Gram-negative Bacterial Component Research Articles

β-lactam-induced OMV release promotes polymyxin tolerance in Salmonella enterica sv. Typhi.

The rise of multidrug-resistant bacteria is a global concern, leading to a renewed reliance on older antibiotics like polymyxins as a last resort. Polymyxins, cationic cyclic peptides synthesized nonribosomally, feature a hydrophobic acyl tail and positively charged residues. Their antimicrobial mechanism involves initial interaction with Gram-negative bacterial outer-membrane components through polar and hydrophobic interactions. Outer membrane vesicles (OMVs), nano-sized proteoliposomes secreted from the outer membrane of Gram-negative bacteria, play a crucial role in tolerating harmful molecules, including cationic peptides such as polymyxins. Existing literature has documented environmental changes' impact on modulating OMV properties in Salmonella Typhimurium. However, less information exists regarding OMV production and characteristics in Salmonella Typhi. A previous study in our laboratory showed that S. Typhi ΔmrcB, a mutant associated with penicillin-binding protein (PBP, a β-lactam antibiotic target), exhibited hypervesiculation. Consequently, this study investigated the potential impact of β-lactam antibiotics on promoting polymyxin tolerance via OMVs in S. Typhi. Our results demonstrated that sub-lethal doses of β-lactams increased bacterial survival against polymyxin B in S. Typhi. This phenomenon stems from β-lactam antibiotics inducing hypervesiculation of OMVs with higher affinity for polymyxin B, capturing and diminishing its biologically effective concentration. These findings suggest that β-lactam antibiotic use may inadvertently contribute to decreased polymyxin effectivity against S. Typhi or other Gram-negative bacteria, complicating the effective treatment of infections caused by these pathogens. This study emphasizes the importance of evaluating the influence of β-lactam antibiotics on the interaction between OMVs and other antimicrobial agents.

Role of Gram-Negative Bacterial Components in Alzheimer’s Disease Pathology

Role of Gram-Negative Bacterial Components in Alzheimer’s Disease Pathology

Altered Osteogenic Differentiation in Mesenchymal Stem Cells Isolated from Compact Bone of Chicken Treated with Varying Doses of Lipopolysaccharides.

Persistent inflammation biologically alters signaling molecules and ultimately affects osteogenic differentiation, including in modern-day broilers with unique physiology. Lipopolysaccharides (LPS) are Gram-negative bacterial components that activate cells via transmembrane receptor activation and other molecules. Previous studies have shown several pathways associated with osteogenic inductive ability, but the pathway has yet to be deciphered, and data related to its dose-dependent effect are limited. Primary mesenchymal stem cells (MSCs) were isolated from the bones of day-old broiler chickens, and the current study focused on the dose-dependent variation (3.125 micrograms/mL to 50 micrograms/mL) in osteogenic differentiation and the associated biomarkers in primary MSCs. The doses in this study were determined using a cell viability (MTT) assay. The study revealed that osteogenic differentiation varied with dose, and the cells exposed to higher doses of LPS were viable but lacked differentiating ability. However, this effect became transient with lower doses, and this phenotypic character was observed with differential staining methods like Alizarin Red, Von Kossa, and alkaline phosphatase. The data from this study revealed that LPS at varying doses had a varying effect on osteogenic differentiation via several pathways acting simultaneously during bone development.

Anti-Inflammatory Activity of Glyceryl 1,3-Distearate Identified from Clinacanthus nutans Extract against Bovine Mastitis Pathogens

Clinacanthus nutans is widely used as a traditional medicine in Thailand and other countries in Southeast Asia. Although its effectiveness is well documented, its therapeutic use is limited to the treatment of only a few diseases; mostly it is used as an anti-viral agent against varicella-zoster and herpes simplex virus infections. Herein, we demonstrate the therapeutic activity of C. nutans extracts in lowering inflammation in a model of bovine mastitis caused by bacterial infection. Lipopolysaccharide (LPS), a gram-negative bacterial component, caused inflammation activation in bovine endothelial cells (CPAE) through the upregulation of proinflammatory cytokines (IL6 and IL1β) and chemokines (CXCL3 and CXCL8) gene expression, partially leading to cell death. Treatment with C. nutans crude extract significantly diminished these responses in a dose-dependent manner. The solvent fractionation of C. nutans extract revealed that the ethyl acetate (C4H8O2) fractions had a high potential to protect against cell death and diminished IL1β, IL6, CXCL3, and CXCL8 levels to less than 0.45 folds relative to the LPS-treated control. Glyceryl 1,3-distearate (C39H76O5) was identified as a bioactive compound responsible for the anti-inflammation activity but not the anti-cell death activity of C. nutans extract. This study highlighted the efficiency of C. nutans extracts as an alternative therapeutic option for the natural-product sustainable development of bovine mastitis treatment.

10-Residue MyD88-Peptide Adopts β-Sheet Structure, Self-Assembles, Binds to Lipopolysaccharides, and Rescues Mice from Endotoxin-Mediated Lung-Infection and Death.

Naturally occurring cationic antimicrobial peptides (AMPs) mostly adopt α-helical structures in bacterial membrane mimetic environments. To explore the design of novel β-sheet AMPs, we identified two short cationic amphipathic β-strand segments from the crystal structure of the innate immune protein, MyD88. Interestingly, of these, the 10-residue arginine-valine-rich synthetic MyD88-segment, KRCRRMVVVV (M3), exhibited β-sheet structure when bound to the outer membrane Gram-negative bacterial component, LPS. Isothermal titration calorimetric data showed that M3 bound to LPS with high affinity, and the interaction was hydrophobic in nature. Supporting these observations, computational studies indicated strong interactions of multiple and consecutive valine residues of M3 with the acyl chain of LPS. Moreover, M3 adopted nanosheet and nanofibrillar structure in 25% acetonitrile/water and isopropanol, respectively. M3 showed substantial antibacterial activities against both Gram-positive and Gram-negative bacteria which it appreciably retained in the presence of human serum and physiological salts. M3 was non-hemolytic against human red blood cells and non-cytotoxic to 3T3 cells up to 200 μM and to mice in vivo at a dose of 40 mg/kg. Furthermore, M3 neutralized LPS-induced pro-inflammatory responses in THP-1 cells and rat bone marrow-derived macrophages. Consequently, M3 attenuated LPS-mediated lung inflammation in mice and rescued them (80% survival at 10 mg/kg dose) against a lethal dose of LPS. The results demonstrate the identification of a 10-mer LPS-interacting, β-sheet peptide from MyD88 with the ability to form nanostructures and in vivo activity against LPS challenge in mice. The identified M3-template provides scope for designing novel bioactive peptides with β-sheet structures and self-assembling properties.

Biomimetic Magnetic Particles for the Removal of Gram-Positive Bacteria and Lipoteichoic Acid.

Gram+ bacteria are very common in clinical medicine and responsible for a large number of infectious diseases. For example, Gram+ bacteria play a major role in causing bloodstream infections and sepsis. Therefore, the detection of Gram+ bacteria is of great importance for the diagnosis and treatment of infectious diseases. Furthermore, these bacteria are often present in biofilms that cover implants. Recent research work has mainly focused on the biologic activity and removal of Gram-negative bacteria or bacterial components such as lipopolysaccharides (LPS). In contrast, the effects of lipoteichoic acid (LTA) have been less well studied so the relevance of their removal from body fluids is possibly underestimated. To address this topic, we evaluated superparamagnetic iron oxide particles (SPION) carrying different peptides derived from the innate immune receptor (GP-340) for their ability to bind and remove Gram+ bacteria and LTA from different media. Our results show that, beyond S. aureus, effective agglutinating and removing of S. pneumoniae was possible. Furthermore, we were able to show for the first time that this was possible with LTA alone and that the magnetic removal of bacteria was also efficient under flow conditions. We also found that this method was able to capture Stapyhylococcus aureus from platelet concentrates, which can help to enhance the sensitivity of microbiological diagnostics, quality control measures, and blood product safety.

BACTERIAL PATHOGEN-ASSOCIATED MOLECULAR PATTERNS UPREGULATE HUMAN GLUCOCORTICOID RECEPTOR EXPRESSION IN PERIPHERAL BLOOD MONONUCLEAR CELLS.

It is well known that bacterial components (pathogen-associated molecular patterns [PAMPs]) induce a proinflammatory response through pattern recognition receptor signaling. What is not known, however, is how the inflammatory response is downregulated. We hypothesize that bacterial products initiate compensatory anti-inflammatory responses by inducing expression of the human glucocorticoid receptor (hGR). Peripheral blood mononuclear cells (PBMCs) were isolated from leukocytes concentrated from single human donors (Leukopaks). PBMCs were treated with a gram-negative bacterial component, LPS, or gram-positive bacterial components, lipoteichoic acid (LTA) or peptidoglycan (PGN), for 1, 3, or 13 h. Protein expression of hGR was evaluated by Western blot analysis. RNA was extracted from similarly treated cells for reverse transcription-polymerase chain reaction analysis of hGR and cytokine expression. At 13 h after LPS treatment, there was an increase in the reference hGR protein (hGRα) expressed within some but not all PBMCs isolated from Leukopaks. There was also a dose-dependent increase in hGRα expression with increasing concentrations of PGN (10 and 50 μg/mL). LTA, however, did not affect hGRα expression. PGN also increased the mRNA expression of an hGR splice variant, hGR-B(54). The mRNA expression changes for the inflammatory cytokines were Leukopak specific. We found that cell wall components of both gram-positive and gram-negative bacteria can increase the expression of hGRα. Although these PAMPs augment the inflammatory response, it seems that there is a simultaneous upregulation of hGRα expression. Because binding of cortisol to hGRα typically induces anti-inflammatory proteins, the same PAMPs that induce an inflammatory response seem to also initiate a negative feedback system by inducing hGRα expression in PBMCs.

Human alveolar macrophages do not rely on glucose metabolism upon activation by lipopolysaccharide

Most macrophages generate energy to mount an inflammatory cytokine response by increased glucose metabolism through intracellular glycolysis. Previous studies have suggested that alveolar macrophages (AMs), which reside in a glucose-poor natural environment, are less capable to utilize glycolysis and instead rely on other substrates to fuel oxidative phosphorylation (OXPHOS) for energy supply. At present, it is not known whether AMs are capable to use glucose metabolism to produce cytokines when other metabolic options are blocked. Here, we studied human AMs retrieved by bronchoalveolar lavage from healthy subjects, and examined their glucose metabolism in response to activation by the gram-negative bacterial component lipopolysaccharide (LPS) ex vivo. The immunological and metabolic responses of AMs were compared to those of cultured blood monocyte-derived macrophages (MDMs) from the same subjects. LPS stimulation enhanced cytokine release by both AMs and MDMs, which was associated with increased lactate release by MDMs (reflecting glycolysis), but not by AMs. In agreement, LPS induced higher mRNA expression of multiple glycolytic regulators in MDMs, but not in AMs. Flux analyses of [13C]-glucose revealed no differences in [13C]-incorporation in glucose metabolism intermediates in AMs. Inhibition of OXPHOS by oligomycin strongly reduced LPS-induced cytokine production by AMs, but not by MDMs. Collectively, these results indicate that human AMs, in contrast to MDMs, do not use glucose metabolism during LPS-induced activation and fully rely on OXPHOS for cytokine production.

Opioids and cancer survival: are we looking in the wrong place?

There is a controversial narrative in the anaesthetic literature suggesting that anaesthetic technique (including opioids) may be detrimental to survival after tumour resection. The initial observations were retrospective. Several prospective studies are ongoing; one in breast cancer has reported no adverse outcome. The evidence for an effect of opioids stems from three pieces of information: (1) opioids depress the immune system, (2) opioids potentially promote angiogenesis, and (3) opioids potentially support tumour growth. Although the evidence for (2)/(3) is unclear, combinations of these effects are beneficial to tumours and potentially promote metastatic reseeding. Accepted wisdom suggests that opioid effects are driven by opioid receptor activation but the presence of opioid receptors on immune cells for example is unlikely. Immune cells, vascular endothelium and a range of tumour cells express Toll-like receptor 4 (TLR4) receptors (for Gram-negative bacterial wall components), and there is growing evidence for opioids interacting with this alternative receptor; and for some there is paradoxical naloxone sensitivity. Is the focus on opioid receptors and cancer the wrong target? TLR4 receptor activation produces immune activation, stimulates angiogenesis, and supports tumour survival. We know that some opioids are more immune suppressive than others (there is no such comparative information for angiogenesis and tumour survival); this may correlate with TLR4 activation. If there are clusters of opioids that have more opioid than TLR4 profiles and vice versa, this may influence outcome. If this is the case, then evidence-based advice could be given for perioperative use in the oncology-anaesthesia setting.

NLRP3 Inflammasome Negatively Regulates RANKL-Induced Osteoclastogenesis of Mouse Bone Marrow Macrophages but Positively Regulates It in the Presence of Lipopolysaccharides.

In inflammatory bone diseases such as periodontitis, the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3 (NLRP3) inflammasome accelerates bone resorption by promoting proinflammatory cytokine IL-1β production. However, the role of the NLRP3 inflammasome in physiological bone remodeling remains unclear. Here, we investigated its role in osteoclastogenesis in the presence and absence of lipopolysaccharide (LPS), a Gram-negative bacterial component. When bone marrow macrophages (BMMs) were treated with receptor activator of nuclear factor-κB ligand (RANKL) in the presence of NLRP3 inflammasome inhibitors, osteoclast formation was promoted in the absence of LPS but attenuated in its presence. BMMs treated with RANKL and LPS produced IL-1β, and IL-1 receptor antagonist inhibited osteoclastogenesis, indicating IL-1β involvement. BMMs treated with RANKL alone produced no IL-1β but increased reactive oxygen species (ROS) production. A ROS inhibitor suppressed apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) speck formation and NLRP3 inflammasome inhibitors abrogated cytotoxicity in BMMs treated with RANKL, indicating that RANKL induces pyroptotic cell death in BMMs by activating the NLRP3 inflammasome via ROS. This suggests that the NLRP3 inflammasome promotes osteoclastogenesis via IL-1β production under infectious conditions, but suppresses osteoclastogenesis by inducing pyroptosis in osteoclast precursors under physiological conditions.

CD19 regulates innate immunity by the toll-like receptor RP105 signaling in B lymphocytes

AbstractLipopolysaccharide (LPS) is a major gramnegative bacterial component that stimulates innate immune response and also induces B-lymphocyte activation. Recent studies have revealed that common molecular patterns of microorganisms such as LPS are recognized by toll-like receptors (TLRs). B cells have 2 known TLRs that mediate LPS signaling, TLR4 and RP105 (CD180). While TLR4 is expressed on immune cells of various types, RP105 is preferentially expressed on mature B cells. Here we demonstrate that CD19 plays a major role in regulating signal transduction through RP105. Anti-RP105 ligation induced normal proliferation of B cells from mice deficient for MyD88, an adaptor protein that mediates most TLR pathways. By contrast, the loss of CD19 resulted in modest B-cell proliferation against anti-RP105 stimulation as well as LPS stimulation. LPS induced tyrosine phosphorylation of CD19, which was RP105-dependent but TLR4-independent. CD19 formed a complex with Lyn and Vav following RP105 ligation, and CD19 expression was required for optimal Lyn activation and Vav phosphorylation. Consistently, B cells deficient for CD19 exhibited specific defect in the activation of c-Jun N-terminal kinases following RP105 ligation and LPS stimulation. In contrast, CD19 and phosphatidylinositol 3-kinase independently regulated intracellular calcium mobilization induced by anti-RP105 stimulation. Thus, signaling through the B-cell–specific LPS receptor RP105 is uniquely regulated by the B-cell–specific signaling component, Lyn/CD19/Vav complex.

Pharmacological inhibition of focal adhesion kinase 1 (FAK1) and anaplastic lymphoma kinase (ALK) identified via kinome profile analysis attenuates lipopolysaccharide-induced endothelial inflammatory activation

Sepsis is a life-threatening condition often leading to multiple organ failure for which currently no pharmacological treatment is available. Endothelial cells (EC) are among the first cells to respond to pathogens and inflammatory mediators in sepsis and might be a sentinel target to prevent the occurrence of multiple organ failure. Lipopolysaccharide (LPS) is a Gram-negative bacterial component that induces endothelial expression of inflammatory adhesion molecules, cytokines, and chemokines. This expression is regulated by a network of kinases, the result of which in vivo enables leukocytes to transmigrate from the blood into the underlying tissue, causing organ damage. We hypothesised that besides the known kinase pathways, other kinases are involved in the regulation of EC in response to LPS, and that these can be pharmacologically targeted to inhibit cell activation. Using kinome profiling, we identified 58 tyrosine kinases (TKs) that were active in human umbilical vein endothelial cells (HUVEC) at various timepoints after stimulation with LPS. These included AXL tyrosine kinase (Axl), focal adhesion kinase 1 (FAK1), and anaplastic lymphoma kinase (ALK). Using siRNA-based gene knock down, we confirmed that these three TKs mediate LPS-induced endothelial inflammatory activation. Pharmacological inhibition with FAK1 inhibitor FAK14 attenuated LPS-induced endothelial inflammatory activation and leukocyte adhesion partly via blockade of NF-κB activity. Administration of FAK14 after EC exposure to LPS also resulted in inhibition of inflammatory molecule expression. In contrast, inhibition of ALK with FDA-approved inhibitor Ceritinib attenuated LPS-induced endothelial inflammatory activation via a pathway that was independent of NF-κB signalling while it did not affect leukocyte adhesion. Furthermore, Ceritinib administration after start of EC exposure to LPS did not inhibit inflammatory activation. Combined FAK1 and ALK inhibition attenuated LPS-induced endothelial activation in an additive manner, without affecting leukocyte adhesion. Summarising, our findings suggest the involvement of FAK1 and ALK in mediating LPS-induced inflammatory activation of EC. Since pharmacological inhibition of FAK1 attenuated endothelial inflammatory activation after the cells were exposed to LPS, FAK1 represents a promising target for follow up studies.

Irgm2 and Gate-16 cooperatively dampen Gram-negative bacteria-induced caspase-11 response.

Inflammatory caspase-11 (rodent) and caspases-4/5 (humans) detect the Gram-negative bacterial component LPS within the host cell cytosol, promoting activation of the non-canonical inflammasome. Although non-canonical inflammasome-induced pyroptosis and IL-1-related cytokine release are crucial to mount an efficient immune response against various bacteria, their unrestrained activation drives sepsis. This suggests that cellular components tightly control the threshold level of the non-canonical inflammasome in order to ensure efficient but non-deleterious inflammatory responses. Here, we show that the IFN-inducible protein Irgm2 and the ATG8 family member Gate-16 cooperatively counteract Gram-negative bacteria-induced non-canonical inflammasome activation, both in cultured macrophages and invivo. Specifically, the Irgm2/Gate-16 axis dampens caspase-11 targeting to intracellular bacteria, which lowers caspase-11-mediated pyroptosis and cytokine release. Deficiency in Irgm2 or Gate16 induces both guanylate binding protein (GBP)-dependent and GBP-independent routes forcaspase-11 targeting to intracellular bacteria. Our findings identify molecular effectors that fine-tune bacteria-activated non-canonical inflammasome responses and shed light on the understanding of the immune pathways they control.

TRPV4 Mediates Acute Bladder Responses to Bacterial Lipopolysaccharides.

Urinary tract infections (UTI) affect a large proportion of the population, causing among other symptoms, more frequent and urgent micturition. Previous studies reported that the gram-negative bacterial wall component lipopolysaccharides (LPS) trigger acute epithelial and bladder voiding responses, but the underlying mechanisms remain unknown. The cation channel TRPV4 is implicated in the regulation of the bladder voiding. Since TRPV4 is activated by LPS in airway epithelial cells, we sought to determine whether this channel plays a role in LPS-induced responses in urothelial cells (UCs). We found that human-derived UCs display a fast increase in intracellular Ca2+ concentration upon acute application of Escherichia coli LPS. Such responses were detected also in freshly isolated mouse UCs, and found to be dependent on TRPV4, but not to require the canonical TLR4 signaling pathway of LPS detection. Confocal microscopy experiments revealed that TRPV4 is dispensable for LPS-induced nuclear translocation of NF-κB in mouse UCs. On the other hand, quantitative RT PCR determinations showed an enhanced LPS-induced production of proinflammatory cytokines in TRPV4-deficient UCs. Cystometry experiments in anesthetized wild type mice revealed that acute intravesical instillation of LPS rapidly increases voiding frequency. This effect was not observed in TRPV4-deficient animals, but was largely preserved in Tlr4 KO and Trpa1 KO mice. Our results suggest that activation of TRPV4 by LPS in UCs regulates the proinflammatory response and contributes to LPS-induced increase in voiding frequency. These findings further support the concept that TRP channels are sensors of LPS, mediating fast innate immunity mechanisms against gram-negative bacteria.

Molecular cloning

Transcription factor c-Jun is a member of AP-1 transcription complex that can be induced by various pathogens and plays a broad regulatory role in vertebrate immune response. In this study, the complete c-Jun cDNA of large yellow croaker Larimichthys crocea (Lcc-Jun) was cloned, whose open reading frame (ORF) is 984 bp long and encodes a protein of 327 amino acids (aa). The deduced Lcc-Jun protein contains three highly conserved domains, a transactivation domain (TAD, Met1-His118), a DNA binding domain (DBD, Lys218-Arg243), and a Leucine zipper domain (LZD, Leu271-Leu299), as found in other specie c-Jun. Lcc-Jun was constitutively expressed in all examined tissues, with the higher levels in blood, heart, and head kidney. Its transcripts were not only induced in spleen and head kidney by poly (I: C) or LPS, but also up-regulated in primary head kidney leukocytes (PKL), macrophages (PKM), and granulocytes (PKG), suggesting that Lcc-Jun may be involved in immune responses induced by poly (I: C), a viral mimic, and LPS, a Gram-negative bacterial component. Overexpression of Lcc-Jun in PKL increased the expression of cytokines and transcription factors involved in T helper 1 (Th1: TNF-α, IFN-γ, and T-bet) and Th2 (IL-4/13 A/B, IL-6, and GATA3) cell development and differentiation, suggesting that Lcc-Jun may play a role in regulation of Th1/Th2 cell response. These results therefore led us to suggest that the c-Jun-mediated signaling pathways may have an important immune-modulatory function in teleost fish.

An MD2-derived peptide promotes LPS aggregation, facilitates its internalization in THP-1 cells, and inhibits LPS-induced pro-inflammatory responses.

MD2, a 160-residue accessory glycoprotein, is responsible for the recognition and binding of Gram-negative bacterial membrane component, lipopolysaccharide (LPS). Internalization of pathogen inside the mononuclear phagocytes has also been attributed to MD2 which leads to the clearance of pathogens from the host. However, not much is known about the segments in MD2 that are responsible for LPS interaction or internalization of pathogen inside the defense cells. A 16-residue stretch (MD54) from MD2 protein has been identified that possesses a short heptad repeat sequence and four cationic residues enabling it to participate in both hydrophobic and electrostatic interactions with LPS. An MD54 analog of the same size was also designed in which a leucine residue at a heptadic position was replaced with an alanine residue. MD54 but not its analog, MMD54 induced aggregation of LPS and aided in its internalization within THP-1 monocytes. Furthermore, MD54 inhibited LPS-induced nuclear translocation of NF-κB in PMA-treated THP-1 and TLR4/MD2/CD14-transfected HEK-293Tcells and the production of pro-inflammatory cytokines. In addition, in in vivo experiments, MD54 showed marked protection and survival of mice against LPS-induced inflammation and death. Overall, we have identified a short peptide with heptad repeat sequence from MD2 that can cause aggregation of LPS and abet in its internalization within THP-1 cells, resulting in attenuation of LPS-induced pro-inflammatory responses in vitro and in vivo.

The NLRP3 inflammasome modulates sleep and NREM sleep delta power induced by spontaneous wakefulness, sleep deprivation and lipopolysaccharide

Both sleep loss and pathogens can enhance brain inflammation, sleep, and sleep intensity as indicated by electroencephalogram delta (δ) power. The pro-inflammatory cytokine interleukin-1 beta (IL-1β) is increased in the cortex after sleep deprivation (SD) and in response to the Gram-negative bacterial cell-wall component lipopolysaccharide (LPS), although the exact mechanisms governing these effects are unknown. The nucleotide-binding domain and leucine-rich repeat protein-3 (NLRP3) inflammasome protein complex forms in response to changes in the local environment and, in turn, activates caspase-1 to convert IL-1β into its active form. SD enhances the cortical expression of the somnogenic cytokine IL-1β, although the underlying mechanism is, as yet, unidentified. Using NLRP3-gene knockout (KO) mice, we provide evidence that NLRP3 inflammasome activation is a crucial mechanism for the downstream pathway leading to increased IL-1β-enhanced sleep. NLRP3 KO mice exhibited reduced non-rapid eye movement (NREM) sleep during the light period. We also found that sleep amount and intensity (δ activity) were drastically attenuated in NLRP3 KO mice following SD (homeostatic sleep response), as well as after LPS administration, although they were enhanced by central administration of IL-1β. NLRP3, ASC, and IL1β mRNA, IL-1β protein, and caspase-1 activity were greater in the somatosensory cortex at the end of the wake-active period when sleep propensity was high and after SD in wild-type but not NLRP3 KO mice. Thus, our novel and converging findings suggest that the activation of the NLRP3 inflammasome can modulate sleep induced by both increased wakefulness and a bacterial component in the brain.

Differential expression of Toll-like receptor genes (TLR2 and TLR4) across different tissues of riverine buffalo

Toll-like receptors (TLRs) are one of the important pattern recognizing receptors which are responsible for the induction of innate as well as adaptive immune response against a wide range of microbial components. Among different TLRs, TLR2 and TLR4 are expressed on the cell surface identifying Gram-positive and Gram-negative bacterial components. The present study was undertaken to analyze the expression of TLR2 and TLR4 genes in different buffalo tissues by using highly sensitive real-time PCR technique. Although the expression of both the genes was observed in all the 8 different tissues taken for the present study, the maximum expression of TLR2 was seen in blood followed by skin, lungs and spleen and the lowest expression was found in the uterus. TLR4 showed maximum expression in blood followed by skin, lungs, mammary gland of non-lactating, lactating buffalo and heifer and the lowest expression was seen again in the uterus.The findings suggest both the genes having important functions in blood and skin, the first line of protection for pathogenic challenge apart from other organs in buffalo.

Functional expression of TLR5 in murine salivary gland epithelial cells.

Toll-like receptors (TLR) recognize microbe-associated molecular patterns and induce the innate immune response. Among them, TLR5 recognizes the Gram-negative bacterial component flagellin. The aim of this study was to examine the expression of TLR5 in mouse salivary gland (SG). The SG was excised from 8- to 10-week-old female C57BL/6 mice. Salivary gland epithelial cells (SGECs) were purified and subjected to reverse transcription polymerase chain reaction (RT-PCR). Western blotting was performed to detect TLR5 expression at the protein level in several organs. The localization of TLR5 in SG was examined using immunohistochemical staining. The responsiveness of SGECs to flagellin was further examined by evaluating the induction of CXCL1 by real-time PCR and immunoprecipitation followed by Western blotting. TLR5 expression in SG was confirmed at the gene and protein levels. Immunohistochemical staining detected TLR5 in both acinic and ductal cells of the sublingual gland, but not in serous acinic cells of the submandibular gland. Although TLR5 was detected throughout the cytoplasm in ductal cells, positive staining was observed on the basal side of the mucous acinic cells. The purified SGECs responded to flagellin and induced the production of CXCL1. These findings suggest that TLR5 is functionally expressed in the SG and responds to its cognate ligand flagellin. (J Oral Sci 58, 317-323, 2016).

Abstract 3162: NOD1 augments cancer cell metastatic potential through p38 MAP kinase activation

Abstract INTRODUCTION: Strong clinical evidence demonstrates a link between acute bacterial infection and metastasis. We and others have shown that activation of certain membrane pattern recognition receptors (PRRs), such as Toll-like receptors, on cancer cells can be implicated in this process. There is emerging data that a cytoplasmic PRR, the nucleotide oligomerization domain receptor 1 (NOD1), may also play an important and complementary role in the immune response to bacterial infection, however its role in cancer progression is entirely unknown. We sought to determine the influence of NOD1 activation on metastasis. METHODS: NOD1 expression in human and murine colon (HT29, MC38) and lung (A549, H59) cancer cells were confirmed using flow cytometry (FC) and immunoblotting (WB). A series of in vitro and in vivo functional assays, including adhesion, migration and hepatic intravital microscopy (IVM), was conducted to assess the effect of NOD1 activation and inhibition. C12-iE-DAP, a highly selective NOD1 ligand derived from gram-negative bacterial wall, was used to simulate NOD1-activation under infectious condition. ML130, a specific NOD1 inhibitor, was used to block C12-iE-DAP activation. Stable knockdown (KD) of NOD1 in HT29 and A549 cells were constructed with shRNA lentiviral transduction and the functional assays were thus repeated. The predominant signaling pathway, downstream cytokines and cell adhesion molecules of NOD1-activation were identified using WB in the presence of kinase inhibitors. RESULTS: WB and FC showed abundant NOD1 expression in all tested cell lines. Cancer cells stimulated with C12-iE-DAP doubled the in vitro adhesion to collagen I, IV and fibronectin, as well as in vitro migration, an effect completely attenuated with ML130 inhibition and NOD1 knockdown. Using a murine hepatic adhesion assay under IVM, we observed a doubling in the amount of in vivo capture of cancer cells within hepatic sinusoids of C57BL6 mice in the C12-iE-DAP group compared to control, an effect again abrogated by ML130 inhibition and NOD1 knockdown. Immunoblotting using HT29 cells under a panel of kinase inhibitors revealed that NOD1 activation is p38 dependent. Subsequently, the use of p38 inhibitor, BIRB0796, abolished C12-iE-DAP mediated adhesion to collagen I and fibronectin. Using human cytokine quantification array, we determined that NOD1 activation led to increase in IL-8, 15, MCP1 and RANTES, all of which have been shown to link to cancer progression or to chemoattract neutrophils, which we have recently shown to mediate metastasis. CONCLUSION: Our data demonstrate that NOD1 activation by the gram-negative bacterial wall component, C12-iE-DAP is important in enhancing the metastatic potential of cancer cells, and its mechanism is dependent on p38 activation and the upregulation of downstream cytokines. This is the first study to implicate NOD1 in metastasis, and thus identify this receptor as a putative therapeutic target. Citation Format: Henry Jiang, Sara Najmeh, Julie Berube, Arielle Leone, Paul Savage, Betty Giannias, France Bourdeau, Simon Rousseau, Morag Park, Lorenzo Edwin Ferri. NOD1 augments cancer cell metastatic potential through p38 MAP kinase activation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3162. doi:10.1158/1538-7445.AM2015-3162