The role of SCFAs and receptor FFAR2 in neutrophils death induced by Aggregatibacter actinomycetemcomitans

Author(s)

Erica Moraes Sernaglia

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2017-09-06

Examining board members

Marco Aurélio Ramirez Vinolo; Marcelo Bispo de Jesus; Ana Paula Duarte de Souza

• Advisor: Marco Aurélio Ramirez Vinolo

Abstract

Neutrophils are the first cells to migrate during the inflammatory process, acting on the internalization and killing of invading microorganisms. They present a short half-life and once in apoptosis, they are engulfed and removed from the tissues by macrophages through a process called efferocytosis. Short chain fatty acids (SCFAs) are products of bacterial metabolism which modulate the migration, activation and neutrophil effector function. However, little is known about their effect on the death of these cells. In this work, we have analyzed the pathways involved in the action of SCFAs on the death of neutrophils and the implication of this effect in the process of efferocytosis and in the response profile to Aggregatibacter actinomycetemcomitans (Aa), a bacterium that is associated with periodontitis, a chronic inflammatory condition of the tooth supporting tissues in which there is an increase in the local concentration of SCFAs. Experiments were performed with neutrophils isolated from mice incubated with Aa and SCFAs (acetate and butyrate) in vitro. Butyrate induces apoptosis but not neutrophil necrosis. This effect was observed in the presence and absence of the Aa bacterium and involved, in part, the activation of the receptor FFAR2. Acetate, at the concentrations and time points tested, showed no effect on the viability/death of neutrophils. Subsequently, it was verified that butyrate increased the activity of caspases 8 and 9 and that its pro-apoptotic effect was blocked in the presence of caspase inhibitor. Using FFAR2-/- neutrophils, the activation of caspase 8 was maintained. The modulatory effects of butyrate on cytokine production were maintained even in the presence of the caspase inhibitor, indicating that they are independent of the action of these AGCCs on neutrophil apoptosis. When evaluating the neutrophil efferocytosis pre-treated with SCFAs by macrophages in vivo and in vitro, no significant change was observed. However, the production of cytokines by macrophages after efferocytosis was distinct (lower production of TNF-alpha). The addition of neutrophils did not modify the phagocytic capacity of the macrophages. In the model of infection in vivo, a greater number of bacteria were observed in the subcutaneous chambers in which FFAR2+/+ -neutrophils treated with butyrate were inoculated, as well as a reduction of TNF-alpha concentration and an increase in CXCL-1 production. When using neutrophils from FFAR2-/- animals previously incubated with butyrate, the number of viable bacteria was similar between conditions. However, the effect on the production of TNF-alpha(reduction) in the chambers was maintained. The results presented here suggest that the SCFA butyrate induces neutrophil apoptosis, which reduces microbicidal activity in vivo. Other effects on both neutrophils and macrophages (in vitro and in vivo), including their action on the production of inflammatory mediators, are independent of the pro-apoptotic action of butyrate on neutrophils. Thus, we conclude that SCFA butyrate alters the death of neutrophils and thereby modifies the initial response to bacteria such as Aa, which may be relevant for the development of periodontitis (AU)

• FAPESP's process: 14/22909-3 - Role of the short chain fatty acids and their receptor (GPR43) in the neutrophil death induced by anaerobic bacteria
• Grantee: Erica Moraes Sernaglia
• Support Opportunities: Scholarships in Brazil - Master