Cutting Edge: Dysregulated Endocannabinoid-Rheostat For

Cutting Edge: Dysregulated Endocannabinoid-Rheostat for Plasmacytoid Dendritic Cell Activation in a Systemic Lupus Endophenotype This information is current as of September 23, 2021. Oindrila Rahaman, Roopkatha Bhattacharya, Chinky Shiu Chen Liu, Deblina Raychaudhuri, Amrit Raj Ghosh, Purbita Bandopadhyay, Santu Pal, Rudra Prasad Goswami, Geetabali Sircar, Parasar Ghosh and Dipyaman Ganguly

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 6, 2019, doi:10.4049/jimmunol.1801521

Cutting Edge: Dysregulated Endocannabinoid-Rheostat for Plasmacytoid Dendritic Cell Activation in a Systemic Lupus Endophenotype Oindrila Rahaman,*,† Roopkatha Bhattacharya,*,† Chinky Shiu Chen Liu,*,† ,† ,† ,† Deblina Raychaudhuri,* Amrit Raj Ghosh,*x Purbitax Bandopadhyay,* x Santu Pal,*,‡ Rudra Prasad Goswami, Geetabali Sircar, Parasar Ghosh, and Dipyaman Ganguly*,† Systemic lupus erythematosus (SLE) is a systemic auto- produced by the plasmacytoid dendritic cells (pDCs) have immune disease, characterized by loss of tolerance to- been found to be a crucial event in SLE pathogenesis (3, 4). ward self nuclear Ags. Systemic induction of type I Genome-wide association studies have linked multiple genomic Downloaded from IFNs plays a pivotal role in SLE, a major source of type loci with SLE susceptibility, although direct pathogenetic role I IFNs being the plasmacytoid dendritic cells (pDCs). of very few of them have been reported, apart from the genes Several genes have been linked with susceptibility to directly linked to induction and response of type I IFNs (5, 6). SLE in genome-wide association studies. We aimed A recent genome-wide association study had reported associa- at exploring the role of one such gene, a/b-hydrolase tion of a novel gene, a/b-hydrolase domain-containing 6 domain-containing 6 (ABHD6), in regulation of IFN-a (ABHD6), with SLE susceptibility (7). http://www.jimmunol.org/ induction in SLE patients. We discovered a regulatory ABHD6 is a serine hydrolase that catalyzes hydrolysis of role of ABHD6 in human pDCs through modulating endocannabinoids, the endogenous ligands for the G-protein– the local abundance of its substrate, the endocannabinoid coupled cannabinoid receptors (CB1 and CB2), which include 2-arachidonyl glycerol (2-AG), and elucidated a hitherto 2-arachidonyl glycerol (2-AG) and anandamide (AEA) (8). The unknown cannabinoid receptor 2 (CB2)–mediated reg- role of ABHD6, as the major hydrolytic enzyme for 2-AG, was ulatory role of 2-AG on IFN-a induction by pDCs. first documented in microglia cells in the CNS (9). Expression We also identified an ABHD6High SLE endophenotype of ABHD6 with a similar function in macrophages was by guest on September 23, 2021 wherein reduced local abundance of 2-AG relieves the also reported (10). In macrophages, ABHD6-mediated hy- CB2-mediated steady-state resistive tuning on IFN-a drolysis of 2-AG prevented 2-AG–driven inhibition of induction by pDCs, thereby contributing to SLE TLR activation; thus, ABHD6 activity was found to have a pathogenesis. The Journal of Immunology, 2019, 202: proinflammatory role. Systemic immunomodulatory role of 000–000. cannabinoid receptor signaling is widely reported, and a link with autoreactive inflammation is also suggested (11). Apart from modulation of endocannabinoids, the substrate of ystemic autoimmunity in systemic lupus erythematosus ABHD6, function of this enzyme is also reported in various (SLE) is characterized by humoral autoreactivity against other physiological contexts, namely receptor recycling in the S nuclear Ags and immune-complex deposition leading to CNS (12), insulin secretion by pancreatic b cells (13), and pathologies involving multiple organs, namely skin, joints, lipolysis in metabolic tissues (14). Accordingly, deregulation vessels, CNS, and kidneys (1). Due to complex genetic factors of this enzyme has been implicated in various clinical contexts associated with the disease and a dominant systemic type I IFN of CNS disorders and metabolic disorders (13–15), but any response critically linked to the pathogenesis, SLE is a proto- pathogenetic role of ABHD6 in immunological disorders typical member of polygenic IFNopathies (2). Type I IFNs remains unidentified. In this study, we aimed at exploring

*Indian Institute of Chemical Biology–Translational Research Unit of Excellence, Coun- University Grants Commission, India. R.B., C.S.C.L., D.R., and P.B. received fellow- cil of Scientific and Industrial Research–Indian Institute of Chemical Biology, Kolkata, ships from the Council of Scientific and Industrial Research, India. West Bengal 700091, India; †Division of Cancer Biology and Inflammatory Disorders, Address correspondence and reprint requests to Dr. Dipyaman Ganguly, Indian Institute Council of Scientific and Industrial Research–Indian Institute of Chemical Biology, ‡ of Chemical Biology –Translational Research Unit of Excellence, Council of Scientific Kolkata, West Bengal 700091, India; Mass Spectrometry Core Facility, Indian Institute and Industrial Research–Indian Institute of Chemical Biology, CN6, Sector V, Salt of Chemical Biology–Translational Research Unit of Excellence, Council of Scientific Lake, Kolkata, West Bengal 700091, India. E-mail address: [email protected] and Industrial Research–Indian Institute of Chemical Biology, Kolkata, West Bengal x 700091, India; and Department of Rheumatology, Institute of Postgraduate Medical The online version of this article contains supplemental material. Education and Research, Kolkata, West Bengal 700020, India Abbreviations used in this article: ABHD6, a/b-hydrolase domain-containing 6; 2-AG, ORCID: 0000-0002-7786-1795 (D.G.). 2-arachidonyl glycerol; COX2, cycloxygenase-2; ISG, IFN signature gene; pDC, plasmacytoid dendritic cell; PPARa, proliferator-activated receptor a; siRNA, small inter- Received for publication November 19, 2018. Accepted for publication January 20, fering RNA; SLE, systemic lupus erythematosus; SLEDAI, SLE Disease Activity Index. 2019. This work was supported by a Ramanujan Fellowship from the Science and Engineer- Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 ing Research Board, India (to D.G.). O.R. and A.R.G. received fellowships from the

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801521 2 CUTTING EDGE: ENDOCANNABINOID CONTROL OF PLASMACYTOID DCs IN SLE functional link of this gene with type I IFN induction and spectrometry of 2-AG on LTQ Orbitrap XL. The output was analyzed by SLE pathogenesis. Thermo Xcalibur software. Materials and Methods ELISA Blood sample collection from healthy donors and SLE patients ELISA was done to quantify IFN-a and TNF-a from PBMC and pDC culture supernatants, using anti-human IFN-a ELISA kit and anti-human Healthy individuals (n = 76) and SLE patients (n = 90) were recruited for the TNF-a ELISA kit (Mabtech). study; patients were recruited at the Department of Rheumatology at the Institute of Postgraduate Medical Education and Research, Kolkata, India Statistics (Supplemental Fig. 1A, 1B). Peripheral blood samples were collected from SLE patients and healthy donors, after taking written informed consent, in Statistical analyses of all data sets were done on GraphPad Prism 5.0 software. accordance with the Declaration of Helsinki and as recommended and ap- Data were compared between groups using paired, unpaired Student t test or proved by the institutional review boards of both the institutes (Institute of Mann–Whitney U test as specified in figure legends, and correlations were Postgraduate Medical Education and Research and Council of Scientific and done using Spearman rank correlation as specified in respective figure legends. Industrial Research–Indian Institute of Chemical Biology, Kolkata, India). Cell isolation and culture Results and Discussion PBMCs were isolated from whole blood. PBMCs were either kept in TRIzol Higher expression of ABHD6 in PBMCs defines an reagent for subsequent RNA isolation or cultured overnight in RPMI 1640 SLE endophenotype with 10% FBS in a 96-well plate at a density of 0.2 million cells per well and were treated with agonists and/or inhibitors as indicated. Collected plasma An overexpression of ABHD6 in SLE patients was suggested in was stored at 280˚C for future use. In some cases, pDCs were isolated from Downloaded from PBMCs by magnetic immunoselection using BDCA-4 MicroBeads (Miltenyi the previous study, which first identified polymorphisms of Biotec) and cultured overnight in RPMI 1640 with 10% FBS in 96-well ABHD6 as the PXK-locus associated predisposition for SLE plates at a density of 40,000 cells per well. pDCs were stimulated with (7). To examine this, we recruited a cohort of SLE patients 150 nM of CpGA (for IFN-a induction) or 500 nM of CpGB (for TNF-a and checked the expression of ABHD6 in PBMCs and found induction), in the presence of the 2-AG (10 mM), WWL70 (25 mM), or other inhibitors as indicated in figure legends. Cell supernatants were col- that there is significant overexpression of ABHD6 in SLE lected 18 h poststimulation. patients (n = 90, female = 86, male = 4), as compared with http://www.jimmunol.org/ healthy controls (n = 76, female = 32, male = 44) (Fig. 1A). A RNA interference recent report suggested that female hormones can increase the CB2 gene was knocked down in freshly isolated pDCs by nuclear transfection expression of ABHD6 when added to in vitro cultured following the manufacturer’s protocol (Amaxa Lonza 4D nucleofector kit; Lonza) using CB2 small interfering RNA (siRNA) or eGFR siRNA as control. leukocytes (16); however, there was no difference in ABHD6 After 24 h, cells were plated in 96-well plate and treated as indicated. Cells expression in PBMCs between female and male healthy and supernatant were harvested 18 h poststimulation. controls in our cohort (Supplemental Fig. 1C). The data also Gene expression studies revealed two subgroups of SLE patients based on expression of ABHD6—taking median expression in healthy controls as the Total RNA was isolated from cells using TRIzol reagent (Invitrogen). threshold, we categorized SLE patients into ABHD6High and by guest on September 23, 2021 Quantitative PCR was carried out for the specified genes normalized against Low 18S rRNA as the housekeeping gene. ABHD6 groups (Fig. 1A). Of note here, ABHD6 expression did not show any correlation with the disease activity Plasma 2-AG quantification score SLE Disease Activity Index (SLEDAI) in our patient A total of 100 ml of plasma sample or cell supernatant was mixed with 300 ml cohort (Supplemental Fig. 1D) and there was no significant of liquid chromatography–mass spectrometry –grade acetonitrile + 0.1% difference between the ABHD6High and ABHD6Low groups formic acid (1:4 dilution) and incubated on ice for 15 min with intermittent vortexing, followed by centrifugation at 14,000 rpm for 10 min at 4˚C. The in terms of either SLEDAI or Damage Index scores supernatant was extracted in a fresh tube and used for subsequent mass (Supplemental Fig. 1E, 1F).

FIGURE 1. Identiﬁcation of an SLE endophenotype with high ABHD6 expression. (A) ABHD6 expression was compared in PBMCs between SLE patients (n = 90) and healthy controls (n = 76) by two-tailed unpaired Student t test. ABHD6High and ABHD6Low groups were based on median ABHD6 expression of healthy controls. (B) ISG index (ISGi), average of relative expressions of ISGs (IRF7, TRIP14, MX1, XIAPAF1, ISG15, IFI44L), correlated with ABHD6 expression in PBMCs (n = 90) by Spearman rank correlation. (C) ISGi was compared between ABHD6High (n = 54) and ABHD6Low (n = 36) SLE patients by Mann–Whitney U test. (D and E) Correlation between ABHD6 and ISGi values in PBMCs of ABHD6High (D) and of ABHD6Low (E) patients, by Spearman rank correlation. The Journal of Immunology 3

FIGURE 2. Inhibition of ABHD6 modulates TLR9-mediated IFN-a induction. (A) PBMCs stimulated by CpGA with escalating doses of ABHD6 inhibitor WWL70 and supernatant IFN-a levels were measured (n = 4). (B) Analysis of ABHD6 expression in major immune cells from Primary Cell Atlas data set accessible from biogps.org (under PubMed identiﬁcation number 24053356). (C) ABHD6 expression compared between healthy control PBMCs and their corresponding puriﬁed pDCs (n = 32). (D) ABHD6, FAAH, MGLL, and ABHD12 expression in pDCs of healthy controls (n = 10).

Enriched expression of IFN signature gene (ISG) in the immune cells (21, 22). Accordingly, TLR9 ligand–induced Downloaded from peripheral blood is a surrogate marker for all IFNopathies IFN-a from PBMCs is mostly due to TLR9 activation in which closely correlate with disease activity in SLE (17, 18). In pDCs (data not shown). On analysis of a public database on our cohort, the peripheral blood ISG was not correlated with human tissue speciﬁc transcriptome, we found that among the disease activity (viz. SLEDAI) (Supplemental Fig. 1G), per- hematopoietic cells represented in that database, dendritic haps because the SLE patients in our cohort were not treat- cells had the highest expression of ABHD6 (Fig. 2B). Also,

ment naive. But average of relative expression of six major when we assessed ABHD6 expression in isolated pDCs and http://www.jimmunol.org/ ISGs (viz. IRF7, TRIP14, MX1, XIAPAF1, ISG15, and compared it with the same in corresponding total PBMCs, IFI44L) was significantly correlated with ABHD6 expression it was evident that ABHD6 expression is enriched in pDCs (Fig.1B),andtheABHD6High groupwasfoundtohave (Fig. 2C). The other serine hydrolases that have similar ac- significantly higher value compared with the ABHD6Low tivity as that of ABHD6 (viz. FAAH, MGLL, and ABHD12) group (Fig. 1C). We also found that in the ABHD6High (23) had a much lower expression in pDCs (Fig. 2D). group of SLE patients, ABHD6 expression in PBMCs strongly This made us explore whether WWL70 can also abrogate correlated with average expression of those six ISGs, whereas IFN-a induction in purified pDCs ex vivo stimulated with in the ABHD6Low group this was not seen (Fig. 1D, 1E). This CpGA. Interestingly, there was no such inhibition docu- led us to hypothesize that overexpression of ABHD6 in the mented at the same concentration of WWL70 that inhibits by guest on September 23, 2021 ABHD6High SLE patients may play a regulatory role on sys- IFN-a induction from PBMCs (Fig. 3A). We reasoned that temic type I IFN induction, leading to ISG enrichment in these apparently counterintuitive data were due to lack of peripheral blood. direct regulatory effect of ABHD6 on this pathway, and the inhibition of IFN-a induction in response to TLR9 activation ABHD6 disrupts a rheostat mechanism for type I IFN induction was, rather, driven by some ABHD6 substrate, which is by pDCs available in PBMC culture but not in purified pDC culture— To validate that ABHD6 may have a regulatory role on sys- ABHD6 regulates the pathway through affecting the relative temic type I IFN induction, we explored if WWL70, a selective abundance of its substrate. The endocannabinoid 2-AG is the ABHD6 inhibitor (19), can affect IFN-a induction from established endogenous substrate for ABHD6 (23, 24), so we healthy PBMCs in response to the bona fide TLR9 ligand repeated the same experiment in presence of exogenous 2-AG, CpGA oligonucleotides. Indeed, WWL70 interfered with considering the possibility that pDC-intrinsic sourcing of CpGA-induced IFN-a induction in PBMCs in a dose- 2-AG may not be sufficient. Indeed, in the presence of ex- dependent manner (Fig. 2A). TLR9 expression in humans ogenous 2-AG, ABHD6 inhibition by WWL70 abrogated is largely restricted to pDCs and B cells (20), and pDCs are IFN-a induction from purified pDCs stimulated with CpGA the major type I IFN producers among the peripheral blood (Fig. 3B). Thus, we concluded that ABHD6, expressed in

FIGURE 3. ABHD6 modulation of IFN-a production in pDCs mediated by 2-AG. (A) pDCs stimulated with CpGA in presence of WWL70 (n = 4) and (B) 2-AG or 2-AG plus WWL70 (n = 10) and IFN-a measured in supernatant. (C) pDCs stimulated by CpGB in presence of 2-AG and WWL70, and TNF-a measured in supernatant by ELISA (n = 8). The p value was determined by two-tailed paired Student t test. 4 CUTTING EDGE: ENDOCANNABINOID CONTROL OF PLASMACYTOID DCs IN SLE

IFN-a induction, whereas activation of NF-kB lead to induction of cytokines like TNF-a (25, 26). We found that the 2-AG–driven inhibition did not affect TNF-a induction in pDCs (Fig. 3C); thus 2-AG has a specific resistive influence on IFN-a induction from pDCs. We also explored if IFN-a has any influence on expression of ABHD6 in human pDCs as well, which revealed no such regulation (Supplemental Fig. 1H).

ABHD6 regulates abundance of 2-AG in ABHD6High SLE patients As ABHD6 can regulate 2-AG abundance and in SLE patients there is an overexpression of ABHD6, we explored if plasma 2-AG abundance was also different when SLE patients are compared with healthy controls. Plasma from both groups of patients were processed for estimation of 2-AG using liquid chromatography–tandem mass spectrometry (Supplemental Fig. 2). But we found there was no significant difference in plasma 2-AG levels between healthy controls and SLE pa- Downloaded from tients (Fig. 4A). This apparent discrepancy may be explained by the fact that plasma abundance of 2-AG is not regulated solely by its catabolism driven by serine hydrolases like ABHD6 expressed on immune cells—this is also influenced by dynamic pathways leading to 2-AG synthesis, not only in circulating immune cells but also in other tissues in the body. http://www.jimmunol.org/ Interestingly, we found that in the ABHD6High SLE patients there was a notable, although not quite statistically significant, negative correlation between ABHD6 expression in PBMCs and plasma 2-AG levels (Fig. 4B), whereas in the ABHD6Low patients no such relationship was evident (Fig. 4C). We concluded that overexpression of ABHD6 on immune cells has the potential of regulating local, possibly even sys- High temic, levels of 2-AG in the ABHD6 SLE patients. by guest on September 23, 2021 For immune cells, like pDCs, that express ABHD6, this local control of 2-AG abundance is of great relevance as that can affect the cellular functions. To validate if ABHD6, expressed on pDCs, can indeed influence local 2-AG abundance, we added exogenous 2-AG to purified pDC cultures ex vivo and measured residual 2-AG in the culture supernatant after 4 h, in absence and presence of WWL70, the ABHD6 inhibitor. We found a significant increase in 2-AG reten- High A FIGURE 4. ABHD6 patients show reduced abundance of 2-AG. ( ) tion in purified pDC cultures when ABHD6 is inhibited Plasma 2-AG levels quantified in SLE patients (n = 79) and healthy controls (n = 43), compared by two-tailed Mann–Whitney U test. (B) Correlation (Fig. 4D), indicating that pDC-intrinsic ABHD6 activ- between ABHD6 expression in PBMCs and plasma 2-AG levels in ABHD6High ity does regulate local 2-AG abundance and thus dis- (n =43)and(C)ABHD6Low groups (n = 36), by Spearman rank correlation. rupts its resistive tuning of TLR activation and type I IFN (D) pDCs cultured with exogenous 2-AG in absence/presence of WWL70, and induction. residual 2-AG in culture supernatants measured after 4 h (n =14).(E)pDCs We also found that in purified cultures with exogenous from SLE patients (n = 21) and healthy control (n = 12) cultured with exog- 2-AG, pDCs from SLE patients (n = 21) depleted significantly enous 2-AG and residual 2-AG in culture supernatants was measured, compared more 2-AG in 4 h, as compared with healthy donors (n = 12) by two-tailed Mann–Whitney U test. (F) ABHD6 expression in pDCs of patients (n = 12) and controls (n = 11) compared by two-tailed unpaired Student t (Fig. 4E). We also compared ABHD6 expression in purified test. (G) Correlation between ABHD6 expression in pDCs from ABHD6High pDCs from SLE patients (n = 12) and healthy donors (n = 11) patients (n = 8) and percentage of 2-AG hydrolyzed (normalized for 104 pDCs) and found significant overexpression of ABHD6 as well as using Spearman rank correlation. a similar subgrouping of patients between ABHD6High and ABHD6Low groups (Fig. 4F), just like we found with ABHD6 pDCs, regulates the abundance of the endocannabinoid expression in total PBMCs (Fig. 1A). In fact, the extent of 2-AG, which in turn relieves a steady-state resistive effect of 2-AG hydrolysis in purified pDC cultures was found to be 2-AG on TLR activation in pDCs and IFN-a induction. It is strongly correlated with ABHD6 expression levels in the established that TLR activation in human pDCs also leads pDCs in the ABHD6High group (Fig. 4G). This led us to to induction of proinflammatory cytokines other than IFN-a conclude that ABHD6 expression in pDCs can indeed control (e.g., TNF-a). But induction of these other cytokines have a local abundance of the endocannabinoid 2-AG, thus affecting different signaling regulation downstream of the endosomal the 2-AG–driven resistive influence on IFN-a induction in TLRs. Activation and nuclear translocation of IRF7 leads to response to TLR9 activation. The Journal of Immunology 5 Downloaded from

FIGURE 5. 2-AG inhibits IFN-a production in pDCs by CB2 receptor. (A) Different pathways known to mediate 2-AG function in cells. (B) Effect of COX2 inhibitor valdecoxib (n = 8) and (C) PPARa inhibitor MK866 (n = 4) on 2-AG mediated inhibition of IFN-a induction. (D) Expression of CB1 and CB2 in puriﬁed healthy pDCs (n = 5). (E) Effects of CB1 antagonist SLV319 (n = 5) and (F) CB2 antagonist SR144528 (n = 11) on 2-AG mediated inhibition of IFN-a induction. (G) Control or CB2 siRNA transfected pDCs stimulated with CpGA in the absence/presence of 2-AG and WWL70, and IFN-a measured in supernatant (n = 4). The p values were determined by two-tailed paired Student t test. (H) The model denotes 2-AG mediated rheostat mechanism that selectively http://www.jimmunol.org/ regulates IFN-a induction in pDCs, in response to TLR activation, and this immunomodulation is mediated by CB2 receptors. ABHD6 overexpression in pDCs reduces 2-AG and disrupts the rheostat, causing aberrant IFN-a production by pDCs in SLE patients.

2-AG controls type I IFN induction by pDCs through CB2 2-AG working on pDCs through CB2 receptors (Fig. 5H). receptor activation Cellular abundance of the 2-AG–catabolizing enzyme ABHD6 After we identified that local presence of 2-AG can inhibit disrupts this rheostat mechanism, leading to supraphysiologic a IFN-a induction in pDCs on TLR9 activation, we wanted to IFN- induction from human pDCs in response to TLR ac- explore the molecular interface responsible for this resistive tivation. This pathway contributes to the systemic type I IFN by guest on September 23, 2021 effect of 2-AG on pDC activation. 2-AG has been previously induction associated with an SLE endophenotype characterized reported to affect cellular functions in different cell types by higher expression of ABHD6 on the circulating immune through a myriad of receptors (Fig. 5A), for example, through cells, specifically pDCs. This study also establishes hitherto engagement of cycloxygenase-2 (COX2) in myeloid cells by unexplored promising therapeutic targets, namely ABHD6 the 2-AG hydrolysis product arachidonic acid (10), through and CB2, for SLE treatment. peroxisome proliferator-activated receptor a (PPARa)in mesenchymal stem cells (27), and the specific cannabinoid receptors CB1 and CB2 (28). We found that the 2-AG– Disclosures driven resistive effect on pDC activation could not be reversed The authors have no financial conflicts of interest. by the COX2-specific inhibitor valdecoxib (Fig. 5B) or the a PPAR -specific inhibitor MK866 (Fig. 5C), so we hypothe- References sized that resistive influence of 2-AG in pDCs may be me- 1. Tsokos, G. C. 2011. Systemic lupus erythematosus. N. Engl. J. Med. 365: diated by the cannabinoid receptors. When we checked 2110–2121. for expression of the two cannabinoid receptors in purified 2. Ganguly, D. 2018. Do type I interferons link systemic autoimmunities and metabolic syndrome in a pathogenetic continuum? Trends Immunol. 39: 28–43. human pDCs, we found that CB2 is the more abundant 3. Ganguly, D., S. Haak, V. Sisirak, and B. Reizis. 2013. The role of dendritic cells in cannabinoid receptor in human pDCs (Fig. 5D). Accord- autoimmunity. Nat. Rev. Immunol. 13: 566–577. 4. Sisirak, V., D. Ganguly, K. L. Lewis, C. Couillault, L. Tanaka, S. Bolland, ingly, the CB1-specific small molecule antagonist SLV319 V. D’Agati, K. B. Elkon, and B. Reizis. 2014. Genetic evidence for the role of could not reverse the 2-AG–driven inhibition of pDC plasmacytoid dendritic cells in systemic lupus erythematosus. J. Exp. Med. 211: 1969–1976. activation (Fig. 5E), whereas the CB2-specific antagonist 5. Rullo, O. J., and B. P. Tsao. 2013. Recent insights into the genetic basis of systemic SR144528 could significantly reverse this effect of 2-AG lupus erythematosus. Ann. Rheum. Dis. 72(Suppl. 2): ii56–ii61. 6. Niewold, T. B. 2015. Advances in lupus genetics. Curr. Opin. Rheumatol. 27: (Fig. 5F). To gather more evidence in support of critical in- 440–447. volvement of CB2 receptors in mediating the effect of 2-AG 7. Oparina, N. Y., A. M. Delgado-Vega, M. Martinez-Bueno, C. Magro-Checa, on pDCs, we targeted the CB2 gene in pDCs. We found that C. Fernańdez, R. O. Castro, B. A. Pons-Estel, S. D’Alfonso, G. D. Sebastiani, T. Witte, et al. 2015. PXK locus in systemic lupus erythematosus: fine mapping siRNA-mediated knockdown of CB2 in human pDCs could and functional analysis reveals novel susceptibility gene ABHD6. Ann. Rheum. Dis. again significantly reverse the resistive effect of 2-AG on 74: e14. a 8. Lu, H. C., and K. Mackie. 2016. An introduction to the endogenous cannabinoid IFN- induction (Fig. 5G). system. Biol. Psychiatry 79: 516–525. Thus, we identified a hitherto unknown rheostat mechanism 9. Muccioli, G. G., C. Xu, E. Odah, E. Cudaback, J. A. Cisneros, D. M. Lambert, a M. L. Lo´pez Rodrı´guez, S. Bajjalieh, and N. Stella. 2007. Identification of a novel that controls IFN- induction from human pDCs in response endocannabinoid-hydrolyzing enzyme expressed by microglial cells. J. Neurosci. 27: to TLR activation, which is driven by the endocannabinoid 2883–2889. 6 CUTTING EDGE: ENDOCANNABINOID CONTROL OF PLASMACYTOID DCs IN SLE

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Supplemental information

for

‘Dysregulated endocannabinoid-rheostat for plasmacytoid dendritic cell activation in a systemic lupus endophenotype’

by Rahaman O. et al.

Supplemental figure 1. Cohort characteristics. (A) Demography and clinical scores for the SLE patients and healthy controls. (B) SLEDAI score, Damage Index, ANA profile (11 antigens, 1-3 being score for each antigen) and ongoing therapies for ABHD6High SLE patients (N=54) and ABHD6Low SLE patients (N=36). (C) ABHD6 expression compared between female (N=32) and male (N=44) healthy controls by Mann Whitney U test. (D) Correlation between PBMC ABHD6 expression and SLEDAI scores in the ABHD6High subgroup. (E) Comparison of SLEDAI scores between ABHD6High and ABHD6Low subgroups. (F) Comparison of Damage Index among patients from ABHD6High and ABHD6Low subgroups. (G) Correlation between PBMC ISGi and SLEDAI scores for SLE patients. (H) Primary human pDCs were cultured in the absence or presence of recombinant human IFN and after 1 hour expression of ABHD6 was assessed by real time PCR. 2

Supplemental Figure 2. Standards for mass spectrometric measurement of plasma 2-AG on LTQ ORBITRAP XL. Standard solutions of 2-AG (Sigma) were prepared in LC-MS grade acetonitrile. Separation was carried out in Hypersil Gold C18 column with a diameter of 100 X 2.1mm, particle size 1.9µm, using mobile phase of Solution A (H2O +0.1% formic acid) and solution B (acetonitrile + 0.1% formic acid), with a sample injection volume of 25µl. The cut off for detection of intact 2-AG was (m/z) 379.28 and the retention time was 9.48mins, while that of fragmented 2-AG was (m/z) 287.23 and the retention time was 9.55mins. The output is analysed by Thermo Xcalibur software. The peak of 2-AG obtained (A) after LC MS at the m/z 379.2849 and (B) after LC MS/MS at the m/z 287.2380. (C) The intensity of 2-AG after LC-MS/MS is represented at different standard concentrations i.e 8ng/mL (200pg), 4ng/mL (100pg), 2ng/mL (50 pg), 1.4ng/mL (35 pg) and 0.8ng/mL (20 pg).