Does Ta Low-grade Urothelial Carcinoma of the Bladder With Focal High-grade Features Carry Worse Prognosis? The Roswell Park Comprehensive Cancer Center Experience.

OBJECTIVE: To describe the management and outcomes of patients with Ta predominantly low-grade urothelial carcinoma with focal high-grade features (FHG) (<5%), compared to those with Ta low grade (LG) and Ta high grade (HG). METHODS: Retrospective review of all patients who underwent transurethral resection of bladder tumor between 2005 and 2023. Patients with Ta disease were identified and categorized into LG, FHG, and HG. Kaplan Meier method was used to depict high-grade recurrence, T-stage progression, and radical cystectomy-free survival. RESULTS: Four hundred forty-nine patients with Ta disease were identified (LG 48%, FHG 12%, and HG 40%). Patients with FHG (32%) had a second-look transurethral resection of bladder tumor more frequently compared to LG (7%) and HG (29%) (P <.01). They received intravesical therapy more frequently compared to LG (36% vs 20%) but lower than HG (55%) (P <.01). They received radical cystectomy less frequently (7% compared to 20% for HG and 11% for LG, P = .01). HG recurrence-free survival at 1, 3, and 5years was HG (68%, 52%, and 43%), FHG (74%, 53%, and 49%), and LG (87%, 79%, and 73%) (log-rank P <.01). T progression-free survival at 1, 3, and 5years was HG (84%, 77%, and 70%), FHG (92%, 82%, and 82%), and LG (94%, 89%, and 85%) (log-rank P = .02). Cystectomy-free survival at 1, 3, and 5years was HG (92%, 84%, and 80%), FHG (96%, 94%, and 94%), and LG (99%, 95%, and 92%) (log-rank P <.01). CONCLUSION: Patients with Ta FHG seem to behave more like Ta HG disease in terms of high-grade recurrences, but they are less likely to experience T-stage progression and convert to cystectomy.

Intestinal epithelial HDAC3 and MHC class II coordinate microbiota-specific immunity.

Aberrant immune responses to resident microbes promote inflammatory bowel disease and other chronic inflammatory conditions. However, how microbiota-specific immunity is controlled in mucosal tissues remains poorly understood. Here, we found that mice lacking epithelial expression of microbiota-sensitive histone deacetylase 3 (HDAC3) exhibited increased accumulation of commensal-specific CD4+ T cells in the intestine, provoking the hypothesis that epithelial HDAC3 may instruct local microbiota-specific immunity. Consistent with this, microbiota-specific CD4+ T cells and epithelial HDAC3 expression were concurrently induced following early-life microbiota colonization. Further, epithelium-intrinsic ablation of HDAC3 decreased commensal-specific Tregs, increased commensal-specific Th17 cells, and promoted T cell-driven colitis. Mechanistically, HDAC3 was essential for NF-κB-dependent regulation of epithelial MHC class II (MHCII). Epithelium-intrinsic MHCII dampened local accumulation of commensal-specific Th17 cells in adult mice and protected against microbiota-triggered inflammation. Remarkably, HDAC3 enabled the microbiota to induce MHCII expression on epithelial cells and limit the number of commensal-specific T cells in the intestine. Collectively, these data reveal a central role for an epithelial histone deacetylase in directing the dynamic balance of tissue-intrinsic CD4+ T cell subsets that recognize commensal microbes and control inflammation.

Dietary phytate primes epithelial antibacterial immunity in the intestine.

Although diet has long been associated with susceptibility to infection, the dietary components that regulate host defense remain poorly understood. Here, we demonstrate that consuming rice bran decreases susceptibility to intestinal infection with Citrobacter rodentium, a murine pathogen that is similar to enteropathogenic E. coli infection in humans. Rice bran naturally contains high levels of the substance phytate. Interestingly, phytate supplementation also protected against intestinal infection, and enzymatic metabolism of phytate by commensal bacteria was necessary for phytate-induced host defense. Mechanistically, phytate consumption induced mammalian intestinal epithelial expression of STAT3-regulated antimicrobial pathways and increased phosphorylated STAT3, suggesting that dietary phytate promotes innate defense through epithelial STAT3 activation. Further, phytate regulation of epithelial STAT3 was mediated by the microbiota-sensitive enzyme histone deacetylase 3 (HDAC3). Collectively, these data demonstrate that metabolism of dietary phytate by microbiota decreases intestinal infection and suggests that consuming bran and other phytate-enriched foods may represent an effective dietary strategy for priming host immunity.

Commensal segmented filamentous bacteria-derived retinoic acid primes host defense to intestinal infection.

Interactions between the microbiota and mammalian host are essential for defense against infection, but the microbial-derived cues that mediate this relationship remain unclear. Here, we find that intestinal epithelial cell (IEC)-associated commensal bacteria, segmented filamentous bacteria (SFB), promote early protection against the pathogen Citrobacter rodentium, independent of CD4+ T cells. SFB induced histone modifications in IECs at sites enriched for retinoic acid receptor motifs, suggesting that SFB may enhance defense through retinoic acid (RA). Consistent with this, inhibiting RA signaling suppressed SFB-induced protection. Intestinal RA levels were elevated in SFB mice, despite the inhibition of mammalian RA production, indicating that SFB directly modulate RA. Interestingly, RA was produced by intestinal bacteria, and the loss of bacterial-intrinsic aldehyde dehydrogenase activity decreased the RA levels and increased infection. These data reveal RA as an unexpected microbiota-derived metabolite that primes innate defense and suggests that pre- and probiotic approaches to elevate RA could prevent or combat infections.

Microbiota-derived metabolite promotes HDAC3 activity in the gut.

The coevolution of mammalian hosts and their beneficial commensal microbes has led to development of symbiotic host-microbiota relationships1. Epigenetic machinery permits mammalian cells to integrate environmental signals2; however, how these pathways are fine-tuned by diverse cues from commensal bacteria is not well understood. Here we reveal a highly selective pathway through which microbiota-derived inositol phosphate regulates histone deacetylase 3 (HDAC3) activity in the intestine. Despite the abundant presence of HDAC inhibitors such as butyrate in the intestine, we found that HDAC3 activity was sharply increased in intestinal epithelial cells of microbiota-replete mice compared with germ-free mice. This divergence was reconciled by the finding that commensal bacteria, including Escherichia coli, stimulated HDAC activity through metabolism of phytate and production of inositol-1,4,5-trisphosphate (InsP3). Both intestinal exposure to InsP3 and phytate ingestion promoted recovery following intestinal damage. Of note, InsP3 also induced growth of intestinal organoids derived from human tissue, stimulated HDAC3-dependent proliferation and countered butyrate inhibition of colonic growth. Collectively, these results show that InsP3 is a microbiota-derived metabolite that activates a mammalian histone deacetylase to promote epithelial repair. Thus, HDAC3 represents a convergent epigenetic sensor of distinct metabolites that calibrates host responses to diverse microbial signals.

Cell type-specific mechanisms coupling protease-activated receptor-1 to infectious colitis pathogenesis.

BACKGROUND: Protease-activated receptor-1 (PAR-1) plays a major role in multiple disease processes, including colitis. Understanding the mechanisms coupling PAR-1 to disease pathogenesis is complicated by the fact that PAR-1 is broadly expressed across multiple cell types. OBJECTIVE: Determine the specific contributions of PAR-1 expressed by macrophages and colonic enterocytes to infectious colitis. METHODS: Mice carrying a conditional PAR-1 allele were generated and bred to mice expressing Cre recombinase in a myeloid- (PAR-1ΔM ) or enterocyte-specific (PAR-1ΔEPI ) fashion. Citrobacter rodentium colitis pathogenesis was analyzed in mice with global PAR-1 deletion (PAR-1-/- ) and cell type-specific deletions. RESULTS: Constitutive deletion of PAR-1 had no significant impact on weight loss, crypt hypertrophy, crypt abscess formation, or leukocyte infiltration in Citrobacter colitis. However, colonic shortening was significantly blunted in infected PAR-1-/- mice, and these animals exhibited decreased local levels of IL-1ß, IL-22, IL-6, and IL-17A. In contrast, infected PAR-1ΔM mice lost less weight and had fewer crypt abscesses relative to controls. PAR-1ΔM mice had diminished CD3+ T cell infiltration into colonic tissue, but macrophage and CD4+ T cell infiltration were similar to controls. Also contrasting results in global knockouts, PAR-1ΔM mice exhibited lower levels of IL-1ß, but not Th17-related cytokines (ie, IL-22, IL-6, IL-17A). Infected PAR-1ΔEPI mice exhibited increased crypt hypertrophy and crypt abscess formation, but local cytokine elaboration was similar to controls. CONCLUSIONS: These studies reveal complex, cell type-specific roles for PAR-1 in modulating the immune response to Citrobacter colitis that are not readily apparent in analyses limited to mice with global PAR-1 deficiency.

Microbiota Inhibit Epithelial Pathogen Adherence by Epigenetically Regulating C-Type Lectin Expression.

Numerous bacterial pathogens infect the mammalian host by initially associating with epithelial cells that line the intestinal lumen. Recent work has revealed that commensal bacteria that reside in the intestine promote defense against pathogenic infection, however whether the microbiota direct host pathways that alter pathogen adherence is not well-understood. Here, by comparing germ-free mice, we identify that the microbiota decrease bacterial pathogen adherence and dampen epithelial expression of the cell surface glycoprotein C-type lectin 2e (Clec2e). Functional studies revealed that overexpression of this lectin promotes adherence of intestinal bacterial pathogens to mammalian cells. Interestingly, microbiota-sensitive downregulation of Clec2e corresponds with decreased histone acetylation of the Clec2e gene in intestinal epithelial cells. Histone deacetylation and transcriptional regulation of Clec2e depends on expression and recruitment of the histone deacetylase HDAC3. Thus, commensal bacteria epigenetically instruct epithelial cells to decrease expression of a C-type lectin that promotes pathogen adherence, revealing a novel mechanism for how the microbiota promote innate defense against infection.

Microbiota-sensitive epigenetic signature predicts inflammation in Crohn's disease.

Altered response to the intestinal microbiota strongly associates with inflammatory bowel disease (IBD); however, how commensal microbial cues are integrated by the host during the pathogenesis of IBD is not understood. Epigenetics represents a potential mechanism that could enable intestinal microbes to modulate transcriptional output during the development of IBD. Here, we reveal a histone methylation signature of intestinal epithelial cells isolated from the terminal ilea of newly diagnosed pediatric IBD patients. Genes characterized by significant alterations in histone H3-lysine 4 trimethylation (H3K4me3) showed differential enrichment in pathways involving immunoregulation, cell survival and signaling, and metabolism. Interestingly, a large subset of these genes was epigenetically regulated by microbiota in mice and several microbiota-sensitive epigenetic targets demonstrated altered expression in IBD patients. Remarkably though, a substantial proportion of these genes exhibited H3K4me3 levels that correlated with the severity of intestinal inflammation in IBD, despite lacking significant differential expression. Collectively, these data uncover a previously unrecognized epigenetic profile of IBD that can be primed by commensal microbes and indicate sensitive targets in the epithelium that may underlie how microbiota predispose to subsequent intestinal inflammation and disease.

Disruption of Epithelial HDAC3 in Intestine Prevents Diet-Induced Obesity in Mice.

BACKGROUND & AIMS: Intestinal microbiota modulate metabolism and associate closely with epithelial cells in the intestine. In intestinal epithelial cells (IECs), histone deacetylase 3 (HDAC3) integrates microbiota-derived signals to control intestinal homeostasis. We investigated whether HDAC3 in IECs regulates metabolism and the development of obesity in mice. METHODS: Adult C57BL/6 (control) mice and mice with constitutive or inducible IEC-specific disruption of Hdac3 (HDAC3ΔIEC mice) were placed on a standard chow or high-fat diet (HFD, 60% kcal from fat). We measured body composition, weight, glucose tolerance, and energy expenditure. IECs were isolated from small intestine and gene expression, and lipid levels were analyzed. HDAC3 levels were determined in 43 pediatric patient ileal biopsy samples and compared with body weight. RESULTS: Control mice fed an HFD gained weight, became obese, and had reduced glucose tolerance with increased serum insulin, whereas HFD-fed HDAC3ΔIEC mice did not develop obesity. Serum levels of triglycerides were reduced in HDAC3ΔIEC mice, and these mice had less liver fat and smaller adipocytes, compared with HFD-fed control mice. HDAC3ΔIEC mice had similar food intake and activity as control mice, but higher energy expenditure because of increased catabolism. IECs from HDAC3ΔIEC mice had altered expression levels of genes that regulate metabolism in response to the microbiota (such as Chka, Mttp, Apoa1, and Pck1) and accumulated triglycerides compared with IECs from control mice. The microbiota-derived short-chain fatty acid butyrate was decreased in obese mice. Butyrate significantly reduced the activity of HDAC3 and increased Pck1 expression in only control IECs. Administration of butyrate to control mice with diet-induced obesity, but not HDAC3ΔIEC mice, led to significant weight loss. Disruption of HDAC3 in IECs of mice after they became obese led to weight loss and improved metabolic profile. Levels of HDAC3 in intestinal biopsy samples correlated with patient weight. CONCLUSIONS: We found that epithelial HDAC3 promotes development of diet-induced obesity in studies of mice and that butyrate reduces activity of HDAC3 in IECs to prevent diet-induced obesity. This pathway might be manipulated to prevent or reduce obesity-associated disease.

Commensal Fungi Recapitulate the Protective Benefits of Intestinal Bacteria.

Commensal intestinal microbes are collectively beneficial in preventing local tissue injury and augmenting systemic antimicrobial immunity. However, given the near-exclusive focus on bacterial species in establishing these protective benefits, the contributions of other types of commensal microbes remain poorly defined. Here, we show that commensal fungi can functionally replace intestinal bacteria by conferring protection against injury to mucosal tissues and positively calibrating the responsiveness of circulating immune cells. Susceptibility to colitis and influenza A virus infection occurring upon commensal bacteria eradication is efficiently overturned by mono-colonization with either Candida albicans or Saccharomyces cerevisiae. The protective benefits of commensal fungi are mediated by mannans, a highly conserved component of fungal cell walls, since intestinal stimulation with this moiety alone overrides disease susceptibility in mice depleted of commensal bacteria. Thus, commensal enteric fungi safeguard local and systemic immunity by providing tonic microbial stimulation that can functionally replace intestinal bacteria.

Epithelial Histone Deacetylase 3 Instructs Intestinal Immunity by Coordinating Local Lymphocyte Activation.

Mucosal tissues are constantly in direct contact with diverse beneficial and pathogenic microbes, highlighting the need for orchestrating complex microbial signals to sustain effective host defense. Here, we show an essential role for intestinal epithelial cell expression of histone deacetylase 3 (HDAC3) in responding to pathogenic microbes and activating protective innate immunity. Mice lacking HDAC3 in intestinal epithelial cells were more susceptible to Citrobacter rodentium when under tonic stimulation by the commensal microbiota. This impaired host defense reflected significantly decreased IFNγ production by intraepithelial CD8+ T cells early during infection. Further, HDAC3 was necessary for infection-induced epithelial expression of the IFNγ-inducing factor IL-18, and administration of IL-18 restored IFNγ activity to resident CD8+ T cells and reduced infection. Thus, HDAC3 mediates communication between intestinal epithelial cells and resident lymphocytes, revealing that epithelial priming by an epigenetic modifier may direct mucosal regulation of host defense against pathogenic microbes.