Mycophenolic Acid, Active Form of Mycophenolate Mofetil, Interferes With the IRF7 Nuclear Translocation and Type I IFN Production by Plasmacytoid Dendritic Cells

doi:10.21203/rs.3.rs-34087/v1

Download PDF

Research article

Mycophenolic Acid, Active Form of Mycophenolate Mofetil, Interferes With the IRF7 Nuclear Translocation and Type I IFN Production by Plasmacytoid Dendritic Cells

https://doi.org/10.21203/rs.3.rs-34087/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 09 Nov, 2020

Read the published version in Arthritis Research & Therapy →

You are reading this older preprint version

Read the latest preprint version →

Background

Both humoral and cellular immune mechanisms are involved in the onset and progression of the autoimmune responses in systemic lupus erythematosus (SLE). Plasmacytoid dendritic cells (pDCs) play a central role in the pathogenesis of SLEvia the dysregulation of type I interferon (IFN) production;these cells act together with activated myeloid DCs (mDCs), to amplify the vicious pathogenic spiral of autoimmune disorders. Therefore, control of the aberrant DC activation in SLE may provide an alternative treatment strategy against this disease. Mycophenolate mofetil (MMF), which has been used to treat lupus nephritis, specifically blocks the proliferation of B and T lymphocytes via inhibition of inosine-5-monophosphate dehydrogenase. Here, we focus on the effects of MMFin targeting DC functions, especially the IFN response of pDCs.

Methods

We isolated human blood pDCs and mDCs by flow cytometry and examined the effect of mycophenolic acid (MPA), which is a metabolic product of MMF, on the toll-like receptor (TLR) ligand response of DC subsets. Additionally, we cultured pDCs with serum from SLE patients in the presence or absence of MPA and then examined the inhibitory function of MPA on SLE serum-induced IFN-a production.

Results

We found that treatment with 1-10mM of MPA (covering the clinical trough plasma concentration range) dose-dependently downregulated the expression of CD80 and CD86 on mDCs(but not pDCs) without inducing apoptosis, in response to R848 or CpG-ODN, respectively. Notably, MPA significantly suppressed IFN-α production with IRF7 nuclear translocation in pDCsand IL-12 production with STAT4 expression in mDCs. We further identified that MPA had an inhibitory effect on SLE serum-induced IFN-α production by pDCs.

Conclusions

Our data suggest that MPA can interrupt the vicious pathogenic spiral of autoimmune disordersby regulating the function of DC subsets. This work unveiled a novel mechanism for the therapeutic ability of MMF against SLE.

Internal Medicine

SLE

plasmacytoid DCs

myeloid DCs

type I IFN

MPA

IRF7

Mycophenolate mofetil (MMF), which has been used to treat lupus nephritisas well asused in solid organ transplantation, is an uncompetitive and reversible inhibitor of inosine monophosphate dehydrogenase (IMPDH)(1, 2). In the body, MMF is naturally hydrolyzed into mycophenolic acid (MPA), which is the active form of MMF.MMF/MPA inhibits the de novo pathway of guanosine nucleotide synthesis without incorporation into DNA(3). Because T and Blymphocytes depend onthe de novo synthesis of purines for their proliferation, MMF/MPA has potent cytostatic effects on lymphocytes(2). MMF has been shown to prevent the occurrence of acute rejection in rat models of kidney and heart allotransplantation(4). MMF also decreases antibody production in mice(5).Thus, MMFfunctions as aversatile immune modulatorbecause ofitsbroad range of actions. The biologicalmechanisms underlying the therapeutic effects of MMF/MPAagainstrejection after organ transplantation are known to be mainly mediated by the inhibition of T and B lymphocytes, but the mechanisms by which MMF/MPA improves lupus nephritis are still largely unclear.

Dendritic cells (DCs) orchestrate both innate and adaptive immunity through cytokine-mediated and cell-cell contact mechanisms. In humans, DCs consist of two major subsets: myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). They play distinct roles in innate and adaptive immunitiesthrough theircapability toexpress specialized cytokines and molecules (6-8). Although they account for only a small fraction of cells, pDCs represent a major source of type I interferons (IFN) in the peripheral blood and lymphoid tissues(9). Thus, they play a central role in the innate antiviral immune response via their ability to produce vast amounts of type I IFN. This function is based on their selective expression of toll-like receptor (TLR)7 and TLR9, which respectively sense viral RNA and DNA within early endosomes (10). Additionally, pDCs constitutively express high levels of IFN regulatory factor 7 (IRF7) (11), andthe type I IFN production requires IRF7 to be phosphorylated and translocated into the nucleus through rapid interaction with MyD88 and IRF7 (12-14). Furthermore,phosphoinositide 3-kinase (PI3K) and Ik-kinase-a (IKK-a)are critically involved in type I IFN production(15-17).

Notably, a series of studies have indicated that pDCs also play a pathogenic role in the development of autoimmune diseases, such as systemic lupus erythematosus (SLE) or psoriasis,through their specialized function regarding type I IFN production(18-20). SLE is a heterogeneous disease in which excessive inflammation, autoantibodies, and complement activation lead to multisystem tissue damage. The vicious pathogenic spiral responsible for theautoimmune process in SLE, likely mediated by the continuous secretion of type I IFN (18), is thought to occur as follows:pDC-derived type I IFNrapidly triggers the differentiation and activation of myeloid DCs, which in turn elicit the differentiation of autoreactive CD4⁺ T cells, CD8⁺ T cells, and B cells as antigen-presenting cells through processing self-components. These autoreactive effectors induce tissue damage(thusproducingDNA/RNA fragments) and make auto anti-nuclear antibody. The resultant immune complexes,such as anti-DNA antibody-self-DNA, further activate pDCs through TLRs in a sustained manner to continuously produce type I IFN, which in turn amplifies the vicious autoimmune spiral. Consequently, pDCs and type I IFN represent specific cellular and molecular targets, respectively, in therapeutic strategies against autoimmune diseases such as SLE.

Although there is evidence indicating thatMMF/MPA has immunomodulatory effects on mDCs(21-23), there are no reports showing its effects on pDCs. Therefore, the present work focused on examining the effects of MMF/MPA on the blood DC subsets, especially pDCs.

We here show a novel function of MMF/MPA, i.e., the ability to inhibit IFN-a production by human pDCs upon cellular activation with SLE serum or CpG-ODNvia inhibiting the nuclear translocation of IRF7. In addition, MPAwas found to suppress mDC-derived IL-12 via inhibiting STAT4 activation. Our present results suggest that MMF/MPA can interfere with the vicious pathogenic spiral of autoimmune disorders by regulating the function of DC subsets.

Media and reagents

RPMI-1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 ng/ml streptomycin, and heat-inactivated 10% FBS (Biosource International) was used for cell cultures throughout the experiments. For human cell stimulation, we used 5 mM CpG-ODN 2216(Invivogen),1 mg/mlR848 (InvivoGen), anddifferent doses of MPA (Sigma) was dissolved in methanol;as a vehicle control, methanolwas diluted in parallel.

Cell isolation and culture

Human mDCs and pDCs were isolated from peripheral blood mononuclear cells (PBMCs) from healthy adult donors, as described previously [3,27]. CD11c⁺/BDCA4^-/lineage^-/CD4⁺ cells (defined as mDCs) and CD11c^-/BDCA4⁺/lineage^-/CD4⁺ cells (defined as pDCs) were sorted by FACS Aria^® (BD Biosciences)(Suppl. Fig. 1) to reach greater than 99% purity according to re-staining withanti-BDCA1 and anti-BDCA2antibodies. The purified mDCs and pDCs were incubated for 24 hwithR848 and CpG-ODN, respectively, in flat-bottomed 96-well plates at a concentration of 2×10⁴ cells in a final volume of 200 ml of medium per well,in the presence ofMPA or vehicle.

Stimulation of lupus serum

Experiments using lupus serum to stimulate pDCs were conducted as described previously (24). Lupus sera were obtained from five active SLE patients with low complement levels prior to steroid therapy who satisfied 2019 European League Against Rheumatism(EULAR)/American College of Rheumatology(ACR) classification criteria for SLE. All patients had anti-double-stranded DNA antibody. Written informed consent was obtained from all SLE patients. In these experiments, pDCs were stimulated with 20% lupus serum in flat-bottomed 96-well plates at a concentration of 2´10⁴ cells in 200 ml of medium per well.

Analyses of cells and supernatants

Human DC subsets were stained with PE-labeled CD86 (2331) and FITC-labeled CD80 (L307.4)monoclonal antibodies (all from BD Biosciences) and then analyzed by FACScalibur^® (BD Biosciences). The production of cytokines in the culture supernatants after 24 h was determined by enzyme-linked immunosorbent assay (ELISA). The ELISA kits for detecting human IL-12p40 and TNFwerepurchased from R＆D Systems. ELISA Kit for human IFN-a was purchased from PBL Biomedical Laboratories.

For the viability assay, cells were washed with PBS containing 2 mM EDTA, and viable cells were counted in triplicate,using trypan-blue exclusion to identify the dead cells. Viable cells were also evaluated as the annexin V-negative fraction using an Annexin V–FITC Kit (Sigma).

Real-time PCR

Purified DC subsets were stimulated with R848 or CpG-ODN in the presence of 10 mMMPA for 4h. The cells were then collected for cDNA synthesis using aQuantiTect.Reverse Transcription (RT) kit (QIAGEN). The DNA resulting from each RT reaction was subjected to real-time PCR using the SYBR® Green Master Mix(QIAGEN) for the detection of IRF7 and STAT4. The following primers were purchased from QIAGEN: IRF7 primer (QT00210595) and STAT4 primer (QT00014133). We selected glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the reference gene. Quantification of the mRNA transcription level was determined using the comparative cycle threshold (Ct) method. All samples were examined in duplicate, and the mean Ct value was calculated by Rotor Gene Q software version 2.2.3. The fold change in gene transcription was calculated as the ratio of D/DCt in the treated samples to untreated samples.

Fluorescence microscope

Purified pDCs were stimulated with CpG-ODN in the presence or absence of 10 mMMPA for 3 h. The cells were then seeded on glass slides by cytospin, mounted, and fixed with a BD Transcription Factor Buffer set (Cat. No. 562725).

Samples were labeled with Alexa Fluor-anti-human IRF-7 (G-8; sc-74472 AF488, Santa Cruz Biotechnology) and 4',6'-diamidino-2- phenylindole (DAPI). Images were acquired using a fluorescence microscope (BZ-9000, Keyence). Cells were regarded as negative when the expression level of IRF7 in the cytoplasm was higher and distinguishable from that in the nucleus, shown by DAPI staining. The ratio of nuclear IRF7-negative cells was calculatedbased on 50 cells from each donor.

Statistical analysis

Data were analyzed using paired t-tests. A value of p<0.05 was considered statistically significant. Data analysis was carried out using Prism (GraphPad Software).

Clinical concentrations of MPAdo not affect the survival of human DC subsets.

Clinical pharmacokinetics show that the mean clinical trough plasma MPA concentration of lupus nephritisfemale patients who were administered with 1,500 mg twice a day of oral MMF is10.6mM (3.4mg/L)(25). Another clinical study reportedthat the plasma concentration of MPA at trough in patients with severe lupus nephritis is 7.7 mM (2.47 mg/L) (26).Therefore, we used 1-10 mMMPA (covering the clinical trough plasma concentration range for MMF/MPA) in our experiments for this study. Concentrations of up to 10 mMof MPA did not induce apoptosis of pDCs or mDCs in response to CpG-ODN or R848, respectively(Fig. 1A and B). The clinical trough concentration of MPA dose-dependently downregulated the R848-induced high expression levels of CD80 and CD86on mDCs(Fig. 2).In contrast, pDCs stimulated with CpG-ODN did not substantially upregulate either CD80 nor CD86expression (Fig. 2A), and the addition of MPA did not significantly decreased these expression levels (Fig. 2B).

MPA inhibits IFN-a production from human pDCs.

The secretion of type I IFN is a key molecular event that initiates and promotes the autoimmune process(18), and pDCs are the major source of type I IFN in PBMCs (9). Thus, wenext examined whether MPAcould inhibitthe type I IFN production by pDCs. Notably, MPAdose-dependently inhibitedthe production of both IFN-aand TNF by pDCs in response to stimulation with CpG-ODN(Fig. 3A).

Immune complexes in serum consisting of auto-antibodies and self-DNA area pathogenic trigger forthe induction of aberrant continuous production of type I IFN by blood pDCs,which drives autoimmunityin SLE(18). SLE sera also contain a nuclear protein (high-mobility group box 1 protein [HMGB1]) or an antimicrobial peptideLL37released from damaged tissues or neutrophils(18, 19, 27);these host-derived transporter agents are required for triggering the SLE pathogenic process. Thus, SLE serapossess a complete set of the essentialcomponentsfor inducingpathogenic pDC-derivedtype I IFN.In previous in vitro experiments, the stimulation of PBMCs with serum obtained from a patient with SLE induced IFN-a production (24, 28, 29). Accordingly,we next investigated whether MPAcan inhibit theSLE serum-induced IFN-a production under conditions mimickingthe pathophysiological condition of SLE.We preliminarily tested sera from five patients with active SLE who had anti-double-stranded DNA antibody, from which we selected the two bestsera for inducing IFN-ain healthy PBMCs (data not shown). SLE serum (either serum-1 or serum-2) induced a significant level of IFN-a from pDCs (Fig.3B). Notably,concentrations of MPA of up to10 mMsignificantly repressed the SLE serum-induced IFN-a production in a dose-dependent manner (Fig. 3B). These data suggest that MPA has an inhibitory potential in relation to the IFN-a production triggered by SLE pathogenic conditions.

MPA interferes with the nuclear translocation of IRF7 in pDCs.

Because the essential molecular step in type I IFN production by pDCs has been shown to be the nuclear translocation of the constitutively expressed IRF7(30, 31),we next examined whether MPAalters this process by evaluatingboth the quantitative transcriptional level of IRF-7 and the migration of IRF7intothe nucleus upon activation. First, we performed real-time PCR assays, and the results show that the level ofIRF-7 RNA in pDCs wassignificantly upregulatedfollowing CpG-stimulation (Fig. 4A).Although the transcriptional upregulation of IRF-7 trended lowered following treatment with MPA, this difference did not reach statistical significance. Analysis with immunofluorescence microscopy revealed that IRF7 was localized in the cytoplasmic area of unstimulated pDCs (Fig. 4B). After stimulation with CpG-ODN, IRF7 expressionwas detected in the nucleus, as shown by its colocalization with DAPI nuclear staining, indicating the nuclear translocation of IRF7. This nuclear staining of IRF7 and the colocalization of IRF7 and DAPI staining induced by CpG stimulation was prevented by the presence of 10 mM MPA (Fig. 4B). Thus, MPA helped retain IRF7 in the cytoplasm, indicating its inhibitory effect on IRF7 nuclear translocation in pDCs. To quantify this finding, we counted the numbers of pDCs on the slide with or without nuclear IRF7 expression, as describedin Material and methods and in a previous study [10]. Almostallunstimulated pDCs werefound to be nuclear IRF7-negative (mean ± SEM: 95.6%± 1.2 %),and CpG stimulation significantly reduced the frequency of cells without IRF7 nuclear translocation(12.0%± 1.4 %)(Fig.4C).Treatment with MPA significantly augmented the frequency of nuclear IRF7-negative cellsfollowing stimulation with CpG (63.2%± 3.4 %)(Fig.4C). Thus, our result indicates that MPA acts as an inhibitor of IRF7 nuclear translocation.

MPA inhibits Th1-related responses in mDCs.

In the SLEpathogenic spiral, activated mDCs play a role in the induction ofautoreactive effectors such as CD4⁺ T cells, CD8⁺ T cells, and B cells. When stimulated with TLR ligands, mDCsare able to induce a Th1 response. To investigate the immunomodulatory actions of MPA on the function of human mDCs in Th1-related activation pathways, we analyzed the cytokine production bymDCsthat had been stimulated with R848. MPA inhibited the R848-induced productions of IL-12p40, and TNF in a dose-dependent manner(Fig. 3C). Based on the results ofreal-time PCRonmDCs, R848 significantly upregulated STAT4 mRNA,which is important for DC-mediated Th1 responses and IL-12 production(32, 33). Notably, we found that 10 mM MPAsignificantly downregulated R848-induced STAT4 mRNA (Fig 4D).

In NZB mice, whichdevelop an immune complex disease with nephritisthat is similar to human SLE, MMF has a beneficial effect on both renal and overall survival (34).MMF has been introduced as a novelimmunosuppressiveagent in SLE, often in patients who are intolerantor resistant to conventional immunosuppressive regimens (35, 36). Although outlined responses are described in prior publications, the mechanism of action for MMF/MPA in the SLE pathogenic spiral remains poorly understood, except for the direct suppressive effects on T and B lymphocytes. In patients with active SLE, auto-anti-DNA antibody not only is a quite common and specific marker, but it also correlates with disease activity, such as flares of lupus glomerulonephritis (37-40).Continuous activation of pDCs in the blood byimmune complexesinduces dysregulated type I IFN elevation, as a central molecular event, whichsubsequently causesmDCactivation and a resultant induction of autoreactive lymphocytesin the vicious autoimmune spiral of SLE (18, 19). Here, we focused on the function of human DC subsets involved in this pathogenic spiral and unveiledthe inhibitory potential of MPA against such pDC-derived IFN-a production and mDC function. We also found that MPA exerted aninhibiting action on IRF7 nuclear translocation in pDCs.Based on our results, treatment with MPAmay be able to interrupt theautoimmune pathogenic spiral by limiting the disordered production of type I IFN that occursunder the pathophysiological conditions of SLE. Thus, the present study revealed a possible mechanism underlying the therapeutic potential of MMF/MPA for treating SLE by targeting pDCs.Notably, trough plasma concentrations of MPA did not induce cell apoptosis of eitherpDCs or mDCs, suggesting that MPA at clinical doses does not cause excessive immunosuppression. Hence, MMF may be useful as a long-term maintenance therapy for SLE.

Hydroxychloroquine is aneffectivetherapeutic agent for SLE and has a mechanism similar to that of MPA; it is a strong TLR-signal inhibitor that suppresses the production of type I IFN by pDCs (41). MMF/MPA may exhibit cellular effectsvia mechanisms of action similar to those by which hydroxychloroquine treats autoimmunity (42).Accumulating evidence have substantiated the importance of TLRs in the pathogenesis and progression of autoimmune processes in SLE and its renal component (43). Based on the common properties of these two drugs, inhibition of the pDC-IFN axis may be an effective SLE treatment strategy.In fact, inhibition of TLR9 signaling with synthetic ODN can also exhibit beneficial effects on the progression of lupus nephritis in MRL^lpr/lpr mice (44).

The present study also revealedthe ability of MPA, used at the clinical trough level,to impair the maturation and Th1-inducing capacity of DCs by downregulating STAT4. In support of our findings, a recent in vitrostudy showed that 20 mM MMF can inhibit the LPS-induced expression of MHC-class II, CD86, CD40, CD83, and NF-kB in human blood mDCs(23).Moreover, MMF was reportedtosuppressthe differentiation of human monocyte-derived DCs (21, 22). These immunosuppressive effects of MMF/MPA on mDCs may further contribute to its ability to break the pathogenic spiral of SLE.

Ideally, pDCs from blood of SLE patients would have been used for evaluating the ability of MPA to suppress type I IFN production, triggered by pathogenic conditions. However, the blood of SLE patients has greatly reduced numbers of circulating pDCs (19).Furthermore, the numbers of pDCs in the bone marrow are very lowin lupus-prone mice(45).Therefore, because it was logistically and ethically difficult to perform these experiments using pDCs from SLE patients, we designed an experimental system using SLE sera.

To the best of our knowledge, this is the first study to investigate the effect of MMF/MPA on pDCs. This work focusing on DC functionclarified one of the possible mechanismsby which MPAcould be clinically effective in the treatment of SLE. Our findings suggest that MMF may be able to interrupt vicious pathogenic spiral of autoimmune disorders, not only by inhibiting T and B lymphocytes but alo by regulating the function of DC subsets.

MMF: Mycophenolate mofetil; MPA: mycophenolic acid; BDCA: blood dendritic cell antigen; DC: dendritic cells; IFN: interferon; IKK: IkB kinase; IRF7: IFN regulatory factor 7; PBMC: peripheral blood mononuclear cells pDC: plasmacytoid dendritic cell; SLE: systemic lupus erythematosus; TNF: tumor necrosis factor; TLR: toll-like receptor

Availability of data and materials

The data that support the findings of this study are available on request from the corresponding author (T.I.). The data are not publicly available due to information that could compromise research participant privacy.

Acknowledgement

We thank Katie Oakley, PhD, from Edanz Group (https://en-author-services.edanzgroup.com/) for editing a draft of this manuscript.

Funding

This study was supported in part by a grant (17K09963 to T.I.) from the Grant-in-Aid for Scientific Research (C), Japan Society for the Promotion of Science.

Author information

Affiliations

First Department of Internal Medicine, Kansai Medical University

Authors’ contributions

MS performed the experiments and wrote the paper. TIplanned, designed, and wrote the paper. AS and SN contributed to the experimental planning. MI,TA, KI, and HY supported the experiments. AT, HA, TN, YS, and YO contributed to the acquisition of patient data and samples. All authors read and approved the final manuscript.

Ethics declarations

Ethics approval and consent to participate

All studies involving human samples were performed under institutional review board-approved protocols at Kansai Medical University (No. 2014110 and 2017208). All subjects provided written informed consent in accordance with the recommendations of the International Conference on Harmonization Guidelines for Good Clinical Practice and the Declaration of Helsinki.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Eugui EM, Almquist SJ, Muller CD, Allison AC. Lymphocyte-selective cytostatic and immunosuppressive effects of mycophenolic acid in vitro: role of deoxyguanosine nucleotide depletion. Scand J Immunol. 1991;33(2):161-73.
Fulton B, Markham A. Mycophenolate mofetil. A review of its pharmacodynamic and pharmacokinetic properties and clinical efficacy in renal transplantation. Drugs. 1996;51(2):278-98.
Allison AC, Eugui EM. Mechanisms of action of mycophenolate mofetil in preventing acute and chronic allograft rejection. Transplantation. 2005;80(2 Suppl):S181-90.
Vu MD, Qi S, Xu D, Wu J, Peng J, Daloze P, et al. Synergistic effects of mycophenolate mofetil and sirolimus in prevention of acute heart, pancreas, and kidney allograft rejection and in reversal of ongoing heart allograft rejection in the rat. Transplantation. 1998;66(12):1575-80.
Van Bruggen MC, Walgreen B, Rijke TP, Berden JH. Attenuation of murine lupus nephritis by mycophenolate mofetil. J Am Soc Nephrol. 1998;9(8):1407-15.
Ito T, Wang YH, Duramad O, Hori T, Delespesse GJ, Watanabe N, et al. TSLP-activated dendritic cells induce an inflammatory T helper type 2 cell response through OX40 ligand. J Exp Med. 2005;202(9):1213-23.
Ito T, Yang M, Wang YH, Lande R, Gregorio J, Perng OA, et al. Plasmacytoid dendritic cells prime IL-10-producing T regulatory cells by inducible costimulator ligand. J Exp Med. 2007;204(1):105-15.
Kadowaki N, Antonenko S, Lau JY, Liu YJ. Natural interferon alpha/beta-producing cells link innate and adaptive immunity. J Exp Med. 2000;192(2):219-26.
Siegal FP, Kadowaki N, Shodell M, Fitzgerald-Bocarsly PA, Shah K, Ho S, et al. The nature of the principal type 1 interferon-producing cells in human blood. Science. 1999;284(5421):1835-7.
Liu YJ. IPC: professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors. Annu Rev Immunol. 2005;23:275-306.
Honda K, Yanai H, Negishi H, Asagiri M, Sato M, Mizutani T, et al. IRF-7 is the master regulator of type-I interferon-dependent immune responses. Nature. 2005;434(7034):772-7.
Honda K, Yanai H, Mizutani T, Negishi H, Shimada N, Suzuki N, et al. Role of a transductional-transcriptional processor complex involving MyD88 and IRF-7 in Toll-like receptor signaling. Proc Natl Acad Sci U S A. 2004;101(43):15416-21.
Kawai T, Sato S, Ishii KJ, Coban C, Hemmi H, Yamamoto M, et al. Interferon-alpha induction through Toll-like receptors involves a direct interaction of IRF7 with MyD88 and TRAF6. Nat Immunol. 2004;5(10):1061-8.
Uematsu S, Sato S, Yamamoto M, Hirotani T, Kato H, Takeshita F, et al. Interleukin-1 receptor-associated kinase-1 plays an essential role for Toll-like receptor (TLR)7- and TLR9-mediated interferon-{alpha} induction. J Exp Med. 2005;201(6):915-23.
Cao W, Manicassamy S, Tang H, Kasturi SP, Pirani A, Murthy N, et al. Toll-like receptor-mediated induction of type I interferon in plasmacytoid dendritic cells requires the rapamycin-sensitive PI(3)K-mTOR-p70S6K pathway. Nat Immunol. 2008;9(10):1157-64.
Guiducci C, Ghirelli C, Marloie-Provost MA, Matray T, Coffman RL, Liu YJ, et al. PI3K is critical for the nuclear translocation of IRF-7 and type I IFN production by human plasmacytoid predendritic cells in response to TLR activation. J Exp Med. 2008;205(2):315-22.
Hoshino K, Sugiyama T, Matsumoto M, Tanaka T, Saito M, Hemmi H, et al. IkappaB kinase-alpha is critical for interferon-alpha production induced by Toll-like receptors 7 and 9. Nature. 2006;440(7086):949-53.
Banchereau J, Pascual V. Type I interferon in systemic lupus erythematosus and other autoimmune diseases. Immunity. 2006;25(3):383-92.
Blanco P, Palucka AK, Gill M, Pascual V, Banchereau J. Induction of dendritic cell differentiation by IFN-alpha in systemic lupus erythematosus. Science. 2001;294(5546):1540-3.
Nestle FO, Conrad C, Tun-Kyi A, Homey B, Gombert M, Boyman O, et al. Plasmacytoid predendritic cells initiate psoriasis through interferon-alpha production. J Exp Med. 2005;202(1):135-43.
Colic M, Stojic-Vukanic Z, Pavlovic B, Jandric D, Stefanoska I. Mycophenolate mofetil inhibits differentiation, maturation and allostimulatory function of human monocyte-derived dendritic cells. Clin Exp Immunol. 2003;134(1):63-9.
Litjens NH, Rademaker M, Ravensbergen B, Thio HB, van Dissel JT, Nibbering PH. Effects of monomethylfumarate on dendritic cell differentiation. Br J Dermatol. 2006;154(2):211-7.
Mazzola MA, Raheja R, Regev K, Beynon V, von Glehn F, Paul A, et al. Monomethyl fumarate treatment impairs maturation of human myeloid dendritic cells and their ability to activate T cells. Mult Scler. 2019;25(1):63-71.
Miyamoto R, Ito T, Nomura S, Amakawa R, Amuro H, Katashiba Y, et al. Inhibitor of IkappaB kinase activity, BAY 11-7082, interferes with interferon regulatory factor 7 nuclear translocation and type I interferon production by plasmacytoid dendritic cells. Arthritis Res Ther. 2010;12(3):R87.
Pourafshar N, Karimi A, Wen X, Sobel E, Pourafshar S, Agrawal N, et al. The utility of trough mycophenolic acid levels for the management of lupus nephritis. Nephrol Dial Transplant. 2019;34(1):83-9.
Lertdumrongluk P, Somparn P, Kittanamongkolchai W, Traitanon O, Vadcharavivad S, Avihingsanon Y. Pharmacokinetics of mycophenolic acid in severe lupus nephritis. Kidney Int. 2010;78(4):389-95.
Ronnblom L, Pascual V. The innate immune system in SLE: type I interferons and dendritic cells. Lupus. 2008;17(5):394-9.
Hua J, Kirou K, Lee C, Crow MK. Functional assay of type I interferon in systemic lupus erythematosus plasma and association with anti-RNA binding protein autoantibodies. Arthritis Rheum. 2006;54(6):1906-16.
Lovgren T, Eloranta ML, Bave U, Alm GV, Ronnblom L. Induction of interferon-alpha production in plasmacytoid dendritic cells by immune complexes containing nucleic acid released by necrotic or late apoptotic cells and lupus IgG. Arthritis Rheum. 2004;50(6):1861-72.
Ito T, Kanzler H, Duramad O, Cao W, Liu YJ. Specialization, kinetics, and repertoire of type 1 interferon responses by human plasmacytoid predendritic cells. Blood. 2006;107(6):2423-31.
Honda K, Ohba Y, Yanai H, Negishi H, Mizutani T, Takaoka A, et al. Spatiotemporal regulation of MyD88-IRF-7 signalling for robust type-I interferon induction. Nature. 2005;434(7036):1035-40.
Moser M, Murphy KM. Dendritic cell regulation of TH1-TH2 development. Nat Immunol. 2000;1(3):199-205.
Thieu VT, Yu Q, Chang HC, Yeh N, Nguyen ET, Sehra S, et al. Signal transducer and activator of transcription 4 is required for the transcription factor T-bet to promote T helper 1 cell-fate determination. Immunity. 2008;29(5):679-90.
Corna D, Morigi M, Facchinetti D, Bertani T, Zoja C, Remuzzi G. Mycophenolate mofetil limits renal damage and prolongs life in murine lupus autoimmune disease. Kidney Int. 1997;51(5):1583-9.
Dooley MA, Cosio FG, Nachman PH, Falkenhain ME, Hogan SL, Falk RJ, et al. Mycophenolate mofetil therapy in lupus nephritis: clinical observations. J Am Soc Nephrol. 1999;10(4):833-9.
Gaubitz M, Schorat A, Schotte H, Kern P, Domschke W. Mycophenolate mofetil for the treatment of systemic lupus erythematosus: an open pilot trial. Lupus. 1999;8(9):731-6.
Wu JF, Yang YH, Wang LC, Lee JH, Shen EY, Chiang BL. Antinucleosome antibodies correlate with the disease severity in children with systemic lupus erythematosus. J Autoimmun. 2006;27(2):119-24.
Hylkema MN, van Bruggen MC, ten Hove T, de Jong J, Swaak AJ, Berden JH, et al. Histone-containing immune complexes are to a large extent responsible for anti-dsDNA reactivity in the Farr assay of active SLE patients. J Autoimmun. 2000;14(2):159-68.
Kippner L, Klint C, Sturfelt G, Bengtsson AA, Eriksson H, Truedsson L. Increased level of soluble HLA class I antigens in systemic lupus erythematosus: correlation with anti-DNA antibodies and leukopenia. J Autoimmun. 2001;16(4):471-8.
Okamura M, Kanayama Y, Amastu K, Negoro N, Kohda S, Takeda T, et al. Significance of enzyme linked immunosorbent assay (ELISA) for antibodies to double stranded and single stranded DNA in patients with lupus nephritis: correlation with severity of renal histology. Ann Rheum Dis. 1993;52(1):14-20.
Sacre K, Criswell LA, McCune JM. Hydroxychloroquine is associated with impaired interferon-alpha and tumor necrosis factor-alpha production by plasmacytoid dendritic cells in systemic lupus erythematosus. Arthritis Res Ther. 2012;14(3):R155.
Schrezenmeier E, Dörner T. Mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology. Nat Rev Rheumatol. 2020;16(3):155-66.
Conti F, Spinelli FR, Alessandri C, Valesini G. Toll-like receptors and lupus nephritis. Clin Rev Allergy Immunol. 2011;40(3):192-8.
Patole PS, Zecher D, Pawar RD, Gröne HJ, Schlöndorff D, Anders HJ. G-rich DNA suppresses systemic lupus. J Am Soc Nephrol. 2005;16(11):3273-80.
Scott JL, Wirth JR, EuDaly JG, Gilkeson GS, Cunningham MA. Plasmacytoid dendritic cell distribution and maturation are altered in lupus prone mice prior to the onset of clinical disease. Clin Immunol. 2017;175:109-14.

Download PDF

Journal Publication

published 09 Nov, 2020

Read the published version in Arthritis Research & Therapy →

Editorial decision: Major revision
21 Jul, 2020
Review #2 received at journal
06 Jul, 2020
Reviewer #2 agreed at journal
21 Jun, 2020
Review #1 received at journal
21 Jun, 2020
Reviewer #1 agreed at journal
16 Jun, 2020
Reviewers invited by journal
15 Jun, 2020
Editor assigned by journal
08 Jun, 2020
First submitted to journal
07 Jun, 2020
Submission checks completed at journal
07 Jun, 2020
Editor invited by journal
07 Jun, 2020

You are reading this older preprint version

Read the latest preprint version →

Mycophenolic Acid, Active Form of Mycophenolate Mofetil, Interferes With the IRF7 Nuclear Translocation and Type I IFN Production by Plasmacytoid Dendritic Cells

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Materials And Methods

Media and reagents

Cell isolation and culture

Stimulation of lupus serum

Analyses of cells and supernatants

Real-time PCR

Fluorescence microscope

Statistical analysis

Results

Discussion

Conclusions

List Of Abbreviations

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1