Theranostics 2021; 11(8):3781-3795. doi:10.7150/thno.49288

Research Paper

Granzyme A inhibition reduces inflammation and increases survival during abdominal sepsis

Marcela Garzón-Tituaña1, José L Sierra-Monzón1,6, Laura Comas2, Llipsy Santiago1, Tatiana Khaliulina-Ushakova1,6, Iratxe Uranga-Murillo1, Ariel Ramirez-Labrada1, Elena Tapia7, Elena Morte-Romea1,6, Sonia Algarate1,5,6, Ludovic Couty8, Eric Camerer8, Phillip I Bird9, Cristina Seral1,5,6, Pilar Luque1,6, José R Paño-Pardo1,6, Eva M Galvez2, Julián Pardo1,3,4,5✉*, Maykel Arias2✉*

1. Fundación Instituto de Investigación Sanitaria Aragón (IIS Aragón), Biomedical Research Centre of Aragón (CIBA), 50009, Zaragoza, Spain.
2. Instituto de Carboquímica ICB-CSIC, 50018, Zaragoza, Spain.
3. Aragon I+D Foundation (ARAID), 50018, Zaragoza, Spain.
4. Nanoscience Institute of Aragon (INA), University of Zaragoza, 50018, Zaragoza, Spain.
5. Department of Biochemistry and Molecular and Cell Biology and Department of Microbiology, Preventive Medicine and Public Health, University of Zaragoza, 50009, Zaragoza, Spain.
6. Hospital Clínico Universitario Lozano Blesa, 50009, Zaragoza, Spain.
7. Animal Unit, University of Zaragoza, 50009, Zaragoza, Spain.
8. INSERM U970, Paris Cardiovascular Research Centre, Université de Paris, 75015, Paris, France.
9. Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, 3800, Clayton VIC, Australia.
* These authors share corresponding and senior authorship.

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Citation:
Garzón-Tituaña M, Sierra-Monzón JL, Comas L, Santiago L, Khaliulina-Ushakova T, Uranga-Murillo I, Ramirez-Labrada A, Tapia E, Morte-Romea E, Algarate S, Couty L, Camerer E, Bird PI, Seral C, Luque P, Paño-Pardo JR, Galvez EM, Pardo J, Arias M. Granzyme A inhibition reduces inflammation and increases survival during abdominal sepsis. Theranostics 2021; 11(8):3781-3795. doi:10.7150/thno.49288. Available from https://www.thno.org/v11p3781.htm

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Abstract

Aims: Peritonitis is one of the most common causes of sepsis, a serious syndrome characterized by a dysregulated systemic inflammatory response. Recent evidence suggests that Granzyme A (GzmA), a serine protease mainly expressed by NK and T cells, could act as a proinflammatory mediator and could play an important role in the pathogenesis of sepsis. This work aims to analyze the role and the therapeutic potential of GzmA in the pathogenesis of peritoneal sepsis.

Methods: The level of extracellular GzmA as well as GzmA activity were analyzed in serum from healthy volunteers and patients with confirmed peritonitis and were correlated with the Sequential Organ Failure Assessment (SOFA) score. Peritonitis was induced in C57Bl/6 (WT) and GzmA-/- mice by cecal ligation and puncture (CLP). Mice were treated intraperitoneally with antibiotics alone or in combination serpinb6b, a specific GzmA inhibitor, for 5 days. Mouse survival was monitored during 14 days, levels of some proinflammatory cytokines were measured in serum and bacterial load and diversity was analyzed in blood and spleen at different times.

Results: Clinically, elevated GzmA was observed in serum from patients with abdominal sepsis suggesting that GzmA plays an important role in this pathology. In the CLP model GzmA deficient mice, or WT mice treated with an extracellular GzmA inhibitor, showed increased survival, which correlated with a reduction in proinflammatory markers in both serum and peritoneal lavage fluid. GzmA deficiency did not influence bacterial load in blood and spleen and GzmA did not affect bacterial replication in macrophages in vitro, indicating that GzmA has no role in bacterial control. Analysis of GzmA in lymphoid cells following CLP showed that it was mainly expressed by NK cells. Mechanistically, we found that extracellular active GzmA acts as a proinflammatory mediator in macrophages by inducing the TLR4-dependent expression of IL-6 and TNFα.

Conclusions: Our findings implicate GzmA as a key regulator of the inflammatory response during abdominal sepsis and provide solid evidences about its therapeutic potential for the treatment of this severe pathology.

Keywords: Granzyme A, inflammation, peritonitis, sepsis, cecal ligation and puncture.