Macropinocytosis in Shiga toxin 1 uptake by human intestinal epithelial cells and transcellular transcytosis

Shiga toxin 1 and 2 production is a cardinal virulence trait of enterohemorrhagic Escherichia coli infection that causes a spectrum of intestinal and systemic pathology. However, intestinal sites of enterohemorrhagic E. coli colonization during the human infection and how the Shiga toxins are taken up and cross the globotriaosylceramide (Gb3) receptor-negative intestinal epithelial cells remain largely uncharacterized. We used samples of human intestinal tissue from patients with E. coli O157:H7 infection to detect the intestinal sites of bacterial colonization and characterize the distribution of Shiga toxins. We further used a model of largely Gb3-negative T84 intestinal epithelial monolayers treated with B-subunit of Shiga toxin 1 to determine the mechanisms of non-receptor-mediated toxin uptake. We now report that E. coli O157:H7 were found at the apical surface of epithelial cells only in the ileocecal valve area and that both toxins were present in large amounts inside surface and crypt epithelial cells in all tested intestinal samples. Our in vitro data suggest that macropinocytosis mediated through Src activation significantly increases toxin endocytosis by intestinal epithelial cells and also stimulates toxin transcellular transcytosis. We conclude that Shiga toxin is taken up by human intestinal epithelial cells during E. coli O157:H7 infection regardless of the presence of bacterial colonies. Macropinocytosis might be responsible for toxin uptake by Gb3-free intestinal epithelial cells and transcytosis. These observations provide new insights into the understanding of Shiga toxin contribution to enterohemorrhagic E. coli-related intestinal and systemic diseases.     

Critical role of Toll‐like receptor 2 in Bacteroides fragilis‐mediated immune responses in murine peritoneal mesothelial cells

In this study, the role of Toll‐like receptor 2 (TLR2) in immune responses of murine peritoneal mesothelial cells against Bacteroides fragilis was investigated. Enzyme linked immunosorbent assay was used to measure cytokines and chemokines. Activation of nuclear factor κB (NF‐κB‐α) and mitogen‐activated protein kinases (MAP kinases) was investigated by western blot analysis. B. fragilis induced production of interleukin‐6, chemokine (C‐X‐C motif) ligand 1 (CXCL1) and chemokine (C‐C motif) ligand 2 (CCL2) in wild type peritoneal mesothelial cells; this was impaired in TLR2‐deficient cells. In addition, in response to B. fragilis, phosphorylation of inhibitory NF‐κB‐α and c‐Jun N‐terminal kinase mitogen‐activated protein kinase (MAPK) was induced in wild type mesothelial cells, but not in TLR2‐deficient cells,. Inhibitor assay revealed that NF‐κB and MAPKs are essential for B. fragilis‐induced production of CXCL1 and CCL2 in mesothelial cells. These findings suggest that TLR2 mediates immune responses in peritoneal mesothelial cells in response to B. fragilis.     

Immune complex negatively regulates Toll‐like receptor 9‐mediated immune responses in B cells through the inhibitory Fc‐gamma receptor IIb

Because inappropriate activation of Toll‐like receptor 9 (TLR9) may induce pathological damage, negative regulation of the TLR9‐triggered immune response has attracted considerable attention. Nonpathogenic immune complex (IC) has been demonstrated to have beneficial therapeutic effects in some kinds of autoimmune diseases. However, the role of IC in the regulation of TLR9‐triggered immune responses and the underlying mechanisms remain unclear. In this study, it was demonstrated that IC stimulation of B cells not only suppresses CpG‐oligodeoxynucleotide (CpG‐ODN)‐induced pro‐inflammatory IL‐6 and IgM κ production, but also attenuates CD40 and CD80 expression. Furthermore, our results suggest that the receptor for the Fc portion of IgG (FcγR) IIb is involved in the suppressive effect of IC on TLR9‐mediated CD40, CD80 and IL‐6 expression. Finally, it was found that IC down‐regulates TLR9 expression in CpG‐ODN activated B cells. Our results provide an outline of a new pathway for the negative regulation of TLR9‐triggered immune responses in B cells via FcγRIIb. A new mechanistic explanation of the therapeutic effect of nonpathogenic IC on inflammatory and autoimmune diseases is also provided.     

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.     

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.     

Toll‐like receptor 9 protects non‐immune cells from stress by modulating mitochondrial ATP synthesis through the inhibition of SERCA2

Toll‐like receptor 9 (TLR9) has a key role in the recognition of pathogen DNA in the context of infection and cellular DNA that is released from damaged cells. Pro‐inflammatory TLR9 signalling pathways in immune cells have been well investigated, but we have recently discovered an alternative pathway in which TLR9 temporarily reduces energy substrates to induce cellular protection from stress in cardiomyocytes and neurons. However, the mechanism by which TLR9 stimulation reduces energy substrates remained unknown. Here, we identify the calcium‐transporting ATPase, SERCA2 (also known as Atp2a2), as a key molecule for the alternative TLR9 signalling pathway. TLR9 stimulation reduces SERCA2 activity, modulating Ca²⁺ handling between the SR/ER and mitochondria, which leads to a decrease in mitochondrial ATP levels and the activation of cellular protective machinery. These findings reveal how distinct innate responses can be elicited in immune and non‐immune cells—including cardiomyocytes—using the same ligand‐receptor system.     

Characterization of mesenchymal stem cells under the stimulation of Toll‐like receptor agonists

Infective factors cause the perpetuation of inflammation as a result of the permanent exposure of the immune system to exogenous or endogenous products of virus or bacteria. Mesenchymal stem cells (MSCs) can be exposed to this infective environment, which may change the characteristics and therapeutic potency of these MSCs. MSCs have the ability to repair damaged and inflamed tissues and regulate immune responses. In this study, we demonstrated that MSCs express functional Toll‐like receptors (TLR) 3 and 4, the Toll‐like receptor families that recognize the signals of viral and bacterial mimics, respectively. The specific stimulations did not affect the self‐renewal and apoptosis capabilities of MSCs but instead promoted their differentiation into the adipocytes and osteoblasts with the TLR3 ligand. The reverse of these results were obtained with the TLR4 ligand. The migration of the MSCs to stimulate either of the two specific ligands was inhibited at different times, whereas the immunogenicity and immunosuppressive properties of the MSCs were not weakened unlike in the MSCs group. These results suggest that TLR3 and TLR4 stimulation affect the characterization of MSCs.     

Matrix vesicles originate from apical membrane microvilli of mineralizing osteoblast‐like Saos‐2 cells

In bone, mineralization is tightly regulated by osteoblasts and hypertrophic chondrocytes which release matrix vesicles (MVs) and control extracellular ionic conditions and matrix composition. MVs are the initial sites of hydroxyapatite (HA) mineral formation. Despite growing knowledge about their morphology and function, their biogenesis is not well understood. The purpose of this work was to determine the source of MVs in osteoblast lineage, Saos‐2 cells, and to check whether MVs originated from microvilli. Microvilli were isolated from the apical plasma membrane of Saos‐2 cells. Their morphology, structure, and function were compared with those of MVs. The role of actin network in MV release was investigated by using microfilament perturbing drugs. When examined by electron microscopy MVs and microvillar vesicles were found to exhibit similar morphology with trilaminar membranes and diameters in the same range. Both types of vesicles were able to induce HA formation. Their electrophoretic profiles displayed analogous enrichment in alkaline phosphatase, Na⁺/K⁺ ATPase, and annexins A2 and A6. MVs and microvillar vesicles exhibited almost the same lipid composition with a higher content of cholesterol, sphingomyelin, and phosphatidylserine as compared to plasma membrane. Finally, cytochalasin D, which inhibits actin polymerization, was found to stimulate release of MVs. Our findings were consistent with the hypothesis that MVs originated from cell microvilli and that actin filament disassembly was involved in their biogenesis. J. Cell. Biochem. 106: 127–138, 2009. © 2008 Wiley‐Liss, Inc.     

Comprehensive modeling and functional analysis of Toll‐like receptor ligand‐recognition domains

Toll‐like receptors (TLRs) are innate immune pattern‐recognition receptors endowed with the capacity to detect microbial pathogens based on pathogen‐associated molecular patterns. The understanding of the molecular principles of ligand recognition by TLRs has been greatly accelerated by recent structural information, in particular the crystal structures of leucine‐rich repeat‐containing ectodomains of TLR2, 3, and 4 in complex with their cognate ligands. Unfortunately, for other family members such as TLR7, 8, and 9, no experimental structural information is currently available. Methods such as X‐ray crystallography or nuclear magnetic resonance are not applicable to all proteins. Homology modeling in combination with molecular dynamics may provide a straightforward yet powerful alternative to obtain structural information in the absence of experimental (structural) data, provided that the generated three‐dimensional models adequately approximate what is found in nature. Here, we report the development of modeling procedures tailored to the structural analysis of the extracellular domains of TLRs. We comprehensively compared secondary structure, torsion angles, accessibility for glycosylation, surface charge, and solvent accessibility between published crystal structures and independently built TLR2, 3, and 4 homology models. Finding that models and crystal structures were in good agreement, we extended our modeling approach to the remaining members of the TLR family from human and mouse, including TLR7, 8, and 9.     

Toll‐like receptor‐mediated IRE1α activation as a therapeutic target for inflammatory arthritis

In rheumatoid arthritis (RA), macrophage is one of the major sources of inflammatory mediators. Macrophages produce inflammatory cytokines through toll‐like receptor (TLR)‐mediated signalling during RA. Herein, we studied macrophages from the synovial fluid of RA patients and observed a significant increase in activation of inositol‐requiring enzyme 1α (IRE1α), a primary unfolded protein response (UPR) transducer. Myeloid‐specific deletion of the IRE1α gene protected mice from inflammatory arthritis, and treatment with the IRE1α‐specific inhibitor 4U8C attenuated joint inflammation in mice. IRE1α was required for optimal production of pro‐inflammatory cytokines as evidenced by impaired TLR‐induced cytokine production in IRE1α‐null macrophages and neutrophils. Further analyses demonstrated that tumour necrosis factor (TNF) receptor‐associated factor 6 (TRAF6) plays a key role in TLR‐mediated IRE1α activation by catalysing IRE1α ubiquitination and blocking the recruitment of protein phosphatase 2A (PP2A), a phosphatase that inhibits IRE1α phosphorylation. In summary, we discovered a novel regulatory axis through TRAF6‐mediated IRE1α ubiquitination in regulating TLR‐induced IRE1α activation in pro‐inflammatory cytokine production, and demonstrated that IRE1α is a potential therapeutic target for inflammatory arthritis.     

Dynamics on human Toll‐like receptor 4 complexation to MD‐2: The coreceptor stabilizing function

The interaction between human Toll‐like receptor 4 (hTLR4) and its coreceptor, myeloid differentiation factor 2 (MD‐2), is important in Gram‐negative bacteria lipopolysaccharide (LPS) recognition. In this process, MD‐2 recognizes LPS and promotes the dimerization of the complex hTLR4–MD‐2–LPS, triggering an intracellular immune signaling. In this study, we employed distinct computational methods to explore the dynamical properties of the hTLR4–MD‐2 complex and investigated the implications of the coreceptor complexation to the structural biology of hTLR4. We characterized both global and local dynamics of free and MD‐2 complexed hTLR4, in both (hTLR4–MD‐2)₁ and (hTLR4–MD‐2)₂ states. Both molecular dynamics and normal mode analysis reveled a stabilization of the terminal regions of hTLR4 upon complexation to MD‐2. We are able to identify conserved important residues involved on the hTLR4–MD‐2 interaction dynamics and disclose C‐terminal motions that may be associated to the signaling process upon oligomerization. Proteins 2015; 83:373–382. © 2014 Wiley Periodicals, Inc.     

A role for the non‐receptor tyrosine kinase ACK1 in TNF‐alpha‐mediated apoptosis and proliferation in human intestinal epithelial caco‐2 cells

The roles of tumor necrosis factor alpha (TNF‐alpha) and its mediators in cellular processes related to intestinal diseases remain elusive. In this study, we aimed to determine the biological role of activated Cdc42‐associated kinase 1 (ACK1) in TNF‐alpha‐mediated apoptosis and proliferation in Caco‐2 cells. ACK1 expression was knocked down using ACK1‐specific siRNAs, and ACK1 activity was disrupted using a small molecule ACK1 inhibitor. The Terminal deoxynucleotidyl transferase biotin‐dUTP Nick End Labeling (TUNEL) and the BrdU incorporation assays were used to measure apoptosis and cell proliferation, respectively. ACK1‐specific siRNA and the pharmacological ACK1 inhibitor significantly abrogated the TNF‐alpha‐mediated anti‐apoptotic effects and proliferation of Caco‐2 cells. Interestingly, TNF‐alpha activated ACK1 at tyrosine 284 (Tyr284), and the ErbB family of proteins was implicated in ACK1 activation in Caco‐2 cells. ACK1‐Tyr284 was required for protein kinase B (AKT) activation, and ACK1 signaling was mediated through recruiting and phosphorylating the down‐stream adaptor protein AKT, which likely promoted cell proliferation in response to TNF‐alpha. Moreover, ACK1 activated AKT and Src enhanced nuclear factor‐кB (NF‐кB) activity, suggesting a correlation between NF‐кB signaling and TNF‐alpha‐mediated apoptosis in Caco‐2 cells. Our results demonstrate that ACK1 plays an important role in modulating TNF‐alpha‐induced aberrant cell proliferation and apoptosis, mediated in part by ACK1 activation. ACK1 and its down‐stream effectors may hold promise as therapeutic targets in the prevention and treatment of gastrointestinal cancers, in particular, those induced by chronic intestinal inflammation.     

Toll‐like receptor 4: A promising therapeutic target for pneumonia caused by Gram‐negative bacteria

Gram‐negative bacteria (GNB) emerge as important pathogens causing pulmonary infection, which can develop into sepsis due to bacterial resistance to antibiotics. GNB pneumonia poses a huge social and economic burden all over the world. During GNB infection in the lung, Toll‐like receptor 4 (TLR4) can form a complex with MD2 and CD14 after recognizing lipopolysaccharide of GNB, initiate the MyD88‐ and TRIF‐dependent signalling pathways and stimulate host non‐specific immune response. In this review, we summarize recent progress in our understanding of the role of TLR4 in GNB pneumonia. The latest experimental results, especially in TLR4 knockout animals, suggest a promising potential of targeting TLR4 signalling pathway for the treatment of GNB pneumonia. Furthermore, we highlight the benefits of Traditional Chinese Medicine as novel candidates for the therapy of GNB pneumonia due to the modulation of TLR4 signalling pathway. Finally, we discuss the promise and challenge in the development of TLR4‐based drugs for GNB pneumonia.     

Nramp2 expression is associated with pH-dependent iron uptake across the apical membrane of human intestinal Caco-2 cells

The absorption of dietary non-heme iron by intestinal enterocytes is crucial to the maintenance of body iron homeostasis. This process must be tightly regulated since there are no distinct mechanisms for the excretion of excess iron from the body. An insight into the cellular mechanisms has recently been provided by expression cloning of a divalent cation transporter (DCT1) from rat duodenum and positional cloning of its human homologue, Nramp2. Here we demonstrate that Nramp2 is expressed in the apical membrane of the human intestinal epithelial cell line, Caco 2 TC7, and is associated with functional iron transport in these cells with a substrate preference for iron over other divalent cations. Iron transport occurs by a proton-dependent mechanism, exhibiting a concurrent intracellular acidification. Taken together, these data suggest that the expression of the Nramp2 transporter in human enterocytes may play an important role in intestinal iron absorption.     

Regulation of Toll‐like receptor 2 interaction with Ecgp96 controls Escherichia coli K1 invasion of brain endothelial cells

The interaction of outer membrane protein A (OmpA) with its receptor, Ecgp96 (a homologue of Hsp90β), is critical for the pathogenesis of Escherichia coli K1 meningitis. Since Hsp90 chaperones Toll‐like receptors (TLRs), we examined the role of TLRs in E. coli K1 infection. Herein, we show that newborn TLR2^?/? mice are resistant to E. coli K1 meningitis, while TLR4^?/? mice succumb to infection sooner. In vitro, OmpA+ E. coli infection selectively upregulates Ecgp96 and TLR2 in human brain microvascular endothelial cells (HBMEC), whereas OmpA? E. coli upregulates TLR4 in these cells. Furthermore, infection with OmpA+ E. coli causes Ecgp96 and TLR2 translocate to the plasma membrane of HBMEC as a complex. Immunoprecipitation studies of the plasma membrane fractions from infected HBMEC reveal that the C termini of Ecgp96 and TLR2 are critical for OmpA+ E. coli invasion. Knockdown of TLR2 using siRNA results in inefficient membrane translocation of Ecgp96 and significantly reduces invasion. In addition, the interaction of Ecgp96 andTLR2 induces a bipartite signal, one from Ecgp96 through PKC‐α while the other from TLR2 through MyD88, ERK1/2 and NF‐κB. This bipartite signal ultimately culminates in the efficient production of NO, which in turn promotes E. coli K1 invasion of HBMEC.     

Distinctive probiotic features share common TLR2‐dependent signalling in intestinal epithelial cells

The underlying mechanisms of probiotics and postbiotics are not well understood, but it is known that both affect the adaptive and innate immune responses. In addition, there is a growing concept that some probiotic strains have common core mechanisms that provide certain health benefits. Here, we aimed to elucidate the signalization of the probiotic bacterial strains Lactobacillus paragasseri K7, Limosilactobacillus fermentum L930BB, Bifidobacterium animalis subsp. animalis IM386 and Lactiplantibacillus plantarum WCFS1. We showed in in vitro experiments that the tested probiotics exhibit common TLR2‐ and TLR10‐dependent downstream signalling cascades involving inhibition of NF‐κB signal transduction. Under inflammatory conditions, the probiotics activated phosphatidylinositol 3‐kinase (PI3K)/Akt anti‐apoptotic pathways and protein kinase C (PKC)‐dependent pathways, which led to regulation of the actin cytoskeleton and tight junctions. These pathways contribute to the regeneration of the intestinal epithelium and modulation of the mucosal immune system, which, together with the inhibition of canonical TLR signalling, promote general immune tolerance. With this study we identified shared probiotic mechanisms and were the first to pinpoint the role of anti‐inflammatory probiotic signalling through TLR10.     

Distinct haplotype structure at the innate immune receptor Toll‐like receptor 2 across bank vole populations and lineages in Europe

Parasite‐mediated selection may contribute to the maintenance of genetic variation at host immune genes over long time scales. To date, the best evidence for the long‐term maintenance of immunogenetic variation in natural populations comes from studies on the major histocompatibility complex (MHC) genes, whereas evidence for such processes from other immune genes remains scarce. In the present study, we show that, despite pronounced population differentiation and the occurrence of numerous private alleles within populations, the innate immune gene Toll‐like receptor 2 (TLR2) displays a distinct haplotype structure in 21 bank vole (Myodes glareolus) populations across Europe. Haplotypes from all populations grouped in four clearly differentiated clusters, with the three main clusters co‐occurring in at least three previously described mitochondrial lineages. This pattern indicates that the distinct TLR2 haplotype structure may precede the split of the mitochondrial lineages 0.19–0.56 Mya and suggests that haplotype clusters at this innate immune receptor are maintained over prolonged time in wild bank vole populations.