Osman, Abu

Abstract Oral commensals such as Parvimonas micra are increasingly implicated in sporadic colorectal cancer (CRC). Several findings support an emerging notion that alterations in host DNA methylation constitute a mechanism by which P. micra adapts to the host microenvironment and contributes to pathogenic processes. However, multiple limitations hinder definitive conclusions, including the correlative nature of the human studies and the absence of human microbiota in mouse models. Here, we investigated how a complex human microbiome influences the adaptation and oncogenic potential of P. micra in a spontaneous mouse model of polyposis. Adult mice, following antibiotic conditioning, received fecal microbiota transplants (FMT) from a healthy human donor, with or without supplementation of P. micra. P. micra caused significant reshaping of the gut microbial community and altered co-occurrence network structures, which however largely recovered within 21 days post FMT. Despite the transient nature of these changes, P. micra caused a marked increase in both the number and invasive potential of intestinal polyps. Epigenomic profiling of polyp crypts revealed global DNA hypomethylation alongside gene-specific hypermethylation, accompanied by transcriptional changes in key oncogenic pathways. Affected pathways included Wnt signaling, epithelial-to-mesenchymal transition, circadian rhythm regulation, and host–microbe interactions. These findings demonstrate that P. micra exerts a profound influence on microbial ecology, host epigenetic programming, and tumorigenesis in a genetically predisposed host, highlighting its role as a microbial driver of intestinal carcinogenesis.

Abstract Colorectal cancer (CRC) is one of the most common cancers in the world, yet it remains one of the most difficult to treat. Immunotherapy of CRC using checkpoint inhibitors is only efficacious in approximately 4% of patients with high microsatellite instability (MSI-H). In earlier studies we showed that in patients with inflammatory bowel diseases (IBD) and CRC as well as in the APCΔ468 murine polyposis model, Tregs overexpress β-catenin. Furthermore, we demonstrated that expression of a dominant active β-catenin in Tregs induces RORγt, compromising suppressive activity and promoting TH17-like properties. This results in a scurfy-like phenotype. By contrast loss of TCF-1 upregulated RORγt, but only compromised Treg anti-inflammatory functions. These observations suggested cooperation of β-catenin and TCF-1 in regulating Treg plasticity. We show here that in healthy mice, β-catenin expression is significantly higher in Tregs as compared to conventional CD4+ T-cells. In Tregs that express a dominant active β-catenin, ablation of TCF-1 or HEB provide partial rescue, while ablation of both TCF-1 and HEB recovers Treg numbers and prevents the scurfy-like phenotype. These observations suggest that TCF-1, HEB and β-catenin cooperate to determine Treg functions. In search for additional partners, we show that loss of TCF-1 upregulates SATB-1, a chromatin modifier that is known to determine T-cell development and function. Upregulation of SATB-1 is enhanced with the loss of both TCF-1 and HEB. All three transcription factors upregulate RORγt suggesting that they cooperatively determine Treg function and plasticity. Elucidating this cooperation will enhance our ability to control Treg plasticity in health and disease. NIH/NIAID - R01 AI108682-0A1

Abstract The transcription factor TCF-1 is essential for the development and function of T regulatory (Treg) cells, however its function is poorly understood. Here, we show that TCF-1 primarily suppresses transcription of genes that are co-bound by Foxp3. Single-cell RNA-seq analysis identified effector- and central-memory Treg-cells with differential expression of Klf2 and memory and activation markers. TCF-1 deficiency did not change the core Treg transcriptional signature, but promoted alternative signaling pathways whereby Treg-cells became activated and gained gut-homing and TH17 characteristics. TCF-1-deficient Treg-cells strongly suppressed T-cell proliferation and cytotoxicity, but were compromised in controlling CD4+ T-cell polarization and inflammation. In mice with polyposis, Treg cell-specific TCF-1 deficiency promoted tumor growth. Consistently, tumor-infiltrating Treg cells of colorectal cancer patients showed lower TCF-1 expression and increased TH17 expression signatures compared to adjacent normal tissue and circulating T-cells. Thus, Treg cell-specific TCF-1 expression differentially regulates TH17-mediated inflammation and T-cell cytotoxicity, and can determine colorectal cancer outcome. Supported by NIH R01 AI 108682 (FG & KK), NIH RO1 AI 147652 (FG), NIH R35GM138283 (MK), and Praespero Innovation Award Alberta, Canada (FG & KK)

Abstract TCF1 is essential for the development and function of T regulatory cells (Tregs). However, how TCF1 regulates Treg function in homeostasis or under pathogenic conditions is poorly understood. Here, we ablated TCF1 in Tregs to elucidate its role in Treg specification in healthy mice and mice with colon cancer. RNAseq revealed that TCF1-deficient Tregs maintain their core transcriptional signature, but promote T-cell receptor, Tgfβ receptor, TH17, and Wnt/β-catenin signaling pathways. Single-cell RNAseq identified central-memory-Tregs with low Klf2 or high Mif expression, which upon downregulation of TCF1 gained TH17 characteristics and matured into Maf expressing effector Tregs. Tcf1-deficient Tregs exhibited enhanced suppression of T-cell proliferation and cytotoxicity but were compromised in controlling CD4+ T-cell polarization and inflammation. In mice with polyposis, Tcf1-deficient Tregs promoted inflammation and tumor growth. Thus, TCF1 differentially regulates Treg control of TH17 inflammation and T-cell cytotoxicity, and its action in Tregs determines colorectal cancer outcome.

Abstract The molecular and functional diversity of regulatory T-cells (Tregs) in health and in disease remains unclear. We previously described in colorectal cancer (CRC) patients a subpopulation of RORγt+ Tregs with elevated expression of β-catenin and pro-inflammatory properties. Here we observed progressive expansion of RORγt+ Tregs in inflammatory bowel disease (IBD) patients during inflammation and early dysplasia. Activating Wnt/β-catenin signaling in human and murine Tregs was sufficient to recapitulate the disease-associated increase in frequencies of RORγt+ Tregs expressing IL-17, IFN-γ, and TNFa. We found that binding of the β-catenin interacting partner, TCF-1, to DNA overlapped with Foxp3 binding at enhancer sites of pro-inflammatory pathway genes. Sustained Wnt/β-catenin activation induced newly accessible chromatin sites in these genes and upregulated their expression. These findings indicate that TCF-1 and Foxp3 together limit the expression of pro-inflammatory genes in Tregs. Activation of ꞵ-catenin signaling interferes with this function and promotes the disease-associated RORγt+ Treg phenotype.

Significance We show that distinct subsets of mast cells (MCs) expand with sequential oncogenic events in small bowel cancer. Mucosal mast cells (MMCs) previously detected early during Trichinella spiralis infection expand in adenomatous polyps in an IL-10–dependent manner. Connective tissue mast cells (CTMCs), earlier shown to expand during the resolution of inflammation following clearance of T. spiralis, are independent of IL-10 and associate with the transition of polyps to adenocarcinoma. IL-33 upregulates the CTMC lineage-specific protease murine mast cell protease 6 (mMCP6). Ablation of mMCP6 attenuates tumor growth. Thus, tissue sentinel cells respond to oncogenic events and cellular transformation in effect to help promote cancer. Delineating the types of MCs present at various stages of disease offers actionable cellular targets for therapeutic intervention in disease progression.