The myeloid immune signature of Enterotoxigenic Bacteroides fragilis-induced murine colon tumorigenesis

Enterotoxigenic Bacteroides fragilis (ETBF), a human commensal and candidate pathogen in colorectal cancer (CRC), is a potent initiator of IL-17-dependent colon tumorigenesis in Min mice. In Chapters 2 and 3, we examined the role of IL-17 and ETBF on the differentiation of myeloid cells into myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM), which are known to promote tumorigenesis. The myeloid compartment associated with ETBF-induced colon tumorigenesis in Min mice was defined using flow cytometry and gene expression profiling. Cell sorted immature myeloid cells were functionally assayed for inhibition of T cell proliferation in order to delineate MDSC populations. A comparison of ETBF infection to that with other oncogenic bacteria (Fusobacterium nucleatum or pks+ E. coli) revealed a specific, ETBF-associated colonic immune infiltrate. ETBF-triggered colon tumorigenesis is associated with an IL-17-driven myeloid signature characterized by subversion of steady-state myelopoiesis in favor of the generation of protumoral monocytic (MO)-MDSC. Combined action of the Bacteroides fragilis enterotoxin bft and IL-17 on colonic epithelial cells promoted the differentiation of MO-MDSC, which selectively upregulated Arg1 and Nos2, produced NO and suppressed T cell proliferation. Evidence of a pathogenic inflammatory signature in humans colonized with ETBF may allow for the identification of populations at risk for developing colon cancer. In Chapter 4, we describe targeting MDSC with the aim of suppressing tumorigenesis. CXCR2, an inflammatory chemokine receptor expressed on neutrophils and granulocytic (PMN)-MDSC, was a strong modifier of ETBF-Min tumorigenesis. Inhibition of CXCR2, via genetic knockout or a synthetic peptide antagonist, pepducin, significantly decreased recruitment of PMN-MDSC to the colon, increased numbers of M1 macrophages and was anti-tumoral. Specific inhibitors of MDSC targeting CXC receptors are under development and may emerge as a novel arm in immunotherapy

The microbial metabolite desaminotyrosine enhances T-cell priming and cancer immunotherapy with immune checkpoint inhibitors

Background Inter-individual differences in response to immune checkpoint inhibitors (ICI) remain a major challenge in cancer treatment. The composition of the gut microbiome has been associated with differential ICI outcome, but the underlying molecular mechanisms remain unclear, and therapeutic modulation challenging. Methods We established an in vivo model to treat C57Bl/6j mice with the type-I interferon (IFN-I)-modulating, bacterial-derived metabolite desaminotyrosine (DAT) to improve ICI therapy. Broad spectrum antibiotics were used to mimic gut microbial dysbiosis and associated ICI resistance. We utilized genetic mouse models to address the role of host IFN-I in DAT-modulated antitumour immunity. Changes in gut microbiota were assessed using 16S-rRNA sequencing analyses. Findings We found that oral supplementation of mice with the microbial metabolite DAT delays tumour growth and promotes ICI immunotherapy with anti-CTLA-4 or anti-PD-1. DAT-enhanced antitumour immunity was associated with more activated T cells and natural killer cells in the tumour microenvironment and was dependent on host IFN-I signalling. Consistent with this, DAT potently enhanced expansion of antigen-specific T cells following vaccination with an IFN-I-inducing adjuvant. DAT supplementation in mice compensated for the negative effects of broad-spectrum antibiotic-induced dysbiosis on anti-CTLA-4-mediated antitumour immunity. Oral administration of DAT altered the gut microbial composition in mice with increased abundance of bacterial taxa that are associated with beneficial response to ICI immunotherapy. Interpretation We introduce the therapeutic use of an IFN-I-modulating bacterial-derived metabolite to overcome resistance to ICI. This approach is a promising strategy particularly for patients with a history of broad-spectrum antibiotic use and associated loss of gut microbial diversity

The myeloid immune signature of Enterotoxigenic Bacteroides fragilis-induced murine colon tumorigenesis

Enterotoxigenic Bacteroides fragilis (ETBF), a human commensal and candidate pathogen in colorectal cancer (CRC), is a potent initiator of IL-17-dependent colon tumorigenesis in Min mice. In Chapters 2 and 3, we examined the role of IL-17 and ETBF on the differentiation of myeloid cells into myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM), which are known to promote tumorigenesis. The myeloid compartment associated with ETBF-induced colon tumorigenesis in Min mice was defined using flow cytometry and gene expression profiling. Cell sorted immature myeloid cells were functionally assayed for inhibition of T cell proliferation in order to delineate MDSC populations. A comparison of ETBF infection to that with other oncogenic bacteria (Fusobacterium nucleatum or pks+ E. coli) revealed a specific, ETBF-associated colonic immune infiltrate. ETBF-triggered colon tumorigenesis is associated with an IL-17-driven myeloid signature characterized by subversion of steady-state myelopoiesis in favor of the generation of protumoral monocytic (MO)-MDSC. Combined action of the Bacteroides fragilis enterotoxin bft and IL-17 on colonic epithelial cells promoted the differentiation of MO-MDSC, which selectively upregulated Arg1 and Nos2, produced NO and suppressed T cell proliferation. Evidence of a pathogenic inflammatory signature in humans colonized with ETBF may allow for the identification of populations at risk for developing colon cancer. In Chapter 4, we describe targeting MDSC with the aim of suppressing tumorigenesis. CXCR2, an inflammatory chemokine receptor expressed on neutrophils and granulocytic (PMN)-MDSC, was a strong modifier of ETBF-Min tumorigenesis. Inhibition of CXCR2, via genetic knockout or a synthetic peptide antagonist, pepducin, significantly decreased recruitment of PMN-MDSC to the colon, increased numbers of M1 macrophages and was anti-tumoral. Specific inhibitors of MDSC targeting CXC receptors are under development and may emerge as a novel arm in immunotherapy

Targeting nucleic acid sensors in tumor cells to reprogram biogenesis and RNA cargo of extracellular vesicles for T cell-mediated cancer immunotherapy

Tumor-derived extracellular vesicles (EVs) have been associated with immune evasion and tumor progression. We show that the RNA-sensing receptor RIG-I within tumor cells governs biogenesis and immunomodulatory function of EVs. Cancer-intrinsic RIG-I activation releases EVs, which mediate dendritic cell maturation and T cell antitumor immunity, synergizing with immune checkpoint blockade. Intact RIG-I, autocrine interferon signaling, and the GTPase Rab27a in tumor cells are required for biogenesis of immunostimulatory EVs. Active intrinsic RIG-I signaling governs composition of the tumor EV RNA cargo including small non-coding stimulatory RNAs. High transcriptional activity of EV pathway genes and RIG-I in melanoma samples associate with prolonged patient survival and beneficial response to immunotherapy. EVs generated from human melanoma after RIG-I stimulation induce potent antigen-specific T cell responses. We thus define a molecular pathway that can be targeted in tumors to favorably alter EV immunomodulatory function. We propose “reprogramming” of tumor EVs as a personalized strategy for T cell-mediated cancer immunotherapy

Bacteroides fragilis Toxin Coordinates a Pro-carcinogenic Inflammatory Cascade via Targeting of Colonic Epithelial Cells

Pro-carcinogenic bacteria have the potential to initiate and/or promote colon cancer, in part via immune mechanisms that are incompletely understood. Using ApcMin mice colonized with the human pathobiont enterotoxigenic Bacteroides fragilis (ETBF) as a model of microbe-induced colon tumorigenesis, we show that the Bacteroides fragilis toxin (BFT) triggers a pro-carcinogenic, multi-step inflammatory cascade requiring IL-17R, NF-κB, and Stat3 signaling in colonic epithelial cells (CECs). Although necessary, Stat3 activation in CECs is not sufficient to trigger ETBF colon tumorigenesis. Notably, IL-17-dependent NF-κB activation in CECs induces a proximal to distal mucosal gradient of C-X-C chemokines, including CXCL1, that mediates the recruitment of CXCR2-expressing polymorphonuclear immature myeloid cells with parallel onset of ETBF-mediated distal colon tumorigenesis. Thus, BFT induces a pro-carcinogenic signaling relay from the CEC to a mucosal Th17 response that results in selective NF-κB activation in distal colon CECs, which collectively triggers myeloid-cell-dependent distal colon tumorigenesis

Bacteroides fragilis Toxin Coordinates a Pro-carcinogenic Inflammatory Cascade via Targeting of Colonic Epithelial Cells

Pro-carcinogenic bacteria have the potential to initiate and/or promote colon cancer, in part via immune mechanisms that are incompletely understood. Using ApcMin mice colonized with the human pathobiont enterotoxigenic Bacteroides fragilis (ETBF) as a model of microbe-induced colon tumorigenesis, we show that the Bacteroides fragilis toxin (BFT) triggers a pro-carcinogenic, multi-step inflammatory cascade requiring IL-17R, NF-κB, and Stat3 signaling in colonic epithelial cells (CECs). Although necessary, Stat3 activation in CECs is not sufficient to trigger ETBF colon tumorigenesis. Notably, IL-17-dependent NF-κB activation in CECs induces a proximal to distal mucosal gradient of C-X-C chemokines, including CXCL1, that mediates the recruitment of CXCR2-expressing polymorphonuclear immature myeloid cells with parallel onset of ETBF-mediated distal colon tumorigenesis. Thus, BFT induces a pro-carcinogenic signaling relay from the CEC to a mucosal Th17 response that results in selective NF-κB activation in distal colon CECs, which collectively triggers myeloid-cell-dependent distal colon tumorigenesis

XIAP restrains TNF-driven intestinal inflammation and dysbiosis by promoting innate immune responses of Paneth and dendritic cells

Deficiency in X-linked inhibitor of apoptosis protein (XIAP) is the cause for X-linked lymphoproliferative syndrome 2 (XLP2). About one-third of these patients suffer from severe and therapy-refractory inflammatory bowel disease (IBD), but the exact cause of this pathogenesis remains undefined. Here, we used XIAP-deficient mice to characterize the mechanisms underlying intestinal inflammation. In Xiap-/- mice, we observed spontaneous terminal ileitis and microbial dysbiosis characterized by a reduction of Clostridia species. We showed that in inflamed mice, both TNF receptor 1 and 2 (TNFR1/2) cooperated in promoting ileitis by targeting TLR5-expressing Paneth cells (PCs) or dendritic cells (DCs). Using intestinal organoids and in vivo modeling, we demonstrated that TLR5 signaling triggered TNF production, which induced PC dysfunction mediated by TNFR1. TNFR2 acted upon lamina propria immune cells. scRNA-seq identified a DC population expressing TLR5, in which Tnfr2 expression was also elevated. Thus, the combined activity of TLR5 and TNFR2 signaling may be responsible for DC loss in lamina propria of Xiap-/- mice. Consequently, both Tnfr1-/-Xiap-/- and Tnfr2-/-Xiap-/- mice were rescued from dysbiosis and intestinal inflammation. Furthermore, RNA-seq of ileal crypts revealed that in inflamed Xiap-/- mice, TLR5 signaling was abrogated, linking aberrant TNF responses with the development of a dysbiosis. Evidence for TNFR2 signaling driving intestinal inflammation was detected in XLP2 patient samples. Together, these data point toward a key role of XIAP in mediating resilience of TLR5-expressing PCs and intestinal DCs, allowing them to maintain tissue integrity and microbiota homeostasis