Treatment with incomplete Freund's adjuvant and Listeria monocytogenes delays diabetes via an interleukin‑17‑secretion‑independent pathway

  • Authors:
    • Hai‑Ping Wang
    • Zhi‑Gao He
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  • Published online on: March 3, 2015     https://doi.org/10.3892/etm.2015.2328
  • Pages: 1934-1938
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Abstract

Non‑obese diabetes (NOD) mice are widely used as an animal model in studies of type I diabetes (TID). Treatment with complete Freund's adjuvant (CFA) in pro‑diabetic NOD mice is known to inhibit disease progression by activating CD1d‑specific natural killer (NK) T cells and inducing interleukin (IL)‑17 secretion in innate immune cells. The aim of the present study was to examine the effect of incomplete Freund's adjuvant (IFA) and L. monocytogenes treatment on the development of TID in NOD mice. This combined treatment of IFA and L. monocytogenes, a microbe that infects the liver and is primarily combatted by NK and cytotoxic T lymphocytes, was applied to mimic CFA treatment in pro‑diabetic NOD mice. The combined IFA + L. monocytogenes treatment effectively delayed TID development in the NOD mice. In contrast to CFA, the IFA + L. monocytogenes treatment did not induce T cells or innate immune cells to secrete IL‑17. However, increased levels of regulatory T cells were detected. Furthermore, IFA + L. monocytogenes mice exhibited higher levels of IgG2a, although no notable T helper 1 cell response was observed when compared with the CFA or IFA control treated mice. Therefore, combined IFA + L. monocytogenes treatment was shown to delay TID development in NOD mice via a novel mechanism, which was independent from the secretion of IL‑17 by CFA‑activated NKT cells.

Introduction

Non-obese diabetes (NOD) mice are one of the most commonly used animal models for the study of autoimmune diseases, and exhibit a susceptibility to the spontaneous development of autoimmune insulin-dependent diabetes mellitus (1). A number of factors are associated with the development of diabetes in NOD mice, including the release of self-reactive cytotoxic T lymphocytes (CTL) from negative selection in the thymus (central tolerance) and the loss of regulatory T (Treg) cell function (peripheral tolerance) (2). In addition, natural killer (NK) and NKT cells play a critical role in the progression of type I diabetes (TID) (35).

In 1990, adjuvant immunotherapy was first reported to be effective in preventing the development of TID in pro-diabetic NOD mice (6), and subsequent studies (7,8) suggested that complete Freunds adjuvant (CFA) therapy was effective in treating new-onset NOD mice (9), although the underlying mechanisms remain unclear. Numerous studies have attempted to reveal the underlying immunotherapy mechanism and have found that adjuvant treatment in mice induces the differentiation of regulatory cell populations in vivo, inhibiting the onset of TID (1012). Mice treated with immunological adjuvants exhibit an altered ratio of T helper (Th)1 and Th2 cells, which promotes an antibody response, but prevents Th1-mediated auto-reactive T cell responses to pancreatic β-cell surface antigens (13). Furthermore, upregulation of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, has been reported following adjuvant treatment (14). A previous study found that NOD mice treated with the bacillus Calmette-Guerin (BCG) vaccine were protected against TID (15). CFA and the BCG vaccine contain inactivated M. tuberculosis; however, incomplete Freund's adjuvant (IFA) does not contain M. tuberculosis. Since IFA is unable to prevent TID development in NOD mice as effectively as CFA (16,17), M. tuberculosis has been hypothesized to play an important role in the modulation of the immune response in cases of TID.

A previous study demonstrated that the pro-inflammatory cytokine, interleukin (IL)-17, plays a critical role in the pathogenesis of TID in NOD mice (18). In addition, treatment with CFA or M. tuberculosis has been reported to induce IL-17 expression. However, this increase in IL-17 expression was produced primarily by CD8+ (19) or γδ T cells (20), rather than CD4+ Th17 cells. Further studies have indicated that NKT cells are involved in CFA-mediated protection against TID in NOD mice via the activation of NK cells (21), which are the primary source of interferon (IFN)-γ in the pro-diabetic NOD mice (12,22). Mechanism studies show that these NKT cells are activated directly by M. tuberculosis, possibly via CD1d recognition of specific long fatty acyl chains (23) on the surface of M. tuberculosis. Activated NKT cells, including Vα19 NKT cells, produce IL-17 and other immunoregulatory cytokines, such as IL-4, −10 and IFN-γ (24).

In the present study, NOD mice were treated with a combined therapy of IFA and inactivated L. monocytogenes, a microbe that often infects the liver in humans and mice and whose elimination is in part dependent on activated invariant NKT cells (25). Heat-killed L. monocytogenes has been previously used as an adjuvant to induce strong Th1 responses in mice (26). L. monocytogenes shares numerous characteristics with M. tuberculosis; however, L. monocytogenes cannot induce IL-17 secretion in NKT cells as effectively as M. tuberculosis. The effects of a combined IFA + L. monocytogenes treatment on the development of TID was investigated in a NOD mouse model.

Materials and methods

Mice and immunizations

A total of 108 female NOD mice (aged five weeks; 17–20 g) were purchased from Shanghai Animal Laboratory Center (Shanghai, China) and housed in the East Hospital of Tongji University (Shanghai, China). Mice were immunized by a hypodermic injection into their back with one of the three treatments. The IFA + L. monocytogenes group mice received heat-killed L. monocytogenes (108 bacteria/mouse) in 100 µl IFA. A second group was injected with CFA, while a third IFA-only group received a control injection containing no bacteria. Another 10 mice were administered twice with IFA + L. monocytogenes immunization with the same dose at 5 weeks and 8 weeks of age. Blood sugar levels were measured every three days following immunization and the mice whose blood sugar levels were >11.8 mmol/L were defined as positive for TID. All animal experiments were performed in accordance with protocols approved by the Animal Care and Use Committee of East Hospital of Tongji University. (Shanghai, China).

Fluorescence-activated cell sorting and intracellular staining

Cytokine secretion in the lymphocytes was analyzed using Cytofix/Cytoperm™ Plus (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer's instructions. Spleen cells were collected and incubated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA), 5 µM calcium ionophore A23187 (Sigma-Aldrich) and GolgiStop™(BD Biosciences) at 37°C for 4 h. Surface staining was performed using anti-CD3e-PerCP/Cy5.5 antibodies (BioLegend, Inc., San Diego, CA, USA) for 20 min at 4°C. Cells were subsequently permeabilized with Cytofix/Cytoperm™ solution for 20 min at 4°C, and intracellular cytokine staining was performed with anti-IL-17A-Alexa Fluor 647 (cat. no. 560224; BD Biosciences) and phycoerythrin (PE)-IFN-γ antibodies (cat. no. 557735; BD Biosciences). For Treg staining, spleen cells were fixed and stained using anti-T cell receptor (TCR)b-fluorescein isothiocyanate (cat. no. 553171; BD Biosciences), anti-CD25-PE (cat. no. 553075; BD Biosciences) and intercellular anti-Foxp3-Alexa Fluor 647 (cat. no. 560402; BD Biosciences) antibodies. Antibodies were used in a 1:100 dillution (BioLegend) or 1:50 dillution (BD Biosciences), according to the manufacturer's instructions.

Antibody levels in the blood serum

Total levels of IgG, IgG1 and IgG2a were examined by ELISA. In brief, 96-well plates (Nunc; Thermo Fisher Scientific, Waltham, MA, USA) were coated with 300 ng/well goat anti-mouse IgG antibodies (Life Technologies, Grand Island, NY, USA) in phosphate-buffered saline (PBS) and incubated overnight at 4°C. After blocking with 5% skim milk in PBS-Tween-20, the plates were incubated for 1 h at 37°C with serially-diluted serum samples. Following three washes with PBS-Tween-20, the samples were reacted with sheep anti-mouse IgG, IgG1 or IgG2a antibodies conjugated to horseradish peroxidase (BD Biosciences). Plates were developed by adding tetramethylbenzidine (Endogen®; Pierce Biotechnology, Inc., Rockford, IL, USA) and incubating in the dark. The reaction was stopped using 1 mol/L H2SO4, and the optical densities (OD) were read at 450 nm using an ELISA reader (Thermo Fisher Scientific). ELISA end-point titers were expressed as the reciprocal of the highest sample dilution that yielded an OD two times the mean value of the blank control.

ELISA analysis of the expression of IL-17

A total of 1 × 106 lymphocytes were collected from pancreas-draining lymph nodes and cultured with RPMI-1640 medium with 10% FCS and 200 U/ml mouse IL-2 (cat. no CYT-370; ProSpec-Tany Technogene Ltd. East Brunswick, NJ, USA) in 96-well plates with plate-coated 0.5 µg/ml functional anti-CD3e (cat. no. 16-0031-81; eBioscience, Inc., San Diego, CA, USA) and 0.5 µg/ml soluble CD28 (cat. no. 16-0821-81; eBioscience, Inc.) for two days in the presence of recombinant mouse IL-2 (40 U/ml, R&D Systems, Minneapolis, MN, USA). The expression of IL-17 in the medium was detected using a Mouse IL-17A Platinum ELISA kit (cat. no. M17AF0; eBioscience Inc.), according to the manufacturer's instructions.

Statistical analysis

Data were analyzed using GraphPad Prisms® 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA) and are expressed as the mean ± standard deviation. Differences between the groups were analyzed by one-way analysis of variance with a post-test comparison using the t-test. P<0.05 was considered to indicate a statistically significant difference.

Results

IFA and L. monocytogenes treatment in pro-diabetic NOD mice delays TID development

TID development in NOD mice is associated with numerous factors, including age, diet and living environment. Blood sugar levels of >11.8 mmol/L were observed in the mice aged 8–10 weeks (data not shown). As previously reported (6), treatment with CFA in pro-diabetic (five-week old) NOD mice was able to effectively block TID development, which was confirmed in the present study. In order to determine the effects of alternative immune therapies, heat-killed L. monocytogenes (108 bacteria/mouse) was used instead of M. tuberculosis in CFA. The results indicated that IFA + L. monocytogenes was unable to totally prevent TID development, as >50% of the mice became diabetic within 12 weeks of treatment, while in the CFA treatment group, this figure was <30% (Fig. 1). However, increased blood sugar levels were not detected in the IFA + L. monocytogenes group mice until they were 12 weeks-old (seven weeks after treatment), suggesting that IFA + L. monocytogenes treatment resulted in a delayed disease progression when compared with the IFA-only group. Another 10 pro-diabetic NOD mice received a second administration of IFA + L. monocytogenes at three weeks following the initial immunization (age, eight weeks), in order to study whether an additional immunization was able to further delay TID development. However, no significant change in disease progression was observed in the twice-immunized mice when compared with the once-immunized mice (data not shown).

IFA + L. monocytogenes treatment has no effect on IL-17-producing T cell populations

Spleen cells were collected three weeks after adjuvant treatment and were incubated with PMA/calcium ionophore. The percentage of IL-17-producing spleen cells was between 0.2 and 0.5%, which was relatively low and no significant difference was observed between the CFA, IFA + L. monocytogenes and IFA-only control groups (data not shown). Spleen cells were separated using the T cell-specific surface marker, TCRb, into TCRb+ T cells and TCRbB220 innate cell populations, which consisted primarily of macrophages, dendritic and NK cells. It is generally accepted that γδ T cells are innate immune cells, despite expressing the TCR on their surface. CFA treatment induced significant IL-17 secretion in the innate immune cells when compared with the IFA + L. monocytogenes and IFA-only control groups (Fig. 2). Furthermore, adjuvant treatment appeared to have no influence on the TCRb+ T cell population, since no statistically significant difference was observed between the three groups. In addition, IFN-γ expression was compared among the groups, and adjuvant treatment was shown to induce a notable increase in IFN-γ expression in T cells of the IFA-only control group. However, no significant difference in IFN-γ expression was observed in the T cells of the CFA and IFA + L. monocytogenes groups (data not shown).

CFA treatment induces strong IL-17 expression in the pancreatic draining lymph nodes

Considering that IL-17 is a pro-inflammatory cytokine that is primarily involved in local inflammation, the expression of IL-17 in the pancreatic draining lymph nodes was analyzed. Lymphocytes from pancreatic draining lymph nodes were collected from eight week-old NOD mice (three weeks after treatment) and cultured in vitro for two days, after which secreted IL-17 was captured by plate-coated antibodies. ELISA results revealed that CFA treatment induced strong IL-17 expression in the pancreatic draining lymph nodes when compared with the IFA + L. monocytogenes and IFA-only groups (Fig. 3). The source of this extra IL-17 expression in the CFA group remains unclear, but it may have been secreted by Th17 and/or innate immune cells, such as NKT cells.

IFA + L. monocytogenes treatment promotes Treg proliferation and increases IgG2a levels in the blood serum

IFA + L. monocytogenes treatment delayed disease progression, but did not alter the secretion of cytokines, including IL-17, in the innate immune response, which indicates that alternative mechanisms were involved. The Treg cell populations in the spleen of the mice were analyzed and the IFA + L. monocytogenes group mice were found to exhibit a significantly higher percentage of Treg cells in the CD4+ T cell population when compared with the CFA and IFA-only groups (Fig. 4). Treg cells are the major immunoregulatory cells in the development of TID in NOD mice; thus, the increased Treg cell population was hypothesized to be associated with the protective effects of the IFA + L. monocytogenes treatment against TID. In addition, Th1 and Th2 responses to the treatment were assessed via serum antibody-subtype analysis, which revealed no change in the total serum levels of IgG. However, the IFA + L. monocytogenes group mice exhibited increased levels of the IgG2a subtype when compared with the CFA and IFA-only groups (Fig. 5). In summary, the results indicated that treatment with IFA + L. monocytogenes had a notable effect on T cell differentiation and antibody responses during the development of TID.

Discussion

In the present study, pro-diabetic NOD mice were treated with IFA + L. monocytogenes, which was found to delay the development of TID. However, the treatment was unable to inhibit disease progression indefinitely. The levels of the cytokines, IL-17 and IFN-γ, were examined in T cells and innate immune cells in all three groups. CFA was found to induce the expression of IL-17 in innate immune cells; however, IFA + L. monocytogenes treatment induced only a small increase in IL-17 expression levels when compared with the IFA-only control group. No statistically significant differences were observed in the levels of IL-17-producing T cells and IFN-γ-producing Th1 cells between the CFA and IFA + L. monocytogenes groups. CFA treatment induced the production of IL-17 in innate immune cells, including NKT and γδ T cells, which is consistent with previous studies investigating the effects of CFA on TID development in NOD mice (27). In the present study, IFA + L. monocytogenes treatment delayed disease progression, but did not induce IL-17 secretion in T cells and innate immune cells, which suggests that alternative mechanisms may be involved in L. monocytogenes-mediated protection against TID.

No significant difference was observed between the groups in the levels of IFN-γ-producing Th1 and Th17 cells; therefore, the levels of Treg cells were analyzed. Treg cells are widely considered to play a critical role in the regulation of autoimmune pathologies, such as TID (28). The results showed that the percentage of Treg cells in the IFA + L. monocytogenes-treated mice was higher compared with the CFA and IFA-only groups. Although treatment with CFA and IFA-only had no effect on thymic Treg levels, the IFA + L. monocytogenes treatment contained components, such as the cell wall and microbial DNA, which effectively activated innate immune cells. This activation was hypothesized to induce local pro-inflammatory cytokine secretion through Toll-like receptor signaling pathways, altering T cell differentiation and promoting the proliferation of Treg cells. However, the data from the current study are not sufficient to confirm whether the IFA + L. monocytogenes treatment increased the Treg cell population via this mechanism.

The levels of IgG antibody isotypes in the blood serum were analyzed, and the IFA + L. monocytogenes group mice were found to exhibit increased levels of IgG2a when compared with the CFA and IFA-only groups. However, no statistically significant difference was observed in the other antibody subtypes. These results indicate that IFA + L. monocytogenes treatment altered the Th1/Th2 balance in NOD mice, inducing the production of IgG2a antibodies, which is closely associated with the Th1 response.

In conclusion, treatment with IFA + L. monocytogenes was observed to delay disease progression in pro-diabetic NOD mice. The mechanisms underlying this L. monocytogenes-specific protection differed from those involved in CFA treatment, since L. monocytogenes did not induce IL-17 secretion in innate immune cells. However, IFA + L. monocytogenes treatment was shown to affect the Th cell subsets. Mice treated with IFA + L. monocytogenes exhibited increased levels of Treg cells and IgG2a antibodies in the blood serum, indicating a marked antibody response when compared with the CFA or IFA-only treated mice. However, the mechanisms by which these T cell subsets affect disease progression remain unclear, and further experimental data are required.

Acknowledgements

Experimental support was provided by Shanghai Xuhai Biological Technology Co., Ltd (Shanghai, China).

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Spandidos Publications style
Wang HP and Wang HP: Treatment with incomplete Freund's adjuvant and Listeria monocytogenes delays diabetes via an interleukin‑17‑secretion‑independent pathway. Exp Ther Med 9: 1934-1938, 2015
APA
Wang, H., & Wang, H. (2015). Treatment with incomplete Freund's adjuvant and Listeria monocytogenes delays diabetes via an interleukin‑17‑secretion‑independent pathway. Experimental and Therapeutic Medicine, 9, 1934-1938. https://doi.org/10.3892/etm.2015.2328
MLA
Wang, H., He, Z."Treatment with incomplete Freund's adjuvant and Listeria monocytogenes delays diabetes via an interleukin‑17‑secretion‑independent pathway". Experimental and Therapeutic Medicine 9.5 (2015): 1934-1938.
Chicago
Wang, H., He, Z."Treatment with incomplete Freund's adjuvant and Listeria monocytogenes delays diabetes via an interleukin‑17‑secretion‑independent pathway". Experimental and Therapeutic Medicine 9, no. 5 (2015): 1934-1938. https://doi.org/10.3892/etm.2015.2328