C57BL/6 mice were purchased from Harlan Olac (Bicester, United Kingdom) and bred in the animal facility of the College of Medicine and Health Sciences, UAE University. IFNγ^−/− mice were purchased from Jackson laboratories (USA) and generously provided by Dr Mariam Al-Shamsi at our institution. Female mice aged 8–10 weeks (weight range 20–22 g) were used for the experiments. All studies involving animals were carried out in accordance with, and after approval of, the animal research ethics committee of the College of Medicine and Health Sciences, UAE University.

Paraoxon (Sigma, St. Louis, MO, USA), an organophosphorous compound, is a highly specific, irreversible, inhibitor of AChE (11). A stock solution (10 mmol/l) was prepared in acetone. Working solution for intraperitoneal (i.p.) injection was prepared ex tempore in pyrogen-free saline to a concentration of 80 nmol/ml. The final acetone concentration in the paraxon solution used for i.p. injection was ~108 μM. Each mouse received 40 nmol/day of paraoxon or saline in 0.5 ml volume. STZ (Sigma) was prepared ex tempore in citrate buffer (pH 4.5) and used i.p. at 60 mg/kg/day per mouse, unless otherwise indicated.

To induce autoimmune diabetes, the MLD-STZ model was used (3). C57BL/6 and IFNγ^−/− mice were administered five consecutive daily doses of STZ; control mice received citrate buffer. At different time points post-STZ administration, blood was drawn from the tail vein to determine glucose levels using One-Touch-ultra-strip (Lifescan, Zurich, Switzerland). Hyperglycemia was defined as non-fasting blood glucose >200 mg/dl.

Twenty age-matched mice were randomly assigned into two groups (10 animals per group). Group I served as control and received daily i.p. injection of sterile saline for 3 weeks. Group II mice received daily injection of paraoxon for 3 weeks. Mice were weighed weekly, at which time blood was also collected and analyzed for AChE activity. At the end of treatment, each group was divided randomly into two subgroups, A and B. Groups IA (Saline) and IIA (paraoxon) received daily injections of citrate buffer while groups IB (Saline/STZ) and IIB (paraoxon/STZ) received daily injection of STZ for five consecutive days. Pancreas, spleen, and serum were collected from mice sacrificed (ether exposure) at days 10 and 18, post-STZ administration. In some experiments, mice were followed for survival for up to 60 days.

The detailed procedure for determining AChE enzyme activity in red blood cells (RBC) has been described (36). Briefly, freshly drawn, diluted, venous blood samples were incubated with DTNB (10 mM) and ethopropazine (6 mM) for 20 min at 37°C prior to addition of acetylthiocholine. The change in the absorbance of DTNB was measured at 436 nm. The AChE activity was calculated using an absorption coefficient of TNB⁻ at 436 nm (ε = 10.6 mM⁻¹ cm¹). The values were normalized to the hemoglobin (Hb) content (determined as cyanmethemoglobin) and expressed as mU/μM/Hb (37). All enzyme activities were expressed as percentage of the baseline activity (100%).

Excised pancreata were processed for histological analysis following established protocol (38, 39). Tissue sections were stained with hematoxylin and eosin (H&E), and images were captured using Olympus BX53 microscope equipped with digital camera DP26 (Tokyo, Japan). Indirect immunostaining for insulin was performed using guinea pig polyclonal antibody (Dako, Carpinteria, CA, USA) followed by FITC-conjugated donkey anti-guinea pig IgG (Jackson ImmunoResearch, West Grove, PA, USA). A three-step staining protocol was utilized to detect infiltrating T cells and macrophages. For T lymphocytes, CD3-specific rabbit polyclonal Ab (Dako) was used followed by biotinylated sheep anti-rabbit Ig (AbD Serotec, Hercules, CA, USA) and finally streptavidin-FITC (eBioscience, San Diego, CA, USA). For macrophages, we used rat F4/80 mAb (BMA Biomedicals, Switzerland) followed by streptavidin-HRP and DAB (Dako). Slides with fluorescence were counter-stained with propidium iodide (BD Biosciences, USA) and then examined and photographed under a Nikon C1 laser scanning confocal microscope. Slides stained with DAB were counter-stained with hematoxylin and visualized and photographed with an Olympus BX53. Histological quantification of CD3⁺ and F4/80⁺ cells was done on two to four non-consecutive sections per animal and using four to five mice per experimental group. All islets found in each section were included in the quantification, which was carried out in a blinded fashion.

Analysis of spleen cells was carried out using a multi-color FACS analysis, following a standard procedure (39, 40). Cells were stained with a combination of directly conjugated mAbs, washed, and analyzed using FACSCanto II (BD Biosciences, San Jose, CA, USA). The antibodies used were CD3-FITC, CD4-APC, and CD8-APC-Cy7 (Biolegend, San Diego, CA, USA), CD19-PE-Cy7 and CD11c-PE (eBioscience), and CD11b-PE-Cy7 (BD Biosciences). Non-viable cells staining positive with 7AAD dye (eBioscience) were excluded from the analysis. Data collected on 50,000 cells were analyzed using FACSDiva software (BD Biosciences).

Spleen single cell suspensions were prepared and 2 × 10⁶ cells/ml were seeded in 24 well plates and stimulated with PMA (100 ng/ml)/Ionomycin (1 μg/ml) for 4 h at 37°C in the presence of brefeldin A (GolgiPlug; BDcytofix/cytoperm plus^TM solution kit, BD Biosciences). After stimulation, cells were first stained with CD4-APC and CD8-APC-Cy7 antibodies (Biolegend), resuspended in fixation/permeabilization solution and finally stained with anti-IL-17A-PE and anti-IFNγ-PE-Cy7 antibodies (eBioscience) and run using FACSCanto II (BD Biosciences). Data collected from 50,000 cells were analyzed using FACSDiva software (BD Biosciences).

qRT-PCR was carried out as previously described (40) on RNA extracted from spleen and pancreas from each animal. After RNA extraction and purification, cDNA was synthesized using Taqman reverse transcription reagents (Applied Biosystems, Foster City, CA, USA) following manufacturer’s protocol. TaqMan primers and probes were used to study the expression of the inflammatory markers IL-1β, IL-6, IL-12p40, IL-17A, IFNγ, and insulin (Applied Biosystems), and IL-23 and TNFα (Metabion, Germany) (Table 1). Transcript levels of target genes were normalized according to the dCq method to respective mRNA levels of the housekeeping gene HPRT.

Serum samples were assayed for insulin (Alpco diagnostics, Salem, NH, USA) level by sandwich ELISA and performed according to the manufacturer’s instructions. Assay sensitivity was 188 pg/ml.

Statistical significance between control and treated groups was analyzed using the unpaired, two-tailed Student’s t-test, using the statistical program of GraphPad Prism version 6 software. For multiple comparisons, we used One-way ANOVA with post hoc Tukey’s test (GraphPad Prism). Two-way ANOVA with Bonferroni post-test was used to analyze repeated measures data, as indicated. Survival analysis was performed by Kaplan–Meier survival curves and log-rank test, using the same GraphPad Prism program. Differences between experimental groups were considered significant when p values were <0.05.

Studies were first carried out to optimize the STZ dose necessary to induce hyperglycemia (blood glucose >200 mg/dl) in C57BL/6 mice. Animals were divided into three groups, each receiving either 40, 50, or 60 mg/kg dose of STZ for five consecutive days. As can be seen, the percentage of mice developing hyperglycemia was 0, 50, and 100% by day 21 post administration of STZ at 40, 50, or 60 mg/kg, respectively (Image 1 in Supplementary Material). Hence, for all subsequent studies, a dose of 60 mg/kg STZ was used.

In order to investigate the potential effect of AChE inhibition on experimental diabetes, C57BL/6 mice were pretreated daily with paraoxon or saline for 3 weeks, and then given MLD-STZ (or saline), as described in the Section “Materials and Methods” and followed for the development of hyperglycemia. No changes in glucose levels were observed in control mice (saline or paraoxon only experimental groups) (Figure 1A). Saline-pretreated mice that received STZ developed progressive hyperglycemia, first evident at 7 days post-STZ administration, and that continued for up to 51 days (Figures 1A,B). All animals in this group were diabetic by day 18 post STZ administration (Figure 1B). In contrast, despite an initial mild elevation in blood glucose levels observed at day 7, mice pretreated with paraoxon prior to STZ were resistant to the development of hyperglycemia (Figure 1B). This remained evident for up to 51 days following STZ administration, the maximum period of observation (Figure 1B).

Serum insulin levels were determined on day 10 (Figure 1C) and 18 (Figure 1D) post STZ injection. Similar levels of serum insulin were observed in saline- and paraoxon-treated control mice at both time points (Figures 1C,D). Interestingly, for STZ-treated mice, while serum insulin was at normal level at day 10 post administration (Figure 1C), the level dropped significantly by day 18 to ~64% of control (Figure 1D). In contrast, no reduction in serum insulin was observed in mice pretreated with paraoxon followed by STZ (Figure 1D). The levels of serum insulin of paraoxon/STZ-treated mice tended to be higher than saline control at days 10 and 18 post STZ injection (2.6-fold and 1.6-fold increase, respectively; Figures 1C,D) with the difference being statistically significant at the latter date. Moreover, differences in serum insulin levels between saline/STZ and paraoxon/STZ groups at day 18 were highly significant and inversely correlated with serum glucose levels (compare Figures 1A,D).

Insulin mRNA expression was also analyzed in pancreatic tissue at day 18 post-STZ treatment (Figure 1E). A dramatic 10.6-fold increase in insulin expression was observed in paraoxon-treated mice compared with saline control (Figure 1E). In sharp contrast, STZ treatment (saline/STZ group) led to a 12-fold reduction in the level of insulin mRNA relative to saline control. In mice pretreated with paraoxon prior to STZ (paraoxon/STZ group), insulin mRNA levels were essentially similar to those observed in saline control group (Figure 1E). Immunohistochemical analysis of insulin-producing β cells in pancreatic tissue revealed a gradual loss of insulin positivity in STZ-treated mice (saline/STZ group), which was evident starting at day 10 following STZ administration and continued up to day 60 (Figure 1F). In contrast, mice pretreated with paraoxon (paraoxon/STZ group) showed evidence of islet preservation and protection from STZ-induced loss of β cells, albeit not total, up to day 60 (Figure 1F). These results demonstrate that inhibition of AChE increases the expression of insulin mRNA in pancreatic cells and prevents STZ-induced hyperglycemia.

Hematoxylin and eosin staining of pancreatic tissue showed highly infiltrated islets in saline/STZ group at days 10 and 18 post-STZ administration (Figure 2A). Pretreatment with paraoxon reduced islet cell infiltration at day 10 (paraoxon/STZ group) and, by day 18, the pancreatic islets appeared completely healthy with no evidence of insulitis (Figure 2A). No infiltrating cells were observed in islets of saline or paraoxon control groups (Figures 2A–E). Immunohistological analysis of the islets at days 10 and 18 revealed the presence of CD3⁺ T cells (Figure 2B) and F4/80⁺ myeloid cells (Figure 2C) in STZ-treated mice. However, the degree of T cell infiltration was significantly reduced in mice pretreated with paraoxon (Figures 2B,D). At day 10, T cells were distributed uniformly throughout the islets of both saline/STZ and paraoxon/STZ groups. At day 18, however, only a few T cells located primarily in the periphery of the islets could be observed in paraoxon/STZ mice, while in saline/STZ group these cells were uniformly distributed within the islets (Figure 2B). Administration of STZ also induced a significant increase in intra-islet recruitment of macrophages, regardless of the pretreatment received (Figures 2C,E). No significant differences were found between saline and paraoxon control groups (Figure 2E).

Next, we investigated mRNA expression of proinflammatory cytokines in pancreatic tissue at day 18 post-STZ (Figures 2F,G). STZ administration led to a significant increase (3.1-fold) in IFNγ mRNA expression compared with saline controls (Figure 2F). Interestingly, mice pretreated with paraoxon alone also exhibited a 2.4-fold increase in pancreatic IFNγ expression compared with controls. In contrast, mice pretreated with paraoxon followed by STZ showed similar levels of IFNγ expression to those receiving paraoxon alone (detectable in only 2/6 mice examined; Figure 2F). Expression of pancreatic IL-1β was also assessed in the different experimental groups. The highest level of IL-1β expression was observed in saline/STZ group (Figure 2G). In contrast, IL-1β expression levels dropped significantly in paraoxon (2.5-fold) and paraoxon/STZ (2.7-fold) experimental groups compared with saline/STZ group (Figure 2G). For pancreatic IL-23, expression was almost undetectable in saline and paraoxon groups, was maximal in saline/STZ group and decreased by >50% in paraoxon/STZ-treated mice compared with saline/STZ group; however, this difference was not statistically significant (Figure 2H). Analysis of pancreatic TNFα expression revealed only background levels with no significant differences among the experimental groups (data not shown). Moreover, no cytokines could be detected in pancreatic tissue at day 10 post-STZ treatment in any of the groups (data not shown).

Next, we explored the effect of AChE inhibition on the peripheral immune response in STZ-treated mice. Immunophenotyping revealed no gross differences in the percentage of major spleen cell populations among the four different experimental groups at either day 10 or 18 post STZ administration (Images 2 and 3 in Supplementary Material), the only exception being a small but significant decrease in the frequency of CD8⁺ T cells in the paraoxon/STZ group compared with saline/STZ-treated mice at day 10 post-STZ administration (Images 2 and 3 in Supplementary Material). Given the role of IL-17 and Th17 cells in autoimmune diabetes and their counter regulation by IFNγ (27, 41), mRNA expression levels of IFNγ and IL-17 were determined in spleen cells. Moderate upregulation of IFNγ expression was observed in mice pretreated with AChE inhibitor (paraoxon and paraoxon/STZ groups) compared with saline control (Figure 3A), with the differences being statistically significant between saline/STZ and paraoxon/STZ groups. Conversely, while administration of STZ led to a 4.7-fold increase in IL-17 expression compared with saline control animals (Figure 3B), pretreatment with paraoxon abrogated the IL-17 response (paraoxon/STZ group; Figure 3B). These results suggest that inhibition of AChE led to an increase in IFN-γ production and a reduction in STZ-induced IL-17. Further analysis revealed a reduction in the expression of IL-6 and IL-23, cytokines involved in the differentiation of Th17 cells, in groups receiving paraoxon compared with saline group (Figures 3C,D). Moreover, compared with control animals, IL-6 expression was upregulated in saline/STZ group (Figure 3C). On the other hand, expression levels of IL-12, an IFNγ-promoting cytokine, were found to be upregulated in paraoxon-treated groups compared with saline control (2.7-fold and 1.8-fold increase in paraoxon and paraoxon/STZ groups, respectively; Figure 3E). Significant differences in IL-12 expression were observed between paraoxon-treated mice compared with the saline or saline/STZ group (Figure 3E). No significant differences were observed in the expression of TNFα and IL-1β proinflammatory cytokines among experimental groups (data not shown). These findings suggest that inhibition of AChE may prevent STZ-induced hyperglycemia by modulating the balance between Th1 and Th17 development.

To assess this directly, we determined the percentage of IL-17⁺ and IFNγ⁺ splenic T cells at day 18 post-STZ administration. Ex vivo stimulation with PMA/ionomycin followed by intracellular cytokine staining revealed that pretreatment with paraoxon induced a moderate increase in the percentage of IFNγ⁺CD4⁺ cells (Figure 3F). Saline/STZ group exhibited significant reduction in percentage of IFNγ⁺CD4⁺ and IFNγ⁺CD8⁺, but increased percentage of IL-17⁺CD4⁺, T cells compared with paraoxon and paraoxon/STZ group (Figures 3F–I). Percentage of CD4⁺IL-17⁺ cells in saline/STZ group was significantly increased compared with paraoxon-treated groups (2.4-fold and 2-fold that of paraoxon and paraoxon/STZ groups, respectively) (Figures 3F,I). Expression of IL-17 was detected mainly in CD4⁺, but not CD8⁺ T cells (Figure 3F). These results support the idea that inhibition of AChE leads to a preferential induction of a Th1 response that suppresses STZ-driven Th17 cell differentiation.

To further elucidate the role of IFNγ in the development of hyperglycemia, we utilized the same MLD-STZ model to induce diabetes in IFNγ^−/− mice. Administration of paraoxon induced 50–60% inhibition in AChE activity in both wild-type (WT) C57BL/6 as well as IFNγ^−/− mice (Figures 4A,D). Importantly, pretreatment with paraoxon protected against STZ-triggered hyperglycemia in WT (Figure 4B) but not in IFNγ^−/− mice (Figure 4E). WT mice developed hyperglycemia 7–10 days post STZ administration and this continued up to 7 weeks later (Figure 4B). In contrast, no hyperglycemia was observed in paraoxon-pretreated WT mice after STZ injection (Figure 4B). It is intriguing to point out that paraoxon pretreatment in IFNγ^−/− mice did appear to protect against onset of hyperglycemia during the first 10 days following STZ administration (Figure 4E). Subsequently, however, paraoxon-pretreated IFNγ^−/− mice became hyperglycemic and, by day 49, their blood glucose levels reached 500 mg/dl, identical to saline-pretreated mice (Figure 4E). A moderate degree of weight loss (10–15%) is commonly observed in mice treated with AChE inhibitors (36, 42), and this was observed in both WT and IFNγ^−/− mice particularly during the 3-week (day –21 to day 0) paraoxon pretreatment period (Figures 4C,F). Following the end of paraoxon pretreatment period, the animals’ weights gradually normalized and by day 28 were identical to saline-pretreated mice (Figures 4C,F). As WT mice developed hyperglycemia due to STZ, they begin to loose weight and this was clearly evident by day 35 post STZ injection (Figure 4C). However, paraoxon-pretreated WT mice exhibited no further loss of weight after STZ and, in fact, their weights were higher than saline/STZ group at days 35 and 42 post STZ (Figure 4C). In contrast, although IFNγ^−/− mice also recovered their initial weight after paraoxon treatment, they did not exhibit further weight gain after STZ administration due to their hyperglycemic state (Figures 4E,F). Finally, we also assessed long term survival in STZ-injected mice (C57BL/6 and IFNγ^−/−) following pretreatment with either saline or paraoxon (Figure 4G). Irrespective of the treatment, no deaths were recorded in C57BL/6 mice up to 60 days post STZ administration. In sharp contrast, only about 20% of IFNγ^−/− mice, whether pretreated with saline or paraoxon, survived by day 60 post-STZ (Figure 4G). This is most likely due to the higher levels of hyperglycemia observed in these mice (Figure 4E). These findings confirm the important role that IFNγ plays in cholinergic pathway-mediated inhibition of diabetes development in the MLD-STZ model.

Next, we analyzed the status of insulin-producing cells in pancreatic tissue of IFNγ^−/− mice at days 10 and 18 post STZ administration. Compared with saline controls, saline/STZ group showed a reduction in insulin-producing β cells by day 10 post STZ injection, and this became more pronounced at day 18 post STZ (Figure 5A). Pretreatment with paraoxon appeared to delay the loss of insulin-positive β cells. Moreover, paraoxon-treated IFNγ^−/− mice exhibited higher levels of serum insulin compared with saline control (Figure 5B; paraoxon group). No reduction in serum insulin was observed in paraoxon/STZ-treated mice during the first 10 days post-STZ administration (paraoxon/STZ group). However, by day 18, serum insulin levels dropped to become similar to the ones in the saline/STZ group (Figure 5B). These data suggest that IFNγ does not play a role in the initial control of STZ-induced hyperglycemia.

Hematoxylin and eosin staining of pancreatic tissue of IFNγ^−/− mice showed highly infiltrated islets in the saline/STZ group at day 10 and 18 post STZ administration (Figure 6A; left middle and bottom panels). In contrast, pancreatic islets from paraoxon/STZ group exhibited mild infiltration at day 10, but by day 18 all islets appeared infiltrated although to a lesser extent than in saline/STZ group (Figure 6A; right middle and bottom panels). No apparent cell infiltration could be observed in islets of saline or paraoxon control groups (Figure 6A; top panels). Immunohistological analysis of the islets at day 10 and 18 revealed the presence of fewer CD3⁺ T cells than in WT mice, with highest levels being detected in saline/STZ group (Figures 6B,D). Pretreatment with paraoxon reduced by >60% the extent of T cell infiltration (paraoxon/STZ group) compared to saline/STZ group (Figure 6D). Staining with F4/80 mAb revealed a similar extent of macrophage infiltration in all four experimental groups (Figures 6C,E).

As expected, intracellular staining of splenocytes of IFNγ^−/− mice showed no CD4⁺IFNγ⁺ or CD8⁺IFNγ⁺ T cells (Figure 7A). AChE inhibition induced a partial but not statistically significant reduction in the percentage of CD4⁺IL-17⁺ cells (paraoxon group). STZ administration induced the differentiation of CD4⁺IL-17⁺ cells in saline/STZ group (mean ± SEM = 3.7 ± 0.2%; Figures 7A,B) which represents a 2.9-fold increase in comparison to saline controls. For paraoxon/STZ-treated mice, the percentage of CD4⁺IL-17⁺ cells was slightly, but significantly, reduced (2.3 ± 0.2%) compared to saline/STZ group but was still significantly higher than that of saline and acetylcholinesterase inhibitor (AChEI) groups (Figures 7A,B). Nevertheless, the presence of a significant level of CD4⁺IL-17⁺ T cells in paraoxon/STZ-treated IFNγ^−/− mice is in sharp contrast to the findings in WT mice where similar treatment reduced the ratio of CD4⁺IL-17⁺ T cells to background levels (Figure 3F). These results highlight the immunomodulatory effect of the cholinergic pathway and the important role of IFNγ in preventing the development of STZ-induced diabetes.