Tristetraprolin expression by keratinocytes controls local and systemic inflammation

Abstract

Tristetraprolin (TTP, encoded by the Zfp36 gene) regulates the mRNA stability of several important cytokines. Due to the critical role of this RNA-binding protein in the control of inflammation, TTP deficiency leads to the spontaneous development of a complex inflammatory syndrome. So far, this phenotype has been largely attributed to dysregulated production of TNF and IL‑23 by myeloid cells, such as macrophages or DCs. Here, we generated mice with conditional deletion of TTP in keratinocytes (Zfp36fl/flK14-Cre mice, referred to herein as Zfp36ΔEP mice). Unlike DC-restricted (CD11c-Cre) or myeloid cell-restricted (LysM-Cre) TTP ablation, these mice developed exacerbated inflammation in the imiquimod-induced psoriasis model. Furthermore, Zfp36ΔEP mice progressively developed a spontaneous pathology with systemic inflammation, psoriatic-like skin lesions, and dactylitis. Finally, we provide evidence that keratinocyte-derived TNF production drives these different pathological features. In summary, these findings expand current views on the initiation of psoriasis and related arthritis by revealing the keratinocyte-intrinsic role of TTP.

Keywords: Dermatology; Inflammation.

Figure 1. Loss of tristetraprolin increases the severity of imiquimod-induced skin lesions.

Zfp36-deficient mice (Zfp36^–/–) and their littermates (Zfp36^+/+) were treated topically with imiquimod over 5 consecutive days (n = 5) or not (n = 3). Skin samples were collected 4 hours after the last application for analysis of epidermal thickness by histology (A), cell recruitment by flow cytometry (B), cytokine expression by intracellular protein staining (C), or transcript levels by RTqPCR (D and E). (A) Representative macroscopic images of the abdomen via MGG staining of skin sections (original magnification, ×200) from 13 mock Zfp36^+/+, 21 imiquimod-treated Zfp36^+/+, 13 mock Zfp36^–/–, and 22 imiquimod-treated Zfp36^–/– mice; Ki67 immunostaining (labeling index, n = 3 by group); and epidermal thickness. (B) Representative FACS plots and manual gating strategy for neutrophil and monocyte populations as well as neutrophil (CD45⁺CD19^–CD3^–CD11b⁺Ly6C⁺Ly6G⁺) and monocyte (CD45⁺CD19^–CD3^–CD11b⁺Ly6C⁺Ly6G^–) recruitment into skin. Squares represent gates for neutrophil and monocyte populations. (C) IL-17A and IL-22 production by CD45⁺CD19^–NK1.1^–Gr1^– cells and cytokine production by αβ T cells (CD45⁺CD19^–NK1.1^–Gr1^–CD3⁺γδTCR^–), γδ T cells (CD45⁺CD19^–NK1.1^–Gr1^–CD3⁺γδTCR⁺), and innate lymphoid cells (ILCs) (CD45⁺CD19^–NK1.1^–Gr1^–CD3^–CD90⁺CD127⁺). Each dot represents total cytokine production in an individual mouse. (D and E) Total skin mRNA levels in mock-treated animals (D) (n = 7 mice by group, pooled from 2 experiments) and in imiquimod-treated animals (E) (n = 10 mice by group, pooled from 2 experiments) were normalized against Actb, Gapdh, and Hprt mRNA levels and expressed relative to the Zfp36^+/+ mock group arbitrarily set to 1. Bars represent mean ± SEM. Statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) was assessed by 1-way ANOVA test with Bonferroni correction (A–C) or 2-tailed Mann-Whitney compared with mock-treated Zfp36^+/+ group (D) or compared with imiquimod-treated Zfp36^+/+ group (E). Results are representative of 5 independent experiments. Results observed with ear or dorsum were presented in Supplemental Figure 1A (see also Supplemental Figure 1).

Figure 2. TTP expression by keratinocytes is important for the control of skin inflammation induced by imiquimod.

(A–E) Myeloid-specific Zfp36-deficient mice (Zfp36^ΔM, n = 6), DC-specific Zfp36-deficient mice (Zfp36^ΔDC, n = 6), keratinocyte-specific Zfp36-deficient mice (Zfp36^ΔEP, n = 5), and their littermate controls (Zfp36^fl/fl, n = 12) were topically treated over 5 consecutive days with imiquimod. Skin samples were collected 4 hours after the last application for analysis of epidermal thickness by histology. Original magnification: ×200. (A), cell recruitment by flow cytometry (B and C), cytokine production by intracellular protein staining (D), or transcript levels by RTqPCR (E and F). Results are given as mean ± SEM. Total skin mRNA levels were normalized against Actb, Gapdh, and Hprt mRNA levels and expressed relative to the Zfp36^fl/fl mock group arbitrarily set to 1. Statistical significance (*P < 0.05, **P < 0.01, ****P < 0.0001) was assessed by 1-way ANOVA test with Bonferroni correction compared with the Zfp36^fl/fl mock group (A–D) or 2-tailed Mann-Whitney test compared with the mock-treated Zfp36^fl/fl group (E) or compared with the imiquimod-treated Zfp36^fl/fl group (F). Results are representative of at least 5 experiments. Histology is representative of 29 Zfp36^fl/fl, 6 Zfp36^ΔM, 18 Zfp36^ΔDC, and 24 Zfp36^ΔEP mice, pooled from 4 experiments. Representative histology for the mock group from each genotype is presented in Supplemental Figure 3 (see also Supplemental Figure 2).

Figure 3. Zfp36 deficiency in keratinocytes results in an increase of multiple transcripts encoding proinflammatory cytokines and chemokines.

Zfp36^ΔEP mice and their littermate controls (Zfp36^fl/fl) (n = 5) were topically treated over 5 consecutive days with imiquimod. Skin samples were collected 4 hours after the last application for transcriptomic analysis. (A) Volcano plot showing log₂ fold changes versus log₁₀ P values. As shown from the top of A to the bottom, 1,828 transcripts were significantly upregulated (red) and 1,377 transcripts were significantly downregulated (blue) among all transcripts (gray) in skin from imiquimod-treated Zfp36^fl/fl mice compared with mock-treated Zfp36^fl/fl mice. Distribution of the 1,828 transcripts (red, the positive imiquimod signature) among all transcripts (gray) in skin from Zfp36^ΔEP mice compared with Zfp36^fl/fl mice, both treated by imiquimod, and P values for differential expression (upregulation or downregulation) of the gene set (red) are shown. The same analysis was performed for the 1,377 transcripts (blue, the negative imiquimod signature). (B) Heatmap of expression levels of transcripts from the positive and negative imiquimod signatures (as shown in A) significantly (q < 0.05) increased or decreased, respectively, in skin from imiquimod-treated Zfp36^ΔEP mice compared with imiquimod-treated Zfp36^fl/fl mice. (C) Gene set enrichment analysis of 4 of the most significantly (P < 0.001) enriched KEGG pathways in skin from imiquimod-treated Zfp36^ΔEP mice compared with Zfp36^fl/fl mice. (D) Enrichment score distribution for the KEGG cytokine-cytokine receptor interaction pathway. (P < 0.001, permutation test) (E) Heatmap of expression levels of genes from the core enrichment of the KEGG cytokine-cytokine receptor interaction pathway. Each column corresponds to a separate mouse.

Figure 4. Mice with Zfp36 deficiency in keratinocytes represent a more specific model of human psoriasis.

(A) Volcano plot showing log₂ fold changes versus log₁₀ P values. As shown from the top of A to the bottom, 6,004 transcripts were significantly upregulated (red) and 6,900 transcripts were significantly downregulated (blue) among all transcripts (gray) in laser-microdissected epidermis from skin of patients with psoriasis compared with healthy epidermis from skin from subjects with no diagnosed skin diseases. Distribution of 2,675 mouse orthologs corresponding to the 6,004 human transcripts increased in psoriatic epidermis (red, the positive psoriasis signature) among all genes (gray) in skin from Zfp36^ΔEP mice compared with Zfp36^fl/fl mice, both treated by imiquimod. P values for differential expression (upregulation or downregulation) of the gene set (red) are shown. The same analysis for 2,700 mouse orthologs corresponding to the 6,900 human transcripts decreased in psoriatic epidermis (blue, the negative psoriasis signature). (B) Heatmap of expression levels of genes of the positive and negative psoriasis signature, significantly (q < 0.05) increased or decreased, respectively, in skin from Zfp36^ΔEP mice compared with Zfp36^fl/fl mice, both treated by imiquimod. Each column corresponds to a separate mouse. (C) Expression of ZFP36 in microdissected epidermis from healthy (n = 5) and psoriatic individuals (lesional skin) (n = 10) was analyzed by microarrays. Results are given as mean ± SEM. Statistical significance (*P < 0.05) was assessed by 2-tailed Mann-Whitney test compared with healthy group.

Figure 5. TTP controls the mRNA stability of many proinflammatory mediators in TPA-stimulated primary mouse keratinocytes.

Primary keratinocytes isolated from Zfp36^ΔEP and Zfp36^fl/fl newborns were stimulated for 2 hours with TPA (100 ng/ml), rIL-17A (50 ng/ml), or rIL-22 (50 ng/ml). (A) Expression of Zfp36 was assessed by RTqPCR and Western blot (mock condition). (B) Gene expression in response to TPA stimulation. (C and D) Expression of Tnf (C) and Cxcl2 (D) after rIL-17A or rIL-22 stimulation. Results are given as mean ± SEM of at least 3 independent experiments performed in triplicate. Normalized mRNA levels were expressed relative to the Zfp36^fl/fl untreated condition arbitrarily set to 1. (E) mRNA half-life analysis. Two hours after TPA stimulation, actinomycin D (10 μg/ml) and SB203580 (1 μM) were added for the indicated times. SB203580 was used to abolish the inhibitory effect of p38 MAPK on TTP activation. Total RNA was extracted and analyzed by RTqPCR. Results are given as mean ± SEM of 2 experiments performed in triplicate (a total of 6 wells from each genotype). Statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) was assessed by 1-way ANOVA test with Bonferroni correction (A) or by 2-tailed Mann-Whitney test compared with the Zfp36^fl/fl group (B–E).

Figure 6. TNF produced by keratinocytes is critical for the control of inflammation in imiquimod-induced dermatitis.

Zfp36^ΔEP (n = 8), Tnf^ΔEP (n = 7), and Zfp36^ΔEPTnf^ΔEP mice (n = 10) and their littermate controls, Zfp36^fl/fl (n = 4), Tnf^fl/fl (n = 5), and Zfp36^fl/fTnf^fl/fl (n = 10) were topically treated over 5 consecutive days with imiquimod. Skin samples were collected 4 hours after the last application for analysis of epidermal thickness by histology. Original magnification: ×200 (A), cell recruitment by flow cytometry (B and C), protein production by intracellular protein staining (D), or transcript levels by RTqPCR (E). Results are given as mean ± SEM. Statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) was assessed by 1-way ANOVA test with Bonferroni correction compared with the littermate group (if not specified). Results are pooled from 2 independent experiments. Representative histology for mock group from each genotype is documented in Supplemental Figure 4.

Figure 7. Loss of tristetraprolin expression by keratinocytes leads to the spontaneous development of local and systemic inflammation.

Zfp36^ΔEP (n = 17), Zfp36^–/– (n = 7), and Zfp36^ΔEPTnf^ΔEP mice (n = 7) and their respective littermate controls, Zfp36^flfl (n = 17), Zfp36^+/+ (n = 7), and Zfp36^fl/flTnf ^fl/fl mice (n = 4), were monitored weekly for clinical features. (A) Phenotypical presentation: macroscopic views of representative Zfp36^ΔEP, Zfp36^fl/fl, and Zfp36^–/– mice, showing, from left to right, neck skin, tail, front paw, and back paw. (B) Incidence of skin lesions. (C) Incidence of arthritis. (D and E) Weight curves. (F–H) Features of skin inflammation of 10- to 12-month-old male mice. (F) Histological view of MGG-stained section of upper thoracic skin samples (representative of 17 animals by group). (G) Proportion of neutrophils and (H) IL-17A–producing cells in the skin of Zfp36^ΔEP (n = 13) and Zfp36^fl/fl (n = 10) mice. (I–K) Characteristics of joint inflammation. (I) Histological view of MGG-stained section of a front paw of 10- to 12-month-old male mice. Arrows indicate the entheseal regions (representative of 17 animals by group). (J) High-resolution CT scan and (K) PET-CT scan of the front paw of 6-month-old male mice injected with [¹⁸F]-FDG (1 representative of 4 mice). Arrows indicate bone disruption. (L) G-CSF levels in the serum, (M) proportion of myeloid cells (CD45⁺CD19^–CD3^–CD11b⁺) in the spleen, and (N) N-GAL levels in the sera (note the log scale for y axis) of Zfp36^ΔEP (n = 7) and Zfp36^fl/fl (n = 10) mice or Zfp36^+/+ (n = 9) and Zfp36^–/– (n = 14) mice at 10 to 12 months old. Each dot represents data from an individual mouse. Results are given as mean ± SEM. Statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001) was assessed by 2-tailed Mann-Whitney compared with the Zfp36^fl/fl mock group (if not specified) (**P < 0.01, ***P < 0.001, ****P < 0.0001). Results are representative of 2 experiments (see also Supplemental Figure 5). Original magnification: ×200 (F and I).