Paneth cell dysfunction causes deficiencies in intestinal C-type lectins and antimicrobial peptides, which leads to dysbiosis of the intestinal microbiota, alters the mucosal barrier, and promotes development of inflammatory bowel diseases. We investigated whether transgenic (TG) expression of the human regenerating family member 3 alpha gene (REG3A) alters the fecal microbiota and affects development of colitis in mice. We performed studies with C57BL/6 mice that express human regenerating family member 3 alpha (hREG3A) in hepatocytes, via the albumin gene promoter. In these mice, hREG3A travels via the bile to the intestinal lumen. Some mice were given dextran sodium sulfate (DSS) to induce colitis. Feces were collected from mice and the composition of the microbiota was analyzed by 16S ribosomal RNA sequencing. The fecal microbiome was also analyzed from mice that express only 1 copy of human REG3A transgene but were fed feces from control mice (not expressing hREG3A) as newborns. Mice expressing hREG3A were monitored for DSS-induced colitis after cohousing or feeding feces from control mice. Colitis was induced in another set of control and hREG3A-TG mice by administration of trinitrobenzene sulfonic acid; some mice were given intrarectal injections of the hREG3A protein. Colon tissues were collected from mice and analyzed by histology and immunohistochemistry to detect mucin 2, as well as by 16S ribosomal RNA fluorescence in situ hybridization, transcriptional analyses, and quantitative polymerase chain reaction. We measured levels of reactive oxygen species (ROS) in bacterial cultures and fecal microbiota using 2′,7′-dichlorofluorescein diacetate and flow cytometry. The fecal microbiota of mice that express hREG3A had a significant shift in composition, compared with control mice, with enrichment of Clostridiales (Ruminococcaceae, Lachnospiraceae) and depletion of Bacteroidetes (Prevotellaceae); the TG mice developed less-severe colitis following administration of DSS than control mice, associated with preserved gut barrier integrity and reduced bacterial translocation, epithelial inflammation, and oxidative damage. A similar shift in the composition of the fecal microbiota occurred after a few months in TG mice heterozygous for REG3A that harbored a wild-type maternal microbiota at birth; these mice developed less-severe forms of colitis following DSS administration. Cohoused and germ-free mice fed feces from REG3A-TG mice and given DSS developed less-severe forms of colitis and had reduced lipopolysaccharide activation of the toll-like receptor 4 and increased survival times compared with mice not fed feces from REG3A-TG mice. REG3A TG mice developed only mild colonic inflammation after exposure to 2,4,6-trinitrobenzene sulfonic acid, compared with control mice. Control mice given intrarectal hREG3A and exposed to 2,4,6-trinitrobenzene sulfonic acid showed less colon damage and inflammation than mice not given intrarectal hREG3A. Fecal samples from REG3A-TG mice had lower levels of ROS than feces from control mice during DSS administration. Addition of hREG3A to bacterial cultures reduced levels of ROS and increased survival of oxygen-sensitive commensal bacteria (Faecalibacterium prausnitzii and Roseburia intestinalis). Mice with hepatocytes that express hREG3A, which travels to the intestinal lumen, are less sensitive to colitis than control mice. We found hREG3A to alter the colonic microbiota by decreasing levels of ROS. Fecal microbiota from REG3A-TG mice protect non-TG mice from induction of colitis. These findings indicate a role for reduction of oxidative stress in preserving the gut microbiota and its ability to prevent inflammation.

Neurogenic heterotopic ossification (NHO) is the formation of ectopic bone generally in muscles surrounding joints following spinal cord or brain injury. We investigated the mechanisms of NHO formation in 64 patients and a mouse model of spinal cord injury–induced NHO. We show that marrow from human NHOs contains hematopoietic stem cell (HSC) niches, in which mesenchymal stromal cells (MSCs) and endothelial cells provide an environment supporting HSC maintenance, proliferation, and differentiation. The transcriptomic signature of MSCs from NHOs shows a neuronal imprinting associated with a molecular network required for HSC support. We demonstrate that oncostatin M (OSM) produced by activated macrophages promotes osteoblastic differentiation and mineralization of human muscle-derived stromal cells surrounding NHOs. The key role of OSM was confirmed using an experimental model of NHO in mice defective for the OSM receptor (OSMR). Our results provide strong evidence that macrophages contribute to NHO formation through the osteogenic action of OSM on muscle cells within an inflammatory context and suggest that OSM/OSMR could be a suitable therapeutic target. Altogether, the evidence of HSCs in ectopic bones growing at the expense of soft tissue in spinal cord/brain-injured patients indicates that inflammation and muscle contribute to HSC regulation by the brain-bone-blood triad.

Background: During aging, hematopoietic stem cells (HSC) lose progressively both their self-renewal and differentiation potential. The precise molecular mechanisms of this phenomenon are not well established. To uncover the molecular events underlying this event, we have performed a bioinformatics analysis of 650 single-cell transcriptomes. Methods: Single-cell transcriptome analyses of expression heterogeneity, cell cycle, and cell trajectory in human cell compartment enriched in hematopoietic stem cell compartment were investigated in the bone marrow according to the age of the donors. Identification of aging-related nodules was identified by weighted correlation network analysis in this primitive compartment. Results: The analysis of single-cell transcriptomes allowed to uncover a major upregulation of EGR1 in human-aged lineage−CD34+CD38− cells which present cell cycle dysregulation with reduction of G2/M phase according to less expression of CCND2 during S phase. EGR1 upregulation in aging hematopoietic stem cells was found to be independent of cell cycle phases and gender. EGR1 expression trajectory in aged HSC highlighted a signature enriched in hematopoietic and immune disorders with the best induction of AP-1 complex and quiescence regulators such as EGR1, BTG2, JUNB, and NR41A. Sonic Hedgehog-related TMEM107 transmembrane molecule followed also EGR1 cell trajectory. EGR1-dependent gene weighted network analysis in human HSC-associated IER2 target protein-specific regulators of PP2A activity, IL1B, TNFSF10 ligands, and CD69, SELP membrane molecules in old HSC module with immune and leukemogenic signature. In contrast, for young HSC which were found with different cell cycle phase progression, its specific module highlighted upregulation of HIF1A hypoxic factor, PDE4B immune marker, DRAK2 (STK17B) T cell apoptosis regulator, and MYADM myeloid-associated marker. Conclusion: EGR1 was found to be connected to the aging of human HSC and highlighted a specific cell trajectory contributing to the dysregulation of an inflammatory and leukemia-related transcriptional program in aged human HSCs. EGR1 and its program were found to be connected to the aging of human HSC with dissociation of quiescence property and cell cycle phase progression in this primitive hematopoietic compartment.

The circadian clock coordinates biological and physiological functions to day/night cycles. The perturbation of the circadian clock increases cancer risk and affects cancer progression. Here, we studied how BMAL1 knockdown (BMAL1-KD) by shRNA affects the epithelial–mesenchymal transition (EMT), a critical early event in the invasion and metastasis of colorectal carcinoma (CRC). In corresponding to a gene set enrichment analysis, which showed a significant enrichment of EMT and invasive signatures in BMAL1_high CRC patients as compared to BMAL1_low CRC patients, our results revealed that BMAL1 is implicated in keeping the epithelial–mesenchymal equilibrium of CRC cells and influences their capacity of adhesion, migration, invasion, and chemoresistance. Firstly, BMAL1-KD increased the expression of epithelial markers (E-cadherin, CK-20, and EpCAM) but decreased the expression of Twist and mesenchymal markers (N-cadherin and vimentin) in CRC cell lines. Finally, the molecular alterations after BMAL1-KD promoted mesenchymal-to-epithelial transition-like changes mostly appeared in two primary CRC cell lines (i.e., HCT116 and SW480) compared to the metastatic cell line SW620. As a consequence, migration/invasion and drug resistance capacities decreased in HCT116 and SW480 BMAL1-KD cells. Together, BMAL1-KD alerts the delicate equilibrium between epithelial and mesenchymal properties of CRC cell lines, which revealed the crucial role of BMAL1 in EMT-related CRC metastasis and chemoresistance.

(1) Background: Triple-negative breast cancer (TNBC) is a distinct subgroup of breast cancer presenting a high level of recurrence, and neo-adjuvant chemotherapy is beneficial in its therapy management. Anti-PD-L1 immunotherapy improves the effect of neo-adjuvant therapy in TNBC. (2) Methods: Immune-modulation and ferroptosis-related R-packages were developed for integrative omics analyses under ferroptosis-inducer treatments: TNBC cells stimulated with ferroptosis inducers (GSE173905, GSE154425), single cell data (GSE191246) and mass spectrometry on breast cancer stem cells. Clinical association analyses were carried out with breast tumors (TCGA and METABRIC cohorts). Protein-level validation was investigated through protein atlas proteome experiments. (3) Results: Erastin/RSL3 ferroptosis inducers upregulate CD274 in TNBC cells (MDA-MB-231 and HCC38). In breast cancer, CD274 expression is associated with overall survival. Breast tumors presenting high expression of CD274 upregulated some ferroptosis drivers associated with prognosis: IDO1, IFNG and TNFAIP3. At the protein level, the induction of Cd274 and Tnfaip3 was confirmed in breast cancer stem cells under salinomycin treatment. In a 4T1 tumor treated with cyclophosphamide, the single cell expression of Cd274 was found to increase both in myeloid- and lymphoid-infiltrated cells, independently of its receptor Pdcd1. The CD274 ferroptosis-driver score computed on a breast tumor transcriptome stratified patients on their prognosis: low score was observed in the basal subgroup, with a higher level of recurrent risk scores (oncotypeDx, ggi and gene70 scores). In the METABRIC cohort, CD274, IDO1, IFNG and TNFAIP3 were found to be overexpressed in the TNBC subgroup. The CD274 ferroptosis-driver score was found to be associated with overall survival, independently of TNM classification and age diagnosis. The tumor expression of CD274, TNFAIP3, IFNG and IDO1, in a biopsy of breast ductal carcinoma, was confirmed at the protein level (4) Conclusions: Ferroptosis inducers upregulate PD-L1 in TNBC cells, known to be an effective target of immunotherapy in high-risk early TNBC patients who received neo-adjuvant therapy. Basal and TNBC tumors highly expressed CD274 and ferroptosis drivers: IFNG, TNFAIP3 and IDO1. The CD274 ferroptosis-driver score is associated with prognosis and to the risk of recurrence in breast cancer. A potential synergy of ferroptosis inducers with anti-PD-L1 immunotherapy is suggested for recurrent TNBC.