Discussion
In this study, we sought to determine whether cystatin C reduces the intracellular growth of P. gingivalis in infected macrophages and to evaluate the inflammatory response induced by this bacterium in macrophages. Our results show that cystatin C is internalized in P. gingivalis -infected macrophages, decreasing the intracellular bacterial load. Intracellular localization of cystatin C in infected macrophages showed that cystatin C co-localizes with P. gingivalis. Furthermore, a decrease in the production of inflammatory cytokines and NO was observed. Interestingly, the treatment with cystatin C increases ROS production and decreases the programmed cell death in macrophages infected with the bacterium. These results highlight the important activity of cystatin C against the P. gingivalis , in addition to its immunoregulatory role in infected macrophages.

P. gingivalis is a keystone pathogen and can impair innate immunity and transform a symbiotic microbiota into a dysbiotic state in periodontal tissue ( Hajishengallis, Darveau & Curtis, 2012 ). Conventional treatment against this disease consists of the mechanical elimination of the bacteria by scaling and root planning of the affected tissue ( de Andrade, Almeida-da Silva & Coutinho-Silva, 2019 ) and use of bactericides such as chlorhexidine and antibacterials such as metronidazole ( Rosa et al., 2021 ; Larsen, 2002 ). However, the clinical application of chlorhexidine is limited by its bitter taste and staining of teeth and tongue ( Larsen, 2002 ). On the other hand, although the use of metronidazole is effective against extracellular P. gingivalis , it cannot penetrate infected cells ( Ye et al., 2017 ) and various adverse effects have been reported ( Kafadar et al., 2013 ).

Antimicrobial peptides arise as promising molecules in the treatment of oral infections such as periodontitis due to their microbicidal effect against P. gingivalis and the immunoregulation capacity exerted by them ( Liang & Diana, 2020 ). The extracellular antimicrobial activity of cystatin C on various oral microorganisms has been previously reported. Cystatin C inhibits the growth of Streptococcus mutans and Enterococcus faecalis , through the induction of morphological changes in their cell walls, peptidoglycan, and a decrease in the electron density of the cytoplasm ( Blancas et al., 2021 ). Blankenvoorde et al. (1998) identified that cystatin C binds to the active site of P. gingivalis cysteine protease, inhibiting its proteolytic activity in culture supernatant and decreasing its growth by 50%. Other peptides such as Nal-P-113 and mPE also inhibit P. gingivalis at a concentration of 20 µg/mL and 4 µg/mL, respectively ( Wang et al., 2018 ; Hua, Scott & Diamond, 2010 ). Our study shows that cystatin C decreases the growth of P. gingivalis by 90%, demonstrating that cystatin C has potent antimicrobial activity at low concentrations (2.75 µg/mL) and not cytotoxic effects on macrophages. Cystatin C inhibits growth at a lower concentration than Nal-P-113 and mPE without altering macrophage viability, which suggests cystatin C is an ideal peptide for the treatment of bacteria associated with the oral cavity such as P. gingivalis .

Another advantage of antimicrobial peptides such as cystatin C is that, unlike antibiotics such as metronidazole, this peptide is internalized by endocytosis mechanisms in endothelial cells and monocytes ( Singhrao & Olsen, 2018 ). Interestingly, in this study, we present several lines of evidence to demonstrate that cystatin C is internalized in P. gingivalis -infected macrophages and has intracellular antimicrobial activity against this bacterium. First, by counting colony-forming units, we observed a lowers number of intracellular bacteria in the infected macrophage after treatment with cystatin C. Second, we observed the intracellular localization of cystatin C in infected macrophages and its co-localization with P. gingivalis , suggesting that this peptide could have direct activity against the bacteria, favoring its elimination.

P. gingivalis internalized in monocytes/macrophages or dendritic cells can thus use a “ Trojan horse ” to favor its dissemination to other tissues such as coronary arteries, placenta, brain, and liver, which constitutes a pathogenic mechanism for the development of systemic diseases ( Singhrao & Olsen, 2018 ; Dominy et al., 2019 ). In addition, intracellular persistence is a strategy developed by the bacteria to escape the host’s immune response, which contributes to the pathological process of periodontitis ( Mei et al., 2020 ). In addition to the ability of P. gingivalis to invade host cell, bacterium can escape to the autophagosome-lysosome fusion to avoid death ( Werheim et al., 2020 ; Shiheido-Watanabe et al., 2023 ). Therefore, the intracellular elimination of this bacterium mediated by treatment with cystatin C constitutes an important finding since it could reduce the presence of P. gingivalis in gingival tissue or prevent bacterial dissemination.

In addition to these mechanism, P. gingivalis induces the recruitment of inflammatory cell infiltrates, particularly macrophages, which leads to the destruction of supporting tissues ( Darveau, 2009 ). Macrophages play an important role in the inflammatory response initiation, due to producing pro-inflammatory cytokines and mediating alveolar bone resorption in periodontitis ( Sun et al., 2021 ). Various cytokines produced by macrophages promote the development of periodontitis. The inflammasome activation induces the maturation of two important inflammatory cytokines, IL-1β and IL-18 ( Champaiboon et al., 2014 ). In addition, P. gingivalis promotes the polarization of M1 macrophages which are directly related to bacterial survival and evasion of the macrophage immune responses in macrophage, thereby inducing the progression of the disease ( Lin et al., 2022 ).

We investigated the cystatin C activity in inflammatory cytokines production in P. gingivalis infected macrophages. We found that P. gingivalis induces the production of IL-1β and TNF-α. However, cystatin C stimulation importantly decreased these cytokines. Interestingly, we found that cystatin C has anti-inflammatory activity due to inducing IL-10 production in macrophages challenged with P. gingivalis. This cytokine production is an important finding in our research because previous reports indicated that IL-10, suppresses TNF-α, IL-6, and IL-1 production ( Sun et al., 2019 ). In addition, Il-10 gene polymorphisms may be associated with a predisposition to chronic periodontitis development ( Geng et al., 2018 ), which indicates cystatin C could contribute to the inflammatory process decrease by IL-10 production. In a previous study, we demonstrated that cystatin C down-regulates the production of IL-1β and TNF-α in HGFs co-incubated with P. gingivalis , whereas IL-10 production increases ( Blancas-Luciano et al., 2022 ). This could represent an important mechanism to inhibit an exacerbated inflammatory response in macrophages to P. gingivalis infections.

The phenomenon observed in both cell types could suggest that cystatin C is a modulator of inflammatory response in both HGF and macrophages. Cystatin C could be participating in the polarization of M2 macrophages infected with P. gingivalis , evidenced by the presence of IL -10. In addition, our findings on the diminished IL-1β and TNF-α production are consistent with the literature, where it is demonstrated that cystatin C down-regulates the production of both cytokines in monocytes stimulated with bacterial LPS ( Gren et al., 2016 ). The downregulated production induced by cystatin C can contribute to the limitation of immune damage in periodontal tissues. A balance of inflammatory cytokines and anti-inflammatory cytokines is critical for homeostasis in the host. Therefore, an appropriate immune response must be developed to resist periodontopathic bacteria without excessive damage to periodontal tissues ( Sztukowska et al., 2002 ; Mydel et al., 2006 ; Kikuchi et al., 2005 ; Smalley, Birss & Silver, 2000a ; Smalley, Birss & Silver, 2000b ). In additionally to cystatin C, other salivary peptides have been described, like histatin 5 and 1, that participate in downregulating inflammatory cytokines, such as TNF-α, IL-1β, IL-6, and IL-8 in macrophages and fibroblasts ( Imatani et al., 2000 ; Lee et al., 2021 ).

In addition to cytokine production, macrophages infected with P. gingivalis produce microbicidal mediators such as NO and ROS ( Wang et al., 2014 ; Sun et al., 2010 ). We found that P. gingivalis induces the production of ROS. An additional increase of ROS was also detected after macrophage stimulation with cystatin C. This finding suggests that the increased ROS production in infected macrophages could represent a microbicidal mechanism exerted by the peptide to reduce the intracellular bacterial load. Antimicrobial peptides, like LL-37, can mediate intracellular bactericidal activity against M. tuberculosis by ROS production in a THP-1 cell line ( Gren et al., 2016 ). Additionally, α-defensins LL-37, CG117-136, and protegrins (PG-1) decrease NO and TNF-α production ( Zughaier, Shafer & Stephens, 2005 ). Our findings demonstrate that infected macrophages stimulated with cystatin C show an increase of ROS production, together with a decrease of NO, suggesting that ROS production could represent a cystatin C-mediated microbicidal mechanism that helps to reduce the intracellular bacterial load, while NO decrease could inhibit the inflammatory response and tissue damage.

When evaluating NO production in macrophages infected with P. gingivalis , we observed an increase in NO production. Interestingly, co-incubation with cystatin C decreased the production of NO importantly. Ghosh et al. (2008) demonstrated that NO and its regulatory enzyme iNOS are a key role in the pathogenesis of periodontal disease. Furthermore, diverse studies have reported that high levels of NO and iNOS are relational with periodontal disease severity in patients and rodent models ( Menaka et al., 2009 ; Di Paola et al., 2004 ; Gyurko et al., 2003 ).

Nitric oxide can be anti- or pro-apoptotic ( Sharma, Al-Omran & Parvathy, 2007 ). It has been reported that NO release by oral keratinocytes infected with P. gingivalis may contribute to their resistance to apoptosis. Apoptosis plays a critical role in gingival tissue homeostasis ( Bugueno et al., 2018 ). However, chronic inflammatory diseases such as periodontitis, can is associated with tissue destruction. Listyarifah et al. (2017) show that in biopsies of patients with periodontitis, there is an expression of caspase-3 in the lamina propria cells and professional phagocytic cells (CD68 + ) ( Listyarifah et al., 2017 ).

P. gingivalis induces apoptosis on fibroblasts ( Graves et al., 2001 ; Imatani et al., 2004 ; Palm, Khalaf & Bengtsson, 2015 ), endothelial cells ( Sheets et al., 2005 ), cardiac myoblasts ( Lee et al., 2006 ), lymphocytes ( Geatch et al., 1999 ), monocytes ( Lee et al., 2006 ), and polymorphonuclear neutrophils ( Hiroi et al., 1998 ; Murray & Wilton, 2003 ; Preshaw & Taylor, 2011 ). P. gingivalis induces apoptosis after invading the host cell, which could con-tribute to the immune dysregulation observed during chronic periodontitis. P. gingivalis induces apoptosis in a time and dose-dependent manner, by regulating pro-apoptotic molecules such as caspase-3, -8, -9, Bid and Bax ( Desta & Graves, 2007 ). In this study, we showed that P. gingivalis induces apoptosis through the increase of caspase 3 activity in P. gingivalis -infected macrophages. In contrast, cystatin C stimulation in infected macrophages reduces cell death. These results agree with the report by Lee et al. (2006) , who demonstrated that histatin 5 induces inhibition of apoptosis in human gingival fibroblasts infected with P. gingivalis .