Widespread discrepancy in Nnt genotypes and genetic backgrounds complicates granzyme A and other knockout mouse studies

Abstract

Granzyme A (GZMA) is a serine protease secreted by cytotoxic lymphocytes, with Gzma-/- mouse studies having informed our understanding of GZMA’s physiological function. We show herein that Gzma-/- mice have a mixed C57BL/6J and C57BL/6N genetic background and retain the full-length nicotinamide nucleotide transhydrogenase (Nnt) gene, whereas Nnt is truncated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated in Gzma-/- mice; however, the presence of Nnt and the C57BL/6N background, rather than loss of GZMA expression, was responsible for this phenotype. A new CRISPR active site mutant C57BL/6J GzmaS211A mouse provided the first insights into GZMA’s bioactivity free of background issues, with circulating proteolytically active GZMA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that ≈27% of Run Accessions and ≈38% of BioProjects listing C57BL/6J as the mouse strain had Nnt sequencing reads inconsistent with a C57BL/6J genetic background. Nnt and C57BL/6N background issues have clearly complicated our understanding of GZMA and may similarly have influenced studies across a broad range of fields.

Mouse models of COVID-19 recapitulate inflammatory pathways rather than gene expression

“…The genetic background can thus influence SARS-CoV-2 induced inflammatory immunopathology in the K18-hACE2 model (Table 2); an observation consistent with a previous report that showed such a mixed background can also significantly affect an arthritic inflammatory immunopathology (52). The Nnt gene may, at least in part, be responsible as NNT controls mitochondrial reactive oxygen species via the glutathione and thioredoxin pathways, and thus generally exerts an anti-inflammatory influence (52). No significant differences in tissues titres were observed (Figure 5E), consistent with previous studies which also showed no detectable influence of such a mixed background on antiviral activity against an alphavirus (52).…”

“…Weight loss, disease scores and survival were all significantly worse in K18-hACE2 6J mice than in the K18-hACE2 6N/6J mice (Figure 5B-D). The genetic background can thus influence SARS-CoV-2 induced inflammatory immunopathology in the K18-hACE2 model (Table 2); an observation consistent with a previous report that showed such a mixed background can also significantly affect an arthritic inflammatory immunopathology (52). The Nnt gene may, at least in part, be responsible as NNT controls mitochondrial reactive oxygen species via the glutathione and thioredoxin pathways, and thus generally exerts an anti-inflammatory influence (52).…”

“…The Nnt gene may, at least in part, be responsible as NNT controls mitochondrial reactive oxygen species via the glutathione and thioredoxin pathways, and thus generally exerts an anti-inflammatory influence (52). No significant differences in tissues titres were observed (Figure 5E), consistent with previous studies which also showed no detectable influence of such a mixed background on antiviral activity against an alphavirus (52).…”

“…The K18-hACE2 founder line was created on a mixed C57BL/6J x SJL/J background (51). Perhaps under-appreciated is that C57BL/6J (6J) mice contain a unique loss-of-function deletion of exons 5 to 9 of the Nicotinamide nucleotide transhydrogenase ( Nnt gene), whereas most mouse strains (including SJL/J mice) encode a full length Nnt gene (52). An exact-match k-mer method targeting exon 2 and 9 of the Nnt gene was used to interrogate the RNA-Seq data from the Suhrbier and Winkler studies.…”

“…Mouse RNA-Seq data were interrogated for the presence of exon two and nine of the nicotinamide nucleotide transhydrogenase ( Nnt ) gene as described (52) using Repair and BBduk from the BBmap package v38.90. Reads containing at least one 31-mer exactly matching either exon were counted as belonging to that exon.…”