Determination of the molecular and cellular mechanisms related to the immune response associated with the susceptibility of isogenic mice to sepsis

Author(s)

Guilherme Cesar Martelossi Cebinelli

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2023-04-14

Examining board members

Fernando de Queiroz Cunha; Marcelo Torres Bozza; Diego Luís Costa; Reinaldo Salomão

• Advisor: Fernando de Queiroz Cunha

Abstract

Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. There is estimate an annual occurrence of 48.9 million cases of sepsis causing potentially 11 million deaths. Despite the attempt to implement immunotherapies modulating important molecules in the pathogenesis of sepsis, no approach showed efficacy in the clinics. This suggests that the pathogenesis of sepsis is not completely understood and that new methodologies may contribute to the identification of new therapeutic targets. In this context, the present work aimed to identify new therapeutic targets for sepsis analysis the molecular and cellular mechanisms related to the immune response responsible for the different outcomes of mice submitted to sepsis. Initially, by inducing the cecal ligation and puncture sepsis (CLP) in mice genetically equal and having the same grown-up environment, we observe different responses to sepsis in which half of the mice survive and the other half die from sepsis. Although we did not observe differences in the plasma concentrations of cytokines after 6 hours of sepsis induction between surviving and non-surviving mice, at 12 h, 24 h and 48 h, mice that survived sepsis showed reduced concentrations of these cytokines, while in non-survived mice these concentrations remained higher. Unsurprisingly, nonsurviving mice also showed increased plasma concentrations of biomarkers of liver, kidney and heart damage and bacteremia. Furthermore, these mice showed increased concentrations of chemokines and cytokines in the lungs, kidneys, heart, liver and also in the primary focus of infection (peritoneal cavity) 24h after CLP. Further, we isolated leukocytes from survived and non-survived mice after 12 h of sepsis induction and performed single cell RNA sequencing analysis. The evaluation of the data obtained by dimensional reduction revealed several populations of leukocytes, and we observed an increase in the frequency of immature neutrophils in non-survived mice. Furthermore, we observed that mature neutrophils from non-survived mice showed increased production of pro-inflammatory mediators and expression of Hif1a and Cd274, the gene that encodes the PD-L1 protein. In the literature it is described that these molecules are associated with increased neutrophil survival and increased production of neutrophil extracellular traps, which cause organ damage. Furthermore, it is described that interferon gamma (IFN-gamma) is able to induce PD-L1 expression in neutrophils and lead to increased promotion of lung injury in a model of endotoxemia by LPS. In this sense, we demonstrated that INF-gamma induced the expression of Hif1a and PD-L1 in neutrophils, and that Ifng-deficient mice are more resistant to sepsis and showed less induction of PD-L1 in neutrophils. In conclusion, we observed that isogenic mice grown under the same environmental conditions interestingly show different susceptibility to CLP sepsis. In this context, non-survived mice show maintenance of high systemic inflammation and increased tissue damage. Furthermore, neutrophils from non-survived mice show increased expression of PD-L1 and Hif1a, which can be promoted by IFN-gamma signaling. Thus, the negative modulation of IFN-gamma, PD-L1 and Hif1a may be potential therapeutic targets for sepsis. (AU)

• FAPESP's process: 19/15070-0 - Determination of molecular and cellular mechanisms associated with Sepsis outcome using single cell RNA sequencing
• Grantee: Guilherme Cesar Martelossi Cebinelli
• Support Opportunities: Scholarships in Brazil - Doctorate