RNA-Seq analysis of chikungunya virus infection and identification of granzyme A as a major promoter of arthritic inflammation

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Abstract
Chikungunya virus (CHIKV) is an arthritogenic alphavirus causing epidemics of acute and chronic arthritic disease. Herein we describe a comprehensive RNA-Seq analysis of feet and lymph nodes at peak viraemia (day 2 post infection), acute arthritis (day 7) and chronic disease (day 30) in the CHIKV adult wild-type mouse model. Genes previously shown to be up-regulated in CHIKV patients were also up-regulated in the mouse model. CHIKV sequence information was also obtained with up to ≈8% of the reads mapping to the viral genome; however, no adaptive viral genome changes were apparent. Although day 2, 7 and 30 represent distinct stages of infection and disease, there was a pronounced overlap in up-regulated host genes and pathways. Type I interferon response genes (IRGs) represented up to ≈50% of up-regulated genes, even after loss of type I interferon induction on days 7 and 30. Bioinformatic analyses suggested a number of interferon response factors were primarily responsible for maintaining type I IRG induction. A group of genes prominent in the RNA-Seq analysis and hitherto unexplored in viral arthropathies were granzymes A, B and K. Granzyme A-/- and to a lesser extent granzyme K-/-, but not granzyme B-/-, mice showed a pronounced reduction in foot swelling and arthritis, with analysis of granzyme A-/- mice showing no reductions in viral loads but reduced NK and T cell infiltrates post CHIKV infection. Treatment with Serpinb6b, a granzyme A inhibitor, also reduced arthritic inflammation in wild-type mice. In non-human primates circulating granzyme A levels were elevated after CHIKV infection, with the increase correlating with viral load. Elevated granzyme A levels were also seen in a small cohort of human CHIKV patients. Taken together these results suggest granzyme A is an important driver of arthritic inflammation and a potential target for therapy. Trial Registration: ClinicalTrials.gov NCT00281294 The largest chikungunya virus (CHIKV) epidemic ever recorded began in 2004 in Africa and spread across Asia reaching Europe and recently the Americas, with millions of cases reported. We undertook a detailed analysis of the mRNA expression profile during acute and chronic arthritis in an adult wild-type mouse model of CHIKV infection and disease. Gene induction profiles showed a high concordance with published human data, providing some validation of the mouse model. The host response was overwhelmingly dominated by type I interferon response genes, even after type I interferon induction was lost. The analysis also provided information on CHIKV RNA, with no adaptive viral genome changes identified. An important goal of the analysis was to identify new players in arthritic inflammation. Granzyme A was prominent in the RNA-Seq data and granzyme A deficient mice showed reduced arthritis, with no effects on viral loads. Arthritic disease could also be ameliorated in wild-type mice with a granzyme A inhibitor. Elevated circulating granzyme A levels were seen in non-human primates infected with CHIKV and in human CHIKV patients. Granzyme A thus emerges to be a major driver of CHIKV-mediated arthritic inflammation and a potential target for anti-inflammatory interventions.
Funding Information
  • National Health and Medical Research Council (AU) (APP613622)
  • University of Queensland (Australian Postgraduate Award)
  • Queensland Government (Advance Queensland Research Fellowship)
  • National Health and Medical Research Council (AU) (Principal Research Fellow)
  • Queensland Tropical Health Alliance (BSL3 equipment)
  • Programme d'Investissements d'Avenir' (ANR-11-INBS-0008; Intergrated Chikungunya Research project 261202)

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