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. 2020 Jun;107(6):941-952.
doi: 10.1002/JLB.3MA1219-398R. Epub 2020 Jan 27.

RIP1 kinase activity is critical for skin inflammation but not for viral propagation

Joshua D Webster  1 , Youngsu C Kwon  2 , Summer Park  2 , Hua Zhang  2 , Nick Corr  3 , Nina Ljumanovic  3 , Adeyemi O Adedeji  3 , Eugene Varfolomeev  4 , Tatiana Goncharov  4 , Jessica Preston  1 , Sara F Santagostino  3 , Snahel Patel  5 , Min Xu  2 , Jonathan Maher  3 , Brent S McKenzie  2 , Domagoj Vucic  4
Affiliations

RIP1 kinase activity is critical for skin inflammation but not for viral propagation

Joshua D Webster et al. J Leukoc Biol. 2020 Jun.

Abstract

Receptor interacting protein kinase 1 (RIP1) is a critical effector of inflammatory responses and cell death activation. Cell death pathways regulated by RIP1 include caspase-dependent apoptosis and caspase-independent necroptosis. The kinase activity of RIP1 has been associated with a number of inflammatory, neurodegenerative, and oncogenic diseases. In this study, we use the RIP1 kinase inhibitor GNE684 to demonstrate that RIP1 inhibition can effectively block skin inflammation and immune cell infiltrates in livers of Sharpin mutant (Cpdm; chronic proliferative dermatitis) mice in an interventional setting, after disease onset. On the other hand, genetic inactivation of RIP1 (RIP1 KD) or ablation of RIP3 (RIP3 KO) or MLKL (MLKL KO) did not affect testicular pathology of aging male mice. Likewise, infection with vaccinia virus or with mouse gammaherpesvirus MHV68 resulted in similar viral clearance in wild-type, RIP1 KD, and RIP3 KO mice. In summary, this study highlights the benefits of inhibiting RIP1 in skin inflammation, as opposed to its lack of relevance for testicular longevity and the response to certain viral infections.

Keywords: MLKL; RIP1; RIP3; RIPK1; RIPK3; caspase.

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Figures

Figure 1
Figure 1
RIP1 kinase inhibition resolves dermatitis in Cpdm mice. (A) resolution of dermatitis and alopecia is observed in the Cpdm mouse treated with GNE684 (left) compared to the vehicle treated Cpdm mouse (right). (B) Histologic scores of dermatitis in wild‐type (N = 18), GNE684 treated Cpdm (N = 8), and vehicle treated Cpdm (N = 8) mice. Asterisks indicate P = 0.0003. Bar depicts mean with sd, P‐value was determined by Mann‐Whitney test. (C) Representative H&E stained, and cleaved caspase‐3 and phospho RIP3 (pRIP3) immunolabeled sections of mice from (B) demonstrating decreased dermal infiltrates, epidermal hyperplasia crusting, or ulceration, and decreased cleaved caspase‐3 and RIP3 phosphorylation in Cpdm mice following treatment with GNE684. The arrow indicates a pRIP3 positive cell
Figure 2
Figure 2
RIP1 kinase inhibition reduces liver, but not esophageal or intestinal, inflammatory infiltrates in Cpdm mice. (A–C) Histologic sections of liver (A), esophagus (B), and colon (C) demonstrated inflammatory infiltrates in Cpdm mice, although these infiltrates are relatively minimal in the colon. (D) Histologic scores of hepatic, esophageal, and colonic inflammation demonstrate abatement of inflammation in the liver, but not in the esophagus or colon, in Cpdm mice following treatment with GNE684 (G684). Asterisks indicate P = 0.0054. Bars depict mean with sd, P‐values were determined by Mann‐Whitney test. Wild‐type, N = 18; GNE684 treated Cpdm, N = 8; vehicle treated Cpdm, N = 8
Figure 3
Figure 3
RIP1 KD, RIP3 KO, and MLKL KO mice have no evidence of differential age‐related testicular degeneration. (A–C.) Body weights (A), seminal vesicle weights (B), and testis weights (C) in RIP1 KD (N = 16), MLKL KO (N = 7), RIP3 KO (N = 5), and wild‐type mice (RIP1 WT, N = 11; MLKL WT, N = 4; RIP3 WT, N = 2) at study termination (18 mo). No group differences were noted. (D) Quantification of seminiferous tubules with loss of germ cells. Variable tubular degeneration was observed across genotypes with no clear genotype‐associated differences noted. (E) H&E stained sections of testes demonstrating similar histomorphologic features across genotypes
Figure 4
Figure 4
Loss of RIP1 kinase activity does not affect vaccinia virus clearance. (A‐C) Vaccinia virus plaque forming units in the liver (A), spleen (B), and ovary (C) measured 2 and 14 d post‐inoculation. Bars depict geometric mean, P‐values were determined by Mann‐Whitney test. N = 8 per group
Figure 5
Figure 5
Loss of RIP1 kinase activity does not affect MHV68 virus clearance. Acute (A) and latent (B) MHV68 infections were assessed in RIP1 KD and WT mice by viral plaque assays (acute infection) or by PCR (latent infection). (C‐D) Levels of IFNγ (C) and CXCL10 (D) from animals analyzed in (A) and (B) were assessed by Luminex ELISA. (E‐F) Populations of B (E) and T‐helper (F) cells in the peripheral blood from animals analyzed in (A) and (B) were assessed by flow cytometry. WT, N = 4/ time point; RIP1 KD, N = 5/ time point; uninfected WT, N = 2. Bars depict mean with sd, P‐values were determined by multiple t tests
Figure 6
Figure 6
Genetic ablation of RIP3 does not affect MHV68 virus clearance. Acute (A) and latent (B) MHV68 infections were assessed in RIP3 KO and WT mice by viral plaque assays (acute infection) or by PCR (latent infection). (C‐D) Levels of IFNγ (C) and CXCL10 (D) from animals analyzed in (A) and (B) were assessed by Luminex ELISA. (E‐F) Populations of B (E) and T‐helper (F) cells in the peripheral blood from animals analyzed in (A) and (B) were assessed by flow cytometry. Day 4, N = 3/ group; other time points, N = 4/ group. Uninfected WT, N = 2. Bars depict mean with sd, P‐values were determined by multiple t tests
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