Brucellosis is a zoonosis caused by bacteria of the genus Brucella of worldwide distribution. Great advance has been made in the control of brucellosis in recent years. However, in many regions of the world, Brucella infection in domestic animals still persists, leading to frequent transmission to the human population. In regions such as the Mediterranean countries of Europe, like Portugal, Italy and Greece, brucellosis is still considered an important human disease and it is often neglected (Corbel, 1997).

Innate immunity is an important arm of the immune system involved in the control of Brucella infection. In previous studies performed by our research group, we demonstrated that receptors and adaptor molecules such as TLR9, AIM2, MyD88, and STING are important components in the protective response against Brucella infection (Macedo et al., 2008; Gomes et al., 2016; Costa Franco et al., 2018, 2019). In addition, we and others have shown that pyroptosis triggered upon activation of inflammasomes, is also an important mechanism in restricting in vivo infection against Brucella abortus (Cerqueira et al., 2018; Lacey et al., 2018). In recent study, we showed that caspase-11/GSDMD-dependent pyroptosis process triggered by B. abortus contributed to the restriction of infection in vivo by assisting in the recruitment and activation of immune cells such as neutrophils, macrophages, and dendritic cells (Cerqueira et al., 2018).

Programmed cell death (PCD) is a intricate circuit that involves the cross-talk among different caspases, and their substrates. Caspase-3 and caspase-7 are considered executioner molecules triggering host cell apoptosis (Kim et al., 2005). Recent study demonstrated that Brucella inhibited the PCD in early stage of infection to allow bacterial replication in host cells and promoted apoptosis in the later stage during infection in macrophages (Zhang et al., 2022). In the literature, there are some controversies in the role of caspase-7 during bacterial infections. Caspase-7 has been implicated in resistance to L. pneumophila through the NLRC4 inflammasome (Akhter et al., 2009). In contrast, Gonçalves et al. (Goncalves et al., 2019) demonstrated that mice with a single deletion in caspase-7 are not fully susceptible to L. pneumophila. The Casp7^−/− did not phenocopy the susceptibility to L. pneumophila infection as observed in Nlrc4^−/− animals.

Caspase-8, early on classified as an apoptotic caspase, has lately been shown to have a role in several inflammatory processes. Caspase-8 is involved in the inflammasome pathway and can be activated by NLRP3, AIM2 and NLRC4 in macrophages (Man et al., 2013; Sagulenko et al., 2013) and NLRP3 inflammasome in dendritic cells (Antonopoulos et al., 2015). Caspase-8 can act by controlling NF-kB signaling, influencing the positive regulation of components of the inflammasome, such as the NLRP3 and pro-IL-1β (Weng et al., 2014). It can also activate the inflammasome pathway in response to C. albicans β-glucans (Ganesan et al., 2014). Interestingly, upon TLR or death receptor activation, active caspase-8 can cleave the IL-1β precursor into its bioactive fragment at the same site as caspase-1 (Shenderov et al., 2014), and can directly cleave GSDMD into its N-terminal fragment, triggering pyroptosis during Yersinia infection (Sarhan et al., 2018). In addition, once the inflammasome is activated, but pyroptosis is impaired, caspase-8 can act leading to a cell death program. This has been demonstrated in studies with intracellular bacteria such as L. pneumophila and S. Typhimurium in the absence of caspase-1 or GSDMD (Mascarenhas et al., 2017; Lee et al., 2018).

To the best of our knowledge, the role of caspase-7 and caspase-8 in Brucella infection has not been addressed so far. Therefore, in order to expand the understanding of the mechanisms involved in the innate immune response and inflammatory cell death, we investigated the participation of the caspase-7 and caspase-8 molecules during B. abortus infection.

Programmed cell death (PCD) can be activated in response to different stimuli (Nagata, 2018). Apoptosis, necroptosis and pyroptosis are three types of cell death that have major involvement in immune response and disease control (Schwarzer et al., 2020). Apoptosis helps in the destruction and removal of infected cells during bacterial infections (Speir et al., 2016). In the case of Brucella, some studies have already established that a virulent strain inhibits cell death in macrophages to allow bacterial replication (Chen et al., 2011). In contrast, in dendritic cells, astrocytes and T lymphocytes, the smooth strain induced apoptotic cell death (Garcia Samartino et al., 2010; Velasquez et al., 2012). Caspase-7, like caspase-3, is an executing caspase, both of which are activated by caspases-8 and -9 during death receptor-induced apoptosis, under certain conditions (Lamkanfi et al., 2002). Recently, some studies show that caspase-7 has a distinct role from caspase-3 during activation of apoptosis and also has a role in the inflammatory response against bacterial pathogens (Slee et al., 2001). Studies using Salmonella typhymurium infection of macrophages or cells stimulated with LPS and ATP, showed that caspase-7 activation was caspase-1-dependent. Additionally, Akhter et al. have observed in the absence of caspase-7 impaired ability of macrophages to restrict intracellular replication of Legionella pneumophila (Akhter et al., 2009). In contrast, Gonçalves et al. (Goncalves et al., 2019) demonstrated that lack of caspase-7 is not required to control L. pneumophila replication in vitro and in vivo. In another study, using an in vivo septic shock model, caspase-7-deficient mice were shown to be resistant to lethality induced by intraperitoneal injections of LPS (Lamkanfi et al., 2009). However, in our study caspase-7 does not appear to participate in the control of B.abortus infection. Inflammasome activation with cleavage of caspase-1 and secretion of IL-1β is not affected in the absence of caspase-7, as well as induction of cell death and in vivo susceptibility to infection. We have previously shown the participation of caspase-1, caspase-11 and GSDMD in controlling B. abortus (Cerqueira et al., 2018). Therefore, susceptibility to Brucella infection observed in Casp7/1/11^−/− and Casp7/Gsdmd^−/− animals is not due to lack of caspase-7 but rather the absence of caspase-1/11 and GSDMD.

Necroptosis is a programmed cell death pathway, with inflammatory features, that involves the kinases RIPK1 and RIPK3 and the pore-forming pseudokinase MLKL. When RIPK3 is phosphorylated, oligomerization of MLKL is initiated and subsequently inserts into the plasma membrane of the cell, leading to pore formation and cell rupture (Tummers and Green, 2017). Caspase-8 is involved in apoptosis and pyroptosis mechanisms of cell death, and when caspase-8 is inhibited RIPK1 interacts with RIPK3 leading to necroptosis (Pandian and Kanneganti, 2022). Pyroptosis is another type of inflammatory programmed cell death triggered by inflammasome activation, and for a long time, it was considered to be caspase-1-mediated in response to bacterial challenge. However, when caspase-11 was shown to detect intracellular LPS and also serve as a trigger to pyroptosis, the role of pyroptosis expanded widely (Kayagaki et al., 2011). Herein, we observed that in WT infected cells LDH release occurs, corroborating with data from our previous study where we showed that B. abortus infection triggers pyroptosis, and this phenomenon is GSDMD-dependent (Cerqueira et al., 2018). Additionally, in this study, we demonstrated that cell death induced by Brucella was also shown to be caspase-8-dependent. Caspase-8 contributes to activation of canonical and noncanonical inflammasomes. During Salmonella infection, caspase-8 can be recruited to the NLRC4 inflammasome regulating IL-1β secretion, but not playing a role in cell death (Man et al., 2013). In contrast, in Yersinia infection model, like we observed in this study, caspase-8 activates GSDMD to induce cell death (Sarhan et al., 2018). Furthermore, we observed here that mice deficient for caspase-8 and GSDMD are more susceptible to Brucella infection in vivo compared to wild type animals, suggesting that pyroptosis triggered during B. abortus infection is an important mechanism to control infection.

Several studies have already identified cellular functions for the GSDMD-mediated pore, such as secretion of molecules such as IL-1β and IL-1α and eicosanoids, which are important for recruiting neutrophils to the site of infection and promoting phagocytosis of infected cells and contributing to infection restriction (Jorgensen et al., 2016). Herein, reduced IL-1β secretion and pyroptosis observed in Casp8/RIPK3^−/− mice are possible mechanisms that may contribute to increased susceptibility to infection. Although we did not investigate cell recruitment in this study, we hypothesize that innate cells recruitment to the site of infection may be impaired by the absence of pyroptosis in caspase-8 deficient animals, which could in part explain the increased bacterial load observed in these animals. Recently, caspase-8 was involved in Aspergillus fumigatus keratitis being critical in the recruitment of inflammatory cells and the clearance of the fungus (Wang et al., 2022). In summary, we suggest that caspase-8 plays an important role in cell death induced during B.abortus infection, contributing to inflammation and infection control in mice.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by the procedures for animal experimentation were approved by the Ethics Committee for the Use of Animals of the Federal University of Minas Gerais-CEUA/UFMG under protocol number 69/2020.

RS performed all the experiments and wrote the manuscript. DC participated in the design of this study, provided assistance with data acquisition, data analysis, and statistical analysis. DZ participated in the design of this study and provided reagents to perform the experiments. SO participated in the design of this study, provided assistance with data acquisition and wrote and reviewed the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico to SO (CNPq, www.cnpq.br; grant# 303044/2020-9), Fundação de Amparo a Pesquisa do Estado de Minas Gerais to SO (FAPEMIG, www.fapemig.br; grants# APQ #01945/17 and Rede Mineira de Imunobiológicos #00140-16), National Institutes of Health to SO (NIH, www.nih.gov; grant# R01 AI116453).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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