Uremic Toxin Indoxyl Sulfate Promotes Macrophage-Associated Low-Grade Inflammation and Epithelial Cell Senescence.

In this study, we investigated the impact of the uremic toxin indoxyl sulfate on macrophages and tubular epithelial cells and its role in modulating the response to lipopolysaccharide (LPS). Indoxyl sulfate accumulates in the blood of patients with chronic kidney disease (CKD) and is a predictor of overall and cardiovascular morbidity/mortality. To simulate the uremic condition, primary macrophages and tubular epithelial cells were incubated with indoxyl sulfate at low concentrations as well as concentrations found in uremic patients, both alone and upon LPS challenge. The results showed that indoxyl sulfate alone induced the release of reactive oxygen species and low-grade inflammation in macrophages. Moreover, combined with LPS (proinflammatory conditions), indoxyl sulfate significantly increased TNF-α, CCL2, and IL-10 release but did not significantly affect the polarization of macrophages. Pre-treatment with indoxyl sulfate following LPS challenge induced the expression of aryl hydrocarbon receptor (Ahr) and NADPH oxidase 4 (Nox4) which generate reactive oxygen species (ROS). Further, experiments with tubular epithelial cells revealed that indoxyl sulfate might induce senescence in parenchymal cells and therefore participate in the progression of inflammaging. In conclusion, this study provides evidence that indoxyl sulfate provokes low-grade inflammation, modulates macrophage function, and enhances the inflammatory response associated with LPS. Finally, indoxyl sulfate signaling contributes to the senescence of tubular epithelial cells during injury.

Candida albicans Shields the Periodontal Killer Porphyromonas gingivalis from Recognition by the Host Immune System and Supports the Bacterial Infection of Gingival Tissue.

Candida albicans is a pathogenic fungus capable of switching its morphology between yeast-like cells and filamentous hyphae and can associate with bacteria to form mixed biofilms resistant to antibiotics. In these structures, the fungal milieu can play a protective function for bacteria as has recently been reported for C. albicans and a periodontal pathogen-Porphyromonas gingivalis. Our current study aimed to determine how this type of mutual microbe protection within the mixed biofilm affects the contacting host cells. To analyze C. albicans and P. gingivalis persistence and host infection, several models for host-biofilm interactions were developed, including microbial exposure to a representative monocyte cell line (THP1) and gingival fibroblasts isolated from periodontitis patients. For in vivo experiments, a mouse subcutaneous chamber model was utilized. The persistence of P. gingivalis cells was observed within mixed biofilm with C. albicans. This microbial co-existence influenced host immunity by attenuating macrophage and fibroblast responses. Cytokine and chemokine production decreased compared to pure bacterial infection. The fibroblasts isolated from patients with severe periodontitis were less susceptible to fungal colonization, indicating a modulation of the host environment by the dominating bacterial infection. The results obtained for the mouse model in which a sequential infection was initiated by the fungus showed that this host colonization induced a milder inflammation, leading to a significant reduction in mouse mortality. Moreover, high bacterial counts in animal organisms were noted on a longer time scale in the presence of C. albicans, suggesting the chronic nature of the dual-species infection.

Porphyromonas gingivalis in Alzheimer's disease brains: Evidence for disease causation and treatment with small-molecule inhibitors.

Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, was identified in the brain of Alzheimer's disease patients. Toxic proteases from the bacterium called gingipains were also identified in the brain of Alzheimer's patients, and levels correlated with tau and ubiquitin pathology. Oral P. gingivalis infection in mice resulted in brain colonization and increased production of Aß1-42, a component of amyloid plaques. Further, gingipains were neurotoxic in vivo and in vitro, exerting detrimental effects on tau, a protein needed for normal neuronal function. To block this neurotoxicity, we designed and synthesized small-molecule inhibitors targeting gingipains. Gingipain inhibition reduced the bacterial load of an established P. gingivalis brain infection, blocked Aß1-42 production, reduced neuroinflammation, and rescued neurons in the hippocampus. These data suggest that gingipain inhibitors could be valuable for treating P. gingivalis brain colonization and neurodegeneration in Alzheimer's disease.

Aristolochic acid I determine the phenotype and activation of macrophages in acute and chronic kidney disease.

Acute and chronic kidney injuries are multifactorial traits that involve various risk factors. Experimental animal models are crucial to unravel important aspects of injury and its pathophysiological mechanisms. Translating knowledge obtained from experimental approaches into clinically useful information is difficult; therefore, significant attention needs to be paid to experimental procedures that mimic human disease. Herein, we compared aristolochic acid I (AAI) acute and chronic kidney injury model with unilateral ischemic-reperfusion injury (uIRI), cisplatin (CP)- or folic acid (FA)-induced renal damage. The administration of AAI showed significant changes in serum creatinine and BUN upon CKD. The number of neutrophils and macrophages were highly increased as well as AAI-induced CKD characterized by loss of tubular epithelial cells and fibrosis. The in vitro and in vivo data indicated that macrophages play an important role in the pathogenesis of AA-induced nephropathy (AAN) associated with an excessive macrophage accumulation and an alternative activated macrophage phenotype. Taken together, we conclude that AA-induced injury represents a suitable and relatively easy model to induce acute and chronic kidney injury. Moreover, our data indicate that this model is appropriate and superior to study detailed questions associated with renal macrophage phenotypes.

Lack of cathelicidin processing in Papillon-Lefèvre syndrome patients reveals essential role of LL-37 in periodontal homeostasis.

BACKGROUND: Loss-of-function point mutations in the cathepsin C gene are the underlying genetic event in patients with Papillon-Lefèvre syndrome (PLS). PLS neutrophils lack serine protease activity essential for cathelicidin LL-37 generation from hCAP18 precursor. AIM: We hypothesized that a local deficiency of LL-37 in the infected periodontium is mainly responsible for one of the clinical hallmark of PLS: severe periodontitis already in early childhood. METHODS: To confirm this effect, we compared the level of neutrophil-derived enzymes and antimicrobial peptides in gingival crevicular fluid (GCF) and saliva from PLS, aggressive and chronic periodontitis patients. RESULTS: Although neutrophil numbers in GCF were present at the same level in all periodontitis groups, LL-37 was totally absent in GCF from PLS patients despite the large amounts of its precursor, hCAP18. The absence of LL-37 in PLS patients coincided with the deficiency of both cathepsin C and protease 3 activities. The presence of other neutrophilic anti-microbial peptides in GCF from PLS patients, such as alpha-defensins, were comparable to that found in chronic periodontitis. In PLS microbial analysis revealed a high prevalence of Aggregatibacter actinomycetemcomitans infection. Most strains were susceptible to killing by LL-37. CONCLUSIONS: Collectively, these findings imply that the lack of protease 3 activation by dysfunctional cathepsin C in PLS patients leads to the deficit of antimicrobial and immunomodulatory functions of LL-37 in the gingiva, allowing for infection with A. actinomycetemcomitans and the development of severe periodontal disease.

Inhibition of GSK3 abolishes bacterial-induced periodontal bone loss in mice.

The tissue destruction that characterizes periodontitis is driven by the host response to bacterial pathogens. Inhibition of glycogen synthase kinase 3ß (GSK3ß) in innate cells leads to suppression of Toll-like receptor (TLR)-initiated proinflammatory cytokines under nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 transcriptional control and promotion of cyclic adenosine monophosphate response element-binding (CREB)-dependent gene activation. Therefore, we hypothesized that the cell permeable GSK3-specific inhibitor, SB216763, would protect against alveolar bone loss induced by the key periodontal pathogen, Porphyromonas gingivalis (P. gingivalis), in a murine model. B6129SF2/J mice either were infected orally with P. gingivalis ATCC 33277; or treated with SB216763 and infected with P. gingivalis; sham infected; or exposed to vehicle only (dimethyl sulfoxide [DMSO]); or to GSK3 inhibitor only (SB216763). Alveolar bone loss and local (neutrophil infiltration and interleukin [IL]-17) and systemic (tumor necrosis factor [TNF], IL-6, Il-1ß and IL-12/IL-23 p40) inflammatory indices also were monitored. SB216763 unequivocally abrogated mean P. gingivalis-induced bone resorption, measured at 14 predetermined points on the molars of defleshed maxillae as the distance from the cementoenamel junction to the alveolar bone crest (p < 0.05). The systemic cytokine response, the local neutrophil infiltration and the IL-17 expression were suppressed (p < 0.001). These data confirm the relevance of prior in vitro phenomena and establish GSK3 as a novel, efficacious therapeutic preventing periodontal disease progression in a susceptible host. These findings also may have relevance to other chronic inflammatory diseases and the systemic sequelae associated with periodontal infections.