Introduction
Periodontitis is a chronic inflammatory disease that progressively affects the integrity of tooth-supporting tissues ( Hajishengallis & Chavakis, 2021 ). Although periodontitis has a multifactorial etiological factor, including local, systemic, and genetic factors, Porphyromonas gingivalis has long been described as the main etiological agent involved in the pathogenesis of this disease, in 75.8% of patients with periodontitis ( Rafiei et al., 2017 ). Furthermore, it has been reported that P. gingivalis induces experimental periodontitis in the murine model ( Hariyani et al., 2021 ).

P. gingivalis is a gram-negative rod-shaped, anaerobic, strictly facultative, and asaccharolytic bacterium ( Smalley & Olczak, 2017 ). During the subgingival sulcus infection, P.gingivalis expresses various virulence factors such as cysteine proteases, known as gingipains, hemagglutinins, lipopolysaccharide (LPS), nucleoside-diphosphate-kinase (NDK), and fimbriae ( Mendez et al., 2019 ; Jia et al., 2019 ; Atanasova et al., 2016 ), which contribute to host-invasion, intracellular persistence, induction of the inflammatory response, and apoptosis of the host cells ( Stathopoulou et al., 2009 ). The clinicopathological consequence is inflicting irreversible damage to the periodontal connective tissues ( Sun et al., 2021 ; Lam et al., 2016 ). Histopathology of gingiva samples and analysis of gingival crevicular fluid from patients with chronic gingivitis suggest that macrophages are pivotal in the development of a long-lasting inflammatory microenvironment ( Fageeh, Fageeh & Patil, 2021 ).

The inflammatory infiltrate in patients with periodontitis includes 5 to 30% of macrophage populations ( Papadopoulos et al., 2017 ). These cells play different roles during the different stages of the disease. The chronic phase is associated with the presence of M1 and M2 phenotypes in the inflammatory infiltrate, being M1 macrophages the most abundant cells ( Blancas-Luciano et al., 2023 ; Yang et al., 2018 ). Circulating blood monocytes reach the inflammatory infiltrate and differentiate into M1 macrophages, induced by cytokines such as IFN-γ and TNF-α, or by direct stimulation of TLRs, exposed to pathogen-associated molecular patterns (PAMPs) ( Fageeh, Fageeh & Patil, 2021 ). LPS from P. gingivalis promotes M1 macrophage differentiation ( Liu et al., 2020 ; Holden et al., 2014 ), inducing the production of inflammatory mediators such as IL-1β, IL-6, IL-8, TNF-α, and the inducible nitric oxide synthase (iNOS) ( Parisi et al., 2018 ). These mediators amplify the inflammatory response by increasing local blood flow, favoring leukocyte recruitment, osteoclast activation, and subsequent bone loss ( Pignatelli et al., 2020 ; Gurav, 2014 ).

P. gingivalis has evolved abilities to evade or subvert macrophages immune response. This bacterium promotes the polarization of macrophages and inflammasome activation in different microenvironments ( Lin et al., 2022 ). Additionally, P. gingivalis uses macrophages as host cells, which allows it to survive intracellularly. Bacterial virulence factors, such as fimbriae, contribute to this phenomenon. Bacterial fimbriae are recognized by TLR2, which induces the activation of a Myd88-independent pathway, into which the Mal/TIRAP/adapter protein is recruited. This pathway prevents phagosome-lysosome fusion in macrophages, promoting the survival of internalized bacteria ( Makkawi et al., 2017 ). Subsequently, macrophages and lymphocytes produce matrix metalloproteinases (MMPs), such as MMP-1, MMP-13, MMP-8, and MMP-9, and inflammatory factors, such as IL 1-β and TNF-α ( Parkhill et al., 2000 ). All these molecules, and the increase in oxidative stress due to the production of ROS, and the others mentioned above, have a critical role in periodontal tissue destruction ( Song, Jeon & Kim, 2021 ; Cheng et al., 2020 ).

Furthermore, diverse cytokines produced by macrophages promote the development of periodontitis. Inflammasome activation induces the maturation of two vital pro-inflammatory cytokines, IL-1α and IL-18 ( Champaiboon et al., 2014 ). The upregulation of the inflammasome components caspase-1 and NLRP3, together with the lack of melanoma 2 (AIM2) in gingival epithelial cells and macrophages of periodontitis patients, suggest their inflammatory role ( Park et al., 2014 ). Therefore, many inflammatory molecules could be crucial in the development of early periodontal disease, including the presence of NO, the main inflammatory mediator in this disease ( Pacher, JS & Liaudet, 2007 ).

It has been shown that high NO concentrations could be cytotoxic to periodontal tissue and related to probing depth and bone resorption ( Pansani et al., 2016 ; Wang, Huang & He, 2019 ; Reher et al., 2007 ). In addition, it has been shown that NO has a side effect on the periodontal tissue, favoring vasodilation and platelet aggregation diminishes, which can contribute to gingival bleeding, aside from having cytotoxic effects on the surrounding tissue, increasing the severity of the periodontitis ( Boutrin et al., 2012 ). The increase of NO levels in patients with periodontitis is related to high levels of iNOS expression in periodontal tissue cells during inflammation. Additionally, NO can function both as an apoptotic and an anti-apoptotic molecule, depending on its concentration and its interactions with other cellular molecules ( Sharma, Al-Omran & Parvathy, 2007 ; Shibata et al., 2001 ).

On the other hand, ROS could be involved in the pathogenesis of many diseases, such as rheumatoid arthritis, chronic pulmonary disease, atherosclerosis, and recently in periodontitis ( Filippin et al., 2008 ; Soory, 2007 ; Tavakoli & Asmis, 2012 ; Waddington, Moseley & Embery, 2000 ). Moreover, phagocytosed P. gingivalis by neutrophils is resistant to oxidative burst killing, partially by producing antioxidant enzymes such as superoxide dismutase, thiol peroxidase, and rubrerythrin ( Sztukowska et al., 2002 ; Mydel et al., 2006 ; Kikuchi et al., 2005 ). Additionally, P. gingivalis accumulates a hemin layer on the cell surface that protects from oxidative stress ( Smalley, Birss & Silver, 2000a ; Smalley, Birss & Silver, 2000b ). P. gingivalis can also survive within epithelial cells and induced the production of ROS in the early stages of infection ( Choi et al., 2012 ). Other studies suggest that ROS production induced by bacteria can activate JAK2 signaling ( Forget, Gregory & Olivier, 2005 ) and increase levels of secreted IL-6 and IL-1 cytokines ( Wang et al., 2014 ). However, it has also also been reported the advantages of using both mediators as cytotoxic molecules for P. gingivalis . The addition of nitric oxide and hydrogen peroxide has shown to kill P. gingivalis , ( Takada et al., 2017 ). Zou et al. (2022) demonstrated that ROS production induces the viability loss of planktonic P. gingivalis and decreases its biofilm formation capacity.

P. gingivalis also can modulate cell death in various types of cells. LPS of P. gingivalis induces cell death in peripheral blood mononuclear cells (PBMCs) by early and late apoptosis, as well as necrosis through upregulation of Fas-FasL and activation of caspase-3 ( Trindade et al., 2012 ; Carvalho-Filho et al., 2019 ). The gingipains of the bacterium promote apoptosis of human gingival fibroblasts through intracellular proteolytic activation of caspase-3 ( Stathopoulou et al., 2009 ). Other cells, as endothelial cells ( Sheets et al., 2005 ), cardiomyoblasts ( Lee et al., 2006 ), lymphocytes ( Geatch et al., 1999 ), monocytes ( Ozaki & Hanazawa, 2001 ), and neutrophils ( Hiroi et al., 1998 ; Murray & Wilton, 2003 ; Preshaw & Taylor, 2011 ), can be also induced to apoptosis by P. gingivalis. However, P. gingivalis also uses diverse mechanisms to modulate cell death pathways and ensure its intracellular survival. P. gingivalis promotes host-cell survival by inhibiting caspase-3 activation and balancing the expression of pro-apoptotic Bax and anti-apoptotic Bcl-2 ( Nakhjiri et al., 2001 ; Mao et al., 2007 ). P. gingivalis , can replicate to high intracellular levels and does not induce host-cell death, in infected gingival epithelial cells ( Yilmaz et al., 2004 ; Mao et al., 2007 ).

The oral antimicrobial peptides are the first line of innate defense in the oral. The antimicrobial capacity of saliva permits colonization of the oral cavity but prevents extensive microbial colonization of the oral and oropharyngeal tissues. The oral antimicrobial peptides also exert different immunomodulatory functions, including the regulation of the innate immune response ( Vray, Hartmann & Hoebeke, 2002 ). Cystatin C is an antimicrobial peptide that decreases the growth of P. gingivalis by inhibiting proteolytic activity in the culture supernatant ( Blankenvoorde et al., 1998 ). Some studies have shown that Ds-cystatin, a cystatin C homolog isolated from the tick Dermacentor silvarum , is internalized in LPS-stimulated mouse macrophages from Borrelia burgdorferi, inducing a decrease in inflammatory cytokines, such as IL-1β, IFN-γ, TNF-α, and IL-6 ( Sun et al., 2018 ; Gren et al., 2016 ). In addition, cystatin C secreted by Schistosoma japonicum induces M2 macrophage polarization, favoring the production of anti-inflammatory cytokines such as IL-10 and TGF-β ( Yang et al., 2021 ).

Due to the importance of controlling the inflammatory response during the development of periodontitis, in the present study, we investigated the effect of cystatin C on P. gingivalis -infected macrophages. We demonstrated that cystatin C has an important role in the intracellular antimicrobial activity and immunoregulatory activity on P. gingivalis -infected macrophages.