RP105 Attenuates Ischemia/Reperfusion-Induced Oxidative Stress in the Myocardium via Activation of the Lyn/Syk/STAT3 Signaling Pathway.

Although our previous studies have established the crucial role of RP105 in myocardial ischemia/reperfusion injury (MI/RI), its involvement in regulating oxidative stress induced by MI/RI remains unclear. To investigate this, we conducted experiments using a rat model of ischemia/reperfusion (I/R) injury. Adenovirus carrying RP105 was injected apically at multiple points, and after 72 h, the left anterior descending coronary artery was ligated for 30 min followed by 2 h of reperfusion. In vitro experiments were performed on H9C2 cells, which were transfected with recombinant adenoviral vectors for 48 h, subjected to 4 h of hypoxia, and then reoxygenated for 2 h. We measured oxidative stress markers, including superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, as well as malondialdehyde (MDA) concentration, using a microplate reader. The fluorescence intensity of reactive oxygen species (ROS) in myocardial tissue was measured using a DHE probe. We also investigated the upstream and downstream components of the signal transducer and activator of transcription 3 (STAT3). Upregulation of RP105 increased SOD and GSH-Px activities, reduced MDA concentration, and inhibited ROS production in response to I/R injury in vivo and hypoxia reoxygenation (H/R) stimulation in vitro. The overexpression of RP105 led to a decrease in the myocardial enzyme LDH in serum and cell culture supernatant, as well as a reduction in infarct size. Additionally, left ventricular fraction (LVEF) and fractional shortening (LVFS) were improved in the RP105 overexpression group compared to the control. Upregulation of RP105 promoted the expression of Lyn and Syk and further activated STAT phosphorylation, which was blocked by PP2 (a Lyn inhibitor). Our findings suggest that RP105 can inhibit MI/RI-induced oxidative stress by activating STAT3 via the Lyn/Syk signaling pathway.

The innate immune receptor RP105 promotes metabolic syndrome by altering gut microbiota composition and intestinal barrier function.

Radioprotective 105 (RP105) plays a key role in the development of high-fat diet (HFD)-induced metabolic disorders; however, the underlying mechanisms remain to be understood. Here, we aimed to uncover whether RP105 affects metabolic syndrome through the modification of gut microbiota. We confirmed that body weight gain and fat accumulation by HFD feeding were suppressed in Rp105-/- mice. Fecal microbiome transplantation from HFD-fed donor Rp105-/- mice into HFD-fed recipient wild-type mice significantly improved various abnormalities associated with metabolic syndrome, including body weight gain, insulin resistance, hepatic steatosis, macrophage infiltration and inflammation in the adipose tissue. In addition, HFD-induced intestinal barrier dysfunction was attenuated by fecal microbiome transplantation from HFD-fed donor Rp105-/- mice. A 16S rRNA sequence analysis indicated that RP105 modified gut microbiota composition and was involved in the maintenance of its diversity. Thus, RP105 promotes metabolic syndrome by altering gut microbiota composition and intestinal barrier function.

Evolution of LPS recognition and signaling: The bony fish perspective.

Fish are the most diverse and successful group of vertebrate animals, with about 30,000 species. The study of fish immunity is of great importance for understanding the evolution of vertebrate immunity, as they are the first animals to show both innate and adaptive immune responses. Although fish immunity is similar to that of mammals, there are obvious differences, such as their dependence of ambient temperature, their poor antibody response, and lack of antibody switching and lymph nodes. In addition, several important differences have also been found between the innate immune responses of fish and mammals. Among these, we will discuss in this review the high resistance of fish to the toxic effects of lipopolysaccharide (LPS) which can be explained by the absence of a Toll-like receptor 4 (Tlr4) ortholog in most fish species or by the inability of the Tlr4/Md2 (Myeloid differentiation 2) complex to recognize LPS, together with the presence of a negative regulator of the LPS signaling complex formed by the TLR-like molecule Rp105 (Radioprotective 105) and Md1. Taken together, these data support the idea that, although TLR4 and RP105 arose from a common ancestor to fish and tetrapods, the TLR4/MD2 receptor complex for LPS recognition arose after their divergence about 450 million years ago.

Distinct contributions of the innate immune receptors TLR2 and RP105 to formation and architecture of structured lung granulomas in mice infected with Mycobacterium tuberculosis.

Granulomas are key histopathological features of Mycobacterium tuberculosis (Mtb) infection, with complex roles in pathogen control and dissemination. Thus, understanding drivers and regulators of granuloma formation is important for improving tuberculosis diagnosis, treatment, and prevention. Yet, molecular mechanisms underpinning granuloma formation and dynamics remain poorly understood. Here we used low-dose Mtb infection of C57BL/6 mice, which elicits structured lung granulomas composed of central macrophage clusters encased by a lymphocyte mantle, alongside the disorganized lymphocyte and macrophage clusters commonly observed in Mtb-infected mice. Using gene-deficient mice, we observed that Toll-like receptor (TLR) 2 and the TLR-related Radioprotective 105 kDa protein (RP105) contributed to the extent and spatial positioning of pathology in infected lung tissues, consistent with functional cooperation between TLR2 and RP105 in the innate immune recognition of Mtb. In mice infected with the highly virulent Mtb clinical isolate HN878, TLR2, but not RP105, positively regulated the extent of central macrophage regions within structured granulomas. Moreover, RP105, but not TLR2, promoted the formation of structured lung granulomas, suggesting that the functions of RP105 as an innate immune sensor for Mtb reach beyond its roles as TLR2 co-receptor. TLR2 and RP105 contributions to lung pathology are governed by Mtb biology, as neither receptor affected the frequency or architecture of structured granulomas in mice infected with the reference strain Mtb H37Rv. Thus, by revealing distinctive as well as cooperative functions of TLR2 and RP105 in lung pathology, our data identify TLRs as molecular determinants of TB granuloma formation and architecture, and expand understanding of how interactions between innate immune receptors and Mtb shape TB disease manifestation.

Cell surface expression of human RP105 depends on N-glycosylation of MD-1.

For its cell surface expression, radioprotective 105 (RP105) - an orphan Toll-like receptor - must form a complex with a soluble glycoprotein called myeloid differentiation 1 (MD-1). The number of RP105-negative cells is significantly increased in patients with systemic lupus erythematosus (SLE); however, to elucidate the mechanism underlying this increase, how RP105 is expressed on the cell surface depending on MD-1 should be investigated. We demonstrated that RP105 exhibits two forms depending on MD-1 and its two N-glycosylation sites, N96 and N156. Cell surface expression of RP105 decreased in the presence of mutant MD-1 (N96Q/N156Q). Nonglycosylated MD-1 decreased the de novo cell surface expression of RP105 but not pre-expressed RP105. Thus, the N-glycans of MD-1 may represent targets for SLE therapy.

RP105 protects against ischemic and septic acute kidney injury via suppressing TLR4/NF-κB signaling pathways.

Acute kidney injury (AKI) is a critical and severe clinical disease caused by a variety of factors. Toll-like receptors (TLRs) play a crucial role in pathogenesis of AKI. Radioprotective 105 kDa protein (RP105) is a member of the TLR family, but the role of RP105 in AKI is unknown. In this study, we overexpressed RP105 in renal tissue and cultured proximal tubular cells in which we then induced ischemic and septic AKI. Renal structure injuries were examined by hematoxylin eosin staining, while renal function was assessed by measuring serum blood urea nitrogen (BUN) and creatinine (SCr) levels. The TUNEL assay was used to detect apoptosis induced changes in the expression of RP105, and nuclear factor κB (NF-κB) in renal tissue detected by Western blot. Inflammatory cytokines including iNOS, IL-1ß, IL-6, and TNF-α were detected by quantitative real-time PCR. The inflammatory indicators, F4/80 and MPO, were identified by IHC staining. The results showed that expression of the TLR4/NF-kB signaling pathway was enhanced in renal ischemia-reperfusion injury and septic renal injury, and that overexpression of RP105 in renal tissue alleviated ischemic and septic AKI. Moreover, RP105 gene delivery was associated with reduced renal inflammatory cells infiltration and inflammatory cytokines after AKI. RP105 overexpression also inhibited nuclear translocation of NF-κB after AKI in both in vitro and in vivo, and blunted the interaction between Myeloid Differentiation factor 2 (MD2) and TLR4. These results indicated that RP105 protected against renal ischemic and septic AKI injury by suppressing inflammatory responses mediated by TLR4 signaling pathways. This study suggests that the anti-inflammatory roles of RP105 have potential for preventing and treating renal ischemic and septic AKI.

Piperine protects against pyroptosis in myocardial ischaemia/reperfusion injury by regulating the miR-383/RP105/AKT signalling pathway.

miRNA-mediated pyroptosis play crucial effects in the development of myocardial ischaemia/reperfusion (I/R) injury (MIRI). Piperine (PIP) possesses multiple pharmacological effects especially in I/R condition. This study focuses on whether PIP protects MIRI from pyroptosis via miR-383-dependent pathway. Rat MIRI model was established by 30 minutes of LAD ligation and 4 hours of reperfusion. Myocardial enzymes, histomorphology, structure and function were detected to evaluate MIRI. Recombinant adenoviral vectors for miR-383 overexpression or miR-383 silencing or RP105 knockdown were constructed, respectively. Luciferase reporter analysis was used to confirm RP105 as a target of miR-383. Pyroptosis-related markers were measured by Western blotting assay. The results showed that I/R provoked myocardial injury, as shown by the increases of LDH/CK releases, infarcted areas and apoptosis as well as worsened function and structure. Pyroptosis-related mediators including NLRP3, cleaved caspase-1, cleaved IL-1ß and IL-18 were also reinforced after MIRI. However, PIP treatment greatly ameliorated MIRI in parallel with pyroptotic repression. In mechanistic studies, MIRI-caused elevation of miR-383 and decrease of RP105/PI3K/AKT pathway were reverted by PIP treatment. Luciferase reporter assay confirmed RP105 as a miR-383 target. miR-383 knockdown ameliorated but miR-383 overexpression facilitated pyroptosis and MIRI. Moreover, the anti-pyroptotic effect from miR-383 silencing was verified to be relied on the RP105/PI3K/AKT signalling pathway. Additionally, our present study further indicated the miR-383/RP105/AKT-dependent approach resulting from PIP administration against pyroptosis in MIRI. Therefore, PIP treatment attenuates MIRI and pyroptosis by regulating miR-383/RP105/AKT pathway, and it may provide a therapeutic manner for the treatment of MIRI.

A Novel Gene Delivery Vector of Agonistic Anti-Radioprotective 105 Expressed on Cell Membranes Shows Adjuvant Effect for DNA Immunization Against Influenza.

Radioprotective 105 (RP105) (also termed CD180) is an orphan and unconventional Toll-like receptor (TLR) that lacks an intracellular signaling domain. The agonistic anti-RP105 monoclonal antibody (mAb) can cross-link RP105 on B cells, resulting in the proliferation and activation of B cells. Anti-RP105 mAb also has a potent adjuvant effect, providing higher levels of antigen-specific antibodies compared to alum. However, adjuvanticity is required for the covalent link between anti-RP105 mAb and the antigen. This is a possible obstacle to immunization due to the link between anti-RP105 mAb and some antigens, especially multi-transmembrane proteins. We have previously succeeded in inducing rapid and potent recombinant mAbs in mice using antibody gene-based delivery. To simplify the covalent link between anti-RP105 mAb and antigens, we generated genetic constructs of recombinant anti-RP105 mAb (αRP105) bound to the transmembrane domain of the IgG-B cell receptor (TM) (αRP105-TM), which could enable the anti-RP105 mAb to link the antigen via the cell membrane. We confirmed the expression of αRP105-TM and the antigen hemagglutinin, which is a membrane protein of the influenza virus, on the same cell. We also found that αRP105-TM could activate splenic B cells, including both mature and immature cells, depending on the cell surface RP105 in vitro. To evaluate the adjuvanticity of αRP105-TM, we conducted DNA immunization in mice with the plasmids encoding αRP105-TM and hemagglutinin, followed by challenge with an infection of a lethal dose of an influenza virus. We then obtained partially but significantly hemagglutinin-specific antibodies and observed protective effects against a lethal dose of influenza virus infection. The current αRP105-TM might provide adjuvanticity for a vaccine via a simple preparation of the expression plasmids encoding αRP105-TM and of that encoding the target antigen.

Toll-Like Receptor Mediated Activation of Natural Autoantibody Producing B Cell Subpopulations in an Autoimmune Disease Model.

Altered expression and function of the Toll-like receptor (TLR) homologue CD180 molecule in B cells have been associated with autoimmune disorders. In this study, we report decreased expression of CD180 at protein and mRNA levels in peripheral blood B cells of diffuse cutaneous systemic sclerosis (dcSSc) patients. To analyze the effect of CD180 stimulation, together with CpG (TLR9 ligand) treatment, on the phenotype defined by CD19/CD27/IgD/CD24/CD38 staining, and function (CD69 and CD180 expression, cytokine and antibody secretion) of B cell subpopulations, we used tonsillar B cells. After stimulation, we found reduced expression of CD180 protein and mRNA in total B cells, and CD180 protein in B cell subpopulations. The frequency of CD180+ cells was the highest in the CD19+CD27+IgD+ non-switched (NS) B cell subset, and they showed the strongest activation after anti-CD180 stimulation. Furthermore, B cell activation via CD180 induced IL-6 and natural autoantibody secretion. Treatment with the combination of anti-CD180 antibody and CpG resulted in increased IL-6 and IL-10 secretion and natural autoantibody production of B cells. Our results support the role of CD180 in the induction of natural autoantibody production, possibly by NS B cells, and suggest an imbalance between the pathologic and natural autoantibody production in SSc patients.

The Role of RP105 in Cardiovascular Disease through Regulating TLR4 and PI3K Signaling Pathways.

Raidoprotective 105 (RP105) was first discovered on the surface of mouse B cells and it has been demonstrated that RP105 can function as an inflammatory regulator in cardiovascular disease (CVD), such as myocardial ischemic reperfusion injury (MI/RI), atherosclerosis and myocardial infarction (MI). As a member of Toll-like receptor (TLR) homolog which is capable of regulating toll-like receptor (TLR4) signaling pathway, RP105 is implicated in various biological processes. Mounting evidence suggests that RP105 regulates the function of TLR4 and phosphoinositide 3-kinase (PI3K) signaling pathways. Here, we review the effect of RP105 on CVD through regulating TLR4/PI3K signaling pathways.

CEACAM1 regulates the IL-6 mediated fever response to LPS through the RP105 receptor in murine monocytes.

BACKGROUND: Systemic inflammation and the fever response to pathogens are coordinately regulated by IL-6 and IL-1ß. We previously showed that CEACAM1 regulates the LPS driven expression of IL-1ß in murine neutrophils through its ITIM receptor. RESULTS: We now show that the prompt secretion of IL-6 in response to LPS is regulated by CEACAM1 expression on bone marrow monocytes. Ceacam1-/- mice over-produce IL-6 in response to an i.p. LPS challenge, resulting in prolonged surface temperature depression and overt diarrhea compared to their wild type counterparts. Intraperitoneal injection of a 64Cu-labeled LPS, PET imaging agent shows confined localization to the peritoneal cavity, and fluorescent labeled LPS is taken up by myeloid splenocytes and muscle endothelial cells. While bone marrow monocytes and their progenitors (CD11b+Ly6G-) express IL-6 in the early response (< 2 h) to LPS in vitro, these cells are not detected in the bone marrow after in vivo LPS treatment perhaps due to their rapid and complete mobilization to the periphery. Notably, tissue macrophages are not involved in the early IL-6 response to LPS. In contrast to human monocytes, TLR4 is not expressed on murine bone marrow monocytes. Instead, the alternative LPS receptor RP105 is expressed and recruits MD1, CD14, Src, VAV1 and ß-actin in response to LPS. CEACAM1 negatively regulates RP105 signaling in monocytes by recruitment of SHP-1, resulting in the sequestration of pVAV1 and ß-actin from RP105. CONCLUSION: This novel pathway and regulation of IL-6 signaling by CEACAM1 defines a novel role for monocytes in the fever response of mice to LPS.

Down-Regulation of miR-327 Alleviates Ischemia/Reperfusion-Induced Myocardial Damage by Targeting RP105.

BACKGROUND/AIMS: Micro RNAs (miRNAs) play a very important role in myocardial ischemia/ reperfusion injury (MIRI), including in inflammation, apoptosis, and angiogenesis. Previous studies have demonstrated up-regulation of miR-327 in renal ischemia/reperfusion injury and MIRI. Via TargetScan, we found RP105 is a possible target gene of miR-327; our previous studies have also confirmed that RP105 acted as a cardioprotective protein in MIRI by reducing inflammation. However, the regulatory effect of miR-327 on RP105 has not previously been proposed. In our study, we aimed to identify the regulatory effect of miR-327 on RP105 protein in MIRI rats. METHODS: Sixty male Sprague-Dawley rats were randomly divided into five groups, which were pre-treated with saline (sham and ischemia/reperfusion group), adenovirus-expressing miR-327-RNAi (Ad-miR-327-i group), control (Ad-NC group), or pri-miR-327 (Ad-miR-327 group) treatments. Three days later, the rat MIRI model was established by ischemia for 30 min, followed by reperfusion for 3 h. Myocardium and plasma were harvested and assessed. RESULTS: miR-327 was increased by nearly 3-fold both in myocardium and plasma, which down-regulated RP105 in a 3'-untranslated region-dependent manner, and down-regulation of miR-327 via adenovirus transfection indirectly suppressed the TLR4/ TLR2-MyD88-NF-κB signaling axis activation via up-regulation of RP105, which subsequently resulted in reduced myocardial infarct size, attenuated cardiomyocyte destruction, and alleviated inflammation. In contrast, up-regulation of miR-327 induced the opposite effect. CONCLUSION: Down-regulation of miR-327 exerts a cardioprotective effect against MIRI by reducing inflammation, which may constitute a promising molecular therapeutic target for treating MIRI.

Immunoregulatory Effects Triggered by Lactic Acid Bacteria Exopolysaccharides: New Insights into Molecular Interactions with Host Cells.

Researchers have demonstrated that lactic acid bacteria (LAB) with immunomodulatory capabilities (immunobiotics) exert their beneficial effects through several molecules, including cell wall, peptidoglycan, and exopolysaccharides (EPS), that are able to interact with specific host cell receptors. EPS from LAB show a wide heterogeneity in its composition, meaning that biological properties depend on the strain and. therefore, only a part of the mechanism of action has been elucidated for these molecules. In this review, we summarize the current knowledge of the health-promoting actions of EPS from LAB with special focus on their immunoregulatory actions. In addition, we describe our studies using porcine intestinal epithelial cells (PIE cells) as a model to evaluate the molecular interactions of EPS from two immunobiotic LAB strains and the host cells. Our studies showed that EPS from immunobiotic LAB have anti-inflammatory capacities in PIE cells since they are able to reduce the production of inflammatory cytokines in cells challenged with the Toll-like receptor (TLR)-4-agonist lipopolysaccharide. The effects of EPS were dependent on TLR2, TLR4, and negative regulators of TLR signaling. We also reported that the radioprotective 105 (RP105)/MD1 complex, a member of the TLR family, is partially involved in the immunoregulatory effects of the EPS from LAB. Our work described, for the first time, that LAB and their EPS reduce inflammation in intestinal epithelial cells in a RP105/MD1-dependent manner. A continuing challenge for the future is to reveal more effector-receptor relationships in immunobiotic-host interactions that contribute to the beneficial effects of these bacteria on mucosal immune homeostasis. A detailed molecular understanding should lead to a more rational use of immunobiotics in general, and their EPS in particular, as efficient prevention and therapies for specific immune-related disorders in humans and animals.

Asparagine reduces the mRNA expression of muscle atrophy markers via regulating protein kinase B (Akt), AMP-activated protein kinase α, toll-like receptor 4 and nucleotide-binding oligomerisation domain protein signalling in weaning piglets after lipopolysaccharide challenge.

Pro-inflammatory cytokines are critical in mechanisms of muscle atrophy. In addition, asparagine (Asn) is necessary for protein synthesis in mammalian cells. We hypothesised that Asn could attenuate lipopolysaccharide (LPS)-induced muscle atrophy in a piglet model. Piglets were allotted to four treatments (non-challenged control, LPS-challenged control, LPS+0·5 % Asn and LPS+1·0 % Asn). On day 21, the piglets were injected with LPS or saline. At 4 h post injection, piglet blood and muscle samples were collected. Asn increased protein and RNA content in muscles, and decreased mRNA expression of muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1). However, Asn had no effect on the protein abundance of MAFbx and MuRF1. In addition, Asn decreased muscle AMP-activated protein kinase (AMPK) α phosphorylation, but increased muscle protein kinase B (Akt) and Forkhead Box O (FOXO) 1 phosphorylation. Moreover, Asn decreased the concentrations of TNF-α, cortisol and glucagon in plasma, and TNF-α mRNA expression in muscles. Finally, Asn decreased mRNA abundance of muscle toll-like receptor (TLR) 4 and nucleotide-binding oligomerisation domain protein (NOD) signalling-related genes, and regulated their negative regulators. The beneficial effects of Asn on muscle atrophy may be associated with the following: (1) inhibiting muscle protein degradation via activating Akt and inactivating AMPKα and FOXO1; and (2) decreasing the expression of muscle pro-inflammatory cytokines via inhibiting TLR4 and NOD signalling pathways by modulation of their negative regulators.

Inflammatory responses increase secretion of MD-1 protein.

Radioprotective 105 (RP105) is a type I transmembrane protein, which associates with a glycoprotein, MD-1. Monoclonal antibody (mAb)-mediated ligation of RP105/MD-1 robustly activates B cells. RP105/MD-1 is structurally similar to Toll-like receptor 4 (TLR4)/MD-2. B-cell responses to TLR2 and TLR4/MD-2 ligands are impaired in the absence of RP105 or MD-1. In addition to RP105/MD-1, MD-1 alone is secreted. The structure of MD-1 shows that MD-1 has a hydrophobic cavity that directly binds to phospholipids. Little is known, however, about a ligand for MD-1 and the role of MD-1 in vivo To study the role of RP105/MD-1 and MD-1 alone, specific mAbs against MD-1 are needed. Here, we report the establishment and characterization of two anti-MD-1 mAbs (JR2G9, JR7G1). JR2G9 detects soluble MD-1, whereas JR7G1 binds both soluble MD-1 and the cell surface RP105/MD-1 complex. With these mAbs, soluble MD-1 was detected in the serum and urine. The MD-1 concentration was altered by infection, diet and reperfusion injury. Serum MD-1 was rapidly elevated by TLR ligand injection in mice. The quantitative PCR and supernatant-precipitated data indicate that macrophages are one of the sources of serum soluble MD-1. These results suggest that soluble MD-1 is a valuable biomarker for inflammatory diseases.

RP105 protects against myocardial ischemia-reperfusion injury via suppressing TLR4 signaling pathways in rat model.

Myocardial ischemia-reperfusion (I/R) injury severely impacts the postoperative survival rate of coronary atherosclerotic heart disease. Radioprotective 105 kDa protein (RP105) is a regulator of Toll-like receptor 4 (TLR4), an inflammatory factor whose functions have been reported in myocardial I/R injury. To investigate the roles of RP105 in mediating myocardial I/R injury, we overexpressed RP105 by injecting its adenovirus vectors, and induced myocardial I/R injury rat model in this study. Myocardial structure injuries of rat hearts were examined by hematoxylin eosin staining, and myocardial infarct area was calculated after Evans blue and triphenyltetrazolium chloride dual staining. Expression changes of TLR4, myeloid differentiation factor 88 (MyD88), and nuclear factor κB (NF-κB) in myocardia were detected by quantitative real-time PCR and Western blot. Amount changes of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) were detected by enzyme-linked immunosorbent assay. Results showed that RP105 attenuated myocardial injuries and effectively reduced myocardial infarct area after I/R (P<0.05). RP105 was also proved to significantly inhibit TLR4 and downstream inflammatory factors MyD88, NF-κB, TNF-α and IL-6 (P<0.05), whose expression levels were up-regulated by I/R induction. These results indicated that RP105 could protect against myocardial I/R injury via suppressing inflammatory responses mediated by TLR4 signaling pathways. This study revealed the anti-inflammatory roles of RP105 and its potential in preventing and treating myocardial I/R injury.

Comparative Geometrical Analysis of Leucine-Rich Repeat Structures in the Nod-Like and Toll-Like Receptors in Vertebrate Innate Immunity.

The NOD-like receptors (NLRs) and Toll-like receptors (TLRs) are pattern recognition receptors that are involved in the innate, pathogen pattern recognition system. The TLR and NLR receptors contain leucine-rich repeats (LRRs) that are responsible for ligand interactions. In LRRs short ß-strands stack parallel and then the LRRs form a super helical arrangement of repeating structural units (called a coil of solenoids). The structures of the LRR domains of NLRC4, NLRP1, and NLRX1 in NLRs and of TLR1-5, TLR6, TLR8, TLR9 in TLRs have been determined. Here we report nine geometrical parameters that characterize the LRR domains; these include four helical parameters from HELFIT analysis. These nine parameters characterize well the LRR structures in NLRs and TLRs; the LRRs of NLR adopts a right-handed helix. In contrast, the TLR LRRs adopt either a left-handed helix or are nearly flat; RP105 and CD14 also adopt a left-handed helix. This geometrical analysis subdivides TLRs into four groups consisting of TLR3/TLR8/TLR9, TLR1/TLR2/TRR6, TLR4, and TLR5; these correspond to the phylogenetic tree based on amino acid sequences. In the TLRs an ascending lateral surface that consists of loops connecting the ß-strand at the C-terminal side is involved in protein, protein/ligand interactions, but not the descending lateral surface on the opposite side.

Retinoic acid-induced IgG production in TLR-activated human primary B cells involves ULK1-mediated autophagy.

In the present study we have established a vital role of autophagy in retinoic acid (RA)-induced differentiation of toll-like receptor (TLR)-stimulated human B cells into Ig-secreting cells. Thus, RA enhanced autophagy in TLR9- and CD180-stimulated peripheral blood B cells, as revealed by increased levels of the autophagosomal marker LC3B-II, enhanced colocalization between LC3B and the lysosomal marker Lyso-ID, by a larger percentage of cells with more than 5 characteristic LC3B puncta, and by the concomitant reduction in the level of SQSTM1/p62. Furthermore, RA induced expression of the autophagy-inducing protein ULK1 at the transcriptional level, in a process that required the retinoic acid receptor RAR. By inhibiting autophagy with specific inhibitors or by knocking down ULK1 by siRNA, the RA-stimulated IgG production in TLR9- and CD180-mediated cells was markedly reduced. We propose that the identified prominent role of autophagy in RA-mediated IgG-production in normal human B cells provides a novel mechanism whereby vitamin A exerts its important functions in the immune system.

The toll-like receptor family protein RP105/MD1 complex is involved in the immunoregulatory effect of exopolysaccharides from Lactobacillus plantarum N14.

The radioprotective 105 (RP105)/MD1 complex is a member of the Toll-like receptor (TLR) family. It was reported that RP105/MD1 cooperates with the lipopolysaccharide (LPS) receptor TLR4/MD2 complex and plays a crucial role in the response of immune cells to LPS. This work evaluated whether RP105, TLR4 or TLR2 were involved in the immunoregulatory capacities of Lactobacillus plantarum N14 (LP14) or its exopolysaccharides (EPS). EPS from LP14 were fractionated into neutral (NPS) and acidic (APS) EPS by anion exchange chromatography. Experiments with transfectant HEK(RP105/MD1) and HEK(TLR2) cells demonstrated that LP14 strongly activated NF-κB via RP105 and TLR2. When we studied the capacity of APS to activate NF-κB pathway in HEK(RP105/MD1) and HEK(TLR4) cells; we observed that APS strongly stimulated both transfectant cells. Our results also showed that LP14 and APS were able to decrease the production of pro-inflammatory cytokines (IL-6, IL-8 and MCP-1) in porcine intestinal epithelial (PIE) cells in response to enterotoxigenic Escherichia coli (ETEC) challenge. In order to confirm the role of TLR2, TLR4 and RP105 in the immunoregulatory effect of APS from LP14, we used small interfering RNA (siRNA) to knockdown these receptors in PIE cells. The capacity of LP14 and APS to modulate pro-inflammatory cytokine expression was significantly reduced in PIE(RP105-/-) cells. It was also shown that LP14 and APS were capable of upregulating negative regulators of the TLR signaling in PIE cells. This work describes for the first time that a Lactobacillus strain and its EPS reduce inflammation in intestinal epithelial cells in a RP105/MD1-dependend manner.