The Effect of Immunosuppressive Adjuvant Kynurenine on Type 1 Diabetes Vaccine

Abstract

Inducing antigen-specific tolerance is a promising treatment for preventing or reversing Type 1 diabetes (T1D). In contrast to a vaccine that induces immune responses against pathogens, a tolerogenic vaccine can suppress immunity against antigens causing diseases by administrating a mixture of self-antigens with an adjuvant that decreases the strength of antigen-specific response. Kynurenine (Kyn) is an endogenous substance that can inhibit the natural killer cell and T cell proliferation and promote the differentiation of naïve T cells into regulatory T cells (T_regs). In this study, we evaluated the efficacy of Kyn as a novel suppressive adjuvant. Kyn was co-immunized with GAD65 phage vaccine to induce T_reg cells and tolerogenic responses for the prevention of T1D in NOD mouse model. Mice were subcutaneously immunized two times with 10¹¹ Pfu (100μL,10¹² Pfu/ml) GAD65 phage vaccine doses mixed with 200 μg of Kyn. Serum antibodies and cytokines were detected by ELISA and electrochemiluminescence, respectively. Flow cytometry assay was used to analyze DC and Treg. MTS was used for the analysis of spleen lymphocyte proliferation. RNA sequencing was used to investigate mRNA and miRNA expression profiles in spleen lymphocytes. Compared to GAD65 phage vaccine alone, co-immunization of Kyn and GAD65 phage vaccine resulted in the prevention of hyperglycemia in 60% of mice for at least one month. Further, Kyn enhances GAD65-specific Th2-mediated immune responses; regulates the Th1/Th2 imbalance and increases the secretion of Th2 cytokines and the number of CD4⁺CD25⁺Foxp3⁺T cells; suppresses DC maturation and GAD65-specific T lymphocyte proliferation. Moreover, we integrated Kyn related miRNA and mRNA expression profiles obtained from the spleen lymphocyte RNA-sequencing which was stimulated by Kyn in vitro. These data provide an important basis for understanding the mechanisms underlying Kyn as an immunosuppressive adjuvant which regulated the immune response. These findings suggest that Kyn can serve as an effective suppressive adjuvant candidate for Type 1 diabetes vaccines.

Keywords: Type 1 diabetes vaccine; adjuvant; immunosuppressive; kynurenine; vaccine.

Figure 1

Schematic Map of immunization schedule.

Figure 2

NOD mice were immunized with phage vaccine with or without KYN on weeks 10 and 12. (A, C) Blood glucose/body weight was measured on weeks 6, 8, 10, 12, 14, 16, 18, and 20. The body weight of mice grew when treated with GAD65 phage vaccine with KYN. (B) Mice immunization of GAD65 phage vaccine with KYN were significantly remission from hyperglycemia in 60% at least one month. (D) The survival curves.

Figure 3

Sera samples were collected to detect cytokines with MSD electochemiluminescence. GAD65 phage vaccine co-immunized with KYN generated significantly lower secretion of IL-2, IFN-γ than the GAD65 phage group did, and higher secretion of IL-10, IL-4, TGF-β1, than any other groups did (*P < 0.05).

Figure 4

Effects of suppressive adjuvant Kyn on the GAD65 specific T cell proliferation. The proliferative response to GAD65 was significantly lower in spleen lymphocytes isolated from GAD65 phage vaccine + Kyn immunized mice than those from the GAD65 phage vaccine immunized mice (*P < 0.05).

Figure 5

Kyn suppressed mouse dendritic cell maturation. At the 16th week, mice spleen lymphocytes were stained and immediately analyzed on Flow Cytometer. Compared with GAD65 phage vaccine immunization alone, significantly lower percentages of CD80⁺CD11c⁺ DC cells were observed in GAD65 phage vaccine co-immunized with KYN, which suggested that KYN may suppress dendritic cell maturation (A, B). At the same time, immatured-dendritic cells in the co-immunized group secreted more IL10 than the control group did (C, D) (*P < 0.05).

Figure 6

Kyn induced CD4⁺CD25⁺Foxp3⁺ Treg cells. At the 16th week, mice spleen lymphocytes were stained and immediately analyzed on Flow Cytometer. Notably, Kyn still strongly enhanced the percentages of CD4⁺CD25⁺Foxp3⁺ Treg cells when co-immunized with GAD65 phage vaccine (*P < 0.05).

Figure 7

The top 25 significantly enriched GO terms of the overlapping target genes of differentially expressed miRNAs in spleen lymphocytes of Balb/C mice (Kyn stimulated vs negative control). (A) Up gene Go analysis. (B) Down gene Go analysis.

Figure 8

The top 25 significantly enriched KEGG-pathway analyses of overlapped target genes of differentially expressed miRNAs in spleen lymphocytes of Balb/C mice (Kyn stimulated vs negative control). (A) Up gene KEGG analysis. (B) Down gene KEGG analysis.

Figure 9

miRNA–mRNA-pathway-net. Squares indicate identified miRNAs, while circles represent the corresponding target genes. Blue indicates down-regulated miRNAs or mRNAs while red indicates up-regulated miRNAs or mRNAs. The relationship between miRNAs and genes is shown connected by gray lines. Eleven significantly enriched pathways associated with splenocytes stimulated by Kyn were depicted using the gray triangles. More details about immune related miRNA–mRNA-pathway are shown in Supplementary Figure 3 .

Figure 10

miRNA–mRNA interaction network. The size of the point represents the regulatory capacity of a given miRNA. Squares indicate identified miRNAs, while circles represent the corresponding target genes. Blue indicates down-regulated miRNAs or mRNAs while red indicates up-regulated miRNAs or mRNAs. The relationship between miRNAs and genes is shown connected by gray lines.