FOXO3 Deficiency in Neutrophils Drives Colonic Inflammation and Tumorigenesis.

Inflammatory bowel disease (IBD), characterized by infiltration of polymorphonuclear neutrophils (PMNs), increases the risk of colon cancer. PMN activation corresponds to the accumulation of intracellular Lipid Droplets (LDs). As increased LDs are negatively regulated by transcription factor Forkhead Box O3 (FOXO3), we aim to determine the significance of this regulatory network in PMN-mediated IBD and tumorigenesis. Affected tissue of IBD and colon cancer patients, colonic and infiltrated immune cells, have increased LDs' coat protein, PLIN2. Mouse peritoneal PMNs with stimulated LDs and FOXO3 deficiency have elevated transmigratory activity. Transcriptomic analysis of these FOXO3-deficient PMNs showed differentially expressed genes (DEGs; FDR < 0.05) involved in metabolism, inflammation, and tumorigenesis. Upstream regulators of these DEGs, similar to colonic inflammation and dysplasia in mice, were linked to IBD and human colon cancer. Additionally, a transcriptional signature representing FOXO3-deficient PMNs (PMN-FOXO3389) separated transcriptomes of affected tissue in IBD (p = 0.00018) and colon cancer (p = 0.0037) from control. Increased PMN-FOXO3389 presence predicted colon cancer invasion (lymphovascular p = 0.015; vascular p = 0.046; perineural p = 0.03) and poor survival. Validated DEGs from PMN-FOXO3389 (P2RX1, MGLL, MCAM, CDKN1A, RALBP1, CCPG1, PLA2G7) are involved in metabolism, inflammation, and tumorigenesis (p < 0.05). These findings highlight the significance of LDs and FOXO3-mediated PMN functions that promote colonic pathobiology.

The ClC-2 Chloride Channel Activator, Lubiprostone, Improves Intestinal Barrier Function in Biopsies from Crohn's Disease but Not Ulcerative Colitis Patients.

The prostone analog, lubiprostone, is approved to manage constipation-predominant irritable bowel syndrome. Lubiprostone also protects intestinal mucosal barrier function in animal models of colitis. The aim of this study was to determine if lubiprostone improves barrier properties in isolated colonic biopsies from Crohn's disease (CD) and ulcerative colitis (UC) patients. Sigmoid colon biopsies from healthy subjects, CD and UC patients in remission, and CD patients with active disease were mounted in Ussing chambers. Tissues were treated with lubiprostone or vehicle to determine the effects on transepithelial electrical resistance (TER), FITC-dextran 4kD (FD4) permeability, and electrogenic ion transport responses to forskolin and carbachol. Localization of the tight junction protein, occludin, was determined by immunofluorescence. Lubiprostone significantly increased ion transport across control, CD and UC remission biopsies but not active CD. Lubiprostone selectively improved TER in both CD remission and active disease biopsies but not in control or UC biopsies. The improved TER was associated with increased membrane localization of occludin. Lubiprostone selectively improved barrier properties of biopsies from CD patients vs. UC and independent of an ion transport response. These data indicate that lubiprostone has potential efficacy in improving mucosal integrity in Crohn's disease.

FOXO3 Expression in Macrophages Is Lowered by a High-Fat Diet and Regulates Colonic Inflammation and Tumorigenesis.

Obesity, characterized by augmented inflammation and tumorigenesis, is linked to genetic predispositions, such as FOXO3 polymorphisms. As obesity is associated with aberrant macrophages infiltrating different tissues, including the colon, we aimed to identify FOXO3-dependent transcriptomic changes in macrophages that drive obesity-mediated colonic inflammation and tumorigenesis. We found that in mouse colon, high-fat-diet-(HFD)-related obesity led to diminished FOXO3 levels and increased macrophages. Transcriptomic analysis of mouse peritoneal FOXO3-deficient macrophages showed significant differentially expressed genes (DEGs; FDR < 0.05) similar to HFD obese colons. These DEG-related pathways, linked to mouse colonic inflammation and tumorigenesis, were similar to those in inflammatory bowel disease (IBD) and human colon cancer. Additionally, we identified a specific transcriptional signature for the macrophage-FOXO3 axis (MAC-FOXO382), which separated the transcriptome of affected tissue from control in both IBD (p = 5.2 × 10−8 and colon cancer (p = 1.9 × 10−11), revealing its significance in human colonic pathobiologies. Further, we identified (heatmap) and validated (qPCR) DEGs specific to FOXO3-deficient macrophages with established roles both in IBD and colon cancer (IL-1B, CXCR2, S100A8, S100A9, and TREM1) and those with unexamined roles in these colonic pathobiologies (STRA6, SERPINH1, LAMB1, NFE2L3, OLR1, DNAJC28 and VSIG10). These findings establish an important understanding of how HFD obesity and related metabolites promote colonic pathobiologies.

Elevated ATGL in colon cancer cells and cancer stem cells promotes metabolic and tumorigenic reprogramming reinforced by obesity.

Obesity is a worldwide epidemic associated with increased risk and progression of colon cancer. Here, we aimed to determine the role of adipose triglyceride lipase (ATGL), responsible for intracellular lipid droplet (LD) utilization, in obesity-driven colonic tumorigenesis. In local colon cancer patients, significantly increased ATGL levels in tumor tissue, compared to controls, were augmented in obese individuals. Elevated ATGL levels in human colon cancer cells (CCC) relative to non-transformed were augmented by an obesity mediator, oleic acid (OA). In CCC and colonospheres, enriched in colon cancer stem cells (CCSC), inhibition of ATGL prevented LDs utilization and inhibited OA-stimulated growth through retinoblastoma-mediated cell cycle arrest. Further, transcriptomic analysis of CCC, with inhibited ATGL, revealed targeted pathways driving tumorigenesis, and high-fat-diet obesity facilitated tumorigenic pathways. Inhibition of ATGL in colonospheres revealed targeted pathways in human colonic tumor crypt base cells (enriched in CCSC) derived from colon cancer patients. In CCC and colonospheres, we validated selected transcripts targeted by ATGL inhibition, some with emerging roles in colonic tumorigeneses (ATG2B, PCK2, PGAM1, SPTLC2, IGFBP1, and ABCC3) and others with established roles (MYC and MUC2). These findings demonstrate obesity-promoted, ATGL-mediated colonic tumorigenesis and establish the therapeutic significance of ATGL in obesity-reinforced colon cancer progression.

Ulcerative colitis immune cell landscapes and differentially expressed gene signatures determine novel regulators and predict clinical response to biologic therapy.

The heterogeneous pathobiology underlying Ulcerative Colitis (UC) is not fully understood. Using publicly available transcriptomes from adult UC patients, we identified the immune cell landscape, molecular pathways, and differentially expressed genes (DEGs) across patient cohorts and their association with treatment outcomes. The global immune cell landscape of UC tissue included increased neutrophils, T CD4 memory activated cells, active dendritic cells (DC), and M0 macrophages, as well as reduced trends in T CD8, Tregs, B memory, resting DC, and M2 macrophages. Pathway analysis of DEGs across UC cohorts demonstrated activated bacterial, inflammatory, growth, and cellular signaling. We identified a specific transcriptional signature of one hundred DEGs (UC100) that distinctly separated UC inflamed from uninflamed transcriptomes. Several UC100 DEGs, with unidentified roles in UC, were validated in primary tissue. Additionally, non-responders to anti-TNFα and anti-α4ß7 therapy displayed distinct profiles of immune cells and pathways pertaining to inflammation, growth, and metabolism. We identified twenty resistant DEGs in UC non-responders to both therapies of which four had significant predictive power to treatment outcome. We demonstrated the global immune landscape and pathways in UC tissue, highlighting a unique UC signature across cohorts and a UC resistant signature with predictive performance to biologic therapy outcome.

STAT1 regulates interferon-γ-induced angiotensinogen and MCP-1 expression in a bidirectional manner in primary cultured mesangial cells.

OBJECTIVE: Intrarenal interferon-γ significantly contributes to the development of glomerular injury in which angiotensinogen and monocyte chemoattractant protein 1 levels are elevated. However, the exact nature of the role that interferon-γ plays in regulating angiotensinogen and monocyte chemoattractant protein 1 expression has not been fully delineated. Therefore, the aim of this study was to investigate the role that interferon-γ plays in angiotensinogen and monocyte chemoattractant protein 1 expression. METHODS: Primary cultured rat mesangial cells were treated with 0-20 ng/mL interferon-γ for 2, 8 or 24 hours. Expression levels of angiotensinogen, monocyte chemoattractant protein 1, suppressors of cytokine signaling 1, an intracellular suppressor of Janus kinase-signal transducers and activators of transcription signaling and activity of the Janus kinase-signal transducers and activators of transcription pathway were evaluated by reverse transcriptase polymerase chain reaction and western blot analysis. RESULTS: Interferon-γ increased angiotensinogen expression in mesangial cells with maximal augmentation observed following 5 ng/mL interferon-γ at 8 hours of treatment (1.87 ± 0.05, mRNA, relative ratio). Further increases were reduced or absent using higher concentrations of interferon-γ. Following treatments, monocyte chemoattractant protein 1 expression was induced in a linear dose-dependent manner (6.85 ± 0.62-fold by 20 ng/mL interferon-γ at 24 hours). In addition, interferon-γ induced STAT1 phosphorylation and suppressors of cytokine signaling 1 expression in a linear dose-dependent manner. The suppression of STAT1 and suppressors of cytokine signaling 1 expression by small interference RNAs facilitated an increase in interferon-γ-induced angiotensinogen expression, indicating that these two factors negatively regulate angiotensinogen expression. In contrast, the increase in interferon-γ-induced monocyte chemoattractant protein 1 expression was attenuated in STAT1-deficient mesangial cells, suggesting that STAT1 positively regulates monocyte chemoattractant protein 1 expression in mesangial cells. CONCLUSION: These results demonstrate that while interferon-γ increases both angiotensinogen and monocyte chemoattractant protein 1 expression, STAT1 plays an opposing role in the regulation of each factor in mesangial cells.

Bacterial TLR4 and NOD2 signaling linked to reduced mitochondrial energy function in active inflammatory bowel disease.

Inflammatory bowel disease (IBD) has been linked to active signaling with bacterial components and reduced mitochondrial ATP production; however, synergism between both of these disease characteristics remains unclear. We aimed to determine in human IBD transcriptomes the link between a transcriptional signature unique to intestinal cells (ICs) with reduced mitochondrial ATP production (Mito-0) and bacteria triggered signaling using a bioinformatics approach. We generated an IC Mito-0 panel comprised of 199 differentially expressed (DE) transcripts mediated by reduced mitochondrial ATP function (DEGseq, log2 fold-change > |2|, p < .001). Transcripts from this panel were involved in diverse biological functions including regulation of mitochondrial energy (lower ATP), extracellular matrix, cell-cell contact, cytoskeleton, growth, metabolism, and inflammation. Next, unsupervised hierarchical clustering showed that the Mito-0 panel distinctly separated inflamed IBD from non-inflamed transcriptomes, which was also supported by principal component analysis (PCA) revealing distinct variation between sample types based on presence of the Mito-0 signature (PCA, p = 8.77e-09). Utilizing three independent IBD cohorts, we validated that 60 novel transcripts from the Mito-0 panel were significantly increased in inflamed tissue. Subsequently, KEGG generated bacterial TLR4 and NOD2 transcriptional signatures strongly associated with inflamed IBD transcriptomes and with the Mito-0 signature as determined by Spearman's analysis (coefficient of correlation, r = 0.92, p < .05). Herein, using a comprehensive analysis we demonstrated existence of an axis between bacteria triggered signaling and reduced mitochondrial energy function. Furthermore, we identified and validated novel transcripts within this axis as potential drivers and therapeutic targets for human IBD.

Loss of Forkhead Box O3 Facilitates Inflammatory Colon Cancer: Transcriptome Profiling of the Immune Landscape and Novel Targets.

BACKGROUND & AIMS: Diminished forkhead box O3 (FOXO3) function drives inflammation and cancer growth; however, mechanisms fostering these pathobiologies are unclear. Here, we aimed to identify in colon loss of FOXO3-dependent cellular and molecular changes that facilitate inflammation-mediated tumor growth. METHODS: FOXO3 knockout (KO) and wild-type (WT) mice were used in the AOM/DSS model of inflammation-mediated colon cancer. Bioinformatics were used for profiling of mRNA sequencing data from human and mouse colon and tumors; specific targets were validated in human colon cancer cells (shFOXO3). RESULTS: In mice, FOXO3 deficiency led to significantly elevated colonic tumor burden (incidence and size) compared with WT (P < .05). In FOXO3 KO colon, activated molecular pathways overlapped with those associated with mouse and human colonic inflammation and cancer, especially human colonic tumors with inflammatory microsatellite instability (false discovery rate < 0.05). FOXO3 KO colon, similar to tumors, had increased neutrophils, macrophages, B cells, T cells, and decreased natural killer cells (false discovery rate < 0.05). Moreover, in KO colon differentially expressed transcripts were linked to activation of inflammatory nuclear factor kappa B, tumorigenic cMyc, and bacterial Toll-like receptor signaling. Among differentially expressed transcripts, we validated altered expression of integrin subunit alpha 2 (ITGA2), ADAM metallopeptidase with thrombospondin type 1 motif 12, and ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 5 in mouse WT and FOXO3 KO colon and tumors (P < .05). Similarly, their altered expression was found in human inflammatory bowel disease and colon cancer tissues and linked to poor patient survival. Ultimately, in human colon cancer cells, FOXO3 knockdown (shFOXO3) led to significantly increased ITGA2, and silencing ITGA2 (siRNA) alone diminished cell growth. CONCLUSIONS: We identified the loss of FOXO3-mediated immune landscape, pathways, and transcripts that could serve as biomarkers and new targets for inflammatory colon cancer treatment.

High-fat diet induced leptin and Wnt expression: RNA-sequencing and pathway analysis of mouse colonic tissue and tumors.

Obesity, an immense epidemic affecting approximately half a billion adults, has doubled in prevalence in the last several decades. Epidemiological data support that obesity, due to intake of a high-fat, western diet, increases the risk of colon cancer; however, the mechanisms underlying this risk remain unclear. Here, utilizing next generation RNA sequencing, we aimed to determine the high-fat diet (HFD) mediated expression profile in mouse colon and the azoxymethane/dextran sulfate sodium model of colon cancer. Mice on HFD had significantly higher colonic inflammation, tumor burden, and a number of differentially expressed transcripts compared to mice on regular diet (RD). We identified 721 transcripts differentially expressed in mouse HFD colon that were in a shared pattern with colonic tumors (RD and HFD). Importantly, in mouse colon, HFD stimulated an expression signature strikingly similar to human colon cancer, especially those with inflammatory microsatellite instability. Furthermore, pathway analysis of these transcripts demonstrated their association with active inflammation and colon cancer signaling, with leptin and Wnt as the top two transcripts elevated in mouse HFD colon shared with tumors. Moreover, in mouse colon, HFD-stimulated tumorigenic Wnt pathway activation was further validated by upregulation of ß-catenin transcriptional targets. Finally, in human colon cancer, upregulation of leptin pathway members was shown with a large network of dysregulated transcripts being linked with worse overall survival.

Reduced mitochondrial activity in colonocytes facilitates AMPKα2-dependent inflammation.

Intestinal inflammation is associated with low levels of mucosal ATP, highlighting the importance of mitochondrial function associated with ATP production in the pathophysiology of the disease. In the inflamed colon of humans and mice, we found decreased levels of mitochondrial complex cytochrome c oxidase I/IV and lower ATP levels. Thus, we generated colonic ρ0 cells with reduced mitochondrial function linked to ATP production by selective depletion of mitochondrial DNA. In these cells, RNA sequencing revealed a substantial number of differentially expressed transcripts, among which 240 belonged to inflammatory pathways activated in human inflamed colon and TNF-α-treated cells (false discovery rate < 0.05). TNF-α treatment of colonic ρ0 cells augmented IL-8 expression by 9-fold (P < 0.01) via NF-κB compared to TNF-α-treated control. Moreover, reduced mitochondrial function facilitated TNF-α-mediated NF-κB luciferase promoter activity as a result of lowered inhibitory IκBα (nuclear factor of κ light polypeptide gene enhancer in B-cell inhibitor, α), leading to elevated NF-κB. In cells with reduced mitochondrial function, TNF-α facilitated AMPKα2 activation by 8-fold (P < 0.01), which was involved in NF-κB-dependent IL-8 expression. Last, in human and mouse colon, anti-TNF-α treatment restored reduced mitochondria-dependent inflammation. We propose that selective targeting of this novel mechanism provides new treatment opportunities for intestinal inflammation.-Heller, S., Penrose, H. M., Cable, C., Biswas, D., Nakhoul, H., Baddoo, M., Flemington, E., Crawford, S. E., Savkovic, S. D. Reduced mitochondrial activity in colonocytes facilitates AMPKα2-dependent inflammation.

In colonic ρ0 (rho0) cells reduced mitochondrial function mediates transcriptomic alterations associated with cancer.

BACKGROUND: Mitochondrial reprogramming has emerged as a hallmark of cancer pathobiology. Although it is believed this reprogramming is essential for cancer cells to thrive, how it supports cancer pathobiology is unclear. We previously generated colonic ρ0 (rho0) cells with reduced mitochondrial energy function and acquired their transcriptional signature. Here, we utilized a bioinformatics approach to identify their changes linked to cancer pathobiology. METHODS: Human colon cancer HCT116 cells, control and ρ0, were used for qPCR. Bioinformatics analysis: GeneCards, Kaplan-Meier Survival, GENT, cBioPortal. RESULTS: The colonic ρ0 transcriptome was linked with proliferation, DNA replication, survival, tumor morphology, and cancer. Among differentially expressed transcripts, 281 were regulators or biomarkers of human colon cancer especially those with inflammatory microsatellite instability (MSI). We identified and validated novel transcripts in ρ0 cells with altered expression in human colon cancer. Among them DGK1, HTR7, FLRT3, and ZBTB18 co-occurred with established regulators of human colon cancer pathobiology. Also, increased levels of DGKI, FLRT3, ZBTB18, and YPEL1 as well as decreased levels of HTR7, and CALML6 were linked to substantially poorer patient survival. CONCLUSION: We identified established and novel regulators in colon cancer pathobiology that are dependent on mitochondrial energy reprogramming and linked to poorer patient survival.

VSL#3 Probiotic Stimulates T-cell Protein Tyrosine Phosphatase-mediated Recovery of IFN-γ-induced Intestinal Epithelial Barrier Defects.

BACKGROUND: VSL#3 is a probiotic compound that has been used in the treatment of inflammatory bowel disease. T-cell protein tyrosine phosphatase (TCPTP) is the protein product of the inflammatory bowel disease candidate gene, PTPN2, and we have previously shown that it protects epithelial barrier function. The aim of this study was to investigate whether VSL#3 improves intestinal epithelial barrier function against the effects of the inflammatory bowel disease-associated proinflammatory cytokine, interferon-gamma (IFN-γ) through activation of TCPTP. METHODS: Polarized monolayers of T84 intestinal epithelial cells were treated with increasing concentrations of VSL#3 to determine effects on TCPTP expression and enzymatic activity. Therapeutic effects of VSL#3 against barrier disruption by IFN-γ were measured by transepithelial electrical resistance and fluorescein isothiocyanate-dextran permeability. A novel TCPTP-deficient HT-29 intestinal epithelial cell line was generated to study the role of TCPTP in mediating the effects of VSL#3. Tight junction protein distribution was assessed with confocal microscopy. RESULTS: VSL#3 increased TCPTP protein levels and enzymatic activity, correlating with a VSL#3-induced decrease in IFN-γ signaling. VSL#3 corrected the decrease in transepithelial electrical resistance and the increase in epithelial permeability induced by IFN-γ. Moreover, the restorative effect of VSL#3 against IFN-γ signaling, epithelial permeability defects, altered expression and localization of the tight junction proteins claudin-2, occludin, and zonula occludens-1, were not realized in stable TCPTP/(PTPN2)-deficient HT-29 intestinal epithelial cells. CONCLUSIONS: VSL#3 reduces IFN-γ signaling and IFN-γ-induced epithelial barrier defects in a TCPTP-dependent manner. These data point to a key role for TCPTP as a therapeutic target for restoration of barrier function using probiotics.

Spermidine stimulates T cell protein-tyrosine phosphatase-mediated protection of intestinal epithelial barrier function.

The gene locus encoding protein-tyrosine phosphatase non-receptor type 2 (PTPN2) has been associated with inflammatory bowel disease. Expression of the PTPN2 gene product, T cell protein-tyrosine phosphatase (TCPTP), in intestinal epithelial cells has been shown to play an important role in the protection of epithelial barrier function during periods of inflammation by acting as a negative regulator of the proinflammatory cytokine IFN-γ. Therefore, agents that increase the activity of TCPTP are of general interest as modifiers of inflammatory signaling events. A previous study demonstrated that the small molecule spermidine is a selective activator of TCPTP in vitro. The aim of this study was to investigate whether activation of TCPTP by spermidine was capable of alleviating IFN-γ-induced, proinflammatory signaling and barrier dysfunction in human intestinal epithelial cells. Studies revealed that treatment of T84 and HT29/cl.19A colonocytes with spermidine increased both TCPTP protein levels and enzymatic activity, correlating with a decrease in the phosphorylation of the signal transducers and activators of transcription 1 and 3, downstream mediators of IFN-γ signaling, upon coadministration of spermidine to IFN-γ-treated cells. On a functional level, spermidine protected barrier function in the setting of inflammation, restricting the decrease in transepithelial electrical resistance and the increase in epithelial permeability induced by IFN-γ in coincubation experiments. These data implicate spermidine as a potential therapeutic agent to treat conditions associated with elevated IFN-γ signaling and a faulty mucosal barrier.