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mt-Nd2(a) Modifies resistance against autoimmune type 1 diabetes in NOD mice at the level of the pancreatic β-cell.

Chen J, Gusdon AM, Piganelli J, Leiter EH, Mathews CE - Diabetes (2010)

Bottom Line: NIT-4 cells with mt-Nd2(a) were also more resistant to AI4-mediated destruction in vitro than NIT-1 cells.Subtle nonhematopoietic type 1 diabetes resistance was observed during adoptive transfer experiments with T-cell clones.This study confirms that genetic polymorphisms in mitochondria can modulate β-cell sensitivity to autoimmune T-cell effectors.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, Gainesville, USA.

ABSTRACT

Objective: To investigate whether a single nucleotide polymorphism (SNP) in the mitochondrial gene for NADH dehydrogenase 2 (mt-Nd2) can modulate susceptibility to type 1 diabetes in NOD mice.

Research design and methods: NOD/ShiLtJ mice conplastic for the alloxan resistant (ALR)/Lt-derived mt-Nd2(a) allele (NOD.mt(ALR)) were created and compared with standard NOD (carrying the mt-Nd2(c) allele) for susceptibility to spontaneous autoimmune diabetes, or to diabetes elicited by reciprocal adoptive splenic leukocyte transfers, as well as by adoptive transfer of diabetogenic T-cell clones. β-Cell lines derived from either the NOD (NIT-1) or the NOD.mt(ALR) (NIT-4) were also created to compare their susceptibility to cytolysis by diabetogenic CD8(+) T-cells in vitro.

Results: NOD mice differing at this single SNP developed spontaneous or adoptively transferred diabetes at comparable rates and percentages. However, conplastic mice with the mt-Nd2(a) allele exhibited resistance to transfer of diabetes by the CD4(+) T-cell clone BDC 2.5 as well as the CD8(+) AI4 T-cell clones from T-cell receptor transgenic animals. NIT-4 cells with mt-Nd2(a) were also more resistant to AI4-mediated destruction in vitro than NIT-1 cells.

Conclusions: Conplastic introduction into NOD mice of a variant mt-Nd2 allele alone was not sufficient to prevent spontaneous autoimmune diabetes. Subtle nonhematopoietic type 1 diabetes resistance was observed during adoptive transfer experiments with T-cell clones. This study confirms that genetic polymorphisms in mitochondria can modulate β-cell sensitivity to autoimmune T-cell effectors.

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The mt-Nd2a allele confers resistance to killing by diabetogenic CD8+ AI4 T-cells in vivo and in vitro. A: Type 1 diabetes onset is significantly retarded following A14 transfer into Rag1 recipients conplastic for the mt-ND2a allele (NOD.mtALR-Rag). Splenocytes from young (3–5 weeks old) NOD-AI4a/b F1 hybrids were collected and erythrocytes removed using hypotonic solution treatment. Cells were injected intravenously (tail vein) at 2 × 107 cells/mouse into age-matched female recipients. Onset of diabetes was monitored using Diastix, with a diagnosis of type 1 diabetes called after positive tests on 2 sequential days. B and C: AI4-induced CML is significantly reduced in NIT-4 cells compared with NIT-1 cells in vitro. Effector cells, splenocytes from NOD-AI4a/b F1 mice were isolated, erythrocytes removed, and the T-cells activated for 3 days with 0.1 μmol/l AI4 mimotope (amino acid sequence YFIENYLEL) and 25 units/ml interleukin-2 in RPMI1640 supplemented with 10% FBS, 2 mmol/l l-glutamine, 1.5g/l sodium bicarbonate, 10 mmol/l HEPES, and 1.0 mmol/l sodium pyruvate. Target NIT-1 and NIT-4 cells were seeded at 1 × 105 cells/well in 96-well culture plates, labeled with 51Cr (Perkin Elmer) for 3 h, and washed; then activated AI4-effector cells were added at increasing effector-to-target (E:T) ratios, in triplicate. B: Cr-51–labeled NIT-1 and NIT-4 cells were cultured with preactivated AI4 cells at different E:T ratio for 16 h. Specific lysis of NIT-4 cells at the highest E:T ratio is only 40% that of NIT-1. C: NIT-1 and NIT-4 cells were primed with 1,000 U/ml IFN-γ for 24 h before adding T-cells. Both cell types were more sensitive to AI4 T-cell killing compared with unprimed. At the highest E:T ratio, NIT-4–specific lysis reached 61% that of NIT-1. **P = 0.0009; *P = 0.0048. AT, adoptive transfer; NS, not statistically significant.
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Figure 3: The mt-Nd2a allele confers resistance to killing by diabetogenic CD8+ AI4 T-cells in vivo and in vitro. A: Type 1 diabetes onset is significantly retarded following A14 transfer into Rag1 recipients conplastic for the mt-ND2a allele (NOD.mtALR-Rag). Splenocytes from young (3–5 weeks old) NOD-AI4a/b F1 hybrids were collected and erythrocytes removed using hypotonic solution treatment. Cells were injected intravenously (tail vein) at 2 × 107 cells/mouse into age-matched female recipients. Onset of diabetes was monitored using Diastix, with a diagnosis of type 1 diabetes called after positive tests on 2 sequential days. B and C: AI4-induced CML is significantly reduced in NIT-4 cells compared with NIT-1 cells in vitro. Effector cells, splenocytes from NOD-AI4a/b F1 mice were isolated, erythrocytes removed, and the T-cells activated for 3 days with 0.1 μmol/l AI4 mimotope (amino acid sequence YFIENYLEL) and 25 units/ml interleukin-2 in RPMI1640 supplemented with 10% FBS, 2 mmol/l l-glutamine, 1.5g/l sodium bicarbonate, 10 mmol/l HEPES, and 1.0 mmol/l sodium pyruvate. Target NIT-1 and NIT-4 cells were seeded at 1 × 105 cells/well in 96-well culture plates, labeled with 51Cr (Perkin Elmer) for 3 h, and washed; then activated AI4-effector cells were added at increasing effector-to-target (E:T) ratios, in triplicate. B: Cr-51–labeled NIT-1 and NIT-4 cells were cultured with preactivated AI4 cells at different E:T ratio for 16 h. Specific lysis of NIT-4 cells at the highest E:T ratio is only 40% that of NIT-1. C: NIT-1 and NIT-4 cells were primed with 1,000 U/ml IFN-γ for 24 h before adding T-cells. Both cell types were more sensitive to AI4 T-cell killing compared with unprimed. At the highest E:T ratio, NIT-4–specific lysis reached 61% that of NIT-1. **P = 0.0009; *P = 0.0048. AT, adoptive transfer; NS, not statistically significant.

Mentions: All NOD-Rag1 recipients developed diabetes after transfer of activated AI4a/b CD8+ T-cells (Fig. 3A). Not surprisingly, ALR-Rag1 mice were resistant to disease transfer by AI4 T-cells (Fig. 3A), consistent with published data showing that ALR/LtJ islets resist lysis by AI4 cells (11). NOD.mtALR-Rag1 mice, although clearly susceptible to AI4-mediated diabetes transfer, nevertheless exhibited a statistically significant retardation in disease development compared with NOD-Rag1 controls (P = 0.0036). Thus, when the T-cell attack is limited to recognition of a single β-cell autoantigen (AI4 recognizes an epitope of dystonia myotonica kinase), the ALR-derived mitochondrial genome confers some resistance, albeit incomplete in the absence of additional protective ALR-derived nuclear genes.


mt-Nd2(a) Modifies resistance against autoimmune type 1 diabetes in NOD mice at the level of the pancreatic β-cell.

Chen J, Gusdon AM, Piganelli J, Leiter EH, Mathews CE - Diabetes (2010)

The mt-Nd2a allele confers resistance to killing by diabetogenic CD8+ AI4 T-cells in vivo and in vitro. A: Type 1 diabetes onset is significantly retarded following A14 transfer into Rag1 recipients conplastic for the mt-ND2a allele (NOD.mtALR-Rag). Splenocytes from young (3–5 weeks old) NOD-AI4a/b F1 hybrids were collected and erythrocytes removed using hypotonic solution treatment. Cells were injected intravenously (tail vein) at 2 × 107 cells/mouse into age-matched female recipients. Onset of diabetes was monitored using Diastix, with a diagnosis of type 1 diabetes called after positive tests on 2 sequential days. B and C: AI4-induced CML is significantly reduced in NIT-4 cells compared with NIT-1 cells in vitro. Effector cells, splenocytes from NOD-AI4a/b F1 mice were isolated, erythrocytes removed, and the T-cells activated for 3 days with 0.1 μmol/l AI4 mimotope (amino acid sequence YFIENYLEL) and 25 units/ml interleukin-2 in RPMI1640 supplemented with 10% FBS, 2 mmol/l l-glutamine, 1.5g/l sodium bicarbonate, 10 mmol/l HEPES, and 1.0 mmol/l sodium pyruvate. Target NIT-1 and NIT-4 cells were seeded at 1 × 105 cells/well in 96-well culture plates, labeled with 51Cr (Perkin Elmer) for 3 h, and washed; then activated AI4-effector cells were added at increasing effector-to-target (E:T) ratios, in triplicate. B: Cr-51–labeled NIT-1 and NIT-4 cells were cultured with preactivated AI4 cells at different E:T ratio for 16 h. Specific lysis of NIT-4 cells at the highest E:T ratio is only 40% that of NIT-1. C: NIT-1 and NIT-4 cells were primed with 1,000 U/ml IFN-γ for 24 h before adding T-cells. Both cell types were more sensitive to AI4 T-cell killing compared with unprimed. At the highest E:T ratio, NIT-4–specific lysis reached 61% that of NIT-1. **P = 0.0009; *P = 0.0048. AT, adoptive transfer; NS, not statistically significant.
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Figure 3: The mt-Nd2a allele confers resistance to killing by diabetogenic CD8+ AI4 T-cells in vivo and in vitro. A: Type 1 diabetes onset is significantly retarded following A14 transfer into Rag1 recipients conplastic for the mt-ND2a allele (NOD.mtALR-Rag). Splenocytes from young (3–5 weeks old) NOD-AI4a/b F1 hybrids were collected and erythrocytes removed using hypotonic solution treatment. Cells were injected intravenously (tail vein) at 2 × 107 cells/mouse into age-matched female recipients. Onset of diabetes was monitored using Diastix, with a diagnosis of type 1 diabetes called after positive tests on 2 sequential days. B and C: AI4-induced CML is significantly reduced in NIT-4 cells compared with NIT-1 cells in vitro. Effector cells, splenocytes from NOD-AI4a/b F1 mice were isolated, erythrocytes removed, and the T-cells activated for 3 days with 0.1 μmol/l AI4 mimotope (amino acid sequence YFIENYLEL) and 25 units/ml interleukin-2 in RPMI1640 supplemented with 10% FBS, 2 mmol/l l-glutamine, 1.5g/l sodium bicarbonate, 10 mmol/l HEPES, and 1.0 mmol/l sodium pyruvate. Target NIT-1 and NIT-4 cells were seeded at 1 × 105 cells/well in 96-well culture plates, labeled with 51Cr (Perkin Elmer) for 3 h, and washed; then activated AI4-effector cells were added at increasing effector-to-target (E:T) ratios, in triplicate. B: Cr-51–labeled NIT-1 and NIT-4 cells were cultured with preactivated AI4 cells at different E:T ratio for 16 h. Specific lysis of NIT-4 cells at the highest E:T ratio is only 40% that of NIT-1. C: NIT-1 and NIT-4 cells were primed with 1,000 U/ml IFN-γ for 24 h before adding T-cells. Both cell types were more sensitive to AI4 T-cell killing compared with unprimed. At the highest E:T ratio, NIT-4–specific lysis reached 61% that of NIT-1. **P = 0.0009; *P = 0.0048. AT, adoptive transfer; NS, not statistically significant.
Mentions: All NOD-Rag1 recipients developed diabetes after transfer of activated AI4a/b CD8+ T-cells (Fig. 3A). Not surprisingly, ALR-Rag1 mice were resistant to disease transfer by AI4 T-cells (Fig. 3A), consistent with published data showing that ALR/LtJ islets resist lysis by AI4 cells (11). NOD.mtALR-Rag1 mice, although clearly susceptible to AI4-mediated diabetes transfer, nevertheless exhibited a statistically significant retardation in disease development compared with NOD-Rag1 controls (P = 0.0036). Thus, when the T-cell attack is limited to recognition of a single β-cell autoantigen (AI4 recognizes an epitope of dystonia myotonica kinase), the ALR-derived mitochondrial genome confers some resistance, albeit incomplete in the absence of additional protective ALR-derived nuclear genes.

Bottom Line: NIT-4 cells with mt-Nd2(a) were also more resistant to AI4-mediated destruction in vitro than NIT-1 cells.Subtle nonhematopoietic type 1 diabetes resistance was observed during adoptive transfer experiments with T-cell clones.This study confirms that genetic polymorphisms in mitochondria can modulate β-cell sensitivity to autoimmune T-cell effectors.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, Gainesville, USA.

ABSTRACT

Objective: To investigate whether a single nucleotide polymorphism (SNP) in the mitochondrial gene for NADH dehydrogenase 2 (mt-Nd2) can modulate susceptibility to type 1 diabetes in NOD mice.

Research design and methods: NOD/ShiLtJ mice conplastic for the alloxan resistant (ALR)/Lt-derived mt-Nd2(a) allele (NOD.mt(ALR)) were created and compared with standard NOD (carrying the mt-Nd2(c) allele) for susceptibility to spontaneous autoimmune diabetes, or to diabetes elicited by reciprocal adoptive splenic leukocyte transfers, as well as by adoptive transfer of diabetogenic T-cell clones. β-Cell lines derived from either the NOD (NIT-1) or the NOD.mt(ALR) (NIT-4) were also created to compare their susceptibility to cytolysis by diabetogenic CD8(+) T-cells in vitro.

Results: NOD mice differing at this single SNP developed spontaneous or adoptively transferred diabetes at comparable rates and percentages. However, conplastic mice with the mt-Nd2(a) allele exhibited resistance to transfer of diabetes by the CD4(+) T-cell clone BDC 2.5 as well as the CD8(+) AI4 T-cell clones from T-cell receptor transgenic animals. NIT-4 cells with mt-Nd2(a) were also more resistant to AI4-mediated destruction in vitro than NIT-1 cells.

Conclusions: Conplastic introduction into NOD mice of a variant mt-Nd2 allele alone was not sufficient to prevent spontaneous autoimmune diabetes. Subtle nonhematopoietic type 1 diabetes resistance was observed during adoptive transfer experiments with T-cell clones. This study confirms that genetic polymorphisms in mitochondria can modulate β-cell sensitivity to autoimmune T-cell effectors.

Show MeSH
Related in: MedlinePlus