prevents the development of PNAS PLUS autoimmune diabetes

Ricardo Villaresa,1, Dimitri Kakabadsea,1, Yasmina Juarranzb, Rosa P. Gomarizb, Carlos Martínez-Aa, and Mario Melladoa,2

aDepartment of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, E-28049 Madrid, Spain; and bDepartment of Cell Biology, Faculty of Biology, Complutense University of Madrid, E-28040 Madrid, Spain

Edited by Tak W. Mak, The Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute at Princess Margaret Hospital, University Health Network, Toronto, ON, Canada, and approved October 18, 2013 (received for review August 9, 2013) Evidence supports a relationship between the neuroendocrine and develop type 1 diabetes, and show normal glycemia throughout the immune systems. Data from mice that overexpress or are their lives. Our histological analyses indicated that these mice deficient in growth hormone (GH) indicate that GH stimulates T develop periinsulitis, but show little or no islet infiltration or and B-cell proliferation and Ig synthesis, and enhances maturation β-cell destruction. The mechanism involved specific GH-medi- of myeloid progenitor cells. The effect of GH on autoimmune ated effects on β-cells, where it influenced proliferation and pathologies has nonetheless been little studied. Using a murine apoptosis, and others that modulate the immune system. GH model of type 1 diabetes, a T-cell–mediated autoimmune disease affected Th17/Th1 plasticity, M1/M2 macrophage differentia- characterized by immune cell infiltration of pancreatic islets and tion, and Treg cell function. Our findings show an unanticipated β destruction of -producing -cells, we observed that sus- GH effect on tolerization mechanisms that reduce type 1 di- tained GH expression reduced prodromal disease symptoms and abetes development, and underline the importance of neuroen- eliminated progression to overt diabetes. The effect involves sev- docrine regulation of the immune system. eral GH-mediated mechanisms; GH altered the cytokine environ- ment, triggered anti-inflammatory macrophage (M2) polarization, Results maintained activity of the suppressor T-cell population, and lim- Sustained GH Expression Precludes Development of Overt Diabetes in ited Th17 cell plasticity. In addition, GH reduced apoptosis and/or NOD Mice. In our animal facility, >85% of virgin female NOD increased the proliferative rate of β-cells. These results support mice develop overt diabetes before 40 wk of age (Fig. 1A). To a role for GH in immune response regulation and identify a unique study the effects of sustained high levels of circulating GH, we target for therapeutic intervention in type 1 diabetes. obtained a mouse strain transgenic for bGH under the control of the rat phosphoenolpyruvate carboxykinase (PEPCK) promoter beta cells | Tregs (17), on the NOD background. In this strain, as in the parental C57BL/6-Tg bGH strain, circulating GH levels are constant rowth hormone (GH) is a pleiotropic hormone that affects (∼5 μg/mL). Our mice were healthy and showed no external signs Ga broad spectrum of physiological functions, from carbohy- of other autoimmune diseases or tumor development throughout drate and lipid metabolism to the immune response (1). Several their lives. Histological examination showed mild sialitis. We studies have linked GH with autoimmune diseases, although its monitored glycemia in female NOD-Tg bGH mice and control effects on the immune system are still debated. Whereas some littermates for 60 wk, and found that the transgenic mice were reports using GH-deficient mice indicate that it does not affect immune competence (2), others suggest that GH is necessary for fi correct immune system development (1, 3). The GH Signi cance (GHR) is expressed by several lymphocyte subpopulations (4). GH stimulates in vitro T and B-cell proliferation (5) and Ig Although the relationship between endocrine and immune synthesis (6); enhances human myeloid progenitor cell matura- systems is well documented, few studies have been performed tion (7); and modulates in vivo Th1/Th2 (8) and humoral im- on autoimmune disorders other than those that are sex hor- mune responses (1). In addition, therapeutic activation of the mone-related. We studied a murine model of autoimmune fi GH/STAT5B axis is postulated as a target for restoring mucosal diabetes, showing that growth hormone (GH) modi es the tolerance in Crohn disease (9, 10). A single point mutation in immune response to render diabetic mice resistant to disease development. The mechanism involves a GH-mediated effect STAT5B limits its DNA binding activity as well as maintenance β of FOXP3 expression by Treg cells in nonobese diabetic (NOD) on -cell survival and/or proliferation and a direct effect on immune cells. GH triggers a cytokine environment that pro- mice (11). These mice develop type 1 diabetes, which is char- motes anti-inflammatory macrophage polarization, maintains acterized by autoimmune destruction of pancreatic β-cells due to the activity of the suppressor T cells, and limits Th17 cell plas- the effect of environmental factors on genetically predisposed ticity. This study provides evidence of the importance of endo- individuals (12, 13). Although this murine model does not crine control of immune functions and indicates that therapies completely mimic the human disease, most steps in the patho- based on GH analogs should be considered for treatment of genesis, including prodromal and clinical symptoms, are closely autoimmune diabetes.

comparable (14). IMMUNOLOGY

Despite the interdependence of GH and insulin regulation Author contributions: R.V., C.M.-A., and M.M. designed research; R.V., D.K., Y.J., and and the known effects of GH and insulin-like growth factor 1 R.P.G. performed research; R.V., D.K., Y.J., R.P.G., and M.M. analyzed data; and R.V. and (IGF1) on pancreatic β-cell survival, proliferation and neo- M.M. wrote the paper. genesis (15, 16), hormone influences have not been described in The authors declare no conflict of interest. type 1 diabetes; no specific studies have addressed the con- This article is a PNAS Direct Submission. sequences of long-term GH replacement therapy in this disease. 1R.V. and D.K. contributed equally to this work. Here we show the effects of long-term GH supplementation as 2To whom correspondence should be addressed. E-mail: [email protected]. β a tool to modulate autoimmune attack on pancreatic -cells. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. NOD mice transgenic for bovine GH (NOD-Tg bGH) do not 1073/pnas.1314985110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1314985110 PNAS | Published online November 11, 2013 | E4619–E4627 Downloaded by guest on October 1, 2021 β A B 500 for GH in protection of -cells from apoptosis and stimulation of their proliferative capacity. 1.0 100 400 NOD NOD-Tg bGH Mice Have Normal Delayed Type Hypersensitivity 0.8 80 NOD-TgbGH Responses. Some reports indicate a role for GH as an immune 300 response activator (5, 20); we thus characterized immune cell 0.6 60 populations in blood, spleen, and peripheral lymph nodes of 200 NOD and NOD-Tg bGH mice. Flow cytometry analysis showed 0.4 40 Glycemia (mg/dl) similar immune cell populations in both mouse lines (Table S1). Survival rate (%) Diabetes incidence No differences were observed in activation markers (CD25, 100 0.2 20 CD69, CD44, and CD62L) in CD3+ cells isolated from NOD and NOD Tg-bGH lymph nodes (Fig. S2). 0.0 0 0 To obtain an overview of immune function in NOD-Tg bGH 0 10 20 30 40 50 60 10 20 30 40 50 60 Age (weeks) Age (weeks) mice, we evaluated T-cell responses in a delayed type hyper- CD sensitivity (DTH) assay of sensitization and challenge with allo- geneic splenocytes. NOD-Tg bGH mice and control littermates were sensitized by i.v. injection of C57BL/6 splenocytes and challenged 6 d later by inoculating splenocytes into the right hind footpad. Inflammation was measured every 24 h until remission by comparing thickness of the inoculated to the contralateral, vehicle-inoculated footpad. Both mouse groups reacted similarly, with no significant differences in inflammation grade or resolu-

500 m 500 m tion time, with only a slight remission delay in the transgenic mice (Fig. 2A). E F NOD-Tg bGH Mice Have Altered Serum Cytokine Levels. In type 1 diabetes, a Th1-to-Th2 shift in the immune response is postu- lated to be protective for pancreatic islets (21). Indeed, the conversion of Th17 to Th1 is necessary to induce diabetes effi- ciently (22). To determine whether the GH-protective effect is associated with changes in circulating cytokines, we used a Luminex assay and detected higher peripheral blood levels of IFN-γ, IL-4, IL-13, IL-17A, and TNF-α in sera from NOD-Tg 100 m 100 m bGH mice than from NOD littermates (Fig. 2B). The Th2/Th1 and Th17/Th1 cytokine ratios suggested a GH-mediated bias Fig. 1. NOD-Tg bGH mice are protected against type 1 diabetes de- against a Th1 response in the transgenic mice (Fig. 2C). velopment. (A) Cumulative diabetes incidence in female NOD (continuous red line, n = 90) and NOD-Tg bGH mice (continuous green line, n = 89). Mice Preclinical Type 1 Diabetes Symptoms in NOD-Tg bGH Mice. Early in were maintained and glucose levels determined as described in Materials fl and Methods. The survival rate of transgenic mice after 60 wk in the absence type 1 diabetes progression, an in ammatory environment is of diabetes was 76% (dotted green line). The NOD mouse survival curve established around the islets of Langerhans, usually interpreted (dotted red line) reflects mouse sacrifice at disease onset. (B) Tendency to as a result of anomalous macrophage activity during postnatal lower blood glucose levels with age in NOD-Tg bGH mice (green line; n =10 remodeling of the endocrine pancreas (23). Mononuclear cells mice per group, Pearson’s correlation coefficient, r = −0.61, P < 0.01). Each are recruited into the pancreas and situate around the pancreatic glucose measurement is plotted (twice monthly per mouse). Red, NOD mice; islets shortly after weaning. The insular parenchyma is invaded green, NOD-Tg bGH mice. (C–F) Immunohistochemistry of pancreas from 12- progressively by immune cells, which destroy insulin-producing wk-old NOD (C and E) and NOD-Tg bGH (D and F) mice stained for laminin β-cells. On serial pancreas sections from NOD-Tg bGH, NOD and counterstained with hematoxylin. (C and D) Low-magnification images (6×) showing mononuclear infiltrates (arrows) surrounding islets. (Scale bar: littermates, and control C57BL/6 mice at different ages, we used μ fi × H&E staining to evaluate islet size as well as insular and peri- 500 m.) (E and F) High-magni cation (40 ) images showing damage in- fi C–F duced in the NOD mouse in the laminin sheet (arrows) surrounding islets and insular in ltration (Fig. 1 ). In NOD-Tg bGH mice, insular compared with its preservation in NOD-Tg bGH mice. (Scale bar: 100 μm.) infiltration was delayed and most β-cells were conserved over time (Fig. 3A). The islet-cell mass thus diminished progressively in NOD mice, as predicted, whereas it increased continuously almost completely resistant to diabetes development (Fig. 1A), in NOD-Tg bGH mice (Fig. 3B). These data concur with the as reflected by their higher survival rate compared with NOD reported GHR-dependent islet hyperplasia (24) and the com- mice (Fig. 1A). The results indicated significant lowering of cir- pensatory hyperinsulinemia mechanism associated with GH- culating glucose levels in the transgenic mice with age (Fig. 1B), dependent insulin resistance (25). resulting in relative hypoglycemia. This observation contrasts Using immunohistochemistry and flow cytometry, we analyzed with the susceptibility of C57BL/6-Tg bGH mice to type 2 di- infiltrate composition in pancreata from 3- to 5-mo-old mice. abetes in a high-fat diet study (18). In histological analyses, Snap-frozen organs were sectioned and stained with anti-CD4, NOD-Tg bGH mice showed giant islets with anomalous mor- -CD8, -F4/80, -CD11c, and -B220 antibodies. Although in- phology and a periinsular mononuclear cell infiltrate that char- dividual variation was broad, the inflammation grade in NOD-Tg acterizes the initial stages of diabetes (Fig. 1 C–F). The irregular bGH mouse pancreas was lower than that of NOD mice (Fig. islet morphology suggests β-cell hyperproliferation and islet co- 3A). Infiltrate composition was nonetheless grossly similar, with alescence (Fig. 1D), in agreement with the β-cell hyperpro- a predominance of T cells, mainly CD4+ (Fig. S3). We observed liferation observed in the presence of (19). no immune cell infiltration in NOD-Tg bGH islet parenchyma, Although the results were not statistically significant, the number even in older mice in which infiltration was massive, but always + of apoptotic β-cells was reduced and Ki-67 cells were increased restricted to the islet periphery (Fig. 3C). This inflammation in NOD Tg-bGH pancreas (Fig. S1). These data suggest a role coincides with no apparent degradation of the periinsular laminin

E4620 | www.pnas.org/cgi/doi/10.1073/pnas.1314985110 Villares et al. Downloaded by guest on October 1, 2021 AC140 PNAS PLUS 135 IL13/TNF- 130 IL13/IFN- 125 ** 120 IL4/TNF- 115 IL4/IFN- **

110 Cytokines 105 IL17/TNF- **

Footpad swelling (%) 100 IL17/IFN- 95 ** 24 48 72 96 0 1 2 Hours post challenge Relative ratio B IL4 IL13 IL17 TNF- IFN- 8 400 300 1000 60 *** *** *** * *

6 300 225 750 45

4 200 150 500 30

pg/ml

2 100 75 250 15

0 0 0 0 0 NOD Tg NOD Tg NOD Tg NOD Tg NOD Tg

Fig. 2. T-cell response in NOD-Tg bGH mice. (A) NOD-Tg bGH mice show standard DTH responses. Footpad swelling at several times postchallenge in NOD-Tg bGH (dark gray, n = 6) and NOD mice (light gray, n = 6). Swelling was calculated as the percentage of footpad thickness compared with the baseline at t =0. Values shown are mean ± SD. Student t test showed no significant differences at any time. (B) Circulating cytokine levels in 4-mo-old NOD (n = 12) and NOD- Tg bGH (Tg) (n = 11) mice. Individual and mean values are shown. Student t test, *P < 0.05, ***P < 0.001. (C) Ratios between Th2/Th1 and Th17/Th1 profiles, assessed by circulating cytokines quantification, in NOD (light gray bars, n = 12) and NOD-Tg bGH (dark gray, n = 11) mice. Values have been normalized to NOD mice data. Student t test, **P < 0.01.

layer, which defines the basal lamina of the sheet of Schwann NOD mice from disease development (Fig. 4A), suggesting that cells thought to be the first target of autoimmune attack (26) NOD-Tg bGH regulatory cells had insufficient suppressive ca- (Fig. 1 E and F). In some cases, we found intrainsular foci in mature pacity in NOD mice. In addition, cells from the transgenic mice mice (Fig. 3 C and D), although they must be considered peri- did not accelerate diabetes in sublethally irradiated NOD mice vascular, because they are surrounded by an intact laminin sheet. (Fig. 4B), indicating a lack of diabetogenic effector cells in the Because type 1 diabetes is also characterized by development NOD-Tg bGH splenocyte population. of a humoral response to islet antigens, we evaluated antiinsulin Radiomimetic drugs trigger type 1 diabetes in NOD mice by antibodies in prediabetic NOD-Tg bGH and control prediabetic targeting the CD4+CD25+FoxP3+ T-cell population and impair- NOD mice. Whereas NOD mice had high antiinsulin antibody ing their recovery in pancreas infiltrates (29). To determine the titers, indicative that insulin is the primary antigen for type 1 role of Treg cells in our model, we transferred splenocytes (2 × diabetes in these mice (27), as it is in humans (28), NOD-Tg bGH 107) from a pool of three overtly diabetic 6-mo-old NOD mice mice had much lower titers (barely detectable even at 12 wk; into 6-wk-old sublethally (7 Gy) irradiated NOD-Tg bGH mice. Fig. 3E). Spleen B cells from NOD and NOD Tg-bGH mice were Diabetes did not develop in the recipients (Fig. 4C), whereas di- activated in vitro with anti-mouse IgM antibody (10 μg/mL, 180–360 abetes was accelerated in irradiated control NOD littermates. min, 37 °C) alone or with exogenous GH (5 μg/mL); activation was These data suggest a resistant suppressive mechanism in irradiated similar, as demonstrated by flow cytometry using anti-CD69 and NOD-Tg bGH mice that blocked NOD effector cells. anti-CD86 antibodies (Fig. S4). These findings rule out B-cell ac- Because Treg cells are reported to be relatively radioresistant tivation defects in NOD Tg-bGH mice. (30), we transplanted diabetogenic splenocytes into lethally ir- In addition to the maintenance of normal glycemia, the lack of radiated (12 Gy) NOD and NOD-Tg bGH recipients for a 3-wk antiinsulin antibodies and the absence of islet-infiltrating, puta- radioprotection/accelerated diabetes assay. Recipient mice were tive antigen-specific CD4+ T cells in NOD-Tg bGH mice sug- rescued from lethality and, though NOD mice became hyper- – gests that GH modulates the adaptive immune response in type glycemic within 7 10 d, transgenic mice remained normoglyce- IMMUNOLOGY 1 diabetes. mic throughout the experiment (Fig. 4D). Untransferred mice of both genotypes, used as a lethality control, died during the first Circulating GH Levels Are Responsible for the Protective Phenotype. week. The results imply a suppressive mechanism for diabetogenic For detailed characterization of the role of the adaptive immune cells in NOD-Tg bGH mice, which are resistant even to a high response in NOD-Tg bGH mice, we evaluated diabetogenic cell dose of full-body irradiation. The relative radioresistance of Treg populations and suppressor cell activity. We transferred total cells and monocytes might account for this suppression. splenocytes from NOD-Tg bGH or NOD mice into untreated We tested whether transgenic splenocytes protect mice in an or sublethally (7 Gy) irradiated NOD or NOD-Tg bGH mice. accelerated diabetes model. Two groups of 12-wk-old NOD/SCID Splenocytes from NOD-Tg bGH mice did not protect untreated (severe combined immunodeficiency) mice were inoculated i.v.

Villares et al. PNAS | Published online November 11, 2013 | E4621 Downloaded by guest on October 1, 2021 function during the prediabetic stage is not well understood (31– 34). CD4+CD25+ Treg cells control disease progression through various potential mechanisms, inhibiting activation, proliferation, and/or migration of islet-specific T cells in lymph nodes and in pancreas (35). Because Treg cell suppressive potential is associ- ated with FoxP3 levels (36), we used flow cytometry to determine FoxP3 expression on CD4+CD25+ peripheral blood lymphocytes from C57BL/6, NOD, and NOD-Tg bGH mice at 2 mo of age, before hyperglycemia was detected (Fig. 5A), and at 5 mo, when NOD mice were hyperglycemic (Fig. 5B). We found no differ- ences in FoxP3 expression in any 2-mo-old mice (Fig. 5 A and C), whereas CD4+CD25+ lymphocytes from 5-mo-old NOD mice showed a clear reduction in FoxP3 levels compared with those from B6 mice; this down-regulation was not observed for NOD- Tg bGH CD4+CD25+ Treg cells (Fig. 5 B and C). The results suggest a GH effect on the maintenance of Treg cell activity. In 2-mo-old mice, the percentage of CD4+CD25+FoxP3+ cells was higher in C57BL/6 than in NOD-Tg bGH mice or NOD

A B 100 100

80 80

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40 40

20 20 NS

0 0 C 0 10 20 30 40 D 0 10 20 30 40 100 100

Fig. 3. Type 1 diabetes symptoms are largely suppressed in NOD-Tg bGH 80 80 mice. (A) Severity of insulitis and destructive lesions in NOD, NOD-Tg bGH, 60 60 and C57BL/6 (control) mice. After H&E staining, ∼50–100 random islets per pancreas were evaluated. Three mice were analyzed for each genotype and 40 40 age group (13, 20, and 24 wk). The degree of mononuclear cell infiltration was graded independently by two observers as follows: 0, normal (white); 1, 20 20

mild periinsulitis (light gray); 2, severe periinsulitis (dark gray); or 3, insulitis 0 0 (black; see Materials and Methods for details). (B) Islet mass estimated from E 0 2 4 6 8 10 12 14 0 10 20 30 40 percentage of endocrine area (relative to total pancreas surface) as survivalCumulative (%) 100 a function of age for NOD (gray), NOD-Tg bGH (black), and C57BL/6 mice 80 (white; n = 3 for each age group). (C and D)Lateinfiltration in islets from NOD-Tg bGH mice. Frozen pancreas sections of 7-mo-old NOD-Tg bGH mice. 60 Islets (I) and infiltrates (M) are labeled. (Scale bar, 100 μm.) (C)Merged image of H&E staining showing giant coalescent islets. Original magnifi- 40 cation, 6×.(D) Section stained with antilaminin antibody to identify the 20 NS basal layer of pancreatic acini, blood vessels, and periinsular sheet of Schwann cells. Hematoxylin counterstain shows perivascular and islet 0 infiltrates. Mononuclear infiltrates were surrounded by this laminin sheet 0 10 20 30 40 and hence did not invade the islet parenchyma. Original magnification, Age (weeks) 10×.(E) Evaluation of prodromal antiinsulin antibodies (NOD-Tg bGH, n =5; NOD, n =7;C57BL/6control,n = 3) at 10 (light gray), 12 (dark gray), and 14 Fig. 4. Kaplan–Meyer analysis for adoptive transfer protocols. (A) Seven- 7 wk (black). ELISA for human insulin did not detect autoantibodies in NOD- week-old NOD mice (continuous line, n = 4) were inoculated with 2 × 10 Tg bGH mice. Background optical density was subtracted. Student t test, NOD-Tg bGH mouse splenocytes; inoculated NOD-Tg bGH mice were used as **P < 0.01. controls (dashed line). No delay in hyperglycemia was observed compared with the normal behavior of NOD mice. (B) Seven-week-old NOD mice (continuous line, n = 6) were sublethally irradiated and inoculated with 2 × 7 with 2 × 107 splenocytes from a pool of two 5-mo-old diabetic 10 NOD-Tg bGH mouse splenocytes. No delay was observed in manifesta- NOD mice. One group also received 2 × 107 splenocytes from tion of overt diabetes compared with untreated NOD control mice (dashed E line). (C) Six-week-old NOD-Tg bGH mice (dashed line, n = 5) were sub- a pool of two 5-mo-old transgenic mice (Fig. 4 ). All mice that lethally irradiated and inoculated with 2 × 107 diabetic NOD mouse sple- received NOD splenocytes alone developed hyperglycemia by nocytes. Irradiated NOD littermates (continuous line, n = 4) were used as 4 wk posttransfer. The group inoculated with splenocytes from controls. Accelerated diabetes development was observed in NOD controls, NOD + NOD-Tg bGH mice showed a slight, nonsignificant whereas NOD-Tg bGH mice remained fully resistant. (D) The experiment in C delay in hyperglycemia (log- test P = 0.094), suggesting loss was repeated using lethally irradiated NOD-Tg bGH mice (dashed line, n =4). of a hypothetical protective mechanism in the absence of circu- NOD mice (n = 2) were used as controls for accelerated diabetes development, and uninoculated mice (NOD, n = 2; NOD-Tg bGH, n = 2) as controls of lethality; lating GH. all four untransferred mice died within 2 wk of irradiation. (E) NOD/SCID mouse sensitivity to diabetes development after splenocyte transfer (NOD, continuous Suppressive Potential in NOD-Tg bGH Mice. Although Treg cells are line; NOD + NOD-Tg bGH, dashed line; 12 wk old, n = 4 for both groups). Log- broadly implicated in type 1 diabetes development, their precise rank test P < 0.05 was considered significant. NS, not significant.

E4622 | www.pnas.org/cgi/doi/10.1073/pnas.1314985110 Villares et al. Downloaded by guest on October 1, 2021 AB We used an in vitro suppression assay to test for a correlation PNAS PLUS between reduced FoxP3 expression on CD4+CD25+ lympho- cytes and a reduction in their suppressive capacity. Coculture of − CD4+CD25 NOD T cells with varying proportions of CD4+CD25+ T cells from 5-mo-old NOD or NOD-Tg bGH mice led to slight but significantly higher suppressive activity by NOD-Tg bGH- derived cells (Fig. 5E). These results indicate that GH participates in maintenance of the suppressive potency of Treg cells as well as of the relatively + + FoxP3 high FoxP3 levels in CD4 CD25 cells. This effect might be potentiated in vivo, because NOD-Tg bGH cells are continu- CD ously exposed to high levels of circulating GH. Treg cells in vivo 700 16 might also be activated locally by antigen-presenting cells (37) 600 * ** fi 500 12 speci cally charged with islet antigens, which were absent in our 400 * in vitro experiments. 300 8 FoxP3 MFI 200 NOD NOD-Tg bGH 4 GH Effect on Macrophage Polarization. Macrophages have a key 100 C57BL/6 role in pancreas remodeling (23) and are also present in pan- 0 FoxP3+ in CD4+ (%) 0 fi 2 m 5 m 2 m 5 m creas in ltrate in NOD and NOD-Tg bGH mice. There are two ) Ct main macrophage types: M1 or inflammatory macrophages, E F  3 8 * characterized by high NOS2 (inducible nitric oxide synthase)

) fl 4 6 2 expression, and M2 or anti-in ammatory macrophages charac- * terized by arginase-1 expression. We used quantitative real-time 4 1 PCR to quantify M1/M2 macrophage markers in pancreatic cpm (x 10 2 * lymph nodes from 3-mo-old NOD and NOD-Tg bGH mice. We 0 0 found lower NOS2 and higher arginase-1 RNA levels in nodes 2:1 4:1 8:1 C Relative expression (2- ARG1 iNOS from NOD-Tg bGH than from NOD mice (Fig. 5F). These data G H were confirmed by immunohistochemical identification of argi- nase-1–producing cells in NOD and NOD-Tg bGH mouse pan- creas (Fig. 5 G and H). The results indicate a GH effect on macrophage polarization toward the M2 phenotype in NOD-Tg I I bGH mice.

GH Modulates Th17 Cell Plasticity. Diabetes development is reported to be associated with acquisition of a Th1-like phenotype by Th17 cells (38), which then express T-bet and secrete IFN-γ. We tested the GH effect on Th17/Th1 plasticity using RT-PCR to determine IL-17, IFN-γ, IL-2, IL-22, and GM-CSF mRNA levels in total Fig. 5. Bias toward a regulatory phenotype in NOD-Tg bGH mice. Down- pancreas of age-matched NOD-Tg bGH and prediabetic NOD regulation of FoxP3 in NOD Treg cells. Blood samples from 2-mo-old (A) and mice, as well as of ROR-γT and T-bet transcription factors as 5-mo-old mice (B) of C57BL/6, NOD, and NOD-Tg bGH genotypes were la- specific markers of Th17 and Th1 polarization, respectively (Fig. beled for CD4, CD25, and FoxP3; Treg cells were gated by light-scatter 6A). To avoid interference due to different numbers of infiltrating properties (forward scatter and side scatter), CD25 and CD4. One represen- cells in the pancreas, we compared the ratio for messages char- tative sample of nine is shown. C57BL/6, black line; NOD, red line; NOD-Tg acteristic of Th17 and Th1 cells, which is more indicative of the + + fl bGH, green line. (C) FoxP3 expression in CD4 CD25 cells assessed by ow relative abundance of nonpathogenic Th17 cells than are in- cytometry. FoxP3 mean fluorescence intensity in blood samples from 2- and + + dividual Th17 or Th1 values (39). The results showed a higher 5-mo-old mice. (D) Relative abundance of FoxP3 cells in the blood CD4 RORC/Tbx21 B γ C population, assessed by flow cytometry, at 2 and 5 mo. (C and D) NOD (light (Fig. 6 ) and IL-17/IFN- (Fig. 6 )ratiosin gray), NOD-Tg bGH (dark gray), and C57BL/6 (white) mice mean values + SD NOD-Tg bGH mouse pancreas. In addition, we detected higher for four experiments (total n = 9 for each mouse group). Student t test, *P < IFN-γ, IL-2, IL-22, and GM-CSF mRNA levels in NOD mouse 0.5; **P < 0.1. (E) Suppressive activity of CD4+CD25+ splenocytes from NOD pancreas (Fig. 6A). These data indicate a significant reduction of − (light gray) and NOD-Tg bGH (dark gray) mice at various ratios of CD4+CD25 pathogenic Th17 cells infiltration and their plasticity to Th1 in 3 effector cells (letter C indicates no suppressor cells added). [ H]thymidine NOD-Tg bGH mouse pancreas. incorporation after coculture is shown as a percentage of the value for cultured pure effector cells. (F) M1 and M2 macrophage marker expression. Discussion Quantitative RT-PCR was used to quantify relative levels of NOS2 and arginase- −ΔΔ 1 mRNA in pancreatic lymph nodes from 3-mo-old mice. Values (2 Ct)are Type 1 diabetes is a multifactorial disease caused by the con- relative to the mean level of each message in samples from NOD mice. (G and currence of genetic and environmental factors that include in- H) Immunohistochemistry showing arginase-1 expression within the peri- fectious agents, diet, and illness (40). Endocrinopathies charac- insular infiltrate in young mice (8 wk). Anti–arginase-1 antibody was visualized terized by chronic overproduction of hormones whose action

with horseradish peroxidase and diaminobenzidine (arrows); hematoxylin was opposes that of insulin, such as epinephrine, glucagon, cortisol, or IMMUNOLOGY used as counterstain (G,NOD;H, NOD-Tg bGH). Original magnification, 20×. GH, generally cause diabetes by triggering insulin resistance (41); nonetheless, very little is known of their potential to influence autoimmune diabetes. Here we observed that a transgenic mouse littermates, with no difference between the last two groups. The strain on the NOD background that expresses bGH under the + + + percentage of CD4 CD25 FoxP3 cells was higher in 5-mo-old control of the rat PEPCK promoter (NOD-Tg bGH) did not NOD-Tg bGH mice than in NOD littermates, although in both develop type 1 diabetes. The pancreatic inflammatory phenotype cases it was lower than that in C57BL/6 mice (Fig. 5D), which characteristic of the NOD background was severely reduced in suggests higher suppressive T-cell activity in NOD-Tg bGH than the presence of GH. These mice also showed almost complete in NOD mice. suppression of the adaptive immune response. We detected no

Villares et al. PNAS | Published online November 11, 2013 | E4623 Downloaded by guest on October 1, 2021 Relative expression Th2 response and Ig production in NOD-Tg bGH mice. Over- A 0% 20% 40% 60% 80% 100% 120% expression of bGH in C57BL/6 mice alters the humoral response IL10 * to egg albumin by reducing Th2 cytokine production (1). We IL17 NS did not observe such Th2 defects on the NOD background, as GM-CSF assessed by measurement of circulating cytokine levels. In any * β IFNG *** case, antiislet antibodies are not directly linked to -cell de- IL2 struction, and the role of β-cells appears to be restricted to their *** β IL22 * antigen-presenting activity (49). -cells from NOD and NOD-Tg Tbx21 *** bGH mice showed no defects in their in vitro activation by anti- RORC * IgM antibodies. We previously observed that after stimulation with conventional antigens, the antigen response is reduced in Tg B RORC/Tbx21 C IL-17/IFN- bGH mice, with a IgG1 to IgG2 isotype shift (1). These findings implicate GH in altering T-cell function. 250 * )

et Transferred NOD-Tg bGH splenocytes did not protect NOD  40 b

T- mice from diabetes, and NOD splenocytes did not promote di-

200 FN- A 30 abetes when transferred into NOD-Tg bGH mice, suggesting

150 ** RNA I that control of T-cell responses in NOD-Tg bGH mice is de- t/mRN /m

 pendent on circulating GH levels. 7 20 1 100 - At 5 mo of age, with a well-developed inflammatory environ- ment, CD4+CD25+ Treg cell numbers are maintained at stable

NA ROR 10

50 RNA IL low levels in NOD mice, whereas they rise in NOD-Tg bGH R m m mice. In NOD-Tg bGH mice, we did not observe the down- 0 0 regulation of FoxP3 expression found in hyperglycemic NOD mice. Because FoxP3 expression is directly linked to the regu- Fig. 6. Cytokine expression in the pancreas of NOD prediabetic and NOD-Tg bGH diabetes-resistant mice. RNA from 13-wk-old mouse whole pancreas latory action of Treg cells (50), it is thus possible that GH reg- was quantified by quantitative RT-PCR. Student t test (n = 6); *P < 0.05; **P < ulates FoxP3 activation via STAT5b or STAT3. A single point 0.01; ***P < 0.001. (A) Relative expression (2−ΔΔCt) of characteristic Th1 and mutation in STAT5b, which encodes a transcription factor in- Th17 transcription factors and cytokines in NOD and NOD-Tg bGH mice volved in GH signaling (51), limits FoxP3 expression by Treg pancreas, relative to mean values in NOD. The ratios between mRNA expression cells (11). Sustained activation of STAT3 (another transcription of RORC/Tbx21 (B) and IL-17/IFN-γ (C) in NOD (light gray) and NOD-Tg bGH (dark factor involved in signaling through the GHR) is needed to main- gray) mice are shown. tain FoxP3 expression by Treg cells (52). Through STAT5B or STAT3 activation, GH might thus increase FoxP3 levels, regu- lating Treg cell activity. prodromal antiislet antibody production or diabetogenic cell ex- In the adoptive transfer experiments, sublethal and even lethal pansion; hence, there was no islet destruction in these mice. irradiation did not alter the protective effect of GH expression in There is considerable evidence for distinct GH effects on NOD-Tg bGH mice. GH promotes radioprotection in a variety pancreatic β-cells (16), some direct and others through its main of cell types, and radiation sensitivity differs in some immune mediator, IGF1 (42). Exogenous GH in rat islet cultures thus system niches. For example, GH and IGF1 enhance hemato- stimulates DNA synthesis and insulin production (43); IGF1 and poietic stem cell radioresistance and proliferation (53). These GH signaling have mitogenic effects on INS-1 cells (44). We resistance mechanisms nonetheless appear to be insufficient for detected a large β-cell mass in NOD-Tg bGH mice that corre- β the rapid expansion of suppressive cells that would be needed to lated with the higher proliferation rate and lower apoptotic -cell control disease in an accelerated diabetes model. It is more likely numbers detected in the pancreas of these mice, which could that GH increases the radioresistance of the Treg cell pop- explain in part the lack of hyperglycemia in these mice. By ac- ulation; lethally irradiated wild-type hosts transferred with scurfy β tivating the JAK/STAT pathway, GH can also stimulate -cell bone marrow cells did not develop autoimmune disease, due to survival. The JAK/STAT-activated suppressors of cytokine sig- suppression of sf-derived T cells by radioresistant host FoxP3+ γ naling block damage triggered by cytokines such as IFN- or Treg cells (30). α TNF- (45), and expression of a constitutively active form of We tested the in vitro suppressive capacity of Treg cells from β STAT5b has a protective effect on -cells in a model of strep- NOD and NOD-Tg bGH mice and found slight but significant tozotocin-induced diabetes (46). differences. These differences might be considered insufficient to NOD-Tg bGH mice showed periinsulitis, although we found explain complete resistance to diabetes development; nonethe- no sign of degradation of the periinsular laminin layer, and no less, T-cell differentiation and activity are dependent on antigen- antiinsulin antibodies in serum. The results confirm a GH effect presenting cell type and the microenvironment in which pre- on the immune system in addition to its effect on β-cells. sentation occurs, and Treg cells might be induced only locally by Although differences in immune cell activity are associated M2 macrophages (37). Macrophages have two distinct pheno- with high levels of circulating GH (47), we detected no major types (54): inflammatory (M1) macrophages participate in anti- differences in any of the circulating cell populations in either gen recognition and secretion of inflammatory cytokines, and + + + + + + mouse type; B220 , CD3 , CD4 , CD8 , CD11b , or Gr1 cell noninflammatory (M2) macrophages are involved in tissue repair numbers were similar, as were the lymph node T-cell activation and remodeling (55). M1 polarization is induced by IFN-γ and markers (CD25, CD69, CD44, and CD62L). This observation characterized by high NOS2 expression and by secretion of concurs with the similar response in a DTH assay of NOD and proinflammatory cytokines IL-1 and IL-12, whereas M2 are in- NOD-Tg bGH mice to immunization with allogeneic splenocytes, duced by IL-4 and IL-13 and are characterized by high arginase-1 ruling out general defects in the T-cell response and/or antigen expression and by IL-10 secretion. Our data for pancreatic lymph presentation. It is nonetheless postulated that APC, antigen pre- nodes and pancreata from prediabetic NOD mice showed the senting cell, defects are responsible for the lack of adequate reg- presence of M1 macrophages (arginase-1lowNOS2high), whereas ulatory potential in NOD mice (48). It could be argued that the age-matched NOD-Tg bGH mouse macrophages had an M2 phe- halt observed at the periinsulitis checkpoint is due to a reduced notype (arginase-1highNOS2low). This difference might be the result

E4624 | www.pnas.org/cgi/doi/10.1073/pnas.1314985110 Villares et al. Downloaded by guest on October 1, 2021 of the high circulating levels of IL-17 (56), and could also influence magnification. Composite images were generated with the photomerge PNAS PLUS increased local Treg cell activity. function of Adobe Photoshop CS5. On merged images, a grid was super- β Autoimmune diabetes is a well-characterized Th1 pathology. imposed and -cell mass calculated from the ratio of intersections in endo- We detected high IFN-γ and IL-2 mRNA levels in NOD mouse crine vs. total pancreas and pancreas weight (67). Separation between grid lines was 50 μm. The same slides were used to determine infiltration level in pancreas, which were lower in NOD-Tg bGH pancreas. Both islets. Insulitis was scored by the following criteria: insulitis (grade 3), in- mouse models also had high IL-17 mRNA levels, suggesting filtration in the islet parenchyma; severe periinsulitis (grade 2), three or Th17 cell involvement in type 1 diabetes. Th17 cells are found in more rows of mononuclear cell infiltrate surrounding the islet; mild peri- some autoimmune diseases such as experimental autoimmune insulitis (grade 1), less than three rows of periinsular infiltrating cells, and no encephalomyelitis (57) and rheumatoid arthritis (58); NOD insulitis (grade 0), absence of cell infiltration. Apparent intraislet area was and NOD-Tg bGH mouse pancreas also showed high ROR-γT sometimes counted as periinsular when the insular parenchyma was not mRNA levels. invaded, as determined by the integrity of Schwann cell basal lamina. Recent evidence suggests that depending on the microenvi- ronment, Th17 cells can alter their differentiation program to Delayed Type Hypersensitivity. C57BL/6J splenocytes were isolated by me- fl chanical disaggregation and erythrocyte lysis with NH4Cl, washed once with induce protective or proin ammatory responses (59, 60). We PBS + 0.1% BSA and twice with PBS. The 6-wk-old mice were sensitized by fi γ 5 found a signi cant reduction in mRNA levels of IFN- , IL-2, and i.v. injection of 2 × 10 C57BL/6J splenocytes, and challenged on day 6 in the GM-CSF, characteristic mediators for Th17 pathogenic cells right footpad with 1.5 × 107 cells in 50 μL PBS. Control left footpads received (59), in NOD-Tg bGH pancreas compared with that of NOD 50 μL PBS. Footpad thickness was measured with a vernier caliper (Mitutoyo mice. Although, increasing evidences points to the role of Th17 Japan) at 24, 48, and 72 h after challenge. Results of footpad swelling were cells in NOD mice, it seems that the conversion of this cell subset calculated as the difference between challenged vs. prechallenged footpad, fl expressed as a percentage. Student t test P values >0.05 were considered into Th1 is more important (22, 38). How the in ammatory fi microenvironment modulates this Th17 differentiation is not nonsigni cant. completely understood, although our results suggest that GH fi Adoptive Transfer. Donor spleens were processed as above. The indicated maintains a nonpathogenic pro le of Th17 cells and reduces number of splenocytes was injected i.v. into the tail of recipient mice. When their Th1 potential in the pancreas. indicated, host mice were previously irradiated lethally (12 Gy) or sublethally We cannot rule out that GH might also contribute to trans- (7 Gy) with a single full-body dose from a 137Cs source. SPSS Statistics software genic islet resistance to immune attack via STAT3 activation. was used for statistical analyses. STAT3 activation in Treg cells, associated with type 1 diabetes resistance in NOD mice (61), controls the macrophage IL-10– Cytokine Measurement. Circulating cytokines were quantified in serum with mediated anti-inflammatory response (62) and is an important a Bio-Plex and analyzer (BioRad). Serum samples were stored at −80 °C survival factor in β-cells (63, 64). Our data show that GH- until use. Antiinsulin and total antibodies in serum were measured by standard ELISA at 495 nm. Plates were coated with 100 μL per well with mediated interference in type 1 diabetes development involves μ β human insulin (20 g/mL; Novo-Nordisk) or goat anti-mouse Ig kappa chain an increase in -cell mass, protection of the periinsular laminin (50 μg/mL) in PBS. Plates were incubated (overnight, 4 °C), washed with PBS, layer, and a direct effect on immune cells—mainly macrophages, 0.1% Tween 20, and blocked with 1% BSA, 0.05% Tween-20 in PBS [1 h, Th17, and possibly Treg cells. These results demonstrate the room temperature (RT)]. Serial serum dilutions were added to washed plates importance of endocrine control of immune functions, and in- and incubated (2 h, RT); after washing, peroxidase-conjugated anti-Ig dicate that therapies based on GH analogs and/or their signaling (1:2,000; Dako) was added. Plates were incubated (1 h, RT), washed, and 100 cascades should be considered for treatment of autoimmune μL orthophenylenediamine substrate solution (Sigma) was added. The re- diabetes. action was terminated after 20 min and measured. Materials and Methods Cell Purification and Flow Cytometry. To prepare single-cell suspensions, spleens and lymph nodes were harvested and minced on a 40-μm nylon mesh Mice. Mice transgenic for bGH under the control of the phosphoenolpyruvate in RPMI medium 1640 (Lonza) supplemented with 10% FBS, 2 mM L-gluta- carboxykinase promoter on a C57BL/6J × C3H/J hybrid background (65) were mine, and 50 μg/mL penicillin/streptomycin. For APC preparations, spleens crossed on the NOD background until NOD polymorphic alleles were stabi- were predigested with collagenase A and DNase I. CD11c+, CD4+CD25+,and lized (F0; NOD-Tg PEPCK-bGH/Ccnb), as assessed by the length of single se- + − CD4 CD25 cell populations were enriched by CD11c Microbeads Mouse quence repeats (66). The transgenic strain was maintained by continuous (Miltenyi Biotech), Dynabeads Flow Comp Mouse CD4+CD25+ Treg Cells Kit backcrosses on NOD females. Experimental results were obtained from F5 (Invitrogen), and an AutoMACS Cell Sorter (Miltenyi Biotech). When re- onward, always using strict littermates. Mice were fed a standard laboratory quired, murine B cells were purified using mouse pan-T Dynabeads (Invi- diet (Global Diet 2918, Harlan Iberica; 18.5% , 5.5% oils and trogen) and T cells with mouse T-cell negative isolation kit (Dynal). Purity of fat) and tap water ad libitum. The mice were monitored twice a week with all cell preparations was routinely >95%. Blood samples were lysed with Accutrend kits (Roche Diagnostics) for development of hyperglycemia, and VersaLyse (Beckman Coulter). > declared diabetic when glucose was 200 mg/dL in two consecutive mea- Single-cell suspensions of lymphoid organs or blood leukocytes were surements. Overtly diabetic mice were killed. Mice were handled according prepared and blocked with anti-CD16/32 (BD Pharmingen) to impede Fc- to national and European Union guidelines, and experiments were ap- mediated nonspecific antibody binding. Samples were stained with antibody proved by the Comité Ético de Experimentación Animal, Centro Nacional de conjugates by a standard procedure, using FITC anti-CD25, FITC anti-CD11b, Biotecnología. and SPRD anti-Gr1 (Pharmingen); SPRD anti-CD4 (eBiosciences); FITC anti- CD3, FITC anti-CD8, PE anti-CD44, FITC anti-CD69, FITC anti-CD45, and APC Immunohistochemistry. Pancreata were embedded in optimal cutting tem- anti-B220 (Beckman Coulter); PE anti-CD86 (BioLegend); and PE anti-CD62L perature freezing medium (Sakura) and snap-frozen in cooled isopentane. (Southern). FoxP3 expression was determined after permeabilization and Sections (7 μm) were cut, air-dried, and fixed in cold acetone. When stored intracellular staining with a PE-labeled antibody (FoxP3 staining set; eBio- at −80 °C, sections were postfixed in ethanol/acetone (1:3) before staining. – fi sciences). When necessary, naïve B cells (92 95% pure) were rst activated IMMUNOLOGY Primary antibodies were guinea pig anti-porcine insulin antibody (Dako), anti– with 10 μg/mL goat anti-mouse IgM Ab (Jackson ImmunoResearch; 3 or 6 h, arginase-1 (BD Biosciences), rabbit anti-laminin (Sigma), and rabbit anti-Ki67 37 °C), alone or with exogenous human GH (5 μg/mL, Genotonorm; Pfizer). (Novocastra antibodies); immunodetected with the tyramide signal amplifica- Stained samples were analyzed on a flow cytometer (Cytomics FC 500; Beck- tion indirect staining kit (Perkin-Elmer); and visualized with diaminobenzidine. man Coulter). FACS data were analyzed with FlowJo and CytoSpec software. Hematoxylin was used for counterstaining. − In Vitro Suppression Assay. CD4+CD25 NOD splenocytes (5 × 104)were β-Cell Mass Determination and Infiltration Level Count. Pancreata from mice of cocultured with variable ratios of CD4+CD25+ cells (2:1, 4:1, and 8:1) and with different ages (three per group) were extracted, weighed, formaldehyde- NOD spleen CD11c+ cells (5 × 104;previouslyγ-irradiated, 15 Gy) in the pres- fixed, and paraffin-embedded. For quantification, one 5-μm section was ence of anti-CD3 (1 μg/mL). Cultures were prepared in triplicate in U-bottom analyzed every 100 μm. Sections were H&E stained and photographed at 10× 96-well plates (Nunclon Surface) with RPMI-1640 medium supplemented with

Villares et al. PNAS | Published online November 11, 2013 | E4625 Downloaded by guest on October 1, 2021 10% FBS, 2 mM L-glutamine, 1 mM pyruvate, and 50 μM β-mercaptoethanol Prism HT7900 sequence detection system (Applied Biosystems). For relative −ΔΔ (72 h, 37 °C), and cells were pulsed (16 h) with 1 μCi [3H]thymidine (Perkin- quantification, we used the equation 2 Ct. We normalized each sample Elmer) per well. The 3H incorporation was measured by liquid scintillation with β-actin (ΔCt), and ΔΔCt represents the difference between the Ct from spectrometry using CytoScint mixture (MP Biomedical) and a 1450 MicroBeta each expression of NOD at 13 wk and each datum. counter (Perkin-Elmer). ACKNOWLEDGMENTS. We are grateful for the gift of Genotonorm (Pfizer Semiquantitative Real Time PCR. cDNA sequences were obtained from the España). We thank R. Barroso and G. Cascio for technical support, L. Gómez for GenBank database. PCR primers were designed from the cDNA sequences animal handling, C. Bastos for secretarial assistance, and C. Mark for editorial assistance. Support for this work was provided by Comunidad de Madrid using Primer-BLAST (68) (Table S2). RNA (5 μg) was used for reverse tran- Contract S2011/BMD-2502 (to R.V.); a La Caixa Fellowship (to D.K.); Spanish scription. cDNA was obtained by SuperScript II reverse transcriptase (Invi- Ministry of Science and Innovation Grant SAF 2011-27370; European Union fi trogen). Then cDNA was ampli ed by PCR analysis, using Power SYBR Green FP7-integrated project Masterswitch 223404; Fondo de Investigación Sanitaria, PCR Master Mix (Applied Biosystems), 0.3 μM of primers, and three serial Instituto de Salud Carlos III RD12/009/009 and RD12/009/002; and the Comunidad dilutions of RT products. Triplicate samples were quantified using the ABI de Madrid S2010/BMD-2350.

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