Search

[Display omitted] • Microglia are a major target of PGN in gut microbiota-brain signalling. • Physiological doses of MDP result in long-lasting transcriptional changes in microglial Nod2 signaling, cytokines and chemokines. • Increased TNF-α and CCL5 gene expression is reflected in elevated protein secretion. • In IMG cells MDP signalling via NF-κB is MAPK dependent. • IMG cells show a strong correlation in gene expression to primary microglia, making them a useful in vitro model to study microglia. Bacterial peptidoglycan (PGN) fragments are commonly studied in the context of bacterial infections. However, PGN fragments recently gained recognition as signalling molecules from the commensal gut microbiota in the healthy host. Here we focus on the minimal bioactive PGN motif muramyl dipeptide (MDP), found in both Gram-positive and Gram-negative commensal bacteria, which signals through the Nod2 receptor. MDP from the gut microbiota translocates to the brain and is associated with changes in neurodevelopment and behaviour, yet there is limited knowledge about the underlying mechanisms. In this study we demonstrate that physiologically relevant doses of MDP induce rapid changes in microglial gene expression and lead to cytokine and chemokine secretion. In immortalised microglial (IMG) cells, C–C Motif Chemokine Ligand 5 (CCL5/RANTES) expression is acutely sensitive to the lowest physiologically prevalent dose (0.1 µg/ml) of MDP. As CCL5 plays an important role in memory formation and synaptic plasticity, microglial CCL5 might be the missing link in elucidating MDP-induced alterations in synaptic gene expression. We observed that a higher physiological dose of MDP elevates the expression of cytokines TNF-α and IL-1β, indicating a transition toward a pro-inflammatory phenotype in IMG cells, which was validated in primary microglial cultures. Furthermore, MDP induces the translocation of NF-κB subunit p65 into the nucleus, which is blocked by MAPK p38 inhibitor SB202190, suggesting that an interplay of both the NF-κB and MAPK pathways is responsible for the MDP-specific microglial phenotype. These findings underscore the significance of different MDP levels in shaping microglial function in the CNS and indicate MDP as a potential mediator for early inflammatory processes in the brain. It also positions microglia as an important target in the gut microbiota-brain-axis pathway through PGN signalling. [ABSTRACT FROM AUTHOR]

There is growing awareness that infections may contribute to the development of senile dementia including Alzheimer's disease (AD), and that immunopotentiation is therefore a legitimate target in the management of diseases of the elderly including AD. In Part I of this work, we provided a historical and molecular background to how vaccines, adjuvants, and their component molecules can elicit broad-spectrum protective effects against diverse agents, culminating in the development of the tuberculosis vaccine strain Bacille Calmette–Guérin (BCG) as a treatment for some types of cancer as well as a prophylactic against infections of the elderly such as pneumonia. In Part II, we critically review studies that BCG and other vaccines may offer a measure of protection against dementia development. Five studies to date have determined that intravesicular BCG administration, the standard of care for bladder cancer, is followed by a mean ∼45% reduction in subsequent AD development in these patients. Although this could potentially be ascribed to confounding factors, the finding that other routine vaccines such as against shingles (herpes zoster virus) and influenza (influenza A virus), among others, also offer a degree of protection against AD (mean 29% over multiple studies) underlines the plausibility that the protective effects are real. We highlight clinical trials that are planned or underway and discuss whether BCG could be replaced by key components of the mycobacterial cell wall such as muramyl dipeptide. We conclude that BCG and similar agents merit far wider consideration as prophylactic agents against dementia. [ABSTRACT FROM AUTHOR]

Vaccines such as Bacille Calmette–Guérin (BCG) can apparently defer dementia onset with an efficacy better than all drugs known to date, as initially reported by Gofrit et al. (PLoS One14, e0224433), now confirmed by other studies. Understanding how and why is of immense importance because it could represent a sea-change in how we manage patients with mild cognitive impairment through to dementia. Given that infection and/or inflammation are likely to contribute to the development of dementias such as Alzheimer's disease (Part II of this work), we provide a historical and molecular background to how vaccines, adjuvants, and their component molecules can elicit broad-spectrum protective effects against diverse agents. We review early studies in which poxvirus, herpes virus, and tuberculosis (TB) infections afford cross-protection against unrelated pathogens, a concept known as 'trained immunity'. We then focus on the attenuated TB vaccine, BCG, that was introduced to protect against the causative agent of TB, Mycobacterium tuberculosis. We trace the development of BCG in the 1920 s through to the discovery, by Freund and McDermott in the 1940 s, that extracts of mycobacteria can themselves exert potent immunostimulating (adjuvant) activity; Freund's complete adjuvant based on mycobacteria remains the most potent immunopotentiator reported to date. We then discuss whether the beneficial effects of BCG require long-term persistence of live bacteria, before focusing on the specific mycobacterial molecules, notably muramyl dipeptides, that mediate immunopotentiation, as well as the receptors involved. Part II addresses evidence that immunopotentiation by BCG and other vaccines can protect against dementia development. [ABSTRACT FROM AUTHOR]

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 pGSK3β and β‐catenin in the distal femur of OVX mice was lower than that in the distal femur of sham‐operated mice. Yet, the expression of pGSK3β and β‐catenin was increased in MDP‐administered OVX mice compared with OVX mice. In addition, MDP increased the expression and transcriptional activity of β‐catenin in osteoblasts. MDP inhibited the proteasomal degradation of β‐catenin via the down‐regulation of its ubiquitination by GSK3β inactivation. When osteoblasts were pretreated with Wnt signaling inhibitors, DKK1 or IWP‐2, the induction of pAKT, pGSK3β, and β‐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. [ABSTRACT FROM AUTHOR]

Context Chinese herb Huangqin decoction (HQD) can regulate intestinal flora in ulcerative colitis (UC) mice. Objective Our study clarifies the mechanism of HQD in regulating the intestinal flora of UC mice. Materials and methods Male C57BL/6 mice were randomly divided into six groups: Control, Model (3% DSS), Sulfasalazine (500 mg/kg), HQD-L (250 mg/kg), HQD-M (500 mg/kg), and HQD-H (1000 mg/kg) groups. Measurement of body weight, colon length, DAI, and haematoxylin–eosin staining were conducted. FISH and 16S rDNA detected colonic bacterial infiltration and intestinal flora changes. The expression of RegIIIγ and PRRs (NOD2, TLR5, TLR4) were detected by FCM and WB, respectively. In addition, WB, qPCR, or IHC were used to detect the expression of NOD2, MyD88, RIP2, and NF-κB p65 in the colon. ELISA was used to determine cytokines. Results Compared with the model group (DAI score, 2.38 ± 0.05; histological score, 4.08 ± 0.54), HQD treatment significantly reduced the DAI score (L, 2.16 ± 0.09; M, 1.45 ± 0.05; H, 1.18 ± 0.05) and histological score (L, 3.16 ± 0.82; M, 2.50 ± 0.81; H, 1.51 ± 0.76); restored the weight, the colonic length (p

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. [ABSTRACT FROM AUTHOR]

According to the WHO, cancer is the second leading cause of death in the world. This is an important global problem and a major challenge for researchers who have been trying to find an effective anticancer therapy. A large number of newly discovered compounds do not exert selective cytotoxic activity against tumorigenic cells and have too many side effects. Therefore, research on muramyl dipeptide (MDP) analogs has attracted interest due to the urgency for finding more efficient and safe treatments for oncological patients. MDP is a ligand of the cytosolic nucleotide-binding oligomerization domain 2 receptor (NOD2). This molecule is basic structural unit that is responsible for the immune activity of peptidoglycans and exhibits many features that are important for modern medicine. NOD2 is a component of the innate immune system and represents a promising target for enhancing the innate immune response as well as the immune response against cancer cells. For this reason, MDP and its analogs have been widely used for many years not only in the treatment of immunodeficiency diseases but also as adjuvants to support improved vaccine delivery, including for cancer treatment. Unfortunately, in most cases, both the MDP molecule and its synthesized analogs prove to be too pyrogenic and cause serious side effects during their use, which consequently exclude them from direct clinical application. Therefore, intensive research is underway to find analogs of the MDP molecule that will have better biocompatibility and greater effectiveness as anticancer agents and for adjuvant therapy. In this paper, we review the MDP analogs discovered in the last 10 years that show promise for antitumor therapy. The first part of the paper compiles the achievements in the field of anticancer vaccine adjuvant research, which is followed by a description of MDP analogs that exhibit promising anticancer and antiproliferative activity and their structural changes compared to the original MDP molecule. [ABSTRACT FROM AUTHOR]

Receptor-interacting serine/threonine protein kinase 2 (RIPK2) is a kinase that plays an essential role in the modulation of innate and adaptive immune responses. As a downstream signaling molecule for nucleotide-binding oligomerization domain 1 (NOD1), NOD2, and Toll-like receptors (TLRs), it is implicated in the signaling triggered by recognition of microbe-associated molecular patterns by NOD1/2 and TLRs. Upon activation of these innate immune receptors, RIPK2 mediates the release of pro-inflammatory factors by activating mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB). However, whether RIPK2 is essential for downstream inflammatory signaling following the activation of NOD1/2, TLRs, or both remains controversial. In this study, we examined the role of RIPK2 in NOD2-and TLR4-dependent signaling cascades following stimulation of microglial cells with bacterial muramyl dipeptide (MDP), a NOD2 agonist, or lipopolysaccharide (LPS), a TLR4 agonist. We utilized a highly specific proteolysis targeting chimera (PROTAC) molecule, GSK3728857A, and found dramatic degradation of RIPK2 in a concentration- and time-dependent manner. Importantly, the PROTAC completely abolished MDP-induced increases in iNOS and COX-2 protein levels and pro-inflammatory gene transcription of Nos2 , Ptgs2 , Il-1β , Tnfα , Il6 , Ccl2 , and Mmp9 . However, increases in iNOS and COX-2 proteins and pro-inflammatory gene transcription induced by the TLR4 agonist, LPS, were only slightly attenuated with the GSK3728857A pretreatment. Further findings revealed that the RIPK2 PROTAC completely blocked the phosphorylation and activation of p65 NF-κB and p38 MAPK induced by MDP, but it had no effects on the phosphorylation of these two mediators triggered by LPS. Collectively, our findings strongly suggest that RIPK2 plays an essential role in the inflammatory responses of microglia to bacterial MDP but not to LPS.

Introduction: In responses to antibiotics exposure, gut dysbiosis is a risk factor not only for pathogen infection but also for facilitating pathobiont expansion, resulting in increased inflammatory responses in the gut and distant organs. However, how this process is regulated has not been fully elucidated., Objectives: In this study, we investigated the role of sialic acid, a host-derived carbohydrate, in the pathogenesis of gut dysbiosis-derived inflammation in distant organs., Methods: Ampicillin (Amp)-induced gut dysbiotic mice were treated with N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac) for three weeks to assess the role of sialic acids in mastitis. The underlying mechanism by which sialic acids regulate mastitis was explored using 16S rRNA sequencing, transcriptomics and employed multiple molecular approaches., Results: Administration of Neu5Ac and Neu5Gc exacerbated gut dysbiosis-induced mastitis and systemic inflammation. The gut dysbiosis caused by Amp was also aggravated by sialic acid. Notably, increased Enterococcus expansion, which was positively correlated with inflammatory markers, was observed in both Neu5Ac- and Neu5Gc-treated gut dysbiotic mice. Treatment of mice with Enterococcus cecorum (E. cecorum) aggravated gut dysbiosis-induced mastitis. Mechanically, sialic acid-facilitated E. cecorum expansion promoted muramyl dipeptide (MDP) release, which induced inflammatory responses by activating the NOD2-RIP2-NF-κB axis., Conclusions: Collectively, our data reveal a role of sialic acid-facilitated postantibiotic pathobiont expansion in gut dysbiosis-associated inflammation, highlighting a potential strategy for disease prevention by regulating the MDP-NOD2-RIP2 axis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Production and hosting by Elsevier B.V.)

According to the WHO, cancer is the second leading cause of death in the world. This is an important global problem and a major challenge for researchers who have been trying to find an effective anticancer therapy. A large number of newly discovered compounds do not exert selective cytotoxic activity against tumorigenic cells and have too many side effects. Therefore, research on muramyl dipeptide (MDP) analogs has attracted interest due to the urgency for finding more efficient and safe treatments for oncological patients. MDP is a ligand of the cytosolic nucleotide-binding oligomerization domain 2 receptor (NOD2). This molecule is basic structural unit that is responsible for the immune activity of peptidoglycans and exhibits many features that are important for modern medicine. NOD2 is a component of the innate immune system and represents a promising target for enhancing the innate immune response as well as the immune response against cancer cells. For this reason, MDP and its analogs have been widely used for many years not only in the treatment of immunodeficiency diseases but also as adjuvants to support improved vaccine delivery, including for cancer treatment. Unfortunately, in most cases, both the MDP molecule and its synthesized analogs prove to be too pyrogenic and cause serious side effects during their use, which consequently exclude them from direct clinical application. Therefore, intensive research is underway to find analogs of the MDP molecule that will have better biocompatibility and greater effectiveness as anticancer agents and for adjuvant therapy. In this paper, we review the MDP analogs discovered in the last 10 years that show promise for antitumor therapy. The first part of the paper compiles the achievements in the field of anticancer vaccine adjuvant research, which is followed by a description of MDP analogs that exhibit promising anticancer and antiproliferative activity and their structural changes compared to the original MDP molecule.

Decay-accelerating factor (DAF) is an essential member of the complement regulatory protein family that plays an important role in immune response and host homeostasis in mammals. However, the immune function of DAF has not been well characterized in bony fish. In this study, a complement regulatory protein named Ci DAF was firstly characterized from Ctenopharyngodon idella and its potential roles were investigated in intestine following bacterial infection. Similar to mammalian DAFs, Ci DAF has multiple complement control protein (CCP) functional domains, suggesting the evolutionary conservation of DAFs. CiDAF was broadly expressed in all tested tissues, with a relatively high expression level detected in the spleen and kidney. In vivo immune challenge experiments revealed that CiDAF strongly responded to bacterial pathogens (Aeromonas hydrophila and Aeromonas veronii) and PAMPs (lipopolysaccharide (LPS) or muramyl dipeptide (MDP)) challenges. In vitro RNAi experiments indicated that knockdown of CiDAF could upregulate the expression of complement genes (C4b , C5 and C7) and inflammatory cytokines (TNF-α , IL-1β and IL-8). Moreover, 2000 ng/mL of CiDAF agonist progesterone effectively alleviated LPS- or MDP-induced intestinal inflammation by regulating expression of complement factors, TLR/PepT1 pathway genes and inflammatory cytokines. Overall, these findings revealed that Ci DAF may act as a negative regulator of intestinal complement pathway and immune response to bacterial challenge in grass carp. • The first experimental evidence for presence of a functional Ci DAF in grass carp. • Transcript levels of Ci DAF in intestine could be significantly induced by bacterial pathogens and PAMPs challenge. • Knockdown of Ci DAF could upregulate the intestinal expression of classic complement genes and inflammation cytokines. • 2000 ng/ml of the Ci DAF agonist progesterone effectively alleviated LPS- or MDP-induced intestinal inflammation. • Ci DAF may act as a negative regulator of the intestinal complement pathway and immune response to bacterial challenge. [ABSTRACT FROM AUTHOR]

The relationship between chronic bacterial colonization in the brain and Alzheimer’s disease is attracting extensive attention. Recent studies indicated that the components of bacterial biofilm drive the amyloid-β production. Muramyl dipeptide, the minimal bioactive peptidoglycan motif common to all bacteria, contributes to the development of many central inflammatory and neurodegenerative disorders. However, the involvement of Muramyl dipeptide in amyloid-β production is not completely defined. In our present study, wild type mice received an intracerebroventricular injection of normal saline or Muramyl dipeptide. Data showed that the production of Aβ1-42 oligomers was significantly increased after Muramyl dipeptide injection in the wild type mice or incubation of the SH-SY5Y cells with Muramyl dipeptide. Moreover, the action of Muramyl dipeptide was dose- and time-dependent. The above results suggested a possibility that the Muramyl dipeptide -induced Aβ1-42 oligomer production might be related to the NOD2/p-p38 MAPK/BACE1 pathway. To confirm this, the SH-SY5Y cells were transfected with siRNA NOD2. Data showed that the transfected SH-SY5Y cells exhibited decreased expression of Aβ1-42 oligomer, NOD2, p-p38 MAPK, and BACE1 after treatment with Muramyl dipeptide. Finally, SH-SY5Y cells were pretreated with SB203580, an inhibitor of the p-38-MAPK pathway. The results indicated that these pretreated SH-SY5Y cells exhibited decreased expression of Aβ1-42 oligomer, p-p38 MAPK, and BACE1 after treatment with Muramyl dipeptide. In conclusion, these results suggested that Muramyl dipeptide was the trigger factor for Aβ1-42 oligomer production, which probably acts via the NOD2/p-p38 MAPK/BACE1 signaling pathway.

Commensal bacteria boost serum IgG production in response to oral immunization with antigen and cholera toxin (CT) in a manner that depends on Nod2 (nucleotide-binding oligomerization domain-containing protein 2). In this study, we examined the role of intestinal lysozyme (Lyz1) in adjuvant activity of CT. We found that Lyz1 released Nod2 ligand(s) from bacteria. Lyz1 deficiency reduced the level of circulating Nod2 ligand in mice. Lyz1 deficiency also reduced the production of IgG and T-cellspecific cytokines after oral immunization in mice. Supplementing Lyz1-deficient mice with MDP restored IgG production. Furthermore, overexpression of Lyz1 in intestinal epithelium boosted the antigen-specific IgG response induced by CT. Collectively, our results indicate that Lyz1 plays an important role in mediating the immune regulatory effect of commensal bacteria through the release of Nod2 ligand(s). [ABSTRACT FROM AUTHOR]

Nucleotide-binding oligomerization domain 2 (NOD2) is a receptor of the innate immune system that is capable of perceiving bacterial and viral infections. Muramyl dipeptide (MDP, N -acetyl muramyl L-alanyl- d -isoglutamine), identified as the minimal immunologically active component of bacterial cell wall peptidoglycan (PGN) is recognized by NOD2. In terms of biological activities, MDP demonstrated vaccine adjuvant activity and stimulated non-specific protection against bacterial, viral, and parasitic infections and cancer. However, MDP has certain drawbacks including pyrogenicity, rapid elimination, and lack of oral bioavailability. Several detailed structure-activity relationship (SAR) studies around MDP scaffolds are being carried out to identify better NOD2 ligands. The present review elaborates a comprehensive SAR summarizing structural aspects of MDP derivatives in relation to NOD2 agonistic activity. [Display omitted] • Muramyl dipeptide (MDP) is the minimal peptidoglycan structure that activates NOD2. • A detailed SAR on MDP analogs is elaborated. • Desmuramylpeptides lacking sugar moiety are emerging as new class of NOD2 agonists. • Bioconjugation approach is explored to strengthen the immune system against tumor. [ABSTRACT FROM AUTHOR]

• Mac-1 signaling is essential for MDP-induced PGE 2 production in human monocytes. • MDP/NOD2 interaction leads to NF-kB binding to the COX2 promoter. • Mac-1 triggered by T cell-derived GPIbα activates MAPK. • The two signals contribute to binding of p300 HAT and Pol II to the COX2 promoter. Fever and inflammatory responses were observed in some subjects in early clinical trials of vaccines adjuvanted with muramyl dipeptide (MDP), a NOD2 agonist. Biosynthesis of Prostaglandin E2 (PGE 2) that transmits febrile signals to the brain is controlled by an inducible enzyme, Cyclooxygenase 2 (COX-2). MDP alone was not sufficient to induce expression of COX-2 and PGE 2 production in vitro. Conditioned medium prepared from Peripheral Blood Mononuclear Cells (PBMCs)-derived CD3-bead purified human T cells (TCM) dramatically increased COX2 gene transcription, COX-2 protein expression, and PGE 2 production in MDP-treated monocytes. We explored epigenetic changes at the COX2 promoter using Chromatin Immunoprecipitation assay (ChIP). Increase in COX2 transcription correlated with increased recruitment of RNA polymerase II (Pol II) and p300 histone acetyl transferase (HAT) to the COX2 promoter in monocytes activated with MDP and TCM. The role of p300 HAT was confirmed by using C646, an inhibitor of p300, that reduced binding of acetylated H3 and H4 histones at the COX2 promoter, COX2 transcription, and PGE 2 production in monocytes. Binding of p300, Nuclear Factor Kappa B (NF-κB), and Pol II to the COX2 promoter was also sensitive to inhibitors of Mitogen-Activated Protein Kinase (MAPK) pathway and to antibodies against Macrophage-1 (Mac-1) integrin in MDP/TCM-treated monocytes. Importantly, recombinant Glycoprotein Ib alfa (GPIbα), the recently identified factor in TCM, increased binding of NF-κB, p300, and of Pol II to the COX2 promoter and COX2 transcription in MDP-treated monocytes. Our findings suggest that a second signal through Mac-1 and MAPK is triggered by a T cell derived soluble GPIbα protein leading to the assembly of the transcription machinery at the COX2 promoter and production of PGE 2 in human monocytes in response to MDP/NOD2 activation. [ABSTRACT FROM AUTHOR]

The c-Jun NH2-terminal kinases (JNKs) are an evolutionarily conserved family of serine/threonine protein kinases that play critical roles in the pathological process in species ranging from insects to mammals. However, the function of JNKs in bacteria-induced intestinal inflammation is still poorly understood. In this study, a fish JNK (CiJNK) pathway was identified, and its potential roles in bacterial muramyl dipeptide (MDP)-induced intestinal inflammation were investigated in Ctenopharyngodon idella. The present CiJNK was found to possess a conserved dual phosphorylation motif (TPY) in a serine/threonine protein kinase (S_TKc) domain and to contain several potential immune-related transcription factor binding sites, including nuclear factor kappa B (NF-κB), activating protein 1 (AP-1), and signal transducer and activator of downstream transcription 3 (STAT3), in its 5′ flanking regions. Quantitative real-time PCR results revealed that the mRNA levels of the JNK pathway genes in the intestine were significantly upregulated after challenge with a bacterial pathogen (Aeromonas hydrophila) and MDP in a time-dependent manner. Additionally, the JNK pathway was found to be involved in regulating the MDP-induced expression levels of inflammatory cytokines (IL-6, IL-8, and TNF-α) in the intestine of grass carp. Moreover, the nutritional dipeptide carnosine and Ala–Gln could effectively alleviate MDP-induced intestinal inflammation by regulating the intestinal expression of JNK pathway genes and inflammatory cytokines in grass carp. Finally, fluorescence microscopy and dual-reporter assays indicated that CiJNK could associate with CiMKK4 and CiMKK7 involved in the regulation of the AP-1 signaling pathway. Overall, these results provide the first experimental demonstration that the JNK signaling pathway is involved in the intestinal immune response to MDP challenge in C. idella, which may provide new insight into the pathogenesis of inflammatory bowel disease.

The current study investigates anti-neoplastic and immunomodulatory activities of co-treatment based on bovine lactoferrin (bLF) and/or muramyl dipeptide (MDP) with or without cisplatin (Cis) in tumor-bearing mice. In the present study, bLF (100 mg/kg; orally) and MDP (0.5 mg/kg; subcutaneously) was administered alone or together.MDP or bLF was co-treated with Cis (1 mg/kg; intraperitoneally) in mice-bearing Ehrlich solid carcinoma. Tumor size, tumor mass proliferation, apoptosis using immunohistochemistry, the alteration in spleen cell proliferation, phenotype using f low cytometry and white blood cells total and differential counts were detected. Treatment with Cis or (bLF and MDP) significantly reduced tumor size, upregulated the pro-apoptotic p53 expression and downregulated the anti-apoptotic Bcl-2 and proliferative marker PCNA expression compared to non-treated tumor-bearing animals. Moreover, co-treatment of MDP and Cis significantly potentiated the reduction of the tumor size, downregulated the Bcl-2 and PCNA expression and upregulated the p53 expression compared to Cis-treated animals. While bLF and Cis co-treatment positively controlled PCNA and p53 expression compared to tumor-bearing animals, it significantly potentiated the reduction of the tumor size and downregulated the Bcl-2 expression compared to Cis-treated animals. Co-treatment of (bLF and MDP), (bLF and Cis) or (MDP and Cis) increased the spleen cell proliferation and altered the immunological profile of the CD3+CD4+, CD3+CD8+, CD3+CD4+CD69+, CD3+CD8+CD69+ and CD11b+Ly6G+ cells to achieve better immune response against tumor. In conclusion, co-treatments based on bLF and/or MDP are promising therapies against cancer, through their potency to control proliferation, enhance apoptosis and improve the immune status against tumor cells. [ABSTRACT FROM AUTHOR]

The c-Jun NH2-terminal kinases (JNKs) are an evolutionarily conserved family of serine/threonine protein kinases that play critical roles in the pathological process in species ranging from insects to mammals. However, the function of JNKs in bacteria-induced intestinal inflammation is still poorly understood. In this study, a fish JNK (Ci JNK) pathway was identified, and its potential roles in bacterial muramyl dipeptide (MDP)-induced intestinal inflammation were investigated in Ctenopharyngodon idella. The present Ci JNK was found to possess a conserved dual phosphorylation motif (TPY) in a serine/threonine protein kinase (S_TKc) domain and to contain several potential immune-related transcription factor binding sites, including nuclear factor kappa B (NF-κB), activating protein 1 (AP-1), and signal transducer and activator of downstream transcription 3 (STAT3), in its 5′ flanking regions. Quantitative real-time PCR results revealed that the mRNA levels of the JNK pathway genes in the intestine were significantly upregulated after challenge with a bacterial pathogen (Aeromonas hydrophila) and MDP in a time-dependent manner. Additionally, the JNK pathway was found to be involved in regulating the MDP-induced expression levels of inflammatory cytokines (IL-6, IL-8, and TNF-α) in the intestine of grass carp. Moreover, the nutritional dipeptide carnosine and Ala–Gln could effectively alleviate MDP-induced intestinal inflammation by regulating the intestinal expression of JNK pathway genes and inflammatory cytokines in grass carp. Finally, fluorescence microscopy and dual-reporter assays indicated that Ci JNK could associate with Ci MKK4 and Ci MKK7 involved in the regulation of the AP-1 signaling pathway. Overall, these results provide the first experimental demonstration that the JNK signaling pathway is involved in the intestinal immune response to MDP challenge in C. idella , which may provide new insight into the pathogenesis of inflammatory bowel disease. [ABSTRACT FROM AUTHOR]

N-Acetylmuramyl-L-alanyl-D-isoglutamine α-phenylglycoside was synthesized from α-phenyl-D-glucosaminide peracetate, which was obtained by fusing β-D-glucosamine pentaacetate with phenol and zinc chloride followed by deacetylation. α-Phenyl-N-acetylglucosaminide was converted in two steps into 4,6-O-isopropylidene-N-acetyl-D-muramic acid α-phenylglycoside, which was condensed with L-Ala-D-iGln benzyl ether using the activated ester method. Sequential removal of glycopeptide protecting groups via acid hydrolysis and catalytic hydrogenolysis gave the target product. [ABSTRACT FROM AUTHOR]

The nucleotide-binding oligomerization domain-containing protein 2 (NOD2) agonist muramyl dipeptide (MDP), a peptidoglycan motif common to all bacteria, supports leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)+ intestinal stem cell (ISC) survival through NOD2 activation upon an otherwise lethal oxidative stress-mediated signal. However, the underlying protective mechanisms remain unknown. Here, using irradiation as stressor and primarily murine-derived intestinal organoids as a model system, we show that MDP induced a significant reduction of total and mitochondrial reactive oxygen species (ROS) within ISCs, which was associated with mitophagy induction. ATG16L1 knockout (KO) and NOD2 KO organoids did not benefit from the MDP-induced cytoprotection. We confirmed the MDP-dependent induction of ISC mitophagy upon stress in vivo. These findings elucidate the NOD2-mediated mechanism of cytoprotection involving the clearance of the lethal excess of ROS molecules through mitophagy, triggered by the coordinated activation of NOD2 and ATG16L1 by a nuclear factor κB (NF-κB)-independent pathway. [ABSTRACT FROM AUTHOR]

Abstract Mitogen-activated protein kinase kinases (MKKs) are a class of evolutionarily conserved signalling intermediates of the MAPK signalling pathway that can be activated by a diverse range of pathogenic stimuli and are crucial for the regulation of host immune defence. In this study, two fish MKK genes (Ci MKK4 and Ci MKK7) were first identified and characterized from grass carp (Ctenopharyngodon idella). Similar to other reported MKKs, the present Ci MKK4 and Ci MKK7 contained a conserved serine/threonine protein kinase (S_TKc) domain and a canonical dual phosphorylation motif. Quantitative real-time PCR results showed that Ci MKK4 and Ci MKK7 were broadly transcribed in all selected tissues and developmental stages of grass carp. The mRNA expression levels of Ci MKK4 and Ci MKK7 in the intestine were significantly induced by bacterial muramyl dipeptide (MDP) challenge in a time-dependent manner (P < 0.01). Additionally, the stimulatory effects of bacterial MDP on Ci MKK4 and Ci MKK7 expression in the intestine were inhibited by the bioactive dipeptide β-alanyl- l -histidine (carnosine) and alanyl-glutamine (Ala-Gln) (P < 0.05). Moreover, overexpression analysis revealed that Ci MKK4 and Ci MKK7 were localized throughout the entire cell and could significantly enhance AP-1 reporter gene activation in HEK293T cells. Taken together, these results provide the first experimental demonstration that Ci MKK4 and Ci MKK7 are involved in the intestinal immune response to MDP challenge in C. idella , which may provide new insight into the bacterial-induced intestinal inflammation of bony fishes. *Highlights • The first experimental evidence for presence of MKK4 and MKK7 in Ctenopharyngodon idella. • Ci MKK4 and Ci MKK7 mRNA were broadly expressed in all tested tissues and developmental stages. • Transcript levels of Ci MKK4 and Ci MKK7 could be significantly induced by bacterial muramyl dipeptide challenge. • The stimulating effects of MDP on Ci MKK4 and Ci MKK7 were inhibited by dipeptide carnosine and Ala-Gln. • Ci MKK4 and Ci MKK7 were cytoplasm- and nucleus-localized proteins which could enhance AP-1-Luc activation. [ABSTRACT FROM AUTHOR]

Interactions between pattern recognition receptors (PRRs) shape innate immune responses to particular classes of pathogens. Here, we review interactions between TLRs and nucleotide‐binding oligomerization domain 1 and 2 (NOD1 and NOD2) receptors, two major groups of PRRs involved in innate recognition of bacteria. Most of experimental data both in vitro and in vivo suggest that NODs and TLRs synergize with each other at inducing the production of cytokines and antimicrobial peptides. Molecular mechanisms of this synergy remain poorly understood, although several scenarios can be proposed: (i) direct interactions of signaling pathways downstream of NODs and TLRs; (ii) mutual transcriptional regulation of unique components of NOD‐dependent and TLR‐dependent signaling pathways; and (iii) interactions at the post‐transcriptional level. Potential practical implications of NOD‐TLR synergy are dual. In sepsis, where synergistic effects probably contribute to excessive proinflammatory cytokine production, blockade of NOD1, and/or NOD2 in addition to TLR4 blockade may be required to achieve therapeutic benefit. On the other hand, synergistic combinations of relatively small doses of NOD and TLR agonists administered before infection could be used to boost innate resistance against bacterial pathogens. NOD1/NOD2 and TLRs synergize at induction of innate immune responses, which can be beneficial (boosting protective immunity) or deleterious (in sepsis). [ABSTRACT FROM AUTHOR]

Adjuvants enhance immunogenicity of vaccines through either targeted antigen delivery or stimulation of immune receptors. Three cationic nanoparticle formulations were evaluated for their potential as carriers for a DNA vaccine, and muramyl dipeptide (MDP) as immunostimulatory agent, to induce and increase immunogenicity of Mycobacterium tuberculosis antigen encoding plasmid DNA (pDNA). The formulations included (1) trimethyl chitosan (TMC) nanoparticles, (2) a squalene-in-water nanoemulsion, and (3) a mineral oil-in-water nanoemulsion. The adjuvant effect of the pDNA-nanocomplexes was evaluated by serum antibody analysis in immunized mice. All three carriers display a strong adjuvant effect, however, only TMC nanoparticles were capable to bias immune responses towards Th1. pDNA naturally contains immunostimulatory unmethylated CpG motifs that are recognized by Toll-like receptor 9 (TLR-9). In mechanistic in vitro studies, activation of TLR-9 and the ability to enhance immunogenicity by simultaneously targeting TLR-9 and NOD-like receptor 2 (NLR-2) was determined by proinflammatory cytokine release in RAW264.7 macrophages. pDNA in combination with MDP was shown to significantly increase proinflammatory cytokine release in a synergistic manner, dependent on NLR-2 activation. In summary, novel pDNA-Ag85A loaded nanoparticle formulations, which induce antigen specific immune responses in mice were developed, taking advantage of the synergistic combinations of TLR and NLR agonists to increase the adjuvanticity of the carriers used.

The development of new, effective, and safe vaccines necessarily requires the identification of new adjuvant(s) to enhance the potency and longevity of antigen-specific immune responses. In the present study, we compare the antibody-mediated and cell-mediated immune (CMI) responses within groups of mice vaccinated subcutaneously with ovalbumin (OVA; as an experimental antigen) plus polyphosphazene (an innate immune modulator), Polyinosinic:polycytidylic acid (poly-I:C; (an RNA mimetic) and glycopeptide ARC5 (which is a Toll-like receptor (TLR), TLR2 ligand and PAM3CSK4 analogue) formulated together in a soluble vaccine. We also investigated the effect of a polymeric nanoparticle of ARC4 and ARC7 (which are a novel muramyl dipeptide analogue and a monophosophoryl lipid A (MPLA) analogue, respectively) plus OVA +/− ARC5 as a subcutaneous vaccine in mice. OVA+ARC4/ARC7 nanoparticle +/− ARC5 triggered a robust and balanced Th1/Th2-type humoral response with significant anti-OVA IgA in serum, and significant interferon (IFN)-γ and interleukin (IL)-17 production in splenocytes after 35 days relative to the controls. Formulation of OVA with ARC4/ARC7 nanoparticles should be investigated for inducing protective immunity against infectious pathogens in mice and other species.

The host’s intestinal microbiota contributes to endocrine and metabolic responses, but a dysbiosis in this environment can lead to obesity and insulin resistance. Recent work has demonstrated a role for microbial metabolites in the regulation of gut hormones, including the metabolic hormone, glucagon-like peptide-1 (GLP-1). Muramyl dipeptide (MDP) is a bacterial cell wall component which has been shown to improve insulin sensitivity and glucose tolerance in diet-induced obese mice by acting through the nucleotide oligomerization domain 2 (NOD2) receptor. The purpose of this study was to understand the effects of MDP on GLP-1 secretion and glucose regulation. We hypothesized that MDP enhances glucose tolerance by inducing intestinal GLP-1 secretion through NOD2 activation. First, we observed a significant increase in GLP-1 secretion when murine and human L-cells were treated with a fatty acid MDP derivative (L18-MDP). Importantly, we demonstrated the expression of the NOD2 receptor in mouse intestine and in L-cells. In mice, two intraperitoneal injections of MDP (5 mg/kg body weight) caused a significant increase in fasting total GLP-1 in chow-fed mice, however this did not lead to an improvement in oral glucose tolerance. When mice were exposed to a high-fat diet, they eventually lost this MDP-induced GLP-1 release. Finally, we demonstrated in L-cells that hyperglycemic conditions reduce the mRNA expression of NOD2 and GLP-1. Together these findings suggest MDP may play a role in enhancing GLP-1 during normal glycemic conditions but loses its ability to do so in hyperglycemia.

The aim of this study was to evaluate whether apoptosis of lymphocytes is modulated by stimulation by lipopolysaccharide (LPS) of Escherichia coli or muramyl dipeptide (MDP). Cell populations were obtained by lavaging of the mammary glands 24, 48, 72, and 168 h following intramammary induced inflammation. The portion of apoptotic lymphocytes peaked at 48 h after treatment with LPS or MDP. The analysis of CD44 expression of the same cell populations showed a higher percentage of CD44-positive lymphocytes 24- and 48-h following induction of inflammation by LPS or MDP. The results demonstrate that during both experimental infection of bovine mammary glands with LPS or MDP, apoptosis of lymphocytes was induced in the initial phase of the inflammatory response and CD44 was also overexpressed at the beginning of inflammation. These data suggest a connection of lymphocyte apoptosis with the expression of CD44 receptors.

Influenza A virus (IAV) is recognized to cause severe pulmonary illnesses in humans, particularly in elderly and children. One of the features associated with IAV infection is an excessive lung inflammation due to an uncontrolled immune response. The nucleotide-binding oligomerization domain 2 (NOD2) receptor is known to recognize ssRNA viruses such as IAV, but its role in the inflammatory process during viral infections remains to be clarified. In a previous report, we have shown that activation of NOD2 with muramyl dipeptide (MDP) significantly reduces both viral loads and lung inflammation and also improves pulmonary function during IAV infection. These findings prompted us to further investigate whether NOD2 receptor may contribute to regulate inflammation during viral infection. In the present study, we show that administration of MDP to mice infected with IAV stimulates the migration of regulatory T (Treg) cells to the lungs. Such a presence of Treg cells was also accompanied with a reduction of neutrophils in the lungs during IAV infection, which correlated, with a significant decrease of Th17 cells. In our model, Treg cell recruitment is dependent of CXCL12 and CCL5 chemokines. Moreover, we show that the presence of Ly6Clow patrolling monocytes is required for Treg cells mobilization to the lung of mice treated with MDP. In fact, following monocyte depletion by administration of clodronate liposome, mobilization of Treg cells to the lungs of treated mice was found to occur when circulating Ly6Clow monocytes begin to reemerge. In addition, we also detected an increased production of TGF-β, a cytokine contributing to Treg activity when blood Ly6Clow monocytes are restored. Together, our results demonstrate that MDP treatment can promote an anti-inflammatory environment through the mobilization of Treg cells to the lung, a mechanism that requires the presence of Ly6Clow monocytes during IAV infection. Overall, our results suggest that activation of NOD2 receptor could be an appealing approach to control pulmonary inflammation in patients infected with IAV.

The aetiology of Crohn's disease and paratuberculosis are the subjects of intensive study and also frequently, of dispute. However, a number of other nosological entities have a similar history, namely type 1 diabetes, multiple sclerosis, sarcoidosis, asthma, psoriasis, spondylarthritis, Blau syndrom etc. The zoonotic risk of Mycobacterium avium subsp. paratuberculosis (MAP) has been discussed for more than one hundred years. "The problem remains open, further research is needed", is the sentence which seems to be obligatory in the conclusions of many scientific articles. A number of hypotheses have been suggested, all with a grain of truth in them. The infection hypothesis has many supporters and opponents, but it does not fit to all Crohn's disease cases. The contribution of the genetic factor has been admitted a long time ago and has been experimentally confirmed by recent excellent studies. An environmental factor is expected and has been often mentioned, but has yet to be discovered. Muramyl dipeptide, derived from peptidoglycans of the bacterial cell wall is one of the triggers, mentioned in connection with chronic inflammatory diseases. The immunomodulatory ability of this compound has been recognised for decades and is exploited in Freund's adjuvant. A critical amount of muramyl dipeptide can affect immunity during some bacterial infections but the long latent period between infection and onset of the clinical form of the disease could explain why a causative relationship between the primary infection and chronic inflammation is not considered. Different species of mycobacteria can be found in the environment, in water, dust, soil and aerosol. Although severe infections with mycobacteria have been described, these species are not thought to be typical zoonotic pathogens. Muramyl dipeptide derived from mycobacteria obviously plays a starring role as a bacterial trigger in the aetiology of many autoimmune and autoinflammatory diseases. Paratuberculosis in cattle and other ruminants is a source of enormous contamination of the environment but also of milk and meat by MAP. Muramyl dipeptide from mycobacteria, namely MAP, and Crohn's disease as a representative of diseases often called civilization threats, are important pieces of the gigantic puzzle. Mycobacteria in the environment and foodstuffs have to be acknowledged as a public health risk, which can never be completely eliminated. There is no reason to push the panic button, but we must learn how to live together with this microorganism, how the pool of immunomodulator sources can be diminished, and how the pathogenic relationship between triggers and target tissues can be disrupted. The dissemination of knowledge, the availability of rapid and inexpensive tools for identification of mycobacteria in different matrices, and the establishment of a maximal allowed limit for mycobacteria in milk and meat should contribute to food safety and consumer protection.

Our study proved that intracellular NOD2 in DCs could be activated by MDP, which could promote maturation and the ability to prime Th0 cells to Th2 cells of DCs exposed to P. gingivalis-LPS. Background: To study the roles of muramyl dipeptide (MDP) in dendritic cells (DCs) exposed to Porphyromonas gingivalis lipopolysaccharide (P. gingivalis-LPS) on maturation and antigen-presenting functions of DCs. To explore the possible mechanism of DCs in periodontitis. Methods: Flow cytometry was used to detect CD11c, MHC-II, CD80, CD86, and CD40 expression, and ELISA was used to detect IL-12, IFN-γ, IL-10, and IL-13 secreted by DCs. RT-PCR analysis was used to detect NOD2, TLR2, and TLR4 mRNA expression in DCs. CCK8 was used to assay CD4+T cell proliferation. Results: MDP had a weak ability to stimulate DC maturation but MDP could promote DC maturation stimulated by P. gingivalis-LPS. MDP could facilitate TLR2 mRNA expression in DCs exposed to P. gingivalis-LPS. The ability of MDP to promote DCs secreting cytokines was far below P. gingivalis-LPS but MDP could promote the functions of Th2 cell-promoting DCs induced by P. gingivalis-LPS. MDP could promote CD4+T cell proliferation primed by DCs exposed to P. gingivalis-LPS and elevate the ability of DCs exposed to P. gingivalis-LPS to prime Th0 cells to Th2 cells. Conclusions: Intracellular NOD2 in DCs could be activated by MDP, which could promote maturation and the ability to prime Th0 cells to Th2 cells of DCs exposed to P. gingivalis-LPS. [ABSTRACT FROM AUTHOR]