CD8(+) T cells mediate RAS-induced psoriasis-like skin inflammation through IFN-γ

Abstract

The RAS signaling pathway is constitutively activated in psoriatic keratinocytes. We expressed activated H-RAS(V12G) in suprabasal keratinocytes of adult mice and observed rapid development of a psoriasis-like skin phenotype characterized by basal keratinocyte hyperproliferation, acanthosis, hyperkeratosis, intraepidermal neutrophil microabscesses, and increased T helper type 1 (Th1)/Th17 and T cell type 1 (Tc1)/Tc17 skin infiltration. The majority of skin-infiltrating CD8(+) T cells coexpressed IFN-γ and IL-17A. When RAS was expressed on a Rag1-/- background, microabscess formation, inducible nitric oxide synthase expression, and keratinocyte hyperproliferation were suppressed. Depletion of CD8(+), but not CD4(+), T cells reduced cutaneous and systemic inflammation, the RAS-induced increase in cutaneous Th17 and IL-17(+) γδ T cells, and epidermal hyperproliferation to levels similar to a Rag1-/- background. Reconstitution of Rag1-/- inducible RAS mice with purified CD8(+) T cells restored microabscess formation and epidermal hyperproliferation. Neutralization of IFN-γ, but not of IL-17A, in CD8(+) T-cell-reconstituted Rag1-/- mice expressing RAS blocked CD8-mediated skin inflammation, inducible nitric oxide synthase expression, and keratinocyte hyperproliferation. These results show that CD8(+) T cells can orchestrate skin inflammation with psoriasis-like pathology in response to constitutive RAS activation in keratinocytes, and this is primarily mediated through IFN-γ.

Figure 1. Suprabasal expression of H-RAS^V12G in adult epidermis causes psoriasis-like phenotype with intraepidermal microabscesses

(a) Photographs of representative shaved DT mice on dox (right) or off dox (left) for 7 days. Representative skin sections from ST mice (b,g,i) or DT mice off dox for 7 days (c–f,h,j). (b) H&E stained ST skin. (c) H&E stained DT section (arrow indicates intraepidermal microabscesses). IHC detection of (d) proliferation (anti-BrdU), (e) leukocytes (anti-CD45), (f) neutrophils (anti-myeloperoxidase), (g,h) mast cells (toluidine blue) and (i,j) macrophages (anti-F4/80) (20x). Arrows indicate representative stained cell. (k) FACS profile (n=5 mice) of Ly6G⁺/CD11b⁺ cells from ST or DT skin gated on viable CD45⁺ cells. (l) Flow histogram of H₂DCFDA fluorescence in neutrophils from ST and DT mice. (m) In vitro cytotoxicity assay of sorted splenic Ly6G⁺/CD11b⁺ cells using SP1 papilloma cells as target. Data is from 3 independent experiments performed in triplicate, error bars = ⁺/− SEM * significantly different from ST Ly6G⁺ cells. ST:single transgenic, DT: double transgenic – dox. Scale bars = 20 μm (b–f), 100μm (g–j)

Figure 2. RAS triggers skin infiltration of CD4⁺ and CD8⁺ T cells expressing IFN-γ and IL-17A

(a) Top: CD4⁺ and TCRβ⁺/CD8⁺ T cell counts in skin of ST and DT mice. N= 5 ST and 6 DT mice from two experiments. Bottom: IHC localization of CD4⁺ T cell infiltration (left) in the dermis and CD8⁺ T cell infiltration (right) in the epidermis of DT mice. (b, c, e) Intracellular FACS analysis for IL-17A and IFN-γ obtained from stimulated skin single cell suspensions gated on viable/CD45⁺ cells and CD4⁺ (b), γΔ TCR⁺ (c), and CD8⁺ (e) cells. (d) FoxP3 IHC in ST and DT skin. (f) Perforin/granzyme B FACS on IFN-γ⁺/IL-17⁺/CD8⁺ T cells. CD8⁺ T cells from ST mice were too scarce for analysis. Means +/-SEM from at least 6 mice per group. * p < 0.05 relative to control ST mice. ST:single transgenic, DT: double transgenic – dox. Arrows point to representative positively stained cells. Scale bars = 50 μm

Figure 3. Lymphocyte ablation blocks RAS-induced neutrophil infiltration and suppresses epidermal proliferation

(a, b) Skin histology from (a) DTRag1+/+ and (b) DTRag1−/− mice. anti-MPO IHC in DTRag1+/+ (c) and DTRag1−/− mice (d), (40X). (e) toluidine blue stain and (f) anti-F4/80 IHC in DTRag1−/− mice (20x). (g) top: FACS of neutrophils from skin of DTRag1+/+ or −/− mice gated on live CD45⁺/CD11b⁺ cells; n =10; bottom: mast cells in ST or DTRag1+/+ and −/− mice off dox 7 days, n=5 mice per group. (h) Relative levels of iNOS mRNA expression from skin of DTRag1+/+ and −/− mice. (i) Quantitation of BrdU⁺ basal keratinocytes in DTRag1 +/+ and −/− mice from 5 mice per group. (j) Epidermal hyperplasia in skin of ST and DTRag1+/+ and −/− mice. Means +/− SEM from 3 ST and 8 DT mice of each genotype. Error bars = ⁺/− SEM, * significantly different from control or indicated group. ST:single transgenic, DT: double transgenic – dox. Scale bars = 50 μm (a,b,e,f) 100 μm (c,d)

Figure 4. CD8⁺ T cells are necessary and sufficient to cause neutrophil inflammation, microabscess formation and enhance keratinocyte proliferation

(a–c) Histology from DT mice injected with (a) control IgG, (b) α-CD4 or (c) α-CD8 depleting antibodies before RAS induction. (d) FACS quantitation of skin Ly6G⁺ cells gated on the viable CD45⁺/CD11b⁺ population, n = 7, repeated twice. (e) BrdU⁺ basal keratinocytes from DT mice treated with indicated antibodies. (f, g) Histology of DTRag1−/− mice reconstituted with (f) saline or (g) CD8⁺ T cells. (h) FACS quantitation of skin Ly6G⁺ neutrophils gated on viable CD45⁺/CD11b⁺ cells from DTRag1 −/− reconstituted with saline (6 mice) or CD8⁺ T cells (7 mice), 3 experiments. (i) BrdU⁺ basal keratinocytes in saline or CD8⁺ T cell repleted DTRag1−/− mice. All mice were analyzed 7 days post-dox removal. Error bars = ⁺/− SEM, * significantly different from control. ST:single transgenic, DT: double transgenic – dox. Scale bars = 50 μm.

Figure 5. CD8⁺ T cells are necessary for LN CD4 activation and increase in skin IL-17⁺ T cells

(a) Percent CD44^hi/CD62L^lo of CD4⁺ T cells in SDLN and (b) CD4⁺ T cells in skin from ST or DT mice injected with IgG isotype control or α-CD8. Means and SEM were calculated from 5 mice in each group. (c) IL-17A expression in ex vivo stimulated skin CD4⁺ or γΔ T cells isolated from ST or DT mice injected with IgG or α-CD8 depleting antibody. (d) Percent CD44^hi/CD62L^lo of CD8⁺ cells in SDLN of IgG or α-CD4 treated ST and DT mice. Error bars = ⁺/− SEM, * significantly different from indicated group. All mice analyzed 7 days post-dox removal. ST:single transgenic, DT: double transgenic - dox

Figure 6. IFN-γ neutralization suppresses CD8-driven skin inflammation and epidermal proliferation

Ly6G⁺/CD11b⁺ percentages from skin of DTRag1−/− mice reconstituted with saline or CD8⁺ T cells followed by injections with α-IL-17A (a), α-IFN-γ (b) or relevant control IgG prior to and during dox removal. Each symbol represents an individual mouse. * significantly different from CD8 reconstituted + IgG. (c) Histology of CD8⁺ T cell-repleted DTRag1^−/− mice administered IgG or α-IFN-γ. (d) Quantitation of BrdU⁺ basal keratinocytes in CD8⁺ T cell reconstituted DTRag1^−/− mice given α-IFN-γ. (e) qRT-PCR analysis of iNOS expression from skin RNA of CD8⁺ T cell or saline reconstituted DTRag1 −/− mice administered IgG or IFN-γ neutralizing antibody. Error bars = ⁺/− SEM, * significantly different from saline, ** significantly different from CD8 reconstituted + IgG, p<.05. Scale bars = 50 μm.