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DOI: 10.1002/eji.201344264

Eur. J. Immunol. 2014. 44: 3056–3067

Dietary gluten increases natural killer cell cytotoxicity and cytokine secretion Jesper Larsen, Morten Dall, Julie Christine Antvorskov, Christian Weile, K˚ are Engkilde, Knud Josefsen and Karsten Buschard The Bartholin Institute, Rigshospitalet, Copenhagen, Denmark Dietary gluten influences the development of type 1 diabetes in nonobese diabetic (NOD) mice and biobreeding rats, and has been shown to influence a wide range of immunological factors in the pancreas and gut. In the present study, the effects of gluten on NK cells were studied in vitro and in vivo. We demonstrated that gliadin increased direct cytotoxicity and IFN-γ secretion from murine splenocytes and NK cells toward the pancreatic beta-cell line MIN6 cells. Additionally, stimulation of MIN6 cells led to a significantly increased proportion of degranulating C57BL/6 CD107a+ NK cells. Stimulation of C57BL/6 pancreatic islets with gliadin significantly increased secretion of IL-6 more than ninefold. In vivo, the gluten-containing diet led to a higher expression of NKG2D and CD71 on NKp46+ cells in all lymphoid organs in BALB/c and NOD mice compared with the gluten-free diet. Collectively, our data suggest that dietary gluten increases murine NK-cell activity against pancreatic beta cells. This mechanism may contribute to development of type 1 diabetes and explain the higher disease incidence associated with gluten intake in NOD mice.

Keywords: C56BL/6 mice



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Gliadin

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Gluten

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NK cells

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NOD mice

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Type 1 diabetes

Additional supporting information may be found in the online version of this article at the publisher’s web-site

Introduction Dietary gluten is the initiating factor in the development of celiac disease (CD), however, an association between CD and type 1 diabetes (T1D) has been established. A gluten-free (GF) diet has been shown to have a protective effect against the development of T1D. It has been demonstrated that a GF diet in nonobese diabetic (NOD) mice reduces diabetes incidence from 64 to 15% [1], and similar results have been obtained with biobreeding rats [2]. Increased intestinal permeability occurs prior to the onset of T1D in both spontaneous animal models and human disease [3, 4]. Patients with both CD and T1D diseases carry high-risk HLA alleles [5], and approximately 10% of T1D patients have an

Correspondence: Jesper Larsen e-mail: [email protected]

 C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

increased prevalence of CD [6]. Moreover, T1D rarely develops after diagnosed CD, which may be because of the protective effect of a GF diet [7]. A recently published case report describes a boy with T1D who was able to maintain a low fasting blood glucose level without insulin therapy for more than 20 months after the time of diagnosis by adhering to a GF diet [8]. Lately, it has also been suggested that a family of gluten proteins known as gliadins may contribute directly to beta-cell hyperactivity, as enzymatically digested gliadin and a 33-mer gliadin fragment increase insulin secretion from beta cells by affecting the KATP channel current [9]. This process could induce increased Ag expression of the beta-cell surface and prevent beta-cell rest [10]. Gliadin has been shown to stimulate several components of the adaptive immune system, such as Treg cells, Th17 cells, and DCs [11, 12]. Gliadin fragments have also been shown to stimulate TLR4 and activate innate cells such as monocytes, macrophages, and DCs [13], and to regulate the NK-cell DC crosstalk by

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stabilizing HLA-E on the cell surface [14]. The exact role of dietary gluten and gliadin fragments in NK-cell activity and T1D has not yet been elucidated. NK cells are lymphocytes that play an important role in the defense against pathogen infections and tumor development, but they have also been shown to participate in autoimmunity [15]. In T1D, NK cells infiltrate the islets at disease onset in both human and murine studies [16, 17], but their exact role is still controversial because of conflicting results. It is well known that NK cells from NOD mice have impaired function and defects in activation [18, 19]. Moreover, altered NK-cell numbers and activity were found in both recent-onset and long-standing diabetes patients [20]. It has also been shown that NK cells can lyze murine and rat islet cells [21, 22], as well as primary human beta cells [23], in vitro. Blocking the activating NK-cell receptor NKp46 protects against disease development in NOD mice, and NK cells kill both murine and human beta cells by recognizing an unknown ligand for NKp46 on the beta-cell surface [23, 24]. Finally, NK cells contribute to diabetes in a coxsackievirus B4-induced T1D model [25]. On the other hand, no effect of depleting NK cells in NOD mice has been reported, and it has even been suggested that the protective effect of complete Freund’s adjuvant in NOD mice is mediated by more active NK cells [26, 27]. Furthermore, it is known that NK cells infiltrate the prediabetic NOD mouse pancreas before T cells do, and they have a distinct phenotype compared with spleen and pancreatic lymph node (PLN) [17]. However, it is speculated that NK cells function as regulators of autoimmunity [15]. This balance might be affected by environmental factors such as dietary gluten. In the present study, we show that in vitro stimulation with enzyme-digested gliadin increases NK-cell cytotoxicity against the MIN6 beta-cell line, and increases IFN-γ secretion in C57BL/6 and NOD mouse splenocytes from mice on a standard glutencontaining diet (STD). Likewise, purified NK cells increase the production of several cytokines after stimulation with enzymedigested gliadin. We further show that gliadin induces production of insulin, IL-6, and CCL2 in isolated C57BL/6 pancreatic islets. Finally, we show that an STD diet increases the expression of activation marker CD71 and activating receptor NKG2D on NKp46+ cells in BALB/c and NOD mice.

Results Gliadin increases C57BL/6 NK-cell activity against the MIN6 pancreatic beta-cell line We observed a significant increase in cytotoxicity of polyI:Cactivated C57BL/6 splenocytes against MIN6 cells in the presence of gliadin digest (Fig. 1A, p < 0.0001). We used C57BL/6 for the in vitro study, since MIN6 cells originate from a transgenic C57BL/6 mouse [28]. Furthermore, we performed a CD107a degranulation assay on MACS-purified, polyI:C-activated C57BL/6 NK cells (Supporting Information Fig. 1 for gating strategy and Supporting Information Fig. 2 for enrichment of NK cells). We observed a 15% increase  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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in degranulation toward MIN6 cells preincubated for 24 h with enzyme-digested gliadin, relative to MIN6 cells preincubated with enzyme control (Fig. 1B, p = 0.04). No difference was seen relative to enzyme control in YAC-1 or HeLa cells preincubated with gliadin digest (Fig. 1B). Hence, gliadin digest both increases NK-cell degranulation and splenocyte cytotoxicity against MIN6 cells.

Gliadin increases splenocyte and NK-cell IFN-γ secretion in the presence of MIN6 cells To look further into gliadin’s effect on NK-cell function, we measured IFN-γ secretion from C57BL/6 and NOD mouse splenocytes incubated for 24 h with MIN6 cells and gliadin digest. Splenocytes were used instead of purified NK cells to keep different T-cell subsets, APCs, and other potentially NK-stimulating cells in the assay. In the absence of MIN6 cells, gliadin digest did not affect IFN-γ secretion. However, in the presence of MIN6 cells, IFN-γ secretion was increased by 165% relative to C57BL/6 splenocytes incubated with enzyme control alone (Fig. 1C, p = 0.0004). The addition of gliadin digest further increased IFN-γ by 51% relative to splenocytes incubated with MIN6 cells and enzyme control (Fig. 1C, p = 0.0042). The MIN6 cells themselves were not responsible for this effect, as pure MIN6 culture incubation for 24 h, with gliadin digest in concentrations ranging from 30 to 600 μg/mL, only led to negligible IFN-γ secretion (not shown). The IFN-γ secretion of IL-2-stimulated MIN6 cells was below the detection limit of 10 pg/mL. NOD mouse splenocytes stimulated with enzyme digest showed the same basal level of IFN-γ secretion as C57BL/6 mice splenocytes. Co-culturing with MIN6 cells increases IFN-γ secretion by 263% compared with splenocytes grown alone (Fig. 1C, p = 0.0002). The addition of gliadin digest to NOD mouse splenocytes co-cultured with MIN6 cells further increases IFN-γ secretion by 60% compared with enzyme digest (Fig. 1C, p = 0.0067). Comparing the different mice strains, splenocytes cultured with gliadin and MIN6 cells showed a 38% higher IFN-γ secretion in NOD mice relative to C57BL/6 mice (Fig. 1C, 38% increase, p = 0.024). We repeated the experiment and analyzed NK cells specifically by flow cytometry and intracellular staining for IFN-γ in NKp46+ CD3− cells (Supporting Information Fig. 3 for gating strategy). Correlating with the ELISA of splenocytes, the amount of IFN-γ+ NK cells was increased by 17% in the presence of MIN6 cells (Fig. 1D, p = 0.031). In the presence of MIN6 cells gliadin further increased IFN-γ+ NK cells by 17% (Fig. 1D, p = 0.0075). Surprisingly, gliadin treatment resulted in a 14% decrease in IFN-γ+ NK cells in the absence of MIN6 cells (Fig. 1D, p = 0.03).

Gliadin increases surface expression of NKp46 receptor ligand on MIN6 cells To test if gliadin increases the presently unknown ligand for NKp46, we used a fusion protein consisting of Ig and the ligand-binding domain of the receptor NKp46D2 on MIN6 cells incubated with gliadin digest. We observed 8.5% increased www.eji-journal.eu

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Figure 1. Gliadin-stimulated NK-cell and splenocyte responses toward MIN6 cells (A) The cytotoxicity of C57BL/6 splenocytes was assessed using standard 4-h chromium-release assay. MIN6 cells were labeled with 51 Cr and co-cultured with freshly prepared splenocytes from polyI:C-stimulated mice in E:T ratios of 12.5:1, 25:1, 50:1, and 100:1. Enzyme control or gliadin digest were present in the incubation medium. Results were analyzed by one-way ANOVA (p < 0.0001). Data are presented as means ± SD pooled from five independent experiments using one mouse in each experiment. (B) Degranulation was measured by flow cytometry using NK-enriched spleen cells isolated from polyI:C-stimulated C57BL/6 mice. NK-enriched splenocytes were cultured together with gliadin-preincubated YAC-1, HeLa, or MIN6 cells (E:T ratio of 1:2). Cells were stained with mAbs for NKp46 and CD107a. CD107a+ cells are presented as a percentage of total NKp46+ cells. Gating strategy is shown in Supporting Information Fig. 1. NK-cell enrichment is shown in Supporting Information Fig. 2. Data are presented as means + SD of three samples pooled from each of four independent experiments with cells from one mouse in each.(C) IFN-γ production was measured by ELISA from IL-2 (500 U/mL) activated C57BL/6 and NOD splenocytes co-cultured with MIN6 cells for 24 h in the presence of enzyme control or gliadin digest. Data are presented as means ± SD of supernatants from 5–7 mice. Each symbol represents an individual mouse. (D) Intracellular IFN-γ staining of splenocytes cultured with enzyme control or gliadin digest for 24 h. Cells were stimulated with PMA/ionomycin for the final 6 h and Golgi Plug for the final 3 h, staining with mAb against CD3, NKp46, and IFN-γ. Data are represented as percentage IFN-γ positive cells of total NKp46+ CD3– cells (left) or as geometric mean of IFN-γ in the NKp46+ CD3– population (right). Data are presented as means + SD pooled from four independent experiments with one mouse in each. (B–D) Data were analyzed by two-tailed Student’s t-test; ࢩ p < 0.05, ࢩࢩ p < 0.01, ࢩࢩࢩ p < 0.001.

expression of the NKp46 ligand on the surface of MIN6 cells treated for 24 h with gliadin digest, as compared with enzyme control (Fig. 2A, p = 0.027). On YAC-1 cells we found a 15% decrease in NKp46 ligand expression in the presence of gliadin (Fig. 2A, p = 0.047). The NKG2D ligands H60, RAE-1, and MULT-1 were also expressed on the surface of MIN6 cells, but we observed no difference in gliadintreated cells compared with control (Fig. 2B–D). H60 is expressed at a level 23% lower on gliadin-treated YAC-1 cells compared with control (Fig. 2B, p < 0.0001). The LFA-1 receptor ligand ICAM-1 is expressed at a level 7.6% lower on gliadin-treated YAC-1 cells (Fig. 2, p = 0.044), while no change was observed on MIN6. MHC-1, which is recognized by NK Killer-cell immunoglobulinlike receptor (KIR) receptors, was expressed at very low levels on both cell lines and no difference between enzyme control and gliadin digest was seen (Fig. 2F).  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Gliadin induces production of cytokines from NK-cell-enriched splenocytes To profile the production of cytokines from NK cells cultured with gliadin, we performed a multiplex cytokine analysis of cell culture supernatant. MACS NK-cell-enriched splenocytes from polyI:Cactivated C57BL/6 mice were cultured alone or with MIN6 cells, and in the presence of enzyme control, gliadin digest or 33-mer for 2 and 24 h before analysis of supernatants on an Meso Scale Discovery (MSD) multiplex TH1/TH2 9-plex plate (Meso Scale Discovery). No significant changes were seen in the cytokine levels of NK cells cultured alone for 2 h and stimulated with gliadin digest or 33-mer. For NK-cell-enriched splenocytes co-cultured with MIN6 cells for 2 h and stimulated with gliadin digest, a significant increase was seen in TNF-α production (Fig. 3E, 67%, p = 0.016). www.eji-journal.eu

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Figure 2. Expression profile of NK-cell ligands on MIN6 cells. MIN6 and YAC-1 cells were treated with enzyme control or gliadin digest for 24 h, stained for surface expression of NKp46 ligand, H60, Rae-1, MULT-1, MHC-1, ICAM-1, and analyzed by flow cytometry. (A) Surface expression of NKp46 ligand was measured by binding of NKp46-D2-Ig fusion protein followed by FITC-conjugated Fcγ Ab. Histogram shows binding of secondary Ab alone. Data are presented as means + SD pooled from five to ten independent experiments with one sample in each. (B) H60 expression on MIN6 (left) and YAC-1 (right) cells. (C) Rae-1 expression on MIN6 (left) and YAC-1 (right) cells. (D) MULT-1 expression on MIN6 (left) and YAC-1 (right) cells. (E) MHC-1 expression on MIN6 (left) and YAC-1 (right). (F) ICAM-1 expression on MIN6 (left) and YAC-1 (right) cells. In all histograms: gray shaded area — unstained cells; open blue lines — staining of enzyme control treated cells; open red lines — staining of gliadin digest treated cells. Data were analyzed by two-tailed Student’s t-test and are indicated, ࢩ p < 0.05, ࢩࢩ p < 0.01, ࢩࢩࢩ p < 0.001. Histograms are representative of six independent experiments and (B–D) data are shown as means + SD pooled from six independent experiments with one sample in each.  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Figure 3. Cytokine production by gliadin-stimulated NK-cell-enriched splenocytes after 2 and 24 h of MIN6 co-culture. Freshly NK-cell-enriched splenocyte cells from polyI:C-activated C57BL/6 mice were incubated with enzyme control, gliadin, or 33-mer in the presence or absence of MIN6 cells. Cytokine concentrations in supernatants were determined after 2 (left panels) and 24 h (right panels) of incubation by using MSD mouse TH1/TH2 9-plex assay. The dotted lines represent the limits of detection. Data were analyzed by two-tailed Student’s t-test and are indicated, ࢩ p < 0.05, ࢩࢩ p < 0.01, ࢩࢩࢩ p < 0.001. Data are presented as means + SD pooled from five independent experiments with enriched cells from one mouse in each.

For the same setup stimulated with 33-mer, significant increases were seen in IL-2 (Fig. 3B, 150% increase, p = 0.033), IL-10 (Fig. 3C, 100% increase, p = 0.0028), and TNF-α (Fig. 3E, 83.8% increase, p = 0.019). As a control, MIN6 cells alone were stimulated with enzyme control and gliadin digest for 24 h. Yet, for all cytokines except IL-4, the levels were below detection limits and no effects were seen for gliadin digest and enzyme stimulation (data not shown). Culturing for 24 h generally led to increased secretion of most cytokines, while for cells co-cultured with MIN6 cells there was an increase in the levels of IL-2 (Fig. 3B, 1.34-fold increase, p = 0.016), IL-10 (Fig. 3C, threefold increase, p = 0.0084), and CXCL1 (Fig. 3F, 24-fold increase, p = 0.0016). Stimulation with gliadin and 33-mer of NK cells co-cultured with MIN6 for 24 h led to significant increases in IL-2 (Fig. 3B, 32.6% increase, p = 0.047 and 77% increase, p = 0.034) and CXCL1 (Fig. 3F, 92% increase, p = 0.0086 and 132% increase, p = 0.026). Gliadin digest did not stimulate secretion of IL-4, IL-5, IL-12 (not shown), or IFN-γ.

 C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Gliadin increases secretion of insulin, IL-6, and CCL2 in C57BL/6 islets NK cells are possibly recruited to the pancreas by islet cytokine production during the pathogenesis of T1D. Isolated C57BL/6 islets treated with gliadin digest were screened using an MSD multiplex TH1/TH2 9-plex plate (data not shown). As they were highly responsive to gliadin stimulation in our screening, IL-6 and IL-10 were chosen as candidates for further investigation, along with CCL2/MCP-1, a chemokine that is produced by pancreatic islets and is known to attract monocytes, DCs, and NK cells [29–32]. We also investigated the effect of gliadin on insulin secretion in murine islets. Incubation of islets with gliadin digest for 24 h increased production of IL-6 more than ninefold (Fig. 4A, p = 0.015), CCL2 (Fig. 4C, 7.2-fold increase, p = 0.019), and insulin (Fig. 4D, 3.5-fold increase, p = 0.032) relative to enzyme control. No significant change in IL-10 secretion was observed with gliadin stimulation (Fig. 4B).

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in NOD mice receiving the STD diet (Fig. 5E, p = 0.0075). IL-10 decreased by 29% and IFN-γ decreased by 57% in BALB/c mice receiving the STD diet (Fig. 5E, p = 0.049 and p = 0.04). No significant changes were seen in the systemic levels of IL-5, IL-12, TNF-α, and CXCL1 (Fig. 5E). IL-1β and IL-4 levels were below detection range (not shown).

Expression of NKp46 in intestine from NOD mice is upregulated in mice receiving the STD diet Figure 4. Gliadin increases islet cell secretion of IL-6, CCL2, and insulin. C57BL/6 islets were isolated from 15 mice/experiment and cultured for 24 h in the presence of enzyme control or gliadin digest and ELISAs for IL-6, IL-20. CCL2 and insulin were performed on the cell supernatants. Data were analyzed by one sample t-test; ࢩ p < 0.05, ࢩࢩ p < 0.01, ࢩࢩࢩ p < 0.001. Data are presented as means + SD of triplicates cultures pooled from each of three to five independent experiments.

NKG2D and CD71 are upregulated on NKp46+ cells in mice receiving the STD diet To investigate the effect of gluten consumption on NK cells in vivo, we analyzed the distribution of NK cells in spleen, PLN and, auricular lymph node (ALN) from 13-week-old BALB/c and NOD mice on an STD gluten-containing diet or a GF diet. For full panel of NKp46 and CD71 stainings, see Supporting Information Fig. 4 and 5. ALN was included as a control distant lymphoid organ. We found a decrease in NK cells in BALB/c spleen receiving the GF diet compared with the STD diet (p = 0.0486), but no substantial differences in NK-cell percentages were found in NOD mice or in the PLN and ALN of either strain (Fig. 4B and D). Gating on NKp46+ cells, we analyzed the expression of the activating receptor NKG2D on NK cells from gluten-consuming and GF animals by comparing the geometric mean. NKG2D expression on NK cells increased 15% in the spleen of NOD mice receiving the STD diet (Fig. 5B, p = 0.0076). An even larger increase (74%) of NKG2D was seen in the PLN of BALB/c mice receiving the STD diet (Fig. 5C, p = 0.024). The same tendency was observed in all tissues in both BALB/c and NOD mice receiving the STD diet, although differences were not significant. We also analyzed the expression of the transferrin receptor CD71, a glycoprotein involved in iron uptake and expressed on proliferating lymphocytes [33, 34]. Gating on NKp46+ cells, we found a significant increase of CD71+ cells in the spleens of BALB/c (114% increase, Fig. 5B, p = 0.0002) mice receiving the STD diet as opposed to the GF diet. Similar effects were seen in the PLN of BALB/c (164% increase in STD mice, Fig. 5C, p=0.032) and NOD mice receiving the STD diet, and in the ALN from both strains (Fig. 5B–D).

The systemic cytokine profile is slightly altered in NOD and BALB/c mice receiving the STD diet Serum from mice used in the flow cytometric analysis of NKp46+ cells was used for a multiplex cytokine analysis, in order to investigate the systemic response to gluten. IL-2 increased by 6.6-fold  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

To further investigate the effect of a GF diet in BALB/c and NOD mice, we used qPCR to analyze the expression of NKp46 in spleen, PLN, intestine, and pancreas of mice receiving the STD and GF and strictly GF (SGF) mice bred from GF mothers. Relative NKp46 expression levels were significantly reduced in intestinal tissue from NOD mice kept on the SGF diet as compared with mice receiving the STD diet (Supporting Information Fig. 6). Relative NKp46 expression in PLN and spleen from NOD mice did not show significant changes, although there was a tendency toward a reduction with the SGF diet (Supporting Information Fig. 6). Relative NKp46 expression in islets from BALB/c and NOD mice showed no significant changes (Supporting Information Fig. 6).

Discussion We have demonstrated that gliadin stimulation increases NK-cell activity against the MIN6 beta-cell line by increasing cytotoxicity, degranulation, and cytokine production. We further showed that an STD gluten-containing diet increases expression of the proliferation marker CD71 and the activating receptor NKG2D on NKp46+ cells in lymphoid tissues in BALB/c and NOD mice, when compared with a GF diet. To understand these results it is important to keep in mind that NK cells may encounter gliadin in several locations. Gliadin fragments have longer intestinal passage time, are resistant to digestion by intestinal enzymes [35], and cross the intestinal barrier [36]. Zonulin, which can induce tight junction disassembly and increase intestinal permeability, is upregulated in T1D, and gliadin induces zonulin upregulation in the intestinal cell line Caco2 [37]. This may result in direct stimulation of NK cells present in the lamina propia by gliadin or APCs [38, 39]. It has also been suggested that gluten peptides might be presented by DCs sampling the intestinal lumen [40], which may increase the crosstalking with NK cells [14], thus making it possible for gliadin to activate NK cells even in the absence of a leaky gut epithelium. It is well known that priming of lymphocytes in gut-associated lymphoid tissue is important in T1D [41], and a similar mechanism may be important for gut-homing NK cells. Furthermore, increased islet endothelial permeability has been observed in prediabetic mice [42], and gliadin fragments have been detected in breast milk from healthy women [43]. We have recently found intact gliadin 33-mer peptides in the blood of BALB/c mice 30 min to 1 h after oral administration (Bruun et al., submitted). It is thus possible www.eji-journal.eu

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Figure 5. Effects of a gluten-containing diet on CD71 and NKG2D expression on NKp46+ cells in BALB/c and NOD mice. Flow cytometry analysis was performed using freshly isolated single-cell suspensions of spleens, pancreatic lymph nodes (PLNs), and auricular lymph nodes (ALNs) from BALB/c, and NOD mice fed the STD or GF diet. Cells were treated with LIVE/DEAD stain before staining with the indicated mAbs and fixation (A) Representative plot of SSC singlet gate, FSC singlet gate, lymphocyte gate, live gate, NKp46+ gate, and CD71+ gate in PLNs from BALB/c mice fed an STD diet. Bottom panels: histograms showing expression of NKG2D and CD71 on NKp46+ cells in BALB/c STD PLNs. Gray shaded area: mice fed a GF diet. Open lines: mice fed an STD diet. Flow cytometry data are representative of three independent experiments. (B) Percentages of NKp46+ cells (left panel) and CD71+ cells (center panel) in the NKp46+ gate and geometric mean of NKG2D (right panel) gated on NKp46+ cells from spleens of BALB/c and NOD mice. Data are represented as means + SD of pooled cells from three independent experiments with cells from three mice in each experiment. Black bars: STD diet. White bars: GF diet. (C) Same representation as in (B), but using PLNs. (D) Same representation as in (B), but using ALNs. (E) Multiplex cytokine analysis of blood serum from mice used in the flow cytometry described in (A–D). Each symbol represents an individual blood serum sample and data are shown as means ± SD of triplicates pooled from each of three independent experiments with serum from three mice in each experiment. Data were analyzed by two-tailed Student’s t-test and are indicated, ࢩ p < 0.05, ࢩࢩ p < 0.01, ࢩࢩࢩ p < 0.001.

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that gliadin fragments are found in the highly vascularized islets, where NK cells could also be stimulated. Our results show that gliadin affects both NK cells and the beta cells directly in vitro in ways that result in increased NK-cell activity and subsequent killing of MIN6 cells. Gliadin may affect NK-cell activation by directly influencing the NK cells, or by changing the composition of NK ligands on the MIN6 surface, as both direct stimulation and preincubation of the MIN6 cells increase NK-cell activity. Both gliadin and 33-mer stimulate NK-cell cytokine production after just 2 h of co-culturing with MIN6 cells. In particular, IL-2 and TNF-α production are increased. Secretion of the most prominent NK cytokine, IFN-γ, is not increased after 2 h, even in the presence of MIN6 cells; and after 24 h, secretion levels are only slightly elevated. However, it is well known that IFN-γ secretion requires a very strong stimulus, long stimulation time, and activation by various cytokines, notably IL-2, IL-12, or IL-18 [44]. Hence, our results are not surprising. LPS stimulation of IFN-γ secretion in human NK cells also required high doses of IL-2 (500 U/mL) [45]. In this study, IL-2-activated splenocytes produced high amounts of IFN-γ in the presence of MIN6 cells, and the effect was substantially increased with gliadin. Furthermore, NOD mouse splenocytes responded more strongly to stimulation with gliadin than did C57BL/6 splenocytes. The molecular mechanism by which gliadin increases NK-cell activity is uncertain, but could be due to binding on CXRC3 or TLR4 receptors on either beta cells or NK cells. Both CXCR3 and TLR4 are expressed on beta cells [46, 47] and subsets of NK cells [45, 48], and gliadin is known to interact with both receptors [47, 49]. Interestingly, a recent study has shown that amylase/trypsin inhibitor family members present in the ω-gliadin fraction are able to induce strong innate responses in human and murine macrophages, monocytes, and DCs via TLR4 [49], through a mechanism that could potentially induce IFN-γ secretion from NK cells [45]. We also investigated the effect of gliadins directly on beta cells. C57BL/6 islets incubated with gliadin increase production of insulin, IL-6, and the chemokine CCL2. Islet production of IL-6 was increased more than ninefold. We have earlier shown that IL-6 is produced by glucose-stimulated rat islets, and IL-6 participate in the formation of insulitis [50]. This raised beta-cell activity may contribute to an increase in surface Ags recruiting immune cells to the islets, along with increased secretion of cytokines. These findings correlate with a previous study showing that gliadin increases insulin secretion in rat islets and the INS-1E cell line [9]. In vivo, we found several gluten-induced effects in immunocompetent BALB/c mice and diabetes-prone NOD mice. We showed that an STD gluten-containing diet increased the amount of NKp46+ cells in the PLN of BALB/c mice, and the same tendency is seen in NOD mice. In the spleen there were no changes to the percentage of NKp46+ cells, which indicates a more local than systemic effect of dietary gluten. This finding is further supported by the observation that only small changes are found in serum levels of cytokines. The NK-activating cytokine IL-2 is significantly higher systemic in NOD mice receiving the STD diet, which may  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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affect NK-cell activity locally in the pancreas. IFN-γ and IL-10 are significantly lower in BALB/c mice receiving the STD diet, which may also affect the local environment in the pancreas. On the transcriptional level NKp46 is also upregulated as a response to the STD diet. Most interestingly, the STD diet resulted in a significant increase of the activation marker CD71 on NKp46+ cells in all animals and tissues, and an increase of the NKG2D receptor on NKp46+ cells in both BALB/c and NOD mouse pancreas, BALB/c PLN, and NOD ALN. In NOD mouse PLN and BALB/c mouse ALN we also saw a tendency toward an increase. This implies that a gluten-containing STD diet increases NK-cell proliferation and activation in lymphoid tissues. Intestinal expression of NKp46 was significantly reduced in NOD mice receiving the SGF diet compared with STD diet, but smaller differences were found in mice receiving the GF diet. This highlights the importance of maternal and early-life GF environment. We have previously shown that a SGF diet reduces incidence of T1D in NOD mice to 6% compared to 15% in GF [1, 51]. Recently, it was shown in NOD mice that a maternal GF diet reduces diabetes incidence in the offspring [52]. Thus, our data indicate that a GF diet may prevent diabetes, by generally reducing the number of NKp46+ NK cells, cellular proliferation, and downregulating the activating receptor NKG2D on NK cells, making them less autoreactive. A counterargument to this hypothesis may be that a clinical study has shown that children with T1D have reduced numbers of NK cells, aberrant NKG2D signaling, and lower NK-cell activity than healthy subjects [53]. Similar results are seen in NOD mice [54], indicating that decreased NK-cell activity, in fact, contributes to the pathogenesis. Conversely, another study has shown that the onset period is characterized by unusually active IFN-γ-secreting NK cells in some patients [20]. Furthermore, reduced NK-cell activation and cytotoxicity is only seen in longstanding patients, implying that it might only be a consequence of the disease [20, 55], and the NKG2D ligand RAE-1 is expressed in the prediabetic NOD mouse pancreas [56]. Activated NK cells could also affect pathology indirectly by NK-mediated DC death [57]. Even though several adverse effects of gliadin have been described, most people can still consume wheat products daily with no symptoms. Both T1D and CD are multifactorial diseases, where genetic predisposition and the mode, timing, and dose of gluten introduction are important for the development [58]. Most people thus tolerate gluten even if several immune stimulating effects have been described in healthy BALB/c and C57BL/6 mice [11, 49, 59]. Interestingly, a disorder called nonceliac gluten sensitivity has been described, reporting about a growing number of cases of self-diagnosed patients who observe improvements in several parameters after reducing gluten intake [60]. The changes described in this article may not be specific only in the diabetogenic process, but imply that gluten activates the immune system in healthy animal models. A GF diet has a dramatic effect on diabetes incidence in NOD mice [1], and we believe that in susceptible individuals, gliadin peptides may contribute to the pathogenesis of T1D, in collaboration with other factors such as gut microbiota or virus infections by the mechanism described in this study. www.eji-journal.eu

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In conclusion, our findings show that gliadin increases the in vitro activity, cytokine secretion, and numbers of NK cells, and that a gluten-containing diet increases NK-cell activation in vivo in BALB/c and NOD mice. This represents a possible mechanism for the effect of gluten on T1D, and may help to explain the beneficial effects of a GF diet, as seen in human disease animal models of T1D.

Materials and methods

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Fragments were used for stimulation in a final concentration of 100 μM for 24 h. Mouse recombinant IL-2 in RPMI 1640 was used for stimulation of splenocytes (500 U/mL, eBioscience, San Diego, CA, USA).

Islet isolation and culture Islets of Langerhans were isolated from single C57BL/6, BALB/c, or NOD mice using collagenase digestion, as previously described [10].

Animals Mice were purchased from Taconic Europe A/S, Ejby, Denmark, and kept in a specific pathogen-free animal facility (temperature 22 ± 2°, 12-h light cycle, air changed 16 times per hour, humidity 55 ± 10%) with free access to water and food. For islet isolation for in vitro stimulation and NK cell in vitro assays, C57BL/6-JbomTac mice were used. For the diet study, NOD and BALB/c mice were used. The mice were put on GF (n = 9) or STD (n = 9) diets upon arrival at the age of 4 weeks. These animals were also used in [61]. The animals received either the standard (STD) nonpurified Altromin diet, or a GF, modified Altromin diet (Altromin, Lage, Germany). Both experimental diets were nutritionally adequate, with similar levels of protein, amino acids, minerals, vitamins, and trace elements. Only the protein sources differed between the diets. The exact compositions of the STD and GF diets are given in [1, 51]. The weights of the mice were continuously monitored and both diet groups showed similar weight distributions.

Cell culture MIN6 cells [28] (kindly provided by J. Miyazaki, Osaka University) were grown at 37°C and 5% CO2 in RPMI 1640 medium (Lonza, Basel, Switzerland), supplemented with 10% FCS (Life Technologies, Carlsbad, CA, USA), 1% Na pyruvate, 1% HEPES, and 50 μM mercaptoethanol in T75 cell culture flasks. Cells of passage number 10–30 were used. For the experiments, 4 × 105 cells/well were seeded in 12-well plates and 4 × 104 cells/well were seeded in 96-well plates. After 24 h, the medium was replaced with RPMI 1640 supplemented with 0.5% FCS and relevant stimulants. YAC-1 cells (ATCC, Manassas, VA, USA) were cultured under the same conditions in RPMI 1640 supplemented with 10% FCS and 2 mM glutamine. HeLa cells (kindly provided by Lars H. Engelholm, Rigshospitalet, Copenhagen, Denmark) were cultured in DMEM (Lonza) supplemented with 10% FCS. Gliadin was enzymatically digested, prepared, and tested as described in [9]. In gliadin stimulation experiments, the cells were exposed to 300 μg/mL gliadin digest for 24 h, unless otherwise noted. As a control, a mixture of the heat-inactivated digestion enzymes was used. Gliadin deamidated 33-mer (LQLQPFPQPELPYPQPEL PYPQPELPYPQPQPF) was synthesized by Schafer-N (Copenhagen, Denmark), and purity was confirmed by HPLC analysis.  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

ELISA Concentrations of IL-1β, IL-2, IL-4, IL-5, IL-10, IL-12 total, TNF-α, IFN-γ, and CXCL1 in cell culture supernatant and serum were measured by an ultrasensitive mouse TH1/TH2 9-plex tissue culture from MSD (Gaithersburg, MD, USA), according to the manufacturer’s instructions. Conventional ELISA was used for the cytokines and chemokines IL-6, IL-10, IFN-γ, CCL-2 (Pierce, Thermo Fischer Scientific, Rockford, IL, USA), and insulin (Mercodia, Uppsala, Sweden) in islet cell culture supernatant.

51

Cr-release assay

Cytotoxicity was measured by conventional 51 Cr-release assay. MIN6 target cells were labeled with 100 μCi sodium chromate (51 Cr) (Perkin Elmer) for 1 h before use in cytotoxicity tests. Target cells were seeded in 96-well round bottomed microtiter plates, with polyI:C-activated C57BL/6 splenocytes as effector cells, to obtain final E:T ratios of 12.5:1, 25:1, 50:1, and 100:1. Cells were incubated for 4 h with enzyme control or gliadin digest. Spontaneous release was measured separately for enzyme control and gliadin. Supernatants were collected in aliquots of 50 μL and assayed for radioactivity in a gamma counter (Packard Topcount NXT). The percentage of specific lysis was calculated as ((experimental release − spontaneous release)/(max release − spontaneous release)) × 100. The spontaneous release never exceeded 20%.

CD107a mobilization Murine NK cells were isolated from the spleens of C57BL/6 mice activated with polyI:C (200 μL (1 μg/μL) i.p.) using the MACS NK-cell isolation kit I (Miltenyi Biotec, Bergisch, Gladbach, Germany) according to the manufacturer’s instructions. Purified NK cells were mixed with YAC-1, HeLa, and MIN6 target cells, all preincubated with gliadin or enzyme control at E:T ratio of 1:2, and incubated for 2 h. Analysis of cell surface mobilized CD107a was done as previously described [62], through staining with FITC-conjugated rat anti-mouse CD107a (553793, BD) and Alexa Fluor 647-conjugated rat anti-mouse NKp46 (560755, BD). Flow cytometry was performed on a FACSCanto (BD). www.eji-journal.eu

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Intracellular IFN-γ staining

Statistics and data analysis

Single-cell suspensions were obtained from C57BL/6 spleens and stimulated with gliadin or enzyme control for 24 h in the presence or absence of MIN6 cells. For stimulation, 100 ng/mL PMA (Sigma) and 250 ng/mL ionomycin (Sigma) were added for the final 6 h. For the final 3 h of incubation Golgi Plug was added (BD Biosciences). Cells were treated with Fc block, surface stained with NKp46 and CD3, before treatment with Cytofix/Cytoperm buffer (BD Biosciences) and intracellular staining of IFN-γ.

Data was analyzed using Graphpad Prism. Where indicated, a Student’s t-test or two-way ANOVA was used to compare mean values. A one sample t-test was used when groups were compared against a control group with a defined value of 1; p < 0.05 was considered statistically significant. The following symbols were used for the figures: ࢩ p < 0.05, ࢩࢩ p < 0.01, ࢩࢩࢩ p < 0.001.

Fusion protein Fusion proteins were generated in COS-7 cells stably expressing NKp46D2-Ig [24] (kind gift from Professor Ofer Mandelboim, Hebrew University Hadassah Medical School, Jerusalem), and purified on a HiTrapTM Protein G HP Column (GE Healthcare, Little Chalfont, UK). MIN6 cells were stained with 5 μg fusion protein for 2 h on ice. For secondary staining for flow cytometry, we used an FITC-conjugated goat anti-human IgG, Fcγ fragment specific Ab (109-096-098; Jackson Immunoresearch).

Antibodies and flow cytometry The following mAbs were purchased from BD Pharmigen: PerCP-Cy5.5-conjugated rat anti-mouse CD335 (NKp46) (560800), PE-conjugated rat anti-mouse IFN-γ (554412), FITC-conjugated rat anti-mouse CD107a (553793), and Alexa Fluor 647-conjugated rat anti-mouse CD335 (NKp46) (560755). The following mAbs were purchased from eBioscience: eFluor 450-conjugated rat anti-mouse CD49b (DX5) (48-5971-82), FITC-conjugated rat anti-mouse ICAM-1 (11-0541-82), PE-Cy7-conjugated hamster anti-mouse CD3e (25-0031-82) mAb, allophycocyanin-conjugated hamster anti-mouse CD11c (17-0114-82), and allophycocyanin-conjugated rat anti-mouse MHC Class 1 (H-2Kd) (17-5957-82). The following mAbs) were purchased from R&D Systems: PE-conjugated rat anti-mouse ULBP-1/MULT-1 (FAB2588P), PE-conjugated rat anti-mouse H60 (FAB1155P), and PE-conjugated rat anti-mouse Rae-1 (FAB17582P). Live/dead discrimination was performed before  R fixation, using LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (L34957, Invitrogen) according to the manufacturer’s protocol. Cells were preincubated with Fc block (CD16/CD32) before staining with relevant mAb to reduce Fc receptor mediated binding (BD 553141). As compensation controls for the fixable aqua-fluorescent reactive dye and antibodies, the ArC Amine Reactive Compensation Bead Kit (Invitrogen) and the AbC anti-Rat/Hamster Bead Kit (Invitrogen) were used, respectively. Single-cell suspensions were prepared from spleens (S), PLNs, and ALNs from mice in each diet group. Cells from three mice were pooled for each organ, and cell suspensions were prepared. All Ab stains were performed on ice and cells were fixed in 2% PFA. The labeled cells were analyzed by flow cytometry using an LSR II (BD Biosciences), and data were analyzed using FACSDiva (BD Biosciences) and Flowlogic (Inivai Technologies) software.  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Acknowledgments: This work was funded by Kirsten and Freddy Johansen’s Foundation. We gratefully thank Professor Ofer Mandelboim for excellent technical advice and the NKp46-D2-Ig fusion protein. We gratefully thank Signe Goul Svendsen and Caroline Benedicte Madsen, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Denmark, and Morten Schou, The Bartholin Institute, Rigshospitalet, Denmark, for excellent technical support.

Conflict of interest: The authors declare no commercial or financial conflict of interest.

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Gluten-free but also gluten-enriched (gluten+) diet prevent diabetes in

Abbreviations: ALN: auricular lymph node · CD: celiac disease · GF:

NOD mice; the gluten enigma in type 1 diabetes. Diabetes Metab. Res. Rev.

gluten-free · MSD: Meso Scale Discovery · NOD: nonobese diabetic ·

2008. 24: 59–63.

PLN: pancreatic lymph node · SGF: strictly GF · STD: standard gluten-

52 Hansen, C. H., Krych, Ł., Buschard, K., Metzdorff, S. B., Nellemann, C., Hansen, L. H., Nielsen, D. S. et al., A maternal gluten-free diet reduces inflammation and diabetes incidence in the offspring of NOD mice. Diabetes 2014. 63: 2821–2832. 53 Qin, H., Lee, I.-F., Panagiotopoulos, C., Wang, X., Chu, A. D., Utz, P. J.,

containing diet · T1D: type 1 diabetes Full correspondence: Jesper Larsen, The Bartholin Institute, Rigshospitalet 3733, Copenhagen Biocenter, Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark e-mail: [email protected]

Priatel, J. J. et al., Natural killer cells from children with type 1 diabetes have defects in NKG2D-dependent function and signaling. Diabetes 2011. 60: 857–866. 54 Ogasawara, K., Hamerman, J. A., Hsin, H., Chikuma, S., Bour-Jordan, H., Chen, T., Pertel, T. et al., Impairment of NK cell function by NKG2D modulation in NOD mice. Immunity 2003. 18: 41–51.

 C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Received: 7/11/2013 Revised: 23/5/2014 Accepted: 4/7/2014 Accepted article online: 14/7/2014

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