Cystatin C: immunoregulation role in macrophages infected with Porphyromonas gingivalis.

Background: Periodontitis is a chronic infectious disease, characterized by an exacerbated inflammatory response and a progressive loss of the supporting tissues of the teeth. Porphyromonas gingivalis is a key etiologic agent in periodontitis. Cystatin C is an antimicrobial salivary peptide that inhibits the growth of P. gingivalis. This study aimed to evaluate the antimicrobial activity of this peptide and its effect on cytokine production, nitric oxide (NO) release, reactive oxygen species (ROS) production, and programmed cell death in human macrophages infected with P. gingivalis. Methods: Monocyte-derived macrophages generated from peripheral blood were infected with P. gingivalis (MOI 1:10) and stimulated with cystatin C (2.75 µg/ml) for 24 h. The intracellular localization of P. gingivalis and cystatin C was determined by immunofluorescence and transmission electron microscopy (TEM). The intracellular antimicrobial activity of cystatin C in macrophages was assessed by counting Colony Forming Units (CFU). ELISA assay was performed to assess inflammatory (TNFα, IL-1ß) and anti-inflammatory (IL-10) cytokines. The production of nitrites and ROS was analyzed by Griess reaction and incubation with 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), respectively. Programmed cell death was assessed with the TUNEL assay, Annexin-V, and caspase activity was also determined. Results: Our results showed that cystatin C inhibits the extracellular growth of P. gingivalis. In addition, this peptide is internalized in the infected macrophage, decreases the intracellular bacterial load, and reduces the production of inflammatory cytokines and NO. Interestingly, peptide treatment increased ROS production and substantially decreased bacterial-induced macrophage apoptosis. Conclusions: Cystatin C has antimicrobial and immuno-regulatory activity in macrophages infected with P. gingivalis. These findings highlight the importance of understanding the properties of cystatin C for its possible therapeutic use against oral infections such as periodontitis.

Macrophages immunomodulation induced by Porphyromonas gingivalis and oral antimicrobial peptides.

Porphyromonas gingivalis is a keystone pathogen associated with periodontitis development, a chronic inflammatory pathology characterized by the destruction of the supporting teeth structure. Macrophages are recruited cells in the inflammatory infiltrate from patients with periodontitis. They are activated by the P. gingivalis virulence factors arsenal, promoting an inflammatory microenvironment characterized by cytokine production (TNF-α, IL-1ß, IL-6), prostaglandins, and metalloproteinases (MMPs) that foster the tissular destruction characteristic of periodontitis. Furthermore, P. gingivalis suppresses the generation of nitric oxide, a potent antimicrobial molecule, through its degradation, and incorporating its byproducts as a source of energy. Oral antimicrobial peptides can contribute to controlling the disease due to their antimicrobial and immunoregulatory activity, which allows them to maintain homeostasis in the oral cavity. This study aimed to analyze the immunopathological role of macrophages activated by P. gingivalis in periodontitis and suggested using antimicrobial peptides as therapeutic agents to treat the disease.

Antimicrobial and anti-inflammatory activity of Cystatin C on human gingival fibroblast incubated with Porphyromonas gingivalis.

Background: Periodontal disease is considered one of the most prevalent chronic infectious diseases, often leading to the disruption of tooth-supporting tissues, including alveolar bone, causing tooth mobility and loss. Porphyromonas gingivalis is considered the major etiological agent of this disease, having a plethora of virulence factors, including, lipopolysaccharides (LPS), hemolysins, and proteinases. Antimicrobial peptides are one of the main components of the innate immune response that inhibit the growth of P. gingivalis. The aim of this study was to analyze the antimicrobial activity of cystatin C and to assess the effect on the inflammatory and anti-inflammatory cytokines, the production of reactive oxygen species, and in the release of nitric oxide by human gingival fibroblasts incubated with P. gingivalis in the presence and absence of cystatin C. Methods: P. gingivalis ATCC 33277 was exposed to cystatin C for 24h and co-cultured with human gingival fibroblasts (HGFs) ATCC CRL-2014. The effect of cystatin on growth of P. gingivalis and HGFs was evaluated. Pro-inflammatory (TNFα, IL-1ß) and anti-inflammatory (IL-10) cytokines were determined by ELISA in the supernatants of HGFs incubated with P. gingivalis exposed to cystatin C. Additionally, nitrites and reactive oxygen species (ROS) production were evaluated. Results: Cystatin Cinhibited the growth of P. gingivalis without affecting HGFs. Incubation of HGFs with P. gingivalis led to a significant increase of TNF-α and IL-1ß. In contrast, HGFs incubated with P. gingivalis exposed to cystatin C showed a decreased production of both cytokines, whereas IL-10 was enhanced. Incubation of HGFs with P. gingivalis led to an increase of nitric oxide (NO) and ROS production, which was reduced in the presence of the peptide. Conclusions: Cystatin C inhibits the growth of P. gingivalis and decreases the inflammatory cytokines, ROS, and NO production during infection of HGFs with P. gingivalis. Knowledge on the antimicrobial and immunomodulatory properties of cystatin C could aid in the design of new therapeutic approaches to facilitate the elimination of this bacterium to improve the treatment of periodontal disease.

Antimicrobial peptides properties beyond growth inhibition and bacterial killing.

Antimicrobial peptides (AMPs) are versatile molecules with broad antimicrobial activity produced by representatives of the three domains of life. Also, there are derivatives of AMPs and artificial short peptides that can inhibit microbial growth. Beyond killing microbes, AMPs at grow sub-inhibitory concentrations also exhibit anti-virulence activity against critical pathogenic bacteria, including ESKAPE pathogens. Anti-virulence therapies are an alternative to antibiotics since they do not directly affect viability and growth, and they are considered less likely to generate resistance. Bacterial biofilms significantly increase antibiotic resistance and are linked to establishing chronic infections. Various AMPs can kill biofilm cells and eradicate infections in animal models. However, some can inhibit biofilm formation and promote dispersal at sub-growth inhibitory concentrations. These examples are discussed here, along with those of peptides that inhibit the expression of traits controlled by quorum sensing, such as the production of exoproteases, phenazines, surfactants, toxins, among others. In addition, specific targets that are determinants of virulence include secretion systems (type II, III, and VI) responsible for releasing effector proteins toxic to eukaryotic cells. This review summarizes the current knowledge on the anti-virulence properties of AMPs and the future directions of their research.