The innate immune system is critical for clearing infection, and is tightly regulated to avert excessive tissue damage. Nod1/2-Rip2 signaling, which is essential for initiating the innate immune response to bacterial infection and ER stress, is subject to many regulatory mechanisms. In this study, we found that LRRK2, encoded by a gene implicated in Crohn's disease, leprosy and familial Parkinson's disease, modulates the strength of Nod1/2-Rip2 signaling by enhancing Rip2 phosphorylation. LRRK2 deficiency markedly reduces cytokine production in macrophages upon Nod2 activation by muramyl dipeptide (MDP), Nod1 activation by D-gamma-Glu-meso-diaminopimelic acid (iE-DAP) or ER stress. Our biochemical study shows that the presence of LRRK2 is necessary for optimal phosphorylation of Rip2 upon Nod2 activation. Therefore, this study reveals that LRRK2 is a new positive regulator of Rip2 and promotes inflammatory cytokine induction through the Nod1/2-Rip2 pathway.
Animals ; Cytokines ; genetics ; immunology ; HEK293 Cells ; Humans ; Immunity, Innate ; genetics ; Inflammation ; genetics ; immunology ; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 ; genetics ; immunology ; Mice ; Mice, Knockout ; Nod1 Signaling Adaptor Protein ; genetics ; immunology ; Nod2 Signaling Adaptor Protein ; genetics ; immunology ; Phosphorylation ; genetics ; immunology ; Receptor-Interacting Protein Serine-Threonine Kinase 2 ; genetics ; immunology ; Receptor-Interacting Protein Serine-Threonine Kinases ; genetics ; immunology ; Signal Transduction ; genetics ; immunology

Autoinflammatory disease (AID) is a newly proposed category of disorders characterized by unprovoked episodes of inflammation without any infectious or autoimmune evidence. We aimed to characterize the clinical and genetic features of patients who had recurrent fever and multi-system inflammation but remain unclassified for any established AIDs. Medical records of 1,777 patients who visited our Rheumatology Clinic between March 2009 and December 2010 were reviewed to identify those who met the following criteria; 1) presence of fever, 2) inflammation in two or more organ systems, 3) recurrent nature of fever or inflammation, 4) no evidence of infection or malignancy, 5) absence of high titer autoantibodies, and 6) failure to satisfy any classification criteria for known AIDs. Genotyping was performed for common missense variants in MEFV, NOD2/CARD15, and TNFRSF1A. A small number of patients (17/1,777, 0.95%) were identified to meet the above criteria. Muco-cutaneous and musculoskeletal features were most common, but there was a considerable heterogeneity in symptom combination. Although they did not satisfy any established classification criteria for AIDs, substantial overlap was observed between the clinical spectrum of these patients and known AIDs. According to the newly proposed Eurofever criteria for periodic fevers, eleven of them were classified as TNF receptor-associated periodic syndrome and two as mevalonate kinase deficiency. However, no examined genetic variants including those in TNFRSF1A were found in these patients. A new set of classification criteria needs to be developed and validated for Asian patients with unclassified AIDs.
Adolescent ; Adult ; Cytoskeletal Proteins/genetics ; Female ; Fever/*etiology ; Genotype ; Hereditary Autoinflammatory Diseases/classification/*diagnosis/genetics ; Humans ; Inflammation/*etiology ; Male ; Middle Aged ; Mutation, Missense ; Nod2 Signaling Adaptor Protein/genetics ; Polymorphism, Single Nucleotide ; Receptors, Tumor Necrosis Factor, Type I/genetics ; Recurrence ; Republic of Korea ; Retrospective Studies ; Young Adult

Autoinflammatory disease (AID) is a newly proposed category of disorders characterized by unprovoked episodes of inflammation without any infectious or autoimmune evidence. We aimed to characterize the clinical and genetic features of patients who had recurrent fever and multi-system inflammation but remain unclassified for any established AIDs. Medical records of 1,777 patients who visited our Rheumatology Clinic between March 2009 and December 2010 were reviewed to identify those who met the following criteria; 1) presence of fever, 2) inflammation in two or more organ systems, 3) recurrent nature of fever or inflammation, 4) no evidence of infection or malignancy, 5) absence of high titer autoantibodies, and 6) failure to satisfy any classification criteria for known AIDs. Genotyping was performed for common missense variants in MEFV, NOD2/CARD15, and TNFRSF1A. A small number of patients (17/1,777, 0.95%) were identified to meet the above criteria. Muco-cutaneous and musculoskeletal features were most common, but there was a considerable heterogeneity in symptom combination. Although they did not satisfy any established classification criteria for AIDs, substantial overlap was observed between the clinical spectrum of these patients and known AIDs. According to the newly proposed Eurofever criteria for periodic fevers, eleven of them were classified as TNF receptor-associated periodic syndrome and two as mevalonate kinase deficiency. However, no examined genetic variants including those in TNFRSF1A were found in these patients. A new set of classification criteria needs to be developed and validated for Asian patients with unclassified AIDs.
Adolescent ; Adult ; Cytoskeletal Proteins/genetics ; Female ; Fever/*etiology ; Genotype ; Hereditary Autoinflammatory Diseases/classification/*diagnosis/genetics ; Humans ; Inflammation/*etiology ; Male ; Middle Aged ; Mutation, Missense ; Nod2 Signaling Adaptor Protein/genetics ; Polymorphism, Single Nucleotide ; Receptors, Tumor Necrosis Factor, Type I/genetics ; Recurrence ; Republic of Korea ; Retrospective Studies ; Young Adult

Brazilein is reported to have immunosuppressive effect on cardiovascular and cerebral-vascular diseases. The essential roles of innate immunity in cerebral ischemia are increasingly identified, but no studies concerning the influence of brazilein on the innate immunity receptors have been reported. The present study was designed to investigate the regulation of NOD2 (Nucleotide-binding oligomerization domain-containing protein 2) by brazilein for its protection of neuron in cerebral ischemia in vivo and oxygen-glucose deprivation in vitro. The results showed that brazilein could reverse the elevated expression of NOD2 and TNFα (tumor necrosis factor alpha) elicited by cerebral ischemia and reperfusion. This reduction could also be detected in normal mice and C17.2 cells, indicating that this suppressive effect of brazilein was correlated with NOD2. The results from GFP reporter plasmid assay suggested brazilein inhibited NOD2 gene transcription. In conclusion, brazilein could attenuate NOD2 and TNFα expression in cerebral ischemia and NOD2 may be one possible target of brazilein for its immune suppressive effect in neuro-inflammation.
Animals ; Benzopyrans ; administration & dosage ; Brain Ischemia ; drug therapy ; genetics ; immunology ; metabolism ; Cells, Cultured ; Drugs, Chinese Herbal ; administration & dosage ; Glucose ; metabolism ; Humans ; Indenes ; administration & dosage ; Male ; Mice ; Mice, Inbred ICR ; Neurons ; drug effects ; immunology ; Nod2 Signaling Adaptor Protein ; genetics ; metabolism ; Oxygen ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; immunology

OBJECTIVE: To explore role of Nods (nucleotide-binding oligomerization domain Nod Like receptors) kind of pattern recognition receptors (PRR) in patients with allergic rhinitis. METHOD: The mRNA and protein of Nod1, Nod2 of Nalp3 were analyzed in the turbinate mucosa of patients with allergic rhinitis, nasal septum deviation (NSD) nasal mucosa of patients and nasal polyp mucosa with Real-Time RT-PCR, Western blot and immunohistochemistry respectively, and Nod1 expression changes was explored in PBMC with wad explored Western-blot and then the level of IL-4, IL-6, IL-10, IFN-γ were detected in serum of AR after desensitization treatment. RESULT: These Nods like receptors, mainly found in nasal mucosa epithelial cells, glandular epithelium and inflammatory cells (e. g. plasma cells, eosinophils), were expressed in the nasal mucosa tissues. In AR group, Nod1 (mRNA and protein) expression were lower than NSD group (P<0.05), Nalp3 expression were higher than (P<0.05), while, there was no significant difference of Nod2 (mRNA and protein) between groups. After 6 months desensitization therapy, the change of Nod1 in PBMC was negatively correlated with the change of IL-10 in the peripheral blood, r=-0.88, P<0.05; while, change of Nod1 was positively correlated, with the change of IL-6, r=0.57, P>0.05. CONCLUSION Nod1, Nod2 and Nalp3 expression were seen in the two groups,and the Nod1 expression in allergic rhinitis group was lower than other two groups, while, the Nalp3 was higher than other two groups. It showed Nod1, Nalp3 may be involved in the pathogenesis of allergic rhinitis. Expression of Nod1 in PBMC reduced after sublingual desensitization treatment. Besides, the change of Nod1 was negatively correlated with the change of IL-10 in PBMC. So,it seemed that Nod1 may regulate IL-10 changes and be involved in sublingual desensitization therapy.
Carrier Proteins ; metabolism ; Humans ; Interferon-gamma ; blood ; Interleukin-10 ; blood ; Interleukin-4 ; blood ; Interleukin-6 ; blood ; Leukocytes, Mononuclear ; metabolism ; NLR Family, Pyrin Domain-Containing 3 Protein ; Nasal Mucosa ; metabolism ; Nasal Polyps ; metabolism ; Nod1 Signaling Adaptor Protein ; metabolism ; Nod2 Signaling Adaptor Protein ; metabolism ; Receptors, Pattern Recognition ; metabolism ; Rhinitis, Allergic ; metabolism ; Turbinates ; metabolism

Bacterial biofilms have emerged as potential critical triggers in the pathogenesis of bisphosphonate (BP)-related osteonecrosis of the jaw (ONJ) or BRONJ. BRONJ lesions have shown to be heavily colonized by oral bacteria, most of these difficult to cultivate and presents many clinical challenges. The purpose of this study was to characterize the bacterial diversity in BRONJ lesions and to determine host immune response. We examined tissue specimens from three cohorts (n=30); patients with periodontal disease without a history of BP therapy (Control, n=10), patients with periodontal disease having history of BP therapy but without ONJ (BP, n=5) and patients with BRONJ (BRONJ, n=15). Denaturing gradient gel electrophoresis of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments revealed less bacterial diversity in BRONJ than BP and Control cohorts. Sequence analysis detected six phyla with predominant affiliation to Firmicutes in BRONJ (71.6%), BP (70.3%) and Control (59.1%). Significant differences (P<0.05) in genera were observed, between Control/BP, Control/BRONJ and BP/BRONJ cohorts. Enzyme-linked immunosorbent assay (ELISA) results indicated that the levels of myeloperoxidase were significantly lower, whereas interleukin-6 and tumor necrosis factor-alpha levels were moderately elevated in BRONJ patients as compared to Controls. PCR array showed significant changes in BRONJ patients with downregulation of host genes, such as nucleotide-binding oligomerization domain containing protein 2, and cathepsin G, the key modulators for antibacterial response and upregulation of secretory leukocyte protease inhibitor, proteinase 3 and conserved helix-loop-helix ubiquitous kinase. The results suggest that colonization of unique bacterial communities coupled with deficient innate immune response is likely to impact the pathogenesis of ONJ.
Actinobacteria ; classification ; Bacteria ; classification ; Bacteroidetes ; classification ; Biofilms ; Bisphosphonate-Associated Osteonecrosis of the Jaw ; immunology ; microbiology ; Bone Density Conservation Agents ; therapeutic use ; Cathepsin G ; analysis ; Cohort Studies ; Down-Regulation ; Female ; Fusobacteria ; classification ; Gram-Negative Bacteria ; classification ; Host-Pathogen Interactions ; immunology ; Humans ; I-kappa B Kinase ; analysis ; Immunity, Innate ; immunology ; Interleukin-6 ; analysis ; Male ; Middle Aged ; Mouth ; immunology ; microbiology ; Myeloblastin ; analysis ; antagonists & inhibitors ; Nod2 Signaling Adaptor Protein ; analysis ; Periodontal Diseases ; microbiology ; Peroxidase ; analysis ; Proteobacteria ; classification ; Tumor Necrosis Factor-alpha ; analysis

OBJECTIVEBlau syndrome (BS), an autosomal dominant inherited autoinflammatory disease, is caused by NOD2 mutations. This study aimed to analyze NOD2 gene of suspected BS patients to make definite diagnosis, find NOD2 mutation types and clinical features of Chinese BS cases, and find some clinical indications to identify BS by comparing BS and non-BS cases.

METHODEighteen suspected BS children (7 boys and 11 girls, age of first visit was from 1 y 8 m to 9 y 6 m) who visited Peking Union Medical College Hospital from 2006 to 2014 and their parents's DNA were extracted from 4 ml blood specimens. PCR was performed for exon 4 of NOD2 and PCR products were purified by 2% gel electrophoresis and sequenced directly. Role of novel missense mutations in pathogenicity was analyzed by SIFT and sequencing NOD 2 of fifty normal controls. Clinical data of BS children diagnosed by NOD2 analysis were summarized and compared with the data of non-BS group.

RESULT(1) Twelve of eighteen suspected BS children were diagnosed as BS by NOD2 analysis, and the remaining 6 were excluded. Seven missense mutations were detected, 4 were reported before: c.1000C>T, p. Arg 334Trp; c.1001G>A, p. Arg334Gln; c.1538T>C, p. Met513Thr; c.1759C>T, p. Arg587Cys. Three novel mutations were found: c. 1147 G>C, p.Glu383Gln; c.1471A>T, p. Met491Leu; c.2006A>G, p.His669Arg. (2) Chronic symmetric arthritis and multi-joints periarticular hydatoncus, which were painless with fluctuation, were found in all 12 BS children with NOD2 mutations. Skin rash, chronic symmetric arthritis, and recurrent uveitis were identified in 7 patients. Three patients had no skin rash, while 1 had no uveitis, 1 only had symmetric arthritis and multi-joints periarticular hydatoncus. Four children inherited the disease from father. (3) Compared with other 6 non-BS children, BS children had such different clinical characteristic (P < 0.05): All the BS cases had multiple periarticular hydatoncus, which always had no persistent fever, most had no elevated CRP, while non-BS group always had no hydatoncus, most had persistent fever, all had elevated CRP.

CONCLUSIONThe 12 BS children were diagnosed by NOD2 analysis; 7 missense mutations were detected, 3 were novel mutations, adding new findings to human NOD2 mutations. Although classic BS was characterized by skin rash, arthritis, and eye involvement, some presented with less than 3 of the classic features. Chronic symmetric arthritis and multi-joints periarticular hydatoncus were the most comment fetures. Comparing with non-BS group, all BS cases had multi hydatoncus surrounding multi-joints, always had no persistent fever, most had no elevated CRP. Those features may distinguish BS in clinical settings.

Arthritis ; etiology ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Case-Control Studies ; Child ; Child, Preschool ; Cranial Nerve Diseases ; complications ; genetics ; Exanthema ; etiology ; Exons ; genetics ; Female ; Humans ; Infant ; Male ; Mutation ; genetics ; Mutation, Missense ; Nod2 Signaling Adaptor Protein ; genetics ; Synovitis ; complications ; genetics ; Uveitis ; complications ; etiology ; genetics

Activation pattern recognition receptors can cause the startup of downstream signaling pathways, the expression of inflammatory factors, and finally immunological inflammatory reaction. Either exogenous pathogenic microorganisms or endogenous tissue components can activate these pattern recognition receptors as ligands at varying degrees, and then cause the immunological inflammatory reaction. Therefore, it is of great significance to inhibit relevant receptors, as well as the immunological inflammatory reaction, in order to avoid tissue injury during the course of disease. Baicalin is able to specifically inhibit the expression of TLR2/4-NOD2, inhibit the expression of inflammatory factors IL-1beta, IL-6 and TNF-alpha, and thereby reducing the injury of the tissue cells during the course of disease. This effect is non-specific with tissues, which is of great theoretical and practical significance in druggability. In addition, the drug metabolism and toxicity of baicalin are also discussed for its druggability in this article.
Animals ; Flavonoids ; pharmacology ; Humans ; Nod2 Signaling Adaptor Protein ; metabolism ; Toll-Like Receptor 2 ; metabolism ; Toll-Like Receptor 4 ; metabolism

The purpose of this study was to explore the effect of NOD2 signalling pathway activated by muramyl dipeptide (MDP) on the immunomodulation effect of human monocyte-derived dendritic cells (DC) loaded with leukemia cell lysates. Peripheral blood mononuclear cells (PBMNC) were isolated by density gradient centrifugation, These cells were cultured with three cytokines for 7 days to induce their maturation. On the 5th day, cells were loaded with leukemia cell HL-60 lysates. NOD2 expression was detected by RT-PCR and Western blot. The phenotype of DC were analyzed by flow cytometry, and ELISA was used to assay levels of IL-12 (p40) . The results showed that MDP could trigger NOD2 mRNA and protein expression in different groups of DC, especially in sensitized DC+MDP group, which was significantly higher than that in the DC+MDP group and sensitized DC without MDP stimulation, the difference was statistically significant (P < 0.05). Besides, the expression of surface molecules (HLA-DR, CD80, CD83, CD86, CD40) in the group of DC loaded with leukemia cell lysate and stimulated by MDP (sensitized DC+MDP) reached the highest level, followed by the group of DC loaded with leukemia cell lysate without MDP and DC only stimulated by MDP, non-treated DC were the lowest (P < 0.05). Similarly, compared with untreated unstimulated DC, after loading with HL-60 lysates or only stimulating with MDP, the secretion of IL-12p40 increased, but IL-12p40 level (573.86 ± 32.09 pg/ml) in DC+MDP group was higher than that in group of sensitized DC (365.03 ± 28.86 pg/ml) (P < 0.05), and it in sensitized DC+MDP group reached the highest (898.30 ± 61.08) pg/ml, compared to other groups (P < 0.05). It is concluded that MDP can significantly enhance the NOD2 mRNA and protein expression in sensitized DC, promote the expression of HLA-DR, synergistic costimulatory molecules and adhesion molecules of DC, at the same time, MDP can increase secretion of inflammatory factors IL-12p40. This study will provide a new ideas for DC application in leukemia immunotherapy.
Acetylmuramyl-Alanyl-Isoglutamine ; pharmacology ; Cells, Cultured ; Dendritic Cells ; immunology ; metabolism ; HL-60 Cells ; Humans ; Interleukin-12 Subunit p40 ; secretion ; Leukemia ; immunology ; metabolism ; Leukocytes, Mononuclear ; metabolism ; Membrane Proteins ; metabolism ; Nod2 Signaling Adaptor Protein ; metabolism

OBJECTIVE: To investigate the expression of NOD1 and NOD2 in the nasal mucosa of healthy individuals and allergic rhinitis(AR), and explored the regulation of glucocorticoids on them. METHOD: RT-PCR and immunohistochemistry were used to detect the expression of NOD1 and NOD2 in nasal mucosa from healthy control and AR. Nasal explant culture was used to explore the effect of glucocorticoids on NOD1 and NOD2 expression. RESULT: NOD1 and NOD2 mRNA expression level was significantly increased in AR compared with control. Immunohistochemical staining demonstrated that NOD1 and NOD2 were mainly expressed by epithelial cells and some unknown cells in lamina propria and there were significantly more positive staining cells were observed in AR tissue when compared with control. Glucocorticoids down-regulated NOD1 and NOD2 expression in AR. CONCLUSION NOD1 and NOD2 as two PRRs may take part in the pathogenesis of AR, glucocorticoids may play a therapeutical role on allergic rhinitis through down-regulated the expression of NOD1 and NOD2.
Adult ; Case-Control Studies ; Female ; Gene Expression Regulation ; Glucocorticoids ; pharmacology ; Humans ; Male ; Middle Aged ; Nasal Mucosa ; drug effects ; metabolism ; Nod1 Signaling Adaptor Protein ; metabolism ; Nod2 Signaling Adaptor Protein ; metabolism ; Rhinitis, Allergic ; Rhinitis, Allergic, Perennial ; metabolism ; Young Adult