Fig 1: Summary of chikungunya virus (CHIKV) foot swelling and granzyme A (GZMA) bioactivity, and the poor concordance between the two.(a) Summary of foot swelling results. 6N and Gzma-/- mice have a 6N or mixed 6J/6N background and have an intact Nnt gene and show reduced CHIKV-induced foot swelling. MitoTEMPO treatment also reduced foot swelling. 6J, GzmaS211A, and 6N?Nnt8-12 mice are all missing exons 8–12 of Nnt and show increased foot swelling. (b) Summary of GZMA bioactivity. Using polyinosinic:polycytidylic acid (poly(I:C)) to induce high levels of GZMA secretion from NK cells, studies in GzmaS211A mice illustrated that proteolytically active circulating GZMA promotes certain immune-stimulating/pro-inflammatory responses (dominant transcription factor upstream regulators (USRs) are shown; Figure S11a). No clear consensus has emerged regarding the molecular target(s) of GZMA (??); two potential extracellular candidate targets are shown; protease activated receptors and pro-IL-1. (c) Low overlap between CHIKV and GZMA induced differentially expressed genes (DEGs). DEGs upregulated in feet by CHIKV infection of 6J mice on days 2 and 7 (a low stringency filter of q < 0.05 was applied to the all gene lists in Supplementary file 2f to provide these DEGs) were compared with the DEGs upregulated in feet by active GZMA in 6J mice 6 hr after poly(I:C) treatment (i.e., downregulated in GzmaS211A mice; Supplementary file 6b). Overlapping genes (n = 27 and 20) are listed in Supplementary file 6i. The mean peak levels of serum GZMA for each group are shown below the Venn diagrams. (d) Cytokine USRs for GZMA vs. CHIKV. A series of cytokine USRs were induced by proteolytically active GZMA (i.e., upregulated for 6J + polyIC vs. GzmaS211A + polyIC; Supplementary file 6g, column R). The same USRs were significantly more upregulated by CHIKV infection (Supplementary file 2i). Data for GZMA (orange) and CHIKV (yellow) are plotted with bubble size representing number of molecules in dataset.
Fig 2: Nnt deletion in 6J but not Gzma-/- mice.(a) Whole-genome sequencing (WGS) of Gzma-/- mice aligned to the 6J (MM10) reference genome build, illustrating the insertion site of the neomycin cassette into the Gzma gene to create the knockout. (b) As for (a) but showing the site of the Nnt deletion, with the additional 12 nucleotides present in the 6J genome. (c) WGS of Gzma-/- and 6J mice aligned to the C3H/HeJ reference genome, illustrating that the Nnt deletion present in 6J mice is absent in Gzma-/- mice. The deletion is in chromosome 13; position 120,695,141–120,711,874 (C3H/HeJ numbering). (d) Reads from RNA-Seq of chikungunya virus (CHIKV)-infected 6J and Gzma-/- mice aligned to the C3H/HeJ and 6J (MM10) reference genomes showing the Sashimi plot (Integrative Genomics Viewer) for the Nnt gene. (e) RT-PCR of testes using primers either side of exons 8–12 in the Nnt mRNA.
Fig 3: Polyinosinic:polycytidylic acid (poly(I:C)) injection into GzmaS211A, 6J mice, and Ifnar-/-.(a) GzmaS211A, 6J, and Ifnar-/- mice were injected i.v. with 250 µg of poly(I:C) in 150 µl of PBS, and serum samples were taken at the indicated times and assayed for GZMA concentration using a capture ELISA kit (6J n = 5–8, Ifnar-/- n = 5–6 and GzmaS211A n = 3 mice per time point). (b) GzmaS211A and 6J mice were injected i.v. with 250 µg of poly(I:C) and feet removed 6 hr later and analyzed by RNA-Seq. The differentially expressed genes (DEGs) (Supplementary file 6b) were analyzed by Cytoscape and Ingenuity Pathway Analysis (IPA). The full gene list (Supplementary file 6a) was analyzed using the Molecular Signature Database (MSigDB).
Fig 4: RNA-Seq of Gzma-/- + chikungunya virus (CHIKV) vs. 6J + CHIKV day 6 feet.(a) Selected Ingenuity Pathway Analysis (IPA) Diseases and Function annotations for the 469 downregulated differentially expressed genes (DEGs) for Gzma-/- + CHIKV vs. 6J + CHIKV (full set of annotations shown in Supplementary file 2c). (b) IPA cytokine upstream regulators (USRs) downregulated in CHIKV-infected feet of Gzma-/- mice (Gzma-/-+CHIKV vs. 6J + CHIKV; Supplementary file 2e) plotted by p-value and z-score. Black circles – minor USRs for Gzma-/- + CHIKV vs. 6J + CHIKV not identified for 6J + CHIKV vs. 6J mock (see also Figure 4e). (c) RNA-Seq identified 1557 DEGs upregulated in feet for 6J + CHIKV vs. 6J mock infection (Supplementary file 2g). RNA-Seq of Gzma-/- + CHIKV vs. 6J + CHIKV day 6 feet identified 469 downregulated DEGs in Gzma-/- mice associated with the reduced foot swelling (Supplementary file 2b). Only 19 of these DEGs were shared by these datasets. (d) Gene Set Enrichment Analysis (GSEA) of downregulated DEGs from Gzma-/- + CHIKV vs. 6J + CHIKV day 6 feet (Supplementary file 2b) vs. all genes (preranked by fold change) from feet 6J + CHIKV vs. 6J mock infection (Supplementary file 2f).
Fig 5: Chikungunya virus (CHIKV) infection in Gzma-/-, GzmaS211A, 6N, and 6J mice.(a) Percent increase in foot swelling for the indicated mouse strains. Data is from 2 to 4 independent experiments with 5–6 mice (10–12 feet) per group per experiment. From day 3 to day 10, feet from GzmaS211A and 6J mice were significantly more swollen than feet from Gzma-/- and 6N mice (Kolmogorov–Smirnov tests, p<0.002). (b) Viremia for the mice in (a) (6N n = 12, GzmaS211A n = 18, Gzma-/- n = 15, 6J n = 17).
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