Bacterial Pattern Recognition Research Articles

A30 MICROBIOTA AND NOD2 REGULATE SMALL INTESTINAL RESTITUTION THROUGH FETAL-LIKE INTESTINAL STEM CELLS

Abstract Background Inflammatory bowel disease is characterized by chronic inflammation of the gastrointestinal tract, resulting in recurrent injury to the intestinal epithelium. Restitution of the small intestinal epithelium is a coordinated response that involves the dedifferentiation of epithelial cell lineages, proliferation of Lgr5+ intestinal stem cells, and fetal-like stem cell reversion. While gut microbiota are critical mediators of intestinal inflammation, their impact on epithelial restitution remains unclear. Aims We aim to identify the how microbes regulate small intestinal epithelial restitution following damage. Our hypothesis is that gut microbiota accelerate restitution through pattern recognition receptor-driven signals following fetal-like stem cell reversion. Methods Irradiation (IR, 12Gγ) was used to induce a synchronized small intestinal epithelial restitution response in mice. Intestinal restitution kinetics were assessed transcriptionally (scRNA-Seq, qPCR) and histologically. Small intestinal organoids were used to assess epithelial restitution kinetics in vitro. Results ScRNA-Seq of small intestinal epithelial cells following IR from germ-free mice (GF), and specific pathogen-free mice (SPF) mice revealed that microbiota induced greater expression of fetal-like stem cell reversion markers, Ly6a and Clu, and greater expression of the proliferation marker, Pcna. These results were supported histologically as irradiated SPF mice observed an increase in fetal-like stem cells marked by Ly6a and Clu and an increase BrdU+ proliferating cells. ScRNA-seq and in situ hybridization highlighted that fetal-like intestinal stem cells upregulate expression of Nod2, a bacterial pattern recognition receptor. Using intestinal organoids to assess the function of Nod2, we observed that muramyl dipeptide driven Nod2-signlaing potentiates an interferon gene signature following IFNγ and TNFα co-stimulation. Further supporting the role for Nod2 in intestinal restitution, intestinal epithelium specific Nod2KO mice decreased BrdU+ proliferating cells post-IR compared to littermate controls. Conclusions Microbiota promote small intestinal restitution following IR through Nod2-signaling in fetal-like intestinal stem cells. Funding Agencies CIHR

Soluble LPS receptor CD14 is increased in saliva of patients with geographic tongue

Objectives The soluble bacterial pattern recognition receptor, sCD14 augments inflammatory responses in oral cavity. The aim of the study was to investigate whether patients with geographic tongue (GT) with and without fissured tongue (FT) have impaired inflammatory regulation, manifesting as increased levels of sCD14 in the saliva. Material and Methods An enzyme-linked immunosorbent assay was used to measure the amount of sCD14 in whole and parotid saliva of patients diagnosed with GT (GT whole, n = 21; GT parotid, n = 23) and control subjects (GT whole, n = 25; GT parotid, n = 18). The levels of sCD14 were also evaluated according to our previous clinical assessment of GT based on the number of lesions detected on the tongue, as ‘mild’ (a single lesion), ‘moderate’ (2–5 lesions), or ‘severe’ (≥6 lesions). Diagnosis of FT was established when multiple grooves or fissures were observed on the dorsal and lateral surfaces of the tongue. Results GT patients had significantly higher sCD14 levels in whole (p<.05) and parotid saliva (p<.001), compared with controls. GT patients with FT had significantly increased sCD14 levels only in parotid saliva. A gradual increase in sCD14 levels in parotid and unstimulated saliva was seen in GT patients with multiple tongue lesions compared with single lesions. Conclusions GT patients had increased sCD14 in both parotid and unstimulated saliva. sCD14 seems to increase local inflammatory responses, which suggests its involvement in the pathophysiology of GT.

Silica Induction of Diverse Inflammatory Proteome in Lungs of Lupus-Prone Mice Quelled by Dietary Docosahexaenoic Acid Supplementation.

Repeated short-term intranasal instillation of lupus-prone mice with crystalline silica (cSiO2) induces inflammatory gene expression and ectopic lymphoid neogenesis in the lung, leading to early onset of systemic autoimmunity and rapid progression to glomerulonephritis. These responses are suppressed by dietary supplementation with the ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA). Here, we tested the hypothesis that dietary DHA supplementation suppresses cSiO2-induced inflammatory proteins in bronchoalveolar alveolar lavage fluid (BALF) and plasma of lupus-prone mice. Archived tissue fluid samples were used from a prior investigation in which 6 wk-old lupus-prone female NZBWF1 mice were fed isocaloric diets containing 0 or 10 g/kg DHA for 2 wks and then intranasally instilled with 1 mg cSiO2 or vehicle once weekly for 4 wks. Cohorts were terminated at 1, 5, 9 or 13 wk post-instillation (PI). BALF and plasma from each cohort were analyzed by high density multiplex array profiling of 200 inflammatory proteins. cSiO2 time-dependently induced increases in the BALF protein signatures that were highly reflective of unresolved lung inflammation, although responses in the plasma were much less robust. Induced proteins in BALF included chemokines (e.g., MIP-2, MCP-5), enzymes (e.g., MMP-10, granzyme B), adhesion molecules (e.g., sE-selectin, sVCAM-1), co-stimulatory molecules (e.g., sCD40L, sCD48), TNF superfamily proteins (e.g., sTNFRI, sBAFF-R), growth factors (e.g., IGF-1, IGFBP-3), and signal transduction proteins (e.g., MFG-E8, FcgRIIB), many of which were blocked or delayed by DHA supplementation. The BALF inflammatory proteome correlated positively with prior measurements of gene expression, pulmonary ectopic lymphoid tissue neogenesis, and induction of autoantibodies in the lungs of the control and treatment groups. Ingenuity Pathway Analysis (IPA) revealed that IL-1β, TNF-α, and IL-6 were among the top upstream regulators of the cSiO2-induced protein response. Furthermore, DHA’s effects were associated with downregulation of cSiO2-induced pathways involving i) inhibition of ARE‐mediated mRNA decay, ii) bacterial and viral pattern recognition receptor activation, or iii) TREM1, STAT3, NF-κB, and VEGF signaling and with upregulation of PPAR, LXR/RXR and PPARα/RXRα signaling. Altogether, these preclinical findings further support the contention that dietary DHA supplementation could be applicable as an intervention against inflammation-driven autoimmune triggering by cSiO2 or potentially other environmental agents.

Up-regulation of AIM2 and TLR4 and down-regulation of NLRC4 are associated with septicemia

PurposeInnate immunity receptors play key roles in recognition of bacterial associated molecular patterns. Inflammasomes and toll like receptors (TLRs) are the important innate immunity receptors. In this project transcription levels of TLR4, a TLR member, absent in melanoma 2 (AIM2) and NLR family CARD domain-containing protein 4 (NLRC4), as inflammasomes, in the patients suffering from septicemia. MethodsAIM2, NLRC4 and TLR4 mRNA levels were evaluated in the 40 patients suffering from septicemia and 40 healthy controls using Real-Time PCR technique. ResultsData analysis revealed that, although NLRC4 expression decreased, TLR4 and AIM2 levels significantly increased in the patients suffering from septicemia. Gender and infection with various bacteria did not affect expression of AIM2, NLRC4 and TLR4. ConclusionsIt appears that septicemia can be limited by immune responses in AIM2 and TLR4 dependent manner. The potential roles played by bacteria to down-regulation of NLRC4 need to be evaluated by further investigations.

Weighted Gene Co-Expression Network Analysis Identifies Key Modules and Hub Genes Associated with Mycobacterial Infection of Human Macrophages.

Tuberculosis (TB) is still a leading cause of death worldwide. Treatments remain unsatisfactory due to an incomplete understanding of the underlying host–pathogen interactions during infection. In the present study, weighted gene co-expression network analysis (WGCNA) was conducted to identify key macrophage modules and hub genes associated with mycobacterial infection. WGCNA was performed combining our own transcriptomic results using Mycobacterium aurum-infected human monocytic macrophages (THP1) with publicly accessible datasets obtained from three types of macrophages infected with seven different mycobacterial strains in various one-to-one combinations. A hierarchical clustering tree of 11,533 genes was built from 198 samples, and 47 distinct modules were revealed. We identified a module, consisting of 226 genes, which represented the common response of host macrophages to different mycobacterial infections that showed significant enrichment in innate immune stimulation, bacterial pattern recognition, and leukocyte chemotaxis. Moreover, by network analysis applied to the 74 genes with the best correlation with mycobacteria infection, we identified the top 10 hub-connecting genes: NAMPT, IRAK2, SOCS3, PTGS2, CCL20, IL1B, ZC3H12A, ABTB2, GFPT2, and ELOVL7. Interestingly, apart from the well-known Toll-like receptor and inflammation-associated genes, other genes may serve as novel TB diagnosis markers and potential therapeutic targets.

Effect of human breast milk on innate immune response: Up-regulation of bacterial pattern recognition receptors and innate cytokines in THP-1 monocytic cells

Human breast milk (HBM) contains many bioactive components that protect infants from various microorganisms. Pattern recognition receptors on phagocytic cells recognize microbial pathogens and promote the innate immune system. This study aimed to evaluate the effect of HBM on the expression of pattern recognition receptors and innate cytokines in the monocytic cell line THP-1 and the phagocytic activity of RAW264.7 macrophages. Expression levels of specific mRNAs in THP-1 cells were quantitated using reverse transcription-polymerase chain reaction. Phagocytic activity was measured by fluorescence microscopy to detect the uptake of fluorescent dye-labeled carboxylate-modified polystyrene latex beads in RAW264.7 macrophages. HBM stimulated the phagocytic activity of RAW264.7 macrophages. HBM increased mRNA expression of pattern recognition receptors, including the cluster of differentiation 14 and toll-like receptor 2 and 4, and various innate cytokines, including tumor necrosis factor α, interleukin-1β, C-X-C motif chemokine 8, and C-C motif chemokine ligand 2, in THP-1 monocytic cells. Furthermore, milk oligosaccharides in HBM, such as lacto- N-fucopentaose I, enhanced the expression of pattern recognition receptors and various innate cytokines. HBM is able to modulate the innate immune response by upregulating the expression of pattern recognition receptors and various innate cytokines in monocytes/macrophages.

Burkholderia pseudomallei triggers canonical inflammasome activation in a human primary macrophage-based infection model.

Most of the current knowledge on Burkholderia pseudomallei-induced inflammasome activation and cell death in macrophages is derived from murine systems. Little is known about the involved bacterial structures and mechanisms in primary human macrophages. This is of particular relevance since murine and human macrophages as well as primary cells and cell lines differ in many aspects of inflammasome activation, including the proteins involved in the recognition of bacterial patterns. In this study, we therefore aimed (i) to establish an in vitro B. pseudomallei infection model with human monocyte-derived primary macrophages from single donors as these cells more closely resemble macrophages in the human host and (ii) to analyze B. pseudomallei-triggered cell death and bacterial elimination in those cells. Our results show that B. pseudomallei-infected primary human macrophages not only release the inflammasome-independent pro-inflammatory cytokines IL-8 and TNF-α, but are also engaged in canonical inflammasome activation as evidenced by caspase-1 and gasdermin D processing. Absence of the B. pseudomallei T3SS-3 needle protein BsaL, a potent activator of the canonical inflammasome, abolished lytic cell death, reduced IL-1β release, and caspase-1 and gasdermin D processing. IFN-γ, known to promote non-canonical inflammasome activation, did not influence pyroptosis induction or IL-1β release from infected primary human macrophages. Nevertheless, it reduced intracellular B. pseudomallei loads, an effect which was partially antagonist by the inhibition of NADPH oxidase. Overall, our data implicate T3SS-3 dependent inflammasome activation and IFN-γ induced immune mechanisms as critical defense mechanisms of human macrophages against B. pseudomallei. In addition, our infection model provides a versatile tool to study human host-pathogen interactions and has the potential to elucidate the role of human individual genetic variations in B. pseudomallei infections.

The involvement of toll-like receptors 2 and 4 in human platelet signalling pathways

In addition to haemostasis, platelets play an essential role in mechanisms of inflammation and in immunological reactions. Platelets express various toll-like receptors (TLR) on their surface, among them TLR2 and TLR4, which are important for the recognition of bacterial patterns. This study compared TLR2- and TLR4-dependent platelet signalling and their effect on platelet function.Platelet-rich-plasma and washed platelets were prepared from peripheral blood samples of healthy donors. Pam3CSK4 or LPS (lipopolysaccharides from Escherichia coli) were used for stimulation of TLR2 and TLR4. Intracellular signalling pathways were investigated by Western blot. TLR2- and TLR4-mediated specific transcription factor DNA binding activity was measured by the nuclear factor kappa B (NFκB) transcription factor assay kit. Platelet adhesion and glycoprotein Ib function were assessed by immunofluorescence staining and analysis of ristocetin-induced agglutination.Both, Pam3CSK4 and LPS were able to induce NFκB-mediated and classical activating platelet signalling with a higher stimulatory capacity of TLR2. In addition, TLR2 and TLR4 activation led to a similar activation of inhibitory pathways. In contrast to TLR2, stimulation of TLR4 resulted in decreased Akt/protein kinase B phosphorylation conditioned by enhanced protein phosphatase 2A activity. TLR4-mediated signalling induced platelet adhesion and facilitated ristocetin-induced platelet agglutination.In conclusion, Pam3CSK4 directly induces aggregation via classical activation cascades, whereas LPS enhances platelet adhesion and glycoprotein receptor Ib-dependent platelet agglutination.

An immune-responsive PGRP-S1 regulates the expression of antibacterial peptide genes in diamondback moth, Plutella xylostella (L.)

Peptidoglycan recognition proteins (PGRPs) are family of pattern recognition receptors (PRRs) and triggers the innate immune system (IIS) against the microbial infection. Although PGRPs have been intensively studied in model insects, they remain uncharacterized in most of the non-model insects. Here, we cloned and characterized a full-length cDNA of PGRP, from P. xylostella (PxPGRP-S1), which encodes a protein of 239 amino acids with PGRP domain, Ami2 domain and transmembrane region. The phylogenetic analysis revealed that the PxPGRP-S1 was closely related to the unigene of Plutella xylostella. Quantitative real-time PCR and immunohistochemistry revealed that PxPGRP-S1 is mainly expressed in the fat body of the healthy larva. The expression of PxPGRP-S1 was significantly upregulated in the midgut at 24 h postinfection by Bacillus thuringiensis. Silencing of the PxPGRP-S1 expression by RNAi, significantly decrease the expression of the antimicrobial peptides (AMPs) in the 4th instar larvae of P. xylostella. Similarly injection of an anti-PxPGRP-S1 serum caused the low expression of the AMPs in P. xylostella. Additionally, PxPGRP-S1 depleted P. xylostella by oral administration of bacterial expressed dsRNA decreased the resistance against B. thuringiensis challenge, leads to high mortality. Together, our result indicates that PxPGRP-S1, served as a bacterial pattern recognition receptor (PRR) and triggers the expression of AMPs in P. xylostella.

Comparative transcriptomics between species attributes reactogenicity pathways induced by the capsular group B meningococcal vaccine, 4CMenB, to the membrane-bound endotoxin of its outer membrane vesicle component

The capsular group B meningococcal (MenB) four component vaccine (4CMenB) has been licensed for the prevention of invasive disease caused by MenB. The vaccine causes fever in infants, particularly when given in combination (concomitant) with other routinely-administered vaccines (routine), such as the standard diphtheria, tetanus, pertussis (DTP)-containing vaccine. To assess the suitability of a mouse immunisation model to study this phenomenon, we monitored temperature in mice after a second dose of routine vaccines, with or without 4CMenB, and compared the results with those in humans. Using this mouse model, we explored the reactogenicity of 4CMenB components by measuring changes in temperature, cytokines, and gene expression induced by 4CMenB, one of its components, wild-type or attenuated endotoxin outer membrane vesicles (OMVs), or lipopolysaccharide (LPS). A significant rise (p < 0.01) in temperature was observed in mice immunised with 4CMenB, wild-type OMVs, and LPS. RNA-sequencing of mouse whole blood revealed a gene signature shared by the 4CMenB, OMV, and LPS groups consisting of bacterial pattern recognition receptors and neutrophil activation marker genes. Sequencing of neutrophils isolated after concomitant 4CMenB identified cells expressing the OMV-associated genes Plek and Lcp1. Immunisation with 4CMenB or OMVs led to increased IL-6 in serum and significant upregulation (p < 0.0001) of prostaglandin-synthesising enzymes on brain tissue. These data demonstrate the suitability of a mouse model for assessing vaccine reactogenicity and strongly indicate that the fever following vaccination with 4CMenB in human infants is induced by endotoxin contained in the OMV component of the vaccine.

The Role of Human Platelet Preparation for Toll-Like Receptors 2 and 4 Related Platelet Responsiveness.

Background Like immune cells, platelets express the repertoire of toll-like receptors (TLR), among them TLR2 and TLR4, which are important for the recognition of bacterial patterns. Receptor-mediated functional effects in platelets have been investigated, but reliable conclusions are tampered due to heterogeneous study designs with variable platelet preparation methods. This study compares TLR2- and TLR4-dependent platelet responsiveness in platelet-rich plasma (PRP) and in washed platelets (WPs). Material and Methods Fresh peripheral blood samples from healthy donors served for the preparation of PRP and WP. Basal and agonist-stimulated TLR2 and TLR4 expression levels were evaluated by flow cytometry. Light transmission aggregometry was used to investigate functional effects of TLR2 and TLR4 stimulation with Pam3CSK4 or LPS (lipopolysaccharides from Escherichia coli ) as ligands. The capacity of chemokine release was determined by immunoassays. Results Pam3CSK4 and LPS (in combination with thrombin) were able to induce aggregation in WP, but not in PRP, with threshold concentrations of 15 µg/mL. Basal expression levels of TLR2 and TLR4 were higher in WP than in PRP, increasing several-fold rapidly and persistently upon platelet activation with potent agonists. Pam3CSK4 (15 µg/mL) or LPS led to the submaximal release of RANTES, PF4, PDGF, NAP-2, and sCD40L from WP. In PRP, secretory effects are less pronounced for RANTES, PDGF, or PF4, and not detectable for NAP-2 or sCD40L. Conclusion The effects mediated by TLR2 and TLR4 stimulation are dependent on platelet preparation, an important issue for experimental designs and for manufacturing of platelet concentrates in transfusion medicine.

Effects of TLR Ligands on the Expression of Cytokines and Possible Role of NFκB in its Process in the Theca of Chicken Follicles.

The aim of this study was to determine the effects of Toll-like receptor (TLR) ligands on the expression of cytokines in chicken follicular theca and to investigate whether nuclear factor-κB (NFκB) was involved in their expression. The follicular theca was collected from the largest follicle of laying hens. In experiment 1, the expression of TLRs in the theca interna and externa was confirmed using RT-PCR. The theca tissues were then incubated with or without Pam3CSK4 (TLR2 ligand), poly I:C (TLR3 ligand), LPS (TLR4 ligand), flagellin (TLR5 ligand), R837 (TLR7 ligand), and CpG-ODN (TLR21 ligand) for 3 h, after which cytokine expression (IL-1β, IL-6, TNFSF15, CXCLi2, IFN-α, and IFN-β) was analyzed by real-time PCR. In experiment 2, the theca tissues were incubated in a medium containing Pam3CSK4, poly I:C, LPS, or CpG-ODN with or without BAY 11-7085 (an inhibitor of NFκB) for 3 h. The results of experiment 1 revealed that all TLRs, namely TLR1 (type 1 and 2), TLR2 (type 1 and 2), 3–5, 7, 15, and 21, were expressed in the follicular theca, although the PCR products of TLR1 (type 2) and TLR21 were faint. Moreover, Pam3CSK4 and LPS upregulated the expression of all detected cytokines, except for IFN-α, whose expression was not upregulated by LPS. Poly I:C upregulated the expression of IL-6, CXCLi2, and IFN-β, while CpG-ODN upregulated IL-1β. Flagellin and R837 did not significantly affect cytokine expression. In experiment 2, the expression of IL-1β, IL-6, CXCLi2 and IFN-β in tissues incubated with LPS was downregulated by BAY 11-7085. These results suggest that the innate immune system, including pattern recognition by TLRs and cytokine synthesis, occur in the theca; whereas, functions for recognition of bacterial patterns is more developed than that of viral ones.

Turning the light on.

Richard Locksley describes a 2004 study by Margaret McFall-Ngai and colleagues that showed the role of bacterial pattern recognition in driving tissue homeostasis and function.

Cyclic dinucleotide detection by RECON controls innate immune responses

Abstract Host cells have evolved a sophisticated arsenal of germ-line encoded pattern recognition receptors that detect a vast array of microorganisms in distinct tissues and cellular compartments. Cytoplasmic sensors monitor for microbes that gain access to the host cell cytosol following breach of the plasma membrane or intracellular invasion. This sensing is achieved through detection of invariant molecular patterns associated with microorganisms or cell stress responses that indicate the presence of a pathogen. Cyclic dinucleotides (cdNs) of both bacterial and host origin have emerged as important molecules that are sensed during infection. Details about the receptors cdNs engage in host cells and the responses those interactions elicit are just coming to light. We have recently discovered the first bacterial cdN-specific pattern recognition receptor, the oxidoreductase RECON. Our work on this receptor has revealed its central role in directing innate immune responses when engaged by bacterial cdNs. These findings reveal a new pattern recognition receptor specific for bacterial cdNs that orchestrates cytosolic immune surveillance by shaping downstream inflammatory gene activation.

Aberrant Expression of Bacterial Pattern Recognition Receptor NOD2 of Basophils and Microbicidal Peptides in Atopic Dermatitis.

Atopic dermatitis (AD) is a chronically relapsing inflammatory skin disease, associated with basophil infiltration into skin lesions and Staphylococcus aureus (S. aureus)-induced inflammation. Pattern recognition receptors (PRRs), including microbicidal peptide human neutrophil α-defensins (HNP) and dermcidin, can exert immunomodulating activity in innate immunity and skin inflammation. We investigated the plasma concentration of HNP and dermcidin, the expression of bacterial toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD)-like receptors of basophils and plasma concentration and ex vivo induction of AD-related inflammatory cytokines and chemokines using ELISA and flow cytometry, in AD patients and control subjects. Plasma concentrations of HNP, dermcidin and AD-related Th2 chemokines CCL17, CCL22 and CCL27 were significantly elevated in AD patients compared with controls (all p < 0.05). Plasma concentrations of CCL27 and CCL22 were found to correlate positively with SCORing atopic dermatitis (SCORAD), objective SCORAD, % area affected, lichenification and disease intensity, and CCL27 also correlated positively with pruritus in AD patients (all p < 0.05). Protein expressions of NOD2 but not TLR2 of basophils were significantly down-regulated in AD patients compared with controls (p = 0.001). Correspondingly, there were lower ex vivo % inductions of allergic inflammatory tumor necrosis factor-α, IL-6 and CXCL8 from peripheral blood mononuclear cells upon NOD2 ligand S. aureus derived muramyl dipeptide stimulation in AD patients comparing with controls. The aberrant activation of bacterial PRRs of basophils and anti-bacterial innate immune response should be related with the allergic inflammation of AD.

Vitamin D [1,25(OH)2D3] Differentially Regulates Human Innate Cytokine Responses to Bacterial versus Viral Pattern Recognition Receptor Stimuli

Vitamin D plays multiple roles in regulation of protective and maladaptive immunity. Although epidemiologic studies link poor in vivo 25(OH)D status to increased viral respiratory infections, we poorly understand how vitamin D affects viral pattern recognition receptor (PRR)-driven cytokine production. In this study, we hypothesized that the biologically active metabolite of vitamin D, 1,25(OH)2D3, inhibits human proinflammatory and anti-inflammatory innate cytokine responses stimulated by representative bacterial or viral PRR ligands. Fresh PBMCs or CD14(+) monocytes were stimulated with TLR4, TLR7/8-selective ligands, or respiratory syncytial virus (RSV) ± 1,25(OH)2D3. Proinflammatory and anti-inflammatory responses resulting from TLR4 stimulation were inhibited ∼50% in the presence of 1,25(OH)2D3. Conversely, its usage at physiologic through pharmacologic concentrations inhibited neither proinflammatory nor anti-inflammatory responses evoked by viral PRR ligands or infectious RSV. This differential responsiveness was attributed to the finding that TLR7/8, but not TLR4, stimulation markedly inhibited vitamin D receptor mRNA and protein expression, selectively reducing the sensitivity of viral PRR responses to modulation. 1,25(OH)2D3 also enhanced expression of IkBa, a potent negative regulator of NF-κB and cytokine production, in TLR4-stimulated monocytes while not doing so upon TLR7/8 stimulation. Thus, 1,25(OH)2D3 inhibits both proinflammatory and a broad panel of anti-inflammatory responses elicited by TLR4 stimulation, arguing that the common view of it as an anti-inflammatory immune response modifier is an oversimplification. In viral responses, it consistently fails to modify TLR7/8- or RSV-stimulated innate cytokine production, even at supraphysiologic concentrations. Collectively, the data call into question the rationale for increasingly widespread self-medication with vitamin D supplements.

Yeast-containing feed additive alters gene expression profiles associated with innate immunity in whole blood of a rodent model.

Feeding a yeast-containing additive (YCA; OmniGen-AF) improves immune responses in ruminant livestock and reduces subsequent production losses. The objective was to identify molecular pathways by which dietary YCA may modify immune responses using a rodent model. Thirty-seven healthy, unchallenged CD rats received a diet containing 0 (control; n = 5, only 28 d), 0.5% (n = 15) or 1% (n = 17) YCA for 7 (n = 4/group), 14 (n = 3 or 4/group), 21 (n = 3 or 4/group) or 28 (n = 5/group) d. At the end of the feeding periods, whole blood was collected and the isolated RNA was analyzed for the expression of 84 genes involved in innate and cell-mediated adaptive immune responses. Three bacterial pattern recognition receptors TLR1 (0.5%: + 2.01; 1%: + 2.38), TLR6 (0.5%: + 2.11; 1%: + 2.34) and NOD2 (0.5%: + 2.32; 1%: + 2.23), two APC surface receptors CD1D1 (0.5%: + 1.75; 1%: + 2.33) and CD80 (0.5%: +2.45; 1%: +3.00), and the cell signaling molecule MAPK8 (0.5%: +1.87; 1%: +2.35) were significantly up-regulated by YCA at both inclusion rates. In conclusion, feeding YCA may potentially increase recognition and responses to bacterial pathogens and T-cell activation and differentiation and thereby maintain health and prevent production losses.

Cdx2 Expression and Intestinal Metaplasia Induced by H. pylori Infection of Gastric Cells Is Regulated by NOD1-Mediated Innate Immune Responses.

Chronic infection with the bacterial Helicobacter pylori is a major cause of gastric and duodenal ulcer disease, gastric mucosal atrophy, and cancer. H. pylori-induced expression of the intestinal epithelial-specific transcription factor caudal-related homeobox 2 (Cdx2) contributes to intestinal metaplasia, a precursor event to gastric cancer. Given a role for the bacterial pattern recognition molecule nucleotide-binding oligomerization domain 1 (NOD1) in the innate immune response to bacterial infection, we investigated mechanisms used by NOD1 to regulate H. pylori infection and its propensity towards the development of intestinal metaplasia. We found that Cdx2 was induced by H. pylori infection in both normal and neoplastic gastric epithelial cells in a manner that was inversely related to NOD1 signaling. Mechanistic investigations revealed that Cdx2 induction relied upon activation of NF-κB but was suppressed by NOD1-mediated activation of TRAF3, a negative regulator of NF-κB. In vivo, prolonged infection of NOD1-deficient mice with H. pylori led to increased Cdx2 expression and intestinal metaplasia. Furthermore, gastric epithelial cells from these mice exhibited increased nuclear expression of the NF-κB p65 subunit and decreased expression of TRAF3. Overall, our findings illuminated a role for NOD1 signaling in attenuating H. pylori-induced Cdx2 expression in gastric epithelial cells, suggesting a rationale to augment NOD1 signaling in H. pylori-infected patients to limit their risks of accumulating precancerous gastric lesions.

Bacterial and fungal pattern recognition receptors in homologous innate signaling pathways of insects and mammals.

In response to bacterial and fungal infections in insects and mammals, distinct families of innate immune pattern recognition receptors (PRRs) initiate highly complex intracellular signaling cascades. Those cascades induce a variety of immune functions that restrain the spread of microbes in the host. Insect and mammalian innate immune receptors include molecules that recognize conserved microbial molecular patterns. Innate immune recognition leads to the recruitment of adaptor molecules forming multi-protein complexes that include kinases, transcription factors, and other regulatory molecules. Innate immune signaling cascades induce the expression of genes encoding antimicrobial peptides and other key factors that mount and regulate the immune response against microbial challenge. In this review, we summarize our current understanding of the bacterial and fungal PRRs for homologous innate signaling pathways of insects and mammals in an effort to provide a framework for future studies.

Analysis of nucleotide-binding oligomerization domain proteins in a murine model of pneumococcal meningitis.

BackgroundThe innate immune system recognizes pathogens via its pattern recognition receptors. The objective of this study was to investigate the role of the nucleotide-binding oligomerization domain (NOD) proteins, a family of the novel bacterial pattern recognition receptors, in host responses to the gram-positive bacteria Streptococcus pneumoniae.MethodsSprague–Dawley rats were infected via intracisternal injections of viable S. pneumoniae, and rats in the control group were injected with sterile saline. After infection, real-time PCR was performed to determine the presence of mRNAs encoding NOD1 and NOD2. Quantitative analyses of the NOD1, NOD2 and NF-kB proteins were also performed western blotting following challenge infections with viable S. pneumoniae. The TNF-α and IL-6 levels in brain homogenates were evaluated using enzyme-linked immunosorbent assays (ELISAs).ResultsThe results revealed up-regulations of the mRNA and protein levels of NOD2 within the CNS of rats with S. pneumoniae meningitis. Moreover, the activation of NF-κB in the brain tissues following infection with live S. pneumoniae was also significantly increased, which indicates that NOD2 mediated NF-κB activation in experimental pneumococcal meningitis. Similarly, TNF-α and IL-6 levels were increased in the brain following in vivo S. pneumoniae administration.ConclusionsThese results suggest that NOD2 is involved in the host response to the gram-positive bacteria S. pneumoniae in the CNS and that NOD2 might play an important role in the initiation and/or progression of CNS inflammation associated with pneumococcal meningitis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-014-0648-3) contains supplementary material, which is available to authorized users.