Site‐specific contribution of Toll‐like receptor 4 to intestinal homeostasis and inflammatory disease

Toll‐like receptor 4 (TLR4) is a highly conserved protein of innate immunity, responsible for the regulation and maintenance of homeostasis, as well as immune recognition of external and internal ligands. TLR4 is expressed on a variety of cell types throughout the gastrointestinal tract, including on epithelial and immune cell populations. In a healthy state, epithelial cell expression of TLR4 greatly assists in homeostasis by shaping the host microbiome, promoting immunoglobulin A production, and regulating follicle‐associated epithelium permeability. In contrast, immune cell expression of TLR4 in healthy states is primarily centred on the maturation of dendritic cells in response to stimuli, as well as adequately priming the adaptive immune system to fight infection and promote immune memory. Hence, in a healthy state, there is a clear distinction in the site‐specific roles of TLR4 expression. Similarly, recent research has indicated the importance of site‐specific TLR4 expression in inflammation and disease, particularly the impact of epithelial‐specific TLR4 on disease progression. However, the majority of evidence still remains ambiguous for cell‐specific observations, with many studies failing to provide the distinction of epithelial versus immune cell expression of TLR4, preventing specific mechanistic insight and greatly impacting the translation of results. The following review provides a critical overview of the current understanding of site‐specific TLR4 activity and its contribution to intestinal/immune homeostasis and inflammatory diseases.     

Rab11‐FIP2 Interaction with MYO5B Regulates Movement of Rab11a‐Containing Recycling Vesicles

A tripartite association of Rab11a with both Rab11‐FIP2 and MYO5B regulates recycling endosome trafficking. We sought to define the intermolecular interactions required between Rab11‐FIP2 and MYO5B. Using a random mutagenesis strategy, we identified point mutations at S229P or G233E in Rab11‐FIP2 that caused loss of interaction with MYO5B in yeast two‐hybrid assays as well as loss of interaction of Rab11‐FIP2(129‐356) with MYO5B tail when expressed in HeLa cells. Single mutations or the double S229P/G233E mutation failed to alter the association of full‐length Rab11‐FIP2 with MYO5B tail in HeLa cells. While EGFP‐Rab11‐FIP2 wild type colocalized with endogenous MYO5B staining in MDCK cells, EGFP‐Rab11‐FIP2(S229P/G233E) showed a significant decrease in localization with endogenous MYO5B. Analysis of Rab11a‐containing vesicle movement in live HeLa cells demonstrated that when the MYO5B/Rab11‐FIP2 association is perturbed by mutation or by Rab11‐FIP2 knockdown, vesicle movement is increased in both speed and track length, consistent with an impairment of MYO5B tethering at the cytoskeleton. These results support a critical role for the interaction of MYO5B with Rab11‐FIP2 in stabilizing the functional complex with Rab11a, which regulates dynamic movements of membrane recycling vesicles.      

Class I Rab11‐family interacting proteins are binding targets for the Rab14 GTPase

Background information. Rab11 and Rab14 are two related Rab GTPases that are believed to function in endosomal recycling and Golgi/endosome transport processes. We, and others, have identified a group of proteins that interact with Rab11 and function as Rab11 effectors, known as the Rab11‐FIPs (family interacting proteins). This protein family has been sub‐classified into two groups—class I FIPs [FIP2, RCP (Rab coupling protein) and Rip11 (Rab11‐interacting protein)] and class II FIPs (FIP3 and FIP4). Results. In the present study we identify the class I FIPs as dual Rab‐binding proteins by demonstrating that they also interact with Rab14 in a GTP‐dependent manner. We show that these interactions are specific for the class I FIPs and that they occur via their C‐terminal regions, which encompass the previously described RBD (Rab11‐binding domain). Furthermore, we show that Rab14 significantly co‐localizes with the TfnR (transferrin receptor) and that Rab14 Q70L co‐localizes with Rab11a and with the class I FIPs on the ERC (endosomal recycling compartment) during interphase. Additionally, we show that during cytokinesis Rab14 localizes to the cleavage furrow/midbody. Conclusions. The data presented in the present study, which identifies the class I FIPs as the first putative effector proteins for the Rab14 GTPase, indicates greater complexity in the Rab‐binding specificity of the class I FIP proteins.     

Toll‐like receptor 4 regulates spontaneous intestinal tumorigenesis by up‐regulating IL‐6 and GM‐CSF

Inflammation is as an important component of intestinal tumorigenesis. The activation of Toll‐like receptor 4 (TLR4) signalling promotes inflammation in colitis of mice, but the role of TLR4 in intestinal tumorigenesis is not yet clear. About 80%–90% of colorectal tumours contain inactivating mutations in the adenomatous polyposis coli (Apc) tumour suppressor, and intestinal adenoma carcinogenesis in familial adenomatous polyposis (FAP) is also closely related to the germline mutations in Apc. The Apc^Min/+ (multiple intestinal neoplasia) model mouse is a well‐utilized model of FAP, an inherited form of intestinal cancer. In this study, Apc^Min/+ intestinal adenoma mice were generated on TLR4‐sufficient and TLR4‐deficient backgrounds to investigate the carcinogenic effect of TLR4 in mouse gut by comparing mice survival, peripheral blood cells, bone marrow haematopoietic precursor cells and numbers of polyps in the guts of Apc^Min/+ WT and Apc^Min/+ TLR4^?/? mice. The results revealed that TLR4 had a critical role in promoting spontaneous intestinal tumorigenesis. Significant differential genes were screened out by the high‐throughput RNA‐Seq method. After combining these results with KEGG enrichment data, it was determined that TLR4 might promote intestinal tumorigenesis by activating cytokine‐cytokine receptor interaction and pathways in cancer signalling pathways. After a series of validation experiments for the concerned genes, it was found that IL6, GM‐CSF (CSF2), IL11, CCL3, S100A8 and S100A9 were significantly decreased in gut tumours of Apc^Min/+ TLR4^?/? mice compared with Apc^Min/+ WT mice. In the functional study of core down‐regulation factors, it was found that IL6, GM‐CSF, IL11, CCL3 and S100A8/9 increased the viability of colon cancer cell lines and decreased the apoptosis rate of colon cancer cells with irradiation and chemical treatment.     

Neutralization of pro‐inflammatory monocytes by targeting TLR2 dimerization ameliorates colitis

Monocytes have emerged as critical driving force of acute inflammation. Here, we show that inhibition of Toll‐like receptor 2(TLR2) dimerization by a TLR2 transmembrane peptide (TLR2‐p) ameliorated DSS‐induced colitis by interfering specifically with the activation of Ly6C⁺ monocytes without affecting their recruitment to the colon. We report that TLR2‐p directly interacts with TLR2 within the membrane, leading to inhibition of TLR2–TLR6/1 assembly induced by natural ligands. This was associated with decreased levels of extracellular signal‐regulated kinases (ERK) signaling and reduced secretion of pro‐inflammatory cytokines, such as interleukin (IL)‐6, IL‐23, IL‐12, and IL‐1β. Altogether, our study provides insights into the essential role of TLR2 dimerization in the activation of pathogenic pro‐inflammatory Ly6C^hi monocytes and suggests that inhibition of this aggregation by TLR2‐p might have therapeutic potential in the treatment of acute gut inflammation.     

Abnormal Rab11‐Rab8‐vesicles cluster in enterocytes of patients with microvillus inclusion disease

Microvillus inclusion disease (MVID) is a congenital enteropathy characterized by accumulation of vesiculo‐tubular endomembranes in the subapical cytoplasm of enterocytes, historically termed “secretory granules.” However, neither their identity nor pathophysiological significance is well defined. Using immunoelectron microscopy and tomography, we studied biopsies from MVID patients (3× Myosin 5b mutations and 1× Syntaxin3 mutation) and compared them to controls and genome‐edited CaCo2 cell models, harboring relevant mutations. Duodenal biopsies from 2 patients with novel Myosin 5b mutations and typical clinical symptoms showed unusual ultrastructural phenotypes: aberrant subapical vesicles and tubules were prominent in the enterocytes, though other histological hallmarks of MVID were almost absent (ectopic intra‐/intercellular microvilli, brush border atrophy). We identified these enigmatic vesiculo‐tubular organelles as Rab11‐Rab8‐positive recycling compartments of altered size, shape and location harboring the apical SNARE Syntaxin3, apical transporters sodium‐hydrogen exchanger 3 (NHE3) and cystic fibrosis transmembrane conductance regulator. Our data strongly indicate that in MVID disrupted trafficking between cargo vesicles and the apical plasma membrane is the primary cause of a defect of epithelial polarity and subsequent facultative loss of brush border integrity, leading to malabsorption. Furthermore, they support the notion that mislocalization of transporters, such as NHE3 substantially contributes to the reported sodium loss diarrhea.      

Recycling of the human prostacyclin receptor is regulated through a direct interaction with Rab11a GTPase

The human prostacyclin receptor (hIP) undergoes agonist-induced internalization but the mechanisms regulating its intracellular trafficking and/or recycling to the plasma membrane are poorly understood. Herein, we conducted a yeast-two-hybrid screen to identify proteins interacting with the carboxyl-terminal (C)-tail domain of the hIP and discovered a novel interaction with Rab11a. This interaction was confirmed by co-immunoprecipitations in mammalian HEK293 and was augmented by cicaprost stimulation. The hIP co-localized to Rab11-containing recycling endosomes in both HEK293 and endothelial EA.hy 926 cells in a time-dependent manner following cicaprost stimulation. Moreover, over-expression of Rab11a significantly increased recycling of the hIP, while the dominant negative Rab11^S25N impaired that recycling. Conversely, while the hIP co-localized to Rab4-positive endosomes in response to cicaprost, ectopic expression of Rab4a did not substantially affect overall recycling nor did Rab4a directly interact with the hIP. The specific interaction between the hIP and Rab11a was dependent on a 22 amino acid (Val²⁹⁹–Gln³²⁰) sequence within its C-tail domain and was independent of isoprenylation of the hIP. This study elucidates a critical role for Rab11a in regulating trafficking of the hIP and has identified a novel Rab11 binding domain (RBD) within its C-tail domain that is both necessary and sufficient to mediate interaction with Rab11a.     

Up‐regulated TLR4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

It remains unclear whether and how cardiomyocytes contribute to the inflammation in chronic heart failure (CHF). We recently reviewed the capacity of cardiomyocytes to initiate inflammation, by means of expressing certain immune receptors such as toll‐like receptors (TLRs) that respond to pathogen‐ and damage‐associated molecular patterns (PAMP and DAMP). Previous studies observed TLR4‐mediated inflammation within days of myocardial infarction (MI). This study examined TLR4 expression and function in cardiomyocytes of failing hearts after 4 weeks of MI in rats. The increases of TLR4 mRNA and proteins, as well as inflammatory cytokine production, were observed in both the infarct and remote myocardium. Enhanced immunostaining for TLR4 was observed in cardiomyocytes but not infiltrating leucocytes. The injection of lentivirus shRNA against TLR4 into the infarcted heart decreased inflammatory cytokine production and improved heart function in vivo. Accordingly, in cardiomyocytes isolated from CHF hearts, increases of TLR4 mRNA and proteins were detected. More robust binding of TLR4 with lipopolysaccharide (LPS), a PAMP ligand for TLR4, and heat shock protein 60 (HSP60), a DAMP ligand for TLR4, was observed in CHF cardiomyocytes under a confocal microscope. The maximum binding capacity (B_max) of TLR4 was increased for LPS and HSP60, whereas the binding affinity (Kd) was not significantly changed. Furthermore, both LPS and HSP60 induced more robust production of inflammatory cytokines in CHF cardiomyocytes, which was reduced by TLR4‐blocking antibodies. We conclude that the expression, ligand‐binding capacity and pro‐inflammatory function of cardiomyocyte TLR4 are up‐regulated after long‐term MI, which promote inflammation and exacerbate heart failure.     

Homology modeling of human Toll‐like receptors TLR7, 8, and 9 ligand‐binding domains

Toll‐like receptors (TLRs) play a key role in the innate immune system. The TLR7, 8, and 9 compose a family of intracellularly localized TLRs that signal in response to pathogen‐derived nucleic acids. So far, there are no crystallographic structures for TLR7, 8, and 9. For this reason, their ligand‐binding mechanisms are poorly understood. To enable first predictions of the receptor–ligand interaction sites, we developed three‐dimensional structures for the leucine‐rich repeat ectodomains of human TLR7, 8, and 9 based on homology modeling. To achieve a high sequence similarity between targets and templates, structural segments from all known TLR ectodomain structures (human TLR1/2/3/4 and mouse TLR3/4) were used as candidate templates for the modeling. The resulting models support previously reported essential ligand‐binding residues. They also provide a basis to identify three potential receptor dimerization mechanisms. Additionally, potential ligand‐binding residues are identified using combined procedures. We suggest further investigations of these residues through mutation experiments. Our modeling approach can be extended to other members of the TLR family or other repetitive proteins.     

Metal allergens nickel and cobalt facilitate TLR4 homodimerization independently of MD2

Development of contact allergy requires cooperation of adaptive and innate immunity. Ni²⁺ stimulates innate immunity via TLR4/MD2, the bacterial LPS receptor. This likely involves receptor dimerization, but direct proof is pending and it is unclear if related haptens share this mechanism. We reveal Co²⁺ as second metal stimulating TLR4 and confirm necessity of H456/H458 therein. Experiments with a new TLR4 dimerization mutant established dimerization as a mechanism of metal‐ and LPS‐induced TLR4 activation. Yet, in interaction studies only LPS‐ but not metal‐induced dimerization required MD2. Consistently, soluble TLR4 expressed without MD2 inhibited metal‐ but not LPS‐induced responses, opening new therapeutic perspectives.     

Endocytic tubules regulated by Rab GTPases 5 and 11 are used for envelopment of herpes simplex virus

Enveloped viruses employ diverse and complex strategies for wrapping at cellular membranes, many of which are poorly understood. Here, an ultrastructural study of herpes simplex virus 1 (HSV1)‐infected cells revealed envelopment in tubular membranes. These tubules were labelled by the fluid phase marker horseradish peroxidase (HRP), and were observed to wrap capsids as early as 2 min after HRP addition, indicating that the envelope had recently cycled from the cell surface. Consistent with this, capsids did not colocalise with either the trans‐Golgi network marker TGN46 or late endosomal markers, but showed coincidence with the transferrin receptor. Virus glycoproteins were retrieved from the plasma membrane (PM) to label wrapping capsids, a process that was dependent on both dynamin and Rab5. Combined depletion of Rab5 and Rab11 reduced virus yield to <1%, resulting in aberrant localisation of capsids. These results suggest that endocytosis from the PM into endocytic tubules provides the main source of membrane for HSV1, and reveal a new mechanism for virus exploitation of the endocytic pathway.     

Robust intestinal homeostasis relies on cellular plasticity in enteroblasts mediated by miR‐8–Escargot switch

The intestinal epithelium is remarkably robust despite perturbations and demand uncertainty. Here, we investigate the basis of such robustness using novel tracing methods that allow simultaneously capturing the dynamics of stem and committed progenitor cells (called enteroblasts) and intestinal cell turnover with spatiotemporal resolution. We found that intestinal stem cells (ISCs) divide “ahead” of demand during Drosophila midgut homeostasis. Their newborn enteroblasts, on the other hand, take on a highly polarized shape, acquire invasive properties and motility. They extend long membrane protrusions that make cell–cell contact with mature cells, while exercising a capacity to delay their final differentiation until a local demand materializes. This cellular plasticity is mechanistically linked to the epithelial–mesenchymal transition (EMT) programme mediated by escargot, a snail family gene. Activation of the conserved microRNA miR‐8/miR‐200 in “pausing” enteroblasts in response to a local cell loss promotes timely terminal differentiation via a reverse MET by antagonizing escargot. Our findings unveil that robust intestinal renewal relies on hitherto unrecognized plasticity in enteroblasts and reveal their active role in sensing and/or responding to local demand.     

Down‐regulation of mitogen‐activated protein kinases and nuclear factor‐κB signaling is involved in rapamycin suppression of TLR2‐induced inflammatory response in monocytic THP‐1 cells

Tripalmitoyl‐S‐glycero‐Cys‐(Lys) 4 (Pam3CSK4) interacted with TLR2 induces inflammatory responses through the mitogen‐activated protein kinases (MAPKs) and nuclear factor‐κB (NF‐κB) signal pathway. Rapamycin can suppress TLR‐induced inflammatory responses; however, the detailed molecular mechanism is not fully understood. Here, the mechanism by which rapamycin suppresses TLR2‐induced inflammatory responses was investigated. It was found that Pam3CSK4‐induced pro‐inflammatory cytokines were significantly down‐regulated at both the mRNA and protein levels in THP‐1 cells pre‐treated with various concentrations of rapamycin. Inhibition of phosphatidylinositol 3‐kinase/protein kinase‐B (PI3K/AKT) signaling did not suppress the expression of pro‐inflammatory cytokines, indicating that the immunosuppression mediated by rapamycin in THP1 cells is independent of the PI3K/AKT pathway. RT‐PCR showed that Erk and NF‐κB signal pathways are related to the production of pro‐inflammatory cytokines. Inhibition of Erk or NF‐κB signaling significantly down‐regulated production of pro‐inflammatory cytokines. Additionally, western blot showed that pre‐treatment of THP‐1 cells with rapamycin down‐regulates MAPKs and NF‐κB signaling induced by Pam3CSK4 stimulation, suggesting that rapamycin suppresses Pam3CSK4‐induced pro‐inflammatory cytokines via inhibition of TLR2 signaling. It was concluded that rapamycin suppresses TLR2‐induced inflammatory responses by down‐regulation of Erk and NF‐κB signaling.     

Rac1‐Rab11‐FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T‐cell activation

The immunological synapse generation and function is the result of a T‐cell polarization process that depends on the orchestrated action of the actin and microtubule cytoskeleton and of intracellular vesicle traffic. However, how these events are coordinated is ill defined. Since Rab and Rho families of GTPases control intracellular vesicle traffic and cytoskeleton reorganization, respectively, we investigated their possible interplay. We show here that a significant fraction of Rac1 is associated with Rab11‐positive recycling endosomes. Moreover, the Rab11 effector FIP3 controls Rac1 intracellular localization and Rac1 targeting to the immunological synapse. FIP3 regulates, in a Rac1‐dependent manner, key morphological events, like T‐cell spreading and synapse symmetry. Finally, Rab11‐/FIP3‐mediated regulation is necessary for T‐cell activation leading to cytokine production. Therefore, Rac1 endosomal traffic is key to regulate T‐cell activation.     

MRP8/14 mediates macrophage efferocytosis through RAGE and Gas6/MFG‐E8, and induces polarization via TLR4‐dependent pathway

Myeloid‐related protein 8/14 (MRP8/14) participates in various inflammatory responses, however, its effect on macrophage efferocytosis remains unclear. Here, we demonstrate that MRP8/14 significantly inhibits the efferocytosis of apoptotic thymocytes by mouse bone marrow‐derived macrophages (BMDMs), which later proves to be associated with the receptor for advanced glycation end products (RAGE) or for reducing the expression of growth arrest‐specific protein 6 and milk fat globule epidermal growth factor 8, independent of RAGE. Furthermore, MRP8/14 promotes polarization of BMDMs from the M₂‐ to M₁‐like phenotype by upregulating expression of M₁‐related surface receptor proteins and signature M₁‐marker genes and by downregulating signature M₂‐marker gene expression, which depends on Toll‐like receptor 4 and p38 mitogen‐activated protein kinase/nuclear factor κB pathways. Thus, we report a significant inhibitory effect of MRP8/14 on macrophage efferocytosis and MRP8/14‐mediated phenotypic polarization, which may be helpful in developing novel therapeutic strategies leading to inflammation resolution.     

A novel TLR2‐triggered signalling crosstalk synergistically intensifies TNF‐mediated IL‐6 induction

Toll‐like receptors (TLR) recognize pathogens and trigger the production of vigorous pro‐inflammatory cytokines [such as tumour necrosis factor (TNF)] that induce systemic damages associated with sepsis and chronic inflammation. Cooperation between signals of TLR and TNF receptor has been demonstrated through the participation of TNF receptor 1 (TNFR) adaptors in endotoxin tolerance. Here, we identify a TLR2‐mediated synergy, through a MyD88‐independent crosstalk, which enhances subsequent TNF‐mediated nuclear factor‐kappa B activation and interleukin‐6 induction. Membrane‐associated adaptor MAL conduces the link between TNF receptor‐associated factor 6 (TRAF6) and TNFR‐associated death domain, leading to a distinctive K63‐ubiquitinylated TRAF6 recruitment into TNFR complex. In summary, our results reveal a novel route of TLR signal that synergistically amplifies TNF‐mediated responses, indicating an innovative target for inflammation manipulation.     

Crystal structure of the C‐terminal domain of mouse TLR9

Toll‐like receptors (TLRs) are important pattern recognition receptors that function in innate immunity. Elucidating the structure and signaling mechanisms of TLR9, a sensor of foreign and endogenous DNA, is essential for understanding its key role in immunity against microbial pathogens as well as in autoimmunity. Abundant evidence suggests that the TLR9‐CTD (C‐terminal domain) by itself is capable of DNA binding and signaling. The crystal structure of unliganded mouse TLR9‐CTD is presented. TLR9‐CTD exhibits one unique feature, a cluster of stacked aromatic and arginine side chains on its concave face. Overall, its structure is most related to the TLR8‐CTD, suggesting a similar mode of ligand binding and signaling. Proteins 2014; 82:2874–2878. © 2014 Wiley Periodicals, Inc.