RiteeshGrandhi/sciclaimeval-shared-task huggingface dataset:
Conclusions
P. gingivalis could induce the recruitment, activation, and disruption of many macrophage functions. These mechanisms contribute to escape macrophage killing and trigger an inflammatory response, which contributes to the periodontitis progression.

Cystatin C is an antimicrobial peptide that can be internalized in macrophages. In addition, this peptide has dual activity, since it inhibits the intracellular bacterial load of P. gingivalis and regulates the inflammatory response. The immunoregulatory role was shown through a decrease in inflammatory cytokines and an increase in anti-inflammatory cytokines. Cystatin C decreased NO production and cell apoptosis. The increase in ROS production can explain its potent antimicrobial effect. These findings could aid in a better understanding of this peptide’s properties and its possible application in infectious and inflammatory oral diseases.
Supplemental Information
a) Lysates of macrophages infected with P. gingivalis . Lysates of macrophages infected with P. gingivalis and stimulated with Cystatin C, for b)24 h, c)48 h, d)96h
Figure 1: Effect of cystatin C on P. gingivalis growth and macrophage viability.
(A) 5 × 106 CFU/mL of P. gingivalis were cultured in the presence of cystatin C at different concentrations, for 1, 12, 24, 48, and 96 h. Cells without stimulation were used as a control group. when compared to the control group. The significance represents a p < 0.05 (*). (B) 1 × 105 cells were co-incubatedat different concentrations of cystatin C for 24 h. No significant differences were found when comparing different concentrations of cystatin C with control group p& γ τ 0.05 (ns). Data represent the mean ± SD of five independent experiments (n = 5).

  Download full-size image
DOI: 10.7717/peerj.17252/fig-1
Figure 2: P. gingivalis intracellular survival assay.
Macrophages were infected with P. gingivalis (MOI: 1:10) for 1, 3, 24, 48, and 96 h.Infected macrophages were stimulated with cystatin C for 24 h and treated with metronidazole (200 µg/mL) and gentamicin (300 µg/mL) to eliminate all extracellular bacteria. Macrophages were lysed at the indicated times and lysates were plated on anaerobic blood agar plates to determine CFU/mL. Macrophages lysates without bacterium and without stimulation were used as a control groups. Statistical differences were observed between macrophages infected with P. gingivalis, and macrophages infected with P. gingivalis plus cystatin C after 24, 48 and 96 h (p < 0.05) and are represented with asterisks (*). No statistical differences were observed after 48 and 96 h (p > 0.05). Data represent the mean ± SD of five independent experiments (n = 5).

  Download full-size image
DOI: 10.7717/peerj.17252/fig-2
Figure 3: Localization of cystatin c in macrophages infected with P. gingivalis and stimulated with cystatin C for 24 hours.
Macrophages were stained with: DAPI(blue); (A), (I), (M), (P), anti-cystatin C (Anti Cy3- bright red) (B), (F), (J), (N), (Q). P. gingivalis was stained with CFDA (green) (C), (G), (K), (ñ), (R). Merge (D), (H), (L), (O), (S). Red arrows exhibit: Intracellular bacteria in the macrophage. White arrows show localization of cystatin C in the macrophage. Scale bar 20 µm.

  Download full-size image
DOI: 10.7717/peerj.17252/fig-3
Figure 4: Cytokine production in macrophages infected with P. gingivalis and stimulated with cystatin C.
Macrophages were infected with P. gingivalis (MOI 1:10) for 3 h and stimulated with cystatin C for 24 h. Basal and LPS-stimulated cells were used as controls. Supernatants from experimental and control groups were analyzed by ELISA. (A) TNF-α expression, (B) IL-1β expression, (C) IL-10 expression. Statistical differences in the expression of TNF-α and IL-1β comparing control group with P. gingivalis p < 0.05. Statistically significant were also observed when comparing control group with P. gingivalis plus cystatin C (p < 0.05). Significant differences comparing P. gingivalis with P. gingivalis plus cystatin C were observed (p < 0.05). No statistical differences were observed when comparing control group with cystatin C (p& γ τ ;0.05). Statistical differences in the expression of IL-10 were observed when comparing control group with cystatin C (p < 0.05), and when comparing control group with P. gingivalis plus cystatin C (p < 0.05). Furthermore, significant differences were observed when comparing P. gingivalis with P. gingivalis plus Cystatin C (p < 0.05). The statistical difference are represented with asterisks (*). Data represent the mean ± SD of five independent experiments (n = 5).

  Download full-size image
DOI: 10.7717/peerj.17252/fig-4
Figure 5: Production of reactive oxygen species and nitric oxide in macrophages infected with P. gingivalis and stimulated with cystatin C.
Macrophages were stimulated with P. gingivalis (MOI 1:10) for 3 h and stimulated with cystatin C for 24 h. Basal cells and cells stimulated with LPS were used as controls. (A) ROS-production for flow cytometry in experimental and control groups. Statistical differences in the production of ROS were observed when comparing control group with cystatin C and between Porphyromonas gingivalis and with P. gingivalis plus cystatin C (p < 0.05). Significant differences were also observed when comparing P. gingivalis, with P. gingivalis plus cystatin C (p < 0.05). (B) Expression of nitrites in supernatants of experimental groups and controls incubated for 48 h. Statistical differences were observed in the NO production when comparing control group with P. gingivalis, and with P. gingivalis plus cystatin C (p < 0.05). Significant differences were also observed when comparing P. gingivalis with P. gingivalis plus cystatin C (p < 0.05). The statistical difference are represented with asterisks (*). No statistical differences were observed when comparing control group with cystatin C (p < 0.05). Data represent the mean ± SD of five independent experiments (n = 5).

  Download full-size image
DOI: 10.7717/peerj.17252/fig-5
Figure 6: Transmission electron microscopy (TEM) of macrophages infected with P. gingivalis and treated with cystatin C.
Cell pellets of cystatin C-stimulated infected macrophage for standard transmission electron microscopy were processed. (A) Basal macrophages. Macrophages infected with P. gingivalis during (B) 24 h, (C) 48 h, (D) 96 h. Macrophages infected with P. gingivalis and stimulated with cystatin C for (E) 24 h, (F) 48, (G) 96 h. Black arrows indicates the presence of P. gingivalis. (H) Note bacterium entering the macrophages (arrow).

  Download full-size image
DOI: 10.7717/peerj.17252/fig-6
Figure 7: Effect of cystatin C on apoptotic activation of macrophages infected with P. gingivalis.
TUNEL staining scatter plots after 96 h of incubation of (A) basal macrophages, (B) macrophages incubated with DNase I (600U/mL), (C) macrophages stimulated with cystatin C, (D) macrophages infected with P. gingivalis, (E) macrophages infected with P. gingivalis and stimulated with cystatin C. (F) Percentage of TUNEL positive cells. Cells without stimulation and stimulated with DNase I as a control group, were used. Scatter plots of Annexin V staining after 96 h incubation of (G) basal macrophages, (H) camptothecin (4 µg/mL) incubated macrophages, (I) cystatin C-stimulated macrophages, (J) P. gingivalis-infected macrophages, (K) macrophages infected with P. gingivalis and stimulated with cystatin C. (Annexin-V2PI2 [viable cells], Annexin V+PI+ [late or necrotic apoptosis), Annexin V+PI3 [cellular apoptosis]. (L) Percentage of cells Annexin V positive cell. Cells without stimulation and stimulated with camptothecin are used as control group. (M) Activity of Caspase-3 on macrophages infected with P. gingivalis. Macrophages were infected with P. gingivalis (MOI 1:10) for 96 h and stimulated with cystatin C for 24 h. Cells without stimulation and stimulated with camptothecin are used as control group. Data represent the mean ± SD of five independent experiments (n = 5). The significance represents a p < 0.05 (*) with respect to the control group.

  Download full-size image
DOI: 10.7717/peerj.17252/fig-7