Need for standardization of Influenza A virus-induced cell death in vivo to improve consistency of inter-laboratory research findings

Abstract

The involvement of necroptosis in the control of influenza A virus (IAV) infection has been reported in multiple studies. Downstream of the nucleic acid sensor ZBP1, RIPK3 kinase activity is critically involved in the induction of necroptotic cell death by phosphorylating MLKL, while RIPK3 as a scaffold can induce apoptosis. Paradoxically, RIPK3-deficiency of mice may result in increased or decreased susceptibility to IAV infection. Here, we critically review the published reports on the involvement of RIPK3 in IAV infection susceptibility and try to identify differences in experimental settings that could explain seemingly conflicting outcomes. Analysis of the experimental reports revealed differences in the IAV challenge dose, the IAV inoculum preparation, IAV titer assessment, as well as the route of inoculation between studies. Furthermore, differences were noticed in the inclusion of littermate controls, which show high variance in viral sensitivity. Our evaluation argues for a standardized setup for IAV infection experiments including the preparation of the IAV virus, the use of different IAV infectious doses description and the proper experimental genetic controls of the mouse strains to increase inter-laboratory consistency in this field. Workflow for IAV infection studies in vivo: Viral preparation and titer assessment should be as standardized as possible with the use of a universal repository (such as BEI resources). Infection studies in genetically modified mice and littermate controls should include dose-response experimentation, following a defined infection route and inoculation volume. Data are generated by consistent analysis methods.

Workflow for IAV infection studies in vivo: Viral preparation and titer assessment should be as standardized as possible with the use of a universal repository (such as BEI resources). Infection studies in genetically modified mice and littermate controls should include dose-response experimentation, following a defined infection route and inoculation volume. Data are generated by consistent analysis methods.

Fig. 1. Survival of genetically modified mice compared to different littermate controls (pooled) at medium IAV challenge dose and comparison of survival different littermates challenged at different IAV doses.

Pooled littermate controls (Ripk3^+/+, Ripk3 KD-KI^+/+, Mlkl^+/+, and Ripk3^+/+Fadd ^+/+ mice) compared with the Ripk3^−/−, Ripk3 KD-KI^K51A/K51A, Mlkl^−/−, and Ripk3^−/−Fadd ^−/−. A Mice were challenged with different IAV doses (here 0.2x LD₅₀/16 pfu). Survival curves were plotted for indicated groups and evaluated statistically according to Kaplan–Meier (GraphPad Prism 8). ns, not significant; ****p < 0.0001 [16]. Survival of littermate controls (B−D): Ripk3^+/+, Ripk3 KD-KI^+/+, Mlkl^+/+, and Ripk3^+/+Fadd ^+/+ mice at different IAV challenge doses Mice were challenged with different IAV doses (0.1 × LD₅₀/8 pfu; 0.2 × LD₅₀/16 pfu; 0.5 × LD₅₀/40 pfu). Survival curves were plotted for indicated groups [16]. Mice: Ripk3^−/− were kindly provided by Dr. Vishva Dixit (Genentech, San Francisco) [44], Mlkl^−/− by Dr. Alexander Warren and Dr. James Murphy [45] and Ripk3 KD-KI^K51A/K51A mice by Dr. John Bertin by GlaxoSmithKline [20]. The Ripk3^−/− animals were congenic to the C57BL/6 N background, while all other strains were of the C57BL/6 J background, and were therefore compared with the appropriate littermate controls. Ripk3^−/− mice were housed in individually ventilated cages in a conventional animal house. The other mice were bred and housed in the SPF facility in individually ventilated cages. Three weeks prior to the experiment all mice were transferred to the conventional animal house and allowed to go through a quarantine and accommodation period of minimum 3 weeks before the infection experiment. Littermate controls of Ripk3^−/−, Mlkl^−/−, Ripk3^−/−Fadd^−/− and Ripk3 KD-KI^K51A/K51A were used in each experiment. In all experiments, 10–15- week-old mice were used. All animal experiments were done under conditions specified by law (European Directive and Belgian Royal Decree of November 14, 1993) and approved by the Institutional Ethics Committee on Experimental Animals. Viral infection: Age-matched mice were anesthetized with a cocktail of 87,5 mg/kg ketamine and 12,5 mg/kg xylazine intraperitoneally and infected intranasally with 50 μl/20 g phosphate-buffered saline (PBS) containing different doses of influenza virus A/PR/8/3443, as described in the legends. The plaque-forming units (pfu) were determined by plaque assay on Madin-Darby Canine Kidney (MDCK) cells [31]. The LD₅₀ of the viral batch was determined on BALB/c mice and 1x LD₅₀ represented 80 pfu, as determined in the lab of Prof. Saelens. Although the LD₅₀ is not referring to 50% of death in the mice that were used in this study, the nomenclature is used together with the pfu to have a supplementary information regarding the power of the virus in vivo. This terminology is often used in the papers cited here. Age- and sex-matched mice were challenged with 0.05x LD₅₀ (4 pfu), 0.1x LD₅₀ (8 pfu), 0.2x LD₅₀ (16 pfu) or 0.5x LD₅₀ (40 pfu) and monitored for survival and weight loss over a period of at least 18 days. We used the following 4 scores of clinical symptoms: 0 = no visible signs of disease; 1 = slight ruffling of fur; 2 = ruffled fur, reduced mobility; 3 = ruffled fur, reduced mobility, rapid breathing; 4 = ruffled fur, minimal mobility, huddled appearance, rapid and/or labored breathing indicative of pneumonia and body temperature below 32 °C. For the combination of body weight loss by 30% and a clinical score 4 the mice were considered moribund and euthanized by CO₂ asphyxiation or cervical dislocation (EC2016–17). The lethality includes an ethical endpoint during which euthanasia was performed. This ethical endpoint is a combination of 30% weight loss and a clinical score of 4. Survival curves: All the survival data were plotted using the Kaplan–Meier survival analysis with the software Prism 10.1.0.