A closer look at ligand specificity for cellular activation of NOD2 with synthetic muramyl dipeptide analogues.

To further understand the specificity of muramyl dipeptide (MDP) sensing by NOD2, we evaluated the compatibility of synthetic MDP analogues for cellular uptake and NAGK phosphorylation, the pre-requisite steps of intracellular NOD2 activation. Our results revealed that these two prior steps do not confer ligand stereoselectivity; yet NAGK strictly discriminates against the disaccharide NOD2 agonists for phosphorylation in vitro, despite it being indispensable for the cellular NOD2-stimulating effects of these analogues, implying potential glycosidase cleavage as a novel intermediate step for cellular activation of NOD2.

Dietary fat differentially modulates the response of bone marrow-derived macrophages to TLR4 and NOD2 agonists.

Consumption of diets high in fat has been linked to the development of obesity and related metabolic complications. Such associations originate from the enhanced, chronic, low-grade inflammation mediated by macrophages in response to translocated bacteria, bacterial products, or dietary constituents such as fatty acids (FAs). Nucleotide-binding Oligomerization Domain 2 (NOD2) senses muramyl dipeptide (MDP), a component of bacterial peptidoglycan. The inability to sense peptidoglycan through NOD2 has been demonstrated to lead to dysbiosis, increased bacterial translocation, inflammation and metabolic dysfunction. Currently, it is unknown how consumption of HFDs with different FA compositions might influence NOD2-dependent responses. In this study, we subjected WT mice to a control diet or to HFDs comprised of various ratios of unsaturated to saturated fats and determined the macrophage response to TLR4 and NOD2 agonists. A HFD with equal ratios of saturated and unsaturated fats enhanced subsequent responsiveness of macrophages to LPS but not to MDP. However, a high-unsaturated fat diet (HUFD) or a high-saturated fat diet (HSFD) both decreased the responsiveness to NOD2 agonists compared to that observed in control diet (CD) fed mice. These data suggest that dietary fatty acid composition can influence the subsequent macrophage responsiveness to bacterial products.

Inhibition of Bacterial Peptidoglycan Cytopathy by Retina 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.

NOD2 in monocytes negatively regulates macrophage development through TNFalpha.

Objective: It is believed that intestinal recruitment of monocytes from Crohn's Disease (CD) patients who carry NOD2 risk alleles may repeatedly give rise to recruitment of pathogenic macrophages. We investigated an alternative possibility that NOD2 may rather inhibit their differentiation from intravasating monocytes. Design: The monocyte fate decision was examined by using germ-free mice, mixed bone marrow chimeras and a culture system yielding macrophages and monocyte-derived dendritic cells (mo-DCs). Results: We observed a decrease in the frequency of mo-DCs in the colon of Nod2-deficient mice, despite a similar abundance of monocytes. This decrease was independent of the changes in the gut microbiota and dysbiosis caused by Nod2 deficiency. Similarly, the pool of mo-DCs was poorly reconstituted in a Nod2-deficient mixed bone marrow (BM) chimera. The use of pharmacological inhibitors revealed that activation of NOD2 during monocyte-derived cell development, dominantly inhibits mTOR-mediated macrophage differentiation in a TNFα-dependent manner. These observations were supported by the identification of a TNFα-dependent response to muramyl dipeptide (MDP) that is specifically lost when CD14-expressing blood cells bear a frameshift mutation in NOD2. Conclusion: NOD2 negatively regulates a macrophage developmental program through a feed-forward loop that could be exploited for overcoming resistance to anti-TNF therapy in CD.

A probiotic bi-functional peptidoglycan hydrolase sheds NOD2 ligands to regulate gut homeostasis in female mice.

Secreted proteins are one of the direct molecular mechanisms by which microbiota influence the host, thus constituting a promising field for drug discovery. Here, through bioinformatics-guided screening of the secretome of clinically established probiotics from Lactobacillus, we identify an uncharacterized secreted protein (named LPH here) that is shared by most of these probiotic strains (8/10) and demonstrate that it protects female mice from colitis in multiple models. Functional studies show that LPH is a bi-functional peptidoglycan hydrolase with both N-Acetyl-ß-D-muramidase and DL-endopeptidase activities that can generate muramyl dipeptide (MDP), a NOD2 ligand. Different active site mutants of LPH in combination with Nod2 knockout female mice confirm that LPH exerts anti-colitis effects through MDP-NOD2 signaling. Furthermore, we validate that LPH can also exert protective effects on inflammation-associated colorectal cancer in female mice. Our study reports a probiotic enzyme that enhances NOD2 signaling in vivo in female mice and describes a molecular mechanism that may contribute to the effects of traditional Lactobacillus probiotics.

Design, synthesis and biological evaluation of novel lipophilic 2, 5-disubstituted tetrazole analogues of muramyl dipeptide as NOD2 agonists.

A focused library of six new 2, 5-disubstituted tetrazole (2, 5-DST) analogues of N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP) as potential immunomodulators were synthesized by the bioisosteric replacement of α-amide of d-isoglutamine with 5-substituted tetrazole (5-ST). Another parameter 'lipophilicity' was also considered to improve the pharmacological properties of MDP through the alkylation of 5-substituted tetrazole during synthesis. In total, six 2, 5-DST analogues of MDP were synthesized and bio-evaluated for the study of human NOD2 stimulation activity in the innate immune response. Interestingly, among the varied lengths of the alkyl chain in 2, 5-disubstituted tetrazole derivatives, the tetrazole analogues 12b bearing the -Butyl (C4) and 12c having -Octyl (C8) chain showed the best NOD2 stimulation potency equivalent with reference compound MDP. These analogues were evaluated for their adjuvanticity against dengue antigen and analogues 12b and 12c have elicited a potent humoral and cell mediated response.

Muramyl dipeptide CD10 monoclonal antibody immunoconjugates inhibited acute leukemia in nude mice.

Minimal residual disease (MRD) is one of the causes of leukemia recurrence. Previously, we developed anti-CD10 mAb conjugated to muramyl dipeptide immunoconjugate (MDP-Ab) for immune enhancement. The present study aimed to investigate anti-leukemia effect of MDP-Ab administered via different methods in leukemia ectopic graft nude mouse model. BALB/c nude mice were injected with Nalm-6 cells subcutaneously to establish leukemia xenografts in nude mice as a model. MDP-Ab or/and human lymphocytes (LYM) was injected into different sites of the nude mice. Immunohistochemistry staining of CDs in the bone marrow, liver and spleen was performed. IFN-γ was detected by ELISA. We detected the metastasis of leukemia cells to the liver, spleen and bone marrow in nude mouse leukemia model. MDP-Ab and LYM inhibited the growth of tumors, and simultaneous injection of MDP-Ab and LYM into the tumor inhibited the growth of tumors. IFN-γ levels in MDP-Ab (ca) + h-LYM (ca) group, MDP-Ab (ca) + h-LYM (ip) group, MDP-Ab (iv) + h-LYM (ip) group and PBS (ca) + h-LYM (ca) group were significantly higher than those in control group, while IFN-γ level in MDP-Ab (ca) + h-LYM (ca) group was the highest. Moreover, MDP-Ab and h-LYM promoted the expression of hCD4 and hCD8, with the highest expression in MDP-Ab (ca) + h-LYM (ca) group. In conclusion, MDP-Ab effectively promoted the production of IFN-γ, enhanced the antitumor immunity of T lymphocytes and inhibited leukemia.

Effects of a co-stimulation with S-PRG filler eluate and muramyl dipeptide (MDP) on matrix metalloproteinase-1 production by human dental pulp fibroblast-like cells.

The present study investigated the effects of a co-stimulation with surface reaction-type pre-reacted glass-ionomer (S-PRG) filler eluate and muramyl dipeptide (MDP) on matrix metalloproteinase (MMP)-1 production by human dental pulp fibroblast-like cells (hDPFs). S-PRG filler eluate contains 6 ions (F, Na, Al, B, Sr, and Si) released from S-PRG filler. Each S-PRG filler eluate and MDP stimulation enhanced MMP-1 production by hDPFs. The co-stimulation with S-PRG filler eluate and MDP enhanced MMP-1 production more than the MDP stimulation alone. A similar stimulation induced the phosphorylation of ERK 1/2. The increased secretion of MMP-1 and enhanced phosphorylation of ERK 1/2 by the co-stimulation with S-PRG filler eluate and MDP were suppressed by the selective and potent CaSR antagonist NPS 2143. Since strontium binds to CaSR, these results suggest that the enhanced production of MMP-1 by the co-stimulation with S-PRG filler eluate and MDP was due to the effects of strontium.

Muramyl dipeptide alleviates estrogen deficiency-induced osteoporosis through canonical Wnt signaling.

Wnt signaling is a positive regulator of bone formation through the induction of osteoblast differentiation and down-regulation of osteoclast differentiation. We previously reported that muramyl dipeptide (MDP) increases bone volume by increasing osteoblast activity and attenuating osteoclast activity in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoporotic model mice. In this study, we investigated whether MDP could alleviate post-menopausal osteoporosis through Wnt signaling regulation in an ovariectomy (OVX)-induced mouse osteoporosis model. MDP-administered OVX mice exhibited higher bone volume and bone mineral density than mice of the control group. MDP significantly increased P1NP in the serum of OVX mice, implying increased bone formation. The expression of pGSK3ß and ß-catenin in the distal femur of OVX mice was lower than that in the distal femur of sham-operated mice. Yet, the expression of pGSK3ß and ß-catenin was increased in MDP-administered OVX mice compared with OVX mice. In addition, MDP increased the expression and transcriptional activity of ß-catenin in osteoblasts. MDP inhibited the proteasomal degradation of ß-catenin via the down-regulation of its ubiquitination by GSK3ß inactivation. When osteoblasts were pretreated with Wnt signaling inhibitors, DKK1 or IWP-2, the induction of pAKT, pGSK3ß, and ß-catenin was not observed. In addition, nucleotide oligomerization domain-containing protein 2-deficient osteoblasts were not sensitive to MDP. MDP-administered OVX mice exhibited fewer tartrate-resistant acid phosphatase (TRAP)-positive cells than did OVX mice, attributed to a decrease in the RANKL/OPG ratio. In conclusion, MDP alleviates estrogen deficiency-induced osteoporosis through canonical Wnt signaling and could be an effective therapeutic for the treatment of post-menopausal bone loss. © 2023 The Pathological Society of Great Britain and Ireland.

Microbe-mediated intestinal NOD2 stimulation improves linear growth of undernourished infant mice.

The intestinal microbiota is known to influence postnatal growth. We previously found that a strain of Lactiplantibacillus plantarum (strain LpWJL) buffers the adverse effects of chronic undernutrition on the growth of juvenile germ-free mice. Here, we report that LpWJL sustains the postnatal growth of malnourished conventional animals and supports both insulin-like growth factor-1 (IGF-1) and insulin production and activity. We have identified cell walls isolated from LpWJL, as well as muramyl dipeptide and mifamurtide, as sufficient cues to stimulate animal growth despite undernutrition. Further, we found that NOD2 is necessary in intestinal epithelial cells for LpWJL-mediated IGF-1 production and for postnatal growth promotion in malnourished conventional animals. These findings indicate that, coupled with renutrition, bacteria cell walls or purified NOD2 ligands have the potential to alleviate stunting.

Conjunctival epitheliopathy induced by topical exposure to bacterial peptidoglycan, muramyl dipeptide.

Noninfectious exudative conjunctivitis can be experimentally produced in rabbits by application of the apoptogenic bacterial cell wall peptidoglycan, muramyl dipeptide (MDP) to the ocular surface. The purpose of this study was to investigate the acute conjunctival cytopathology induced by unilateral ocular surface exposure to MDP. Hematoxylin and eosin staining assessed bilateral tear cytopathology and conjunctival histopathology. The caspases levels in conjunctival tissue and tears were measured in standard assays utilizing p-nitroanaline tagged caspase-specific substrates. Immunofluorescent antibody identified intracellular caspase-3, nuclear factor-κß (NF-κß), and oxidative DNA damage (8-OHdG; 8-oxo-2'-deoxyguanosine) in tear and conjunctiva cells. DNA extracted from conjunctival tissues and pooled tear fluids were visualized by ethydium bromide agarose gel electrophoresis. Onset of ipsilateral conjunctivitis was due to an epitheliopathy characterized by loss of conjunctival epithelial cell adherence, exuviation of conjunctival epithelial cells, and neutrophil infiltration. Caspase-3 levels were significantly higher in exuviated cells in ipsilateral than contralateral tear (p's ≤ 0.001) collected at 3-5 h post MDP. Significantly higher caspase-2, -3, -6, -8 and -9 (p's ≤ 0.03) levels were detected in ipsilateral than contralateral conjunctival tissue at 5 h. Polymeric DNA was detected in ipsilateral but not contralateral conjunctival tissue and tears. Caspase-3, NF-κß, and 8-OHdG positive neutrophils were detected in bilateral conjunctiva and tear. The caspase-3/NF-κß epithelial cells and polymeric DNA in conjunctival tissue and shedding of caspase positive cells and polymeric DNA into ipsilateral tears support MDP induction of acute programmed cell death in vivo. The results suggest that ipsilateral exudative conjunctivitis is due to acute caspase-mediated conjunctival epitheliopathy induced by topical exposure to the bacterial peptidoglycan MDP.

A Protective Role of NOD2 on Oxazolone-induced Intestinal Inflammation Through IL-1ß-mediated Signalling Pathway.

BACKGROUND AND AIMS: NOD2 has emerged as a critical player in the induction of both Th1 and Th2 responses for potentiation and polarisation of antigen-dependent immunity. Loss-of-function mutations in the NOD2-encoding gene and deregulation of its downstream signalling pathway have been linked to Crohn's disease. Although it is well documented that NOD2 is capable of sensing bacterial muramyl dipeptide, it remains counter-intuitive to link development of overt intestinal inflammation to a loss of bacterial-induced inflammatory response. We hypothesised that a T helper bias could also contribute to an autoimmune-like colitis different from inflammation that is fully fledged by Th1 type cells. METHODS: An oedematous bowel wall with a mixed Th1/Th2 response was induced in mice by intrarectal instillation of the haptenating agent oxazolone. Survival and clinical scoring were evaluated. At several time points after instillation, colonic damage was assessed by macroscopic and microscopic observations. To evaluate the involvement of NOD2 in immunochemical phenomena, quantitative polymerase chain reaction [PCR] and flow cytometry analysis were performed. Bone marrow chimera experimentation allowed us to evaluate the role of haematopoietic/non-hematopoietic NOD2-expressing cells. RESULTS: Herein, we identified a key regulatory circuit whereby NOD2-mediated sensing of a muramyl dipeptide [MDP] by radio-resistant cells improves colitis with a mixed Th1/Th2 response that is induced by oxazolone. Genetic ablation of either Nod2 or Ripk2 precipitated oxazolone colitis that is predominantly linked to a lack of interferon-gamma. Bone marrow chimera experiments revealed that inactivation of Nod2 signalling in non-haematopoietic cells is causing a biased M1-M2 polarisation of macrophages and a decreased frequency of splenic regulatory T cells that correlates with an impaired activation of CD4 + T cells within mesenteric lymph nodes. Mechanistically, mice were protected from oxazolone-induced colitis upon administration of MDP in an interleukin-1- and interleukin-23-dependent manner. CONCLUSIONS: These findings indicate that Nod2 signalling may prevent pathological conversion of T helper cells for maintenance of tissue homeostasis.

Activation of nucleotide-binding oligomerization domain 2 by muramyl dipeptide negatively regulates Toll-like receptor 9-mediated colonic inflammation through the induction of deubiquitinating enzyme A expression.

Mutations in nucleotide-binding oligomerization domain 2 (NOD2) are associated with Crohn's disease (CD). Although NOD2 activation contributes to the maintenance of intestinal homeostasis through the negative regulation of pro-inflammatory cytokine responses mediated by Toll-like receptors (TLRs), the effects of NOD2 activation on interferon (IFN)-α responses induced by TLR9 have been poorly defined. To explore the cross-talk between NOD2 and TLR9, human monocytes or dendritic cells (DCs) were stimulated with NOD2 and/or TLR9 ligands to measure IFN-α production. The severity of dextran sodium sulfate (DSS)-induced colitis was compared in mice treated with NOD2 and/or TLR9 ligands. Expression of IFN-α and IFN-stimulated genes (ISGs) was examined in the colonic mucosa of patients with inflammatory bowel disease (IBD). NOD2 activation reduced TLR9-induced IFN-α production by monocytes and DCs in a deubiquitinating enzyme A (DUBA)-dependent manner. Activation of DUBA induced by the co-stimulation of TLR9 and NOD2 inhibited Lys63-linked polyubiquitination of TRAF3 and suppressed TLR9-mediated IFN-α production. NOD2 activation in hematopoietic cells protected mice from TLR9-induced exacerbation of DSS-induced colitis by down-regulating IFN-α responses and up-regulating DUBA expression. Colonic mucosa of patients with active and remitted IBD phases was characterized by the enhanced and reduced expression of ISGs, respectively. Expression levels of IFN-α and IL-6 positively correlated in the active colonic mucosa of patients with ulcerative colitis and CD, whereas DUBA expression inversely correlated with that of IFN-α in patients with CD. Collectively, these data suggest that DUBA-dependent negative effect of NOD2 on TLR9-mediated IFN-α responses contributes to the maintenance of intestinal homeostasis.

Two novel mollusk short-form ApeC-containing proteins act as pattern recognition proteins for peptidoglycan.

The Apextrin C-terminal (ApeC) domain is a new protein domain largely specific to aquatic invertebrates. In amphioxus, a short-form ApeC-containing protein (ACP) family is capable of binding peptidoglycan (PGN) and agglutinating bacteria via its ApeC domain. However, the functions of ApeC in other phyla remain unknown. Here we examined 130 ACPs from gastropods and bivalves, the first and second biggest mollusk classes. They were classified into nine groups based on their phylogenetics and architectures, including three groups of short-form ACPs, one group of apextrins and two groups of ACPs of complex architectures. No groups have orthologs in other phyla and only four groups have members in both gastropods and bivalves, suggesting that mollusk ACPs are highly diversified. We selected one bivalve ACP (CgACP1; from the oyster Crossostrea gigas) and one gastropod ACP (BgACP1; from the snail Biomphalaria glabrata) for functional experiments. Both are highly-expressed, secreted short-form ACPs and hence comparable to the amphioxus ACPs previously reported. We found that recombinant CgACP1 and BgACP1 bound with yeasts and several bacteria with different affinities. They also agglutinated these microbes, but showed no inhibiting or killing effects. Further analyses show that both ACPs had high affinities to the Lys-type PGN from S. aureus but weak or no affinities to the DAP-type PGN from Bacillus subtilis. Both recombinant ACPs displayed weak or no affinities to other microbial cell wall components, including lipopolysaccharide (LPS), lipoteichoic acid (LTA), zymosan A, chitin, chitosan and cellulose, as well as to several PGN moieties, including muramyl dipeptide (MDP), N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). Besides, CgACP1 had the highest expression in the gill and could be greatly up-regulated quickly after bacterial challenge. This is reminiscent of the amphioxus ACP1/2 which serve as essential mucus lectins in the gill. Taken together, the current findings from mollusk and amphioxus ACPs suggest several basic common traits for the ApeC domains, including the high affinity to Lys-type PGN, the bacterial binding and agglutinating capacity, and the role as mucus proteins to protect the mucosal surface.

NOD2-mediated HDAC6/NF-κb signalling pathway regulates ferroptosis induced by extracellular histone H3 in acute liver failure.

Acute liver failure (ALF) is life-threatening and often associated with high mortality rates. The aim of the present study was to investigate whether extracellular histone H3 could induce ferroptosis in hepatic macrophages in ALF and explore its potential mechanism. RAW264.7 macrophages and C57BL/6 mice were used in this study. LPS, D-galactosamine (D-Gal), histone H3, histone H3 antibody, NOD2 agonist Muramyl Dipeptide (MDP) and HDAC6-siRNA were administered in this study. The key molecules of ferroptosis, NOD2, HDAC6 and the NF-κb pathway, were detected. In vitro, histone H3 was released into the extracellular environment from cell nucleus after LPS exposure. In addition, histone H3 could induce ferroptosis in RAW264.7 macrophages with increased level of Fe2+ and ROS and decreased levels of GPX4 and GSH. MDP further aggravated ferroptosis in RAW264.7 macrophages stimulated by histone H3, which was accompanied by elevated NOD2, HDAC6, p-P65 and IκBα. HDAC6-siRNA ameliorated ferroptosis in RAW264.7 macrophages induced by histone H3, which was accompanied by decreased levels of HDAC6, p-P65 and IκBα. However, HDAC6-siRNA did not alter NOD2 levels in RAW264.7 macrophages administered histone H3. In vivo, the levels of NOD2, HDAC6 the NF-κb pathway and ferroptosis were increased in ALF mice, which were downregulated by histone H3 antibody and upregulated by histone H3. Extracellular histone H3 could induce ferroptosis in hepatic macrophages in ALF by regulating theNOD2-mediated HDAC6/NF-κb signalling pathway.

Blau syndrome NOD2 mutations result in loss of NOD2 cross-regulatory function.

The studies described here provide an analysis of the pathogenesis of Blau syndrome and thereby the function of NOD2 as seen through the lens of its dysfunction resulting from Blau-associated NOD2 mutations in its nucleotide-binding domain (NBD). As such, this analysis also sheds light on the role of NOD2 risk polymorphisms in the LRR domain occurring in Crohn's disease. The main finding was that Blau NOD2 mutations precipitate a loss of canonical NOD2 signaling via RIPK2 and that this loss has two consequences: first, it results in defective NOD2 ligand (MDP)-mediated NF-κB activation and second, it disrupts NOD2-mediated cross-regulation whereby NOD2 downregulates concomitant innate (TLR) responses. Strong evidence is also presented favoring the view that NOD2-mediated cross-regulation is under mechanistic control by IRF4 and that failure to up-regulate this factor because of faulty NOD2 signaling is the proximal cause of defective cross-regulation and the latter's effect on Blau syndrome inflammation. Overall, these studies highlight the role of NOD2 as a regulatory factor and thus provide additional insight into its function in inflammatory disease. Mutations in the nucleotide binding domain of the CARD15 (NOD2) gene underlie the granulomatous inflammation characterizing Blau syndrome (BS). In studies probing the mechanism of this inflammation we show here that NOD2 plasmids expressing various Blau mutations in HEK293 cells result in reduced NOD2 activation of RIPK2 and correspondingly reduced NOD2 activation of NF-κB. These in vitro studies of NOD2 signaling were accompanied by in vivo studies showing that BS-NOD2 also exhibit defects in cross-regulation of innate responses underlying inflammation. Thus, whereas over-expressed intact NOD2 suppresses TNBS-colitis, over-expressed BS-NOD2 does not; in addition, whereas administration of NOD2 ligand (muramyl dipeptide, MDP) suppresses DSS-colitis in Wild Type (WT) mice it fails to do so in homozygous or heterozygous mice bearing a NOD2 Blau mutation. Similarly, mice bearing a Blau mutation exhibit enhanced anti-collagen antibody-induced arthritis. The basis of such cross-regulatory failure was revealed in studies showing that MDP-stimulated cells bearing BS-NOD2 exhibit a reduced capacity to signal via RIPK2 as well as a reduced capacity to up-regulate IRF4, a factor shown previously to mediate NOD2 suppression of NF-κB activation. Indeed, TLR-stimulated cells bearing a Blau mutation exhibited enhanced in vitro cytokine responses that are quieted by lentivirus transduction of IRF4. In addition, enhanced anti-collagen-induced joint inflammation in mice bearing a Blau mutation was accompanied by reduced IRF4 expression in inflamed joint tissue and IRF4 expression was reduced in MDP-stimulated cells from BS patients. Thus, inflammation characterizing Blau syndrome are caused, at least in part, by faulty canonical signaling and reduce IRF4-mediated cross-regulation.

Effects of Microencapsulated Ferulic Acid or Its Prodrug Methyl Ferulate on Neuroinflammation Induced by Muramyl Dipeptide.

Ferulic acid (Fer) is known for its antioxidant and anti-inflammatory activities, which are possibly useful against neurodegenerative diseases. Despite the ability of Fer to permeate the brain, its fast elimination from the body does not allow its therapeutic use to be optimized. The present study proposes the preparation and characterization of tristearin- or stearic acid-based solid lipid microparticles (SLMs) as sustained delivery and targeting systems for Fer. The microparticles were produced by conventional hot emulsion techniques. The synthesis of the methyl ester of Fer (Fer-Me) allowed its encapsulation in the SLMs to increase. Fer-Me was hydrolyzed to Fer in rat whole blood and liver homogenate, evidencing its prodrug behavior. Furthermore, Fer-Me displayed antioxidant and anti-inflammatory properties. The amount of encapsulated Fer-Me was 0.719 ± 0.005% or 1.507 ± 0.014% in tristearin or stearic acid SLMs, respectively. The tristearin SLMs were able to control the prodrug release, while the stearic acid SLMs induced a significant increase of its dissolution rate in water. Jointly, the present results suggest that the tristearin SLMs loaded with Fer-Me could be a potential formulation against peripheral neuropathic pain; conversely, the stearic acid SLMs could be useful for Fer-Me uptake in the brain after nasal administration of the formulation.

Gut microbial DL-endopeptidase alleviates Crohn's disease via the NOD2 pathway.

The pattern-recognition receptor NOD2 senses bacterial muropeptides to regulate host immunity and maintain homeostasis. Loss-of-function mutations in NOD2 are associated with Crohn's disease (CD), but how the variations in microbial factors influence NOD2 signaling and host pathology is elusive. We demonstrate that the Firmicutes peptidoglycan remodeling enzyme, DL-endopeptidase, increased the NOD2 ligand level in the gut and impacted colitis outcomes. Metagenomic analyses of global cohorts (n = 857) revealed that DL-endopeptidase gene abundance decreased globally in CD patients and negatively correlated with colitis. Fecal microbiota from CD patients with low DL-endopeptidase activity predisposed mice to colitis. Administering DL-endopeptidase, but not an active site mutant, alleviated colitis via the NOD2 pathway. Therapeutically restoring NOD2 ligands with a DL-endopeptidase-producing Lactobacillus salivarius strain or mifamurtide, a clinical analog of muramyl dipeptide, exerted potent anti-colitis effects. Our study suggests that the depletion of DL-endopeptidase contributes to CD pathogenesis through NOD2 signaling, providing a therapeutically modifiable target.

Synthesis and Immunological Evaluation of Mannosylated Desmuramyl Dipeptides Modified by Lipophilic Triazole Substituents.

Muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine, MDP) is the smallest peptidoglycan fragment able to trigger an immune response by activating the NOD2 receptor. Structural modification of MDP can lead to analogues with improved immunostimulating properties. The aim of this work was to prepare mannosylated desmuramyl peptides (ManDMP) containing lipophilic triazole substituents to study their immunomodulating activities in vivo. The adjuvant activity of the prepared compounds was evaluated in the mouse model using ovalbumin as an antigen and compared to the MDP and referent adjuvant ManDMPTAd. The obtained results confirm that the α-position of D-isoGln is the best position for the attachment of lipophilic substituents, especially adamantylethyl triazole. Compound 6c exhibited the strongest adjuvant activity, comparable to the MDP and better than referent ManDMPTAd.

Chemoenzymatic Probes Reveal Peptidoglycan Recognition and Uptake Mechanisms in Candida albicans.

Candida albicans, the major fungal pathogen in humans, is under the strong influence of bacterial peptidoglycan fragments to undergo the yeast-to-hyphae transition, a key virulent step in C. albicans pathogenesis and infections. However, due to the synthetic difficulties of obtaining peptidoglycan fragments for biological studies, mechanistic details of how C. albicans recognizes and uptakes these peptidoglycan fragments have not been well elucidated. Notably, previous works have solely focused on the synthetic peptidoglycan ligand, muramyl dipeptide (MDP), despite its poor hyphal-inducing activity in C. albicans. In this work, we isolated and purified natural peptidoglycan fragments via enzymatic degradation of bacteria cell wall sacculi and chemoenzymatically installed a series of functional d-amino acids into the natural muropeptide, creating peptidoglycan probes that bear photoaffinity, bio-orthogonal, or fluorescent functionality. Using these chemoenzymatic peptidoglycan probes, we established that natural peptidoglycan fragments, which are potent hyphal-inducers, interact with the C. albicans Cyr1 sensor protein in the in-gel fluorescence assay as well as in in vitro pulldown studies. Moreover, we established that bacterial peptidoglycan probes enter C. albicans cells via an energy-dependent endocytic process.