Understanding the mechanism and functional effects of caspase-11 regulated nitric oxide production during inflammation and cancer

Abstract

Inflammatory caspases are a group of cysteine aspartic proteinases essential for the inflammatory process, but when dysregulated are implicated in inflammatory diseases and cancer-associated chronic inflammatory events. Human caspases-4 and -5 (and their murine orthologue caspase-11) induce a form of inflammatory cell death, termed pyroptosis, resulting in the secretion of potent pro-inflammatory cytokines, IL-1? and IL-1?. Caspase-11 has also been reported to regulate STAT1 activation, a key transcriptional activator of inducible nitric oxide synthase (iNOS) which is central to macrophage polarisation. iNOS mediated nitric oxide (NO) production alters macrophage metabolism via modulation of mitochondrial morphology and Kreb?s cycle impairment. This study shows that caspase-11 functions to alter mitochondrial metabolism via regulation of NO production. Understanding the molecular mechanisms underpinning immunometabolism are important for investigating its dysfunction during disease. Investigation of the potential mechanism regulating caspase-11 mediated NO production revealed that caspase-11 inhibits PINK1-induced mitophagy during inflammatory responses, identifying a potential link between caspase-11 mediated suppression of mitophagy and type I IFN driven NO production. The impact of caspase-11 mediated NO production during macrophage metabolic reprogramming in response to M1 stimuli was also examined. A role for caspase-11 during macrophage polarization was identified, regulating iNOS expression and NO production, thereby driving metabolic rewiring towards glycolytic commitment in inflammatory macrophages. Metabolic analysis and measurement of metabolites revealed that caspase-11 driven NO production is required for glycolytic commitment during inflammatory responses, as oxidative phosphorylation (OXPHOS) remains intact in inflammatory M1 polarised Casp11-/- BMDM. Breast cancer is a commonly diagnosed cancer in females and the second leading cause of cancer related death among women. It is a highly heterogeneous disease with distinct molecular patterns and treatment availabilities. Breast cancer development is associated with increased leukocyte infiltration and an inflammatory microenvironment, conducive for tumour development. Breast cancers are classified into 4 molecular subtypes, of which basal-like breast cancer is the most aggressive and difficult to treat, with increased risk of recurrence and metastatic potential. Current investigations of its tumour microenvironment (TME) are underway to uncover targetable features. Immunotherapy is emerging as a viable therapeutic alternative for TNBC patients, with macrophage re-education becoming a targetable component of cancer therapeutics. Examination of inflammatory caspase expression levels between tumour and normal adjacent breast tissue revealed that inflammatory caspase-5 was more highly expressed in tumours. Further immunohistochemical analysis of a breast cancer tumour microarray revealed significantly higher caspase-5 expression in the stroma of estrogen receptor negative (ER-) tissue. Pyroptosis is closely associated with tumour development and metastasis, however it has both pro-tumour and anti-tumour roles. This study suggests that caspase-5 mediated pyroptosis in tumour associated macrophages (TAMs) is important during breast cancer initiation, which may support immune cell infiltration and activity. Secreted factors from an ER- cell line were shown to induce caspase-11 dependent IL-1? secretion from primary murine macrophages. Dysregulated pyroptosis impairs tumour clearance as disease advances. As CASP5 expression decreases with tumour stage, during advanced disease pyroptosis may become negatively regulated to aid tumour development. COX-2 is associated with poor overall survival in TNBC patients, and increased expression with tumour stage inversely correlates with CASP5. The COX-2 product PGE2 inhibits caspase-11 mediated pyroptosis, therefore we hypothesise that once caspase-5 contributes to TNBC initiation, PGE2 inhibits caspase-11 mediated pyroptosis to escape immunosurveillance.