Study On The Effect And Mechanism Of Complement C3a/C4a And Invariant Nature Killer T Cells On Osteoclasts In Patients With Multiple Myeloma

Objective:To explore the effect and mechanism of complement C3a/C4a and invariant nature killer T(iNKT)cells on osteoclasts in patients with multiple myeloma(MM).Contents:The effects of C3a/C4a and iNKT cells on the occurrence of myeloma bone disease(MBD)were preliminarily investigated by detecting the serum levels of C3a/C4a and the number of iNKT cells in both newly diagnosed multiple myeloma(NDMM)patients and healthy controls,and analyzing the correlation with the bone disease stages,the number of osteoblast precursors(OBPs)and osteoclast precursors(OCPs)and biological indicators of MBD.By adding C3a/C4a or?-galactosylceramide(?-GalCer)(specific activation amplifiers of iNKT cells)to culture system of osteoclasts induced from mononuclear cells in vitro,and the expression of related gene/proteins,number and function of osteoclasts were detected,so as to further clarify the effect and potential mechanism of C3a/C4a and iNKT cells on osteoclasts in patients with MM.Methods:Part 1 studies on the effect and mechanism of complement C3a/C4a on osteoclasts in patients with MM1.To explore the correlation between the serum levels of C3/C4/C4a in patients with NDMM and the severity of bone disease:The serum levels of C3 and C4 were detected by rate scattering method.Serum levels of C4a and tartrate-rsistant acid phosphatase isoform-5b(TRACP-5b)were detected by enzyme-linked immunosorbent assay(ELISA).Serum levels of osteocalcin(OCN)/procollagen I amino-terminal propeptide(PINP)/carboxy-terminal cross-linking telopeptide of type I collagen(CTX)were detected by enzyme-linked immunosorbent assay(ECLIA).Flow cytometry was used to detect the number of OBPs/OCPs.Grade the severity of bone disease in NDMM.The serum levels of complement C3,C4 and C4a were analysed the correlation with the grading of bone disease,number of OBPs/OCPs,levels of the biomarker OCN/PINP related to bone reconstruction and the biomarker CTX/TRACP-5b related to bone destruction in NDMM.2.To explore the effect of C3a/C4a on the formation and function of osteoclasts in patients with NDMM:Bone marrow mononuclear cells from NDMM patients were divided into experimental group(adding C3a/C4a)and control group(the same volume of dimethylsulfoxide(DMSO)),using the macrophage colony stimulating factor(M-CSF)and Receptor Activator for Nuclear Factor-?B Ligand(RANKL)inducing osteoclasts from mononuclear cells,osteoclasts area per view were observed and analysed by light microscope after the identification by tartrate resistant acid phosphatase(TRAP)staining.The mRNA expression levels of RANKL/Osteoclast Associated Receptor(OSCAR)/TRAP/Cathepsin K genes from osteoclasts were detected and analysed by qRT-PCR.Osteoclast resorption pit were induced in sterilization bovine cortical bone slices,and the resorption pit area per view was observed and compared by scanning electron microscopy.3.To investigate and verify the relevant signaling pathways of C3a and C4a on osteoclasts in patients with NDMM:The bone marrow mononuclear cells from NDMM patients was divided into the experimental group(adding C3a/C4a)and control group(the same volume of DMSO).Osteoclasts were induced by using M-CSF and RANKL and total RNA of osteoclasts was extracted.RNA-Seq was used to detect the differentially expressed genes on osteoclasts between the complement group and control group,and the possible signal pathways were analyzed.Quantitative RT-PCR,Western blot and pathway inhibitors were used for further validation.Part 2 studies on the effect and mechanism of iNKT cells on osteoclasts in patients with MM1.To explore the correlation between iNKT cells or cytokines secreted by iNKT cells and the severity of bone disease in NDMM patients:Flow cytometry was used to detect the percent of iNKT cells and their subsets,OBPs/OCPs,as well as the levels of IFN-?/TNF/IL-4/IL-13 secreted by iNKT cells in peripheral blood of NDMM patients and healthy controls.Serum levels of OCN/PINP/CTX were detected by ECLIA.Grade the severity of bone disease in NDMM.The number of iNKT cells and the levels of IFN-?/TNF/IL-4/IL-13 were analysed the correlation with the grading of bone disease,number of OBPs/OCPs and levels of the biomarker OCN/PINP related to bone reconstruction and the biomarker CTX related to bone destruction in NDMM.2.To investigate the proliferation effects of iNKT cells stimulated by?-GalCer and the effects on osteoclast regulated by iNKT cells in NDMM patients and healthy controls in vitro:The peripheral blood mononuclear cells from NDMM patients and healthy controls was divided into the experimental group(adding?-GalCer)and control group(the same volume of DMSO).The number of iNKT cells was detected by flow cytometry on 07/14 days after culture for 14 days.Osteoclasts were induced from mononuclear cells by using M-CSF and RANKL.After the identification by TRAP staining,osteoclasts was observed using light microscope.The number of osteoclasts was counted and compared.3.To explore the possible mechanism of the effect of iNKT cells on osteoclast:The levels of cytokines in the co-culture supernatant were detected by ELISA.The peripheral blood mononuclear cells from NDMM patients and healthy controls was divided into the experimental group(adding?-GalCer or/and human recombinant cytokines or cytokine blockers)and control group(the same volume of DMSO).Osteoclasts were induced from mononuclear cells by using M-CSF and RANKL.After the identification by TRAP staining,osteoclasts was observed using light microscope.The number of osteoclasts was counted and compared.The mRNA expression levels of TRAP/OSCAR/RANKL genes on osteoclasts were detected and compared by qRT-PCR.Results:Part 1 studies on the effect and mechanism of complement C3a/C4a on osteoclasts in patients with MM1.The serum levels of C3,C4 and C4a in NDMM patients were significantly positive correlated with the severity of bone disease,the number of OCPs,and levels of the biomarker CTX/TRACP-5b related to bone destruction.2.In vitro,the osteoclasts area per view induced by C3a 1?g/ml(52.794±13.027%)and 10?g/ml(51.797±12.464%)was significantly increased than control group(0?g/ml)(33.668±8.427%)(P<0.001;P<0.001),the mRNA relative expression of osteoclasts related genes OSCAR/TRAP/RANKL/Cathepsin K induced by C3a1?g/ml(median 5.041;3.726;1.638;4.752)and 10?g/ml(median 5.140;3.702;2.250;5.172)was significantly increased than control group(0?g/ml)(median 3.137;2.004;0.573;2.257)(1?g/ml P=0.001;P=0.003;P<0.001;P=0.008;1?g/ml P<0.001;P=0.019;P<0.001;P=0.002),and the absorption area of osteoclast resorption pit per view induced by C3a 1?g/ml(51.464±11.983%)and 10?g/ml(50.219±12.067%)was also significantly increased than control group(0?g/ml)(33.845±8.331%)(P<0.001;P<0.001)in NDMM patients.There was no difference among the osteoclasts area,relative expression of osteoclasts related genes and absorption area of osteoclast resorption pit between C4a(1?g/ml and 10?g/ml)group and control group(0?g/ml).3.RNA-Seq was performed on total RNA of osteoclasts induced by C3a in 1?g/ml group and 0?g/ml group of 4 patients with NDMM.There were 184 differentially expressed genes that were detected by RNA-Seq analysis.KEGG Pathway enrichment bubble chart shows C3a may through Phosphoinositide 3-kinase(PI3K)signaling pathways(including PI3K-Akt pathway and AKT-independent signaling pathway)promotes the proliferation of osteoclast.Upregulated differentially expressed genes in this pathway among at least 3 patients with sequencing were validated by qRT-PCR and Western Blot.It was found that the relative expression level of Phosphoinositide dependent kinase-1(PDK1)/Serum and glucocorticoid inducible protein kinases(SGK3)genes(median 2.078;4.428)in C3a group(1?g/ml)was significantly higher than control group(0?g/ml)(median 1.336;1.714)(P<0.001;P=0.001).The relative grayscale levels of PDK1/P-SGK3 protein(1.785±0.323;2.190±0.274)in C3a group(1?g/ml)was significantly stronger than control group(0?g/ml)(0.8653±0.588;0.176±0.152)(P=0.034;P<0.001).Under the action of C3a in patients with NDMM,osteoclasts area per view in SGK inhibitor(EMD638683)1?M group(39.244±9.089%)and 10?M group(39.299±9.587%)significantly reduced than control group(0?M)(54.884±12.837%)(P<0.001;P<0.001),the relative expression of osteoclast related genes OSCAR/RANKL/TRAP/Cathepsin K in EMD638683 1?M group(median 0.869;1.097;0.902;1.328)and 10?M group(median 0.703;1.391;0.843;1.418)significantly decreased than control group(0?M)(median 2.270;3.024;2.208;3.237)(1?M P=0.015;P=0.002;P=0.003;P=0.015;10?M P=0.012;P=0.006;P<0.001;P=0.017),and the absorption area per view of osteoclast resorption pit in EMD638683 1?M(35.383±7.794%)group and 10?M group(32.886±8.993%)significantly reduced than control group(0?M)(49.358±11.856%)(P<0.001;P<0.001).Part 2 studies on the effect and mechanism of iNKT cells on osteoclasts in patients with MM1.The number of iNKT cells and subsets CD4~-CD8~+and DN iNKT cells decreased significantly,and the levels of Th1 factor(IFN-?and TNF)secreted by iNKT cells decreased,while the levels of Th2 factor(IL-4 and IL-13)increased in patients with NDMM.The number of iNKT cells(including CD4~-CD8~+iNKT cells)and the levels of IFN-?secreted by iNKT cells in NDMM patients who are at stage C of bone disease were significantly lower than at stage A/B of bone disease and healthy controls.Moreover,the number of iNKT cells and the level of IFN-?secreted by iNKT cells were significantly negative correlated with the level of the biomarker CTX related to bone destruction and the number of OCPs.2.On day 7 and 14,the number of iNKT cells amplified by?-GalCer in the healthy controls was significantly higher than control group(the same volume of DMSO)(on day 7:2.359±0.267%vs 0.451±0.046%,P<0.001;on day 14:4.584±0.362%vs1.490±0.188%,P<0.001),and the amplification effect of iNKT cells stimulated by?-GalCer in NDMM patients was not significant.The number of osteoclasts produced under the stimulation of?-GalCer decreased significantly(512±32 per 2cm~2 vs 758±55 per 2cm~2,P=0.001)than control group(with the same volume of DMSO)in healthy controls.The number of osteoclasts produced under the stimulation of?-GalCer than control group(with the same volume of DMSO)has a tendency to reduce,but no statistical significance in NDMM patients.3.In the healthy controls,the levels of IFN-?in the supernatant of the osteoclasts co-cultured with iNKT cells activated by?-GalCer were increased than control group(with the same volume of DMSO)on the 7/14th day.In the healthy controls,the number of osteoclasts induced by the addition of?-GalCer and IFN-?blocker(anti-IFN-?/?-GalCer group)(719±45 per 2cm~2)were significantly increased than adding?-GalCer(?-GalCer group)(542±31 per 2cm~2)(P=0.009),the relative expression levels of TRAP/OSCAR/RANKL mRNA of osteoclast related genes in the anti-IFN-?/?-GalCer group(median 1.443/1.307/6.132)were also significantly increased than?-GalCer group(median 0.238/0.322/0.642)(P=0.007,P=0.002,P=0.025).In patients with NDMM,the number of osteoclasts induced in IFN-?/?-GalCer group(529±37 per 2cm~2)were significantly reduced than?-GalCer group(765±36 per 2cm~2)(P<0.001)and control group(805±44 per 2cm~2)(P<0.001),the relative expression level of RANKL mRNA of osteoclast related genes in IFN-?/?-GalCer group(median 0.076)was also significantly decreased than?-GalCer group(median 5.807)(P=0.036)and control group(11.433)(P=0.029).Conclusions:Complement C3a activates osteoclasts by enhancing SGK3phosphorylation on the PI3K signaling pathway in patients with MM,thus leading to the occurrence of bone diseases.SGK inhibitor has a significant inhibitory effect on osteoclasts in patients with MM.Invariant NKT cells inhibit the osteoclastogenesis by secretion of IFN-?and this function was impaired in patients with NDMM on account of quantitative and functional deficiency of iNKT cell,thus giving rise to the occurrence of MBD.These studies provide important evidences for the search for new therapeutic targets and strategies for MBD patients.