INTRODUCTION
The innate immune response is the first line of host defence against a virus. Double-stranded RNA (dsRNA), making up the genome of the virus or formed as an intermediate during single-stranded RNA virus replication, triggers the production of type I interferons (IFN-a/b) and other pro-inflammatory cytokines by recognition by toll-like receptor 3 (TLR-3) in the endosomal compartments [Alexopoulou et al., 2001] or intracellularly by the cytoplasmic helicases retinoic acidinducible gene-I (RIG-I) [Yoneyama et al., 2004] and melanoma differentiation-associated gene 5 (MDA5) [Kato et al., 2006] . The relative importance of different components of the innate immune system for virus recognition is not fully clarified and varies among different viruses and cell types. Recognition and induction of pro-inflammatory cytokines by members of the family Picornaviridae, have been shown to be mediated by MDA5 in mice [Kato et al., 2006; Wang et al., 2009] and in various cell lines [Pichlmair et al., 2009] . Several viruses in this family, in particular Coxsackie B viruses (CVB) belonging to the human enterovirus (EV) B species, have long been implicated in the etiology of type 1 diabetes [Gamble et al., 1969; Yoon et al., 1979; King et al., 1983; Frisk et al., 1985; Yin et al., 2002] , a disease characterized by the destruction of insulin-producing b cells in the pancreas. Most interestingly, a recent genetic study found four rare variants of MDA5 that confer protection against type 1 diabetes [Nejentsev et al., 2009] , suggesting a role for MDA5 in the development of the disease, and also providing a possible new link between type 1 diabetes and infection with EV. EV can infect human islets in vitro [Frisk and Diderholm, 2000; Roivainen et al., 2000] , and has been found in islets of infants who died of fulminant EV infection [Ylipaasto et al., 2004] , as well as in islets of patients with a recent onset of type 1 diabetes [Dotta et al., 2007; Richardson et al., 2009; Tanaka et al., 2009] , and in islets from a ''pre-diabetic'' patient with islet cell autoantibodies [Oikarinen et al., 2008] . Thus, understanding the innate immune response triggered by EV in human pancreatic islets is essential since it provides an immediate response to a viral challenge, while also controlling the later antigen-specific adaptive immune response, possibly contributing to the b-cell destruction in type 1 diabetes.
INTRODUCTION
There is an excess of males in the prevalence of type 1 diabetes, beginning at the time of puberty [Nyström et al., 1992] . The female sex hormone 17b-estradiol has been demonstrated to protect mice from streptozotocin-induced diabetes [Le May et al., 2006] , and to protect human pancreatic islets from apoptosis induced by proinflammatory cytokines in vitro [Contreras et al., 2002] . 17b-Estradiol has also been shown to modulate inflammation by suppressing chemokines induced by synthetic dsRNA in human endometrial epithelial cells [Lesmeister et al., 2005] , and to protect mice from CVB3-induced myocarditis [Huber, 2008] . In this study, the ability of 17b-estradiol to modulate the innate immune response induced in human islets by infection with EV or exposure to synthetic dsRNA was evaluated. Islet secretion of the chemokine IFN-inducible peptide-10/CXCL10 (IP-10/CXCL10) was measured as a sign of innate immune activation. IP-10/CXCL10 has been shown previously to be induced by CVB infection of human islets [Berg et al., 2006; Moell et al., 2009] , induce b-cell death [Schulthess et al., 2009] , and to be elevated in serum [Shimada et al., 2001; Nicoletti et al., 2002] and in pancreatic islets [Tanaka et al., 2009] from patients with type 1 diabetes at onset of the disease. Synthetic dsRNA, polyinosinic:polycytidylic acid (poly(I:C)), is used often to mimic viral infection. When poly(I:C) is administered in vitro to b cells extracellularly to the culture medium, it induces innate immunity in a TLR3-dependent fashion [Dogusan et al., 2008; Garcia et al., 2009 ], while MDA5 is essential for detection of poly(I:C) in mice [Kato et al., 2006] . Poly(I:C) can induce production of type 1 IFN in mice, protecting against EV infection [Liu et al., 2005] . In this study, the ability of poly(I:C) to induce an antiviral state in human islets was evaluated.
INTRODUCTION
Most present knowledge of virus-host interactions relies on studies in mice or in immortalized cell lines, although innate immune signaling varies heavily between different human primary cell types [Lundberg et al., 2007] . In this study, primary human islets were used, enabling the study of innate immune signaling in a relevant system, that is, a model for the possible induction of type 1 diabetes by EV. The aims were to evaluate the ability of 17b-estradiol to modulate the innate immune response induced in human islets by infection with EV or exposure to synthetic dsRNA, as well as to evaluate the ability of poly(I:C) to induce an antiviral state in human islets by the induction of genes involved in antiviral innate immunity.
MATERIALS AND METHODS

Isolation and Culture of Human Islets
Human pancreases were obtained from 19 organ donors (8 females and 11 males) free of any known pancreatic disease, from transplantation units in Sweden, Norway, and Finland. The donors had an average age of 56 AE 9.2 years (range: 34-77) and an average BMI of 25.8 AE 2.5 kg/m 2 (range: 19.7-30.9). Islets of Langerhans were isolated in Uppsala, Sweden, using a protocol approved by the Local Ethics Committee, as described previously [Goto et al., 2004] . Islet preparations were of good quality and available for research only because the total islet volume was too low for clinical transplantation. The islets were kept in culture bags (Baxter Medical AB, Kista, Sweden) with 200 ml CMRL-1066 (ICN Biomedicals, Costa Mesa, CA) supplemented with 10 mM HEPES, 2 mM L-glutamine, 50 mg/ml Gentamycin, 0.25 mg/ml Fungizone (Gibco BRL, Invitrogen Ltd, Paisley, UK), 20 mg/ml Ciproxfloxacin (Bayer Healthcare AG, Leverkusen, Germany), 10 mM nicotinamide, and 10% heat-inactivated human serum at 378C in 5% CO 2 and humidified air for 1-7 days. The culture medium was changed on day 1 and then every other day until the islet experiments were performed.
Virus
A 1000 tissue culture infectious dose-50 (TCID 50 )/ 200 ml stock of Adr, an EV strain isolated from a boy at onset of type 1 diabetes [Elshebani et al., 2007] and genotyped as Coxsackie virus B5 (GenBank accession number FJ374273) was used. Studies have shown that this strain replicates well in islet cells resulting in a pronounced destruction of the islets, impaired islet function [Elshebani et al., 2007] , and induction of chemokines [Moell et al., 2009] . All virus infections were performed by addition of 200 ml of the 1000 TCID 50 CVB5/Adr stock, yielding a virus concentration of 100 TCID 50 .
Virus
Chemicals 17b-Estradiol (Sigma-Aldrich, St Louis, CA) was dissolved in DMSO and added to 50 hand-picked islets per well at a final 17b-estradiol concentration of 1 mM and a final DMSO concentration of 0.001%. Islets were exposed to synthetic dsRNA, by addition of poly(I:C) (Sigma-Aldrich) at a final concentration of 1 mg/ml.
Islet Experiments
Isolated human islets had a purity ranging from 40% to 95%, and were purified further for islet experiments by handpicking with a micropipette under an inverted light microscope. All experiments were performed on 50 hand-picked islets per well, cultured in six-well plates (Sarstedt AB, Landskrona, Sweden) in 2 ml RPMI-1640 containing 5.5 mM glucose (SVA, Uppsala, Sweden), supplemented with 5% fetal bovine serum and 2 mM L-glutamine.
Islet Experiments
Islets from nine donors were cultured with or without addition of 17b-estradiol for 24 hr before infection with CVB5/Adr or exposure to poly(I:C) (Sigma-Aldrich). Mock infection with 200 ml vehicle (medium only) was used as negative control. Samples of culture medium were collected from all wells, directly after infection/ exposure and on the indicated days post-infection (dpi)/exposure, and stored at À208C for subsequent virus titration and chemokine measurement. Islets from three of these experiments were lysed at 5 dpi for analysis of the intracellular chemokine content.
Islet Experiments
Islets from one donor were infected with CVB5/Adr in six separate wells (50 islets/well), washed with PBS, and stored in RNAlater (Qiagen, Sollentuna, Sweden) at À808C at 0, 0.7, 1, 3, 4, and 6 dpi for virus titration and analysis of gene expression over time.
Islet Experiments
Islets from nine donors were exposed to poly(I:C) 24 hr before or 24 hr post-infection with CVB5/Adr or mock infection, and samples of culture medium from all wells were collected at the time of infection and at 1, 2, 4, and 6 dpi, for virus titrations. Islets from three of these donors were washed with PBS at 24 hr post-poly(I:C) exposure and stored in RNAlater (Qiagen) at À808C for subsequent RNA extraction and real-time RT-PCR analysis. Islets from one donor were exposed to poly(I:C) in six separate wells (50 islets/well), washed with PBS, and stored in RNAlater (Qiagen) at À808C at 0, 0.125, 1, 3, 4, and 6 dpi.
Analysis of Virus Replication
Virus replication was determined by TCID 50 titrations of samples of culture medium as described previously [Moell et al., 2009] . The samples were collected from the infected islet cultures on the indicated dpi.
Cytokine Measurement
Culture media, sampled on the indicated time points, from CVB5/Adr-infected, poly(I:C)-exposed, or mockinfected human islets, cultured with or without 17b-estradiol, were analyzed for IP-10/CXCL10 content using Human IP-10 Cytoscreen ELISA (Biosource, Nivelles, Belgium) with a detection limit of 2 pg/ml.
Islet Function Test
Insulin secretion in response to glucose stimulation was assessed in a dynamic perifusion system on day 6 of culture with or without 17b-estradiol in the culture medium (n ¼ 2). Islets were perifused with two glucose concentrations (1.67 and 16.7 mmol/L and then 1.67 mmol/L again). Fractions were collected at 6-min intervals for 120 min and the insulin concentrations were determined by ELISA (Mercodia, Uppsala, Sweden).
RNA Extraction and Real-Time RT-PCR
Total RNA from islets stored in RNAlater (Qiagen) was extracted with RNeasy Mini kit with on-column DNaseI digestion (Qiagen). RNA quantity and quality were determined using spectrophotometry and gel electrophoresis. Fifty nanograms of total RNA per sample were primed with oligo(dT) 12 ). PCR specificity was verified by melt curve analysis of the final PCR products.
Statistical Analysis
Differences in virus titer and chemokine secretion were analyzed with Wilcoxon rank-sum test. P-values <0.05 were considered statistically significant. Data are presented as mean þ SEM.
RESULTS

17b-Estradiol Reduces Enterovirus-Induced IP-10/CXCL10 Secretion From Human Islets
To study the effect of the female sex hormone, 17b-estradiol on EV infection of pancreatic islets, isolated human islets were cultured with or without the addition of 17b-estradiol to the culture medium prior to infection with CVB5/Adr. CVB5/Adr replicated well in the islets and induced the secretion of IP-10/CXCL10, measured as accumulation of IP-10/CXCL10 in the culture medium during the first 5 or 6 dpi (n ¼ 6; Fig. 1a) . When islets were cultured in the presence of 17b-estradiol prior to infection, the accumulation of IP-10/CXCL10 in the culture medium (Fig. 1a ) was reduced significantly (P ¼ 0.037, n ¼ 6). The intracellular IP-10/CXCL10 content was reduced correspondingly (n ¼ 3, data not shown). The reduced secretion of IP-10/CXCL10 was not due to any antiviral activity, since 17b-estradiol did not affect viral replication (data not shown). To ensure that the used dose (1 mM) of 17b-estradiol did not affect islet function, the ability of islets, untreated or treated with 10 or 1 mM of 17b-estradiol, to respond to glucose stimulation was compared. Insulin release in response to glucose was not affected by any of the tested concentrations of 17b-estradiol (n ¼ 2; Fig. 1c ).
17b-Estradiol Does Not Affect Poly(I:C)-Induced IP-10/CXCL10 Secretion From Human Islets
It has been suggested that picornaviral dsRNA and poly(I:C) trigger antiviral responses partly by separate pathways [Kato et al., 2006] . To test if poly(I:C) could induce IP-10/CXCL10 in human islets, and whether such an induction could be reduced by 17b-estradiol like that induced by CVB5/Adr infection, human islets were cultured for 24 hr with or without the addition of 17b-estradiol and exposed to poly(I:C). Analysis of IP-10/ CXCL10 that accumulated in the culture medium during 3 days post-exposure to poly(I:C) (1 mg/ml) showed that IP-10/CXCL10 secretion was induced by poly(I:C). However, this secretion was not affected by pre-treatment of the islets with 17b-estradiol (n ¼ 5; Fig. 1b) . Also, a 10 times lower dose of poly(I:C) (0.1 mg/ml) induced IP-10/CXCL10 secretion that was not reduced by 17b-estradiol (n ¼ 1, data not shown).
Synthetic dsRNA Reduces or Inhibits EV Replication in Human Islets
Several antiviral genes are upregulated in human islets upon infection with picornavirus [Ylipaasto et al., 2005] . To study the induction of an antiviral response by CVB5/Adr, human islets were infected in vitro, and mRNA expression of three antiviral genes was analyzed on several time points post-infection. IFN-b and MDA5 mRNA levels were upregulated 3 dpi, and increased further on 4 and 6 dpi. TLR3 was not expressed until 6 dpi, when a low-level expression could be detected (Fig. 2a) . Exposure of human islets to poly(I:C) led to a transient increase in expression of MDA5, IFN-b, and TLR3, peaking at 24 hr postexposure (Fig. 2b) . IFN-a/b has been shown to inhibit picornavirus replication in human islets [Hultcrantz et al., 2007] , so in order to replicate, the virus needs to counteract the induction of an antiviral state. To study if synthetic dsRNA can induce an antiviral state in human islets, poly(I:C) was added to the culture media of islets from nine donors. When the islets were exposed to poly(I:C) for 24 hr prior to infection with CVB5/Adr, the mean viral replication was reduced significantly (n ¼ 9, P ¼ 0.004; Fig. 3a) . Addition of poly(I:C) 24 hpi also reduced viral replication (Fig. 3b) . The antiviral activity of poly(I:C) was not affected by addition of 17b-estradiol to the culture medium prior to exposure to poly(I:C) (data not shown), supporting the finding that 17b-estradiol does not affect poly(I:C)-induced antiviral cytokines. The variations between individual donors were large, and exposure Fig. 1 . 17b-Estradiol reduces enterovirus-but not poly(I:C)-induced IP-10/CXCL10 secretion from human islets. Fifty hand-picked human islets were cultured for 24 hr, with or without the addition of 17b-estradiol (1 mM) to the culture medium, before infection with enterovirus (Adr) (A) or exposure to poly(I:C) (B). Accumulation of IP-10/ CXCL10 in the culture media was analyzed by ELISA at 3 days postexposure to 1 mg/ml poly(I:C) or at 5-6 dpi with enterovirus. Exposure of islets to 17b-estradiol did not affect their ability to respond to high glucose, as tested in a dynamic perifusion system (C). Isolated islets were cultured for five days either untreated (&), or in the presence of 1 (*) or 10 mM (~) 17b-estradiol. The figure shows insulin release after perifusion with glucose (1.67, 16.7, and 1.67 mmol/L). to poly(I:C) either blocked (5 donors), reduced (3 donors), or did not affect (1 donor) CVB5/Adr replication (Fig. 3b) .
Induction Levels of IFN-b and MDA5 Gene Expression in Islets Upon Poly(I:C) Exposure Vary Greatly Between Individual Islet Donors
To evaluate if the degree of antiviral effect of poly(I:C) in islets from different donors correlated with the expression levels of antiviral genes induced by exposure to poly(I:C), total RNA was extracted from islets in which poly(I:C) blocked CVB5/Adr replication completely (donors #4 and #8), and from islets in which poly(I:C) reduced CVB5/Adr replication only partly (donor #7; Fig. 4) . mRNA expression levels of IFN-b and MDA5 were analyzed by real-time RT-PCR. Untreated islets expressed basal levels of MDA5 (Fig. 4b) , whereas IFN-b expression could not be detected (Fig. 4a) . After 24 hr of culture in the presence of poly(I:C), islets from all three tested donors expressed IFN-b (Fig. 4a) , and increased levels of MDA5 (Fig. 4b) . The expression levels of IFN-b as well as of MDA5 after 24 hr of culture with poly(I:C) varied greatly between the three donors, but did not correlate with the degree of inhibition of CVB5/Adr replication.
DISCUSSION
The present study highlights important pathways of induction of innate immunity in primary human pancreatic islets. It confirms the previous finding that proinflammatory chemokine, IP-10/CXCL10, is induced by infection of human islets with EV in vitro [Berg et al., 2006; Moell et al., 2009] , which indicates viral recognition and activation of innate immunity. The female sex hormone 17b-estradiol, was shown previously to modulate inflammation induced by poly(I:C) in an endometrial epithelial cell line in vitro [Lesmeister et al., 2005] , and by infection of mice with EV in vivo [Huber, 2008] . In the present study performed on isolated human pancreatic islets, 17b-estradiol reduced IP-10/ CXCL10 secretion induced by infection with EV, whereas it had no effect on IP-10/CXCL10 secretion induced by exposure to poly(I:C), suggesting that in primary human islets of Langerhans, poly(I:C) and EV induce secretion of IP-10/CXCL10 by two separate pathways. These divergent results further emphasize the importance of being careful when comparing data from studies performed in separate systems. Since poly(I:C) introduced to cells extracellularly is likely taken up by endocytosis and recognized by TLR3 in endsomes, while dsRNA produced intracellularly as an intermediate during EV infection is more likely recognized by intracellular helicases [Dogusan et al., 2008] , it is tempting to speculate that 17b-estradiol interferes with a step in the pathway leading from MDA5 activation to IP-10/CXCL10 transcription that is not shared by the TLR3 pathway. However, untreated islets in this study contained TLR3 mRNA levels close to, or below, the RT-PCR detection limit, and it is therefore questionable whether the basal TLR3 expression can be solely responsible for sensing poly(I:C) in human islets. Other pathogen recognition receptors, such as RIG-I or MDA5, may contribute to the initial islet response to poly(I:C). Signaling pathways initiated by dsRNA binding to MDA5, RIG-I, or TLR3 are mediated by NF-kB, MAPK, and IRF-3, but the response pattern differs between human cells of separate lineages [Lundberg et al., 2007] . 17b-Estradiol has been suggested to exert its effects on cytokine and chemokine production by binding to the nuclear estrogen receptor-a (ER-a) [Lesmeister et al., 2005] present in both male and female islets [Alonso-Magdalena et al., 2008] , but the exact mechanism is unknown. In addition, recent studies have identified an intracellular G-protein-coupled ER, GPR30 [Filardo et al., 2000; Prossnitz and Maggiolini, 2009] . ER-a [Duan et al., 2001 [Duan et al., , 2002 as well as GPR30 [Prossnitz and Maggiolini, 2009] binding can mediate the activation of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K), both of which may interact with steps in induction of innate immunity.
DISCUSSION
In vitro infection of human islets with EV in this study led to increased transcription of the antiviral genes IFNb, MDA5, and TLR3, indicating that the virus was recognized in the islet cells. Type I IFNs induce the transcription of more than 300 IFN stimulated genes (ISGs), many of which are known to have antiviral effects [de Veer et al., 2001] , and although the 2A pro of EV has been shown to allow some replication in cells pre-treated with type I IFN, at least at a high-infectious dose [Morrison and Racaniello, 2009 ], IFN-a pre-treatment inhibits replication of CVB3 in human islets [Hultcrantz et al., 2007] . Since EV replication in islets in this present study continued even after the observed induction of IFN-b, the virus must in some way have counteracted the antiviral effects of IFN-b. One possibility, which will be addressed in a future study, is that the translation of IFN-b mRNA or secretion of IFN-b protein may be inhibited by viral proteins. When islets were exposed to poly(I:C) prior to infection with EV, replication was blocked completely in islets from 56% of the donors, suggesting that poly(I:C) induced an antiviral state in the islets that the EV could not counteract. However, in islets from 44% of the donors a productive infection with EV could be established despite pre-treatment with poly(I:C). This individual variation could not be explained by donor differences in the ability of islets to transcribe IFN-b, MDA5, or TLR3 in response to poly(I:C) exposure, since, even though induction levels of these genes varied, they did not correlate with the level of antiviral activity. Possibilities that need further studying are that other genes could be involved and induced differently, or there may be genotype-specific differences in the genes degraded by 2A pro . The finding that poly(I:C) reduced replication of EV, also when added 24 hpi, could suggest that EV infection cannot counteract poly(I:C)-induced production of type I IFN. However, intact human islets contain between 1,000 and 2,000 cells, all of which may not be permissive to infection. Thus, it is highly plausible that uninfected cells, in which poly(I:C) could induce IFN expression, were available even 24 hpi when poly(I:C) was added. The antiviral effect of poly(I:C) in isolated human pancreatic islets may suggest that local administration of poly(I:C) could be exploited as an antiviral strategy. However, when virus-induced auto-destructive immune responses are suspected to contribute to the pathology of a disease, such as in type 1 diabetes [Richardson et al., 2011] , additional stimulation of innate immunity is unlikely to be beneficial.
DISCUSSION
To conclude, this study shows for the first time that the female sex hormone, 17b-estradiol modulates induction of the antiviral chemokine IP-10/CXCL10 in human islets infected with a CVB5 strain isolated from a patient at onset of type 1 diabetes, introducing one possible contributor to the high male to female ratio in the incidence of this disease after puberty. Interestingly, 17b-estradiol does not affect secretion of IP-10/CXCL10 induced by exposure of islets to poly(I:C), suggesting that in human islets different signaling cascades are involved in induction of chemokines by EV and synthetic dsRNA, respectively. This highlights further the importance of being careful when interpreting results of studies performed in different cell systems. Finally, this study shows that poly(I:C) can induce a form of antiviral state in human islets, probably by the induction of antiviral innate immunity that cannot be counteracted by the virus, and that this is likely dependent on the genetic background of the islet donor.

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