Molecular Mechanism Of ORMDL3 Promoting B Cells Autophagy In The Pathogenesis Of SLE

Systemic lupus erythematosus (SLE) (OMIM152700) is a clinically common and heterogeneous autoimmune disease with a widespread involvement of multi-systems and organs. It is characterized by autoantibody production, complement activation and immune complex deposition. Epidemiological evidence supports a female predominance of SLE with a female to male ratio of approximately 10:1, and the incidence in Chinese populations being around 31-70/100000. SLE is uniformly conceived as an inherited disease with a heritability as high as 43.6%. Prior studies have supported a risk of individuals with diseased siblings 8-29 times higher than that of the general population; and twin studies indicated an elevated risk of monozygotic twins (24-65%) than that of dizygotic twins (2-9%). Due to its pathogenesis largely elusive and clinical manifestations universally complicated, SLE remains one of the greatest challenges for human health, with the scarcity of effective diagnostic and treatment modalities. Hence, it is of vital significance to explore the detailed molecular mechanism underlying the SLE pathogenesis and the development of targeted therapeutic drugs.Dysregulated immune responses have long been recognized in SLE that include overactive B lymphocyte function, autoreactive T lymphocyte proliferation, autoantibody production, immune complex clearance impairment and cytokine production abnormality. Among these, it is gaining great attention that aberrant B cell development and differentiation lead to the impaired immune tolerance and B cell over-activation. Besides, accumulating evidence has pointed towards the relevance of autophagy in the initiation and progression of SLE.Autophagy is an evolutionarily conserved catabolic process degrading aggregated protein and damaged organelles, and in the meantime, sustaining the cells with an alternative source of material and energy at stressed conditions. At the physiological level, autophagy is crucial in maintaining cellular homeostasis and survival, and also in modulating innate and adaptive immune responses. It has been recently illuminated that autophagy participates in CD4+T cell selection and central immune tolerance establishment and regulates the differentiation of Treg cells in the context of autoimmune diseases. Besides,autophagy also plays a role in the differentiation to plasma cells and humoral immune responses. With the advent of genome-wide association study (GWAS),an array of autophagy-associated genes, such as ATG5, ATG7,UVRAG and IRGM has been found associated with SLE susceptibility and additionally, a large amount of autophagosomes and elevated autophagic activity have been demonstrated in the B and T cells from SLE cases and lupus mice.With the aid of GWAS multiple susceptibility genes have been found associated with SLE among which the 17q21 region has also been suggested associated with several other autoimmune diseases including Crohn’s Disease, rheumatic arthritis and ulcerative colitis. Within 17q21, the IKZF3-ZPBP2 and GSDMB-ORMDL3 region has been potentially recognized as one of the susceptibility genes/loci.ORMDL3 gene belongs to the orosomucoid 1-like gene family, maps in 17q21 and encodes an endoplasmic reticulum (ER)-anchored protein consisting of 153 animoacid. Evidence is emerging that ORMDL3 suppresses the activity of serine palmitoyltransferase (SPT), the very first rate-limiting enzymein sphingolipid biosynthesis pathway.Thus it is a possible negative regulator of sphingolipid biosynthesis that participates in inhibiting the NK and T cell function in SLE pathogenesis. Besides,ORMDL3 co-localizes with ER-anchored calcium pump (SERCA) and therefore may promote ER stress (ERS) and unfolded protein response (UPR) via disturbing pump activity. And ERS is a common stimulator for autophagy. In addition,ORMDL3 elevates the secretion of IL-17, indicating that ORMDL3 is likely to be a regulator in T cell development in early stage.The relevant investigation regarding the association of ORMDL3 with complex diseases is just in the initiating phase. What have been confirmed are that ORMDL3, as a robustly verified susceptibility gene for asthma, can lead to airway remodeling and hyperresponsiveness, cause eosinophil migration and activation and influence T cell responses, through impairing intracellular calcium homeostasis and promoting ERS. In the meantime, ORMDL3 has been demonstrated associated with type I diabetes, Crohn’s Disease, rheumatic arthritis and ankylosing spondylitis. Given the role of ORMLD3 in immunity and autoimmune diseases, it is reasonable to postulate that ORMDL3 confers SLE predisposition by activating ERS, inducing autophagy, promoting B cell proliferation and fine-tuning the development and differentiation of B cells in SLE pathogenesis. In this study, we will in vitro and in vivo clarify the above-mentioned hypothesis in three separated sections below. Section One Generation and characterization of Ormdl3 knockout mice and Pristane induced lupus miceDue to the particular relevance of ORMDL3 gene with inflammatory autoimmune diseases and our previous findings that the expression of ORMDL3 gene is increased in SLE patients and Faslpr/lpr lupus mice, it is reasonable to postulate the involvement of oRMDL3 in the pathogenesis of SLE. Despite several reports regarding the role of oRMDL3 in mediating immune cells and cytokines, it remains largely undetermined that whether or not oRMDL3 could influence the development of immune cells. Thus we established the Ormdl3-/- mice in order to in vivo investigate how oRMDL3 might modulate the immune system.First we obtained the ormdl3+/-mice based on TALEN targeted gene knockdown strategy and further got Ormdl3-/- mice through mating. Due to the high homology among ORM family members and the difficulty of differentiating Ormdl3 from other members by antibody, we finally used qPCR and semi-quantitative PCR to evaluate the knockdown efficiency and found relatively high efficiency in spleen and lung tissue. Then we compared the Ormdl3-/-mice with wild type mice in terms of body weight and spleen weight and we found lower weights and smaller sizes of spleen (P<0.01) but not body weight in Ormdl3-/- mice compared to WT mice. Next we observed relatively normal structure of lung from Ormdl3-/- mice but smaller white pulparea of Ormdl3-/- mice compared to WT mice (probably due to the spleen of Ormdl3-/- mice was diminished).We found no significant difference after comparing the proportions of all kinds of T lymphocytes subsets of thymus and spleen from Ormdl3-/- mice and WT mice at the age of 6-24 weeks. And also we observed no difference in the proportions of Pro-B cells (B220+CD43+) and Pre-B cells (B220+CD43-) between Ormdl3-/- mice and WT mice in bone marrow. However,a significant decrease was noted in total B cell (CD19+), mature B cell (CD19+IgM+IgDhi) and Transitional 2 B cell(CD19+IgMhiIgDhi) in spleen but not in T1 B cell(CD19+IgMhiIgDlo) and bone marrow B cell from Ormdl3-/- mice compared to WT mice. Because immature and mature B cells all develop from Pro-B cells (B220+CD43+) and Pre-B cells (B220+CD43-), the results above indicated that lack of Ormdl3 could result in dysregulation of cell emigration from bone marrow to peripheral lymphoid organ, hence, lead to disproportionality of different type B cells in spleen. The development of B cell from T1 B to T2 B cell in spleen depend on BAFF, so next we detected the expression level of Baff in Ormdl3-/-mice and WT mice. The results showed that lack of Ormdl3 significantly decrease the expression of Baff in spleen lymphocyte cells and serum concentration of Baff, meanwhile, the secretion of IgM and IgG decreased heavily, which indicating ORMDL3 may upregulate the expression of BAFF and affect the development or survival of B cells.Besides, we noted the high expression of Ormdl3 in B cells of all stages from bone marrow and spleen with the expression reaching the peak in immature B cells from spleen. We also demonstrated the significant difference of Bla cells (B220loCD5+)but not B1b cells (B220loCD5-) from abdominal cavity perfusion fluid between the Ormdl3-/- mice and WT mice.Next we used flow cytometry to measure the proliferation and cell cycle of spleen cells from Ormdl3-/- mice and WT mice at the age of 6-8 weeks and we observed no effect of ORMDL3 knockdown on cell proliferation and cell cycle.We found an enhanced apoptosis in Ormdl3-/-mice after comparing the apoptotic rates of the total lymphocytes and CD19+B cells of spleen but not the bone marrow, from Ormdl3-/- mice compared to WT mice.This might lead to the decrease of the immature and mature B cells of spleen from Ormdl3-/- mice.On account of regulation of ORMDL3 in the development of B cells, lack of Ormdl3 may relieve the symptoms of SLE model mice.Therefore, we had constructed the model by intraperitoneal injection of 0.5ml Pristane in Ormdl3+/+mice and Ormdl3/-/ mice, respectively. Compared with Pristane injected WT mice, Pristane injected Ormdl3-/- mice showed milder splenomegaly, lower concentration of urine protein, milder renal pathologicaldamage and lower proportion of CD4+/CD8+. Although these results would not be enough to confirm the effects of Ormdl3 in SLE-like mice model, at least it pointed out that Ormdl3 may have a role in the pathogenesis of SLE.Taken together, we successfully established the Ormdl3/-/ mice in which we induced the lupus phenotype using pristane and in vivo affirmed the mechanism by which Ormdl3 modulates B cell development. The results of this section suggested the possibility of ORMDL3 as a novel therapeutic target in treating SLE. Section Two ORMDL3 promotes autophagy and suppresses apoptosisORMDL3, as an ER-anchored protein, can inhibit the ER calcium activity and then lead to ER calcium imbalance and ERS. And persistent ERS can potently induce the autophagy. An array of prior studies have demonstrated large amounts of autophagosomes in immune cells from SLE patients and lupus model mice. And our work found a high level of expression of ORMDL3 gene in peripheral blood of SLE patients and in spleen of lupus mice in which the autophagy-associated genes were also consistently induced. Hence we assume that ORMDL3 might positively regulate autophagy by promoting autophagy in SLE and we used Raji cell line (human Burkitt’s lymphoma) and HEK293 to in vitro prove it. In the meantime, we also used the Ormdl3-/- mice to in vivo confirm the hypothesis.First, to knockdown and overexpress ORMDL3 gene, we established stably transfected cell lines and then measured the autophagy-related protein expression using western blot. Our results demonstrated that overexpression of ORMDL3 could promote the expression of Beclinl and LC3II, both dose-dependently and time-dependently.When rapamycin was used to facilitate autophagic flow and chloroquine for blocking autophagic flow, overexpression of ORMDL3 could lead to further accumulation of LC3II, indicating the positive role of ORMDL3 in autophagy. To verify the above-mentioned results, we applied immunofluorescence to determine the formation of LC3 puncta in ORMDL3-overexpressed HEK293 cells. And we demonstrated that overexpression of ORMDL3 could promote the formation of LC3 puncta. Furthermore, we used electron microscope to view ORMDL3-overexpressed HEK293 and Raji cells and we noticed overexpression of ORMDL3 lead to pronounced autophagosomes (including autophagosomes and autolysosomes). All these evidence pointed towards the role of ORMDL3 promoting autophagy in HEK293 and Raji cells.We next aimed to confirm the vital molecular mechanisms by which ORMDL3 regulates autophagy. Small interfering RNA (siRNA) was used to knockdown Beclinl and ATG5 in ORMDL3-overexpressed HEK293 cell lines and we found that silencing Beclinl or ATG5 could alleviate the promotion of LC3II conversion by ORMDL3 overexpression mainly through Beclinl.Autophagy and apoptosis orchestrate a complex interplay in cellular homeostasis and survival and we observed enhanced apoptosis of lymphocytes from spleen and B cells in Ormdl3-/- mice. Next we sought to investigate the role of ORMDL3 in apoptosis and we measured the apoptosis-related proteins in Raji cells and found that overexpression of ORMDL3 significantly inhibited the cleavage of Caspase 3, Caspase 9 and PARP, and the results of flow cytometry yielded consistent results. This is not discordant with what we observed in lymphocytes from spleen and B cells in Ormdl3-/- mice.It enlightened a notion that ORMDL3 is crucial in resisting cellular apoptosis and we further assumed that the apoptosis-inhibitory effect of ORMDL3 was via the induction of autophagy.To sum up, the above-mentioned evidence indicated that ORMDL3 could promote autophagy and suppress apoptosis to facilitate the survival of cells.Section ThreeORMDL3 induces autophagy via up-regulating ATF6Former investigation demonstrated that ORMDL3 could inhibit ER calcium pump, lead to calcium efflux and ultimately cause ERS, an effective autophagy inducer. In the second section, we have uncovered that ORMDL3 is a positive regulator of autophagy. It still remains unresolved that whether or not ORMDL3 promotes autophagy via ERS, and if yes, which pathway among ERS regulatory networks is specifically responsible for it.First we tested the mRNA expression of several ERS markers in ORMDL3-overexpressed and ORMDL3-knockdown Raji cells. We found that overexpression of ORMDL3 significantly unregulated the mRNA expression of Binding protein (BiP) to activate ERS and the expression of Bip was significantly decreased in lymphocytes from spleen in Ormdl3-/- mice. Next, we verified that ORMDL3 modulates ERS mainly through ATF6 pathway, but not PERK and IRE1-XBP1. Specifically, overexpression of ORMDL3 resulted in increased expression of ATF6 (90KDa) and enhanced cleavage of 50KDa fragment. However, ORMDL3 seemed to have no effect on the phosphorylation of eIF2a and splice of XBP1. And we verified the results in Ormdl3-/- mice.To further confirm the regulatory role of ORMDL3 on ATF6, we used immunofluorescence to determine the translocation of ATF6 from cytoplasm to nucleus in ORMDL3-overexpressed HEK293 cells. And our results showed that overexpression of ORMDL3 promoted the ATF6 nucleus translocation, and the phenomenon was further augmented when we treated the cells with thapsigargin (TG,a potent ERS inducer). Thus we concluded that ORMDL3 could initiate ERS through ATF6 pathway.Subsequently, we aimed to explore whether or not ORMDL3 could modulate autophagy via induction of ATF6 pathway. To resolve this question, we first had to confirm that whether or not ATF6 could influence autophagic activity. "We showed that overexpression of ATF6 significantly upregulated the expression of Beclinl and the ratio of LC3II to I, while knockdown of ATF6 yielded opposite results. We further silenced the expression of ATF6 in ORMDL3-overexpressed HEK293 cells and we noticed that knockdown of ATF6 partially alleviated the up-regulation of Beclinl and LC3II by ORMDL3 overexpression. The results of immunofluorescence were also not discordant and further proved that ORMDL3 regulates autophagy via induction of ATF6.We next applied rescue experiment in which we transiently transfected the ORMDL3-knockdown HEK293 cells with pEGFP-ORMDL3 expression vector and measured the alterations of ATF6 and autophagy-related protein. The results showed that the supplement of ORMDL3 by pEGFP-ORMDL3 vector in ORMDL3-knockdown HEK293 cells could effectively reverse the downregulation of ATF6, Beclinl and LC3II (and also autophagic activity) by ORMDL3 silencing.The results of immunofluorescence also supported the evidence above-mentioned.Besides, we found the ORMDL3-inducing factor IL-13 could also promote autophagy and inhibit apoptosis. However, it still remains unsolved that whether or not the effects of IL-13 on autophagy and apoptosis are through ORMDL3.In this part we verified a possible mechanism by which ORMDL3 upregulates autophagy via activation of ATF6 pathway in ERS.