Diabetes Volume 63, December 2014 4239

Yudong Wang,1,2 Yang Xiao,3 Ling Zhong,1,2 Dewei Ye,1,2,4 Jialiang Zhang,1,2 Yiting Tu,3 Stefan R. Bornstein,5 Zhiguang Zhou,3 Karen S.L. Lam,1,2 and Aimin Xu1,2,4

Increased and and Augmented NETosis Are Closely Associated With b-Cell Autoimmunity in Patients With Type 1 Diabetes

Diabetes 2014;63:4239–4248 | DOI: 10.2337/db14-0480 MUOOYADTRANSPLANTATION AND IMMUNOLOGY

Type 1 diabetes (T1D) is an autoimmune disease resulting activities in the pathogenesis of b-cell au- from the self-destruction of insulin-producing b-cells. Re- toimmunity and also suggest that circulating NE and PR3 duced neutrophil counts have been observed in patients may serve as sensitive biomarkers for the diagnosis of T1D. with T1D. However, the pathological roles of in the development of T1D remain unknown. Here we show that circulating protein levels and enzymatic activities of The global incidence of type 1 diabetes (T1D), an autoim- (NE) and proteinase 3 (PR3), both of mune disease caused by an interactive combination of which are neutrophil serine proteases stored in neutrophil genetic and environmental factors, has more than doubled primary granules, were markedly elevated in patients with in the past two decades (1,2). Although the triggering fac- T1D, especially those with disease duration of less than tors that are involved in the initiation of T1D remain un- 1 year. Furthermore, circulating NE and PR3 levels in- clear, it is widely accepted that organ-specific autoimmune creased progressively with the increase of the positive destruction of the insulin-producing b-cells in the pancre- numbers and titers of the autoantibodies against b-cell atic islets of Langerhans is mediated primarily by autoreac- antigens. An obvious elevation of NE and PR3 was detected tive T cells, which is accompanied by the production of even in those autoantibody-negative patients. Increased different autoantibodies to b-cell antigens, including glutamic NE and PR3 in T1D patients are closely associated with acid decarboxylase autoantibody (GADA), insulinoma- elevated formation of neutrophil extracellular traps. associated protein 2 autoantibody (IA2A), and zinc transporter-8 By contrast, the circulating levels of a1-antitrypsin, an autoantibody (ZnT8A) (3–5). These autoantibodies have endogenous inhibitor of neutrophil serine proteases, are been proven to be instrumental for the prediction and di- decreased in T1D patients. These findings support an early agnosis of T1D but are deemed not to be pathogenic (6,7). role of neutrophil activation and augmented neutrophil A number of other immune cells, including dendritic cells,

1State Key Laboratory of Pharmaceutical Biotechnology, The University of Hong Received 24 March 2014 and accepted 28 July 2014. Kong, Hong Kong, China This article contains Supplementary Data online at http://diabetes 2 Department of Medicine, The University of Hong Kong, Hong Kong, China .diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0480/-/DC1. 3Diabetes Center, Institute of Metabolism and Endocrinology, Second Xiangya Y.W., Y.X., and L.Z. contributed equally to this work. Hospital, Key Laboratory of Diabetes Immunology, Ministry of Education, Central South University, National Clinical Research Center for Metabolic Diseases, © 2014 by the American Diabetes Association. Readers may use this article as Changsha, Hunan, China long as the work is properly cited, the use is educational and not for profit, and 4Department of Pharmacology & Pharmacy, The University of Hong Kong, Hong the work is not altered. Kong, China See accompanying article, p. 4018. 5Department of Medicine, University of Dresden, Dresden, Germany Corresponding author: Aimin Xu, [email protected]; Karen S.L. Lam, [email protected]; or Zhiguang Zhou, [email protected]. 4240 Neutrophil Serine Proteases, NETosis, and T1D Diabetes Volume 63, December 2014 macrophages, B cells, and neutrophils, are also implicated October 2000 to October 2013. Patients with T1D were in the development of insulitis in T1D (8,9). diagnosed according to the criteria of the American Neutrophils, which are the most abundant (40–75%) Diabetes Association (21). All patients were treated with type of white blood cells, have recently been implicated in insulin. The median disease duration of T1D was 4.2 the onset and progression of T1D (10,11). The primary (interquartile range 1.7–7.1) years. functions of neutrophils are to eliminate extracellular patho- A total of 77 age- and sex-matched healthy control gens by multiple strategies, including phagocytosis, degran- subjects were recruited from children in the community ulation to release lytic , and neutrophil extracellular participating in a health screening at the Children Health traps (NETs) that are formed through a unique cell death Center of the Second Xiangya Hospital, Central South process clearly differentiated from apoptosis and necrosis, University, using the following inclusion criteria: fasting termed “NETosis” (12–14). However, improper activation of plasma glucose of less than 5.6 mmol/L and 2-h plasma neutrophils may lead to tissue damage during autoimmune glucose of less than 7.8 mmol/L, and no family history of or exaggerated inflammatory responses (15). Notably, circu- diabetes or other autoimmune or chronic diseases. lating neutrophil counts are reduced in patients with T1D A total of 25 adults with type 2 diabetes diagnosed within and in their nondiabetic first-degree relatives but not in 1 year and 25 age- and sex-matched healthy control subjects patients with type 2 diabetes (11). In nonobese diabetic were recruited at the Diabetes Center, Second Xiangya (NOD) mice (a spontaneous model of T1D), neutrophil in- Hospital of Central South University, and the inclusion filtration and NET formation in the islets were observed as criteria were described in our previous study (22). early as 2 weeks after birth, and blockage of neutrophil The Second Xiangya Hospital of Central South Univer- activities with an anti-Ly6G antibody reduced the subse- sity Institutional Review Board approved the study, and quent development of insulitis and diabetes (9). written informed consents were obtained from the patients Neutrophil serine proteases, including neutrophil elas- and healthy control subjects. tase (NE), proteinase 3 (PR3), and G (CG), are the major components of neutrophil azurophilic granules Clinical and Biochemical Assessments that participate in the elimination of engulfed micro- After overnight fasting, a venous blood specimen was organisms (16). Neutrophil activation and degranulation collected in the morning (;0800) for analysis of various can result in the release of neutrophil serine proteases biochemical parameters. Plasma glucose was measured into the extracellular medium and circulation, where they enzymatically on a Hitachi 7170 analyzer (Boehringer not only help to eliminate the invaded pathogens but also Mannheim GmbH, Mannheim, Germany). HbA1c was mea- serve as the humoral regulators of the immune responses sured by automated liquid chromatography (VARIANT II during acute and chronic inflammation, modulating cellular Hemoglobin Testing System; Bio-Rad, Hercules, CA). Serum signaling network by processing chemokines, and activat- levels of C-peptide and C-reactive protein were quantified ing specific cell-surface receptors (17–19). Abnormal activ- using a chemiluminescence immunoassay on a Bayer 180SE ities of neutrophil serine proteases have been implicated in Automated Chemiluminescence System (Bayer AG, Leverkusen, the pathogenesis of several inflammatory and autoimmune Germany) and an immunoturbidimetric assay (Orion diseases, including chronic obstructive pulmonary disease, Diagnostica, Espoo, Finland), respectively. The titers of cystic fibrosis, Wegener granulomatosis, Papillon-Lefèvre GADA, IA2A, and ZnT8A were determined by in-house syndrome, and small-vessel vasculitis (20). However, their radioligand assays, as previously described (22,23). association with T1D has not been explored so far. Circulating protein levels of NE, PR3, and A1AT were In this study, we measured circulating levels of two main measured using ELISA kits established in our laboratory types of neutrophil serine proteases (NE and PR3) and their (Antibody and Immunoassay Services, The University of enzymatic activities in T1D patients with different disease Hong Kong). The limits of detection for the NE, PR3, and duration together with age- and sex-matched healthy control A1AT ELISA kits were 0.156 ng/mL. No cross-reactivity subjects. Furthermore, we explored whether altered NET among these proteins or with other proteins was detected. formation and a1-antitrypsin (A1AT), a major endogenous The intra- and interassay variations were, respectively, inhibitor of neutrophil serine proteases, are associated with 4.5% and 5.1% for the NE ELISA kit, 3.9% and 4.3% for the reduced neutrophil counts and markedly increased activities PR3 ELISA kit, and 4.9% and 5.3% for the A1AT ELISA kit. of neutrophil serine proteases in patients with T1D. We also The combined enzymatic activities of PR3 and NE in measured the dynamic changes of circulating NE/PR3 activ- serum were determined with a chromogen-based assay ities during the development of autoimmune diabetes in using N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide NOD mice. (Sigma-Aldrich, St. Louis, MO) as the substrate, which has 21 21 a catalytic constant Kcat/Km of 33,915 M s for NE 2 2 RESEARCH DESIGN AND METHODS and 499 M 1 s 1 for PR3 (24). Briefly, serum (20 mL) was Study Cohort incubated with 0.1 mol/L Tris-HCl buffer (180 mL; pH A total of 149 patients with T1D were randomly selected 8.0) containing 0.5 mol/L NaCl and 1 mmol/L substrate from children diagnosed at the Diabetes Center, Second at 37°C for 24 h. The amount of p-nitroaniline released Xiangya Hospital of Central South University, from was measured spectrophotometrically at 405 nm. The diabetes.diabetesjournals.org Wang and Associates 4241 enzymatic activities of PR3 and NE were calculated accord- RESULTS ing to the D optical density (OD) values before and after Subject Characteristics 24-h incubation with substrate and expressed as mU/mL The clinical characteristics of T1D patients and their fi serum, where 1 unit was de ned as the amount of PR3 and matched healthy control subjects are described in Table 1. NE that hydrolyze the substrate to yield p-nitroaniline at T1D patients were further divided into three groups by m 1 mol/min at 37°C (25). disease duration, including patients within 1 year from The levels of neutrophil NETosis were measured by diagnosis (n = 28), with a disease duration .1 and ,5 quantifying the amount of circulating years (n = 59), and with duration .5 years (n = 62). (MPO)-DNA complexes, a well-established marker of NET Compared with healthy subjects, T1D patients had higher formation, as previously described (26). Briefly, 5 mg/mL fasting glucose and HbA1c but lower fasting C-peptide mouse anti-MPO monoclonal antibody (ABD Serotec, levels. No significant differences in C-reactive protein Puchheim, Germany) was coated to 96-well microtiter were found among these groups (Table 1). Consistent plates overnight at 4°C. After blocking with 1% BSA, se- with previous reports (10,11), circulating neutrophils rum samples were added per well with the peroxidase- were moderately reduced in T1D patients diagnosed labeled anti-DNA monoclonal antibody (component No. within 1 year compared with the healthy control subjects 2 of the Cell Death Detection ELISA PLUS kit; Roche (median 2.27 [IQR 1.80–3.52] vs. 3.63 [3.02–4.15] 3 Diagnostics, Indianapolis, IN) according to the manufac- 106/mL; P , 0.05), but not in T1D patients with a disease ’ turer s instructions. After incubation for 2 h at room duration of .1 and ,5 years or with duration .5 years temperature on a shaking device (300 rpm), the wells (Table 1). were washed three times and then incubated with the peroxidase substrate at 37°C for 60 min. The OD at wave- Circulating Protein Levels and Enzymatic Activities of NE and PR3 Are Dramatically Increased in T1D length of 405 nm was measured using a mQuant micro- Patients plate reader (BioTek Instruments, Winooski, VT). In contrast to the mild reduction of peripheral neutro- phils, we found that the circulating protein levels of NE Animal Studies and PR3 were dramatically increased in T1D patients NOD/ShiLtJ breeder mice were purchased from The Jackson compared with the healthy control subjects (median NE: Laboratory (Bar Harbor, ME). BALB/c and C57BL/6N mice 1594.7 [IQR 988.4–2284.6] vs. 397.0 [262.2–468.8] were obtained from the University of Hong Kong Animal ng/mL, P , 0.001; PR3: 295.3 [206.0–430.4] vs. 107.4 Unit. All mice were housed in a room under specific [92.5–165.0] ng/mL, P , 0.001). Notably, the magnitude pathogen–free conditions and 12-h light/dark cycles at 22° of increases in protein levels of NE and PR3 was signifi- to 24°C and with ad libitum access to water and standard cantly higher in T1D patients diagnosed within 1 year chow (PicoLab Rodent Diet 20; LabDiet, St. Louis, MO). compared with the other two groups with longer disease Blood was collected weekly from female mice from 2 to duration (all P , 0.01, Fig. 1A and B). Circulating protein 30 weeks of age. Blood glucose was monitored using an levels of NE and PR3 did not differ between boys and girls Accu-Chek Advantage glucose meter (Roche Diagnostics, in patients or control subjects. Indianapolis, IN), and diabetes was defined as two con- To further confirm these findings, we measured the secutive readings above 11.1 mmol/L (9). Circulating NE/ combined enzymatic activities of NE and PR3 using PR3 enzymatic activities were measured as described N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide, a com- above. All experimental procedures were approved by mon substrate for NE and PR3 (24). The results showed the University of Hong Kong Committee on the Use of that circulating NE/PR3 enzymatic activities in T1D Live Animals for Teaching and Research and were per- patients were also substantially higher than those in formed in accordance with the Guide for the Care and healthy individuals (median 0.69 [IQR 0.41–1.03] vs. Use of Laboratory Animals. 0.14 [0.10–0.21] mU/mL, P , 0.001). Likewise, the most significant increase in NE/PR3 enzymatic activities was Statistical Analysis observed in T1D patients within 1 year from diagnosis All analyses were performed with SPSS 16.0 software (SPSS, (Fig. 1C). The correlation coefficient between NE/PR3 en- Chicago, IL). Normality was tested using the Kolmogorov- zymatic activities and circulating protein levels was 0.915 Smirnov test. Data that were not normally distributed were for NE (P , 0.001) and 0.874 for PR3 (P , 0.001). logarithmically transformed before analysis. Differences between groups were assessed by x2 or unpaired Student t Circulating A1AT Levels Are Decreased in T1D Patients test. Comparisons among groups were performed using one- The activities of plasma NE and PR3 are tightly controlled way ANOVA and independent Student t tests. Correlations by their associated endogenous inhibitors, especially A1AT, were analyzed using Pearson correlation or partial correla- an archetype member of the serine inhibitor tion, as appropriate. Data are expressed as mean 6 SD or () superfamily. Because our data showed that the median with the interquartile range (IQR), as appropriate. In amplitude of increases in NE and PR3 enzymatic activities all statistical comparisons, a P value ,0.05 was used to was higher than that of the circulating protein levels, we indicate a statistically significant difference. next investigated whether dysregulated A1AT contributed 4242 Neutrophil Serine Proteases, NETosis, and T1D Diabetes Volume 63, December 2014 to the increased enzymatic activities of NE and PR3 in circulating NE and PR3 protein levels in T1D patients T1D. In contrast to elevated NE and PR3 levels, the are at least partly attributed to enhanced neutrophil circulating concentrations of A1AT in T1D patients di- NETosis. agnosed within 1 year were significantly decreased com- – Circulating NE and PR3 Are Associated With the pared with healthy subjects (median 1.37 [IQR 1.07 1.83] Numbers and Titers of Autoantibodies in T1D Patients – P , vs. 1.80 [1.57 2.07] mg/mL, 0.01), whereas the de- We next explored the relationship between circulating – cline in patients with disease duration for 1 5yearsor neutrophil serine proteases and GADA, IA2A, and ZnT8A, . with disease duration 5 years did not reach statistical the three autoantibodies associated with b-cell autoimmu- fi D signi cance (Fig. 1 ). nity in T1D patients. Among 149 T1D patients, 54 (36%) were negative for autoantibodies, and 61 (41%), 24 (16%), Neutrophil NETosis Is Increased in T1D Patients and 10 patients (7%) were positive for one, two, and three To explore the underlying mechanism responsible for the autoantibodies, respectively. Notably, circulating levels of NE markedly elevated circulating NE and PR3 levels, we and PR3 proteins and their enzymatic activities were in- examined the levels of neutrophil NETosis by quantifying creased progressively with increased numbers of the auto- the amount of circulating MPO-DNA complexes, a well- antibodies detected in these patients (Fig. 3A–C). Even for established marker of NET formation (26). In line with the autoantibody-negative T1D patients, the circulating pro- the increased NE and PR3 levels, a significant elevation of tein levels and enzymatic activities of NE and PR3 were circulating MPO-DNA complexes was observed in T1D much higher than those in healthy control subjects (median patients, especially in T1D patients with the disease du- protein levels for NE: 1154.90 [IQR 770.8–1749.5] vs. 397.0 ration of ,1 year compared with the healthy individuals [262.2–468.8] ng/mL, P , 0.0001; PR3: 237.4 [154.3– (median 0.197 [IQR, 0.049–0.412] vs. 0.026 [0.011– 307.1] vs. 107.4 [92.5–165.0] ng/mL, P , 0.0001; enzy- 0.058] mean OD 405, P , 0.001; Fig. 2A). Furthermore, matic activities: 0.53 [0.37–0.79] vs. 0.14 (0.10–0.21) the amount of MPO-DNA complexes in serum was signif- mU/mL, P , 0.001; Fig. 3A–C). Furthermore, a strong cor- icantly correlated with the circulating protein levels of NE relation between the titers of GADA and the circulating (r = 0.554, P , 0.001) and PR3 (r = 0.575, P , 0.001) as protein levels of NE (r =0.296,P = 0.011) and PR3 (r = well as with NE and PR3 enzymatic activities (r = 0.527, 0.270, P = 0.021) as well as NE and PR3 enzymatic activities P , 0.001; Fig. 2B–D), suggesting that the increased (r = 0.275, P = 0.019) were detected in the GADA-positive

Table 1—Characteristics of healthy control subjects and T1D patients recruited for this study T1D patients from diagnosis Healthy control subjects ,1 year 1–5 years .5 years n =77 n =28 n =59 n =62 Age (years) 13.3 6 5.3 15.4 6 6.9 12.9 6 4.3 14.9 6 3.8 Sex (n) Male 43 12 21 24 Female 34 16 38 38 BMI (kg/m2) 18.35 6 2.70 17.56 6 3.16 17.83 6 3.77 18.46 6 3.05 Duration of diabetes (years) Not applicable 0.4 (0.2–0.7) 2.8 (1.9–3.9) 7.6 (6.4–9.3) Fasting glucose (mmol/L)§ 4.69 (4.41–4.89) 7.85 (6.20–11.93)a 8.4 (6.60–14.20)a 7.80 (5.68–11.83)a a a a HbA1c (%)§ 5.00 (4.80–5.15) 8.05 (6.03–11.15) 7.50 (6.70–10.10) 7.40 (6.48–8.43) a a a HbA1c (mmol/mol)§ 31 (29–33) 64 (42–99) 58 (50–87) 57 (47–69) Fasting C-peptide (pmol/L)§ 445.4 (362.1–678.2) 55.35 (16.92–146.73)a 22.80 (5.50–92.95)a,b 5.50 (4.20–28.43)a,b,c C-reactive protein (mg/L)§ 0.23 (0.13–0.61) 0.24 (0.11–0.51) 0.27 (0.13–0.75) 0.28 (0.15–1.12) Blood cell counts Erythrocytes (3106/mL)§ 4.66 (4.30–4.94) 4.52 (4.06–5.12) 4.83 (4.54–5.17) 4.82 (4.50–5.03) White blood cells (3106/mL)§ 6.85 (5.80–7.78) 4.70 (3.75–7.35)a 5.70 (4.95–6.90) 6.40 (5.20–6.97) Lymphocytes (3106/mL)§ 2.18 (1.55–2.62) 1.74 (1.57–2.08) 1.98 (1.54–2.43) 1.84 (1.64–2.38) Monocytes (3106/mL)§ 0.42 (0.34–0.49) 0.29 (0.17–0.37)a 0.29 (0.22–0.38)a 0.31 (0.23–0.39)a Neutrophils (3106/mL)§ 3.63 (3.02–4.15) 2.27 (1.80–3.52)a 3.29 (2.75–4.15) 3.49 (3.02–4.14) (3106/mL)§ 0.20 (0.12–0.34) 0.13 (0.08–0.20)a 0.15 (0.12–0.23) 0.15 (0.11–0.21) (3106/mL)§ 0.05 (0.03–0.06) 0.03 (0.02–0.07) 0.07 (0.05–0.10)a,b 0.08 (0.06–0.14)a,b Platelets (3106/mL)§ 256 (223–315) 244 (202–285)a 219 (193–265)a 250 (199–280) Data are expressed as mean 6 SD or median (IQR), unless otherwise indicated. §Log-transformed before analysis. aP , 0.05 compared with healthy control subjects. bP , 0.05 compared with T1D patients from diagnosis ,1 years. cP , 0.05 compared with T1D patients from diagnosis 1–5 years. diabetes.diabetesjournals.org Wang and Associates 4243

Figure 1—Circulating protein levels of NE (A) and PR3 (B), NE/PR3 enzymatic activities (C), and A1AT protein levels (D) in healthy control subjects (n = 77) and in T1D patients within 1 year from diagnosis (n = 28), in those with a disease duration >1 and <5 years from diagnosis (n = 59), and in those with a duration >5 years (n = 62) from diagnosis are shown as box-and-whisker plots. The horizontal line in the middle of each box indicates the median value; the top and bottom borders of the boxes represent the 75th and 25th percentiles, respectively; the whiskers represent the 10th and 90th percentiles, respectively; and the dots represent the outliers. **P < 0.01, ***P < 0.001 vs. healthy control subjects; #P < 0.05, ##P < 0.01 vs. T1D patients within 1 year from diagnosis.

T1D patients (n = 73; Fig. 3D–F). Likewise, the titers of IA2A Elevated NE/PR3 Enzymatic Activity Is Closely in IA2A-positive T1D patients (n = 44) were also posi- Associated With the Development of Diabetes in NOD tively associated with the protein levels of NE, PR3, Mice and their enzymatic activities (Supplementary Fig. To further explore the relationship between neutrophil 1A–C). After adjustment for disease duration, the cir- serine proteases and the development of T1D, we de- culating protein levels and enzymatic activities of NE termined the dynamic changes of PR3 and NE in NOD mice and PR3 were still significantly correlated with the (n = 30), a well-established animal model for autoimmune numbers and titers of these autoantibodies (all P , diabetes, from 2 to 30 weeks of age. These mice were ret- 0.05, Supplementary Table 1). However, no significant rospectively assigned to two groups: those that eventually correlation between fasting blood glucose and circulat- developed diabetes (n =22,called“diabetic”) and those that ing protein levels of NE (r = 20.103, P = 0.211) or PR3 did not (n = 8, called “nondiabetic”). The circulating NE/ (r = 20.097, P = 0.237) or NE/PR3 enzymatic activities PR3 enzymatic activities in diabetic mice were markedly (r = 20.078, P = 0.342) was observed in the current study elevated by fourfold as early as 4 weeks after birth com- cohort. We further measured and compared the circulat- pared with those in 2-week-old mice, and the elevation was ing protein levels and enzymatic activities of NE and PR3 sustained for more than 10 weeks before the onset of di- in 25 type 2 diabetes patients within 1 year from diagno- abetes. Afterward, the NE/PR3 activities in diabetic mice sis and in 25 age- and sex-matched healthy control sub- were gradually decreased to the baseline levels, presumably jects (Supplementary Table 2). There was no significant due to the termination of autoimmune responses as a result difference in protein levels or enzymatic activities of NE of complete b-cell destruction (Fig. 4A). Although there was and PR3 or NETosis between the two groups (Supplemen- a transient and modest increase of circulating NE/PR3 ac- tary Table 2). Taken together, these data suggested that tivities in nondiabetic mice between 4 and 5 weeks after elevated NE and PR3 might be closely associated with birth (Fig. 4B), the magnitude and duration of NE/PR3 b-cell autoimmunity but not glycemic status in T1D elevation was substantially lower than in age-matched di- patients. abetic mice (Fig. 4C). In BALB/c and C57BL/6N mice, which 4244 Neutrophil Serine Proteases, NETosis, and T1D Diabetes Volume 63, December 2014

Figure 2—A: Circulating levels of MPO-DNA complexes in healthy control subjects (n = 77) and in T1D patients within 1 year from diagnosis (n = 28), in those with a disease duration >1 and <5 years (n = 59), and in those with a duration >5 years (n = 62) are shown in box-and- whisker plots. The horizontal line in the middle of each box indicates the median value; the top and bottom borders of the boxes represent the 75th and 25th percentiles, respectively; the whiskers represent the 10th and 90th percentiles, respectively; and the dots represent the outliers. Circulating MPO-DNA complexes were significantly correlated with circulating NE (B) and PR3 (C) protein levels and the enzymatic activities of NE and PR3 (D). **P < 0.01, ***P < 0.001 vs. healthy controls; ##P < 0.01 vs. T1D patients within 1 year from diagnosis.

do not develop insulitis and autoimmune diabetes, circulat- with our findings, a previous study in Italy also found ing NE/PR3 activities remained little changed throughout that neutrophil reduction is greatest in individuals with the 30-week observation period (Supplementary Fig. 2). the highest risk of developing T1D (11). After the disease onset, mild neutropenia persists for a few years and then DISCUSSION resolves at 5 years after diagnosis (as determined by a lon- In this study, we demonstrated that a modest reduction gitudinal analysis). In NOD mice with spontaneous devel- of neutrophil counts in patients with T1D at onset is opment of autoimmune diabetes, neutrophil infiltration accompanied by a marked elevation of protein levels and and NET formation in the islets are detected as early as 2 enzymatic activities of NE and PR3, the two major weeks after birth, well before the onset of overt diabetes neutrophil serine proteases. Furthermore, these changes (9). Furthermore, neutrophil depletion at the early stage in T1D patients are closely associated with increased reduces subsequent development of diabetes in NOD mice neutrophil NETosis, as determined by quantification of (9). Taken together, these data support an early role of MPO-DNA complexes in the circulation. These findings neutrophil NETosis, NET formation, and augmented release suggest that the reduction of neutrophil counts in T1D of neutrophil serine proteases in the onset of b-cell auto- patients is partly attributed to augmented NETosis, which immunity in T1D. Indeed, increased neutrophil NETosis in turn leads to increased NET formation and the release and NET formation have been implicated in a number of of NE and PR3 into the blood stream. autoimmune diseases, including small vessel vasculitis, sys- We showed that the amplitude of elevation in circulating temic lupus erythematosus (SLE), and multiple sclerosis NE/PR3 enzymatic activities and NET formation in patients (26–28). with disease duration of ,1 year is substantially higher In SLE, NETs have been demonstrated to stimulate than those with disease duration of .1year.Asignificant plasmacytoid dendritic cells for releasing interferon-a,which reduction in neutrophil counts is observed only in T1D in turn augments the autoreactivity of antigen-presenting patients with a disease duration of ,1 year. Consistent and antibody-producing cells (29,30). NETosis leads to the diabetes.diabetesjournals.org Wang and Associates 4245

Figure 3—Circulating protein levels of NE (A) and PR3 (B) and the enzymatic activities of NE and PR3 (C) in healthy control subjects (n = 77) and in T1D patients negative for autoantibody (n = 54), positive for one autoantibody of GADA, IA2A or ZnT8A (n = 61), positive for two autoantibodies of GADA, IA2A or ZnT8A (n = 24), or positive for three autoantibodies of GADA, IA2A and ZnT8A (n = 10) are shown in box- and-whisker plots. The horizontal line in the middle of each box indicates the median value; the top and bottom borders of the boxes represent the 75th and 25th percentiles, respectively; the whiskers represent the 10th and 90th percentiles, respectively; and the dots represent the outliers. Circulating protein levels of NE (D) and PR3 (E), and enzymatic activities of NE and PR3 (F) were significantly correlated with the titers of GADA in T1D patients with GADA-positive autoantibodies (n = 73). ***P < 0.001 vs. healthy control subjects; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. T1D patients negative for autoantibodies; $P < 0.05 vs. T1D patients positive for one autoantibody.

release of intracellular proteins, including histones and high- the generation of autoantibodies (32). In particular, a grow- mobility group protein B1, the latter of which is implicated ing body of evidence supports a pathogenic role for citrulli- in the initiation and/or perpetuation of autoimmunity in nated autoantigens in triggering autoimmune responses in several types of autoimmune disorders, including T1D SLE, rheumatoid arthritis, and multiple sclerosis (33). How- (30,31). Furthermore, NETs is associated with altered pat- ever, the pathophysiological roles of NETosis and its associ- terns of epigenetic and posttranslational modifications, such ated changes in T1D remain elusive. as methylation, acetylation, and citrullination, which may The current etiopathological diagnosis of autoimmune represent an important source of autoantigens promoting T1D heavily relies on the detection of the autoantibodies 4246 Neutrophil Serine Proteases, NETosis, and T1D Diabetes Volume 63, December 2014

autoantibody measurement in T1D patients is as low as 59–67% (35). To capture the therapeutic window for this disease, identifying new biomarkers for detection of early immunological events that affect human islets is critically important. Our current study demonstrated an approxi- mately fourfold increase of circulating protein levels and more than a fivefold elevation of enzymatic activities of NE and PR3 in T1D patients. Furthermore, elevated NE and PR3 were significantly associated with the positive numbers and titers of the autoantibodies detected in T1D patients. Even in those autoantibody-negative patients, the circulating enzymatic activities of NE and PR3 are still substantially higher than in healthy controls. Using the animal model of T1D, we found that elevated circulating NE/PR3 activities occur well before the onset of hypergly- cemia and diabetes and that their activities gradually decline after the development of overt diabetes. Taken together, our data suggest that circulating NE and PR3 may serve as sensitive biomarkers for early detection of those individuals with high risk of developing T1D. However, we found no significant association between increased levels of NE and PR3 and the severity of hyperglycemia in T1D patients. In fact, although hyper- glycemia becomes more severe with the progression of T1D, circulating levels of NE and PR3 exhibit opposite changes, suggesting that increased neutrophil NETosis and augmented release of NE and PR3 are not the consequence of impaired glycemic controls but are related to b-cell autoimmunity. Indeed, our observation that NETosis and NE/PR3 levels in T1D patients with longer disease duration are much lower than new-onset patients (,1 year) may be attributed to the gradual attenuation of b-cell autoimmunity with the progression of diabetes to an advanced stage. This is also in line with the fact that the number of autoantibodies in new-onset T1D patients was much higher than those with longer disease duration (36). In addition to its classical roles for host defense against infection, neutrophil serine proteases are an impor- tant regulator of inflammation and innate immunity (17,19,37,38). NE and PR3 are both involved in matura- tion and release of proinflammatory cytokines, such as tumor necrosis factor-a, interleukin-1b, and interleukin- 18, and also induces expression and activation of Toll-like Figure 4—Dynamic changes are shown in enzymatic activities of receptors (39–42), all of which are important mediators of circulating NE/PR3 in NOD female mice. Blood samples were col- insulitis and b-cell destruction (43,44). Furthermore, NE lected weekly from 30 NOD female mice from 2 to 30 weeks after birth. Circulating NE/PR3 enzymatic activities and blood glucose and PR3 play an indispensable role in recruiting neutro- levels were measured in mice that developed diabetes (n = 22) (A) phils to the site of inflammation. Notably, neutrophil and in those that remained nondiabetic (n =8)(B) until 30 weeks serine proteases have recently been implicated in high- C after birth. : Comparisons of NE/PR3 enzymatic activities between fat diet–induced obesity, inflammation, and macrophage diabetic mice and nondiabetic mice throughout the observation fi period. #P < 0.05 vs. the NE/PR3 enzymatic activities at 2 weeks in ltration in adipose tissues in mice (45). Injection of of age; *P < 0.05 vs. age-matched nondiabetic mice. recombinant PR3 alone is sufficient to induce hypergly- cemia in mice (46). By contrast, treatment with A1AT, a major endogenous inhibitor of NE and PR3, decreases against several b-cell antigens. However, these autoanti- lymphocyte infiltration in the islets and prevents b-cell bodies are rarely detectable in children before 6 months of loss and diabetes in rodent models of T1D (47,48). These age (34). Moreover, the diagnostic sensitivity of the single animal studies, in conjunction with our clinical and animal diabetes.diabetesjournals.org Wang and Associates 4247

findings, suggest that elevated NE and PR3 may be the 7. Miao D, Yu L, Eisenbarth GS. Role of autoantibodies in type 1 diabetes. direct contributors to the pathogenesis of autoimmune Front Biosci 2007;12:1889–1898 diabetes by early involvement of autoimmune inflamma- 8. Richardson SJ, Willcox A, Bone AJ, Morgan NG, Foulis AK. Immunopa- tory responses in pancreatic islets. thology of the human pancreas in type-I diabetes. Semin Immunopathol 2011;33: 9–21 A1AT, the most abundant circulating serpin secreted 9. Diana J, Simoni Y, Furio L, et al. Crosstalk between neutrophils, B-1a cells from hepatocytes, inhibits neutrophil serine proteases by fi and plasmacytoid dendritic cells initiates autoimmune diabetes. Nat Med 2013; covalent binding to the enzymes (49). De ciency of A1AT 19:65–73 fl has been implicated in a number of in ammatory disor- 10. Harsunen MH, Puff R, D’Orlando O, et al. Reduced blood leukocyte and ders, including chronic obstructive pulmonary disease (50). neutrophil numbers in the pathogenesis of type 1 diabetes. Horm Metab Res Our present study observed a modest but significant re- 2013;45:467–470 duction of circulating A1AT in patients with T1D, suggest- 11. Valle A, Giamporcaro GM, Scavini M, et al. Reduction of circulating neu- ing that augmented circulating NE and PR3 activities may trophils precedes and accompanies type 1 diabetes. Diabetes 2013;62:2072– result from a combination of increased release of these two 2077 enzymes from neutrophil NETosis and decreased produc- 12. Mestas J, Hughes CC. Of mice and not men: differences between mouse – tion of their endogenous inhibitor A1AT. and human immunology. J Immunol 2004;172:2731 2738 Our study has several limitations, including the rel- 13. Kolaczkowska E, Kubes P. Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol 2013;13:159–175 atively small sample size and the cross-sectional design. 14. Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil extracellular traps In addition, because our samples were collected from kill bacteria. Science 2004;303:1532–1535 fi Chinese only, whether the ndings are replicable in other 15. Mócsai A. Diverse novel functions of neutrophils in immunity, inflammation, ethnic group remains to be determined. Further large and beyond. J Exp Med 2013;210:1283–1299 scale, longitudinal studies on different ethnic groups are 16. Korkmaz B, Moreau T, Gauthier F. Neutrophil elastase, proteinase 3 and mandatory to clarify the roles of NE and PR3 in the : physicochemical properties, activity and physiopathological func- initiation and progression of b-cell autoimmunity and to tions. Biochimie 2008;90:227–242 evaluate their clinical value for prediction and early di- 17. Wiedow O, Meyer-Hoffert U. Neutrophil serine proteases: potential key reg- agnosis of T1D. ulators of cell signalling during inflammation. J Intern Med 2005;257:319–328 18. Pham CT. Neutrophil serine proteases fine-tune the inflammatory response. Int J Biochem Cell Biol 2008;40:1317–1333 19. Meyer-Hoffert U, Wiedow O. Neutrophil serine proteases: mediators of in- Funding. This work was supported by The University of Hong Kong (HKU) nate immune responses. Curr Opin Hematol 2011;18:19–24 matching funds for the State Key Laboratory of Pharmaceutical Biotechnology, 20. Korkmaz B, Horwitz MS, Jenne DE, Gauthier F. Neutrophil elastase, pro- the Hong Kong Research Grants Council Collaborative Research Fund (HKU4/ teinase 3, and cathepsin G as therapeutic targets in human diseases. Pharmacol CRF/10R and HKU2/CRF/12R) and Germany/Hong Kong Joint Research Scheme Rev 2010;62:726–759 (G-HK708/13) to A.X., the National Key Technology Research & Development 21. American Diabetes Association. Diagnosis and classification of diabetes Program (2012BAI02B04) to Z.Z., and the National Natural Science Foundation mellitus. Diabetes Care 2013;36(Suppl. 1):S67–S74 of China (81200600) to Y.X. 22. Xiao Y, Xu A, Law LS, et al. Distinct changes in serum fibroblast growth Duality of Interest. No potential conflicts of interest relevant to this article factor 21 levels in different subtypes of diabetes. J Clin Endocrinol Metab 2012; were reported. 97:E54–E58 Author Contributions. Y.W., Y.X., and L.Z. conducted the experiments, 23. Yang L, Luo S, Huang G, et al. The diagnostic value of zinc transporter 8 analyzed data, and wrote the manuscript. D.Y., J.Z., and Y.T. conducted the autoantibody (ZnT8A) for type 1 diabetes in Chinese. Diabetes Metab Res Rev experiments. S.R.B. and K.S.L.L. contributed to data analysis and edited the 2010;26:579–584 manuscript. Z.Z. and A.X. contributed to the experimental design, analyzed 24. Wiesner O, Litwiller RD, Hummel AM, et al. 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