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

Daniel J Rawle,Thuy T Le,Eri Nakayama,Cameron Bishop,Kexin Yan,Phillip I Bird,Andreas Suhrbier,Troy Dumenil

doi:10.7554/elife.70207

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.

Highlights

Granzyme A (GZMA) is a granule trypsin-like serine protease secreted by cytotoxic lymphocytes such as NK cells (Fehniger et al, 2007; Wu et al, 2019), NKT cells (Gordy et al, 2011), and CD8+ cytotoxic T lymphocytes (Suhrbier et al, 1991)
We reported previously that the inflammatory arthritis induced by chikungunya virus (CHIKV) infection was significantly lower in Gzma-/- mice than in C57BL/6J (6J) mice (Wilson et al, 2017); an observation we confirm (Figure 1a)
When infected with CHIKV, viral titers in feet were not significantly different for GzmaS211A and 6J mice (Figure 1—figure supplement 1f), arguing that enzymically active GZMA has no significant antiviral activity against CHIKV. This is consistent with our previous study using Gzma-/- mice that concluded that GZMA has no important antiviral function (Wilson et al, 2017)

Summary

Introduction

Granzyme A (GZMA) is a granule trypsin-like serine protease (trypase) secreted by cytotoxic lymphocytes such as NK cells (Fehniger et al, 2007; Wu et al, 2019), NKT cells (Gordy et al, 2011), and CD8+ cytotoxic T lymphocytes (Suhrbier et al, 1991). In a range of settings, GZMA has been associated with the promotion of inflammation, providing an additional or alternative view of its physiological role, consensus on mechanisms has remained elusive (Metkar et al, 2008; Park et al, 2020; Santiago et al, 2020; Santiago et al, 2017; Schanoski et al, 2019; Shimizu et al, 2019; van Daalen et al, 2020; Wensink et al, 2015; Wilson et al, 2017), with a number of potential intracellular and extracellular targets for GZMA reported These include pro-IL-1β (Hildebrand et al, 2014), SET complex proteins (Mandrup-Poulsen, 2017; Mollah et al, 2017), gasdermin B (Zhou et al, 2020), mitochondrial complex I protein NDUFS3 (Martinvalet et al, 2008), protease activated receptors (Hansen et al, 2005; Sower et al, 1996; Suidan et al, 1994; Suidan et al, 1996), TLR2/4 (van Eck et al, 2017), and TLR9 (Shimizu et al, 2019). GZMA’s bioactivity has generally (Plasman et al, 2014; Schanoski et al, 2019; Zhou et al, 2020), but not always (Shimizu et al, 2019; van Eck et al, 2017), been associated with GZMA’s protease activity, with circulating GZMA in humans shown to be proteolytically active (Spaeny-Dekking et al, 1998)

Results

Discussion

Conclusion