PMC

IL-36β deficient mice develop more severe secondary zosteriform skin lesions. (a) Wild type and IL-36β KO mice were infected with HSV-1 as described in Fig. . Male and female wild type and IL-36β KO mice were anesthetized and photographed at day 6 post-infection. (b) Wild type (open circles, n = 6) and IL-36β KO (black circles, n = 5) mice were infected with HSV-1 as described in Fig.  and progression of secondary zosteriform lesions photo-documented for 15 days as illustrated in a and Fig. . Lesions were sized using ImageJ. †, all mice dead; ***p < 0.001.

Primary skin infection site viral genome copy numbers are indistinguishable in wild type and IL-36β KO mice. Wild type (black circles) and IL-36β^−/− (open triangles) mice were infected with HSV-1 on the flank skin as described in Fig. . The center of primary infection sites were collected with 4 mm punch biopsy tools at the indicated time-points. Viral genome copy numbers were determined by QPCR. Each symbol represents a single mouse. **p < 0.01; ^# p > 0.05.

Mortality is associated with viral dissemination to multiple organs. (a) Representative images of bowels of moribund wild type and IL-36β^−/− (Fig. ) mice are shown. (b) Wild type (black circles) and IL-36β^−/− (open triangles) mice were infected with 1.5 × 10⁶ PFU HSV-1 on the right flank. Organs were collected from moribund mice, homogenized and HSV-1 genome copy numbers determined by QPCR. Each symbol represents a single mouse. Data is pooled from two independent experiments. No statistical significant differences were observed between wild type and IL-36β deficient mice.

Number of IFNγ-producing CD4⁺ cells are similar in wild type and IL-36β KO zosteriform lesions. Wild type (black bars) and IL-36β KO (open bars) mice were infected with HSV-1 (n = 10 per group) or left uninfected (n = 3–5 per group). Skin was collected 6 days post-infection. Primary lesion sites were collected with 8 mm biopsy punches at the center of the initial infection sites. Upper and lower secondary lesions (Fig. ) were excised and pooled. Number of IFNγ-producing CD4 + cell were determined using ELISpot assays. Representative data (means ± SD) from one of two experiments are shown. ^# p > 0.05; *p < 0.05; **p < 0.01.

IL-36 mRNA expression is induced in vivo. (a–d) C57BL/6 mice were infected with HSV-1 (black symbols) or left uninfected (open symbols). At the indicated time-points primary or mock lesions were collected at the center using 4 mm punch biopsies. IL-36 (a–c) and HSV-1 gD (d) mRNA levels determined by real-time PCR using GAPDH as the housekeeping gene against which individual mRNA levels were standardized. Fold changes in IL-36 mRNA levels were calculated against uninfected skin. Fold changes in HSV-1 gD mRNA levels were calculated against levels at day 1. Data shown is pooled from three independent experiments. (e) IL-36α (open bars), IL-36β (blue bars) and IL-36γ (red bars) mRNA levels were recalculated (data from a–c) relative to levels of the IL-36α mRNA in uninfected mice. *p < 0.05; **p < 0.01; ***p < 0.001; ND, not detected.

Human IL-36 mRNAs are differentially induced in response to HSV-1 infection and cytokines. Human primary keratinocytes were infected with HSV-1 (a–c) or treated with medium only, 10 ng/ml IL-1β, 20 ng/ml IFNγ and/or 50 ng/ml TNFα (d) as indicated. IL-36α, IL-36β (isoform 1 and 2 in d) and IL-36γ mRNA levels were determined by real-time PCR and standardized against GAPDH levels. (a–c) Fold changes in IL-36 expression were calculated against medium only treated samples at the same time-point. (d) Proportional expression of IL-36 levels are graphed as log₂(C_t-IL-36−C_t-GAPDH). Representative data from one of at least three independent experiments is shown as means ± SD. *p < 0.05 (compared to medium only at the same time-point); **p < 0.01; ***p < 0.001.

Wild type and IL-36β KO mice have equal numbers of early HSV-1 gB(498-505) specific CD8⁺ cells. Wild type and IL-36β KO mice were infected with HSV-1 (n = 5 per group) as described in Fig.  or left uninfected (n = 2–3 per group). Cells were isolated from spleen and draining inguinal lymph nodes 6 days post-infection and analyzed by flow cytometry. (a) Total number of CD3, CD4 or CD8 positive cells per organs are shown. (b) Cells were examined following initial gating of the CD3⁺ population. The square gates identify the gB(498–505)/CD8 double positive cells. Numbers above the gates indicate percentage gated cells of all CD8⁺ cells. (c) Graphic representation of gB(498–505) specific CD8⁺ T cells (means ± SD) quantified in b. One representative experiment of 3 is shown. ^# p > 0.05; *p < 0.05, **p < 0.01.

Protein sequence alignment of human and mouse IL-36 cytokines. (a) Human (h) and mouse (m) IL-36 cytokines were aligned using Clustal omega. *conserved residues: Gonnet PAM 250 matrix score > 0.5, 0 < Gonnet PAM 250 matrix score ≤ 0.5. Amino acid sequence of human IL-36β isoform 1, which diverge from the other family members, is shown in red. (b) Neighbor-joining phylogenetic tree showing relationships among IL-36 cytokines. The optimal tree with the sum of branch length = 2.69 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. (c) Heat map showing percentage similarity between the human and mouse IL-36 cytokines.

IL-36β, but not IL-36α or IL-36γ, provides protection against lethal outcome of HSV-1 infection. (a) Illustration of inoculation site (yellow circle) and directions of retrograde (red arrows) and anterograde (blue arrows) migration of HSV-1 through neurons. (b) Progression of disease in wild type mice (C57BL/6) infected with 1.5 × 10⁶ PFU HSV-1 on flank skin (white arrowhead) at day 0 is depicted at the indicated time-points. Black arrowhead points to early secondary lesion appearing along sensory neurons (note the linear progression of the lesions). Ruler used for quantification of lesion sizes visible in day 9 image. Image of gravestone can be found at: https://freeclipartnow.com/holidays/halloween/graveyard/R-I-P-gravestone.jpg.html. (c) Wild type (C57BL/6, green), IL-36α^−/− (red), IL-36β^−/− (blue), IL-36γ^−/− (black), and RAG1^−/− (brown) mice were infected with 1.5 × 10⁶ PFU HSV-1 on the right flank on day 0, and survival monitored for 16 days. n indicates number of mice per group. Data shown is pooled from 6 independent experiments. Statistical significance was determined using Mantel-Cox and Gehan-Breslow-Wilcoxon tests; ****p < 0.0001. (d) Wild type (C57BL/6, n = 18, black lines and symbols) and IL-36β^−/− (n = 8, red lines and symbols) mice were infected with HSV-1 as described above. Pooled weight data (means ± SD) of survivors (closed symbols) and mice progressing to become moribund (open symbols) from two independent experiments is shown. *p < 0.05 (comparing survivors to moribund within each strain); **p < 0.01; ***p < 0.001.

IL-36β^−/− mice develop antibodies against HSV-1 at the same time as wild type mice. (a) Wild type and IL-36β^−/− mice were infected with HSV-1 (n = 5 per group) or left uninfected (n = 2–3 per group). Cells were isolated from spleen and draining inguinal lymph nodes 6 days post-infection and analyzed by flow cytometry. Granulocyte, lymphocyte, and antigen presenting cell (APCs) populations were quantified via gates depicted on representative images of forward scatter and side scatter. (b) Graphic representation of data (means ± SD) from a. ^# p > 0.05. Representative experiment of 3. (c and d) Wild type (as labeled in c, black circles in d) and IL-36β^−/− (as labeled in c, open triangles in d) mice were infected with HSV-1 and serum collected at the indicated time-points. (c) Reactivity of mouse sera against HSV-1 infected HaCaT protein lysate was examined by Western blotting. The HaCaT protein lysate was separated by SDS-PAGE using a gel with a single approximately 7 cm wide well. After transfer to a PVDF membrane, multiple strips approximately 2 mm wide were cut and used for incubation with individual sera. Approximate positions of protein markers are shown. (d) Reactivity of mouse sera against HSV-1 gD were determined by direct ELISA. Log₂ reciprocal values of end-point titers are depicted. Gray box indicates background signal against trace E. coli proteins. No statistically significant differences were observed between wild type and IL-36β deficient mice (#) despite significant increases in anti-gD titers in both strains (**p < 0.01 (compared as indicated); ***p < 0.001). Each symbol represents a single mouse.