Regulation Of Innate Immune In Response To S.aureus By Protein Geranylgeranylation And Expression Of PGGT1B In Psoriasis

BackgroundsStaphylococcus aureus is the most common commensal bacteria that colonizes in human nasal mucosa,respiratory tract and skin.Patients with dermatosis carry significantly higher rates of S.aureus than healthy people.Long-term colonization of S.aureus affects the immune system and lead to progression of chronic inflammatory skin diseases.Recently several clinic trails have shown that patients who took statins had lower morbidity and mortality caused by S.aureus infection.Statins are competitive inhibitors of enzyme-3-Hydroxy-3-Methylglutaryl CoA Reductase(HMGCR)of the mevalonate pathway for hypercholesteremia treatment.Mevalonate pathway generate intermediates other than cholesterols,such as geranylgeranyl pyrophosphate(GGPP).Inhibition of HMGCR by statins would also cause shortage of GGPP and inhibition of protein geranylgeranylation.Protein geranylgeranylation is a lipoprotein modification that plays a key role in regulating membrane localization and activation of small GTPase by regulating the attachment of geranylgeranyl group to the carboxy-terminal cysteine residue of CAAX.Deficiency of protein geranylgeranylation has been found to associated with inflammatory diseases such as rheumatoid arthritis,inflammatory bowel disease and type II diabetes.However,no study had been done to elucidate the relationship between deficiency of protein geranylgeranylation and S.aureus infection or inflammatory skin disorders.Objectives1.To study the impact of protein geranylgeranylation on phagocytosis,bacteria killing and reactive oxygen species(ROS)production of macrophages and neutrophils during the infection of S.aureus;2.To explore the production and release of cytokines as well as activation of associated signaling pathways in deficiency of protein geranylgeranylation;3.To clarify the expression of PGGT1B in lesions and peripheral blood mononuclear cell(PBMCs)in patients with psoriasis.Methods1.Bone marrow derived macrophages(BMDM)and bone marrow-derived neutrophils(BMN)were collected from Pggt1b+/+ LyZ2-Cre mice and Pggt1bfl/fl LyZ2-Cre mice.Purity of neutrophils was measured by flow cytometry,and Pggt1b knockdown efficiency was verified by Western Blotting.2.BMDM and BMN were infected with S.aureus,the ability of phagocytosis and killing of S.aureus between wild type and Pggt1b deficient BMDMs and BMNs were compared with Gentamycin Protection Assay and Neutrophil killing assay;the production of ROS between wild type and Pggt1b deficient macrophages and nuetrophils were compare with chemiluminescence method.3.Real-time PCR and ELISA were performed to compare the production and release of cytokines by wild-type and Pggt1b deficient BMDM.The difference in activation of associated signaling pathways and inflammasome between wild-type and Pggt1b deficient BMDM were compared with Western Blotting assay.4.Real-time PCR was used to study the expression of PGGT1B in lesion by comparing with non-lesion and normal control as well as in PBMC by comparing with nonnal control.Results1.PGGT1B expression was significantly decreased in BMDM and BMN from Pggt1bfl/fl LyZ2-Cre mice compared with Pggt1b+/+ LyZ2-Cre,indicating that Pggt1b gene were significantly inhibited in BMDM and BMN in conditioned knockout mice.2.The capacity of phagocytosis and bacteria killing was significantly impaired in Pggt1b deficient BMDM and BMN;the production of ROS was significantly reduced in Pggt1b deficient BMDM and BMN.3.The production and release of pro-inflammatory cytokines(IL-12,IL-6,TNF-a,IL-1β)were significantly increased and production of IL-10 was significantly decreased in Pggt1b deficient BMDM following S.aureus infection;The activation of IKK was not affected,activation of MAPK were delayed but not inhibited in Pggt1b deficient BMDM following S.aureus infection,whereas the activation of PI(3)K-Akt-Gsk3p/mTOR kinase signaling pathway was significantly inhibited in Pggt1b deficient BMDM.4.The activation of inflammasome Caspase-1 was significantly increased,which lead to significantly increased maturation and release of IL-1β following S.aureus infection.5.The expression of PGGT1B was significantly decreased in lesions compared with non-lesion and normal controls;the expression of PGGTIB in PBMC from patients with psoriasis was significantly reduced compared with normal controls.Conclusions1.Deficiency of protein geranylgeranylation inhibits phagocytosis,bacteria killing and ROS production of BMDM and BMN.2.Deficiency of protein geranylgeranylation promote the production of pro-inflammatory cytokines such as IL-1 β and TNF-α and inhibit the production of IL-10 following S.aureus infection.3.Deficiency of protein geranylgeranylation have no impact on the activation of IKK,delay but not inhibit the activation of MAPK and inhibit the activation of PI(3)K-Akt-Gsk3β/mTOR kinase signaling pathway following S.aureus infection;it also promotes excessive activation of inflammasome Caspase-1,leading to significant increased maturation and release of IL-1β.4.PGGTIB expression was significantly reduced in lesions and PBMC from patients with psoriasis.