Background NFκB drives acute vascular inflammation by activating gene expression programs in endothelial cells to promote leukocyte recruitment. Numerous negative feedback regulators of NFκB activation have been defined that promote resolution of inflammation. However, the identities of endogenous suppressors of NFκB transcription are less clear. In macrophages, the transcriptional repressor BCL6 was shown to substantially overlap with NFκB-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. Objective Based 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 NFκB. Methods We analyzed public datasets of RNA and ChIP-Seq, probed BCL6 expression in human tissue, and tested BCL6 knockdown, overexpression and pharmacological manipulation on TNFα induced gene expression in vitro using human primary endothelium isolated from the heart. Results We 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 TNFα, through direct action of NFκB. The BCL6 consensus DNA binding motif overlaps with that of NFκB. BCL6 target genes included endothelial pro-inflammatory chemokines and adhesion molecules, as well as NFκB-related genes themselves. BCL6 knockdown and the degrading BCL6 inhibitor BI-3802 augmented the endothelial cell response to TNFα. Surprisingly, antagonism of the BTB domain of BCL6 with small molecules 79-6, FX1 or BI-3812, blocked leukocyte adherence and accordingly suppressed both NFκB transcriptional activity as well as the expression of many genes in response to TNFα. Lastly, we show that HDAC activity is increased by TNFα, and can be reduced in the presence of BTB domain inhibitors. Conclusions Our results demonstrate that BCL6 is a repressor of NFκB-driven gene expression and inflammation in cardiac endothelial cells. These findings indicate that targeting of BCL6 may enhance vascular inflammation resolution.

The microenvironment, or niche, regulates stem cell fate and improves differentiation efficiency. Human umbilical cord mesenchymal stem cells (hUC-MSCs) are ideal cell source for bone tissue engineering. However, the role of the microenvironments in hUC-MSC-based bone regeneration is not yet fully understood. This study is aimed at investigating the effects of the in vitro culture microenvironment (hUC-MSCs, nano-hydroxyapatite/collagen/poly (L-lactide) (nHAC/PLA), osteogenic media (OMD), and recombinant human bone morphogenetic protein-7 (rhBMP-7)) and the in vivo transplanted microenvironment (ectopic and orthotopic) on bone regeneration ability of hUC-MSCs. The isolated hUC-MSCs showed self-renewal potential and MSCs' characteristics. In the in vitro two-dimensional culture microenvironment, OMD or OMD with rhBMP-7 significantly enhanced hUC-MSCs' osteocalcin immunofluorescence staining, alkaline phosphatase, and Alizarin red staining; OMD with rhBMP-7 exhibited the highest ALP secretion and mineralized matrix formation. In the in vitro three-dimensional culture microenvironment, nHAC/PLA supported hUC-MSCs' adhesion, proliferation, and differentiation; the microenvironment containing OMD or OMD and rhBMP-7 shortened cell proliferation progression and made osteogenic differentiation progression advance; rhBMP-7 significantly attenuated the inhibiting effect of OMD on hUC-MSCs' proliferation and significantly enhanced the promoting effect of OMD on gene expression and protein secretion of osteogenic differentiation markers, calcium and phosphorous concentration, and mineralized matrix formation. The in vitro three-dimensional culture microenvironment containing OMD and rhBMP-7 induced hUC-MSCs to form the most new bones in ectopic or orthotopic microenvironment as proved by microcomputed tomography and hematoxylin and eosin staining, but bone formation in orthotopic microenvironment was significantly higher than that in ectopic microenvironment. The results indicated that the combination of in vitro hUC-MSCs+nHAC/PLA+OMD+rhBMP-7 microenvironment and in vivo orthotopic microenvironment provided a more optimized niche for bone regeneration of hUC-MSCs. This study elucidates that hUC-MSCs and their local microenvironment, or niche, play an important role in hUC-MSC-based bone regeneration. The endogenously produced BMP may serve an important regulatory role in the process.
Copyright © 2021 Lingling E et al.

h4>Background: /h4> Human umbilical cord mesenchymal stem cells (hUC-MSCs) have been suggested as an alternative source of MSCs. However, the studies on its bone formation ability in oral maxillo-facial bone defect are rare. This study investigated the bone formation ability of recombinant human bone morphogenetic protein-7 (rhBMP-7)-induced hUC-MSCs combined with nano-hydroxyapatite/collagen/poly(L-lactide) (nHAC/PLA) in the back of nude mice and jaw bone defect of rabbit. h4>Methods: /h4>: The characteristics of hUC-MSCs were analyzed by plastic adherence, cell phenotype and multilineage differentiation potential. Cell proliferation was examined using a CCK-8 assay. Osteogenic differentiation was evaluated by quantitative calcium concentration, phosphorous concentration, alkaline phosphatase (ALP) activity, osteocalcin (OCN) concentration, mineral formation, and the mRNA levels of ALP, OCN and bone morphogenetic protein (BMP)-2. Scanning electron microscopy was used to observe cell adhesion, growth and differentiation. Bone formation was assessed by immunohistochemical staining, micro-CT and hematoxylin and eosin staining. h4>Results: /h4>: The isolated hUC-MSCs expressed CD105, CD90 and CD73, did not express CD45, CD34, CD11a and HLA-DR, exhibited self-renewal potential, and favored osteogenesis and adipogenesis. The exogenously-added rhBMP-7 attenuated the inhibitory effect of the serum-free osteogenic media (OMD) on the proliferation of hUC-MSCs combined with nHAC/PLA, increased the promoting effect of OMD on osteogenic differentiation of hUC-MSCs in two or three-dimensional culture, and enhanced its heterotopic bone formation and jaw bone defect repair abilities. Furthermore, the bone formation of in situ bone defect was significantly superior to heterotopic bone formation. h4>Conclusions: /h4>: That the microenvironment formed by the regenerative engineered constructs and the in situ bone defect microenvironment closely matched that of the bone tissue in its native state may be essential for sufficient and timely bone regeneration. The endogenously-produced BMPs may serve an important regulatory role in the process. These results paved the way for developing allogeneic hUC-MSCs-based constructs for clinical jaw bone regenerative therapeutic applications.

gammadelta T cells account for up to 10% of T lymphocytes in the peripheral blood of healthy donors. They can be activated by cytokines such as interleukin (IL)-2, IL-12 and IL-15, express natural killer (NK) cell markers such as NKG2D and show cytotoxic activity against several tumour cells, including multiple myeloma. Here, we present activated polyclonal gammadelta T cells from healthy donors with an NK T cell-like phenotype expressing the natural cytotoxicity receptor NKp44. Natural cytotoxicity receptors NKp30, NKp44 and NKp46 have been regarded as specific NK receptors; only two gammadelta T cell clones described so far expressed NKp 44. Isolated polyclonal gammadelta T cells cultured for 7 days according to the cytokine-induced killer cell (CIK) protocol with additional IL-15 revealed a surface expression of NKp44 of 8+/-7% (n=22). This could be confirmed by detection of NKp 44 mRNA by reverse transcription-polymerase chain reaction (RT-PCR). gammadelta T cells exhibited a marked cytotoxic activity against myeloma cells, which could be reduced by inhibition of NKp44. To our knowledge, this is the first description of the expression of NKp44 on polyclonal gammadelta T cells.

Stem cell factor (SCF)/c-kit system is critical for human mast cell development. We thus examined the effects of STI571, an inhibitor of the c-kit tyrosine kinase receptor, on the proliferation and function of human mast cells. STI571 at concentrations of 10(-6) M or higher almost completely abolished the SCF-dependent progeny generation from cord blood-derived cultured mast cells through an inhibition of the tyrosine phosphorylation of c-kit. The compound also suppressed the early phase of mast cell development. The extinction of mast cell growth induced by STI571 may be due largely to apoptosis according to the flow cytometric analysis and gel electrophoresis. Two-hour exposure to STI571 that failed to influence the total viable cell number suppressed adhesion of the cells to fibronectin in the presence of SCF without altering the expressions of integrin molecules. Our results may provide a fundamental insight for the clinical application of STI571 in allergic disorders.