Effects Of Catecholamines On Th17/Treg Imbalance And Joint Inflammation In Mouse Model Of Rheumatoid Arthritis

Objectives:Catecholamines(CAs),including norepinephrine(NE),epinephrine(E)and dopamine(DA),are expressed principally in nerve and endocrine cells.Tyrosine hydroxylase(TH)is a rate-limiting enzyme for synthesis of catecholamines.In addition to regulation of cardiovascular,respiratory and digestive activities,the catecholamines synthesized and released by nerve and endocrine cells also regulate immune function.In recent decade,the reports from other and our laboratories have presented that catecholamines can also be synthesized and secreted by immune cells.The T cell-derived catecholamines modulate differentiation and function of helper T(Th)cells,with an effect of facilitating the shift of Th1/Th2 balance toward Th2 polarization.We hypothesized that this shift toward Th2polarizationisbeneficialforameliorationofimbalancein proinflammatory/antiinflammatory responses in some autoimmune diseases.RA is a chronic autoimmune disease characterized by synovial hyperplasia and inflammatory cell recruitment,intraarticular fibrin deposition and,eventually,cartilage and bone destruction.A few studies showed that T helper(Th)17/regulatory T(Treg)imbalance play a pivotal role in RA.Collagen-induced arthritis(CIA)was employed in this study to clarify the role of TH on Th17/Treg imbalance,inflammation and bone erosion in RA.Methods:1.Male DBA/1 mice of 8-10 weeks were immunized by chicken type II collagen.Mice were observed for clinical score of limbs at the beginning from day 21post-immunization.When mice were sacrificed on day 35 and day 55 after immunization,the thickness of hind paws and ankle joints,histopathological change(HE stain)and serum anti-CII IgG antibody level(ELISA)were detected.2.Expression and secretion of Th17 and Treg-related transcription factor,ROR-γt and Foxp3,cytokines,interleukin(IL)-17,IL-22,transforming growth factor(TGF)-βand IL-10 in ankle joints,joint tissue supernatants,spleen and serum of CIA mice were assessed by real-time PCR,Western blot and ELISA.Expression of TH,vesicular monoamine transporter(VMAT)-2 and monoamine oxidase(MAO)in ankle joints and spleen of CIA mice were detected.Immunostaining was used to observe co-expression of ROR-γt or Foxp3 with TH,VMAT-2 or MAO in mice,respectively.The percentage of IL-17~+TH~+and CD25~+TH~+cells in mouse splenic CD4~+T cells was determined by flow cytometry.3.On the 41th day following first immunization,the CD4~+T cells were isolated from the mouse spleen tissues and sorted using magnetic beads.The constructed plasmids used for TH overexpression or knockdown were transfected into CD4~+T cells.Expression of TH in CD4~+T cells of CIA mice was detected.Expression and secretion of Th17 and Treg-related transcription factors and cytokines in CD4~+T cells and cell supernatants of CIA mice were assessed by real-time PCR,Western blot and ELISA.Number of Th17 and Treg cells in mouse CD4~+T cells was determined by flow cytometry4.On the 33th and 41th day following first immunization,lentivirus of overexpression and knockdown of TH gene were injected into the paw of CIA mice.Mice were observed for clinical score of limbs at the beginning from day 21 post-immunization.When mice were sacrificed on day 69 after immunization,the thickness of hind paws and ankle joints,histopathological change(HE stain)and serum anti-CII IgG antibody level(ELISA)were detected.Expression of TH in ankle joints of CIA mice was detected.Expression and secretion of Th17 and Treg-related transcription factors and cytokines in ankle joints,joint tissue supernatants,spleen and serum of CIA mice were assessed by real-time PCR,Western blot and ELISA.Number of Th17 and Treg cells in mouse splenic CD4~+T cells was determined by flow cytometry.TRAP~+cells were dected by immunohistochemical analyses.The relative mRNA expression of osteoclast and osteoblast-related genes were assessed by real-time PCR.Results:1.Compared with intact mice,the mice that had received CII injection began to rise in clinical score on day 31 after the first immunization,reached a significant increase on day35,and remained high until the last day observed in this study.The thickness of both ankle joints and rear paws was augmented and anti-CII IgG antibody level was notably elevated in CIA mice.Histopathologic observation of ankle joints of CIA mice displayed an evident inflammatory change on day 35 and a major damage to bones on day 55post-immunization.2.Expression and secretion of ROR-γt,Il-17 and IL-22 in ankle joints,joint tissue supernatants,spleen and serum of CIA mice were upregulated.Expression and secretion of Foxp3,TGF-βand IL-10 in ankle joints,joint tissue supernatants and serum of CIA mice were upregulated.The Th17 response tended to decrease and the Treg response tended to be larger with the CIA progression.3.Expression TH,VMAT-2 and MAO in ankle joints and spleen of CIA mice was upregulated.The co-localized cells of ROR-γt or Foxp3 with TH,ROR-γt or Foxp3 with VMAT-2,ROR-γt or Foxp3 with MAO were observed in the synovial tissue of CIA mice and in the spleen of intact and CIA mice.In splenic CD4~+T cells,the cells expressing TH were increased during CIA.These cells that expressed more TH in CIA were mainly Th17cells rather than Treg cells.4.TH gene overexpression in CD4~+T cells from CIA mice reduced Th17 cell percentage as well as Th17-related transcription factor and cytokine expression and secretion,whereas TH gene knockdown enhanced the Th17 cell activity.In contrast,TH gene overexpression increased Treg-related cytokine expression and secretion in CD4~+T cells of CIA mice,while TH gene knockdown decreased the Treg cell changes.5.TH gene overexpression in paw of CIA mice reduced the clinical score,joint inflammation and inflammatory cell infiltration,while the role of TH gene knockdown was opposite.6.TH gene overexpression in paw of CIA mice reduced Th17-related transcription factor and cytokine expression and secretion in ankle joints and joint supernatants,whereas TH gene knockdown enhanced the Th17 cell activity.In contrast,TH gene overexpression increased Treg-related cytokine expression and secretion in ankle joints and joint supernatants of CIA mice,while TH gene knockdown decreased the Treg cell changes.7.TH gene overexpression reduced the expression of osteoclast specific genes,TRAP,Ctsk,Ctr and MMP-9,while TH gene knockdown enhanced the osteoclast activity.TH gene overexpression increased the expression of osteoblast specific genens,ALP,Col1α1and Col1α2,while TH gene knockdown reduced the osteoblast activity.8.TH gene overexpression and knockdown in paw of CIA mice did not affect anti-CII IgG antibody level,expression and secretion of Th17 and Treg-related transcription factors and cytokines and number of Th17 and Treg cells in spleen and serum.Conclusions:Th17/Treg imbalance principally manifesting a striking increase in Th17 frequency and IL-17 and IL-22 release occurs during RA.Synthesis,storage and degradation of catecholamines upregulate in both spleen and ankle joints in RA mice.Th17 and Treg cells can synthetize,store and degrade catecholamies.That the upregulation of synthesis of catecholamines in Th17 cells of CIA mice contributes to inhibit Th17/Treg imbalance.The synthesis of catecholamines represents a compensatorily protective mechanism by which Th17/Treg imbalance,joint inflammation and bone destruction is alleviated.