Nucleotide-binding Oligomerization Domain Receptors Research Articles

Topical N-phosphonacetyl-l-aspartate is a dual action candidate for treating non-melanoma skin cancer.

Each year, 3.3 million Americans are diagnosed with non-melanoma skin cancers (NMSC) and an additional 40 million individuals undergo treatment of precancerous actinic keratosis lesions. The most effective treatments of NMSC (surgical excision and Mohs surgery) are invasive, expensive and require specialised training. More readily accessible topical therapies currently are 5-fluorouracil (a chemotherapeutic agent) and imiquimod (an immune modulator), but these can have significant side effects which limit their efficacy. Therefore, more effective and accessible treatments are needed for non-melanoma cancers and precancers. Our previous work demonstrated that the small molecule N-phosphonacetyl-L-aspartate (PALA) both inhibits pyrimidine nucleotide synthesis and activates pattern recognition receptor nucleotide-binding oligomerization domain 2. We propose that topical application of PALA would be an effective NMSC therapy, by combining the chemotherapeutic and immune modulatory features of 5-fluorouracil and imiquimod. Daily topical application of PALA to mouse skin was well tolerated and resulted in less irritation, fewer histopathological changes, and less inflammation than caused by either 5-fluorouracil or imiquimod. In an ultraviolet light-induced NMSC mouse model, topical PALA treatment substantially reduced the numbers, areas and grades of tumours, compared to vehicle controls. This anti-neoplastic activity was associated with increased expression of the antimicrobial peptide cathelicidin and increased recruitment of CD8+ T cells and F4/80+ macrophages to the tumours, demonstrating both immunomodulatory and anti-proliferative effects. These findings indicate that topical PALA is an excellent candidate as an effective alternative to current standard-of-care NMSC therapies.

Long-term effect of Toll-like receptor-2, -4, -5, -7 and NOD2 stimulation on Na+,K+-ATPase activity and expression in intestinal epithelial cells.

Inappropriate activation of Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain receptors (NOD) is involved in many chronic disorders, including inflammatory bowel disease (IBD). Altered function and/or expression of Na+,K+-ATPase (NKA) and epithelial ion channels are the main cause of electrolyte absorption imbalance in patients with IBD, leading to diarrhea. We aimed to evaluate the effect of TLRs and NOD2 stimulation upon NKA activity and expression in human intestinal epithelial cells (IECs) using RT-qPCR, Western blot, and electrophysiology techniques. TLR2, TLR4, and TLR7 activation inhibited NKA activity [(means ± SE) -20.0 ± 1.2%, -34.0 ± 1.5%, and -24.5 ± 2.0% in T84 cells; and -21.6 ± 7.4%, -37.7 ± 3.5%, and -11.0 ± 2.3% in Caco-2 cells]. On the other hand, activation of TLR5 increased NKA activity (16.2 ± 2.9% in T84 and 36.8 ± 5.2% in Caco-2 cells) and β1-NKA mRNA levels (21.8 ± 7.8% in T84 cells). The TLR4 agonist synthetic monophosphoryl lipid A (MPLAs) reduced α1-NKA mRNA levels in both T84 and Caco-2 cells (-28.5 ± 3.6% and -18.7 ± 2.8%), and this was accompanied by a decrease in α1-NKA protein expression (-33.4 ± 11.8% and -39.4 ± 11.2%). NOD2 activation upregulated NKA activity (12.2 ± 5.1%) and α1-NKA mRNA levels (6.8 ± 1.6%) in Caco-2 cells. In summary, TLR2, TLR4, and TLR7 activation induce downregulation of NKA in IECs, whereas TLR5 and NOD2 activation has the opposite effect. A comprehensive understanding of the cross talk between TLRs, NOD2, and NKA is of utmost relevance for developing better IBD treatments.

From Co-Infections to Autoimmune Disease via Hyperactivated Innate Immunity: COVID-19 Autoimmune Coagulopathies, Autoimmune Myocarditis and Multisystem Inflammatory Syndrome in Children.

Neutrophilia and the production of neutrophil extracellular traps (NETs) are two of many measures of increased inflammation in severe COVID-19 that also accompany its autoimmune complications, including coagulopathies, myocarditis and multisystem inflammatory syndrome in children (MIS-C). This paper integrates currently disparate measures of innate hyperactivation in severe COVID-19 and its autoimmune complications, and relates these to SARS-CoV-2 activation of innate immunity. Aggregated data include activation of Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD) receptors, NOD leucine-rich repeat and pyrin-domain-containing receptors (NLRPs), retinoic acid-inducible gene I (RIG-I) and melanoma-differentiation-associated gene 5 (MDA-5). SARS-CoV-2 mainly activates the virus-associated innate receptors TLR3, TLR7, TLR8, NLRP3, RIG-1 and MDA-5. Severe COVID-19, however, is characterized by additional activation of TLR1, TLR2, TLR4, TLR5, TLR6, NOD1 and NOD2, which are primarily responsive to bacterial antigens. The innate activation patterns in autoimmune coagulopathies, myocarditis and Kawasaki disease, or MIS-C, mimic those of severe COVID-19 rather than SARS-CoV-2 alone suggesting that autoimmunity follows combined SARS-CoV-2-bacterial infections. Viral and bacterial receptors are known to synergize to produce the increased inflammation required to support autoimmune disease pathology. Additional studies demonstrate that anti-bacterial antibodies are also required to account for known autoantigen targets in COVID-19 autoimmune complications.

Changes in expression levels of Nod-like receptors in the spleen of ewes.

Nucleotide-binding oligomerization domain receptors (NOD-like receptors, NLRs) have critical effects on interfaces of the immune and reproductive systems, and the spleen plays a key role in both innate and adaptive immune functions. It is hypothesized that NLR family participates in maternal splenic immune regulation during early pregnancy in sheep. In this study, maternal spleens were collected on day 16 of the estrous cycle, and days 13, 16 and 25 of gestation (n = 6 for each group) in ewes. Expression of NLR family, including NOD1, NOD2, class II transactivator (CIITA), NLR family apoptosis inhibitory protein (NAIP), nucleotide-binding oligomerization domain, Leucine rich repeat and Pyrin domain containing 1 (NLRP1), NLRP3 and NLRP7, was analyzed using quantitative real-time PCR, Western blot and immunohistochemistry analysis. The results revealed that expression levels of NOD1, NOD2, CIITA and NLRP3 were downregulated at days 13 and 16 of pregnancy, but expression of NLRP3 was increased at day 25 of pregnancy. In addition, expression values of NAIP and NLRP7 mRNA and proteins were improved at days 16 and 25 of pregnancy, and NLRP1 was peaked at days 13 and 16 of pregnancy in the maternal spleen. Furthermore, NOD2 and NLRP7 proteins were limited to the capsule, trabeculae and splenic cords. In summary, early pregnancy changes expression of NLR family in the maternal spleen, which may be related with the maternal splenic immunomodulation during early pregnancy in sheep.

Inhibition of Bacterial Peptidoglycan Cytopathy byRetina Pigment Epithelial PGRP2 Amidase.

Peptidoglycan (PGN) recognition protein 2 (PGRP2; N-acetylmuramyl-L-alanine amidase (NAMAA)) activity in corneal epithelial cells is thought to inhibit corneal inflammation by reducing the PGN-induced cytokines. PGRP2 has not been reported in human retinal pigment epithelial (RPE) cells. RPE cell lysate NAMAA activity was measured densitometrically via cleavage of FITC-tagged muramyl dipeptide (FITCMDP). RPE lysate degradation of the cytopathic activity of nucleotide-binding oligomerization domain (NOD) receptor agonists was assessed by caspase-3 activation and DNA ladder detection and quantitation. PGRP2/NAMAA protein was detected in RPE cells by immunofluorescent antibody assay. RPE lysate NAMAA cleaved FITCMDP in a dose- and time-dependent manner. RPE lysate selectively inhibited PGN cytopathic activity of NOD1 agonists containing D-γ-glutamyl-meso-diaminopimelic acid and NOD2 containing L-alanyl-D-isoglutamine. The results suggest RPE PGRP2 amidase selectively degrades PGN that stimulate NOD-mediated cytopathic activity. The failure of RPE NAMAA to degrade pro-inflammatory PGN may play a role in bacterial retinopathies.

Modulation of Nod-like Receptor Expression in the Thymus during Early Pregnancy in Ewes.

Nucleotide-binding oligomerization domain receptors (NOD-like receptors, NLRs) are involved in modulating the innate immune responses of the trophoblast and the placenta in normal pregnancy. The thymus participates in regulation of innate and adaptive immune responses. However, it is unclear whether expression of NLR is modulated in the maternal thymus during early pregnancy. In this study, thymuses were sampled at day 16 of the estrous cycle, and at days 13, 16 and 25 of gestation (n = 6 for each group) from ewes after slaughter. Different stages were chosen because the maternal thymus was under the different effects of interferon-tau and/or progesterone or not. RT-qPCR, Western blot and immunohistochemistry analysis were used to analyze the expression of the NLR family, including NOD1; NOD2; major histocompatibility complex class II transactivator (CIITA); NLR family apoptosis inhibitory protein (NAIP); nucleotide-binding oligomerization domain and Leucine-rich repeat and Pyrin domain containing protein 1 (NLRP1), NLRP3 and NLRP7. The results showed that expression level of NOD1 was changed with the pregnancy stages, and expression levels of NOD2, CIITA, NAIP, NLRP1, NLRP3 and NLRP7 mRNA and proteins were peaked at day 13 of pregnancy. The levels of NOD2 and CIITA were increased during early pregnancy. The stainings for NOD2 and NLRP7 proteins were located in epithelial reticular cells, capillaries and thymic corpuscles. In summary, pregnancy stages changed expression of NLR family in the maternal thymus, which may be related to the modulation of maternal thymic immune responses, and beneficial for normal pregnancy in sheep.

Negative regulator NLRC3: Its potential role and regulatory mechanism in immune response and immune-related diseases.

NLRC3 is a member of the pattern recognition receptors nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) family, and plays a pivotal regulatory role in modulating the activation of immune cells. In macrophages, NLRC3 inhibits the activation of the NF-κB signaling pathway, the STING/TBK1 signaling pathway, and the formation of the inflammasome. In the context of T cells immune response, NLRC3 prevents the activation of T cells by regulating the function of dendritic cells and directly influencing the function of T cells. Different from other pattern recognition receptors, NLRC3 is more closely associated with regulatory activity than pathogens recognition, it influences the fates of cells, for example, prevents proliferation, promotes apoptosis and inhibits pyroptosis. These cellular functions regulated by NLRC3 are involved in the development processes of a variety of diseases, such as infectious disease, sterile inflammatory diseases, and cancer. However, its characteristics, function and regulatory mechanism in immune response and immune-related diseases have not been addressed fully. In this review, we elaborate the potential roles of NLRC3 from several different levels, include molecular mechanism, cellular functions in the immune-related diseases.

Neuroprotective Effects of the Pannexin-1 Channel Inhibitor: Probenecid on Spinal Cord Injury in Rats.

Proinflammatory immune cell subsets constitute the majority in the local microenvironment after spinal cord injury (SCI), leading to secondary pathological injury. Previous studies have demonstrated that inflammasomes act as an important part of the inflammatory process after SCI. Probenecid, an inhibitor of the Pannexin-1 channel, can inhibit the activation of inflammasomes. This article focuses on the effects of probenecid on the local immune microenvironment, histopathology, and behavior of SCI. Our data show that probenecid inhibited the expression and activation of nucleotide-binding oligomerization domain receptor pyrindomain-containing 1 (NLRP1), apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1, interleukin-1β (IL-1β), and caspase-3 proteins associated with inflammasomes, thereby suppressing the proportion of M1 cells. And consequently, probenecid reduced the lesion area and demyelination in SCI. Moreover, the drug increased the survival of motor neurons, which resulted in tissue repair and improved locomotor function in the injured SC. Altogether, existing studies indicated that probenecid can alleviate inflammation by blocking Pannexin-1 channels to inhibit the expression of caspase-1 and IL-1β, which in turn restores the balance of immune cell subsets and exerts neuroprotective effects in rats with SCI.

Chaperoning mechanism of innate immune receptor NOD2 by HSP70

Host pattern‐recognition receptors (PRRs) are the frontline members of the innate immune system. They are responsible for recognizing molecular structures derived from bacteria or damaged cells and producing appropriate innate immune responses against potential threats from pathogenic agents. However, their defective forms can lead to the development of inflammatory diseases. Crohn’s disease (CD), a prevalent and incurable inflammatory bowel disease, has been shown to closely link to three polymorphisms of the intracellular innate receptor nucleotide‐binding oligomerization domain containing 2 (NOD2). Biophysical and biochemical data show that CD‐associated NOD2 mutants have a much shorter half‐life and reduced signaling function compared to the wild‐type, contributing to the downstream chronic inflammation condition found in CD patients. Given NOD2’s involvement in disease development, it has become an attractive target for drug development against CD. Besides the existing effort in developing anti‐inflammatory drugs and immune suppressors, a novel approach focuses on designing peptidomimetics targeting NOD2 based on its positive modulator protein ‐ stress‐induced heat shock protein 70 kDa (HSP70). Overexpression of both full‐length and truncated HSP70 in cells stabilizes NOD2 mutants and rescues their function, indicating that HSP70 can be used as a platform for developing NOD2 peptide agonists. We first determined the direct interactions at a low micromolar affinity between HSP70 and NOD2 by surface plasmon resonance (SPR) and biolayer interferometry (BLI) technologies. The optimized binding assays will further allow screening of potential HSP70‐derived peptides that retain the same affinity for NOD2. To identify the interacting regions on the amino acid level, we will utilize cross‐linking mass spectrometry (XLMS) together with different docking algorithms. The results will give us a glance into the regulatory mechanisms of HSP70 on NOD2 and provide beneficial structural information for structure‐based peptide drug design. In the future, we plan to study the effects of HSP70‐NOD2 interactions on NOD2‐specific immune responses via RNA sequencing and RT‐PCR.

IDF21-0092 Influence of the NLRP3 inflammasome in podocytes on the pathogenesis of diabetes dependent hyperfiltration-induced CKD

Background: Diabetic nephropathy (DN) is one of the most severe complications of Type 1 diabetes mellitus (T1) that is accounting for about 40% of end-stage renal disease (ESRD). The pathology is seen to correlate with inflammation. Amongst others, pattern recognition receptors are important mediators of disease progression, e.g. nucleotide-binding oligomerization domain receptors, together with inflammasomes. Especially the Nod-like receptor protein 3 (NLRP3) inflammasome multiprotein complex seems to be a key mediator in DN. The assembly of this promotes the maturation of proinflammatory cytokines (canonical pathway) such as IL-1β and IL-18. It is assumed that there are non-canonical pathways, that trigger innate inflammatory response. Whereas the role of NLRP3 in specialized immune cells is well understood, its contribution to processes of disease and homeostasis in renal parenchymal cells is controversially discussed in literature. Aim: With this project we expect to get a better understanding of the role of canonical and non-canonical NLRP3 signaling in podocytes. Furthermore, we aim to investigate if an inhibition of the NLRP3 signaling may lead to an ameliorated or even absent hyperfiltration-induced CKD. Method: To verify, if the NLRP3 inflammasome is involved in canonical, proinflammatory signaling, primary human podocytes derived from renal progenitor cells were stimulated in vitro. By using different concentrations of LPS, ATP, glucose and nigericin, metabolic stress was imitated. IL-1β and IL-18 ELISAs were used to verify canonical pathway activation. We further introduced podocyte-specific NLRP3 inflammasome modulations and Streptozotocin dependent diabetes in mice with a NLRP3 overactive variant (Nlrp3A350V fl/wt) to investigate, if NLRP3 overactivation leads to an aggravated phenotype of hyperfiltration-induced CKD. By using pharmaceuticals such as Oridonine and beta-Hydroxybutyrate we examined, if the inhibition of NLRP3 signaling in C57BL/6J wildtype mice with diabetes leads to an ameliorated outcome. To investigate if it is possible to reverse diabetes dependent hyperfiltration-induced CKD in NLRP3 overactive mice, we treated them with beta-Hydroxybutyrate. The primary endpoint of all in vivo experiments was defined as urinary albumin-creatinine-ratio. Furthermore, relevant histopathological alterations were observed. Results: Our data suggest that human derived podocytes are not susceptible for metabolic stress dependent canonical NLRP3 inflammasome signaling compared with THP-1 + PMA cells. Even though mice presented with severe diabetes and polyuria, we did not observe a significant difference in mice with NLRP3 overactivation regarding urinary albumin excretion and histological changes. So far, also the treatment with NLRP3 inhibitors did not ameliorate hyperfiltration-induced CKD. Discussion: Investigation of the NLRP3 functionality in podocytes provided a deeper insight and better understanding of the pathogenesis of diabetes dependent hyperfiltration-induced CKD. Even though it might have been possible that the specific inhibition of the inflammasome associated proteins should have synergistic effects in inflammation in ESRD and might be a future therapeutic target beyond IL-1β inhibition, our data do not support this hypothesis.

Bacillus-Based Direct-Fed Microbial Reduces the Pathogenic Synergy of a Coinfection with Salmonella enterica Serovar Choleraesuis and Porcine Reproductive and Respiratory Syndrome Virus.

ABSTRACTViral respiratory infections predispose lungs to bacterial coinfections causing a worse outcome than either infection alone. Porcine reproductive and respiratory syndrome virus (PRRSV) causes pneumonia in pigs and is often associated with bacterial coinfections. We examined the impact of providing weanling pigs a Bacillus-based direct-fed microbial (DFM) on the syndrome resulting from infection with either Salmonella enterica serotype Choleraesuis alone, or in combination with PRRSV. Nine days after the bacterial challenge, Salmonella was isolated from ileocecal lymph nodes of all challenged pigs regardless of DFM treatment. Compared to the single bacterial challenge, the dual challenge with Salmonella and PRRSV resulted in a pathogenic synergy exhibited by a higher rate of Salmonella colonization in the lung and a more extensive and severe interstitial pneumonia. Provision of DFM to dually challenged pigs reduced the rate of lung colonization by Salmonella, eliminated or reduced the presence of PRRSV in the lung, and reduced the extent and severity of gross lung pathology. Dually challenged pigs that received DFM had increased concentrations of interleukin 1 (IL-1) and IL-8 in lung lavage fluids, accompanied by increased expression in their blood cells of nucleotide-binding oligomerization domain receptor 2 (NOD2) and triggering receptor expressed in myeloid cells 1 (TREM-1) molecules. These changes in pulmonary inflammatory cytokine production and increased expression of NOD2 and TREM-1 suggest that the DFM exerted a systemic modulating effect on innate immunity. These observations are consistent with the notion that tonic stimulation by gut-derived microbial products can poise innate immunity to fight infections in the respiratory tract.

Type I Interferons Are Involved in the Intracellular Growth Control of Mycobacterium abscessus by Mediating NOD2-Induced Production of Nitric Oxide in Macrophages.

Mycobacterium abscessus (MAB) is one of the rapidly growing, multidrug-resistant non-tuberculous mycobacteria (NTM) causing various diseases including pulmonary disorder. Although it has been known that type I interferons (IFNs) contribute to host defense against bacterial infections, the role of type I IFNs against MAB infection is still unclear. In the present study, we show that rIFN-β treatment reduced the intracellular growth of MAB in macrophages. Deficiency of IFN-α/β receptor (IFNAR) led to the reduction of nitric oxide (NO) production in MAB-infected macrophages. Consistently, rIFN-β treatment enhanced the expression of iNOS gene and protein, and NO production in response to MAB. We also found that NO is essential for the intracellular growth control of MAB within macrophages in an inhibitor assay using iNOS-deficient cells. In addition, pretreatment of rIFN-β before MAB infection in mice increased production of NO in the lungs at day 1 after infection and promoted the bacterial clearance at day 5. However, when alveolar macrophages were depleted by treatment of clodronate liposome, rIFN-β did not promote the bacterial clearance in the lungs. Moreover, we found that a cytosolic receptor nucleotide-binding oligomerization domain 2 (NOD2) is required for MAB-induced TANK binding kinase 1 (TBK1) phosphorylation and IFN-β gene expression in macrophages. Finally, increase in the bacterial loads caused by reduction of NO levels was reversed by rIFN-β treatment in the lungs of NOD2-deficient mice. Collectively, our findings suggest that type I IFNs act as an intermediator of NOD2-induced NO production in macrophages and thus contribute to host defense against MAB infection.

TLR or NOD receptor signaling skews monocyte fate decision via distinct mechanisms driven by mTOR and miR-155

Monocytes are rapidly recruited to inflamed tissues where they differentiate into monocyte-derived macrophages (mo-mac) or dendritic cells (mo-DC). At infection sites, monocytes encounter a broad range of microbial motifs. How pathogen recognition impacts monocyte fate decision is unclear. Here, we show, using an in vitro model allowing the simultaneous differentiation of human mo-mac and mo-DC, that viruses promote mo-mac while Mycobacteria favor mo-DC differentiation. Mechanistically, we found that pathogen sensing through toll-like receptor (TLR) ligands increases mo-mac differentiation via mTORC1. By contrast, nucleotide-binding oligomerization domain (NOD) ligands favor mo-DC through the induction of TNF-α secretion and miR-155 expression. We confirmed these results in vivo, in mouse skin and by analyzing transcriptomic data from human individuals. Overall, our findings allow a better understanding of the molecular control of monocyte differentiation and of monocyte plasticity upon pathogen sensing.

IFN-γ Manipulates NOD1-Mediated Interaction of Autophagy and Edwardsiella piscicida to Augment Intracellular Clearance in Fish

Edwardsiella piscicida is an intracellular pathogenic bacterium accounting for significant losses in farmed fish. Currently, cellular and molecular mechanisms underlying E. piscicida-host cross-talk remain obscure. In this study, we revealed that E. piscicida could increase microtubule-associated protein L chain 3 (LC3) puncta in grass carp (Ctenopharyngodon idella) monocytes/macrophages and a carp cell line, Epithelioma papulosum cyprini The autophagic response was confirmed by detecting the colocalization of E. piscicida with LC3-positive autophagosomes and LysoTracker-probed lysosomes in the cells. Moreover, we unveiled the autophagic machinery targeting E. piscicida by which the nucleotide-binding oligomerization domain receptor 1 (NOD1) functioned as an intracellular sensor to interact and recruit autophagy-related gene (ATG) 16L1 to the bacteria. Meanwhile, E. piscicida decreased the mRNA and protein levels of NOD1 and ATG16L1 in an estrogen-related receptor-α-dependent manner, suggesting a possible mechanism for this bacterium escaping autophagy. Subsequently, we examined the effects of various E. piscicida virulence factors on NOD1 expression and found that two of them, EVPC and ESCB, could reduce NOD1 protein expression via ubiquitin-dependent proteasomal degradation. Furthermore, an intrinsic regulator IFN-γ was found to enhance the colocalization of E. piscicida with NOD1 or autophagosomes, suggesting its involvement in the interaction between autophagy and E. piscicida Along this line, a short-time treatment of IFN-γ caused intracellular E. piscicida clearance through an autophagy-dependent mechanism. Collectively, our works demonstrated NOD1-mediated autophagy-E. piscicida dialogues and uncovered the molecular mechanism involving autophagy against intracellular bacteria in fish.

1161-P: NOD1-JNK1 Axis Plays a Causal Role in Palmitate-Induced ß-Cell Dysfunction

Chronic elevation of free fatty acids (FFA) in obesity may cause ß-cell dysfunction leading to type 2 diabetes (T2D). Recent studies have focused on innate immunity receptors due to evidence implicating inflammation in obesity-induced T2D. While several of these receptors have been implicated in FFA-induced ß-cell dysfunction, the nucleotide-binding oligomerization domain (NOD) receptors have not been investigated. Our previous work showed that whole-body NOD1-null mice were protected from palmitate-induced ß-cell dysfunction in vivo. This may be due to lack of NOD1 in ß-cells and/or macrophages infiltrating islets. Also, islets of whole-body NOD1-null and JNK1-null mice were protected from ß-cell dysfunction induced by palmitate but not oleate in vitro, suggesting NOD1 activation is likely in ß-cells, is specific to saturated FFA and may cause ß-cell dysfunction via JNK1. Thus, we hypothesized that ß-cell NOD1 mediates palmitate-induced ß-cell dysfunction in vivo, and that JNK1 is downstream of NOD1 activation. Addition of an ethyl group on palmitate reduces toxicity of unbound palmitate, thus ethylpalmitate (EtPAL) was used. Ethyl esterases in mouse plasma hydrolyze EtPAL into palmitate and ethanol. ß-cell specific NOD1-null (ßKO) mice (using Ins1- and RIP-Cre) and their controls were infused with EtPAL or ethanol vehicle (Veh) for 48 hours, followed by a hyperglycemic clamp to evaluate ß-cell function in vivo from the disposition index (DI). Islets of JNK1-null and wildtype (WT) mice were isolated and treated with a NOD1 activator to investigate the NOD1-JNK1 axis. As expected, EtPAL infusion elevated plasma FFA. EtPAL decreased DI in control mice, while EtPAL-infused ßKO mice had similar DI to Veh-infused mice. Further, the NOD1 activator reduced insulin secretion in WT islets but not in JNK1-null islets. These data implicate ß-cell NOD1-JNK1 axis in palmitate-induced ß-cell dysfunction and reveal a novel pathway that could be targeted for manipulation in order to preserve ß-cell function. Disclosure J. Yung: None. A. Ivovic: None. I. Clark: None. F. Li: None. B. Lin: None. Y. Tan: None. A. Giacca: None. Funding Canadian Institutes of Health Research (CIHR50748)

NLRP3 Inflammasome and Its Critical Role in Gynecological Disorders and Obstetrical Complications.

Inflammasomes, intracellular, multimeric protein complexes, are assembled when damage signals stimulate nucleotide-binding oligomerization domain receptors (NLRs). Several inflammasomes have been reported, including the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), NLRP1, NLRP7, ice protease-activating factor (IPAF), absent in melanoma 2 (AIM2) and NLR family CARD domain-containing protein 4 (NLRC4). Among these inflammasomes, the NLRP3 inflammasome is the most well-studied in terms of structure and function. Unlike other inflammasomes that can only be activated by a finite number of pathogenic microorganisms, the NLRP3 inflammasome can be activated by the imbalance of the internal environment and a large number of metabolites. The biochemical function of NLRP3 inflammasome is to activate cysteine-requiring aspartate proteinase-1 (caspase-1), which converts pro-IL-1β and pro-IL-18 into their active forms, namely, IL-1β and IL-18, which are then released into the extracellular space. The well-established, classic role of NLRP3 inflammasome has been implicated in many disorders. In this review, we discuss the current understanding of NLRP3 inflammasome and its critical role in gynecological disorders and obstetrical complications.

Synthesis of Conformationally Constrained d-Glu-meso-DAP Analogs as Innate Immune Agonists.

The dipeptide d-Glu-meso-DAP (iE-DAP) is the minimal structural fragment capable of activating the innate immune receptor nucleotide-binding oligomerization domain protein (NOD1). The meso-diaminopimelic acid (meso-DAP) moiety is known to be very stringent in terms of the allowed structural modifications which still retain the NOD1 activity. The aim of our study was to further explore the chemical space around the meso-DAP portion and provide a deeper understanding of the structural features required for NOD1 agonism. In order to achieve the rigidization of the terminal amine functionality of meso-DAP, isoxazoline and pyridine heterocycles were introduced into its side-chain. Further, we incorporated the obtained meso-DAP mimetics into the structure of iE-DAP. Collectively, nine innovative iE-DAP derivatives additionally equipped with lauroyl or didodecyl moieties at the α-amino group of d-Glu have been prepared and examined for their NOD1 activating capacity. Overall, the results obtained indicate that constraining the terminal amino group of meso-DAP abrogates the compounds’ ability to activate NOD1, since only compound 6b retained noteworthy NOD1 agonistic activity, and underpin the stringent nature of this amino acid with regard to the allowed structural modifications.

NOD1/2 and the C-Type Lectin Receptors Dectin-1 and Mincle Synergistically Enhance Proinflammatory Reactions Both In Vitro and In Vivo.

PurposePathogens consist of a wide variety of evolutionarily conserved molecular structures that are recognized by pattern recognition receptors (PRRs) of innate immunity. Reasonably assuming that no single PRR is ever likely to be the sole trigger of the immune response during infection, a great deal remains unknown about collaborative mechanisms and consequential crosstalk effects between multiple PRRs belonging to different families. Here, we aimed to investigate inflammatory response to combined stimulation of cytosolic nucleotide-binding oligomerization domain (NOD) receptors: NOD1, NOD2 and membrane-bound C-type lectin receptors (CLRs): Mincle and Dectin-1 in comparison to individual stimulation both in vitro and in vivo.Materials and MethodsFor in vitro studies, we used human monocytic THP-1 cells endogenously expressing NOD1,2, as well as Mincle and Dectin-1 receptors. Using reporter gene and immunoassay approaches, we measured activity of key proinflammatory transcription factors (NF-κB and AP-1) and cytokine production after addition of specific PRR agonists or their pairwise combinations. In vivo NF-κB activity (bioluminescent detection in NF-κB-Luc transgenic mice), as well as cytokine levels in mouse blood serum, was measured 3 hours after intramuscular injection of PRR agonists.ResultsWe detected that combined stimulation of NOD1/2 and C-type lectin receptors (Dectin-1, Mincle) strongly potentiates NF-κB and AP-1 transcription factor activity in human monocytic THP-1 cells, as well as resulting in enhanced levels of IL-8 cytokine production. We demonstrated that RIP2- and Syk-dependent signaling pathways downstream of NOD1/2 and Dectin-1/Mincle, respectively, are essential for the potentiated proinflammatory cell response. Lastly, we confirmed that synergy between NOD and C-type lectin receptors resulting in potentiated levels of NF-κB activation and cytokine (IL-6, KC) production also occurs in vivo.ConclusionThese findings originally indicate cooperation between NODs and CLRs, leading to potentiated levels of proinflammatory immune response both in vitro and in vivo.

Risk factors for aggressive recurrent respiratory papillomatosis in Chinese juvenile patients

Background: Aggressive juvenile-onset recurrent respiratory papillomatosis (JORRP) threatens patient lives if not receives immediate surgical intervene. Even with surgical intervene, complete remission of this disease is not approachable. Therefore, understanding the factors relevant to disease severity and prognosis will do help to the treatment strategy and health management of this disease.Objective: This study aimed to explore the clinic, laboratory and socioeconomic characteristics of patients and evaluate the risk factors for aggressive JORRP.Methods: The information of clinical and socioeconomic status of the patients was reviewed and its association with disease severity was analyzed. Papilloma from JORRP patients undergone surgeries was used to determine HPV subtypes by real-time PCR. The profiles of mRNA expression in the papilloma were assessed using microarray.Results: Age at diagnosis and socioeconomic status were shown to associate with the severity of JORRP. There was no differential severity considering different HPV subtype. The mRNA expression of nucleotide binding oligomerization domain receptor protein 3 (NLRP3) and Gasdermin B (GSDMB) was reduced in papillomas.Conclusions: A younger age at diagnosis and low socioeconomic status were associated with the severity of JORRP. mRNA expression of NLRP3 and GSDMB in the papillomas of JORRP patients was significantly reduced.Abbreviation: JORRP: Juvenile-onset recurrent respiratory papillomatosis; RRP: Recurrent respiratory papillomatosis; OSAS: Obstructive sleep apnea syndrome; NLRP3: Nucleotide binding oligomerization domain receptor protein 3; GSDMB: Gasdermin B.

Pectin Interaction with Immune Receptors is Modulated by Ripening Process in Papayas

Dietary fibers have been shown to exert immune effects via interaction with pattern recognition receptors (PRR) such as toll-like receptors (TLR) and nucleotide-binding oligomerization domain (NOD)-like receptors. Pectin is a dietary fiber that interacts with PRR depending on its chemical structure. Papaya pectin retains different chemical structures at different ripening stages. How this influence PRR signaling is unknown. The aim of this work was to determine how ripening influences pectin structures and their ability to interact with TLR2, 3, 4, 5 and 9, and NOD1 and 2. It was evaluated the interaction of the water-soluble fractions rich in pectin extracted from unripe to ripe papayas. The pectin extracted from ripe papayas activated all the TLR and, to a lesser extent, the NOD receptors. The pectin extracted from unripe papayas also activated TLR2, 4 and 5 but inhibited the activation of TLR3 and 9. The differences in pectin structures are the higher methyl esterification and smaller galacturonan chains of pectin from ripe papayas. Our finding might lead to selection of ripening stages for tailored modulation of PRR to support or attenuate immunity.