Materials & Methods
Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coat cell packages donated for research and no longer usable for humans, obtained from the Blood Bank of the General Hospital of México “Eduardo Liceaga”. PBMCs were isolated from blood by density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA) at 325×g for 20 min at 20 °C. Briefly, after 1:1 dilution with PBS pH 7.2 (Gibco Life Technologies, Waltham, MA, USA), blood samples are slowly and steadily placed on top of Histopaque-1077 (Sigma-Aldrich), in centrifuge tubes and centrifuged at 325×g for 10 min. The cells were carefully harvested from the interphase, washed with cold PBS pH 7.2 buffer, and incubated for 15 min in lytic solution to lyse residual erythrocytes. The PBMCs obtained were washed with PBS pH 7.2 and counted in a hemocytometer (Millipore, Burlington, MA, USA) using the trypan blue dye exclusion method.

The study was performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Universidad Nacional Autónoma de México (reference number FM/DI/038/2021).

Monocytes were isolated from previously obtained PMBC by positive selection with anti-human CD14 microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany), according to the manufacturer’s protocol.

Human CD14 + monocytes were seeded in low adherence 24-well plates (Costar 3473) with a cell density of 1 × 10 6 /well, in RPMI-1640 medium (Sigma-Aldrich, USA, R6504-1L) supplemented with 10%, 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/mL streptomycin and 10% fetal bovine serum (FBS) (GIBCO BRL, Gaithersburg, MD, USA) at 37 °C in a humidified 5% CO 2 incubator for 7 days.

The adherent macrophages were then detached via cold EDTA solution at 0.02% (Sigma-Aldrich, USA), collected in 50 ml tubes, and centrifuged at 325× g for 5 min at 4 °C. The cells were resuspended in RPMI-1640 (R6504-1L; Sigma-Aldrich) supplemented with 10%, 2 mM L-glutamine, 100 U/mL penicillin, and 0.1 mg/mL streptomycin and 10% FBS (GIBCO BRL, Gaithersburg, MD, USA). The recovered macrophages were counted with a hemocytometer (Millipore, Burlington, MA, USA) using the trypan blue dye exclusion method.

Monocytes-derived macrophages purity was evaluated using flow cytometry (FACScan). Monocytes were resuspended in 100 µL buffer, pH 7.2 and stained with 2 µL anti-human CD14-PE (1:50) (BD Pharmigen, San Diego, CA, USA [clone M5E2]) for 30 min in the dark at 4 °C.

Differentiated macrophages were resuspended in 100 µL of buffer, pH 7.2, and stained with 2 µL of anti-human CD68-PE (1:50) (BD Pharmigen [clone M5E2]) for 30 min in the dark at 4 °C. Analysis was performed with a Flow Jo 10 software (BD Biosciences, Heidelberg, Germany).

P. gingivalis strain ATCC 33277 was cultured as previously described ( Blancas-Luciano et al., 2022 ). Briefly P. gingivalis was cultured in brain-heart-infusion (BHI; BD Biox-on, Milan, Italy) containing 5 mg/mL of hemin (Sigma-Aldrich, Munich, Germany) and 1 mg/mL of menadione (Sigma-Aldrich) under anaerobiosis using the anaerobic BBL-GasPak jar system (BD Biosciences), at 37 °C for 24 h.

After 24 h of culturing, bacteria were harvested by centrifugation for 10 min at 8,200 x g and then washed and resuspended in Krebs-Ringer-Glucose (KRG) buffer (120 mM NaCl, 4.9 mM KCl, 1.2 mM MgSO 4 , 1.7 mM KH 2 PO 4 , 8.3 mM Na 2 HPO 4 , 10 mM glucose, and 1.1 mM CaCl 2 , pH 7.3). Bacterial growth was monitored spectrophotometrically (Jenway Genova R0027; Thermo Fischer Scientific, Waltham, MA, USA) at 675 nm. The bacterial density was visually adjusted to a turbidity of 0.5 McFarland (1 × 10 8 colony-forming units); (CFU/mL) ( Bollela, Sato & Fonseca, 1999 ; Emani, Gunjiganur & Mehta, 2014 ).

We used human recombinant Cystatin C expressed in Pichia pastoris (Sigma Aldrich, St. Louis, MO). It was reconstituted with one mL of buffer Tris-Base NaCl (pH 7.4).

The antimicrobial activity of cystatin C was evaluated by microdilution technique ( Eloff, 1998 ; Jadaun et al., 2007 ) using the commercially available growth indicator Presto Blue. Briefly, 5 × 10 5 CFU/mL of P. gingivalis per well were seeded in KRG buffer in 96-well plates (Costar; Corning Life Sciences, Corning, NY, USA). Cystatin C (1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75 µg/mL) was incubated for 1, 12, 24, 48, and 96 h at 37 °C under anaerobic conditions. After the incubation period, 20 µL of Presto Blue Cell Viability Reagent (Invitrogen, Thermo Fisher Scientific) per well was added. The plates were incubated for 30 min at 37 °C in the dark. Finally, the plates were read in a microplate reader (Multiskan SkyHigh Microplate Spectrophotometer; Thermo Fisher Scientific), at a 675 nm wavelength.

Macrophages were seeded in a 96-well (Costar, Corning Life Sciences) plate at a density of 1 × 10 5 cells/well and incubated for 12 h at 37 °C with 5% CO 2 . Next, different concentrations of cystatin C (1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75 µg/mL) were added and incubated for 24 h. At the end of the incubation, 25 µl/well of XTT/PBS solution (4 mg/mL) for 40 min at room temperature in the dark, was added. The samples were read at a wavelength of 450 nm in a microplate spectrophotometer (Multiskan SkyHigh Microplate Spectrophotometer). The method was first used in the methods section from Blancas-Luciano et al. (2022) .

Macrophages were seeded in Costar ® 24-well plate (Corning Life Sciences, Corning, NY, USA) at a cell density of 1 × 10 6 /well in RPMI-1640 medium (R6504-1L; Sigma-Aldrich) supplemented with 10%, 2 mM L-glutamine, for 12 h at 37 °C and 5% CO 2 . At the end of the incubation, the medium was removed and replaced with fresh medium without antibiotics. Cells were infected with P. gingivalis at MOI: 1:10 for 1, 3, 24, 48, and 96 h. The infected macrophages were stimulated with cystatin C for 24 h. Subsequently, the cells were washed three times with PBS and harvested using 0.02% EDTA solution (Sigma-Aldrich).

Invasion of P. gingivalis was quantitated by a standard antibiotic protection assay as described previously ( Lamont et al., 1995 ; Yilmaz, Watanabe & Lamont, 2002 ). Briefly, infected macrophages and stimulated with cystatin C and washed twice and treated with metronidazole (200 µg/ml) and gentamicin (300 µg/ml) for 1 h to eliminate remaining extracellular bacteria. After exposure to the antibiotic, cells were washed twice with PBS pH 7.2 and lysed in one mL of cold sterile distilled water per well for 30 min. Cell lysates were serially diluted and plated on blood agar plates (BHI, Becton Dickinson, Sparks, MD, USA) supplemented with hemin and menadione, and incubated anaerobically at 37 °C for 7 days. Colony forming units per mL were then enumerated ( Supplementary figure ).

Antisera against cystatin C was generated in female CD-1 mice weighing 20 ± 2 g. The mice were immunized for 6 weeks at 8-day intervals with 100 µg/100 µL Cystatin C. The immunizations were administered intraperitoneally for 4 weeks and subcutaneously for 2 week. For the first intraperitoneal immunization, Freund’s complete adjuvant was added, and for the second intraperitoneal immunization, Freund’s incomplete adjuvant was included. Five weeks after the first immunization, mice were anesthetized to obtain whole blood, which was aliquoted and stored at −80 °C until use. Antibody titers were estimated by an enzyme-linked immunosorbent assay, according to Fernández-Presas et al. (2018) . Guidelines for laboratory animal care established by the ethical committee of the School of Medicine, Universidad Nacional Autónoma de México were strictly followed.

Cystatin C-stimulated P. gingivalis -infected macrophages were incubated in 24-well plates containing glass coverslips in RPMI-1640 (R6504-1L; Sigma-Aldrich, USA) supplemented with 10% FBS (GIBCO BRL, Gaithersburg, MD, USA). Briefly, cells attached to coverslips were washed with PBS, fixed with 2% paraformaldehyde (PFA) for 20 min at 4 °C, and washed with 1x Perm/Wash buffer (Biolegend). Staining was done with mouse anti-Cystatin C primary antibodies diluted 1:25 in permeabilization solution for 30 min at 4 °C. Cells were washed again with 1x Perm/Wash. Subsequently, it was stained with mouse IgG secondary antibody (Cy3) diluted 1:25 in permeabilization solution for 30 min at 4 °C. Cell nuclei were counterstained with DAPI (1:1000) for 3 min in fluoroshield mounting medium (Sigma-Aldrich, USA). The cells from experimental groups and controls are visualized by confocal microscopy (Leica, TCS SP5; Leica Microsystems, USA), using LAS X v software 3 for image analysis.

Macrophages were infected with P. gingivalis and stimulated with cystatin C for 24 h at 37 °C with 5% CO 2 . The cytokines IL-1β, TNF-α and IL-10 were quantified by ELISA in the cell supernatants. Human cytokine ELISA kits (BD Biosciences Cytokine ELISA Protocol) were used according to the manufacturer’s instructions. Briefly, anti-TNF-α, an-ti-IL-1β, and anti-IL-10 capture antibodies (BD Bioscience, Pharmingen) were placed in 96-well flat-bottom plates (Costar; Corning Life Sciences, Corning, NY, USA) diluted in 0.1 M Na 2 HPO 4 , pH 6, and incubated overnight at 4 °C. The wells were blocked with PBS solution supplemented with 0.5% casein diluted in 0.1 N NaOH. Cell supernatants and those from the TNF-α standard curve, recombinant IL-1β, and IL-10 (BD Bioscience, Pharmingen) were incubated in a D-MEM culture medium overnight at 4 °C. To detect TNF-α, IL-1β, and IL-10, the detection antibodies anti-TNF-α, anti-IL-1β, and anti-IL-10 biotinylated (BD Bioscience) were diluted in 1% BSA with 0.05% Tween 20 and incubated at room temperature for 1 h. The plate was analyzed using a streptavidin-alkaline phosphatase (AP) conjugate (Thermo Fisher Scientific, Life Technologies) and 0.005 mg/mL phosphatase substrate (Sigma Aldrich). Control groups included macrophages without stimulation or stimulated with LPS (100 ng/mL) (LPS from Escherichia coli O111:B4; Sigma Aldrich) or with a peptidoglycan (10 µg/mL) (peptidoglycan from Staphylococcus aureus , (Sigma-Aldrich). Absorbance was quantified in a spectrophotometer (Multiskan SkyHigh Microplate Spectrophotometer) at 405 nm using Ascent Multiskan V. 2.6 software. Cytokine concentrations in samples were determined from the standard curve by regression analysis.

Macrophages infected with P. gingivalis were stimulated with 2.75 µg/mL cystatin C for 48 h at 37 °C with 5% CO 2 . Subsequently, the supernatants were isolated, and their nitrite concentration was quantified using the Griess Reaction kit (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. Supernatants from macrophages infected with P. gingivalis and stimulated with cystatin C were placed in a 96-well microplate and 20 µL Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthyl ethylenediamine-HCl) were added (Sigma Aldrich). The cells were incubated at room temperature for 30 min. The absorbance was measured at 550 nm with a microplate reader (Multiskan SkyHigh Microplate Spectrophotometer). The culture medium was used as a blank in all experiments. The number of nitrites in the test samples was calculated from a standard curve of sodium nitrite.

Macrophages infected with P. gingivalis and stimulated with cystatin C (2.75 µg/mL) were incubated for 24 h at 37 °C with 5% CO2. Subsequently, the cells were incubated with 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCFDA) at a concentration of 100 µM for 30 min. Cells were washed with PBS pH 7.2 and detached from plates with 0.02% EDTA solution (Sigma Aldrich, St. Louis, MO, USA). The samples were resuspended in PBS pH 7.2 with 1% FBS and analyzed in a BD bioscience FACS Canto II flow cytometer. Data analysis was performed using FlowJo 10 software ( https://www.flowjo.com/solutions/flowjo/downloads ).

The cell pellets of infected macrophages and stimulated with cystatin C were processed for standard transmission electron microscopy as previously described ( Fernández-Presas et al., 2018 ). Briefly, all samples were fixed in a mixture of 1.5% glutaraldehyde and 4% paraformaldehyde, buffered in PBS (pH 7.2), at room temperature for 1 h. The samples were postfixed with 1% osmium tetraoxide for 4 h. All samples were subsequently dehydrated by incubation in a series of ascending concentrations of ethanol and embedded in an epoxy resin (Poly/Bed812/DMP30; Polysciences, Warrington, PA, USA).

Ultrathin sections (50–60 nm) were mounted on formvar-coated copper grids, stained, using the double-contrast technique, with uranyl acetate for 30 min followed by lead citrate for 15 min. Later, the sections were rinsed with deionized water and dried. Subsequently, they were examined at 80 kV with a JEOL l JEM 1200EXII transmission electron microscope (JEOL, Peabody, MA, USA). Depending on the structure of interest, multiple microphotographs were taken with a magnification range of 5, 000 × to 1,20 000 ×.

A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay ( in situ cell death detection kit; Boehringer Mannheim; Ingelheim am Rhein, Germany) was used for the detection of cell death by apoptosis using enzymatic labeling of the DNA strand breaks with fluorescein dUTP and TdT ( Gorczyca, Gong & Darzynkiewicz, 1993 ).

P. gingivalis -infected macrophages stimulated with cystatin C were washed twice in PBS/1% bovine serum albumin and subsequently resuspended in paraformaldehyde (2%) in PBS (pH 7.4) and incubated for 30 min at room temperature. Cells were washed three times (centrifuged at 1,300 g) for 10 min and suspended in permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice. The cells were washed twice in PBS at pH 7.4 and 50 µL TUNEL reaction mix was added. The cells were incubated for 60 min at 37 °C in a humid chamber. Macrophages stimulated with DNase I (Sigma; 600 U/ml in 50 mM Tris–HCl, pH 7.5, 1 mg/ml BSA) for 10 min were used as a positive control. The samples were washed twice in PBS and analyzed by flow cytometry ( Fernández-Presas et al., 2001 ).

Exposure of phosphatidylserine (PS) on the cell surface was analyzed by annexin-V binding and cell membrane integrity by 7-AAD staining, using the Annex-in-V-FLUOS Staining Kit (Roche Diagnostics GmbH, Mannheim, Germany) following the protocol recommended by the manufacturer. Briefly, macrophages infected with P. gingivalis (1.5 × 10 5 ) and stimulated with cystatin C (2.75 µg/mL) were washed twice in PBS, pH 7.4, and subsequently suspended in staining solution (Annexin-V-Fluos/Hepes buffer [10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 5 mM CaCl 2 ], supplemented with propidium iodide [50 µg/mL]) for 15 min at 25 °C in the dark. Cells incubated with 4 µg/mL of camptothecin (Roche) for 3 h, were used as a positive control. Samples were analyzed by flow cytometry on a FACSort with CellQuest software (BD Immunocytometry Systems, San Jose, CA, USA). Data were analyzed using the Flow Jo software (Becton, Dickinson, Milpitas, CA, USA). The results were presented as the percentage of viable (Ann − V − PI − ), early apoptotic (Ann − V + PI − ), or nonviable (Ann − V − PI + ) cells.

The assay was performed as recommended by the manufacturer. Briefly, cells were washed twice with cold PBS and homogenized in standard buffer [100 mM Hepes; 10% (w/v sucrose 0.1% w/v) CHAPS; 10 mM DTT; 1 mM EDTA; 1 mM PMSF; 1 mM Naf; 2 µg/mL aprotinin; 1 µg/mL pepstatin; and 5 µg/mL leupeptin]. Macrophages infected with P. gingivalis and stimulated with cystatin C were transferred to well plates at a density of 1 × 10 4 cells/well. The reaction buffer supplemented with the caspase 3 substrate MOCAc-DEVDAPKb (Dnp)-NH2 (25 µM) was incubated for 4 h at 37 °C. Caspase 3 activity was measured by the release of p-nitroaniline (pNA). The p-nitroaniline was detected at 405 nm with a luminescence spectrometer (Fluoroskan Ascent Thermo Labsystems, Helsinki, Finland). The concentration of pNA released from the substrate was calculated us-ing a calibration curve prepared with pNA standards (pNA standard included in the kit). This method was first used in the Methods section from Fernández-Presas et al. (2010) .

Experimental and control conditions were statistically compared for significance using analysis of variance (ANOVA), followed by Benferroni correction. The predetermined level of significance was p < 0.05. Statistical analysis was performed with the GraphPad, Prism v.6 software (GraphPad Software, Inc., La Jolla, CA, USA).