Biology subjects

Valenzuela, N. M.

Publications and source records attributed to Valenzuela, N. M..

3 recordsLinked to original sources

bioRxiv2022

BCL6 regulates the endothelial pro-immunogenic phenotype relevant to organ transplant rejection

BackgroundIFN{gamma} induces an endothelial cell pro-immunogenic phenotype through the JAK/STAT1 pathway, which can influence alloreactive leukocytes in transplant rejection. Numerous endogenous suppressors of JAK and STAT activation have been described, but regulation of STAT1 at the level of transcription is not well-understood. In immune cells, the DNA binding protein BCL6 controls transcription of lineage and inflammatory genes, including STAT-dependent responses. The goal of this study was to determine if BCL6 also modulates the IFN{gamma}-induced immunogenic phenotype in endothelium. MethodsBCL6 binding to target genes and similarity to STAT1 motifs was analyzed in silico using public datasets. In vitro, primary human aortic endothelial cells were tested for expression of IFN{gamma}-inducible costimulatory molecules, HLA and cytokines, under BCL6 overexpression, depletion and pharmacological targeting. Gene expression was measured by RNA-Seq and protein expression was confirmed by flow cytometry, Luminex and ELISA. Paired biopsies from stable and rejecting human cardiac allografts were compared for expression of BCL6, PD ligands, CXCL chemokines and HLA-DR in the vasculature. ResultsBCL6 expression is increased within human cardiac transplants during rejection, which is positively correlated with expression of interferon response gene HLA-DR in donor blood vessels. Further, BCL6 is IFN{gamma}-inducible via JAK1/2 in endothelium. Next, the consensus DNA binding motif of BCL6 is highly similar to that of STAT1, and numerous interferon response genes harbor BCL6 DNA binding motifs. Depletion of BCL6 in endothelium results in augmentation, while overexpression causes suppression, of MHC class II, chemokine, and PD ligand expression. Unexpectedly, pharmacological targeting of the corepressor domain BTB domain of BCL6 also repressed many interferon response genes, particularly HLA class II, CXCR3 chemokines and PD-L2. On the other hand, BCL6 targeting did not impact inducible expression of any HLA class I genes, PD-L1 or CD40; and the effect is correlated with the presence of BCL6 binding motifs in or near affected genes. ConclusionOur results show for the first time that the transcriptional repressor BCL6 selectively controls the endothelial response to IFN{gamma}. A better understanding of the endogenous mechanisms that regulate donor endothelial activation has the potential to discover new avenues to dampen transplant rejection, with broader relevance to other vascular inflammatory diseases.

immunology↗

bioRxiv2022

The DNA binding protein BCL6 regulates NFκB-controlled endothelial inflammatory gene expression

BackgroundNF{kappa}B drives acute vascular inflammation by activating gene expression programs in endothelial cells to promote leukocyte recruitment. Numerous negative feedback regulators of NF{kappa}B activation have been defined that promote resolution of inflammation. However, the identities of endogenous suppressors of NF{kappa}B transcription are less clear. In macrophages, the transcriptional repressor BCL6 was shown to substantially overlap with NF{kappa}B-driven genes and influence the response to LPS. We identified that the DNA binding protein BCL6 was expressed in endothelial cells. Although the role of BCL6 in adaptive immune cells has been characterized, how BCL6 modifies transcription in endothelial cells has not been studied. ObjectiveBased on prior knowledge that BCL6 represses part of the LPS-induced transcriptome in macrophages, we asked whether BCL6 regulated endothelial pro-inflammatory state by direct interaction with NF{kappa}B. MethodsWe analyzed public datasets of RNA and ChIP-Seq, probed BCL6 expression in human tissue, and tested BCL6 knockdown, overexpression and pharmacological manipulation on TNF induced gene expression in vitro using human primary endothelium isolated from the heart. ResultsWe demonstrate that the DNA binding protein BCL6 is basally expressed in the endothelium, with chromatin marks reflective of a superenhancer, and is particularly enriched in aortic endothelial cells (ECs) compared with ECs from other organs. Although basal expression was relatively low, BCL6 was rapidly upregulated in cardiac endothelium stimulated with TNF, through direct action of NF{kappa}B. The BCL6 consensus DNA binding motif overlaps with that of NF{kappa}B. BCL6 target genes included endothelial pro-inflammatory chemokines and adhesion molecules, as well as NF{kappa}B-related genes themselves. BCL6 knockdown and the degrading BCL6 inhibitor BI-3802 augmented the endothelial cell response to TNF. Surprisingly, antagonism of the BTB domain of BCL6 with small molecules 79-6, FX1 or BI-3812, blocked leukocyte adherence and accordingly suppressed both NF{kappa}B transcriptional activity as well as the expression of many genes in response to TNF. Lastly, we show that HDAC activity is increased by TNF, and can be reduced in the presence of BTB domain inhibitors. ConclusionsOur results demonstrate that BCL6 is a repressor of NF{kappa}B-driven gene expression and inflammation in cardiac endothelial cells. These findings indicate that targeting of BCL6 may enhance vascular inflammation resolution.

immunology↗

bioRxiv2021

Endothelial Resolution of Inflammation is Delayed Following JAK-driven but not NFκB-dependent Activation

Blood endothelial cells actively regulate egress of leukocytes into peripheral tissues in response to inflammatory insult. The resolution of inflammation is critical for healing and return to homeostasis, but the timing and mechanisms involved in return to a non-inflamed state are not well-understood. We examined vascular endothelial activation comparing NF{kappa}B-driven TNF and JAK/STAT-mediated IFN{gamma}. Pro-adhesive gene expression, phenotype and secretome of human endothelial cells from 6 vascular beds were measured under chronic cytokine stimulation, and after short-term cytokine priming followed by withdrawal. The majority of inducible TNF effectors require continuous exposure for reinforcement of the altered phenotype. NF{kappa}B and target genes are quickly down-regulated in the absence of cytokine. In contrast, the consequences of even short exposure to IFN{gamma} are long-lasting and broad, with sustained elevation of adhesion molecules and chemokines up to 48hr later. JAK/STAT and interferon response factor expression are likewise durable, dependent on new transcription and autonomous of continuous IFN{gamma}. Finally, intact persistent STAT expression and JAK signaling in the endothelium is required to maintain a pro-adhesive phenotype after IFN{gamma} withdrawal, which could be prevented by the JAK1/2 inhibitor ruxolitinib. Our results reveal a sustained JAK-dependent perturbation of endothelial function after exposure to IFN{gamma}, but not after NF{kappa}B-driven inflammation.

immunology↗