Effect of nifedipine, a calcium channel blocker, on the generation of nitric oxide and interleukin-1ß by murine macrophages activated by lipopolysaccharide from Prevotella intermedia.

Nifedipine, a calcium channel blocker, has been reported to possess anti-inflammatory and immunosuppressive effects. The current study was undertaken to explore the influence of nifedipine on the generation of proinflammatory mediators by murine macrophages activated by lipopolysaccharide (LPS) prepared from Prevotella intermedia, a putative periodontal pathogen, and associated mechanisms of action as well. LPS was purified by employing phenol-water extraction protocol. Culture supernatants were analyzed for nitric oxide (NO) and interleukin (IL)-1ß. Real-time PCR and immunoblotting were conducted to quantify mRNA and protein expression, respectively. NF-κB-dependent secreted embryonic alkaline phosphatase (SEAP) levels were estimated by reporter assay. Nifedipine markedly suppressed the generation of iNOS-derived NO and IL-1ß together with their mRNA expressions in murine macrophages activated by P. intermedia LPS. LPS-stimulated cells exposed to nifedipine notably increased the mRNA levels of Arg-1, Ym-1, FIZZ1, and TGF-ß, which are typical markers for M2 macrophage polarization. Nifedipine induced HO-1 at both gene and protein levels in cells challenged with P. intermedia LPS, and the nifedipine-mediated inhibition of NO generation was significantly abrogated by adding SnPP, an HO-1 inhibitor. Nifedipine inhibited LPS-evoked generation of NO and IL-1ß in a PPAR-γ-independent manner. In addition, NF-κB activation as well as phosphorylation of STAT1/3 induced by P. intermedia LPS was suppressed by nifedipine. Nifedipine is an inhibitor of P. intermedia LPS-evoked production of NO and IL-1ß in murine macrophages and encourages macrophage polarization toward the M2 phenotype. Nifedipine possibly has potential to be used for host modulation of periodontal disease and is worth being further researched.

Tricarbonyldichlororuthenium(II) dimer, the lipid-soluble carbon monoxide-releasing molecule, attenuates Prevotella intermedia lipopolysaccharide-induced production of nitric oxide and interleukin-1ß in murine macrophages.

Carbon monoxide (CO) is increasingly being appreciated as an important mediator that has pleiotropic biological properties and appears to have a possible therapeutic application for a variety of disorders. Nevertheless, whether this gaseous molecule may be utilized as a therapeutic intervention for periodontal disease is unclear. Here, we examined the potential beneficial effect of CO-releasing molecule-2 (CORM-2), a tricarbonyldichlororuthenium(II) dimer, against the elaboration of proinflammatory mediators by murine macrophages challenged with lipopolysaccharide (LPS) isolated from Prevotella intermedia, a pathogenic bacterium implicated in inflammatory periodontal disease. We found that NO and IL-1ß production, iNOS protein expression and mRNA expressions of iNOS and IL-1ß were significantly down-regulated when LPS-challenged RAW264.7 cells were exposed to CORM-2. In addition, HO-1 expression was upregulated by CORM-2 in cells activated with P. intermedia LPS, and the inhibitory influence of CORM-2 upon NO production was attenuated by tin protoporphyrin IX, an inhibitor of HO activity. PPAR-γ did not function in the attenuation of NO and IL-1ß by CORM-2. JNK and p38 phosphorylation caused by LPS was not altered by CORM-2. CORM-2 reduced NF-κB reporter activity and IκB-α degradation elicited by P. intermedia LPS. Additionally, CORM-2 inhibited LPS-induced phosphorylation of STAT1/3. In conclusion, CORM-2 suppresses NO and IL-1ß production caused by P. intermedia LPS. CORM-2 exerts its effect by a mechanism involving anti-inflammatory HO-1 induction and attenuation of NF-κB and STAT1/3 activation, independently of PPAR-γ as well as JNK and p38. CORM-2 may hold promise as host response modulation agent for periodontal disease, though further research is indicated to verify the therapeutic effect.

Telmisartan, an angiotensin II receptor blocker, attenuates Prevotella intermedia lipopolysaccharide-induced production of nitric oxide and interleukin-1ß in murine macrophages.

Telmisartan, widely prescribed for the treatment of hypertension, has an anti-inflammatory property in addition to being an angiotensin II type 1 receptor antagonist. This study was carried out to explore the influence of telmisartan upon the elaboration of inflammatory mediators in murine macrophages stimulated with lipopolysaccharide (LPS) prepared from Prevotella intermedia, a periodontal pathogen, as well as its molecular mechanisms. Telmisartan significantly inhibited LPS-induced generation of inducible nitric oxide (NO) synthase-derived NO and interleukin-1ß (IL-1ß) as well as their gene expressions in RAW264.7 cells. Telmisartan treatment of LPS-activated cells significantly up-regulated arginase 1 (Arg-1) and chitinase-like 3 (Ym-1), which are specific markers of M2 macrophages. Telmisartan caused a significant increase in heme oxygenase-1 (HO-1) expression in cells stimulated with LPS, and its inhibitory action against NO production was reversed by treatment with SnPP, an HO-1 inhibitor. Phosphorylation of STAT1 and STAT3 induced by LPS was attenuated by telmisartan. Telmisartan inhibited LPS-induced generation of NO and IL-1ß independently of PPAR-γ activation. In addition, activation of NF-κB as well as JNK and p38 signaling induced by LPS was not modulated by telmisartan. In summary, telmisartan is a potent inhibitor of P. intermedia LPS-induced generation of NO and IL-1ß in RAW264.7 cells and promotes macrophage phenotype switching toward the M2 phenotype. Telmisartan may have potential to be developed into host modulatory agent for inflammatory periodontal disease, although additional studies are needed to confirm the therapeutic effect.

A Probiotic Mixture Regulates T Cell Balance and Reduces Atopic Dermatitis Symptoms in Mice.

Atopic dermatitis (AD) is a chronic inflammatory skin disorder with a complex etiology involving the immune response. Recent studies have demonstrated the role of certain probiotics in the treatment and prevention of AD. However, the mechanism by which these probiotics regulate the immune system remains unclear. In this study, we examined the immunomodulatory capacity of Duolac ATP, a mixed formulation of probiotics, both in vitro and in vivo. Results showed that the expression of programmed death-ligand 1(PD-L1) was significantly upregulated on bone marrow-derived dendritic cells (BMDCs) treated with Duolac ATP. Furthermore, the anti-inflammatory cytokines IL-10 and TGF-beta were both upregulated when BMDCs were treated with Duolac ATP. The percentage of proliferated regulatory T cells (Tregs) was enhanced when CD4+ T cells were co-cultured with Duolac ATP-treated BMDCs on plates coated with anti-CD3/CD28 antibodies. Intriguingly, IL-10 secretion from CD4+ T cells was also observed. The AD symptoms, histologic scores, and serum IgE levels in AD mice were significantly decreased after oral treatment with Duolac ATP. Moreover, the Th1-mediated response in AD-induced mice treated with oral Duolac ATP showed upregulation of IL-2 and IFN-gamma as well as of downstream signaling molecules T-bet, STAT-1, and STAT-4. Conversely, Duolac ATP suppressed Th2 and Th17 responses in AD-like mice, as evidenced by the downregulation of GATA-3, C-maf, IL-4, IL-5, and IL-17. Additionally, Duolac ATP increased the number of Tregs found at Peyer's patches (PP) in treated AD mice. These results suggest that Duolac ATP modulates DCs to initiate both Th1 and Treg responses in AD mice. Thus, Duolac ATP represents a potential preventative agent against AD and could serve as an effective immunomodulator in AD patients.

Josamycin suppresses Prevotella intermedia lipopolysaccharide-induced production of nitric oxide and interleukin-1ß in murine macrophages.

AIMS: Josamycin has immunomodulatory properties independent of its antibacterial actions. This study was designed to explore the influences and associated mechanisms of josamycin upon the generation of proinflammatory mediators in murine macrophages activated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogenic bacterium associated with periodontal disease. MAIN METHODS: LPS was purified by employing phenol-water extraction protocol. Culture supernatants were analyzed for nitric oxide (NO) and interleukin (IL)-1ß. Real-time PCR and immunoblotting were conducted to quantify mRNA and protein expression, respectively. The expression levels of IL-1ß were analyzed by confocal laser scanning microscopy. NF-κB-dependent SEAP levels were estimated by reporter assay. KEY FINDINGS: Josamycin significantly attenuated NO production elicited by P. intermedia LPS as well as induction of iNOS protein and mRNA in RAW264.7 cells. While the release of IL-1ß from cells stimulated by LPS was suppressed in the presence of josamycin, josamycin failed to reduce the degree of IL-1ß mRNA expression. Josamycin did not reduce the stability of IL-1ß mRNA induced by P. intermedia LPS, at the same time josamycin also failed to suppress the LPS-induced intracellular IL-1ß expression. Josamycin augmented HO-1 induction in cells exposed to P. intermedia LPS, and SnPP, an inhibitor of HO-1 activity, reversed the suppressive impact of josamycin upon NO generation induced by LPS. Josamycin diminished NF-κB transcriptional activity induced by P. intermedia LPS. Further, josamycin enhanced SOCS1 mRNA level in cells activated with LPS. SIGNIFICANCE: Josamycin suppressed P. intermedia LPS-induced generation of NO and IL-1ß in RAW264.7 macrophages via the inhibition of NF-κB activation as well as HO-1 and SOCS1 induction. Josamycin may have benefits as a host modulatory agent in treating periodontal disease.

Effect of nitric oxide-releasing derivative of indomethacin on Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages.

The purpose of this study was to investigate the influences of NCX 2121, a nitric oxide (NO)-releasing derivative of indomethacin, upon the generation of proinflammatory mediators using murine macrophages activated by lipopolysaccharide (LPS) isolated from Prevotella intermedia, which is one of the pathogens implicated in periodontal diseases. Inducible NO synthase (iNOS)-derived NO, IL-1ß and IL-6 as well as their relevant mRNA were significantly attenuated by NCX 2121 in RAW264.7 cells activated by P. intermedia LPS. NCX 2121 was much more effective than the parental compound indomethacin in reducing these proinflammatory mediators. NCX 2121 triggered induction of heme oxygenase-1 (HO-1) in cells exposed to P. intermedia LPS, and its inhibitory influence upon P. intermedia LPS-elicited NO generation was notably blocked by SnPP treatment. NCX 2121 attenuated NF-κB-dependent SEAP release induced by P. intermedia LPS. NCX 2121 did not display inhibitory action towards IκB-α degradation triggered by LPS. Instead, it significantly diminished nuclear translocation as well as DNA-binding action of NF-κB p50 subunit elicited by P. intermedia LPS. Further, NCX 2121 significantly up-regulated SOCS1 mRNA expression in cells challenged with P. intermedia LPS. In summary, NCX 2121 down-regulates P. intermedia LPS-elicited generation of NO, IL-1ß and IL-6 in murine macrophages in a mechanism that involves anti-inflammatory HO-1 induction as well as decrement of NF-κB activation, which may be associated with SOCS1 expression. NCX 2121 may have potential benefits as a host immunomodulatory agent for the therapy of periodontal disease.

Agomelatine, a MT1/MT2 melatonergic receptor agonist with serotonin 5-HT2C receptor antagonistic properties, suppresses Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages.

OBJECTIVE: This study was performed in an attempt to examine the influence of agomelatine in mitigating the generation of proinflammatory mediators in RAW264.7 murine macrophages exposed to lipopolysaccharide (LPS) obtained from Prevotella intermedia, a gram-negative anaerobic bacterium that is related with various types of periodontal diseases, and the molecular mechanisms behind its effects. DESIGN: LPS from P. intermedia strain ATCC 25611 was prepared employing the conventional phenol-water procedure. Conditioned culture media were analyzed for the levels of nitric oxide (NO), interleukin-1ß (IL-1ß) and IL-6. Real-time PCR analysis was carried out to determine the mRNA levels of inducible NO synthase (iNOS), IL-1ß, IL-6 and SOCS1. Protein expression levels were evaluated by immunoblot test. NF-κB-dependent SEAP reporter assay was performed using a reporter cell line. DNA-binding activities of NF-κB subunits were analyzed utilizing the ELISA-based kits. RESULTS: Agomelatine was found to down-regulate significantly the generation of iNOS-derived NO, IL-1ß and IL-6 as well as the expression of their mRNAs in cells activated with P. intermedia LPS. Agomelatine decreased NF-κB-dependent SEAP release caused by P. intermedia LPS. Agomelatine did not inhibit NF-κB transcription induced by LPS at the level of IκB-α degradation. Instead, LPS-induced nuclear translocation and DNA binding of NF-κB p50 subunit was blocked by agomelatine. P. intermedia LPS-elicited activation of STAT1 and STAT3 was reduced notably by co-treatment with agomelatine. Agomelatine showed a tendency to enhance mRNA level of SOCS1 in LPS-activated cells as well. CONCLUSIONS: Agomelatine merits further evaluation to reveal its usefulness on the host modulation of periodontal disease.

Genistein suppresses Prevotella intermedia lipopolysaccharide-induced inflammatory response in macrophages and attenuates alveolar bone loss in ligature-induced periodontitis.

OBJECTIVE: Genistein is a major isoflavone subclass of flavonoids found in soybean and a potent tyrosine kinase inhibitor. The present study aimed to assess the effect of genistein on the production of proinflammatory mediators in murine macrophages stimulated with lipopolysaccharide (LPS) isolated from Prevotella intermedia, a pathogen associated with different forms of periodontal disease, and to evaluate its possible influence on alveolar bone loss in ligature-induced periodontitis using micro-computed tomography (micro-CT) analysis as well. DESIGN: LPS was isolated from P. intermedia ATCC 25611 by using the standard hot phenol-water method. Culture supernatants were analyzed for nitric oxide (NO) and interleukin-6 (IL-6). Inducible NO synthase (iNOS) protein expression was evaluated by immunoblot analysis. Real-time PCR was carried out to measure iNOS and IL-6 mRNA expression. In addition, effect of genistein on alveolar bone loss was evaluated in a rat model of experimental periodontitis using micro-CT analysis. RESULTS: Genistein significantly attenuated P. intermedia LPS-induced production of iNOS-derived NO and IL-6 with attendant decrease in their mRNA expression in RAW264.7 cells. In addition, when genistein was administered to rats, decreases in alveolar bone height and bone volume fraction induced by ligature placement were significantly inhibited. Genistein administration also prevented ligature-induced alterations in the microstructural parameters of trabecular bone, including trabecular thickness, trabecular separation, bone mineral density and structure model index. CONCLUSIONS: While additional studies are required, we suggest that genistein could be utilized for the therapy of human periodontitis in the future.

NCX 4040, a nitric oxide-donating aspirin derivative, inhibits Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages.

In this study, the effects and underlying mechanisms of NCX 4040, a nitric oxide (NO)-donating aspirin derivative, on the production of proinflammatory mediators were examined using murine macrophages exposed to lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in the etiology of periodontal disease. NCX 4040 significantly reduced P. intermedia LPS-induced production of inducible NO synthase (iNOS)-derived NO, IL-1ß and IL-6 as well as their mRNA expression in RAW264.7 cells. Notably, NCX 4040 was much more effective than the parental compound aspirin in reducing LPS-induced production of inflammatory mediators. NCX 4040 induced the expression of heme oxygenase-1 (HO-1) in cells treated with P. intermedia LPS, and the suppressive effect of NCX 4040 on LPS-induced NO production was significantly reversed by SnPP, a competitive HO-1 inhibitor. NCX 4040 did not influence LPS-induced phosphorylation of JNK and p38. IκB-α degradation as well as nuclear translocation and DNA-binding activities of NF-κB p65 and p50 subunits induced by P. intermedia LPS were significantly reduced by NCX 4040. Besides, LPS-induced phosphorylation of STAT1 and STAT3 was significantly down-regulated by NCX 4040. Further, NCX 4040 elevated the SOCS1 mRNA in cells stimulated with LPS. This study indicates that NCX 4040 inhibits P. intermedia LPS-induced production of NO, IL-1ß and IL-6 in murine macrophages through anti-inflammatory HO-1 induction and suppression of NF-κB, STAT1 and STAT3 activation, which is associated with the activation of SOCS1 signaling. NCX 4040 could potentially be a promising tool in the treatment of periodontal disease, although further studies are required to verify this.

Carbon monoxide-releasing molecule-3 suppresses Prevotella intermedia lipopolysaccharide-induced production of nitric oxide and interleukin-1ß in murine macrophages.

This study was performed to analyze the effect of carbon monoxide (CO)-releasing molecule-3 (CORM-3) in alleviating the production of proinflammatory mediators in macrophages treated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen associated with periodontal disease, and its possible mechanisms of action. LPS was isolated using the hot phenol-water method. Culture supernatants were assayed for nitric oxide (NO) and interleukin-1ß (IL-1ß). Gene expression was quantified by real-time PCR, and protein expression by immunoblotting. DNA-binding activities of NF-κB subunits were determined using an ELISA-based kit. CORM-3 suppressed the production of inducible NO synthase (iNOS)-derived NO and IL-1ß at both gene transcription and translation levels in P. intermedia LPS-activated RAW264.7 cells. CORM-3 enhanced heme oxygenase-1 (HO-1) expression in cells stimulated with P. intermedia LPS, and inhibition of HO-1 activity by SnPP notably reversed the suppressive effect of CORM-3 on LPS-induced production of NO. LPS-induced phosphorylation of p38 and JNK was not affected by CORM-3. CORM-3 did not influence P. intermedia LPS-induced degradation of IκB-α. Instead, nuclear translocation of NF-κB p65 and p50 subunits was blocked by CORM-3 in LPS-treated cells. In addition, CORM-3 reduced LPS-induced p65 and p50 binding to DNA. Besides, CORM-3 significantly suppressed P. intermedia LPS-induced phosphorylation of STAT1. Overall, this study indicates that CORM-3 suppresses the production of NO and IL-1ß in P. intermedia LPS-activated murine macrophages via HO-1 induction and inhibition of NF-κB and STAT1 pathways. The modulation of host inflammatory response by CORM-3 would be an attractive therapeutic approach to attenuate the progression of periodontal disease.