Viral dosing of influenza A infection reveals involvement of RIPK3 and FADD, but not MLKL

Abstract

RIPK3 was reported to play an important role in the protection against influenza A virus (IAV) in vivo. Here we show that the requirement of RIPK3 for protection against IAV infection in vivo is only apparent within a limited dose range of IAV challenge. We found that this protective outcome is independent from RIPK3 kinase activity and from MLKL. This shows that platform function of RIPK3 rather than its kinase activity is required for protection, suggesting that a RIPK3 function independent of necroptosis is implicated. In line with this finding, we show that FADD-dependent apoptosis has a crucial additional effect in protection against IAV infection. Altogether, we show that RIPK3 contributes to protection against IAV in a narrow challenge dose range by a mechanism that is independent of its kinase activity and its capacity to induce necroptosis.

Fig. 1. RIPK3 is partially required for protection at medium IAV dose in vivo.

Survival analysis and body weight loss of age-matched Ripk3^−/− and Ripk3^+/+ mice infected intranasally with IAV is shown at very low dose: 0.05x LD₅₀/4 pfu; (a), low dose: 0.1x LD₅₀/8 pfu p value = 0.3340 (b), medium dose: 0.2x LD₅₀/16 pfu p value= 0.0218 (c) and high dose: 0.5x LD₅₀/40 pfu (d). Data were pooled from 2 (panel A and B) or 3 (c, d) independent experiments. Bodyweight curves are shown as mean ± SD. Survival curves were plotted for indicated groups and evaluated statistically according to Kaplan–Meier. A log-rank test verified significant differences between Ripk3^+/+ and Ripk3^−/− mice (GraphPad Prism 7). *p < 0.05.

Fig. 2. MLKL is not required for partial protection against IAV infection at different viral doses.

Survival analysis and bodyweight loss of age-matched Mlkl^−/− and Mlkl^+/+ infected intranasally with IAV is shown at each infection: low dose: 0.1x LD₅₀/8 pfu (a), medium dose: 0.2x LD₅₀/16 pfu (b) and high dose: 0.5x LD₅₀/40 pfu (c). Data were pooled from 2 (a, c) or 3 (b) independent experiments. Bodyweight curves are shown as mean ± SD. Survival curves were plotted for indicated groups and evaluated statistically according to Kaplan–Meier (GraphPad Prism 7). NS not significant.

Fig. 3. RIPK3 kinase activity is not required for partial protection against IAV infection at different viral doses.

Survival analysis and bodyweight loss of age-matched Ripk3 KD-KI^K51A/K51A and Ripk3 KD-KI^+/+ mice infected intranasally with IAV is shown at each infection dose: low dose: 0.1x LD₅₀/8 pfu, (a), medium dose: 0.2x LD₅₀/16 pfu, (b) and high dose: 0.5x LD₅₀/40 pfu (c). Data were pooled from 2 (panel A), 3 (c) or 5 (b) independent experiments. Bodyweight curves are shown as mean ± SD. Survival curves were plotted for indicated groups and evaluated statistically according to Kaplan–Meier (GraphPad Prism 7). NS not significant.

Fig. 4. FADD is required against low and medium IAV doses in vivo.

Survival analysis and bodyweight loss of age-matched Ripk3^−/−Fadd^−/− DKO and WT controls Ripk3^+/+ Fadd^+/+ as well as single Ripk3^−/− and Ripk3^+/+ mice infected intranasally with PR8 are shown: low dose: 0.1x LD₅₀/8 pfu (a), medium dose: 0.2x LD₅₀/16 pfu (b) and high dose: 0.5x LD₅₀/40 pfu (c). Data were pooled from 2 (a) or 3 independent experiments (b, c). Bodyweight curves are shown as mean ± SD. Survival curves were plotted for indicated groups and evaluated statistically according to Kaplan–Meier (GraphPad Prism 7). NS not significant; *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 5. Graphic summary: involvement of FADD and RIPK3 in protection at different doses of IAV challenge.

After low to medium IAV challenge dose in vivo, intracellular IAV activates FADD to drive apoptosis of infected cells and protects the host (main pathway involved in protection). RIPK3 is not essential to activate FADD-dependent apoptosis post IAV infection. The platform function of RIPK3 can associate with FADD and caspase-8 to drive apoptosis (to a lesser extent than the main pathway which is RIPK3-independent) or other cell death-independent mechanism for protection. The kinase active RIPK3 and downstream MLKL are not involved in the protection against IAV.