Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Endogenous mitochondrial double‐stranded RNA is not an activator of the type I interferon response in human pancreatic beta cells

Endogenous mitochondrial double‐stranded RNA is not an activator of the type I interferon... Background: Type 1 diabetes ( T1D) is an autoimmune disease characterized by the progressive destruction of pancreatic beta cells. Interferon-α (IFNα), an antiviral cytokine, is expressed in the pancreatic islets in early T1D, which may be secondary to viral infections. However, not all patients harboring a type I IFN signature present signals of viral infection, suggesting that this response might be initiated by other “danger signals”. Accumulation of mitochondrial double-stranded RNA (mtdsRNA; a danger signal), secondary to silencing of members of the mitochondrial degrado- some, PNPT1 and SUV3, has been described to activate the innate immune response. Methods: To evaluate whether mtdsRNA represents a “danger signal” for pancreatic beta cells in the context of T1D, we silenced PNPT1 and/or SUV3 in slowly proliferating human insulin-secreting EndoC-βH1 cells and in non-prolif- erating primary human beta cells and evaluated dsRNA accumulation by immunofluorescence and the type I IFN response by western blotting and RT-qPCR. Results: Only the simultaneous silencing of PNPT1/SUV3 induced dsRNA accumulation in EndoC-βH1 cells but not in dispersed human islets, and there was no induction of a type I IFN response. By contrast, silencing of these two genes individually was enough to induce dsRNA accumulation in fibroblasts present in the human islet preparations. Conclusions: These data suggest that accumulation of endogenous mtdsRNA following degradosome knockdown depends on the proliferative capacity of the cells and is not a mediator of the type I IFN response in human pancreatic beta cells. Keywords: Human pancreatic beta cells, Type 1 diabetes, PNPT1, Mitochondrial dsRNA, Type I interferon Background response at the islet level, are associated with increased Type 1 diabetes (T1D) is a chronic autoimmune dis- disease risk [1, 2]. Interferon-α (IFNα), a type I IFN ease characterized by the progressive destruction of involved in innate immunity and the antiviral response, pancreatic beta cells by the immune system and subse- is expressed in islets of patients affected by T1D, as first quent loss of insulin secretion. More than 50 gene vari- described by Foulis et al. who found IFNα in pancreases ants, including several genes that regulate the antiviral of 33/34 patients with T1D, while it was detected in only 4/80 controls [3]. More recently, analysis of laser- captured islets from five patients with recent-onset T1D showed that > 30% of the 84 IFN-stimulated genes *Correspondence: alcooman@ulb.ac.be ULB Center for Diabetes Research, Medical Faculty, Campus Erasme, (ISG) analysed where overexpressed by at least fivefold Université Libre de Bruxelles (ULB), Route de Lennik, 808-CP618, in these patients compared with islets from five non- 1070 Brussels, Belgium diabetic control organ donors [4]. Additionally, T1D Full list of author information is available at the end of the article © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. Coomans de Brachène et al. Autoimmun Highlights (2021) 12:6 Page 2 of 10 patients have higher circulating levels of IFNα than Against this background, we presently evaluated controls. Thus, Chehadeh et  al. detected elevated lev - whether generation of mtdsRNA is part of the potential els of IFNα in plasma of 70% (39/56) of T1D patients endogenous “danger signals” generated in pancreatic and 50% of these IFNα-positive patients were also posi- beta cells that may contribute to trigger a local innate tive for enterovirus RNA, while IFNα-negative patients immune response in the context of T1D. Surprisingly, were negative for enterovirus RNA [5]. Accordingly, we observed that the degradosome silencing-induced enteroviral infection of beta cells, particularly by cox- mtdsRNA accumulation is a cell type specific event, sackievirus B (CVB), has been associated with T1D present in the human insulin-secreting EndoC-βH1 development [1, 6]. Viral infections may stimulate the cells and islet fibroblasts but not in primary human production of IFNα in the vicinity of the beta cells and beta cells, suggesting that the phenomenon depends contribute to trigger autoimmunity in genetically pre- on the proliferative status of cells. In addition, abnor- disposed individuals [1]. Enterovirus RNA or proteins mal accumulation of mtdsRNA does not induce a type are found in blood, stool or pancreatic islets from T1D I IFN signature in human pancreatic beta cells, indicat- patients more frequently than in controls [7, 8], but ing that this is not a direct contributory factor for T1D many patients that develop disease don’t show a clear initiation. correlation with viral infection [9], suggesting the exist- ence of other “danger signals” that may initiate a type I IFN response in the context of T1D. Methods Viral replication may lead to accumulation of cytosolic Culture of human EndoC‑βH1 cells, HeLa cells and human double-stranded RNA (dsRNA), which is recognized by islets and cell treatment sensors such as retinoic acid-inducible gene I (RIG-I)- EndoC-βH1 cells, a human pancreatic beta cell line like receptors (RLRs), including DDX58 (also known as provided by Dr. R. Scharfmann (University of Paris, RIG-I) and IFIH1 (also known as melanoma differenti - France) [16], were cultured in Matrigel-fibronectin- ation-associated protein-5 MDA5) [10]. Other sources coated plates as previously described [17]. These are of dsRNAs, derived from intracellular processes such as slowly proliferating cells, dividing every 5  days [16]. the transcription of repetitive sequences, are also present EndoC-βH1 cells have been shown to be chronically naturally in mammalian cells. This poses a challenge to infected by a xenotropic mouse retrovirus [18]. How- these cells, as they must discriminate between self and ever, our RNAseq data indicate that they do not pre- non-self dsRNA to avoid accidental activation of the type sent an increased basal innate immune response and I IFN signaling. This discrimination is made possible have also a similar immune response when treated by different mechanisms [11], including adenosine-to- with IFNα as compared to primary human islets [19]. inosine (A-to-I) editing of cellular dsRNA by the RNA- The human cervical cancer cells HeLa were grown in editing enzyme Adenosine deaminase RNA-specific Dulbecco’s modified Eagle’s medium (Lonza, Basel, (ADAR). Indeed, mutations in ADAR1 cause the auto- Switzerland) supplemented with 10% FBS and 2% peni- immune Aicardi-Goutières syndrome associated with a cillin–streptomycin (Lonza). These cell lines were free type I IFN signature [12, 13]. Another cellular source of of mycoplasma infection, as determined by monthly dsRNA is the mitochondria, where the circular genome is testing using the MycoAlert Mycoplasma Detection kit transcribed bidirectionally and the RNA generated from (Lonza). the two mitochondrial DNA strands (H and L strands) Human islets from 6 non-diabetic organ donors (see can generate long mitochondrial dsRNA (mtdsRNA). The Additional file  1) were isolated in Pisa, Italy, following a mitochondrial RNA degradosome, formed by the Suv3- protocol previously described [20] with the approval of like RNA helicase SUPV3L1 (also known as SUV3) and the local ethics committee and sent to Brussels for dis- the polynucleotide phosphorylase PNPase (also known persion and experiments [21]. as PNPT1), mediates the rapid decay of the non-coding Each experiment considered as n = 1 corresponds to L-strand transcripts which prevents the formation of one independent biological observation, i.e. EndoC-βH1 potentially immunogenic dsRNA [14]. In line with this, cells and HeLa cells from different passages or human depletion of PNPT1 in HeLa cells led to an abnormal islets from different donors. accumulation of mtdsRNA, its recognition by RIG-I and As positive controls for some of the parameters stud- MDA5, and the subsequent activation of a type I IFN ied, the cells were treated with human IFNα (PeproTech, response [15]. These and other similarly observations Rocky Hill, NJ, USA) at 2000 U/ml or transfected with led to the suggestion that generation of mtdsRNA is an 1  µg/ml of the synthetic dsRNA analog polyinosinic- important mechanism in the triggering of antiviral sign- polycytidylic acid (Sigma-Aldrich, Saint-Louis, MO, aling in humans [15]. USA) as previously described [22]. C oomans de Brachène et al. Autoimmun Highlights (2021) 12:6 Page 3 of 10 RNA interference and 0.005% bromophenol blue). To confirm PNPT1 and EndoC-βH1 cells or dispersed human islets were trans- ADAR knockdown, we used a rabbit polyclonal anti- fected overnight with 30  nmol/l of different siRNAs, PNPT1 antibody diluted 1/2000 (ab96176, Abcam, the medium was then changed, and cells kept in cul- Cambridge, UK) and a rabbit monoclonal anti-ADAR1 ture for 48  h to 6  days to allow gene silencing. Trans- antibody diluted 1/1000 (#14175, Cell Signaling Tech- fection was performed using siRNA targeting PNPT1 nologies, Danvers, MA, USA). To evaluate the type I IFN (PNPT1#1: 5′-GCU GCA CUA CGA GUU UCC UCC UUA activation, we measured STAT1 and STAT2 phospho- U-3′, PNPT1#2: 5′-CCU UUG GUG GUU GACU ACA rylation using rabbit anti-phospho antibodies (pSTAT1: GAC AAA-3′, and PNPT1#3: 5-GGG CAG UAC GAA #9167 and pSTAT2: #8841, Cell signaling Technologies) UAG GAA UAA UUG A-3′; HSS131758, HSS131759 diluted 1/1000, total STAT1 and STAT2 expression using and HSS131760 respectively, Thermo Fisher Scientific, a rabbit anti-STAT1 (sc-346, Santa Cruz Biotechnol- Waltham, MA, USA), SUV3 (5′-GGC CUC UGG ACA ogy, Dallas, USA) and a monoclonal rabbit anti-STAT2 AGA AUG AAG UAA A-3′; HSS110378, Thermo Fisher (#72604, Cell Signaling Technologies) antibody, both Scientific) or ADAR (5′-TTC CGT TAC CGC AGG GAT diluted 1/1000. We have previously shown that total CTA-3′; 1027416, Qiagen, Venlo, The Netherlands) using STAT1 and STAT2 expression are up-regulated in beta Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, cells following exposure to a type I IFN [19]. We normal- USA). In experiments with double transfection, we mixed ized our data for the expression of the housekeeping gene 30 nmol/l of each siRNA used. Allstars Negative Control β-actin using a polyclonal rabbit anti-β-actin antibody siRNA (siQ; Qiagen) was used as a negative control. diluted 1/2000 (#4967, Cell Signaling Technologies). A secondary donkey anti-rabbit antibody coupled with the horseradish peroxidase (711-036-152, Jackson Immu- Viral infection noResearch Laboratories, Wes Grove, PA, USA) was used The prototype strain of enterovirus (CVB5 / Faulkner) to detect the signal. Immunoreactive bands were visual- was obtained from the American Type Culture Collec- ized using the SuperSignal West Femto chemilumines- tion (ATCC, Old Town Manassas, Virginia, USA). GMK cent substrate (Thermo Fisher Scientific), detected using cells were used to amplify viruses and their identity was ChemiDoc XRS + (BIO-RAD), and quantified with the confirmed by a plaque neutralization assay with CVB5 Image Studio Lite v5.2 software (LI-COR Biosciences). anti-sera. EndoC-βH1 cells were inoculated for 1  h at 37  °C with CVB5 at a multiplicity of infection (MOI) of 5 based on our previous studies [23]. The inoculum virus Immunofluorescence was then removed, and cells were cultured in medium After different periods post-siRNA transfection or viral containing FBS during 24 h to allow viral replication. infection, cells were fixed with PFA 4% for 15  min fol - lowed by permeabilization with Triton X-100 0.3% for mRNA extraction and real‑time PCR 10  min. Cells were blocked in PBS-BSA 3%—Tween20 The Dynabeads mRNA DIRECT purification kit (Invit - 0.02% during 30  min and then incubated overnight with rogen) was used to purify polyadenylated mRNA from the monoclonal mouse J2 anti-dsRNA antibody diluted cultured cells, and reverse transcription was performed 1/200 in blocking buffer (10010200, Scicons, Hungary). using the Reverse Transcriptase Core kit (RT-RTCK-03, Cells infected with CVB5 were stained for the viral cap- Eurogentec, Liège, Belgium). We used the SsoAdvanced sid protein VP1 using the enterovirus-specific polyclonal Universal SYBR Green Supermix (BIO-RAD, Hercu- antiserum KTL-510 [24]. In dispersed human islets, les, CA, USA) to perform the real-time PCR amplifica - beta cells were also stained for insulin using a polyclonal tion reactions. We generated standard curves to allow guinea pig anti-insulin ready-to-use antibody (IR00261- adequate quantification of our data, and corrected gene 2, Agilent, Santa Clara, CA, USA) and fibroblasts were expression by the housekeeping gene β-actin, as its stained using the rabbit polyclonal anti-vimentin anti- expression is not affected by the conditions used in this body diluted 1/200 (ab137321, Abcam). The signal was study (data not shown). The list of primers used in this detected using Alexa Fluor 568 rabbit anti-mouse, Alexa study can be found in Additional file 2. Fluor 488 goat anti-guinea pig and Alexa Fluor 647 don- key anti-rabbit (A11061, A11073 and A31573 respec- tively, Thermo Fisher Scientific) secondary antibodies Protein extraction and western blot analysis at 1/200. Hoechst 33342 (Sigma-Aldrich, Saint-Louis, Cells were washed with PBS and lysed using 1X Lae- MO, USA) was used for nuclear staining. The coverslips mmli Sample Buffer (60  mM tris(hydroxymethyl)ami - were mounted with the Glycergel mounting medium nomethane pH 6.8, 10% Glycerol, 1.5% Dithiothreitol, (C056330-2, Agilent) and immunofluorescence was visu - 1.5% 2-mercaptoethanol, 2% Sodium dodecyl sulfate alized using a Zeiss microscope equipped with a camera Coomans de Brachène et al. Autoimmun Highlights (2021) 12:6 Page 4 of 10 (Zeiss-Vision, Munich, Germany). Images were acquired of these dsRNAs by ADAR, we silenced PNPT1 and at ×40 magnification and analysed with the AxioVision ADAR alone or in combination in EndoC-βH1 cells, software. reducing their expression by > 50% at the protein (Fig.  2a–c) and mRNA (Fig.  2d–e) levels. However, the double silencing of PNPT1/ADAR neither induced Statistical analysis accumulation of dsRNA (data not shown) nor activated Data are expressed as means ± SEM. A normality test was a type I IFN response, as evaluated by the phospho- performed to evaluate the distribution of the observed rylation of STAT1 and STAT2 (Fig.  2a, f, g), and by the determinations. Paired or unpaired t test were performed expression of total STAT1 and STAT2 (Fig.  2a, h, i) at to assess statistical significance and results with p < 0.05 the protein level, and IFNβ (Fig.  2j, k), MDA5 (Fig.  2l), were considered statistically significant. HLA-ABC (Fig. 2m), MX1 (Fig. 2n) and CHOP (Fig. 2o) at the mRNA level. IFNα was used as a positive con- Results trol for all genes except for IFNβ, where we used the PNPT1 silencing, alone or together with ADAR silencing, synthetic dsRNA analog polyinosinic-polycytidylic does not induce dsRNA accumulation or a type I IFN acid (PIC) since IFNα does not induce its expression response in EndoC‑βH1 cells (Fig.  2k). Altogether, these data suggest that the silenc- To study the possible role of PNPT1 and mtdsRNA in the ing of PNPT1 for 48 h does not induce accumulation of initiation of the innate immune response in pancreatic immunogenic mtdsRNA in EndoC-βH1 cells. beta cells, as previously described in HeLa cells [15], we used two different siRNAs (siPNPT1#1 and #2). These siRNAs reduced PNPT1 expression by > 50% at both pro- Long‑term (6 days) double silencing of PNPT1 and SUV3 tein (Fig. 1a, b) and mRNA (Fig. 1c) levels in EndoC-βH1 induces dsRNA accumulation in EndoC‑βH1 cells but not a cells. After 48 h of silencing, no accumulation of dsRNA type I IFN response was detected (Fig.  1d), while cells infected with the cox- To evaluate the possible compensation of PNPT1 sackievirus CVB5, used as a positive control, had a strong silencing by SUV3, the other member of the mito- signal for dsRNA in cells positive for the viral capside chondrial degradosome, we silenced these two genes protein VP1 (Fig.  1d). Furthermore, PNPT1 knockdown simultaneously in EndoC-βH1 cells but again did not did not induce a type I IFN response in EndoC-βH1 observe dsRNA accumulation after 48  h and 72  h of cells, as measured by the phosphorylation of STAT1 and knockdown (data not shown). On the other hand, a STAT2 (Fig. 1a, e, f ) and by the expression of key down- prolonged exposure to these 2 siRNAs for 4 additional stream targets of IFNα signaling, namely total STAT1 days after the usual 48  h of silencing (6  days in total) and STAT2 (Fig. 1a, g, h) at the protein level, and MDA5 significantly reduced the expression of both PNPT1 (Fig.  1i), the Human Leukocyte Antigen-ABC (HLA- and SUV3 (Fig.  3a–d) and induced a clearly detect- ABC also known as Major Histocompatibility complex able accumulation of dsRNA in EndoC-βH1 cells class I, MHC class I) (Fig. 1j), the antiviral MX dynamin- (Fig.  3e). These data were confirmed by using a sec - like GTPase 1 (MX1) (Fig.  1k) and the ER stress marker ond siPNPT1 (Additional file  4a–c). Of note, the single CCAAT/enhancer-binding protein homologous protein knockdown of either PNPT1 or SUV3 for 6  days only (CHOP, also known as DDIT3) (Fig.  1l) at the mRNA modestly induced dsRNA accumulation (Fig.  3e and level. We used IFNα as a positive control since we pre- Additional file  4c). Despite the presence of dsRNA in viously showed that it induces a clear up-regulation of EndoC-βH1 cells, there was still no induction of a type these genes in EndoC-βH1 cells [19, 25]. On the other I IFN response as indicated by the absence of STAT1 hand, a clear accumulation of dsRNA was observed in and STAT2 phosphorylation (Fig.  3a, f, g) and the lack the fast proliferating HeLa cells following knockdown of of induction of MDA5 (Fig.  3h and Additional file  4d), PNPT1 with three different siPNPT1 (#1, #2 and #3) as HLA-ABC (Fig. 3i and Additional file  4e), MX1 (Fig. 3j) previously described [15] (Additional file  3a). Surpris- and CHOP (Fig.  3k and Additional file  4f ) expression. ingly, and in spite of the fact that siPNPT1#2 and #3 IFNα was used as a positive control. These findings induced a more marked inhibition of PNPT1 expression support the importance of the collaborative action of than that observed with siPNPT1#1 (the siRNA used the two members of the mitochondrial degradosome, by Dhir et  al., [15]) (Additional file  3b), only siPNPT1#1 PNPT1 and SUV3, in preventing accumulation of mtd- triggered an antiviral response, as determined by the sRNA in pancreatic beta cells. However, and different expression of IFNβ and MDA5 (Additional file 3c, d). from observations in other fast-proliferating cell lines Next, to assess whether PNPT1 silencing-mediated [15], in human pancreatic beta cells these mtdsRNA do mtdsRNA accumulation was masked by a rapid editing not trigger a type I IFN response. C oomans de Brachène et al. Autoimmun Highlights (2021) 12:6 Page 5 of 10 Fig. 1 PNPT1 silencing does not induce dsRNA accumulation and a type I IFN response in EndoC-βH1 cells. EndoC-βH1 cells were transfected with a siRNA control (siQ: white bars) and two different siRNAs targeting PNPT1 (siP#1: grey bars and siP#2: blue bars) and cells were maintained in culture during 48 h. As a positive control for gene expression, in some experiments cells were treated with IFNα (2000 U/ml) for 24 h (red bars), and as a positive control for dsRNA staining, cells were infected with CVB5 (MOI 5). a Protein expression was measured by western blotting and representative images of 2–9 independent experiments are shown. Densitometry results are shown for PNPT1 (b), pSTAT1 (e), pSTAT2 (f), STAT1 (g) and STAT2 (h). d dsRNA accumulation (red) and the presence of the viral capside protein VP1 (green) for CVB5-infected cells was analyzed by immunocytochemistry. Representative images of 2 (siPNPT1#1) or 3 (siPNPT1#2) independent experiments and images of 1 experiment with CVB5 infection are shown (magnification 40×). mRNA expression of PNPT1 (c), MDA5 (i), HLA-ABC (j), MX1 (k) and CHOP (l) were analyzed by RT-qPCR and normalized by β-actin and then by the value of siQ considered as 1. Results are mean ± SEM of 3–9 independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs siQ, Student t test Coomans de Brachène et al. Autoimmun Highlights (2021) 12:6 Page 6 of 10 Fig. 2 PNPT1/ADAR double silencing does not induce a type I IFN response in EndoC-βH1 cells. EndoC-βH1 cells were transfected with a siRNA control (siQ: white bars) and siRNAs targeting PNPT1 (siP#1, grey bars), ADAR (siA, green striped bars), or both (siP/A: grey bars with green stripes) and cells were maintained in culture during 48 h. As a positive control for gene expression, in some experiments cells were treated with IFNα (2000 U/ml) for 24 h (red bars), and as a positive control for IFNβ expression, cells were transfected with PIC (1 µg/ml) for 24 h (k, orange bar). a Protein expression was measured by western blotting and representative images of 3–7 independent experiments are shown. Densitometry results are shown for PNPT1 (b), ADAR (c), pSTAT1 (f), pSTAT2 (g), STAT1 (h) and STAT2 (i). mRNA expression of PNPT1 (d), ADAR (e), IFNβ (j, k), MDA5 (l), HLA-ABC (m), MX1 (n) and CHOP (o) were analyzed by RT-qPCR and normalized by β-actin and then by the value of siQ considered as 1. Results are mean ± SEM of 3–9 independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs siQ, Student t test C oomans de Brachène et al. Autoimmun Highlights (2021) 12:6 Page 7 of 10 Fig. 3 Long-term PNPT1/SUV3 silencing induces dsRNA accumulation in EndoC-βH1 cells but no type I IFN response. EndoC-βH1 cells were transfected with a siRNA control (siQ: white bars) or with siRNAs targeting PNPT1 (siP#1, grey bars), SUV3 (siS, black striped bars) or both (siP/S: grey bars with black stripes), and then maintained in culture for 6 days after transfection. As a positive control for gene expression, in some experiments cells were treated with IFNα (2000 U/ml) for 24 h (red bars). a Protein expression was measured by western blotting and representative images of 2 independent experiments are shown. Densitometry results are shown for PNPT1 (b), pSTAT1 (f) and pSTAT2 (g). e dsRNA accumulation (red) was analyzed by immunocytochemistry. Representative images of 3 independent experiments are shown (magnification 40×). mRNA expression of PNPT1 (c), SUV3 (d), MDA5 (h), HLA-ABC (i), MX1 (j) and CHOP (k) were analyzed by RT-qPCR and normalized by β-actin and then by the value of siQ considered as 1. Results are mean ± SEM of 2–10 independent experiments. *p < 0.05 and ***p < 0.001 vs siQ, Student t test The double silencing PNPT1/SUV3 induces dsRNA Discussion accumulation in fibroblasts but not in dispersed human In the present study, while searching for new triggers of islet cells innate immunity in the context of T1D, we did two major In dispersed human islets, PNPT1 and SUV3 double observations: (1) the accumulation of mtdsRNA upon knockdown for 6  days (the usual 48  h of silencing fol- degradosome silencing seems to be cell-type specific; and lowed by 4 additional days) did not induce dsRNA (2) these mtdsRNA do not trigger a type I IFN response accumulation in the primary non-proliferating insu- in human pancreatic beta cells. lin-positive beta cells (Fig.  4a) as it was observed in In a previous study, and confirmed by our own data EndoC-βH1 cells. There was, however, a clear accu - (Additional file  3), PNPT1 or SUV3 knockdown induced mulation of dsRNA in some, but not all, vimentin-pos- a rapid and massive accumulation of dsRNA in the fast- itive fibroblasts present in the human islet preparations proliferating HeLa cells [15]. However, in slowly prolifer- (Fig.  4b, c and Additional file  5, upper part). In a lesser ating EndoC-βH1 cells PNPT1 silencing for 48 h did not proportion, other non-beta cells negative for vimentin induce dsRNA accumulation. This cannot be explained also contained dsRNA (Fig. 4c and Additional file  5, lower by ADAR editing as a compensatory mechanism for the part). This distinct effect, occurring in cells present in the inhibition of PNPT1, since the double knockdown of same preparation and treated under the same experimen- PNPT1 and ADAR for 48 h also failed to induce dsRNA. tal conditions, highlights the fact that this phenomenon On the other hand, long-term (6  days) double knock- is cell-type dependent and probably related to the prolif- down of the two members of the mitochondrial degrado- erative capacity of the cells (i.e. fibroblasts are fast prolif - some, PNPT1 and SUV3, induced dsRNA accumulation erating cells, while primary human beta cells practically in the slowly proliferating EndoC-βH1 cells but not in the don’t proliferate under basal culture conditions). non-proliferating primary human beta cells. Surprisingly, Coomans de Brachène et al. Autoimmun Highlights (2021) 12:6 Page 8 of 10 Fig. 4 PNPT1/SUV3 knockdown induces dsRNA accumulation in surrounding fibroblasts but not in dispersed human islet cells. Dispersed human islets were transfected with a siRNA control (siQ) or with siRNAs targeting PNPT1 (#1), SUV3 or both (P/S), and then maintained in culture for 6 days after transfection. dsRNA accumulation (red), insulin content for beta cell staining (green), and vimentin for fibroblast staining (purple) were analyzed by immunocytochemistry. Representative images of 4 (a) and 2 (b, c) independent experiments are shown (magnification 40×). c dsRNA and vimentin positivity were evaluated by counting 1400 non-beta cells from 69 images obtained from 2 independent experiments and confirming the cell-specific nature of this process, different from previous observations in HeLa cells [15]. the vimentin-positive fibroblasts present in the human On the other hand, when using the same siPNPT1 (siP- islet preparations stained positively for dsRNA after NPT1#1) used in the study by Dhir and colleagues [15] silencing of PNPT1 and/or SUV3. These data point to a we were able to reproduce the observed mtdsRNA accu- cell-specific effect that could be connected to the prolif - mulation in HeLa cells (Additional file  3). However, two erative status of the cells, as this is the main difference other siPNPT1 that inhibited PNPT1 by > 70% (#2 and between EndoC-βH1 cells and primary human islet cells. #3) failed to trigger the type I IFN response, in spite of Of note, we have recently shown that EndoC-βH1 cells inducing a clear accumulation of dsRNA in HeLa cells or primary human islets treated with IFNα have a similar (Additional file  3). Thus, these data, and the data pre - gene signature as compared to beta cells obtained from sented by Dhir and colleagues [15] using one of the pres- patients affected by T1D [19]. ently tested siRNAs, need to be taken with caution as we In line with the hypothesis that mtdsRNA accumula- can’t exclude that some of the effects observed with siP - tion correlates with the proliferative status of the cells, NPT1#1 are off target effects, unrelated from the actual when studying mitochondrial RNA (mtRNA) extracted inhibition of PNPT1. In any case, none of these siPNPT1, from S or M phase-arrested HeLa cells, the largest frac- alone or combined with SUV3 siRNA, induced a type I tion of mtRNA isolated in the M phase were present as IFN response in EndoC-βH1 cells, despite the presence of duplex RNA [26]. In addition, the analysis of mitochon- dsRNA. This indicates that mtdsRNA accumulating upon drial DNA (mtDNA) replication and transcription dur- degradosome inactivation is probably not a mediator of ing the cell cycle showed a clear coordination between the type I IFN response in human pancreatic islets. mtDNA and nuclear DNA synthesis [27] that may explain the presently observed absence of mtdsRNA in the non- Conclusions proliferating primary beta cells. Our findings support the concept that accumulation of We also noticed that mtdsRNA accumulation in mtdsRNA following mitochondrial degradosome knock- EndoC-βH1 cells fail to induce a type I IFN response, down is not a general cell mechanism but is preferentially C oomans de Brachène et al. Autoimmun Highlights (2021) 12:6 Page 9 of 10 and innovation programme and “EFPIA”, “JDRF” and “The Leona M. and Harry B. present in fast proliferating cells. More importantly, Helmsley Charitable Trust”. accumulation of mtdsRNA is not a general trigger of the innate immune response, as it fails to do so in human Availability of data and materials All data generated or analysed during the current study are available from the pancreatic beta cells. Therefore, mtdsRNA does not seem corresponding author on reasonable request. to be an endogenous “danger signal” for the induction of IFNα in the beta cells in the context of T1D. We cannot Ethics approval and consent to participate The use of human islets was approved by the local ethics committee of Pisa, exclude, however, an indirect effect of these mtdsRNA Italy. in the development of T1D through their release from the beta cells via exosomes, and their subsequent action Consent for publication Not applicable. on immune cells as described in the context of alcoholic liver disease [28]. Competing interests The authors declare that they have no competing interests. Supplementary Information Author details The online version contains supplementary material available at https ://doi. ULB Center for Diabetes Research, Medical Faculty, Campus Erasme, Univer- org/10.1186/s1331 7-021-00148 -2. sité Libre de Bruxelles (ULB), Route de Lennik, 808-CP618, 1070 Brussels, Bel- gium. Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy. Indiana Biosciences Research Institute, Indianapolis, IN, USA. Additional file 1. Characteristics of the human donors providing the islets used in the present study. Received: 28 September 2020 Accepted: 2 January 2021 Additional file 2. List of primers used in the study. Additional file 3. PNPT1 knockdown in HeLa cells induces dsRNA accu- mulation but a type I IFN response is only observed with one siRNA out of three tested. References Additional file 4. A second siPNPT1 confirms the accumulation of dsRNA 1. Op de Beeck A, Eizirik DL. Viral infections in type 1 diabetes mellitus— after the double knockdown of PNPT1 and SUV3 for 6 days in EndoC-βH1 why the β cells? Nat Rev Endocrinol. 2016;12(5):263–73. cells, without a type I IFN response. 2. Eizirik DL, Pasquali L, Cnop M. Pancreatic β-cells in type 1 and type 2 Additional file 5. The double silencing PNPT1/SUV3 induces dsRNA accu- diabetes mellitus: different pathways to failure. Nat Rev Endocrinol. mulation in non-beta cells of the human islet preparations. 2020;16(7):349–62. 3. Foulis AK, Farquharson MA, Meager A. Immunoreactive alpha-interferon in insulin-secreting beta cells in type 1 diabetes mellitus. Lancet Lond Abbreviations Engl. 1987;2(8573):1423–7. CVB: Coxsackievirus B; dsRNA: Double-stranded RNA; ER: Endoplasmic 4. Lundberg M, Krogvold L, Kuric E, Dahl-Jørgensen K, Skog O. Expression reticulum; IFNα: Interferon-α; IFNβ: Interferon-β; MOI: Multiplicity of infection; of interferon-stimulated genes in insulitic pancreatic islets of patients mtDNA: Mitochondrial DNA; mtdsRNA: Mitochondrial double-stranded RNA; recently diagnosed with type 1 diabetes. Diabetes. 2016;65(10):3104–10. PIC: Polyinosinic-polycytidylic acid; siRNA: Small-interfering RNA; T1D: Type 1 5. Chehadeh W, Weill J, Vantyghem MC, Alm G, Lefèbvre J, Wattré P, et al. diabetes. Increased level of interferon-alpha in blood of patients with insulin- dependent diabetes mellitus: relationship with coxsackievirus B infection. Acknowledgements J Infect Dis juin. 2000;181(6):1929–39. The authors are grateful to Dr A. Op de Beeck for help with CVB infection, 6. Richardson SJ, Morgan NG. Enteroviral infections in the pathogenesis and M. Pangerl, M. Depessemier and I. Millard of the ULB Center for Diabetes of type 1 diabetes: new insights for therapeutic intervention. Curr Opin Research, Université Libre de Bruxelles, Belgium, for excellent technical Pharmacol. 2018;43:11–9. support. 7. Chehadeh W, Weill J, Vantyghem M-C, Alm G, Lefèbvre J, Wattré P, et al. Increased level of interferon-α in blood of patients with insulin-depend- Authors’ contributions ent diabetes mellitus: relationship with coxsackievirus B infection. J Infect ACdB contributed to the original idea, design and performance of the Dis. 2000;181(6):1929–39. experiments, investigation and formal analysis, and wrote, revised, and edited 8. Hyöty H. Viruses in type 1 diabetes. Pediatr Diabetes. 2016;17(Suppl the manuscript. AC and AEM performed experiments. LM and PM contrib- 22):56–64. uted with resources. DLE contributed to the original idea and the design, 9. Cinek O, Stene LC, Kramna L, Tapia G, Oikarinen S, Witsø E, et al. Entero- supervision and interpretation of the experiments, wrote, revised and edited virus RNA in longitudinal blood samples and risk of islet autoimmunity the manuscript. All authors have read and approved the final version of the in children with a high genetic risk of type 1 diabetes: the MIDIA study. manuscript. ACdB is the guarantor of this work and, as such, has full access to Diabetologia. 2014;57(10):2193–200. all the data in the study and takes responsibility for the integrity of the data 10. Said EA, Tremblay N, Al-Balushi MS, Al-Jabri AA, Lamarre D. Viruses seen and the accuracy of the data analysis. All authors read and approved the final by our cells: the role of viral RNA sensors. J Immunol Res. 2018;2018:1–14. manuscript. 11. Gebhardt A, Laudenbach BT, Pichlmair A. Discrimination of self and non- self ribonucleic acids. J Interferon Cytokine Res. 2017;37(5):184–97. Funding 12. Rice GI, Kasher PR, Forte GMA, Mannion NM, Greenwood SM, Szynkiewicz D.L.E. acknowledges the support of a grant from the Welbio-FNRS (Fonds M, et al. Mutations in ADAR1 cause Aicardi-Goutières syndrome associ- National de la Recherche Scientifique), Belgium, the Dutch Diabetes Fonds ated with a type I interferon signature. Nat Genet. 2012;44(11):1243–8. (DDFR), Holland, and start up-funds from the Indiana Biosciences Research 13. Yu Z, Chen T, Cao X. RNA editing by ADAR1 marks dsRNA as “self.” Cell Res Institute (IBRI), Indianapolis, Indiana, USA. D.L.E. and P.M. are supported by the déc. 2015;25(12):1283–4. Innovative Medicines Initiative 2 Joint Undertaking under grant agreements 14. Kotrys AV, Szczesny RJ. Mitochondrial gene expression and beyond— No 115797 (INNODIA) and 945268 (INNODIA HARVEST ), supported by the novel aspects of cellular physiology. Cells. 2019;9(1):17. European Union’s Horizon 2020 research and innovation programme. These 15. Dhir A, Dhir S, Borowski LS, Jimenez L, Teitell M, Rötig A, et al. Mitochon- Joint Undertakings receive support from the Union’s Horizon 2020 research drial double-stranded RNA triggers antiviral signalling in humans. Nature. 2018;560(7717):238–42. Coomans de Brachène et al. Autoimmun Highlights (2021) 12:6 Page 10 of 10 16. Ravassard P, Hazhouz Y, Pechberty S, Bricout-Neveu E, Armanet M, 23. Colli ML, Paula FM, Marselli L, Marchetti P, Roivainen M, Eizirik DL, et al. Czernichow P, et al. A genetically engineered human pancreatic β cell Coxsackievirus B tailors the unfolded protein response to favour viral line exhibiting glucose-inducible insulin secretion. J Clin Invest sept. amplification in pancreatic β cells. J Innate Immun. 2019;11(4):375–90. 2011;121(9):3589–97. 24. Hovi T, Roivainen M. Peptide antisera targeted to a conserved sequence 17. Brozzi F, Nardelli TR, Lopes M, Millard I, Barthson J, Igoillo-Esteve M, et al. in poliovirus capsid VP1 cross-react widely with members of the genus Cytokines induce endoplasmic reticulum stress in human, rat and mouse Enterovirus. J Clin Microbiol mai. 1993;31(5):1083–7. beta cells via different mechanisms. Diabetologia. 2015;58(10):2307–16. 25. Marroqui L, Dos Santos RS, Op de Beeck A, Coomans de Brachène A, Mar- 18. Kirkegaard JS, Ravassard P, Ingvarsen S, Diedisheim M, Bricout-Neveu E, selli L, Marchetti P, et al. Interferon-α mediates human beta cell HLA class Grønborg M, et al. Xenotropic retrovirus Bxv1 in human pancreatic β cell I overexpression, endoplasmic reticulum stress and apoptosis, three hall- lines. J Clin Invest. 2016;126(3):1109–13. marks of early human type 1 diabetes. Diabetologia. 2017;60(4):656–67. 19. Colli ML, Ramos-Rodríguez M, Nakayasu ES, Alvelos MI, Lopes M, Hill JLE, 26. Kim Y, Park J, Kim S, Kim M, Kang M-G, Kwak C, et al. PKR senses nuclear et al. An integrated multi-omics approach identifies the landscape of and mitochondrial signals by interacting with endogenous double- interferon-α-mediated responses of human pancreatic beta cells. Nat stranded RNAs. Mol Cell. 2018;71(6):1051-1063.e6. Commun. 2020;11(1):2584. 27. Chatre L, Ricchetti M. Prevalent coordination of mitochondrial DNA 20. Marchetti P, Suleiman M, Marselli L. Organ donor pancreases for the study transcription and initiation of replication with the cell cycle. Nucleic Acids of human islet cell histology and pathophysiology: a precious and valu- Res. 2013;41(5):3068–78. able resource. Diabetologia. 2018;61(4):770–4. 28. Lee J-H, Shim Y-R, Seo W, Kim M-H, Choi W-M, Kim H-H, et al. Mitochon- 21. Marroqui L, Lopes M, dos Santos RS, Grieco FA, Roivainen M, Richardson drial double-stranded RNA in exosome promotes interleukin-17 produc- SJ, et al. Differential cell autonomous responses determine the outcome tion through toll-like receptor 3 in alcohol-associated liver injury. Hepatol of coxsackievirus infections in murine pancreatic α and β cells. eLife. Baltim Md août. 2020;72(2):609–25. 2015;4:e06990. 22. Marroqui L, Dos Santos RS, Fløyel T, Grieco FA, Santin I, Op de Beeck A, Publisher’s Note et al. TYK2, a candidate gene for type 1 diabetes, modulates apoptosis Springer Nature remains neutral with regard to jurisdictional claims in pub- and the innate immune response in human pancreatic β-cells. Diabetes. lished maps and institutional affiliations. 2015;64(11):3808–17. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Autoimmunity Highlights Springer Journals

Endogenous mitochondrial double‐stranded RNA is not an activator of the type I interferon response in human pancreatic beta cells

Loading next page...
 
/lp/springer-journals/endogenous-mitochondrial-double-stranded-rna-is-not-an-activator-of-HYewA59wAZ

References (32)

Publisher
Springer Journals
Copyright
Copyright © The Author(s) 2021
ISSN
2038-0305
eISSN
2038-3274
DOI
10.1186/s13317-021-00148-2
Publisher site
See Article on Publisher Site

Abstract

Background: Type 1 diabetes ( T1D) is an autoimmune disease characterized by the progressive destruction of pancreatic beta cells. Interferon-α (IFNα), an antiviral cytokine, is expressed in the pancreatic islets in early T1D, which may be secondary to viral infections. However, not all patients harboring a type I IFN signature present signals of viral infection, suggesting that this response might be initiated by other “danger signals”. Accumulation of mitochondrial double-stranded RNA (mtdsRNA; a danger signal), secondary to silencing of members of the mitochondrial degrado- some, PNPT1 and SUV3, has been described to activate the innate immune response. Methods: To evaluate whether mtdsRNA represents a “danger signal” for pancreatic beta cells in the context of T1D, we silenced PNPT1 and/or SUV3 in slowly proliferating human insulin-secreting EndoC-βH1 cells and in non-prolif- erating primary human beta cells and evaluated dsRNA accumulation by immunofluorescence and the type I IFN response by western blotting and RT-qPCR. Results: Only the simultaneous silencing of PNPT1/SUV3 induced dsRNA accumulation in EndoC-βH1 cells but not in dispersed human islets, and there was no induction of a type I IFN response. By contrast, silencing of these two genes individually was enough to induce dsRNA accumulation in fibroblasts present in the human islet preparations. Conclusions: These data suggest that accumulation of endogenous mtdsRNA following degradosome knockdown depends on the proliferative capacity of the cells and is not a mediator of the type I IFN response in human pancreatic beta cells. Keywords: Human pancreatic beta cells, Type 1 diabetes, PNPT1, Mitochondrial dsRNA, Type I interferon Background response at the islet level, are associated with increased Type 1 diabetes (T1D) is a chronic autoimmune dis- disease risk [1, 2]. Interferon-α (IFNα), a type I IFN ease characterized by the progressive destruction of involved in innate immunity and the antiviral response, pancreatic beta cells by the immune system and subse- is expressed in islets of patients affected by T1D, as first quent loss of insulin secretion. More than 50 gene vari- described by Foulis et al. who found IFNα in pancreases ants, including several genes that regulate the antiviral of 33/34 patients with T1D, while it was detected in only 4/80 controls [3]. More recently, analysis of laser- captured islets from five patients with recent-onset T1D showed that > 30% of the 84 IFN-stimulated genes *Correspondence: alcooman@ulb.ac.be ULB Center for Diabetes Research, Medical Faculty, Campus Erasme, (ISG) analysed where overexpressed by at least fivefold Université Libre de Bruxelles (ULB), Route de Lennik, 808-CP618, in these patients compared with islets from five non- 1070 Brussels, Belgium diabetic control organ donors [4]. Additionally, T1D Full list of author information is available at the end of the article © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. Coomans de Brachène et al. Autoimmun Highlights (2021) 12:6 Page 2 of 10 patients have higher circulating levels of IFNα than Against this background, we presently evaluated controls. Thus, Chehadeh et  al. detected elevated lev - whether generation of mtdsRNA is part of the potential els of IFNα in plasma of 70% (39/56) of T1D patients endogenous “danger signals” generated in pancreatic and 50% of these IFNα-positive patients were also posi- beta cells that may contribute to trigger a local innate tive for enterovirus RNA, while IFNα-negative patients immune response in the context of T1D. Surprisingly, were negative for enterovirus RNA [5]. Accordingly, we observed that the degradosome silencing-induced enteroviral infection of beta cells, particularly by cox- mtdsRNA accumulation is a cell type specific event, sackievirus B (CVB), has been associated with T1D present in the human insulin-secreting EndoC-βH1 development [1, 6]. Viral infections may stimulate the cells and islet fibroblasts but not in primary human production of IFNα in the vicinity of the beta cells and beta cells, suggesting that the phenomenon depends contribute to trigger autoimmunity in genetically pre- on the proliferative status of cells. In addition, abnor- disposed individuals [1]. Enterovirus RNA or proteins mal accumulation of mtdsRNA does not induce a type are found in blood, stool or pancreatic islets from T1D I IFN signature in human pancreatic beta cells, indicat- patients more frequently than in controls [7, 8], but ing that this is not a direct contributory factor for T1D many patients that develop disease don’t show a clear initiation. correlation with viral infection [9], suggesting the exist- ence of other “danger signals” that may initiate a type I IFN response in the context of T1D. Methods Viral replication may lead to accumulation of cytosolic Culture of human EndoC‑βH1 cells, HeLa cells and human double-stranded RNA (dsRNA), which is recognized by islets and cell treatment sensors such as retinoic acid-inducible gene I (RIG-I)- EndoC-βH1 cells, a human pancreatic beta cell line like receptors (RLRs), including DDX58 (also known as provided by Dr. R. Scharfmann (University of Paris, RIG-I) and IFIH1 (also known as melanoma differenti - France) [16], were cultured in Matrigel-fibronectin- ation-associated protein-5 MDA5) [10]. Other sources coated plates as previously described [17]. These are of dsRNAs, derived from intracellular processes such as slowly proliferating cells, dividing every 5  days [16]. the transcription of repetitive sequences, are also present EndoC-βH1 cells have been shown to be chronically naturally in mammalian cells. This poses a challenge to infected by a xenotropic mouse retrovirus [18]. How- these cells, as they must discriminate between self and ever, our RNAseq data indicate that they do not pre- non-self dsRNA to avoid accidental activation of the type sent an increased basal innate immune response and I IFN signaling. This discrimination is made possible have also a similar immune response when treated by different mechanisms [11], including adenosine-to- with IFNα as compared to primary human islets [19]. inosine (A-to-I) editing of cellular dsRNA by the RNA- The human cervical cancer cells HeLa were grown in editing enzyme Adenosine deaminase RNA-specific Dulbecco’s modified Eagle’s medium (Lonza, Basel, (ADAR). Indeed, mutations in ADAR1 cause the auto- Switzerland) supplemented with 10% FBS and 2% peni- immune Aicardi-Goutières syndrome associated with a cillin–streptomycin (Lonza). These cell lines were free type I IFN signature [12, 13]. Another cellular source of of mycoplasma infection, as determined by monthly dsRNA is the mitochondria, where the circular genome is testing using the MycoAlert Mycoplasma Detection kit transcribed bidirectionally and the RNA generated from (Lonza). the two mitochondrial DNA strands (H and L strands) Human islets from 6 non-diabetic organ donors (see can generate long mitochondrial dsRNA (mtdsRNA). The Additional file  1) were isolated in Pisa, Italy, following a mitochondrial RNA degradosome, formed by the Suv3- protocol previously described [20] with the approval of like RNA helicase SUPV3L1 (also known as SUV3) and the local ethics committee and sent to Brussels for dis- the polynucleotide phosphorylase PNPase (also known persion and experiments [21]. as PNPT1), mediates the rapid decay of the non-coding Each experiment considered as n = 1 corresponds to L-strand transcripts which prevents the formation of one independent biological observation, i.e. EndoC-βH1 potentially immunogenic dsRNA [14]. In line with this, cells and HeLa cells from different passages or human depletion of PNPT1 in HeLa cells led to an abnormal islets from different donors. accumulation of mtdsRNA, its recognition by RIG-I and As positive controls for some of the parameters stud- MDA5, and the subsequent activation of a type I IFN ied, the cells were treated with human IFNα (PeproTech, response [15]. These and other similarly observations Rocky Hill, NJ, USA) at 2000 U/ml or transfected with led to the suggestion that generation of mtdsRNA is an 1  µg/ml of the synthetic dsRNA analog polyinosinic- important mechanism in the triggering of antiviral sign- polycytidylic acid (Sigma-Aldrich, Saint-Louis, MO, aling in humans [15]. USA) as previously described [22]. C oomans de Brachène et al. Autoimmun Highlights (2021) 12:6 Page 3 of 10 RNA interference and 0.005% bromophenol blue). To confirm PNPT1 and EndoC-βH1 cells or dispersed human islets were trans- ADAR knockdown, we used a rabbit polyclonal anti- fected overnight with 30  nmol/l of different siRNAs, PNPT1 antibody diluted 1/2000 (ab96176, Abcam, the medium was then changed, and cells kept in cul- Cambridge, UK) and a rabbit monoclonal anti-ADAR1 ture for 48  h to 6  days to allow gene silencing. Trans- antibody diluted 1/1000 (#14175, Cell Signaling Tech- fection was performed using siRNA targeting PNPT1 nologies, Danvers, MA, USA). To evaluate the type I IFN (PNPT1#1: 5′-GCU GCA CUA CGA GUU UCC UCC UUA activation, we measured STAT1 and STAT2 phospho- U-3′, PNPT1#2: 5′-CCU UUG GUG GUU GACU ACA rylation using rabbit anti-phospho antibodies (pSTAT1: GAC AAA-3′, and PNPT1#3: 5-GGG CAG UAC GAA #9167 and pSTAT2: #8841, Cell signaling Technologies) UAG GAA UAA UUG A-3′; HSS131758, HSS131759 diluted 1/1000, total STAT1 and STAT2 expression using and HSS131760 respectively, Thermo Fisher Scientific, a rabbit anti-STAT1 (sc-346, Santa Cruz Biotechnol- Waltham, MA, USA), SUV3 (5′-GGC CUC UGG ACA ogy, Dallas, USA) and a monoclonal rabbit anti-STAT2 AGA AUG AAG UAA A-3′; HSS110378, Thermo Fisher (#72604, Cell Signaling Technologies) antibody, both Scientific) or ADAR (5′-TTC CGT TAC CGC AGG GAT diluted 1/1000. We have previously shown that total CTA-3′; 1027416, Qiagen, Venlo, The Netherlands) using STAT1 and STAT2 expression are up-regulated in beta Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, cells following exposure to a type I IFN [19]. We normal- USA). In experiments with double transfection, we mixed ized our data for the expression of the housekeeping gene 30 nmol/l of each siRNA used. Allstars Negative Control β-actin using a polyclonal rabbit anti-β-actin antibody siRNA (siQ; Qiagen) was used as a negative control. diluted 1/2000 (#4967, Cell Signaling Technologies). A secondary donkey anti-rabbit antibody coupled with the horseradish peroxidase (711-036-152, Jackson Immu- Viral infection noResearch Laboratories, Wes Grove, PA, USA) was used The prototype strain of enterovirus (CVB5 / Faulkner) to detect the signal. Immunoreactive bands were visual- was obtained from the American Type Culture Collec- ized using the SuperSignal West Femto chemilumines- tion (ATCC, Old Town Manassas, Virginia, USA). GMK cent substrate (Thermo Fisher Scientific), detected using cells were used to amplify viruses and their identity was ChemiDoc XRS + (BIO-RAD), and quantified with the confirmed by a plaque neutralization assay with CVB5 Image Studio Lite v5.2 software (LI-COR Biosciences). anti-sera. EndoC-βH1 cells were inoculated for 1  h at 37  °C with CVB5 at a multiplicity of infection (MOI) of 5 based on our previous studies [23]. The inoculum virus Immunofluorescence was then removed, and cells were cultured in medium After different periods post-siRNA transfection or viral containing FBS during 24 h to allow viral replication. infection, cells were fixed with PFA 4% for 15  min fol - lowed by permeabilization with Triton X-100 0.3% for mRNA extraction and real‑time PCR 10  min. Cells were blocked in PBS-BSA 3%—Tween20 The Dynabeads mRNA DIRECT purification kit (Invit - 0.02% during 30  min and then incubated overnight with rogen) was used to purify polyadenylated mRNA from the monoclonal mouse J2 anti-dsRNA antibody diluted cultured cells, and reverse transcription was performed 1/200 in blocking buffer (10010200, Scicons, Hungary). using the Reverse Transcriptase Core kit (RT-RTCK-03, Cells infected with CVB5 were stained for the viral cap- Eurogentec, Liège, Belgium). We used the SsoAdvanced sid protein VP1 using the enterovirus-specific polyclonal Universal SYBR Green Supermix (BIO-RAD, Hercu- antiserum KTL-510 [24]. In dispersed human islets, les, CA, USA) to perform the real-time PCR amplifica - beta cells were also stained for insulin using a polyclonal tion reactions. We generated standard curves to allow guinea pig anti-insulin ready-to-use antibody (IR00261- adequate quantification of our data, and corrected gene 2, Agilent, Santa Clara, CA, USA) and fibroblasts were expression by the housekeeping gene β-actin, as its stained using the rabbit polyclonal anti-vimentin anti- expression is not affected by the conditions used in this body diluted 1/200 (ab137321, Abcam). The signal was study (data not shown). The list of primers used in this detected using Alexa Fluor 568 rabbit anti-mouse, Alexa study can be found in Additional file 2. Fluor 488 goat anti-guinea pig and Alexa Fluor 647 don- key anti-rabbit (A11061, A11073 and A31573 respec- tively, Thermo Fisher Scientific) secondary antibodies Protein extraction and western blot analysis at 1/200. Hoechst 33342 (Sigma-Aldrich, Saint-Louis, Cells were washed with PBS and lysed using 1X Lae- MO, USA) was used for nuclear staining. The coverslips mmli Sample Buffer (60  mM tris(hydroxymethyl)ami - were mounted with the Glycergel mounting medium nomethane pH 6.8, 10% Glycerol, 1.5% Dithiothreitol, (C056330-2, Agilent) and immunofluorescence was visu - 1.5% 2-mercaptoethanol, 2% Sodium dodecyl sulfate alized using a Zeiss microscope equipped with a camera Coomans de Brachène et al. Autoimmun Highlights (2021) 12:6 Page 4 of 10 (Zeiss-Vision, Munich, Germany). Images were acquired of these dsRNAs by ADAR, we silenced PNPT1 and at ×40 magnification and analysed with the AxioVision ADAR alone or in combination in EndoC-βH1 cells, software. reducing their expression by > 50% at the protein (Fig.  2a–c) and mRNA (Fig.  2d–e) levels. However, the double silencing of PNPT1/ADAR neither induced Statistical analysis accumulation of dsRNA (data not shown) nor activated Data are expressed as means ± SEM. A normality test was a type I IFN response, as evaluated by the phospho- performed to evaluate the distribution of the observed rylation of STAT1 and STAT2 (Fig.  2a, f, g), and by the determinations. Paired or unpaired t test were performed expression of total STAT1 and STAT2 (Fig.  2a, h, i) at to assess statistical significance and results with p < 0.05 the protein level, and IFNβ (Fig.  2j, k), MDA5 (Fig.  2l), were considered statistically significant. HLA-ABC (Fig. 2m), MX1 (Fig. 2n) and CHOP (Fig. 2o) at the mRNA level. IFNα was used as a positive con- Results trol for all genes except for IFNβ, where we used the PNPT1 silencing, alone or together with ADAR silencing, synthetic dsRNA analog polyinosinic-polycytidylic does not induce dsRNA accumulation or a type I IFN acid (PIC) since IFNα does not induce its expression response in EndoC‑βH1 cells (Fig.  2k). Altogether, these data suggest that the silenc- To study the possible role of PNPT1 and mtdsRNA in the ing of PNPT1 for 48 h does not induce accumulation of initiation of the innate immune response in pancreatic immunogenic mtdsRNA in EndoC-βH1 cells. beta cells, as previously described in HeLa cells [15], we used two different siRNAs (siPNPT1#1 and #2). These siRNAs reduced PNPT1 expression by > 50% at both pro- Long‑term (6 days) double silencing of PNPT1 and SUV3 tein (Fig. 1a, b) and mRNA (Fig. 1c) levels in EndoC-βH1 induces dsRNA accumulation in EndoC‑βH1 cells but not a cells. After 48 h of silencing, no accumulation of dsRNA type I IFN response was detected (Fig.  1d), while cells infected with the cox- To evaluate the possible compensation of PNPT1 sackievirus CVB5, used as a positive control, had a strong silencing by SUV3, the other member of the mito- signal for dsRNA in cells positive for the viral capside chondrial degradosome, we silenced these two genes protein VP1 (Fig.  1d). Furthermore, PNPT1 knockdown simultaneously in EndoC-βH1 cells but again did not did not induce a type I IFN response in EndoC-βH1 observe dsRNA accumulation after 48  h and 72  h of cells, as measured by the phosphorylation of STAT1 and knockdown (data not shown). On the other hand, a STAT2 (Fig. 1a, e, f ) and by the expression of key down- prolonged exposure to these 2 siRNAs for 4 additional stream targets of IFNα signaling, namely total STAT1 days after the usual 48  h of silencing (6  days in total) and STAT2 (Fig. 1a, g, h) at the protein level, and MDA5 significantly reduced the expression of both PNPT1 (Fig.  1i), the Human Leukocyte Antigen-ABC (HLA- and SUV3 (Fig.  3a–d) and induced a clearly detect- ABC also known as Major Histocompatibility complex able accumulation of dsRNA in EndoC-βH1 cells class I, MHC class I) (Fig. 1j), the antiviral MX dynamin- (Fig.  3e). These data were confirmed by using a sec - like GTPase 1 (MX1) (Fig.  1k) and the ER stress marker ond siPNPT1 (Additional file  4a–c). Of note, the single CCAAT/enhancer-binding protein homologous protein knockdown of either PNPT1 or SUV3 for 6  days only (CHOP, also known as DDIT3) (Fig.  1l) at the mRNA modestly induced dsRNA accumulation (Fig.  3e and level. We used IFNα as a positive control since we pre- Additional file  4c). Despite the presence of dsRNA in viously showed that it induces a clear up-regulation of EndoC-βH1 cells, there was still no induction of a type these genes in EndoC-βH1 cells [19, 25]. On the other I IFN response as indicated by the absence of STAT1 hand, a clear accumulation of dsRNA was observed in and STAT2 phosphorylation (Fig.  3a, f, g) and the lack the fast proliferating HeLa cells following knockdown of of induction of MDA5 (Fig.  3h and Additional file  4d), PNPT1 with three different siPNPT1 (#1, #2 and #3) as HLA-ABC (Fig. 3i and Additional file  4e), MX1 (Fig. 3j) previously described [15] (Additional file  3a). Surpris- and CHOP (Fig.  3k and Additional file  4f ) expression. ingly, and in spite of the fact that siPNPT1#2 and #3 IFNα was used as a positive control. These findings induced a more marked inhibition of PNPT1 expression support the importance of the collaborative action of than that observed with siPNPT1#1 (the siRNA used the two members of the mitochondrial degradosome, by Dhir et  al., [15]) (Additional file  3b), only siPNPT1#1 PNPT1 and SUV3, in preventing accumulation of mtd- triggered an antiviral response, as determined by the sRNA in pancreatic beta cells. However, and different expression of IFNβ and MDA5 (Additional file 3c, d). from observations in other fast-proliferating cell lines Next, to assess whether PNPT1 silencing-mediated [15], in human pancreatic beta cells these mtdsRNA do mtdsRNA accumulation was masked by a rapid editing not trigger a type I IFN response. C oomans de Brachène et al. Autoimmun Highlights (2021) 12:6 Page 5 of 10 Fig. 1 PNPT1 silencing does not induce dsRNA accumulation and a type I IFN response in EndoC-βH1 cells. EndoC-βH1 cells were transfected with a siRNA control (siQ: white bars) and two different siRNAs targeting PNPT1 (siP#1: grey bars and siP#2: blue bars) and cells were maintained in culture during 48 h. As a positive control for gene expression, in some experiments cells were treated with IFNα (2000 U/ml) for 24 h (red bars), and as a positive control for dsRNA staining, cells were infected with CVB5 (MOI 5). a Protein expression was measured by western blotting and representative images of 2–9 independent experiments are shown. Densitometry results are shown for PNPT1 (b), pSTAT1 (e), pSTAT2 (f), STAT1 (g) and STAT2 (h). d dsRNA accumulation (red) and the presence of the viral capside protein VP1 (green) for CVB5-infected cells was analyzed by immunocytochemistry. Representative images of 2 (siPNPT1#1) or 3 (siPNPT1#2) independent experiments and images of 1 experiment with CVB5 infection are shown (magnification 40×). mRNA expression of PNPT1 (c), MDA5 (i), HLA-ABC (j), MX1 (k) and CHOP (l) were analyzed by RT-qPCR and normalized by β-actin and then by the value of siQ considered as 1. Results are mean ± SEM of 3–9 independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs siQ, Student t test Coomans de Brachène et al. Autoimmun Highlights (2021) 12:6 Page 6 of 10 Fig. 2 PNPT1/ADAR double silencing does not induce a type I IFN response in EndoC-βH1 cells. EndoC-βH1 cells were transfected with a siRNA control (siQ: white bars) and siRNAs targeting PNPT1 (siP#1, grey bars), ADAR (siA, green striped bars), or both (siP/A: grey bars with green stripes) and cells were maintained in culture during 48 h. As a positive control for gene expression, in some experiments cells were treated with IFNα (2000 U/ml) for 24 h (red bars), and as a positive control for IFNβ expression, cells were transfected with PIC (1 µg/ml) for 24 h (k, orange bar). a Protein expression was measured by western blotting and representative images of 3–7 independent experiments are shown. Densitometry results are shown for PNPT1 (b), ADAR (c), pSTAT1 (f), pSTAT2 (g), STAT1 (h) and STAT2 (i). mRNA expression of PNPT1 (d), ADAR (e), IFNβ (j, k), MDA5 (l), HLA-ABC (m), MX1 (n) and CHOP (o) were analyzed by RT-qPCR and normalized by β-actin and then by the value of siQ considered as 1. Results are mean ± SEM of 3–9 independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs siQ, Student t test C oomans de Brachène et al. Autoimmun Highlights (2021) 12:6 Page 7 of 10 Fig. 3 Long-term PNPT1/SUV3 silencing induces dsRNA accumulation in EndoC-βH1 cells but no type I IFN response. EndoC-βH1 cells were transfected with a siRNA control (siQ: white bars) or with siRNAs targeting PNPT1 (siP#1, grey bars), SUV3 (siS, black striped bars) or both (siP/S: grey bars with black stripes), and then maintained in culture for 6 days after transfection. As a positive control for gene expression, in some experiments cells were treated with IFNα (2000 U/ml) for 24 h (red bars). a Protein expression was measured by western blotting and representative images of 2 independent experiments are shown. Densitometry results are shown for PNPT1 (b), pSTAT1 (f) and pSTAT2 (g). e dsRNA accumulation (red) was analyzed by immunocytochemistry. Representative images of 3 independent experiments are shown (magnification 40×). mRNA expression of PNPT1 (c), SUV3 (d), MDA5 (h), HLA-ABC (i), MX1 (j) and CHOP (k) were analyzed by RT-qPCR and normalized by β-actin and then by the value of siQ considered as 1. Results are mean ± SEM of 2–10 independent experiments. *p < 0.05 and ***p < 0.001 vs siQ, Student t test The double silencing PNPT1/SUV3 induces dsRNA Discussion accumulation in fibroblasts but not in dispersed human In the present study, while searching for new triggers of islet cells innate immunity in the context of T1D, we did two major In dispersed human islets, PNPT1 and SUV3 double observations: (1) the accumulation of mtdsRNA upon knockdown for 6  days (the usual 48  h of silencing fol- degradosome silencing seems to be cell-type specific; and lowed by 4 additional days) did not induce dsRNA (2) these mtdsRNA do not trigger a type I IFN response accumulation in the primary non-proliferating insu- in human pancreatic beta cells. lin-positive beta cells (Fig.  4a) as it was observed in In a previous study, and confirmed by our own data EndoC-βH1 cells. There was, however, a clear accu - (Additional file  3), PNPT1 or SUV3 knockdown induced mulation of dsRNA in some, but not all, vimentin-pos- a rapid and massive accumulation of dsRNA in the fast- itive fibroblasts present in the human islet preparations proliferating HeLa cells [15]. However, in slowly prolifer- (Fig.  4b, c and Additional file  5, upper part). In a lesser ating EndoC-βH1 cells PNPT1 silencing for 48 h did not proportion, other non-beta cells negative for vimentin induce dsRNA accumulation. This cannot be explained also contained dsRNA (Fig. 4c and Additional file  5, lower by ADAR editing as a compensatory mechanism for the part). This distinct effect, occurring in cells present in the inhibition of PNPT1, since the double knockdown of same preparation and treated under the same experimen- PNPT1 and ADAR for 48 h also failed to induce dsRNA. tal conditions, highlights the fact that this phenomenon On the other hand, long-term (6  days) double knock- is cell-type dependent and probably related to the prolif- down of the two members of the mitochondrial degrado- erative capacity of the cells (i.e. fibroblasts are fast prolif - some, PNPT1 and SUV3, induced dsRNA accumulation erating cells, while primary human beta cells practically in the slowly proliferating EndoC-βH1 cells but not in the don’t proliferate under basal culture conditions). non-proliferating primary human beta cells. Surprisingly, Coomans de Brachène et al. Autoimmun Highlights (2021) 12:6 Page 8 of 10 Fig. 4 PNPT1/SUV3 knockdown induces dsRNA accumulation in surrounding fibroblasts but not in dispersed human islet cells. Dispersed human islets were transfected with a siRNA control (siQ) or with siRNAs targeting PNPT1 (#1), SUV3 or both (P/S), and then maintained in culture for 6 days after transfection. dsRNA accumulation (red), insulin content for beta cell staining (green), and vimentin for fibroblast staining (purple) were analyzed by immunocytochemistry. Representative images of 4 (a) and 2 (b, c) independent experiments are shown (magnification 40×). c dsRNA and vimentin positivity were evaluated by counting 1400 non-beta cells from 69 images obtained from 2 independent experiments and confirming the cell-specific nature of this process, different from previous observations in HeLa cells [15]. the vimentin-positive fibroblasts present in the human On the other hand, when using the same siPNPT1 (siP- islet preparations stained positively for dsRNA after NPT1#1) used in the study by Dhir and colleagues [15] silencing of PNPT1 and/or SUV3. These data point to a we were able to reproduce the observed mtdsRNA accu- cell-specific effect that could be connected to the prolif - mulation in HeLa cells (Additional file  3). However, two erative status of the cells, as this is the main difference other siPNPT1 that inhibited PNPT1 by > 70% (#2 and between EndoC-βH1 cells and primary human islet cells. #3) failed to trigger the type I IFN response, in spite of Of note, we have recently shown that EndoC-βH1 cells inducing a clear accumulation of dsRNA in HeLa cells or primary human islets treated with IFNα have a similar (Additional file  3). Thus, these data, and the data pre - gene signature as compared to beta cells obtained from sented by Dhir and colleagues [15] using one of the pres- patients affected by T1D [19]. ently tested siRNAs, need to be taken with caution as we In line with the hypothesis that mtdsRNA accumula- can’t exclude that some of the effects observed with siP - tion correlates with the proliferative status of the cells, NPT1#1 are off target effects, unrelated from the actual when studying mitochondrial RNA (mtRNA) extracted inhibition of PNPT1. In any case, none of these siPNPT1, from S or M phase-arrested HeLa cells, the largest frac- alone or combined with SUV3 siRNA, induced a type I tion of mtRNA isolated in the M phase were present as IFN response in EndoC-βH1 cells, despite the presence of duplex RNA [26]. In addition, the analysis of mitochon- dsRNA. This indicates that mtdsRNA accumulating upon drial DNA (mtDNA) replication and transcription dur- degradosome inactivation is probably not a mediator of ing the cell cycle showed a clear coordination between the type I IFN response in human pancreatic islets. mtDNA and nuclear DNA synthesis [27] that may explain the presently observed absence of mtdsRNA in the non- Conclusions proliferating primary beta cells. Our findings support the concept that accumulation of We also noticed that mtdsRNA accumulation in mtdsRNA following mitochondrial degradosome knock- EndoC-βH1 cells fail to induce a type I IFN response, down is not a general cell mechanism but is preferentially C oomans de Brachène et al. Autoimmun Highlights (2021) 12:6 Page 9 of 10 and innovation programme and “EFPIA”, “JDRF” and “The Leona M. and Harry B. present in fast proliferating cells. More importantly, Helmsley Charitable Trust”. accumulation of mtdsRNA is not a general trigger of the innate immune response, as it fails to do so in human Availability of data and materials All data generated or analysed during the current study are available from the pancreatic beta cells. Therefore, mtdsRNA does not seem corresponding author on reasonable request. to be an endogenous “danger signal” for the induction of IFNα in the beta cells in the context of T1D. We cannot Ethics approval and consent to participate The use of human islets was approved by the local ethics committee of Pisa, exclude, however, an indirect effect of these mtdsRNA Italy. in the development of T1D through their release from the beta cells via exosomes, and their subsequent action Consent for publication Not applicable. on immune cells as described in the context of alcoholic liver disease [28]. Competing interests The authors declare that they have no competing interests. Supplementary Information Author details The online version contains supplementary material available at https ://doi. ULB Center for Diabetes Research, Medical Faculty, Campus Erasme, Univer- org/10.1186/s1331 7-021-00148 -2. sité Libre de Bruxelles (ULB), Route de Lennik, 808-CP618, 1070 Brussels, Bel- gium. Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy. Indiana Biosciences Research Institute, Indianapolis, IN, USA. Additional file 1. Characteristics of the human donors providing the islets used in the present study. Received: 28 September 2020 Accepted: 2 January 2021 Additional file 2. List of primers used in the study. Additional file 3. PNPT1 knockdown in HeLa cells induces dsRNA accu- mulation but a type I IFN response is only observed with one siRNA out of three tested. References Additional file 4. A second siPNPT1 confirms the accumulation of dsRNA 1. Op de Beeck A, Eizirik DL. Viral infections in type 1 diabetes mellitus— after the double knockdown of PNPT1 and SUV3 for 6 days in EndoC-βH1 why the β cells? Nat Rev Endocrinol. 2016;12(5):263–73. cells, without a type I IFN response. 2. Eizirik DL, Pasquali L, Cnop M. Pancreatic β-cells in type 1 and type 2 Additional file 5. The double silencing PNPT1/SUV3 induces dsRNA accu- diabetes mellitus: different pathways to failure. Nat Rev Endocrinol. mulation in non-beta cells of the human islet preparations. 2020;16(7):349–62. 3. Foulis AK, Farquharson MA, Meager A. Immunoreactive alpha-interferon in insulin-secreting beta cells in type 1 diabetes mellitus. Lancet Lond Abbreviations Engl. 1987;2(8573):1423–7. CVB: Coxsackievirus B; dsRNA: Double-stranded RNA; ER: Endoplasmic 4. Lundberg M, Krogvold L, Kuric E, Dahl-Jørgensen K, Skog O. Expression reticulum; IFNα: Interferon-α; IFNβ: Interferon-β; MOI: Multiplicity of infection; of interferon-stimulated genes in insulitic pancreatic islets of patients mtDNA: Mitochondrial DNA; mtdsRNA: Mitochondrial double-stranded RNA; recently diagnosed with type 1 diabetes. Diabetes. 2016;65(10):3104–10. PIC: Polyinosinic-polycytidylic acid; siRNA: Small-interfering RNA; T1D: Type 1 5. Chehadeh W, Weill J, Vantyghem MC, Alm G, Lefèbvre J, Wattré P, et al. diabetes. Increased level of interferon-alpha in blood of patients with insulin- dependent diabetes mellitus: relationship with coxsackievirus B infection. Acknowledgements J Infect Dis juin. 2000;181(6):1929–39. The authors are grateful to Dr A. Op de Beeck for help with CVB infection, 6. Richardson SJ, Morgan NG. Enteroviral infections in the pathogenesis and M. Pangerl, M. Depessemier and I. Millard of the ULB Center for Diabetes of type 1 diabetes: new insights for therapeutic intervention. Curr Opin Research, Université Libre de Bruxelles, Belgium, for excellent technical Pharmacol. 2018;43:11–9. support. 7. Chehadeh W, Weill J, Vantyghem M-C, Alm G, Lefèbvre J, Wattré P, et al. Increased level of interferon-α in blood of patients with insulin-depend- Authors’ contributions ent diabetes mellitus: relationship with coxsackievirus B infection. J Infect ACdB contributed to the original idea, design and performance of the Dis. 2000;181(6):1929–39. experiments, investigation and formal analysis, and wrote, revised, and edited 8. Hyöty H. Viruses in type 1 diabetes. Pediatr Diabetes. 2016;17(Suppl the manuscript. AC and AEM performed experiments. LM and PM contrib- 22):56–64. uted with resources. DLE contributed to the original idea and the design, 9. Cinek O, Stene LC, Kramna L, Tapia G, Oikarinen S, Witsø E, et al. Entero- supervision and interpretation of the experiments, wrote, revised and edited virus RNA in longitudinal blood samples and risk of islet autoimmunity the manuscript. All authors have read and approved the final version of the in children with a high genetic risk of type 1 diabetes: the MIDIA study. manuscript. ACdB is the guarantor of this work and, as such, has full access to Diabetologia. 2014;57(10):2193–200. all the data in the study and takes responsibility for the integrity of the data 10. Said EA, Tremblay N, Al-Balushi MS, Al-Jabri AA, Lamarre D. Viruses seen and the accuracy of the data analysis. All authors read and approved the final by our cells: the role of viral RNA sensors. J Immunol Res. 2018;2018:1–14. manuscript. 11. Gebhardt A, Laudenbach BT, Pichlmair A. Discrimination of self and non- self ribonucleic acids. J Interferon Cytokine Res. 2017;37(5):184–97. Funding 12. Rice GI, Kasher PR, Forte GMA, Mannion NM, Greenwood SM, Szynkiewicz D.L.E. acknowledges the support of a grant from the Welbio-FNRS (Fonds M, et al. Mutations in ADAR1 cause Aicardi-Goutières syndrome associ- National de la Recherche Scientifique), Belgium, the Dutch Diabetes Fonds ated with a type I interferon signature. Nat Genet. 2012;44(11):1243–8. (DDFR), Holland, and start up-funds from the Indiana Biosciences Research 13. Yu Z, Chen T, Cao X. RNA editing by ADAR1 marks dsRNA as “self.” Cell Res Institute (IBRI), Indianapolis, Indiana, USA. D.L.E. and P.M. are supported by the déc. 2015;25(12):1283–4. Innovative Medicines Initiative 2 Joint Undertaking under grant agreements 14. Kotrys AV, Szczesny RJ. Mitochondrial gene expression and beyond— No 115797 (INNODIA) and 945268 (INNODIA HARVEST ), supported by the novel aspects of cellular physiology. Cells. 2019;9(1):17. European Union’s Horizon 2020 research and innovation programme. These 15. Dhir A, Dhir S, Borowski LS, Jimenez L, Teitell M, Rötig A, et al. Mitochon- Joint Undertakings receive support from the Union’s Horizon 2020 research drial double-stranded RNA triggers antiviral signalling in humans. Nature. 2018;560(7717):238–42. Coomans de Brachène et al. Autoimmun Highlights (2021) 12:6 Page 10 of 10 16. Ravassard P, Hazhouz Y, Pechberty S, Bricout-Neveu E, Armanet M, 23. Colli ML, Paula FM, Marselli L, Marchetti P, Roivainen M, Eizirik DL, et al. Czernichow P, et al. A genetically engineered human pancreatic β cell Coxsackievirus B tailors the unfolded protein response to favour viral line exhibiting glucose-inducible insulin secretion. J Clin Invest sept. amplification in pancreatic β cells. J Innate Immun. 2019;11(4):375–90. 2011;121(9):3589–97. 24. Hovi T, Roivainen M. Peptide antisera targeted to a conserved sequence 17. Brozzi F, Nardelli TR, Lopes M, Millard I, Barthson J, Igoillo-Esteve M, et al. in poliovirus capsid VP1 cross-react widely with members of the genus Cytokines induce endoplasmic reticulum stress in human, rat and mouse Enterovirus. J Clin Microbiol mai. 1993;31(5):1083–7. beta cells via different mechanisms. Diabetologia. 2015;58(10):2307–16. 25. Marroqui L, Dos Santos RS, Op de Beeck A, Coomans de Brachène A, Mar- 18. Kirkegaard JS, Ravassard P, Ingvarsen S, Diedisheim M, Bricout-Neveu E, selli L, Marchetti P, et al. Interferon-α mediates human beta cell HLA class Grønborg M, et al. Xenotropic retrovirus Bxv1 in human pancreatic β cell I overexpression, endoplasmic reticulum stress and apoptosis, three hall- lines. J Clin Invest. 2016;126(3):1109–13. marks of early human type 1 diabetes. Diabetologia. 2017;60(4):656–67. 19. Colli ML, Ramos-Rodríguez M, Nakayasu ES, Alvelos MI, Lopes M, Hill JLE, 26. Kim Y, Park J, Kim S, Kim M, Kang M-G, Kwak C, et al. PKR senses nuclear et al. An integrated multi-omics approach identifies the landscape of and mitochondrial signals by interacting with endogenous double- interferon-α-mediated responses of human pancreatic beta cells. Nat stranded RNAs. Mol Cell. 2018;71(6):1051-1063.e6. Commun. 2020;11(1):2584. 27. Chatre L, Ricchetti M. Prevalent coordination of mitochondrial DNA 20. Marchetti P, Suleiman M, Marselli L. Organ donor pancreases for the study transcription and initiation of replication with the cell cycle. Nucleic Acids of human islet cell histology and pathophysiology: a precious and valu- Res. 2013;41(5):3068–78. able resource. Diabetologia. 2018;61(4):770–4. 28. Lee J-H, Shim Y-R, Seo W, Kim M-H, Choi W-M, Kim H-H, et al. Mitochon- 21. Marroqui L, Lopes M, dos Santos RS, Grieco FA, Roivainen M, Richardson drial double-stranded RNA in exosome promotes interleukin-17 produc- SJ, et al. Differential cell autonomous responses determine the outcome tion through toll-like receptor 3 in alcohol-associated liver injury. Hepatol of coxsackievirus infections in murine pancreatic α and β cells. eLife. Baltim Md août. 2020;72(2):609–25. 2015;4:e06990. 22. Marroqui L, Dos Santos RS, Fløyel T, Grieco FA, Santin I, Op de Beeck A, Publisher’s Note et al. TYK2, a candidate gene for type 1 diabetes, modulates apoptosis Springer Nature remains neutral with regard to jurisdictional claims in pub- and the innate immune response in human pancreatic β-cells. Diabetes. lished maps and institutional affiliations. 2015;64(11):3808–17. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions

Journal

Autoimmunity HighlightsSpringer Journals

Published: Mar 27, 2021

There are no references for this article.