Suppressor of cytokine signaling 1 expression during LPS-induced inflammation and bone loss in rats

Abstract This study aimed to characterize the dynamics of suppressor of cytokine signaling (SOCS1) expression in a rat model of lipopolysaccharide-induced periodontitis. Wistar rats in the experimental groups were injected three times/week with LPS from Escherichia coli on the palatal aspect of the first molars, and control animals were injected with vehicle (phosphate-buffered saline). Animals were sacrificed 7, 15, and 30 days after the first injection to analyze inflammation (stereometric analysis), bone loss (macroscopic analysis), gene expression (qRT-PCR), and protein expression/activation (Western blotting). The severity of inflammation and bone loss associated with LPS-induced periodontitis increased from day 7 to day 15, and it was sustained through day 30. Significant (p < 0.05) increases in SOCS1, RANKL, OPG, and IFN-γ gene expression were observed in the experimental group versus the control group at day 15. SOCS1 protein expression and STAT1 and NF-κB activation were increased throughout the 30-day experimental period. Gingival tissues affected by experimental periodontitis express SOCS1, indicating that this protein may potentially downregulate signaling events involved in inflammatory reactions and bone loss and thus may play a relevant role in the development and progression of periodontal disease.

Modulation of cell proliferation, survival and gene expression by RAGE and TLR signaling in cells of the innate and adaptive immune response: role of p38 MAPK and NF-KB

Objective: The aim of this study was to evaluate a possible synergism between AGE-RAGE and TLR4 signaling and the role of p38 MAPK and NF-kB signaling pathways on the modulation of the expression of inflammatory cytokines and proliferation of cells from the innate and adaptive immune response. Material and Methods: T lymphocyte (JM) and monocyte (U937) cell lines were stimulated with LPS and AGE-BSA independently and associated, both in the presence and absence of p38 MAPK and NF-kB inhibitors. Proliferation was assessed by direct counting and viability was assessed by a biochemical assay of mitochondrial function. Cytokine gene expression for RAGe, CCL3, CCR5, IL-6 and TNF-α was studied by RT-PCR and RT-qPCR. Results: RAGE mRNA expression was detected in both cell lines. LPS and AGE-BSA did not influence cell proliferation and viability of either cell line up to 72 hours. LPS and LPS associated with AGE induced expression of IL-6 and TNF-α in monocytes and T cells, respectively. Conclusions: There is no synergistic effect between RAGE and TLR signaling on the expression of IL-6, TNF-α , RAGE, CCR5 and CCL3 by monocytes and lymphocytes. Activation of RAGE associated or not with TLR signaling also had no effect on cell proliferation and survival of these cell types. .

Modulação da proliferação, morte celular e expressão gênica de mediadores inflamatórios pela ativação de RAGE e TLR4 em células da resposta imune inata e adaptativa: papel das vias de sinalização p38 MAPK e NF-kB / Modulation of cell proliferation, survival and gene expression of inflammatory mediators by RAGE and TLR signaling in cells of the innate and adaptive immune response: role of p38 MAPK and NF-KB

O diabetes está associado à maior susceptibilidade à infecções e sepsis, demonstrando uma influência desta condição sobre a resposta imune. A doença periodontal é um tipo de infecção crônica, modulada pela resposta imune que apresenta maior prevalência e severidade em pacientes diabéticos. Nosso objetivo foi avaliar um possível sinergismo entre os receptores RAGE e TLR4 na modulação da proliferação celular, atividade metabólica, apoptose e expressão de citocinas inflamatórias em células da resposta imune inata e adaptativa. Como objetivo secundário, avaliamos o papel das vias de sinalização p38 MAPK e NF-kB na expressão dos genes inflamatórios por estas células após estimulação de RAGE e TLR4. Linhagens de células humanas de linfócitos T (JM) e monócitos (U937) foram estimulados com LPS e AGE-BSA tanto de forma independente como associados. A estimulação foi realizada também na presença e na ausência de inibidores bioquímicos para p38 MAPK (SB203580) e NF-kB (Bay 11-7082). A proliferação celular foi determinada por ensaio de exclusão azul de trypan, a apoptose pela via intrínseca e atividade metabólica foi avaliada por um ensaio bioquímico da função mitocondrial, a expressão de citocinas foi estudada por RT-PCR e RTqPCR e a ativação das vias de sinalização de interesse pelos estímulos utilizados foi investigada através de Western blotting. LPS e AGE-BSA não influenciaram a proliferação e sobrevivência celular de monócitos e linfócitos T após 24, 48 e 72 h. LPS, isoladamente ou associado a AGE, induziu a expressão de IL-6 e TNF-α em monócitos e células T, respectivamente. A ativação de p38 MAPK, mas não de NF-kB, foi necessária para a indução por LPS e LPS/AGE de IL-6 e TNF-α por LPS, isolado ou associado a AGE. A expressão de RNAm de RAGE foi detectada em ambos os tipos de célulares. RNAm de CCL3 foi expresso de forma mais marcante em monócitos, particularmente após estimulação com LPS. Não houve sinergismo na sinalização entre RAGE e TLR4 na modulação da expressão de IL-6, TNF-α, RAGE, CCR5 e CCL3 por monócitos e células T

Diabetes is associated to increased susceptibility to infections and sepsis, indicating that this condition modulates the immune response. Periodontal disease is one type of chronic infection, which is modulated by the immune response and presents with increased prevalence and severity in diabetic patients. Our objective was to evaluate a possible synergism between RAGE and TLR4 signaling on the modulation of cell proliferation, metabolic activity, apoptosis and gene expression of inflammatory cytokines by cells of the innate and adaptive immune response. As a secondary objective, we assessed the role of p38 MAPK and NF-kB signaling pathways on the expression of the inflammatory genes by these cells after stimulation of RAGE and TLR4. Human cell lines of T lymphocytes (JM) and monocytes (U937) were stimulated with LPS and AGE-BSA both independently and associated. Stimulation was also performed in the presence and absence of biochemical inhibitors for p38 MAPK (SB203580) and NF-kB (Bay 11-7082). Cell proliferation was determined by trypan blue dye exclusion assay, apoptosis by the intrinsic pathway and metabolic activity were assessed by a biochemical assay of the mitochondrial function, cytokine gene expression was studied by RTPCR and RT-qPCR and the activation of the selected signaling pathways after RAGE and TLR4 activation was investigated by Western blotting. LPS and AGE-BSA did not influence cell proliferation and survival 24, 48 and 72 h after stimulation. LPS, alone or associated with AGE-BSA, induced expression of IL-6 and TNF- mRNA by monocytes and T cells, respectively. Activation of p38 MAPK, but not of NF-kB, was required for LPS and LPS/AGE-induced induction of IL-6 and TNF. RAGE mRNA expression was detected in both cell types. CCL3 mRNA expression levels were higher in monocytes upon stimulation with LPS. There was no synergism between RAGE and TLR4 signaling on the expression of IL-6, TNF-, RAGE, CCR5 and CCL3 by monocytes and T cells