Immune cell infiltration in pancreatic islets of STZ-treated mice.

<p>Paraffin sections of pancreatic islets collected from mice one week after completion of the STZ treatment were stained for CD3 (A) or F4/80 (B). CD3⁺ or F4/80⁺ cells were counted on 30–50 islets per genotype of mice scored with insulitis. <i>Myd88</i>^FL (n = 4), MyD88^CD11c-KO (n = 3), <i>Trif</i>^FL (n = 5) and TRIF^CD11c-KO (n = 6).</p

Development of autoimmune diabetes in NOD mice with myeloid- specific MyD88 or TRIF deficiency.

<p>(A and C) Graphs depicting the incidence of diabetes in mice with the indicated genotypes. (B and D) Representative images and quantification of H&E stained paraffin sections of pancreatic tissue from 10-week-old mice with the indicated genotypes. Graph depicts the percentage of mice with a given histology score per genotype: 0 or I, no islet infiltrates or only small peri-islet infiltrates; II, invasive insulitis (<50% of islet area); III, severe invasive insulitis (>50% of islet area). 20–30 islets per mouse were counted. NOD.MyD88^CD11c-KO (n = 8), NOD.MyD88^LysM-KO (n = 9), NOD.TRIF^CD11c-KO (n = 7) and NOD.TRIF^LysM-KO (n = 9), <i>NOD</i>.<i>Myd88</i>^FL (n = 13), and <i>NOD</i>.<i>Trif</i>^FL (n = 12).</p

Differential effect of MyD88 and TRIF deficiency on <i>Ido</i> expression and Treg induction in PLNs of STZ-treated mice.

<p>(A) The mRNA expression of the indicated genes was measured by qRT-PCR in the PLNs of MyD88^CD11c-KO (n = 5), TRIF^CD11c-KO (n = 9) and their respective littermate control <i>Myd88</i>^FL (n = 4) and <i>Trif</i>^FL (n = 8) mice one week after the completion of STZ injections. Results are depicted as fold increase compared to untreated mice (buffer-only) of each genotype. (B) FACS analysis for Tregs (CD4⁺CD25⁺Foxp3⁺) in the PLNs of STZ-treated mice one week after the completion of the STZ injections. Representative plots of gated live, CD4⁺ PLN cells stained with CD25 and intracellular Foxp3. Bar graph shows quantification of CD4⁺CD25⁺Foxp3⁺ cells in STZ-treated <i>Myd88</i>^FL (n = 4), MyD88^CD111c-KO (n = 6), <i>Trif</i>^FL (n = 5) and TRIF^CD11c-KO (n = 6), as well as untreated <i>Myd88</i>^FL (n = 3), MyD88^CD111c-KO (n = 2), <i>Trif</i>^FL (n = 2) and TRIF^CD11c-KO (n = 2) mice.</p

Differential response of MyD88- or TRIF-deficient DCs to apoptotic cell phagocytosis.

<p>(A) WT, <i>Myd88</i>^-/- and <i>Trif</i>^-/- BMDCs were stimulated with apoptotic splenocytes for 6 or 24 hours and the expression of <i>Tnf</i>, <i>Il10</i> and <i>Ido</i> mRNAs was measured by qRT-PCR. Data shown are representative of three independent experiments. (B) The expression of NF-κB proteins was assessed by immunoblotting with specific antibodies in cytoplasmic and nuclear extracts from BMDCs stimulated with apoptotic splenocytes for 4 hours. Tubulin and HDAC1 were used as loading controls. Blots are representative of three independent experiments. (C) The mRNA expression of the indicated genes was measured by qRT-PCR in peritoneal cells collected from MyD88^CD11c-KO, TRIF^CD11c-KO and Cre negative littermate control mice (n = 4 per genotype) 18 hours after i.p. injection of 10⁷ apoptotic thymocytes or medium-only. Data are representative of two independent experiments. (D) The mRNA and protein expression of IDO was analyzed by qRT-PCR and immunoblotting respectively in WT, <i>Myd88</i>^-/- and <i>Trif</i>^-/- BMDCs that were stimulated with primary apoptotic islets (immunoblot was performed on protein extracts prepared from islets stimulated for 12 hours with STZ). Data are representative of two independent experiments.</p

Effect of pre- or post-HD serum on the expression of TIMP-1 and TIMP-2.

<p>(A): HUVEC were incubated in culture medium supplemented with 20% pre- or post- HD serum. 4 h later the medium was replaced by minimal medium and 8 h or 20 h later the supernatants were analyzed for TIMP-1 and TIMP-2 proteins by SDS-PAGE. (B): HUVEC were incubated with culture medium supplemented with 20% pre- or post- HD serum. 6, 12, or 24 h later total RNA was extracted from the cells, RT-PCR reactions were performed using specific primers for TIMP-1, TIMP-2 or GAPDH mRNAs, the PCR products were analyzed in agarose gels and quantified. Data are expressed as mean ± SEM of three independent experiments. ^* and ^** represent p<0.05 and p<0.01 respectively.</p

Effect of pre- or post-HD serum on the expression of collagen-IV and elastin.

<p>HUVEC were incubated with culture medium supplemented with 20% pre- or post- HD serum. 6, 12, or 24 h later total RNA was extracted from the cells, RT-PCR reactions were performed using specific primers for Collagen IV, Elastin or GAPDH mRNAs, the PCR products were analyzed in agarose gels and quantified. Data are expressed as mean ± SEM of three independent experiments. ^*, ^** and ^*** represent p<0.05, p<0.01 and p<0.001 respectively.</p