<i>Erythropoietin</i> and <i>glucose transporter-1</i> gene expression in isolated primary cardiomyocytes in response to hypoxia.

<p>Isolated rat cardiomyocyte cultures were subjected to basal normoxic conditions and/or hypoxia (1% O₂) for 24 hours. <i>Epo</i> (A) and <i>Glut1</i> (B) expression was then analyzed by RT-PCR and normalized to that of <i>Hprt</i>. The data from four independent experiments are expressed as the change relative to the normoxic values. Statistical significance was assessed using a two-tailed paired t-test (*, p<0.05).</p

Gross appearance of tamoxifen-fed Vhl^floxed-Cre-ERT2 mice.

<p>(A) Vhl^wt-UBC-Cre-ER^T2 (n = 3), Vhl^floxed (n = 9) and Vhl^floxed-UBC-Cre-ER^T2 (n = 10) mice were administered tamoxifen as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022589#pone-0022589-g001" target="_blank">Figure 1</a> and the spleen/body weight ratio was then determined. Statistical significance was assessed using a two-tailed Student's t-test (*, p<0.05; **, p<0.01). Representative images of spleens (B), snouts (C) and paws (D) of Vhl^floxed-UBC-Cre-ER^T2 and control Vhl^floxed mice are shown.</p

<i>Vhl</i> and <i>Hif1α</i> gene expression in tamoxifen-fed Vhl^floxed-UBC-Cre-ER^T2 and Hif1α^floxed-UBC-Cre-ER^T2 mice.

<p>(A) Vhl^wt-UBC-Cre-ER^T2 (n = 3), Vhl^floxed (n = 6) and Vhl^floxed-UBC-Cre-ER^T2 (n = 6) mice were placed on a tamoxifen diet for ten days followed by ten additional days on a normal diet. Gene expression was assessed by RT-PCR in the tissues indicated, the expression of the <i>Vhl</i> gene was normalized to that of <i>Hprt</i> and it was expressed as the change relative to Vhl^floxed mice. (B) Tamoxifen intake was measured over the 10 days of tamoxifen administration in Vhl^wt-UBC-Cre-ER^T2 (n = 3), Vhl^floxed (n = 6) and Vhl^floxed-UBC-Cre-ER^T2 (n = 6) mice. (C,E) Hif1α^wt-UBC-Cre-ER^T2 (n = 4), Hif1α^floxed (n = 3) and Hif1α^floxed-UBC-Cre-ER^T2 (n = 5) mice were administered tamoxifen as indicated above. <i>Hif1α</i> (C) or <i>Vhl</i> (E) gene expression was normalized to that of <i>Hprt</i> and expressed as the change relative to Hif1α^floxed mice. (D) Tamoxifen intake in Hif1α^wt-UBC-Cre-ER^T2 (n = 4), Hif1α^floxed (n = 3) and Hif1α^floxed-UBC-Cre-ER^T2 (n = 5) mice was measured as in B. Total intake per day was expressed relative to the body weight at the end of the tamoxifen treatment and the values represent the mean ± SEM. Statistical significance was assessed using a two-tailed Student's t-test, (*, p<0.05; **, p<0.01) when comparing Vhl^wt-UBC-Cre-ER^T2 or Hif1α^wt-UBC-Cre-ER^T2 with Vhl^floxed-UBC-Cre-ER^T2 or Hif1α^floxed-UBC-Cre-ER^T2 respectively; (^##, p<0.01) when comparing Vhl^floxed or Hif1α^floxed with Vhl^floxed-UBC-Cre-ER^T2 or Hif1α^floxed-UBC-Cre-ER^T2 respectively.</p

<i>Erythropoietin</i> and <i>glucose transporter-1</i> gene expression in HL-1 cardiomyocyte cell line in response to activation of the oxygen-sensing HIF pathway.

<p>(A,B,C) HL-1 cells were transfected with a siRNA for <i>Hif1α</i> (siHIF1α) or a scrambled siRNA control (siSCR) and 24 hours after transfection, the cells were exposed to normoxic or hypoxic (1% O₂) conditions. The expression of <i>Epo</i>, <i>Glut1</i> and <i>Hif1α</i> was measured as described above and the data from three independent experiments are expressed as the change relative to the normoxic values. Statistical significance was assessed using a two-tailed Student's t-test (*, p<0.05).</p

Body weight during and after tamoxifen diet administration in Vhl^floxed-UBC-Cre-ER^T2 and control mice.

<p>Body weight of Vhl^wt-UBC-Cre-ER^T2 (n = 3), Vhl^floxed-UBC-Cre-ER^T2 (n = 6) control Vhl^floxed (n = 6) mice was measured before tamoxifen treatment (TFX 0d), at the end of 10 days on a tamoxifen diet (TFX 10d) and one day after returning to a normal diet (N +1d). Statistical significance was assessed using a two-tailed Student's t-test, (*, p<0.05; **, p<0.01; ns, no significant differences).</p

Sanmarco et al., 2023.pdf

Dendritic cells (DCs) control the generation of self-reactive pathogenic T cells. Thus, DCs are considered attractive therapeutic targets for autoimmune diseases. Using single-cell and bulk transcriptional and metabolic analyses in combination with cell-specific gene perturbation studies we identified a negative feedback regulatory pathway that operates in DCs to limit immunopathology. Specifically, we found that lactate, produced by activated DCs and other immune cells, boosts NDUFA4L2 expression through a mechanism mediated by HIF-1a. NDUFA4L2 limits the production of mitochondrial reactive oxygen species that activate XBP1-driven transcriptional modules in DCs involved in the control of pathogenic autoimmune T cells. Moreover, we engineered a probiotic that produces lactate and suppresses T-cell autoimmunity in the central nervous system via the activation of HIF-1a/NDUFA4L2 signaling in DCs. In summary, we identified an immunometabolic pathway that regulates DC function, and developed a synthetic probiotic for its therapeutic activation. </p