival curves (pool of 3 experiments, n = 14–20 per group). (B) body weight loss (n = 5 per group); Statistical differences are shown for day 7. (C) Clinical score (n = 5 per group). The hatched area indicates ECM related scores. (D) Time-course analysis of parasitemia (n = 5 per group). (E) Parasite-derived bioluminescence. One representative mouse is shown at each time point. Radiance (P/Sec/cm²/Sr), color scale Min = 2×10⁶, Max = 5×10⁷. (F) Mean body bioluminescence (n = 5 per group). (G) Mice infected with PbA-Luc were sacrificed on day 7 and bioluminescence of the brain was recorded. (H) Mean brain bioluminescence per group (n = 6). (I) Representative H&E histopathology of brain vasculature from PBS- or IL-33-treated mice. Magnification ×400. (J) Severity of brain microvascular obstruction and local hemorrhage assessed from a whole-brain section (n = 6 per group). (K) <i>Icam-1</i> mRNA expression (relative to <i>Hprt1</i>) in the brain determined by qPCR (n = 6 per group); Data are mean ± SEM. Representative data from 3 independent experiments were shown. ns, non significant, ***P<0.001.</p

Schematic representation of the pathways by which IL-33 attenuates ECM.

<p>Arrows represent activation/promotion; dotted blunted arrows = proposed mechanism of suppression.</p

IL-33 polarizes M2 macrophages in PbA-infected mice.

<p>C57BL/6 mice were not infected (NI) or infected i.v. with PbA and treated daily for 5 days with PBS or IL-33 and spleen cells were harvested at indicated time points and analysed. (A) Representative dotplots showing percentage of CD11b⁺F4/80⁺ cells among total splenocytes. (B) Percentages and (C) numbers of CD11b⁺F4/80⁺CD11c⁻ macrophages in the spleen. (D) Mean Fluorescence Intensity (MFI) of CD206, CD86, MHC-II and CD40 on macrophages (n = 5 per group). (E) Expression (relative to <i>Hprt1</i>) of <i>Arg-1, Ym1, Fizz1, Nos2</i> and <i>Hmox-1</i> mRNA in the spleen on day 5. Data are mean ± SEM, representative of 2 independent experiments. ns, not significant, *P<0.05, **P<0.01, ***P<0.001 by two-tailed ANOVA. </p