Heat shock factor 1 drives regulatory T-cell induction to limit murine intestinal inflammation.

The heat shock response is a critical component of the inflammatory cascade that prevents misfolding of new proteins and regulates immune responses. Activation of clusters of differentiation (CD)4+ T cells causes an upregulation of heat shock transcription factor, heat shock factor 1 (HSF1). We hypothesized that HSF1 promotes a pro-regulatory phenotype during inflammation. To validate this hypothesis, we interrogated cell-specific HSF1 knockout mice and HSF1 transgenic mice using in vitro and in vivo techniques. We determined that while HSF1 expression was induced by anti-CD3 stimulation alone, the combination of anti-CD3 and transforming growth factor ß, a vital cytokine for regulatory T cell (Treg) development, resulted in increased activating phosphorylation of HSF1, leading to increased nuclear translocation and binding to heat shock response elements. Using chromatin immunoprecipitation (ChIP), we demonstrate the direct binding of HSF1 to foxp3 in isolated murine CD4+ T cells, which in turn coincided with induction of FoxP3 expression. We defined that conditional knockout of HSF1 decreased development and function of Tregs and overexpression of HSF1 led to increased expression of FoxP3 along with enhanced Treg suppressive function. Adoptive transfer of CD45RBHigh CD4 colitogenic T cells along with HSF1 transgenic CD25+ Tregs prevented intestinal inflammation when wild-type Tregs did not. Finally, overexpression of HSF1 provided enhanced barrier function and protection from murine ileitis. This study demonstrates that HSF1 promotes Treg development and function and may represent both a crucial step in the development of induced regulatory T cells and an exciting target for the treatment of inflammatory diseases with a regulatory T-cell component. SIGNIFICANCE STATEMENT: The heat shock response (HSR) is a canonical stress response triggered by a multitude of stressors, including inflammation. Evidence supports the role of the HSR in regulating inflammation, yet there is a paucity of data on its influence in T cells specifically. Gut homeostasis reflects a balance between regulatory clusters of differentiation (CD)4+ T cells and pro-inflammatory T-helper (Th)17 cells. We show that upon activation within T cells, heat shock factor 1 (HSF1) translocates to the nucleus, and stimulates Treg-specific gene expression. HSF1 deficiency hinders Treg development and function and conversely, HSF1 overexpression enhances Treg development and function. While this work, focuses on HSF1 as a novel therapeutic target for intestinal inflammation, the findings have significance for a broad range of inflammatory conditions.

Interplay of gut microbiota and host epithelial mitochondrial dysfunction is necessary for the development of spontaneous intestinal inflammation in mice.

BACKGROUND: Intestinal epithelial cell (IEC) mitochondrial dysfunction involvement in inflammatory bowel diseases (IBD), including Crohn's disease affecting the small intestine, is emerging in recent studies. As the interface between the self and the gut microbiota, IECs serve as hubs of bidirectional cross-talk between host and luminal microbiota. However, the role of mitochondrial-microbiota interaction in the ileum is largely unexplored. Prohibitin 1 (PHB1), a chaperone protein of the inner mitochondrial membrane required for optimal electron transport chain function, is decreased during IBD. We previously demonstrated that mice deficient in PHB1 specifically in IECs (Phb1i∆IEC) exhibited mitochondrial impairment, Paneth cell defects, gut microbiota dysbiosis, and spontaneous inflammation in the ileum (ileitis). Mice deficient in PHB1 in Paneth cells (epithelial secretory cells of the small intestine; Phb1∆PC) also exhibited mitochondrial impairment, Paneth cell defects, and spontaneous ileitis. Here, we determined whether this phenotype is driven by Phb1 deficiency-associated ileal microbiota alterations or direct effects of loss of PHB1 in host IECs. RESULTS: Depletion of gut microbiota by broad-spectrum antibiotic treatment in Phb1∆PC or Phb1i∆IEC mice revealed a necessary role of microbiota to cause ileitis. Using germ-free mice colonized with ileal microbiota from Phb1-deficient mice, we show that this microbiota could not independently induce ileitis without host mitochondrial dysfunction. The luminal microbiota phenotype of Phb1i∆IEC mice included a loss of the short-chain fatty acid butyrate. Supplementation of butyrate in Phb1-deficient mice ameliorated Paneth cell abnormalities and ileitis. Phb1-deficient ileal enteroid models suggest deleterious epithelial-intrinsic responses to ileal microbiota that were protected by butyrate. CONCLUSIONS: These results suggest a mutual and essential reinforcing interplay of gut microbiota and host IEC, including Paneth cell, mitochondrial health in influencing ileitis. Restoration of butyrate is a potential therapeutic option in Crohn's disease patients harboring epithelial cell mitochondrial dysfunction. Video Abstract.

Pancreatic Ductal Adenocarcinoma Cells Regulate NLRP3 Activation to Generate a Tolerogenic Microenvironment.

Defining feature of pancreatic ductal adenocarcinoma (PDAC) that participates in the high mortality rate and drug resistance is the immune-tolerant microenvironment which enables tumors to progress unabated by adaptive immunity. In this study, we report that PDAC cells release CSF-1 to induce nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) activation in myeloid cells. Increased NLRP3 expression was found in the pancreas of patients with PDAC when compared with normal pancreas which correlated with the formation of the NLRP3 inflammasome. Using human primary cells and an orthotopic PDAC mouse model, we show that NLRP3 activation is responsible for the maturation and release of the inflammatory cytokine IL1ß which selectively drives Th2-type inflammation via COX2/PGE2 induction. As a result of this inflammation, primary tumors were characterized by reduced cytotoxic CD8+ T-cell activation and increased tumor expansion. Genetic deletion and pharmacologic inhibition of NLRP3 enabled the development of Th1 immunity, increased intratumoral levels of IL2, CD8+ T cell­mediated tumor suppression, and ultimately limited tumor growth. In addition, we observed that NLRP3 inhibition in combination with gemcitabine significantly increased the efficacy of the chemotherapy. In conclusion, this study provides a mechanism by which tumor-mediated NLRP3 activation exploits a distinct adaptive immunity response that facilitates tumor escape and progression. Considering the ability to block NLRP3 activity with safe and small orally active molecules, this protein represents a new promising target to improve the limited therapeutic options in PDAC. SIGNIFICANT: This study provides novel molecular insights on how PDAC cells exploit NLRP3 activation to suppress CD8 T-cell activation. From a translational perspective, we demonstrate that the combination of gemcitabine with the orally active NLRP3 inhibitor OLT1177 increases the efficacy of monotherapy.

Autophagy in Crohn's Disease: Converging on Dysfunctional Innate Immunity.

Crohn's disease (CD) is a chronic inflammatory bowel disease marked by relapsing, transmural intestinal inflammation driven by innate and adaptive immune responses. Autophagy is a multi-step process that plays a critical role in maintaining cellular homeostasis by degrading intracellular components, such as damaged organelles and invading bacteria. Dysregulation of autophagy in CD is revealed by the identification of several susceptibility genes, including ATG16L1, IRGM, NOD2, LRRK2, ULK1, ATG4, and TCF4, that are involved in autophagy. In this review, the role of altered autophagy in the mucosal innate immune response in the context of CD is discussed, with a specific focus on dendritic cells, macrophages, Paneth cells, and goblet cells. Selective autophagy, such as xenophagy, ERphagy, and mitophagy, that play crucial roles in maintaining intestinal homeostasis in these innate immune cells, are discussed. As our understanding of autophagy in CD pathogenesis evolves, the development of autophagy-targeted therapeutics may benefit subsets of patients harboring impaired autophagy.

Inner mitochondrial membrane protein Prohibitin 1 mediates Nix-induced, Parkin-independent mitophagy.

Autophagy of damaged mitochondria, called mitophagy, is an important organelle quality control process involved in the pathogenesis of inflammation, cancer, aging, and age-associated diseases. Many of these disorders are associated with altered expression of the inner mitochondrial membrane (IMM) protein Prohibitin 1. The mechanisms whereby dysfunction occurring internally at the IMM and matrix activate events at the outer mitochondrial membrane (OMM) to induce mitophagy are not fully elucidated. Using the gastrointestinal epithelium as a model system highly susceptible to autophagy inhibition, we reveal a specific role of Prohibitin-induced mitophagy in maintaining intestinal homeostasis. We demonstrate that Prohibitin 1 induces mitophagy in response to increased mitochondrial reactive oxygen species (ROS) through binding to mitophagy receptor Nix/Bnip3L and independently of Parkin. Prohibitin 1 is required for ROS-induced Nix localization to mitochondria and maintaining homeostasis of epithelial cells highly susceptible to mitochondrial dysfunction.

Targeting Mitochondrial Damage as a Therapeutic for Ileal Crohn's Disease.

Paneth cell defects in Crohn's disease (CD) patients (called the Type I phenotype) are associated with worse clinical outcomes. Recent studies have implicated mitochondrial dysfunction in Paneth cells as a mediator of ileitis in mice. We hypothesized that CD Paneth cells exhibit impaired mitochondrial health and that mitochondrial-targeted therapeutics may provide a novel strategy for ileal CD. Terminal ileal mucosal biopsies from adult CD and non-IBD patients were characterized for Paneth cell phenotyping and mitochondrial damage. To demonstrate the response of mitochondrial-targeted therapeutics in CD, biopsies were treated with vehicle or Mito-Tempo, a mitochondrial-targeted antioxidant, and RNA transcriptome was analyzed. During active CD inflammation, the epithelium exhibited mitochondrial damage evident in Paneth cells, goblet cells, and enterocytes. Independent of inflammation, Paneth cells in Type I CD patients exhibited mitochondrial damage. Mito-Tempo normalized the expression of interleukin (IL)-17/IL-23, lipid metabolism, and apoptotic gene signatures in CD patients to non-IBD levels. When stratified by Paneth cell phenotype, the global tissue response to Mito-Tempo in Type I patients was associated with innate immune, lipid metabolism, and G protein-coupled receptor (GPCR) gene signatures. Targeting impaired mitochondria as an underlying contributor to inflammation provides a novel treatment approach for CD.

Nuclear partitioning of Prohibitin 1 inhibits Wnt/ß-catenin-dependent intestinal tumorigenesis.

The Wnt/ß-catenin signaling pathway is aberrantly activated in the majority of colorectal cancer cases due to somatic mutations in the adenomatous polyposis coli (APC) gene. Prohibitin 1 (PHB1) serves pleiotropic cellular functions with dynamic subcellular trafficking, facilitating signaling crosstalk between organelles. Nuclear-localized PHB1 is an important regulator of gene transcription. Using mice with inducible intestinal epithelial cell (IEC)-specific deletion of Phb1 (Phb1iΔIEC) and mice with IEC-specific overexpression of Phb1 (Phb1Tg), we demonstrate that IEC-specific PHB1 combats intestinal tumorigenesis in the ApcMin/+ mouse model by inhibiting Wnt/ß-catenin signaling. Forced nuclear accumulation of PHB1 in human RKO or SW48 CRC cell lines increased AXIN1 expression and decreased cell viability. PHB1 deficiency in CRC cells decreased AXIN1 expression and increased ß-catenin activation that was abolished by XAV939, a pharmacological AXIN stabilizer. These results define a role of PHB1 in inhibiting the Wnt/ß-catenin pathway to influence the development of intestinal tumorigenesis. Induction of nuclear PHB1 trafficking provides a novel therapeutic option to influence AXIN1 expression and the ß-catenin destruction complex in Wnt-driven intestinal tumorigenesis.

The Synthetic Small Molecule FL3 Combats Intestinal Tumorigenesis via Axin1-Mediated Inhibition of Wnt/ß-Catenin Signaling.

Colorectal cancer exhibits aberrant activation of Wnt/ß-catenin signaling. Many inhibitors of the Wnt/ß-catenin pathway have been tested for Wnt-dependent cancers including colorectal cancer, but are unsuccessful due to severe adverse reactions. FL3 is a synthetic derivative of natural products called flavaglines, which exhibit anti-inflammatory and cytoprotective properties in intestinal epithelial cells, but has not been previously tested in cell or preclinical models of intestinal tumorigenesis. In vitro studies suggest that flavaglines target prohibitin 1 (PHB1) as a ligand, but this has not been established in the intestine. PHB1 is a highly conserved protein with diverse functions that depend on its posttranslational modifications and subcellular localization. Here, we demonstrate that FL3 combats intestinal tumorigenesis in the azoxymethane-dextran sodium sulfate and ApcMin/+ mouse models and in human colorectal cancer tumor organoids (tumoroids) by inhibiting Wnt/ß-catenin signaling via induction of Axin1 expression. FL3 exhibited no change in cell viability in normal intestinal epithelial cells or human matched-normal colonoids. FL3 response was diminished in colorectal cancer cell lines and human colorectal cancer tumoroids harboring a mutation at S45 of ß-catenin. PHB1 deficiency in mice or in human colorectal cancer tumoroids abolished FL3-induced expression of Axin1 and drove tumoroid death. In colorectal cancer cells, FL3 treatment blocked phosphorylation of PHB1 at Thr258, resulting in its nuclear translocation and binding to the Axin1 promoter. These results suggest that FL3 inhibits Wnt/ß-catenin signaling via PHB1-dependent activation of Axin1. FL3, therefore, represents a novel compound that combats Wnt pathway-dependent cancers, such as colorectal cancer. SIGNIFICANCE: Targeting of PHB1 by FL3 provides a novel mechanism to combat Wnt-driven cancers, with limited intestinal toxicity. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/17/3519/F1.large.jpg.

The influence of mitochondrial-directed regulation of Wnt signaling on tumorigenesis.

Mitochondria are dynamic organelles that play a key role in integrating cellular signaling. Mitochondrial alterations are evident in all stages of tumorigenesis and targeting mitochondrial pathways has emerged as an anticancer therapeutic strategy. The Wnt-signaling pathway regulates many fundamental cellular functions such as proliferation, survival, migration, stem-cell maintenance, and mitochondrial metabolism and dynamics. Emerging evidence demonstrates that mitochondrial-induced regulation of Wnt signaling provides an additional mechanism to influence cell-fate decisions. Crosstalk between mitochondria and Wnt signaling presents a feedforward loop in which Wnt activation regulates mitochondrial function that, in turn, drives Wnt signaling. In this mini-review, we will discuss the recent evidence revealing the mitochondrial control of Wnt signaling and its implications for tumorigenesis and anticancer therapeutic targeting.

Effects of high-fat diet on sympathetic neurotransmission in mesenteric arteries from Dahl salt-sensitive rat.

Obesity hypertension is driven by sympathetic neurotransmission to the heart and blood vessels. We tested the hypothesis that high-fat diet (HFD)-induced hypertension is driven by sympathetic neurotransmission to mesenteric arteries (MA) in male but not female Dahl salt-sensitive (Dahl ss) rat. Rats were fed a control diet (CD; 10 kcal% from fat) or HFD (60 kcal% from fat) beginning at 3 weeks (wk) of age; measurements were made at 10-, 17- and 24-wk. Body weight increased with HFD, age and sex. Mean arterial pressure (MAP) was higher in HFD versus CD rats from both sexes at 17- and 24-wk. MA constriction measured using pressure myography, and electrical field stimulation (EFS, 0.2-30 Hz) was greater in HFD versus CD in males at 17-wk; this was not due to changes in α2 autoreceptor or norepinephrine transporter (NET) function. Prazosin (α1-AR antagonist) and suramin (P2 receptor antagonist) inhibited neurogenic MA constriction equally in all groups. Arterial reactivity to exogenous norepinephrine (NE; 10-8 - 10-5 M) was lower in HFD versus CD at 10-wk in males. Female MA reactivity to exogenous ATP was lower at 24-weeks compared to earlier time points. HFD did not affect tyrosine hydroxylase (TH) or the vesicular nucleotide transporter (VNUT) nerve density in MA from both sexes. NE content was lower in MA but higher in plasma at 24-wk compared to 10- and 17-wk in both sexes. In conclusion, HFD-induced hypertension is not driven by increased sympathetic neurotransmission to MA in male and female Dahl ss rats.