Phosphorylation of muramyl peptides by NAGK is required for NOD2 activation.

Bacterial cell wall components provide various unique molecular structures that are detected by pattern recognition receptors (PRRs) of the innate immune system as non-self. Most bacterial species form a cell wall that consists of peptidoglycan (PGN), a polymeric structure comprising alternating amino sugars that form strands cross-linked by short peptides. Muramyl dipeptide (MDP) has been well documented as a minimal immunogenic component of peptidoglycan1-3. MDP is sensed by the cytosolic nucleotide-binding oligomerization domain-containing protein 24 (NOD2). Upon engagement, it triggers pro-inflammatory gene expression, and this functionality is of critical importance in maintaining a healthy intestinal barrier function5. Here, using a forward genetic screen to identify factors required for MDP detection, we identified N-acetylglucosamine kinase (NAGK) as being essential for the immunostimulatory activity of MDP. NAGK is broadly expressed in immune cells and has previously been described to contribute to the hexosamine biosynthetic salvage pathway6. Mechanistically, NAGK functions upstream of NOD2 by directly phosphorylating the N-acetylmuramic acid moiety of MDP at the hydroxyl group of its C6 position, yielding 6-O-phospho-MDP. NAGK-phosphorylated MDP-but not unmodified MDP-constitutes an agonist for NOD2. Macrophages from mice deficient in NAGK are completely deficient in MDP sensing. These results reveal a link between amino sugar metabolism and innate immunity to bacterial cell walls.

Strategies for Using Muramyl Peptides - Modulators of Innate Immunity of Bacterial Origin - in Medicine.

The spread of infectious diseases is rampant. The emergence of new infections, the irrational use of antibiotics in medicine and their widespread use in agriculture contribute to the emergence of microorganisms that are resistant to antimicrobial drugs. By 2050, mortality from antibiotic-resistant strains of bacteria is projected to increase up to 10 million people per year, which will exceed mortality from cancer. Mutations in bacteria and viruses are occurring faster than new drugs and vaccines are being introduced to the market. In search of effective protection against infections, new strategies and approaches are being developed, one of which is the use of innate immunity activators in combination with etiotropic chemotherapy drugs. Muramyl peptides, which are part of peptidoglycan of cell walls of all known bacteria, regularly formed in the body during the breakdown of microflora and considered to be natural regulators of immunity. Their interaction with intracellular receptors launches a sequence of processes that ultimately leads to the increased expression of genes of MHC molecules, pro-inflammatory mediators, cytokines and their soluble and membrane-associated receptors. As a result, all subpopulations of immunocompetent cells are activated: macrophages and dendritic cells, neutrophils, T-, B- lymphocytes and natural killer cells for an adequate response to foreign or transformed antigens, manifested both in the regulation of the inflammatory response and in providing immunological tolerance. Muramyl peptides take part in the process of hematopoiesis, stimulating production of colony-stimulating factors, which is the basis for their use in the treatment of oncological diseases. In this review we highlight clinical trials of drugs based on muramyl peptides, as well as clinical efficacy of drugs mifamurtide, lycopid, liasten and polimuramil. Such a multifactorial effect of muramyl peptides and a well-known mechanism of activity make them promising drugs in the treatment and preventing of infectious, allergic and oncological diseases, and in the composition of vaccines.

Marginal Zone B Cells Assist With Neutrophil Accumulation to Fight Against Systemic Staphylococcus aureus Infection.

In addition to regulating immune responses by producing antibodies that confer humoral immunity, B cells can also affect these responses by producing cytokines. How B cells participate in the clearance of pathogenic infections via functions other than the production of pathogen-specific antibodies is still largely unknown. Marginal zone (MZ) B cells can quickly respond to bacterial invasion by providing the initial round of antibodies. After a bloodborne bacterial infection, neutrophils promptly migrate to the MZ. However, the mechanisms regulating neutrophil accumulation in the MZ during the initial phase of infection also remain obscure. Here, we found that MZ B cell-deficient mice are more susceptible to systemic Staphylococcus aureus (S. aureus) infection compared with wildtype mice. The expression levels of interleukin (IL)-6 and CXCL1/CXCL2 in MZ B cells increased significantly in mice at 3-4 h after infection with S. aureus, then decreased at 24 h post-infection. After systemic S. aureus infection, splenic neutrophils express increased CXCR2 levels. Our results from confocal microscopy imaging of thick-section staining demonstrate that neutrophils in wildtype mice form cell clusters and are in close contact with MZ B cells at 3 h post-infection. This neutrophil cluster formation shortly after infection was diminished in both MZ B cell-deficient mice and IL-6-deficient mice. Blocking the action of CXCL1/CXCL2 by injecting anti-CXCL1 and anti-CXCL2 antibodies 1 h before S. aureus infection significantly suppressed the recruitment of neutrophils to the MZ at 3 h post-infection. Compared with peptidoglycan stimulation alone, peptidoglycan stimulation with neutrophil co-culture further enhanced MZ B-cell activation and differentiation. Using a Förster resonance energy transfer by fluorescence lifetime imaging (FLIM-FRET) analysis, we observed evidence of a direct interaction between neutrophils and MZ B cells after peptidoglycan stimulation. Furthermore, neutrophil depletion in mice resulted in a reduced production of S. aureus-specific immunoglobulin (Ig)M at 24 h post-infection. Together, our results demonstrate that MZ B cells regulate the rapid neutrophil swarming into the spleen during the early phase of systemic S. aureus infection. Interaction with neutrophils assists MZ B cells with their differentiation into IgM-secreting cells and contributes to the clearance of systemic bacterial infections.

Transient systemic inflammation in adult male mice results in underweight progeny.

PROBLEM: While the testes represent an immune-privileged organ, there is evidence that systemic inflammation is accompanied by local inflammatory responses. We therefore examined whether transient systemic inflammation caused any inflammatory and functional consequences in murine testes. METHOD OF STUDY: Using a single systemic administration of Toll-like receptor (TLR) agonists [lipopolysaccharide (LPS) or peptidoglycan (PG) or polyinosinic-polycytidylic acid (polyIC)] in young adult male mice, we assessed testicular immune-inflammatory landscape and reproductive functionality. RESULTS: Our findings demonstrated a significant induction of testicular TNF-α, IL-1ß and IL-6 transcripts within 24 h of TLR agonist injection. By day 6, these cytokine levels returned to baseline. While there was no change in caudal sperm counts at early time points, eight weeks later, twofold decrease in sperm count and reduced testicular testosterone levels were evident. When these mice were subjected to mating studies, no differences in mating efficiencies or litter sizes were observed compared with controls. Nonetheless, the neonatal weights of progeny from LPS/PG/polyIC-treated sires were significantly lower than controls. Postnatal weight gain up to three weeks was also slower in the progeny of LPS/polyIC-treated sires. Placental weights at 17.5 days post-coitum were significantly lower in females mated to LPS- and polyIC-treated males. Given this likelihood of an epigenetic effect, we found lower testicular levels of histone methyltransferase enzyme, mixed-lineage leukaemia-1, in mice given LPS/PG/polyIC 8 weeks earlier. CONCLUSION: Exposure to transient systemic inflammation leads to transient local inflammation in the testes, with persistent sperm-mediated consequences for foetal development.

A C-type lectin (OmCTL) in Onychostoma macrolepis: Binding ability to LPS, PGN and agglutinating activity against bacteria.

C-type lectins (CTLs) are calcium-dependent carbohydrate-binding proteins that mainly bind to carbohydrate-based or other ligands to mediate cell adhesion, recognize pathogens, and play important roles in the immune system. In the present study, a novel C-type lectin (OmCTL) isolated from Onychostoma macrolepis was investigated. The open reading frame of OmCTL comprises 468 bp, encoding a 155 amino acid polypeptide with an 18 amino acid putative signaling peptide. The predicted primary OmCTL structure contains a signal peptide, a single carbohydrate recognition domain (CRD) and an EPN/WND motif required for carbohydrate-binding specificity. Using tissue expression pattern analysis, OmCTL has been shownto be highly expressed in the liver, and is also detected in other tissues. OmCTL was significantly upregulated in the liver and spleen following infection with Aeromonas hydrophila, suggesting its involvement in immune response. The recombinant OmCTL protein (rOmCTL) agglutinated two gram-negative bacteria, Escherichia coli and A. hydrophila, in vitro in the presence of Ca2+, showing that it is a typical Ca2+-dependent carbohydrate-binding protein.Furthermore, rOmCTL purified from E. coli BL21 (DE3) strongly bound to LPS and PGN, as well as all tested bacteria in a Ca2+-independent manner. These results indicate that OmCTL plays a central role in the innate immune response and as a pattern recognition receptor that recognizes diverse pathogens among O. macrolepis.

Effect of D-Ala-Ended Peptidoglycan Precursors on the Immune Regulation of Lactobacillus plantarum Strains.

The resistance of Lactobacillus plantarum to vancomycin depends on its peptidoglycan composition. Vancomycin has poor binding affinity with peptidoglycan precursors ending in D-alanyl-D-lactate (D-Ala-D-Lac) but binds strongly to peptidoglycan precursors ending in D-alanyl-D-alanine (D-Ala-D-Ala), resulting in resistance and sensitivity, respectively. The ligase Ddl, which generates D-Ala-D-Lac or D-Ala-D-Ala incorporated into the peptidoglycan precursor chain, is responsible for this specificity. To study the effect of peptidoglycan precursors on immunity, we constructed several strains of L. plantarum expressing the ddl gene of Lactococcus lactis to change their peptidoglycan precursors. The change in the termini of the peptidoglycan precursors was determined by the sensitivity of the strains to vancomycin. The overexpression of ddl increased the susceptibility of the strains to vancomycin. We further explored the regulation of the macrophage inflammatory response pathway by the wild-type and constructed strains, and found that these strains induced the MyD88-dependent TRAF6/MAPK pathway, and the increase in D-Ala L. plantarum peptidoglycan precursors increased the secretion of the inflammatory factors IL-6, IL-1ß and TNF-α. These results indicate that D-Ala-ended peptidoglycan precursors play a central role in the variable immunomodulatory ability of L. plantarum.

Identification of an IRF10 gene in common carp (Cyprinus carpio L.) and analysis of its function in the antiviral and antibacterial immune response.

BACKGROUND: Interferon (IFN) regulatory factors (IRFs), as transcriptional regulatory factors, play important roles in regulating the expression of type I IFN and IFN- stimulated genes (ISGs) in innate immune responses. In addition, they participate in cell growth and development and regulate oncogenesis. RESULTS: In the present study, the cDNA sequence of IRF10 in common carp (Cyprinus carpio L.) was characterized (abbreviation, CcIRF10). The predicted protein sequence of CcIRF10 shared 52.7-89.2% identity with other teleost IRF10s and contained a DNA-binding domain (DBD), a nuclear localization signal (NLS) and an IRF-associated domain (IAD). Phylogenetic analysis showed that CcIRF10 had the closest relationship with IRF10 of Ctenopharyngodon idella. CcIRF10 transcripts were detectable in all examined tissues, with the highest expression in the gonad and the lowest expression in the head kidney. CcIRF10 expression was upregulated in the spleen, head kidney, foregut and hindgut upon polyinosinic:polycytidylic acid (poly I:C) and Aeromonas hydrophila stimulation and induced by poly I:C, lipopolysaccharide (LPS) and peptidoglycan (PGN) in peripheral blood leucocytes (PBLs) and head kidney leukocytes (HKLs) of C. carpio. In addition, overexpression of CcIRF10 was able to decrease the expression of the IFN and IFN-stimulated genes PKR and ISG15. CONCLUSIONS: These results indicate that CcIRF10 participates in antiviral and antibacterial immunity and negatively regulates the IFN response, which provides new insights into the IFN system of C. carpio.

Peptidoglycan disrupts early embryo-maternal crosstalk via suppression of ISGs expression induced by interferon-tau in the bovine endometrium.

Uterine infection with bacteria and the release of peptidoglycan (PGN), antigenic cell wall components of both Gram-negative and Gram-positive bacteria, can cause early pregnancy losses in ruminants, but the associated mechanisms remain unsolved. Day 7 blastocyst starts to secrete a minute amount of interferon-tau (IFNT) in the uterine horn which is required for early stage of maternal recognition of pregnancy (MRP) in ruminants, and it induces interferon stimulated genes (ISGs) for driving uterine receptivity in cows. This study investigated if PGN disrupts IFNT response through modulation of endometrial ISGs expressions. Cultured bovine endometrial epithelial cells (BEECs) were treated with embryo culture medium (ECM) or IFNT (1 ng/ml) in the presence or absence of a low level of PGN (10 pg/ml) for 24 h. A real-time PCR analyses revealed that the presence of PGN suppressed IFNT-induced ISGs (OAS1 and ISG15) and STAT1 expressions in BEECs. To visualize the impact of PGN in an ex-vivo model that resembles the in vivo status, endometrial explants were treated by IFNT (1 ng/ml) with or without PGN (10 pg/ml) for 12 h. PGN suppressed IFNT-induced gene expressions of the above factors, but not for IFNA receptor type1 (IFNAR1) or type2 (IFNAR2) in explants. Immunofluorescence analysis illustrated that PGN completely suppressed the IFNT-triggered OAS1 protein expression in the luminal epithelium of explants. Of note, PGN did not stimulate pro-inflammatory cytokines (TNFA and IL1B) or TLR2 mRNA expression in both models. These findings indicate that the presence of low levels of PGN suppresses ISGs expression induced by IFNT secreted from early embryo, at the luminal epithelium of the bovine endometrium. This could severely interfere with early stage of MRP processes in cows, leading to pregnancy failure.

Peptidoglycan Switches Off the TLR2-Mediated Sperm Recognition and Triggers Sperm Localization in the Bovine Endometrium.

In mammals, the uterine mucosal immune system simultaneously recognizes and reacts to most bacteria as well as allogenic sperm mainly through the Toll-like receptors (TLR)2/4 signaling pathway. Here, we characterized the impact of pathogen-derived TLR2/4 ligands (peptidoglycan (PGN)/lipopolysaccharide (LPS)) on the immune crosstalk of sperm with the bovine endometrial epithelium. The real-time PCR analysis showed that the presence of low levels of PGN, but not LPS, blocked the sperm-induced inflammatory responses in bovine endometrial epithelial cells (BEECs) in vitro. Immunoblotting analysis revealed that PGN prevented the sperm-induced phosphorylation of JNK in BEECs. Activation or blockade of the TLR2 system in the endometrial epithelium verified that TLR2 signaling acts as a commonly-shared pathway for PGN and sperm recognition. The impairment of endometrial sperm recognition, induced by PGN, subsequently inhibited sperm phagocytosis by polymorphonuclear neutrophils (PMNs). Moreover, using an ex vivo endometrial explant that more closely resembles those in vivo conditions, showed that sperm provoked a mild and reversible endometrial tissue injury and triggered PMN recruitment into uterine glands, while PGN inhibited these events. Of note, PGN markedly increased the sperm attachment to uterine glands, and relatively so in the surface epithelium. However, addition of the anti-CD44 antibody into a PGN-sperm-explant co-culture completely blocked sperm attachment into glands and surface epithelia, indicating that the CD44 adhesion molecule is involved in the PGN-triggered sperm attachment to the endometrial epithelium. Together, these findings demonstrate that, the presence of PGN residues disrupts sperm immune recognition and prevents the physiological inflammation induced by sperm in the endometrial epithelium via the MyD88-dependent pathway of TLR2 signaling, possibly leading to impairment of uterine clearance and subsequent embryo receptivity.

Monocyte procoagulant responses to anthrax peptidoglycan are reinforced by proinflammatory cytokine signaling.

Disseminated intravascular coagulation is a frequent manifestation during bacterial infections and is associated with negative clinical outcomes. Imbalanced expression and activity of intravascular tissue factor (TF) is central to the development of infection-associated coagulopathies. Recently, we showed that anthrax peptidoglycan (PGN) induces disseminated intravascular coagulation in a nonhuman primate model of anthrax sepsis. We hypothesized that immune recognition of PGN by monocytes is critical for procoagulant responses to PGN and investigated whether and how PGN induces TF expression in primary human monocytes. We found that PGN induced monocyte TF expression in a large cohort of healthy volunteers similar to lipopolysaccharide stimulation. Both immune and procoagulant responses to PGN involve intracellular recognition after PGN internalization, as well as surface signaling through immune Fcγ receptors (FcγRs). In line with our hypothesis, blocking immune receptor function, both signaling and FcγR-mediated phagocytosis, significantly reduced but did not abolish PGN-induced monocyte TF expression, indicating that FcγR-independent internalization contributes to intracellular recognition of PGN. Conversely, when intracellular PGN recognition is abolished, TF expression was sensitive to inhibitors of FcγR signaling, indicating that surface engagement of monocyte immune receptors can promote TF expression. The primary procoagulant responses to PGN were further amplified by proinflammatory cytokines through paracrine and autocrine signaling. Despite intersubject variability in the study cohort, dual neutralization of tumor necrosis factor-α and interleukin-1ß provided the most robust inhibition of the procoagulant amplification loop and may prove useful for reducing coagulopathies in gram-positive sepsis.

A Novel Rare Missense Variation of the NOD2 Gene: Evidencesof Implication in Crohn's Disease.

The NOD2 gene, involved in innate immune responses to bacterial peptidoglycan, has been found to be closely associated with Crohn's Disease (CD), with an Odds Ratio ranging from 3⁻36. Families with three or more CD-affected members were related to a high frequency of NOD2 gene variations, such as R702W, G908R, and 1007fs, and were reported in the EPIMAD Registry. However, some rare CD multiplex families were described without identification of common NOD2 linked-to-disease variations. In order to identify new genetic variation(s) closely linked with CD, whole exome sequencing was performed on available subjects, comprising four patients in two generations affected with Crohn's disease without R702W and G908R variation and three unaffected related subjects. A rare and, not yet, reported missense variation of the NOD2 gene, N1010K, was detected and co-segregated across affected patients. In silico evaluation and modelling highlighted evidence for an adverse effect of the N1010K variation with regard to CD. Moreover, cumulative characterization of N1010K and 1007fs as a compound heterozygous state in two, more severe CD family members strongly suggests that N1010K could well be a new risk factor involved in Crohn's disease genetic susceptibility.

Aryl Hydrocarbon Receptor Modulates the Expression of TNF-α and IL-8 in Human Sebocytes via the MyD88-p65NF-κB/p38MAPK Signaling Pathways.

Activation of Toll-like receptor (TLR)-2 and subsequent inflammatory response contribute to lesion development in acne vulgaris. A cross-talk between aryl hydrocarbon receptor (AhR), a cytosolic receptor protein that responds to environmental and physiological stress, and TLRs has recently been reported. In this study, we explored the possible role of AhR in the effects induced on cultured human SZ95 sebocytes by peptidoglycan (PGN), a classic TLR2 agonist. PGN-induced secretion of inflammatory factors TNF-α and IL-8 in human SZ95 sebocytes was suppressed after knockdown of AhR and pretreatment with the AhR antagonist CH223191. In addition, the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) enhanced TNF-α and IL-8 secretion in PGN-pretreated sebocytes. Furthermore, PGN-induced expression of myeloid differentiation factor 88 (MyD88), phospho-p38MAPK (p-p38MAPK), and p-p65NF-κB was strengthened by TCDD and repressed by CH223191. AhR inhibition by transfecting shRNA blocked the ability of PGN to stimulate phosphorylation of p38MAPK and p65NF-κB in SZ95 sebocytes. Overall, these data demonstrate that AhR is able to modulate PGN-induced expression of TNF-α and IL-8 in human SZ95 sebocytes involving the MyD88-p65NF-κB/p38MAPK signaling pathway, which probably indicates a new mechanism in TLR2-mediated acne.

An ML protein from the silkworm Bombyx mori may function as a key accessory protein for lipopolysaccharide signaling.

Lipopolysaccharide (LPS) is a common component of the outermost cell wall in Gram-negative bacteria. In mammals, LPS serves as an endotoxin that can be recognized by a receptor complex of TLR4 (Toll-like receptor 4) and MD-2 (myeloid differentiation-2) and subsequently induce a strong immune response to signal the release of tumor necrosis factor (TNF). In Drosophila melanogaster, no receptors for LPS have been identified, and LPS cannot activate immune responses. Here, we report a protein, BmEsr16, which contains an ML (MD-2-related lipid-recognition) domain, may function as an LPS receptor in the silkworm Bombyx mori. We showed that antibacterial activity in the hemolymph of B. mori larvae was induced by Escherichia coli, peptidoglycan (PGN) and LPS and that the expression of antimicrobial peptide genes was also induced by LPS. Furthermore, both the expression of BmEsr16 mRNA in the fat body and the expression of BmEsr16 protein in the hemolymph were induced by LPS. Recombinant BmEsr16 bound to LPS and lipid A, as well as to PGN, lipoteichoic acid, but not to laminarin or mannan. More importantly, LPS-induced immune responses in the hemolymph of B. mori larvae were blocked when the endogenous BmEsr16 protein was neutralized by polyclonal antibody specific to BmEsr16. Our results suggest that BmEsr16 may function as a key accessory protein for LPS signaling in B. mori.

Regulation of the Peptidoglycan Amidase PGLYRP2 in Epithelial Cells by Interleukin-36γ.

Interleukin-36 (IL-36) cytokines are important regulators of mucosal homeostasis and inflammation. We have previously established that oral epithelial cells upregulate IL-36γ expression in response to the bacterial pathogen Porphyromonas gingivalis Here, we have established that IL-36γ can stimulate the gene expression of mechanistically distinct antimicrobial proteins, including the peptidoglycan amidase PGLYRP2, in oral epithelial cells (e.g., TIGK cells). PGLYRP2 gene expression was not stimulated by either IL-17 or IL-22, thus demonstrating selectivity in the regulation of PGLYRP2 by IL-36γ. The IL-36γ-inducible expression of PGLYRP2 was shown to be mediated by IRAK1- and p38 mitogen-activated protein (MAP) kinase-dependent signaling. Furthermore, our finding that IL-36γ-inducible PGLYRP2 expression was reduced in proliferating TIGK cells but increased in terminally differentiating cells suggests that control of PGLYRP2 expression is associated with the maturation of the oral epithelium. PGLYRP2 expression in TIGK cells can also be directly stimulated by oral bacteria. However, the extracellular gingipain proteases (Kgp and RgpA/B) produced by P. gingivalis, which are critical virulence factors, can antagonize PGLYRP2 expression. Thus, the expression of IL-36γ by oral epithelial cells in response to P. gingivalis might enable the subsequent autocrine stimulation of PGLYRP2 expression. In summary, our data identify how IL-36γ may promote oral mucosal homeostasis by regulating PGLYRP2 expression.

Xenopus laevis macrophage-like cells produce XCL-1, an intelectin family serum lectin that recognizes bacteria.

Xenopus laevis Ca2+ -dependent lectin-1 (XCL-1) is an intelectin family serum lectin that selectively recognizes carbohydrate chains on the bacterial cell surface. Immunofluorescence examination of control spleen tissues from normal X. laevis revealed cells producing XCL-1 (XCL-1+ cells) exclusively in red pulps. Intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) caused a marked increase in the number of XCL-1+ cells in red pulps on day 3, followed by a rapid decrease to near control levels by day 7. XCL-1+ cells were also detected in peripheral blood leukocytes (PBLs) and peritoneal exudate cells (PECs), and their numbers increased upon LPS injection until day 7. The XCL-1+ cells exhibited the morphological characteristics of macrophages, with a large oval or lobulated nucleus and abundant cytoplasm with vacuoles and dendritic projections. Western blot analyses revealed concurrent increases in XCL-1 levels in the spleen, PBLs, and PECs. When LPS-stimulated frogs were intraperitoneally injected with paraformaldehyde-fixed, green fluorescent protein-labeled E. coli cells (GFP-Eco), these were phagocytosed by XCL-1+ PECs. The purified XCL-1 protein agglutinated GFP-Eco in a Ca2+ -dependent manner, which was blocked effectively by xylose and partly by LPS and Staphylococcus aureus peptidoglycan, but not by sucrose. These results indicate that X. laevis macrophage-like cells produce XCL-1 and suggest that XCL-1 promotes the clearance of invaded bacteria by facilitating phagocytosis.

Are the immunomodulatory properties of Lactobacillus rhamnosus CRL1505 peptidoglycan common for all Lactobacilli during respiratory infection in malnourished mice?

Previously, we reported that Lactobacillus rhamnosus CRL1505 peptidoglycan (PG05) improves the innate immune response in immunocompromised-malnourished mice after Streptococcus pneumoniae infection. This study extends those previous findings by demonstrating that the dietary recovery of malnourished mice with nasal administration of PG05 improves not only the innate immune response but the respiratory and systemic adaptive humoral response as well. PG05 enhanced the Th2 response, the recovery of B cells, and the concentration and opsonophagocytic activity of anti-pneumococcal antibodies. In addition, by performing comparative studies with the peptidoglycans from lactobacilli of the same species (L. rhamnosus CRL534) or with similar immunomodulatory properties (L. plantarum CRL1506), we demonstrated here that PG05 has unique immunomodulatory properties that cannot be extended to peptidoglycans from other probiotic strains. However, the knowledge of the molecular characteristics of PG05 is indispensable to understand immunomodulatory abilities of L. rhamnosus CRL1505.

Recombinant flagellin-PAL fusion protein of Legionella pneumophila induced cell-mediated and protective immunity against bacteremia in BALB/c mice.

We report a new recombinant fusion protein composed of full-length Legionella pneumophila flagellin A and peptidoglycan-associated lipoprotein (PAL), rFLA-PAL, capable of inducing protective immunity against L. pneumophila. The recombinant protein was over expressed in Escherichia coli strain BL21 (DE3) using pET-28a (+) expression vector (pET28a-flaA-pal) and purified by Ni2+ exchange chromatography. Immunological properties of rFLA-PAL were assessed in a mouse model. Female BALB/c mice, immunized with rFLA-PAL, exhibited a rapid increase in serum antibody concentration against each of its protein portions. Furthermore, a strong activation of both innate and adaptive cell-mediated immunity was observed as indicated by antigen-specific splenocyte proliferation, IFN-γ and IL-12 production, and early production of TNF-α in the serum and in splenocyte cultures which were separately assessed against PAL and FLA. BALB/c mice were challenged with a lethal dose of L. pneumophila intravenously. In a 10-days follow-up after intravenous lethal challenge with L. pneumophila, a 100% survival rate was observed for mice immunized with rFLA-PAL, same as for those immunized with a sublethal dose of L. pneumophila. Based on the potent immune responses observed in mice immunized with rFLA-PAL, this recombinant fusion protein could be a potential vaccine candidate against the intracellular pathogen L. pneumophila.

The first CUB-domain containing serine protease from Chlamys farreri which might be involved in larval development and immune response.

Serine proteases (SPs) are one of the most well understood enzyme families, which play an important role in regulating many physiological events. In the present study, one CUB-domain containing serine protease was identified from Chlamys farreri (designated as CfCUBSP). The full-length cDNA of CfCUBSP was of 3181 bp with an open reading frame of 2688 bp encoding a polypeptide of 896 amino acids. CfCUBSP shared closer phylogenetic relationship with those multi-domain SPs which consisted of one SP domain, and different numbers of CUB domain and LDLa domain than other SPs. The mRNA transcripts of CfCUBSP were detected in all developmental stages with the highest expression level in fertilized eggs and the lowest in trochophore larvae. In adult scallop, the CfCUBSP mRNA could be detected in all examined tissues with the highest level in hepatopancreas, and CfCUBSP protein was dominantly located in the gills, hepatopancreas, gonad and kidney. The mRNA expression of CfCUBSP in hemocytes was significantly up-regulated after the stimulation of lipopolysaccharide (LPS), peptidoglycan (PGN) and ß-glucan (GLU) (P < 0.05). All the results collectively indicated that CfCUBSP was a primitive member of the invertebrate SPs which might be involved in larval development and immune response against Gram-negative (G-) and Gram-positive (G+) bacteria and fungus in scallop.

The immunomodulatory potential of Leonurus cardiaca extract in relation to endothelial cells and platelets.

The immunomodulatory activity of Leonurus cardiaca L. polyphenol-rich extract (LCE) was tested in vitro on HUVECs to explore its potential therapeutic usefulness in the treatment of inflammatory lesions. The phytochemical composition of LCE, its antioxidant and cytotoxic activity, and the influence of LCE on NO and platelet-activating factor (PAF) secretion by HUVECs and platelet aggregation were all assessed. Total polyphenol contents in LCE reached 137.0 ± 0.8 mg/g, with hydroxycinnamic acid derivatives as the predominant phenolic compounds. LCE expressed antioxidant capacity, which was, however, 13- to 16-fold lower than the antioxidant activity of ascorbic acid. The plant extract was not cytotoxic up to a concentration 4500 µg/ml and did not exhibit proapoptotic activity. LCE significantly increased NO production in HUVECs in a concentration-dependent manner and led to the inhibition of PAF secretion induced by staphylococcal peptidoglycan. The extract used at the concentration of 100 µg/ml significantly reduced platelet aggregation in the presence of arachidonic acid. We provide in vitro data demonstrating the immunomodulatory potential of LCE, which may be beneficial in preventing the development of difficult-to-treat inflammatory lesions within chronically infected tissues.

Epistatic effect of TLR-1, -6 and -10 polymorphisms on organic dust-mediated cytokine response.

Exposure to organic dust from agricultural environments is associated with inflammatory respiratory conditions. The putative causal agents in organic dust include viral, microbial and fungal components, which are recognized by the family of Toll-like receptors (TLRs) and drive host innate and adaptive responses. Our aim in this study was to determine whether responsiveness to organic dust among agricultural workers was dependent on polymorphisms in the TLR10-TLR1-TLR6 gene cluster. We stimulated whole blood from 509 agricultural workers with organic dust, triacyl lipopeptide N-palmitoyl-S-dipalmitoylglyceryl Cys-Ser-(Lys)4 (Pam3CSK4) and the diacyl-lipopeptide peptidoglycan. Several of the tagging polymorphisms and haplotypes conferred hyper-responsiveness to organic dust with an increase in interleukin-6 (IL-6; P<0.005), but not tumor necrosis factor-α (TNF-α), secretion. We conclude that genetic variation in the TLR10-TLR1-TLR6 gene cluster mediates responsiveness to organic dust, but indicates different signaling pathways for IL-6 and TNF-α. These studies provide new insight into the role of the TLR10-TLR1-TLR6 gene cluster and the innate immune response to organic dust.