Limits...
IFN regulatory factors 4 and 8 expression in the NOD mouse.

Besin G, Gaudreau S, Dumont-Blanchette E, Ménard M, Guindi C, Dupuis G, Amrani A - Clin. Dev. Immunol. (2011)

Bottom Line: DCs play a crucial role in the presentation of autoantigen and activation of diabetogenic T cells, and IRF4 and IRF8 are crucial genes involved in the development of DCs.Importantly, levels of IRF4 and IRF8 expression were lower in tolerogenic bone marrow derived DCs (BMDCs) generated with GM-CSF as compared to immunogenic BMDCs generated with GM-CSF and IL-4.Analysis of splenic DCs subsets indicated that high expression of IRF4 was associated with increased levels of CD4(+)CD8α(-)IRF4(+)CD11c(+) DCs but not CD4(-)CD8α(+)IRF8(+)CD11c(+) DCs in NOD mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric, Immunology Division, Faculty of Medicine and Health Sciences, University of Sherbrooke, 3001 12th Avenue North, Sherbrooke, QC, Canada.

ABSTRACT
Dendritic cells (DCs) contribute to islet inflammation and its progression to diabetes in NOD mouse model and human. DCs play a crucial role in the presentation of autoantigen and activation of diabetogenic T cells, and IRF4 and IRF8 are crucial genes involved in the development of DCs. We have therefore investigated the expression of these genes in splenic DCs during diabetes progression in NOD mice. We found that IRF4 expression was upregulated in splenocytes and in splenic CD11c(+) DCs of NOD mice as compared to BALB/c mice. In contrast, IRF8 gene expression was higher in splenocytes of NOD mice whereas its expression was similar in splenic CD11c(+) DCs of NOD and BALB/c mice. Importantly, levels of IRF4 and IRF8 expression were lower in tolerogenic bone marrow derived DCs (BMDCs) generated with GM-CSF as compared to immunogenic BMDCs generated with GM-CSF and IL-4. Analysis of splenic DCs subsets indicated that high expression of IRF4 was associated with increased levels of CD4(+)CD8α(-)IRF4(+)CD11c(+) DCs but not CD4(-)CD8α(+)IRF8(+)CD11c(+) DCs in NOD mice. Our results showed that IRF4 expression was up-regulated in NOD mice and correlated with the increased levels of CD4(+)CD8α(-) DCs, suggesting that IRF4 may be involved in abnormal DC functions in type 1 diabetes in NOD mice.

Show MeSH

Related in: MedlinePlus

Expression of IRF4 in splenic DCs of diabetes-prone NOD mice and diabetes-resistant BALB/c mice. Proteins were extracted from CD11c+-purified splenic DCs from NOD and BALB/c mice and analyzed by Western blotting for (a) IRF4 or (b) IRF8 expression. The relative intensities of the bands were assessed using the NIH Image software and normalized to reference actin bands to establish a ratio of IRF4/actin (a), lower panel) and (b), lower panel) IRF8/actin. The expression levels observed in BALB/c were arbitrarily set as a unitary value. Data are representative of 2 independent experiments (Exp1 and Exp2). (c) and (d) splenocytes from NOD and BALB/c mice were stained for the CD11c, CD4, and CD8α surface markers in combination with intracellular staining with an anti-IRF8 or anti-IRF4 mAb and analyzed by flow cytometry. Data represent the average percentage of CD11c+CD4+CD8α−IRF4+-positive (c) and CD11c+CD4−CD8α+IRF8+-positive (d) cells of two independent experiments. Error bars correspond to the averages ±S.D (*P < .05, **P < .01 and ***P < .001).
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3102445&req=5

fig3: Expression of IRF4 in splenic DCs of diabetes-prone NOD mice and diabetes-resistant BALB/c mice. Proteins were extracted from CD11c+-purified splenic DCs from NOD and BALB/c mice and analyzed by Western blotting for (a) IRF4 or (b) IRF8 expression. The relative intensities of the bands were assessed using the NIH Image software and normalized to reference actin bands to establish a ratio of IRF4/actin (a), lower panel) and (b), lower panel) IRF8/actin. The expression levels observed in BALB/c were arbitrarily set as a unitary value. Data are representative of 2 independent experiments (Exp1 and Exp2). (c) and (d) splenocytes from NOD and BALB/c mice were stained for the CD11c, CD4, and CD8α surface markers in combination with intracellular staining with an anti-IRF8 or anti-IRF4 mAb and analyzed by flow cytometry. Data represent the average percentage of CD11c+CD4+CD8α−IRF4+-positive (c) and CD11c+CD4−CD8α+IRF8+-positive (d) cells of two independent experiments. Error bars correspond to the averages ±S.D (*P < .05, **P < .01 and ***P < .001).

Mentions: To determine whether differential expression of IRF4 and IRF8 in splenocytes of NOD mice resided in splenic DCs, we examined the levels of protein expression of IRF4 and IRF8 by Western blots in CD11c+-purified splenic DCs of 8-week-old NOD and BALB/c mice. Results showed (Figure 3(a)) that the expression of IRF4 was higher in splenic DCs of NOD mice as compared to BALB/c mice (P < .05). In contrast, IRF8 expression was similar in splenic DCs of both strains of mice (Figure 3(b)). Since IRF4 and IRF8 have been shown to be essential for the development of CD4+CD8α−CD11c+ and CD4−CD8α+CD11c+ subsets, respectively [9], we determined whether the changes observed in IRF4 and IRF8 expression in splenic DCs of NOD and BALB/c mice affected the proportions of these two DCs subsets. Results of FACS analysis (Figure 3(c)) showed no difference in the percentage of CD4+CD8α−IRF4+CD11c+ DCs subset in splenocytes of 3-week-old NOD and BALB/c mice (0.42% ± 0.03 as opposed to 0.27% ± 0.03, P > .05). The percentages of splenic CD4+CD8α−IRF4+CD11c+ DCs subset were significantly (P < .05) increased in 7-, 10-, and 20-week-old NOD mice (0.60% ± 0.030 at 7 weeks, 0.63% ± 0.03 at 10 weeks, and 0.60% ± 0.07 at 20 weeks) as compared to 3 weeks old (NOD 0.42% ± 0.03) and BALB/c (0.27% ± 0.0) mice. In addition, there were no significant difference in splenic CD4+CD8α−IRF4+CD11c+ DCs subset in 7-, 10-, and 20-week-old nondiabetic and diabetic NOD mice. In contrast, an absence of differences was noted in the percentage of CD4−CD8α+IRF8+CD11c+ DCs subset in the splenocytes of BALB/c mice and diabetic or nondiabetic NOD mice (Figure 3(d)). Together, these data suggested that high expression of IRF4 in NOD mice was associated with enhanced splenic CD4+CD8α−IRF4+CD11c+ DCs subset but not with splenic CD4−CD8α+IRF8+CD11c+ DCs subset.


IFN regulatory factors 4 and 8 expression in the NOD mouse.

Besin G, Gaudreau S, Dumont-Blanchette E, Ménard M, Guindi C, Dupuis G, Amrani A - Clin. Dev. Immunol. (2011)

Expression of IRF4 in splenic DCs of diabetes-prone NOD mice and diabetes-resistant BALB/c mice. Proteins were extracted from CD11c+-purified splenic DCs from NOD and BALB/c mice and analyzed by Western blotting for (a) IRF4 or (b) IRF8 expression. The relative intensities of the bands were assessed using the NIH Image software and normalized to reference actin bands to establish a ratio of IRF4/actin (a), lower panel) and (b), lower panel) IRF8/actin. The expression levels observed in BALB/c were arbitrarily set as a unitary value. Data are representative of 2 independent experiments (Exp1 and Exp2). (c) and (d) splenocytes from NOD and BALB/c mice were stained for the CD11c, CD4, and CD8α surface markers in combination with intracellular staining with an anti-IRF8 or anti-IRF4 mAb and analyzed by flow cytometry. Data represent the average percentage of CD11c+CD4+CD8α−IRF4+-positive (c) and CD11c+CD4−CD8α+IRF8+-positive (d) cells of two independent experiments. Error bars correspond to the averages ±S.D (*P < .05, **P < .01 and ***P < .001).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102445&req=5

fig3: Expression of IRF4 in splenic DCs of diabetes-prone NOD mice and diabetes-resistant BALB/c mice. Proteins were extracted from CD11c+-purified splenic DCs from NOD and BALB/c mice and analyzed by Western blotting for (a) IRF4 or (b) IRF8 expression. The relative intensities of the bands were assessed using the NIH Image software and normalized to reference actin bands to establish a ratio of IRF4/actin (a), lower panel) and (b), lower panel) IRF8/actin. The expression levels observed in BALB/c were arbitrarily set as a unitary value. Data are representative of 2 independent experiments (Exp1 and Exp2). (c) and (d) splenocytes from NOD and BALB/c mice were stained for the CD11c, CD4, and CD8α surface markers in combination with intracellular staining with an anti-IRF8 or anti-IRF4 mAb and analyzed by flow cytometry. Data represent the average percentage of CD11c+CD4+CD8α−IRF4+-positive (c) and CD11c+CD4−CD8α+IRF8+-positive (d) cells of two independent experiments. Error bars correspond to the averages ±S.D (*P < .05, **P < .01 and ***P < .001).
Mentions: To determine whether differential expression of IRF4 and IRF8 in splenocytes of NOD mice resided in splenic DCs, we examined the levels of protein expression of IRF4 and IRF8 by Western blots in CD11c+-purified splenic DCs of 8-week-old NOD and BALB/c mice. Results showed (Figure 3(a)) that the expression of IRF4 was higher in splenic DCs of NOD mice as compared to BALB/c mice (P < .05). In contrast, IRF8 expression was similar in splenic DCs of both strains of mice (Figure 3(b)). Since IRF4 and IRF8 have been shown to be essential for the development of CD4+CD8α−CD11c+ and CD4−CD8α+CD11c+ subsets, respectively [9], we determined whether the changes observed in IRF4 and IRF8 expression in splenic DCs of NOD and BALB/c mice affected the proportions of these two DCs subsets. Results of FACS analysis (Figure 3(c)) showed no difference in the percentage of CD4+CD8α−IRF4+CD11c+ DCs subset in splenocytes of 3-week-old NOD and BALB/c mice (0.42% ± 0.03 as opposed to 0.27% ± 0.03, P > .05). The percentages of splenic CD4+CD8α−IRF4+CD11c+ DCs subset were significantly (P < .05) increased in 7-, 10-, and 20-week-old NOD mice (0.60% ± 0.030 at 7 weeks, 0.63% ± 0.03 at 10 weeks, and 0.60% ± 0.07 at 20 weeks) as compared to 3 weeks old (NOD 0.42% ± 0.03) and BALB/c (0.27% ± 0.0) mice. In addition, there were no significant difference in splenic CD4+CD8α−IRF4+CD11c+ DCs subset in 7-, 10-, and 20-week-old nondiabetic and diabetic NOD mice. In contrast, an absence of differences was noted in the percentage of CD4−CD8α+IRF8+CD11c+ DCs subset in the splenocytes of BALB/c mice and diabetic or nondiabetic NOD mice (Figure 3(d)). Together, these data suggested that high expression of IRF4 in NOD mice was associated with enhanced splenic CD4+CD8α−IRF4+CD11c+ DCs subset but not with splenic CD4−CD8α+IRF8+CD11c+ DCs subset.

Bottom Line: DCs play a crucial role in the presentation of autoantigen and activation of diabetogenic T cells, and IRF4 and IRF8 are crucial genes involved in the development of DCs.Importantly, levels of IRF4 and IRF8 expression were lower in tolerogenic bone marrow derived DCs (BMDCs) generated with GM-CSF as compared to immunogenic BMDCs generated with GM-CSF and IL-4.Analysis of splenic DCs subsets indicated that high expression of IRF4 was associated with increased levels of CD4(+)CD8α(-)IRF4(+)CD11c(+) DCs but not CD4(-)CD8α(+)IRF8(+)CD11c(+) DCs in NOD mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric, Immunology Division, Faculty of Medicine and Health Sciences, University of Sherbrooke, 3001 12th Avenue North, Sherbrooke, QC, Canada.

ABSTRACT
Dendritic cells (DCs) contribute to islet inflammation and its progression to diabetes in NOD mouse model and human. DCs play a crucial role in the presentation of autoantigen and activation of diabetogenic T cells, and IRF4 and IRF8 are crucial genes involved in the development of DCs. We have therefore investigated the expression of these genes in splenic DCs during diabetes progression in NOD mice. We found that IRF4 expression was upregulated in splenocytes and in splenic CD11c(+) DCs of NOD mice as compared to BALB/c mice. In contrast, IRF8 gene expression was higher in splenocytes of NOD mice whereas its expression was similar in splenic CD11c(+) DCs of NOD and BALB/c mice. Importantly, levels of IRF4 and IRF8 expression were lower in tolerogenic bone marrow derived DCs (BMDCs) generated with GM-CSF as compared to immunogenic BMDCs generated with GM-CSF and IL-4. Analysis of splenic DCs subsets indicated that high expression of IRF4 was associated with increased levels of CD4(+)CD8α(-)IRF4(+)CD11c(+) DCs but not CD4(-)CD8α(+)IRF8(+)CD11c(+) DCs in NOD mice. Our results showed that IRF4 expression was up-regulated in NOD mice and correlated with the increased levels of CD4(+)CD8α(-) DCs, suggesting that IRF4 may be involved in abnormal DC functions in type 1 diabetes in NOD mice.

Show MeSH
Related in: MedlinePlus