NLRP3 Inflammasome as Therapeutic Targets in Inflammatory Diseases

Innate immunity is a first line defence system in the body which is for sensing signals of danger such as pathogenic microbes or host-derived signals of cellular stress. Pattern recognition receptors (PRR’s), which present in the cell memebrane, are suspect the infection through pathogen-associated molecular patterns (PAMP), and activate innate immunity with response to promote inflammation via inflammatory cells such as macrophages and neutrophils, and cytokines. Inflammasome are protein complexes which are part of innate immunity in inflammation to remove pathogens and repair damaged tissues. What is the important role of inflammation in disease? In this review, we are focused on the action mechanism of NLRP3 inflammasome in inflammatory diseases such as asthma, atopic dermatitis, and sepsis.

Cell Autonomous Circadian Systems and Their Relation to Inflammation

All living beings on earth have an important mechanism of 24-h periodicity, which controls their physiology, metabolism, and behavior. In humans, 24-h periodicity is regulated by the superchiasmatic nucleus (SCN) through external and environmental cues.Peripheral organs demonstrate circadian rhythms and circadian clock functions, and these are also observed in cultured cell lines.Every cell contains a CLOCK: BMAL1 loop for the generation of circadian rhythms. In this review, we focused on cell autonomous circadian rhythms in immune cells, the inflammatory diseases caused by disruption of circadian rhythms in hormones, and the role of clock genes in inflammatory diseases.

Repositioned Drugs for Inflammatory Diseases such as Sepsis, Asthma, and Atopic Dermatitis

The process of drug discovery and drug development consumes billions of dollars to bring a new drug to the market. Drug development is time consuming and sometimes, the failure rates are high. Thus, the pharmaceutical industry is looking for a better option for new drug discovery. Drug repositioning is a good alternative technology that has demonstrated many advantages over de novo drug development, the most important one being shorter drug development timelines. In the last two decades, drug repositioning has made tremendous impact on drug development technologies. In this review, we focus on the recent advances in drug repositioning technologies and discuss the repositioned drugs used for inflammatory diseases such as sepsis, asthma, and atopic dermatitis.

Rifampicin Alleviates Atopic Dermatitis-Like Response in vivo and in vitro

Atopic dermatitis (AD) is a common inflammatory skin disorder mediated by inflammatory cells, such as macrophages and mast cells. Rifampicin is mainly used for the treatment of tuberculosis. Recently, it was reported that rifampicin has anti-inflammatory and immune-suppressive activities. In this study, we investigated the effect of rifampicin on atopic dermatitis in vivo and in vitro. AD was induced by treatment with 2, 4-dinitrochlorobenzene (DNCB) in NC/Nga mice. A subset of mice was then treated with rifampicin by oral administration. The severity score and scratching behavior were alleviated in the rifampicin-treated group. Serum immunoglobulin E (IgE) and interleukin-4 (IL-4) levels were also ameliorated in mice treated with rifampicin. We next examined whether rifampicin has anti-atopic activity via suppression of mast cell activation. Rifampicin suppressed the release of β-hexosaminidase and histamine from human mast cell (HMC)-1 cultures stimulated with compound 48/80. Treatment with rifampicin also inhibited secretion of inflammatory mediators, such tumor necrosis factor-α (TNF-α) and prostaglandin D₂ (PGD₂), in mast cells activated by compound 48/80. The mRNA expression of cyclooxygenase 2 (COX-2) was reduced in the cells treated with rifampicin in a concentration-dependent manner. These results suggest that rifampicin can be used to treat atopic dermatitis.

Rifampicin Alleviates Atopic Dermatitis-Like Response in vivo and in vitro

Atopic dermatitis (AD) is a common inflammatory skin disorder mediated by inflammatory cells, such as macrophages and mast cells. Rifampicin is mainly used for the treatment of tuberculosis. Recently, it was reported that rifampicin has anti-inflammatory and immune-suppressive activities. In this study, we investigated the effect of rifampicin on atopic dermatitis in vivo and in vitro. AD was induced by treatment with 2, 4-dinitrochlorobenzene (DNCB) in NC/Nga mice. A subset of mice was then treated with rifampicin by oral administration. The severity score and scratching behavior were alleviated in the rifampicin-treated group. Serum immunoglobulin E (IgE) and interleukin-4 (IL-4) levels were also ameliorated in mice treated with rifampicin. We next examined whether rifampicin has anti-atopic activity via suppression of mast cell activation. Rifampicin suppressed the release of β-hexosaminidase and histamine from human mast cell (HMC)-1 cultures stimulated with compound 48/80. Treatment with rifampicin also inhibited secretion of inflammatory mediators, such tumor necrosis factor-α (TNF-α) and prostaglandin D₂ (PGD₂), in mast cells activated by compound 48/80. The mRNA expression of cyclooxygenase 2 (COX-2) was reduced in the cells treated with rifampicin in a concentration-dependent manner. These results suggest that rifampicin can be used to treat atopic dermatitis.

Chitin from Cuttlebone Activates Inflammatory Cells to Enhance the Cell Migration

Our previous report showed that the extract from cuttlebone (CB) had wound healing effect in burned lesion of rat and the extract was identified as chitin by HPLS analysis. We herein investigated the morphology in CB extract using scanning electron microscope (SEM). Chitin was used as a control. There is no difference in morphology between CB extract and chitin. We also assessed the role of CB extract on the production of inflammatory mediators using murine macrophages and the migration of inflammatory cells. The extract induced the production of nitric oxide (NO) in macrophages. While the extract of CB itself stimulated macrophages to increase the expression of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6, CB extract suppressed the production of those cytokines by LPS. CB extract also induced the production of mouse IL-8 which is related to the cell migration, and treatment with CB enhanced fibroblast migration and invasion. Therefore, our results suggest that CB activates inflammatory cells to enhance the cell migration.

Expression of Nucleotide-oligomerization Domain (NOD) and Related Genes in Mouse Tissues Infected with Mycobacterium leprae

The nucleotide-oligomerization domain (NOD) is an important molecule involved in host defense against bacterial infection. To study the role of NODs in the host response to Mycobacterium leprae, we measured the mRNA levels of NODs and related genes in infected mouse tissues. The mRNA expression of NOD1, NOD2, caspase-1 and ASC was increased in mouse footpads. Whereas NOD2 expression in macrophages was increased at 2 and 24 h post-infection with M. leprae, there was no expression of NOD1 at these time points. An increase in caspase-1 expression was observed at 2 h and continued at 24 h. However, the expression of ASC was increased only at the early time point. The expression of caspase-1 is regulated by NOD2-dependent pathway in established HEK 293. Our results suggest NOD2, rather than NOD1, may be associated with the host response to M. leprae and that caspase-1 activation is essential for the host response.

Chitin from the Extract of Cuttlebone Induces Acute Inflammation and Enhances MMP1 Expression

We previously reported that the extract from cuttlebone (CB) has wound healing effect in burned lesion of rat. In present study, the main component of CB extract was analyzed and its wound healing activity was evaluated by using in vitro acute inflammation model. The extract of CB stimulated macrophages to increase the production of TNF-alpha. The extract also enhanced the production of TGF-beta and VEGF, which were involved in angiogenesis and fibroblast activation. The treatment with CB extract enhanced proliferation of murine fibroblast. CB extract also induced the activation of fibroblast to increase the secretion of matrix metalloproteases 1 (MMP1). The constituent of CB extract which has wound healing activity was identified as chitin by HPLC analysis. The mechanism that the CB extract helps to promote healing of burned lesion is associated with that chitin in CB extracts stimulated wound skins to induce acute inflammation and to promoted cell proliferation and MMP expression in fibroblast. Our results suggest that CB or chitin can be a new candidate material for the treatment of skin wound such as ulcer and burn.

Polyacetylene Compound from Cirsium japonicum var. ussuriense Inhibited Caspase-1-mediated IL-1beta Expression

Our previous report showed that polyacetylene compound, 1-Heptadecene-11, 13-diyne-8, 9, 10-triol (PA) from the root of Cirsium japonicum var. ussuriense has anti-inflammatory activity. In this study we investigated the role of the PA as inhibitor of caspase-1, which converts prointerleukin-1beta (proIL-1beta) to active IL-1beta and is activated by inflammasome involved in the inflammatory process. We tested the effect of PA on the production of pro-inflammatory cytokines, IL-1beta in murine macrophage cell line, RAW264.7. PA inhibited lipopolysaccharide (LPS)-induced IL-1beta production by macrophages at a dose dependent manner. PA also suppressed the activation of caspase-1. The mRNA level of ASC (apoptosis-associated spec-like protein containing a CARD), an important adaptor protein of inflammasome, was decreased in the PA treated group. Therefore our results suggest that the anti-inflammatory effect of PA is due to inhibit the caspase-1 activation.

The Role of the Ethylacetate Fraction from Hydnocarpi Semen in Acute Inflammation In Vitro Model

We previously reported that Hydnocarpi Semen (HS) has a wound healing effect on diabetic foot ulcer lesion in mice. In this study, ethylacetate (EtOAc) fraction from HS extract were evaluated for their wound healing activity by using in vitro acute inflammation model. GC and GC/MS analysis shows that the main constituents in EtOAc fraction are chaulmoogric acid, hydnocarpic acid, and gorlic acid. EtOAc fraction activated macrophages to increase the production of TNF-alpha. The fraction also increased the production of TGF-beta and VEGF, which induced fibroblast activation and angiogenesis. These results suggest that the mechanism that the fraction helps to enhance healing of skin wound is possibly associated with the production of TNF-alpha, as well as secretion of VEGF, TGF-beta and HS may have a new bioactive material for the treatment of skin wound.

Vitamin D Receptor Gene TaqI, BsmI and FokI Polymorphisms in Korean Patients with Tuberculosis

BACKGROUND: The active metabolite (1, 25-dihydroxycholecalciferol) of vitamin D (25-hydroxycholecalciferol) leads to activation of macrophages and deficiency of vitamin D seems to be involved in the risk of tuberculosis. The effects of vitamin D are exerted by interaction with the vitamin D receptor (VDR) and may be influenced by polymorphism in the VDR gene. In this study, variation in the VDR gene was investigated in Korean population with tuberculosis. METHODS: We typed three VDR polymorphisms of restriction endonuclease sites for TaqI, BsmI and FokI in 155 patients with tuberculosis and 105 healthy volunteers. RESULTS: The frequencies of FokI genotypes determined from TB patients were 29.13% for FF, 56.31% for Ff, and 14.56% for ff. We observed 1.4-fold increased prevalence of the Ff genotype in TB patients compared with normal healthy groups (p=0.0857). However, there was no significant association between the genotype groups, TB patient and normal control, for FokI polymorphism. There was also no significant association between VDR gene and tuberculosis in another polymorphism (BsmI and TaqI). CONCLUSION: Three polymorphisms (TaqI, BsmI and FokI) in the VDR gene do not appear to be responsible for host susceptibility to human tuberculosis in Korean population.

Vitamin D Receptor Gene TaqI, BsmI and FokI Polymorphisms in Korean Patients with Tuberculosis

BACKGROUND: The active metabolite (1, 25-dihydroxycholecalciferol) of vitamin D (25-hydroxycholecalciferol) leads to activation of macrophages and deficiency of vitamin D seems to be involved in the risk of tuberculosis. The effects of vitamin D are exerted by interaction with the vitamin D receptor (VDR) and may be influenced by polymorphism in the VDR gene. In this study, variation in the VDR gene was investigated in Korean population with tuberculosis. METHODS: We typed three VDR polymorphisms of restriction endonuclease sites for TaqI, BsmI and FokI in 155 patients with tuberculosis and 105 healthy volunteers. RESULTS: The frequencies of FokI genotypes determined from TB patients were 29.13% for FF, 56.31% for Ff, and 14.56% for ff. We observed 1.4-fold increased prevalence of the Ff genotype in TB patients compared with normal healthy groups (p=0.0857). However, there was no significant association between the genotype groups, TB patient and normal control, for FokI polymorphism. There was also no significant association between VDR gene and tuberculosis in another polymorphism (BsmI and TaqI). CONCLUSION: Three polymorphisms (TaqI, BsmI and FokI) in the VDR gene do not appear to be responsible for host susceptibility to human tuberculosis in Korean population.

The Role of Intracellular Receptor NODs for Cytokine Production by Macrophages Infected with Mycobacterium leprae

The nucleotide-oligomerization domain (NOD) proteins are members of the NOD-like receptor (NLR) family, which are intracellular and cytoplasmic receptors. We analyzed the role of NODs for cytokine production by macrophages infected with intracellular pathogen M. leprae, the causative agent of leprosy. Production of pro-inflammatory cytokines such as IL-1beta and TNF-alpha was inhibited in the presence of cytochalasin D, an agent blocking phagocytosis, suggesting that intracellular signaling was, partially, required for macrophage activation to M. leprae infection. Next, we investigated the role of NOD1 and NOD2 proteins on NF-kappaB activation and cytokine expression. Treatment with M. leprae significantly increased NF-kappaB activation and expression of TNF-alpha and IL-1beta in NOD1- and NOD2-transfected cells. Interestingly, their activation and expression were inhibited by cytochalasin D, suggesting that stimulation of NOD proteins may be associated with the enhancement of cytokine production in host to M. leprae.

Long-term Outcome of Endopyelotomy for the Treatment of Ureteropelvic Junction Obstruction / 대한비뇨기과학회지

Purpose: The treatment outcome of endopyelotomy is now being re-evaluated in the era of laparoscopic pyeloplasty. This study was performed to evaluate the long-term success rate of an endopyelotomy for the treatment of an ureteropelvic junction obstruction (UPJO). Materials and Methods: Between January 1995 and December 2003, 77 patients with an UPJO (mean age 35.2+/-13.89) underwent 85 endopyelotomies, with a percutaneous approach in 10 and a retrograde approach in the other 75 cases. The mean number of the procedure was 1.14, with 69 patients undergoing a single procedure. Endopyelotomies were performed using either a cold knife (n=26), Ho:YAG laser (n=47) or a hook electrode (n=12). Treatment success was defined as symptomatic relief, with radiographic resolution or stabilization of renal function in the excretory and/or diuretic renograms. A Kaplan-Meier curve of the long-term success was generated, and the preoperative hydronephrosis and renal function correlated with the success rate. Results: With a median follow-up of 37.1 months (3-98), the overall success rate was 67.5%. The median time to failure was 7.7 months (1-50). The Kaplan-Meier estimates of success were 87.8, 76.9, 72.2, 68.7, 64.8 and 61.6% at 1, 12, 18, 24, 36 and 60 months, respectively. The success rate was not significantly affected by the etiology, the approach and the incisional methods. The degree of hydronephrosis and renal function showed no significant correlation with the success rate. Conclusions: The success rates of an endopyelotomy decreased with increasing follow-up period. In our conclusion, a long-term follow-up, at least as long as 36 months, is mandatory in patients having undergone an endopyelotomy for an ureteropelvic junction obstruction, although the majority of failures were found within 1 year.

Expression of Bcl-2 and Bax in cis-Diamminedichloroplatinum (II)-Resistant Bladder Cancer Cell Lines / 대한비뇨기과학회지

PURPOSE: Development of drug resistance has been the major obstacle in cis-Diamminedichloroplatinum (II) (cisplatin)-based combination chemotherapy in the treatment of advanced bladder cancer for which a variety of mechanisms has been suggested. We investigated to determine the changes of expression of apoptotic regulator proteins Bcl-2 and Bax in cisplatin-resistant bladder cancer cell lines and the reversibility of chemoresistance with antisense oligonucleotide against Bcl-2. MATERIALS AND METHODS: In T24, J82, 253J, 253J-BV and HT-1376 bladder cancer cell lines, we established cisplatin-resistance using stepwise exposure to cisplatin. The changes of Bcl-2 and Bax proteins in the resistant cell lines were determined by Western blot. Then, after administration of antisense oligonucleotide targeting the Bcl-2 coding sequence to the T24, T24-R1, and T24-R2 cell lines with lipofectamine, changes of Bcl-2 expression were determined along with cisplatin cytotoxicity before and after transfection. RESULTS: We confirmed the acquisition of cisplatin resistance in all 5 cell lines as the percent increase of IC50 in each cell lines were 210%, 175%, 181%, 280% and 153%, respectively. The expression of Bcl-2 protein increased in all 5 cisplatin-resistant cell lines, while the expressions of Bax decreased in 4 of 5 cisplatin-resistant cell lines. Treatment with antisense oligonucleotide significantly enhanced the cytotoxicity of cisplatin in T24, T24-R1 and T24-R2 cell lines. CONCLUSIONS: These results suggest that the up-regulation of Bcl-2 expression as well as down-regulation of Bax expression may be one of the mechanisms of cisplatin resistance in bladder cancer cells, and antisense Bcl-2 oligonucleotide may be helpful in chemotherapy of bladder cancer by reversing cisplatin resistance.

Comparison of rpoT gene of M. leprae strain from korean and foreign leprosy patients / 나학회지

The variance of tandem repeats in the rpoT gene of Mycobacterium leprae was recently demonstrated. The objects of this study was to examine the proportion and distributions of the genotypes of M. leprae in Korea and to compared it with genotypes of M. leprae form foreign leprosy patients using difference of the tandem repeats. Among 101 cases, 72 isolated from Korea and 4 cases from Japan (except Okinawa) demonstrated four copies of the 6 bp tandem repeats in the rpoT gene, and three copies were found in isolates from two korean, 2 cases of Okinawa in Japan, and those from Southeast Asian countries, Peru and Paraguay. These results reveal the genetic diversity of M. leprae and the related genotype-specific distribution in the world. In this study, a more detailed explanation can be also possible regarding the transmission route of M. leprae.

Detection of point mutation at C-terminal region of phagosomal coat protein (TACO) in patients with leprosy / 나학회지

Mycobacteria, which are highly successful pathogen, resist delivary to lysosomes and instead survive within a specialized vacuole, the mycobacterial phagosome. The bacteria survive intracellularly because they are able to actively recruit and retain TACO ( tryptophane aspartate-containing coat protein ) at the mycobacterial phagosome, where it prevents lysosomal delivary in a cholesterol-dependent manner. In this study, we investigated the difference of TACO expression is whether related to mutant in coro1a gene in patients with leprosy and normal volunteer. First, we screened for detection of a mutant in the leucine zipper motif within the exon 11, and then in the exon 9 to 10, and finally in the coiled-coil region. Interestingly, single base substitutions ( point mutation ) presents at assembly site of U1 snRNP, around of 5' splice site in the intron 9, there are a C to T and G to A transition are at 9 bp and 14 bp downstream of 5' splice site, respectively, and both of it. Among the 3 types of polymorphism, frequency of a G to A transition is markedly increased in patients of lepromatous type, which are new cases or relapsed. Both a C to T and G to A transitions are found in 1 case of tuberculoid type and 2 cases in lepromatoue type, but not found in control group. The silent mutation in leucine zipper motif within the exon 11 is located at codon at 454 ( CTG-->CTA), which is 1st leucine from C-terminal among four leucine zipper. In coiled-coil region, no mutation is found in genomic DNA of patients with leprosy. Further, we will do functional study about the identified point mutation and will screen any possible mutation in the region of promotor and WD repeat.

Mutations in folP1, rpoB, and gyrB associated with multiple drug resistance in Mycobacterium leprae isolates from South Korea / 나학회지

The emergence of multiple drug resistant Mycobacterium leprae has emphasized the need for early decision of effective antileprosy drug in the treatment for leprosy patients. Mutations in the genes associated with multiple drug resistance in Mycobacterium leprae isolates from 17 South Korean patients, who were already confirmed to have mutations in folP1 gene, were investigated using a PCR - single strand conformation polymorphism (SSCP) - DNA sequencing assay. Two strains, which has double mutations, were found in the two unrelated patients : one missense mutation in folP1 (Arg 55 for Pro) and in rpoB (Gly 522 for Ser), and in folP1 (Ala 53 for Thr) and in gyrB (Asn 426 for Asp), respectively. The patients were treated with the long monotheraphy of dapsone or with the inappropriate regimen of antileprosy drugs. These results emphasize the importance of multi-drug theraphy in order to prevent mult-idrug resistance and assist in the choice of the appropriate regimens for treating leprosy.

The association study on infection of Mycobacterium leprae and RIPK2 / 나학회지

Receptor-interacting serine/threonine kinase 2(RIPK2) is an adaptor molecule involved in the signal pathway of TLRs. However, there is no report on association between RIPK2 expression and infectious disease including mycobacterial disease in which TLRs play main role on interaction of infection. We evaluated relationship between Mycobacterium leprae and RIPK 2 by real-time RT-PCR. This study revealed that RIPK2 expression was down-regulated in the footpads and skin but was up-regulated in the liver, lymph node, and spleen of Mycobacterium leprae-infected nu/nu mice compared with those of non-infected nu/nu mice. It was observed that the IL-12p40, IFN-gamma, and IL-18 involved in the susceptibility of Mycobacterium leprae were down-regulated in the skin and footpad but up-regulated in the liver. These results suggest that regulation of RIPK2 expression is tissue-specific and is associated with M. leprae infection.

The effect of TLR2 mutation in IL-12 production according to type of leprosy / 나학회지

Recently, many reports suggest that the activation of Toll-like receptor 2 (TLR2) by microbial lipoproteins may initiate innate defense mechanism against infectious pathogens. Especially, TLR2 is critical in the immune response to mycobacterial infections and the mutations in the TLR2 have been shown to confer the susceptibility to severe infection with mycobacteria. A previous study reported that there is a mutation of TLR2 (TLR2-R677W) in lepromatous leprosy. In this study, thus, we performed the functional study on TLR2 by measurement of IL-12 production in serum and monocytes from leprosy patients with TLR2 mutation (R677W) to verify the relation between TLR2 and the susceptibility to leprosy. Tuberculoid leprosy patients showed higher serum levels of IL-12p70 compared with those of lepromatous leprosy. In contrast with the lepromatous leprosy patient with TLR2 mutation, especially, the group with wild-type TLR2 showed 2-fold increase levels of IL-12p70. Functional studies demonstrated that monocytes from patient with the TLR2 mutation, in comparison to the wild-type TLR2, is significantly less responsive to Mycobacterium leprae lysate. Our results reveal that TLR2 has a important role in the IL-12 production from monocytes and the susceptibility of lepromatous leprosy.