Granzyme A Produced by γ9δ2 T Cells Activates ER Stress Responses and ATP Production, and Protects Against Intracellular Mycobacterial Replication Independent of Enzymatic Activity

Mycobacterium tuberculosis (Mtb), the pathological agent that causes tuberculosis (TB) is the number one infectious killer worldwide with one fourth of the world’s population currently infected. Data indicate that γ9δ2 T cells secrete Granzyme A (GzmA) in the extracellular space triggering the infected monocyte to inhibit growth of intracellular mycobacteria. Accordingly, deletion of GZMA from γ9δ2 T cells reverses their inhibitory capacity. Through mechanistic studies, GzmA’s action was investigated in monocytes from human PBMCs. The use of recombinant human GzmA expressed in a mammalian system induced inhibition of intracellular mycobacteria to the same degree as previous human native protein findings. Our data indicate that: 1) GzmA is internalized within mycobacteria-infected cells, suggesting that GzmA uptake could prevent infection and 2) that the active site is not required to inhibit intracellular replication. Global proteomic analysis demonstrated that the ER stress response and ATP producing proteins were upregulated after GzmA treatment, and these proteins abundancies were confirmed by examining their expression in an independent set of patient samples. Our data suggest that immunotherapeutic host interventions of these pathways may contribute to better control of the current TB epidemic.

An inconvenient association between granzyme A and Nicotinamide Nucleotide Transhydrogenase

Granzyme A (GzmA) is a serine protease secreted by cytotoxic lymphocytes, with GzmA-/- mouse studies informing our understanding of GzmAs physiological function. We show herein that GzmA-/- mice have a mixed C57BL/6J and C57BL/6N 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, rather than loss of GzmA, was responsible for this phenotype by constraining lymphocyte infiltration. A new CRISPR active site mutant C57BL/6J GzmAS211A mouse provided the first insights into GzmAs 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 background. The Nnt issue has clearly complicated our understanding of GzmA and may similarly have influenced studies across a broad range of fields.

Heparin-binding protein as a novel biomarker for sepsis-related acute kidney injury

Background Sepsis-related acute kidney injury (AKI) is associated with high morbidity and mortality among patients. Underlying pathomechanisms include capillary leakage and fluid loss into the interstitial tissue and constant exposure to pathogens results in activation of inflammatory cascades, organ dysfunction and subsequently organ damage. Methods To identify novel factors that trigger sepsis-related acute kidney injury, plasma levels of Granzyme A, as representative of a lymphocyte-derived protease, and heparin-binding protein as indicator for neutrophil-derived mediators, were investigated retrospectively in 60 sepsis patients. Results While no association was found between plasma levels of lymphocyte-derived Granzyme A and the incidence of sepsis-related AKI, sepsis patients with AKI had significantly higher plasma levels of heparin-binding protein compared to those without AKI. This applies both to heparin-binding protein peak values (43.30 ± 23.34 vs. 30.25 ± 15.63 pg/mL; p = 0.005) as well as mean values (27.93 ± 14.39 vs. 22.02 ± 7.65 pg/mL; p = 0.021). Furthermore, a heparin-binding protein cut-off value of 23.89 pg/mL was established for AKI diagnosis. Conclusion This study identifies the neutrophil-derived heparin-binding protein as a valuable new biomarker for AKI in sepsis. Beyond the diagnostic perspective, this offers prospect for further research on pathogenesis of AKI and novel therapeutic approaches.

Granzyme A inhibition reduces inflammation and increases survival during abdominal sepsis

ABSTRACTSepsis is a serious syndrome characterised by a dysregulated systemic inflammatory response. Here we have analysed the role and the therapeutic potential of Granzyme A (GzmA) in the pathogenesis of peritoneal sepsis using the Cecal Ligation and Puncture (CLP) polymicrobial sepsis model and samples from humans undergoing abdominal sepsis.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 indicating that GzmA has no role in bacterial control. 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.Our findings implicate GzmA as a key regulator of the inflammatory response during abdominal sepsis, and suggest that it could be targeted for treatment of this severe pathology.