Epithelial Toll-like Receptor Research Articles

Pterostilbene Alleviates Dextran Sodium Sulfate (DSS)-Induced Intestinal Barrier Dysfunction Involving Suppression of a S100A8-TLR-4-NF-κB Signaling Cascade.

Intestinal barrier hemostasis is the key to health. As a resveratrol analogue, pterostilbene (PT) has been reported to prevent dextran sodium sulfate (DSS)-induced intestinal barrier dysfunction mainly associated with the intestinal NF-κB signaling pathway. However, the exact underlying mechanisms are not yet well-defined yet. In this study, we performed RNA-sequencing analysis and unexpectedly found that alarmin S100A8 sensitively responded to DSS-induced intestinal injury. Accordingly, histologic assessments suggested that the high expression of S100A8 was accompanied by increased intestinal infiltration of macrophages, upregulated intestinal epithelial Toll-like receptor 4 (TLR-4), and activated NF-κB signaling pathway. Interestingly, the above phenomena were effectively counteracted upon the addition of PT. Furthermore, by using a coculture system of macrophage THP-1 cells and HT-29 colon cells, we identified macrophage-secreted S100A8 activated intestinal epithelial NF-κB signaling pathway through TLR-4. Taken together, these findings suggested that PT ameliorated DSS-induced intestinal barrier injury through suppression of the macrophage S100A8-intestinal epithelial TLR-4-NF-κB signaling cascade.

Commensal bacteria weaken the intestinal barrier by suppressing epithelial neuropilin-1 and Hedgehog signaling

The gut microbiota influences intestinal barrier integrity through mechanisms that are incompletely understood. Here we show that the commensal microbiota weakens the intestinal barrier by suppressing epithelial neuropilin-1 (NRP1) and Hedgehog (Hh) signaling. Microbial colonization of germ-free mice dampens signaling of the intestinal Hh pathway through epithelial Toll-like receptor (TLR)-2, resulting in decreased epithelial NRP1 protein levels. Following activation via TLR2/TLR6, epithelial NRP1, a positive-feedback regulator of Hh signaling, is lysosomally degraded. Conversely, elevated epithelial NRP1 levels in germ-free mice are associated with a strengthened gut barrier. Functionally, intestinal epithelial cell-specific Nrp1 deficiency (Nrp1ΔIEC) results in decreased Hh pathway activity and a weakened gut barrier. In addition, Nrp1ΔIEC mice have a reduced density of capillary networks in their small intestinal villus structures. Collectively, our results reveal a role for the commensal microbiota and epithelial NRP1 signaling in the regulation of intestinal barrier function through postnatal control of Hh signaling.

Upregulation of TLR5 indicates a favorable prognosis in prostate cancer.

Toll-like receptors (TLRs) are the key sensors of innate immunity for triggering immune responses against infections. TLRs are well known to be expressed and activated in innate immune cells, such as macrophage and dendritic cells, but we and others have found that some TLRs are also functional in epithelial cells. However, the role of an epithelial TLR in prostate cancer remains elusive. TLR5 expression in messenger RNA and protein level in prostate cancer was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). The activation of TLR5 signaling in epithelial cells was detected upon nuclear factor-κB activation by luciferase assay and western blot analysis, and proinflammatory cytokine activation by RT-qPCR. Distinguishing between the TLR5 and NLRC4 pathways, both recognizing flagellin, is determined by small interfering RNA and proinflammatory cytokine activation. The role of TLR5 in prostate cancer was analyzed by IHC and bioinformatics using a general and single-cell database. In the present study, we show that TLR5, among other TLRs, is exceedingly expressed in human prostate cancer cells. This cancer epithelial cell TLR5 functions to activate the TLR5 signaling pathway in human prostate cancer cells, as it does with innate immune cell TLR5. The bacterial protein flagellin induces a robust immune response in prostate cancer cells in a TLR5-dependent but NLRC4-independent manner. TLR5 is highly expressed in prostate cancer patient specimens, and high TLR5 expression in prostate cancer patients indicates a favorable prognosis. TLR5, as an innate immunity receptor, is a functional TLR in human prostate cancer epithelial cells. TLR5 plays an important role in prostate cancer development and is a new potential prognosis biomarker. TLR5 may represent a novel immunotherapy target against prostate cancer.

Redox-Dependent Activation of Lung Epithelial STAT3 Is Required for Inducible Protection against Bacterial Pneumonia.

The lung epithelium is dynamic, capable of considerable structural and functional plasticity in response to pathogen challenges. Our laboratory has demonstrated that an inhaled combination of a Toll-like receptor (TLR) 2/6 agonist and a TLR9 agonist (Pam2ODN) results in robust protection against otherwise lethal pneumonias. We have previously shown that intact epithelial TLR signaling and generation of multisource epithelial reactive oxygen species (ROS) are required for inducible protection. Further investigating the mechanisms underlying this phenomenon of inducible resistance, reverse-phase protein array analysis demonstrated robust STAT3 (signal transducer and activator of transcription 3) phosphorylation following treatment of lung epithelial cells. We show here that Pam2ODN-induced STAT3 phosphorylation is IL-6-independent. We further found that therapeutic epithelial STAT3 activation is required for inducible protection against Pseudomonas aeruginosa pneumonia. Additional studies showed that inhibiting epithelial dual oxidases or scavenging ROS significantly reduced the Pam2ODN induction of STAT3 phosphorylation, suggesting a proximal role for ROS in inducible STAT3 activation. Dissecting these mechanisms, we analyzed the contributions of redox-sensitive kinases and found that Pam2ODN activated epithelial growth factor receptor in an ROS-dependent manner that is required for therapeutically inducible STAT3 activation. Taken together, we demonstrate that epithelial STAT3 is imperative for Pam2ODN's function and describe a novel redox-based mechanism for its activation. These key mechanistic insights may facilitate strategies to leverage inducible epithelial resistance to protect susceptible patients during periods of peak vulnerability.

Intestinal TLR4 deletion exacerbates acute pancreatitis through gut microbiota dysbiosis and Paneth cells deficiency

ABSTRACT Toll-like receptor 4 (TLR4) has been identified as a potentially promising therapeutic target in acute pancreatitis (AP). However, the role of intestinal TLR4 in AP and AP-associated gut injury remains unclear. This study aimed to explore the relationship between intestinal TLR4 and gut microbiota during AP. A mouse AP model was establish by intraperitoneal injection of L-arginine. Pancreatic injury and intestinal barrier function were evaluated in wild-type and intestinal epithelial TLR4 knockout (TLR4ΔIEC) mice. Gut microbiota was analyzed by 16S rRNA sequencing. Quadruple antibiotics were applied to induce microbiota-depleted mice. Differentially expressed genes in gut were detected by RNA sequencing. L. reuteri treatment was carried out in vivo and vitro study. Compared with wild-type mice, AP and AP-associated gut injury were exacerbated in TLR4ΔIEC mice in a gut microbiota-dependent manner. The relative abundance of Lactobacillus and number of Paneth cells remarkably decreased in TLR4ΔIEC mice. The KEGG pathway analysis derived from RNA sequencing suggested that genes affected by intestinal TLR4 deletion were related to the activation of nod-like receptor pathway. Furthermore, L. reuteri treatment could significantly improve the pancreatic and intestinal injury in TLR4ΔIEC mice through promoting Paneth cells in a NOD2-dependent manner. Loss of intestinal epithelial TLR4 exacerbated pancreatic and intestinal damage during AP, which might be attributed to the gut microbiota dysbiosis especially the exhausted Lactobacillus. L. reuteri might maintain intestinal homeostasis and alleviate AP via Paneth cells modulation. Abbreviations: AP Acute pancreatitis, TLR4 Toll-like receptor 4, IL-1β Interleukin-1β, IL-6 Interleukin-6, TNF-α Tumor necrosis factor-α, SIRS Systematic inflammatory response syndrome, LPS Lipopolysaccharides, SPF Specific pathogen-free, ZO-1 Zonula occludens-1, CON Control, H&E Hematoxylin and eosin, FISH Fluorescence in situ hybridization, DAPI 4′,6-diamidino-2-phenylindole, PCoA Principal co-ordinates analysis, SCFA Short chain fatty acid, LEfSe Linear discriminant analysis Effect Size, ANOVA Analysis of variance, F/B Firmicutes/Bacteroidetes, PCA Principal component analysis, NOD2 Nod-like receptor 2, ABX antibiotics, PCNA proliferating cell nuclear antigen

Aesculetin Inhibits Airway Thickening and Mucus Overproduction Induced by Urban Particulate Matter through Blocking Inflammation and Oxidative Stress Involving TLR4 and EGFR.

Particulate matter (PM) is a mixture of solid and liquid air pollutant particles suspended in the air, varying in composition, size, and physical features. PM is the most harmful form of air pollution due to its ability to penetrate deep into the lungs and blood streams, causing diverse respiratory diseases. Aesculetin, a coumarin derivative present in the Sancho tree and chicory, is known to have antioxidant and anti-inflammatory effects in the vascular and immune system. However, its effect on PM-induced airway thickening and mucus hypersecretion is poorly understood. The current study examined whether naturally-occurring aesculetin inhibited airway thickening and mucus hypersecretion caused by urban PM10 (uPM10, particles less than 10 μm). Mice were orally administrated with 10 mg/kg aesculetin and exposed to 6 μg/mL uPM10 for 8 weeks. To further explore the mechanism(s) involved in inhibition of uPM10-induced mucus hypersecretion by aesculetin, bronchial epithelial BEAS-2B cells were treated with 1–20 µM aesculetin in the presence of 2 μg/mL uPM10. Oral administration of aesculetin attenuated collagen accumulation and mucus hypersecretion in the small airways inflamed by uPM10. In addition, aesculetin inhibited uPM10-evoked inflammation and oxidant production in lung tissues. Further, aesculetin accompanied the inhibition of induction of bronchial epithelial toll-like receptor 4 (TLR4) and epidermal growth factor receptor (EFGR) elevated by uPM10. The inhibition of TLR4 and EGFR accompanied bronchial mucus hypersecretion in the presence of uPM10. Oxidative stress was responsible for the epithelial induction of TLR4 and EGFR, which was disrupted by aesculetin. These results demonstrated that aesculetin ameliorated airway thickening and mucus hypersecretion by uPM10 inhalation by inhibiting pulmonary inflammation via oxidative stress-stimulated TLR4 and EGFR. Therefore, aesculetin may be a promising agent for treating airway mucosa-associated disorders elicited by urban coarse particulates.

Tailored Modulation of Cellular Pro-inflammatory Responses With Disaccharide Lipid A Mimetics.

Pro-inflammatory signaling mediated by Toll-like receptor 4 (TLR4)/myeloid differentiation-2 (MD-2) complex plays a crucial role in the instantaneous protection against infectious challenge and largely contributes to recovery from Gram-negative infection. Activation of TLR4 also boosts the adaptive immunity which is implemented in the development of vaccine adjuvants by application of minimally toxic TLR4 activating ligands. The modulation of pro-inflammatory responses via the TLR4 signaling pathway was found beneficial for management of acute and chronic inflammatory disorders including asthma, allergy, arthritis, Alzheimer disease pathology, sepsis, and cancer. The TLR4/MD-2 complex can recognize the terminal motif of Gram-negative bacterial lipopolysaccharide (LPS)—a glycophospholipid lipid A. Although immense progress in understanding the molecular basis of LPS-induced TLR4-mediated signaling has been achieved, gradual, and predictable TLR4 activation by structurally defined ligands has not yet been attained. We report on controllable modulation of cellular pro-inflammatory responses by application of novel synthetic glycolipids—disaccharide-based lipid A mimetics (DLAMs) having picomolar affinity for TLR4/MD-2. Using crystal structure inspired design we have developed endotoxin mimetics where the inherently flexible β(1 → 6)-linked diglucosamine backbone of lipid A is replaced by a conformationally restricted α,α-(1↔1)-linked disaccharide scaffold. The tertiary structure of the disaccharide skeleton of DLAMs mirrors the 3-dimensional shape of TLR4/MD-2 bound E. coli lipid A. Due to exceptional conformational rigidity of the sugar scaffold, the specific 3D organization of DLAM must be preserved upon interaction with proteins. These structural factors along with specific acylation and phosphorylation pattern can ensure picomolar affinity for TLR4 and permit efficient dimerization of TLR4/MD-2/DLAM complexes. Since the binding pose of lipid A in the binding pocket of MD-2 (±180°) is crucial for the expression of biological activity, the chemical structure of DLAMs was designed to permit a predefined binding orientation in the binding groove of MD-2, which ensured tailored and species-independent (human and mice) TLR4 activation. Manipulating phosphorylation and acylation pattern at the sugar moiety facing the secondary dimerization interface allowed for adjustable modulation of the TLR4-mediated signaling. Tailored modulation of cellular pro-inflammatory responses by distinct modifications of the molecular structure of DLAMs was attained in primary human and mouse immune cells, lung epithelial cells and TLR4 transfected HEK293 cells.

Epithelial-Specific TLR4 Knockout Challenges Current Evidence of TLR4 Homeostatic Control of Gut Permeability

Introduction: Toll-like receptor 4 (TLR4) is a highly conserved immunosurveillance protein of innate immunity, displaying well-established roles in homeostasis and intestinal inflammation. Current evidence shows complex relationships between TLR4 activation, maintenance of health, and disease progression; however, it commonly overlooks the importance of site-specific TLR4 expression. This omission has the potential to influence translation of results as previous evidence shows the differing and distinct roles that TLR4 exhibits are dependent on its spatiotemporal expression. Methods: An intestinal epithelial TLR4 conditional knockout (KO) mouse line (Tlr4^ΔIEC, n = 6–8) was utilized to dissect the contribution of epithelial TLR4 expression to intestinal homeostasis with comparisons to wild-type (WT) (n = 5–7) counterparts. Functions of the intestinal barrier in the ileum and colon were assessed with tissue resistance in Ussing chambers. Molecular and structural comparisons in the ileum and colon were assessed via histological staining, expression of tight junction proteins (occludin and zonular occludin 1 [ZO-1]), and presence of CD11b-positive immune cells. Results: There was no impact of the intestinal epithelial TLR4 KO, with no differences in (1) tissue resistance–ileum (mean ± standard error of mean [SEM]): WT 22 ± 7.2 versus Tlr4^ΔIEC 20 ± 5.6 (Ω × cm²) p = 0.831, colon WT 30.8 ± 3.6 versus Tlr4^ΔIEC 45.1 ± 9.5 p = 0.191; (2) histological staining (overall tissue structure); and (3) tight junction protein expression (% area stain, mean ± SEM)–ZO-1: ileum–WT 1.49 ± 0.155 versus Tlr4^ΔIEC 1.17 ± 0.07, p = 0.09; colon–WT 1.36 ± 0.26 versus Tlr4^ΔIEC 1.12 ± 0.18 p = 0.47; occludin: ileum–WT 1.07 ± 0.12 versus Tlr4^ΔIEC 0.95 ± 0.13, p = 0.53; colon–WT 1.26 ± 0.26 versus Tlr4^ΔIEC 1.02 ± 0.16 p = 0.45. CD11b-positive immune cells (% area stain, mean ± SEM) in the ileum were mildly decreased in WT mice: WT 0.14 ± 0.02 versus Tlr4^ΔIEC 0.09 ± 0.01 p = 0.04. However, in the colon, there was no difference in CD11b-positive immune cells between strains: WT 0.53 ± 0.08 versus Tlr4^ΔIEC 0.49 ± 0.08 p = 0.73. Conclusions: These data have 2 important implications. First, these data refute the assumption that epithelial TLR4 exerts physiological control of intestinal physiology and immunity in health. Second, and most importantly, these data support the use of the Tlr4^ΔIEC line in future models interrogating health and disease, confirming no confounding effects of genetic manipulation.

Epithelial TLR4 Signaling Activates DUOX2 to Induce Microbiota-Driven Tumorigenesis

Chronic colonic inflammation leads to dysplasia and cancer in patients with inflammatory bowel disease. We have described the critical role of innate immune signaling via Toll-like receptor 4 (TLR4) in the pathogenesis of dysplasia and cancer. In the current study, we interrogate the intersection of TLR4 signaling, epithelial redox activity, and the microbiota in colitis-associated neoplasia. Inflammatory bowel disease and colorectal cancer data sets were analyzed for expression of TLR4, dual oxidase 2 (DUOX2), and NADPH oxidase 1 (NOX1). Epithelial production of hydrogen peroxide (H2O2) was analyzed in murine colonic epithelial cells and colonoid cultures. Colorectal cancer models were carried out in villin-TLR4 mice, carrying a constitutively active form of TLR4, their littermates, and villin-TLR4 mice backcrossed to DUOXA-knockout mice. The role of the TLR4-shaped microbiota in tumor development was tested in wild-type germ-free mice. Activation of epithelial TLR4 was associated with up-regulation of DUOX2 and NOX1 in inflammatory bowel disease and colorectal cancer. DUOX2 was exquisitely dependent on TLR4 signaling and mediated the production of epithelial H2O2. Epithelial H2O2 was significantly increased in villin-TLR4 mice; TLR4-dependent tumorigenesis required the presence of DUOX2 and a microbiota. Mucosa-associated microbiota transferred from villin-TLR4 mice to wild-type germ-free mice caused increased H2O2 production and tumorigenesis. Increased TLR4 signaling in colitis drives expression of DUOX2 and epithelial production of H2O2. The local milieu imprints the mucosal microbiota and imbues it with pathogenic properties demonstrated by enhanced epithelial reactive oxygen species and increased development of colitis-associated tumors. The inter-relationship between epithelial reactive oxygen species and tumor-promoting microbiota requires a 2-pronged strategy to reduce the risk of dysplasia in colitis patients.

Epithelial Toll-like receptors and their role in gut homeostasis and disease.

The human gastrointestinal tract is colonized by trillions of microorganisms that interact with the host to maintain structural and functional homeostasis. Acting as the interface between the site of the highest microbial burden in the human body and the richest immune compartment, a single layer of intestinal epithelial cells specializes in nutrient absorption, stratifies microorganisms to limit colonization of tissues and shapes the responses of the subepithelial immune cells. In this Review, we focus on the expression, regulation and functions of Toll-like receptors (TLRs) in the different intestinal epithelial lineages to analyse how epithelial recognition of bacteria participates in establishing homeostasis in the gut. In particular, we elaborate on the involvement of epithelial TLR signalling in controlling crypt dynamics, enhancing epithelial barrier integrity and promoting immune tolerance towards the gut microbiota. Furthermore, we comment on the regulatory mechanisms that fine-tune TLR-driven immune responses towards pathogens and revisit the role of TLRs in epithelial repair after injury. Finally, we discuss how dysregulation of epithelial TLRs can lead to the generation of dysbiosis, thereby increasing susceptibility to colitis and tumorigenesis.

20 ELUCIDATING THE ROLE OF FUSOBACTERIUM NUCLEATUM IN INTESTINAL INFLAMMATION

Abstract Background While the direct cause of inflammatory bowel disease (IBD) is unknown, the gut microbiota is speculated to play a key role. The complexity of the microbiome has made it difficult to pinpoint whether bacterial species are specifically associated with IBD exacerbations; although select microbes have emerged as compelling candidates. Several groups have identified increased abundance in Fusobacterium in IBD patients. Fusobacterium nucleatum drives inflammation in the oral cavity, but few studies have examined the potential for F. nucleatum to promote intestinal inflammation. We hypothesize that F. nucleatum secretes Outer Membrane Vesicles (OMVs) that activate epithelial Toll-like receptor 4 (TLR4) to drive inflammation. Methods & Results Given the prevalence of F. nucleatum in IBD specimens, we sought to determine if this pathobiont could promote pro-inflammatory responses in human epithelial cultures. Using fluorescently tagged F. nucleatum, we demonstrate that F. nucleatum subspecies polymorphum adheres to the mucus layer of human colonic HT29-MTX cells. Application of F. nucleatum metabolites to HT29-MTX cells resulted in upregulation of pro-inflammatory cytokines IL-8 and TNF by qPCR and ELISA. Purified OMVs from F. nucleatum alone were able to stimulate IL-8 and TNF production. This demonstrates the robust response of colonic epithelial cells to F. nucleatum. Additionally, we used human jejunum and colon enteroid monolayers treated with F. nucleatum metabolites in an anti-oxidant free enteroid media and found that F. nucleatum secreted products promoted TNF secretion by ELISA. Using the Luminex Magpix Platform we further queried the enteroid system to assess which pathways were activated. Enteroid monolayers treated with F. nucleatum metabolites exhibited increased phosphorylated ERK and CREB, downstream effectors of TLRs. We next sought to address whether F. nucleatum alone could elicit pro-inflammatory responses in a mouse model. Mice harboring a human microbiota, or humanized mice, were treated for 5 days with a cocktail of antibiotics and treated with F. nucleatum (108 CFU) by oral gavage; a regimen designed to mimic IBD patient treatment. Compared to control mice that received antibiotics and PBS vehicle control, mice treated with F. nucleatum exhibited disruption of the colonic architecture, with increased immune infiltrate and depleted mucus layer, resulting in a closer proximity of luminal contents to the epithelium. Analysis of mucosal gene expression revealed increased levels of epithelial TNF and KC (mouse homolog of IL-8), in addition to immune cell derived IL-6 in F. nucleatum-treated mice compared for controls. Conclusions These data provide evidence that F. nucleatum is capable of driving a pro-inflammatory signaling cascade in vitro and in vivo and F. nucleatum may represent a specific target for drug therapy.

20 ELUCIDATING THE ROLE OF FUSOBACTERIUM NUCLEATUM IN INTESTINAL INFLAMMATION

While the direct cause of inflammatory bowel disease (IBD) is unknown, the gut microbiota is speculated to play a key role. The complexity of the microbiome has made it difficult to pinpoint whether bacterial species are specifically associated with IBD exacerbations; although select microbes have emerged as compelling candidates. Several groups have identified increased abundance in Fusobacterium in IBD patients. Fusobacterium nucleatum drives inflammation in the oral cavity, but few studies have examined the potential for F. nucleatum to promote intestinal inflammation. We hypothesize that F. nucleatum secretes Outer Membrane Vesicles (OMVs) that activate epithelial Toll-like receptor 4 (TLR4) to drive inflammation.

Rapid activation of brainstem nuclei following TLR stimulation of the nasal mucosa

We recently identified a novel neuroimmune mechanism in the nasal mucosa, in which activation of neuronal Toll‑like receptor (TLR) 7 results in upregulation of epithelial TLRs, via release of substance P. In the present study, we assessed whether intranasal challenge with the TLR7 agonist R‑837 additionally activated neurons in the central nervous system. Within one hour, R‑837 induced activation of the nucleus of the solitary tract, as well as a small increase in nasal IL‑6, but otherwise in the absence of an overt inflammatory response. It is tempting to speculate that it might be a direct interaction of R‑837 with trigeminal neurons in order to alert the central nervous system of invading pathogens.

Pathobiology of inherited biliary diseases: a roadmap to understand acquired liver diseases.

Bile duct epithelial cells, also known as cholangiocytes, regulate the composition ofbileand its flow. Acquired, congenital and genetic dysfunctions in these cells give rise to asetof diverse and complex diseases, often of unknown aetiology, called cholangiopathies. New knowledge has been steadily acquired about genetic and congenital cholangiopathies, and this has led to a better understanding of the mechanisms of acquired cholangiopathies. This Review focuses on findings from studies on Alagille syndrome, polycystic liver diseases, fibropolycystic liver diseases (Caroli disease and congenital hepatic fibrosis) and cystic fibrosis-related liver disease. In particular, knowledge on the role of Notch signalling in biliary repair and tubulogenesis has been advanced by work on Alagille syndrome, and investigations in polycystic liver diseases have highlighted the role of primary cilia in biliary pathophysiology and the concept of biliary angiogenic signalling and its role in cyst growth and biliary repair. In fibropolycystic liver disease, research has shown that loss of fibrocystin generates a signalling cascade that increases β-catenin signalling, activates the NOD-, LRR- and pyrin domain-containing 3 inflammasome, and promotes production of IL-1β and other chemokines that attract macrophages and orchestrate the process of pericystic and portal fibrosis, which are the main mechanisms of progression in cholangiopathies. In cystic fibrosis-related liver disease, lack of cystic fibrosis transmembrane conductance regulator increases the sensitivity of epithelial Toll-like receptor 4 that sustains the secretion of nuclear factor-κB-dependent cytokines and peribiliary inflammation in response to gut-derived products, providing a model for primary sclerosing cholangitis. These signalling mechanisms may be targeted therapeutically and they offer a possibility for the development of novel treatments for acquired cholangiopathies.

Inducible lung epithelial resistance requires multisource reactive oxygen species generation to protect against bacterial infections.

Pneumonia remains a global health threat, in part due to expanding categories of susceptible individuals and increasing prevalence of antibiotic resistant pathogens. However, therapeutic stimulation of the lungs’ mucosal defenses by inhaled exposure to a synergistic combination of Toll-like receptor (TLR) agonists known as Pam2-ODN promotes mouse survival of pneumonia caused by a wide array of pathogens. This inducible resistance to pneumonia relies on intact lung epithelial TLR signaling, and inducible protection against viral pathogens has recently been shown to require increased production of epithelial reactive oxygen species (ROS) from multiple epithelial ROS generators. To determine whether similar mechanisms contribute to inducible antibacterial responses, the current work investigates the role of ROS in therapeutically-stimulated protection against Pseudomonas aerugnosa challenges. Inhaled Pam2-ODN treatment one day before infection prevented hemorrhagic lung cytotoxicity and mouse death in a manner that correlated with reduction in bacterial burden. The bacterial killing effect of Pam2-ODN was recapitulated in isolated mouse and human lung epithelial cells, and the protection correlated with inducible epithelial generation of ROS. Scavenging or targeted blockade of ROS production from either dual oxidase or mitochondrial sources resulted in near complete loss of Pam2-ODN-induced bacterial killing, whereas deficiency of induced antimicrobial peptides had little effect. These findings support a central role for multisource epithelial ROS in inducible resistance against a bacterial pathogen and provide mechanistic insights into means to protect vulnerable patients against lethal infections.

Endothelial toll-like receptor 4 maintains lung integrity via epigenetic suppression of p16INK4a.

We previously reported that the canonical innate immune receptor toll‐like receptor 4 (TLR4) is critical in maintaining lung integrity. However, the molecular mechanisms via which TLR4 mediates its effect remained unclear. In the present study, we identified distinct contributions of lung endothelial cells (Ec) and epithelial cells TLR4 to pulmonary homeostasis using genetic‐specific, lung‐ and cell‐targeted in vivo methods. Emphysema was significantly prevented via the reconstituting of human TLR4 expression in the lung Ec of TLR4−/− mice. Lung Ec‐silencing of TLR4 in wild‐type mice induced emphysema, highlighting the specific and distinct role of Ec‐expressed TLR4 in maintaining lung integrity. We also identified a previously unrecognized role of TLR4 in preventing expression of p16INK4a, a senescence‐associated gene. Lung Ec‐p16INK4a‐silencing prevented TLR4−/− induced emphysema, revealing a new functional role for p16INK4ain lungs. TLR4 suppressed endogenous p16INK4a expression via HDAC2‐mediated deacetylation of histone H4. These findings suggest a novel role for TLR4 in maintaining of lung homeostasis via epigenetic regulation of senescence‐related gene expression.

P122 EPITHELIAL TLR4 PROMOTES OXIDATIVE STRESS VIA NADPH OXIDASE ACTIVATION AND INDUCES REDOX-SENSITIVE PATHWAYS DURING TUMORIGENESIS

Dysbiosis and accumulation of reactive oxygen species (ROS) are hallmarks of ulcerative colitis (UC) and have been associated with tumor progression. We have previously shown that UC patient biopsies and colorectal adenomas overexpress toll-like receptor 4 (TLR4) in intestinal epithelial cells (IECs). A potential mechanism of TLR4-mediated tumorigenesis is by providing cancer stem cells with redox signals that they efficiently use to outgrow their non-transformed counterparts. We test the hypothesis that epithelial signaling via TLR4 induces the production of ROS through the NADPH oxidases Nox1 and Duox2. We also explore the main redox-sensitive cascades that participate in TLR4-driven tumorigenesis. Colonoids from wild-type (WT), TLR4-KO, and Nox1-KO mice were stimulated with LPS and tested for H2O2 production in the presence of the NADPH oxidase inhibitor, diphenyleneiodonium. IECs isolated from villin-TLR4 mice (which have constitutive activation of epithelial TLR4) and their WT littermates were analyzed for H2O2 production (Amplex Red) and expression of the NADPH oxidases Nox1 and Duox2 (qPCR). Villin-TLR4 and their littermates underwent a model of CAC by azoxymethane injection followed by 2 cycles of 3% dextran sulfate sodium (1.5% in villin-TLR4 mice). Cancer stem cells (aldehyde dehydrogenase positive) were sorted for RNA sequencing. LPS incubation enhanced the expression of Nox1 and Duox2 in WT but not TLR4-KO colonoids, demonstrating that these oxidative enzymes are regulated by TLR4. Consistently, LPS stimulation elicited H2O2 release by WT organoids in a NADPH oxidase-dependent manner. Nox1-KO colonoids showed attenuated production of H2O2 upon LPS stimulation, confirming the participation of this NADPH oxidase in TLR4-mediated production of ROS. Constitutive activation of TLR4 induced the production of H2O2 in IECs, which was accompanied by a marked upregulation of Nox1, Duox2, and DuoxA2. RNAseq done on cancer stem cells shows that cancer stem cells of villin-TLR4 tumors have increased expression of NADPH oxidases, as well as enrichment of redox-sensitive pathways, including PI3K, myc, Rac1, and NF-κB. All of these pathways have been implicated in the promotion of proliferative and antiapoptotic signals. TLR4 controls the expression and function of oxidative enzymes in IECs, enhancing redox signals in cancer stem cells upon bacterial challenge. In the context of inflammation-associated dysbiosis, we speculate that overexpression of TLR4 provides additional activation of proliferative and antiapoptotic pathways, accelerating tumor development and growth. The results of our studies highlight how we might target critical signaling pathways in inflammatory-driven cancer stem cells.

Inducible Lung Epithelial Resistance Requires Multisource Reactive Oxygen Species Generation To Protect against Viral Infections.

ABSTRACTViral pneumonias cause profound worldwide morbidity, necessitating novel strategies to prevent and treat these potentially lethal infections. Stimulation of intrinsic lung defenses via inhalation of synergistically acting Toll-like receptor (TLR) agonists protects mice broadly against pneumonia, including otherwise-lethal viral infections, providing a potential opportunity to mitigate infectious threats. As intact lung epithelial TLR signaling is required for the inducible resistance and as these cells are the principal targets of many respiratory viruses, the capacity of lung epithelial cells to be therapeutically manipulated to function as autonomous antiviral effectors was investigated. Our work revealed that mouse and human lung epithelial cells could be stimulated to generate robust antiviral responses that both reduce viral burden and enhance survival of isolated cells and intact animals. The antiviral protection required concurrent induction of epithelial reactive oxygen species (ROS) from both mitochondrial and dual oxidase sources, although neither type I interferon enrichment nor type I interferon signaling was required for the inducible protection. Taken together, these findings establish the sufficiency of lung epithelial cells to generate therapeutically inducible antiviral responses, reveal novel antiviral roles for ROS, provide mechanistic insights into inducible resistance, and may provide an opportunity to protect patients from viral pneumonia during periods of peak vulnerability.

Intestinal epithelial Toll-like receptor 4 prevents metabolic syndrome by regulating interactions between microbes and intestinal epithelial cells in mice.

Little is known about the pathogenesis of metabolic syndrome, although Toll-like receptor 4 (TLR4) has been implicated. We investigated whether TLR4 in the intestinal epithelium regulates metabolic syndrome by coordinating interactions between the luminal microbiota and host genes that regulate metabolism. Mice lacking TLR4 in the intestinal epithelium (TLR4ΔIEC), but not mice lacking TLR4 in myeloid cells nor mice lacking TLR4 globally, developed metabolic syndrome; these features were not observed in TLR4ΔIEC mice given antibiotics. Metagenomic analysis of the fecal microbiota revealed differences between TLR4ΔIEC and wild type mice, while meta-transcriptome analysis of the microbiota showed that intestinal TLR4 affected the expression of microbial genes involved in the metabolism of lipids, amino acids, and nucleotides. Genes regulated by peroxisome proliferator-activated receptors (PPARs) and the antimicrobial peptide lysozyme were significantly down-regulated in TLR4ΔIEC mice, suggesting a mechanism by which intestinal TLR4 could exert its effects on the microbiota and metabolic syndrome. Supportingly, antibiotics prevented both downregulation of PPAR genes and the development of metabolic syndrome, while PPAR agonists prevented development of metabolic syndrome in TLR4ΔIEC mice. Thus, intestinal epithelial TLR4 regulates metabolic syndrome through altered host-bacterial signaling, suggesting that microbial or PPAR based strategies might have therapeutic potential for this disease.

T84 monolayers are superior to Caco-2 as a model system of colonocytes.

Colonic adenocarcinoma-derived Caco-2 and T84 epithelial cell lines are frequently used as in vitro model systems of functional epithelial barriers. Both are utilised interchangeably despite evidence that differentiated Caco-2 cells are more reminiscent of small intestinal enterocytes than of colonocytes, whereas differentiated T84 cells are less well characterised. The aim of this study was, therefore, to further characterise and compare differentiated Caco-2 and T84 cells. The objectives were to (1) compare the brush border morphology, (2) measure the expression of enterocyte- and colonocyte-specific genes and (3) compare their response to butyrate, which is dependent on the monocarboxylate transporter 1 (MCT1), an apical protein expressed primarily in colonocytes. T84 microvilli were significantly shorter than those of Caco-2 cells, which is a characteristic difference between small intestinal enterocytes and colonocytes. Also, enterocyte-associated brush border enzymes expressed in differentiated Caco-2 cells were not increased during T84 maturation, whereas colonic markers such as MCT1 were more abundant in differentiated T84 cells compared to differentiated Caco-2 cells. Consequently, T84 cells displayed a dose-responsive improvement of barrier function towards butyrate, which was absent in Caco-2 cells. On the other hand, differences in epithelial toll-like receptor expression between Caco-2 and T84 monolayers did not result in a corresponding differential functional response. We conclude that differentiated Caco-2 and T84 cells have distinct morphological, biochemical and functional characteristics, suggesting that T84 cells do not acquire the biochemical signature of mature small intestinal enterocytes like Caco-2 cells, but retain much of their original colonic characteristics throughout differentiation. These findings can help investigators select the appropriate intestinal epithelial cell line for specific in vitro research purposes.