Ognjen Čulić

Anti-inflammatory effects of macrolide antibiotics.

Macrolides are widely used as antibacterial drugs. Clinical and experimental data, however, indicate that they also modulate inflammatory responses, both contributing to the treatment of infective diseases and opening new opportunities for the therapy of other inflammatory conditions. Considerable evidence, mainly from in vitro studies, suggests that leukocytes and neutrophils in particular, are important targets for modulatory effects of macrolides on host defense responses. This underlies the use of the 14-membered macrolide erythromycin for the therapy of diffuse panbronchiolitis. A variety of other inflammatory mediators and processes are also modulated by macrolides, suggesting that the therapeutic indications for these drugs may be extended significantly in future.

Azithromycin modulates neutrophil function and circulating inflammatory mediators in healthy human subjects

Effects on human neutrophils and circulating inflammatory mediators were studied in 12 volunteers who received azithromycin (500 mg/day, p.o.) for 3 days. Blood was taken 1 h before treatment, 2.5, 24 h and 28 days after the last dose. An initial neutrophil degranulating effect of azithromycin was reflected in rapid decreases in azurophilic granule enzyme activities in cells and corresponding increases in serum. The oxidative response to a particulate stimulus was also acutely enhanced. These actions were associated with high plasma and neutrophil drug concentrations. A continuous fall in chemokine and interleukin-6 serum concentrations, within the non-pathological range, accompanied a delayed down-regulation of the oxidative burst and an increase in apoptosis of neutrophils up to 28 days after the last azithromycin dose. Neutrophils isolated from blood at this time point still contained detectable drug concentrations. Acute neutrophil stimulation could facilitate antibacterial effects of azithromycin, while delayed, potentially anti-inflammatory activity may curtail deleterious inflammation.

Azithromycin and Clarithromycin Inhibit Lipopolysaccharide-Induced Murine Pulmonary Neutrophilia Mainly through Effects on Macrophage-Derived Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-1β

Macrolide antibiotics possess immunomodulatory/anti-inflammatory properties. These properties are considered fundamental for the efficacy of macrolide antibiotics in the treatment of chronic inflammatory diseases like diffuse panbronchiolitis and cystic fibrosis. However, the molecular mechanisms and cellular targets of anti-inflammatory/immunomodulatory macrolide activity are still not fully understood. To describe anti-inflammatory effects of macrolides in more detail and to identify potential biomarkers of their activity, we have investigated the influence of azithromycin and clarithromycin on the inflammatory cascade leading to neutrophil infiltration into lungs after intranasal lipopolysaccharide challenge in mice. Azithromycin and clarithromycin pretreatment reduced total cell and neutrophil numbers in bronchoalveolar lavage fluid and myeloperoxidase concentration in lung tissue. In addition, concentrations of several inflammatory mediators, including CCL2, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-1β (IL-1β), tumor necrosis factor α, and sE-selectin in lung homogenates were decreased after macrolide treatment. Inhibition of cytokine production observed in vivo was also corroborated in vitro in lipopolysaccharide-stimulated monocytes/macrophages, but not in an epithelial cell line. In summary, results presented in this article confirm that macrolides can suppress neutrophil-dominated pulmonary inflammation and suggest that the effect is mediated through inhibition of GM-CSF and IL-1β production by alveolar macrophages. Besides GM-CSF and IL-1β, CCL2 and sE-selectin are also identified as potential biomarkers of macrolide anti-inflammatory activity in the lungs.

NLRP3 Inflammasome Is Activated in Fibromyalgia: The Effect of Coenzyme Q10

AIMS Fibromyalgia (FM) is a prevalent chronic pain syndrome characterized by generalized hyperalgesia associated with a wide spectrum of symptoms such as fatigue and joint stiffness. Diagnosis of FM is difficult due to the lack of reliable diagnostic biomarkers, while treatment is largely inadequate. We have investigated the role of coenzyme Q10 (CoQ10) deficiency and mitochondrial dysfunction in inflammasome activation in blood cells from FM patients, and in vitro and in vivo CoQ10 deficiency models. RESULTS Mitochondrial dysfunction was accompanied by increased protein expression of interleukin (IL)-1β, NLRP3 (NOD-like receptor family, pyrin domain containing 3) and caspase-1 activation, and an increase of serum levels of proinflammatory cytokines (IL-1β and IL-18). CoQ10 deficiency induced by p-aminobenzoate treatment in blood mononuclear cells and mice showed NLRP3 inflammasome activation with marked algesia. A placebo-controlled trial of CoQ10 in FM patients has shown a reduced NLRP3 inflammasome activation and IL-1β and IL-18 serum levels. INNOVATION These results show an important role for the NLRP3 inflammasome in the pathogenesis of FM, and the capacity of CoQ10 in the control of inflammasome. CONCLUSION These findings provide new insights into the pathogenesis of FM and suggest that NLRP3 inflammasome inhibition represents a new therapeutic intervention for the disease.

Can Coenzyme Q10 Improve Clinical and Molecular Parameters in Fibromyalgia

Fibromyalgia (FM) is a complex disorder that affects up to 5% of the general population worldwide. Its pathophysiological mechanisms are difficult to identify and current drug therapies demonstrate limited effectiveness. Both mitochondrial dysfunction and coenzyme Q10 (CoQ10) deficiency have been implicated in FM pathophysiology. We have investigated the effect of CoQ10 supplementation. We carried out a randomized, double-blind, placebo-controlled trial to evaluate clinical and gene expression effects of forty days of CoQ10 supplementation (300 mg/day) on 20 FM patients. This study was registered with controlled-trials.com (ISRCTN 21164124). An important clinical improvement was evident after CoQ10 versus placebo treatment showing a reduction of FIQ (p<0.001), and a most prominent reduction in pain (p<0.001), fatigue, and morning tiredness (p<0.01) subscales from FIQ. Furthermore, we observed an important reduction in the pain visual scale (p<0.01) and a reduction in tender points (p<0.01), including recovery of inflammation, antioxidant enzymes, mitochondrial biogenesis, and AMPK gene expression levels, associated with phosphorylation of the AMPK activity. These results lead to the hypothesis that CoQ10 have a potential therapeutic effect in FM, and indicate new potential molecular targets for the therapy of this disease. AMPK could be implicated in the pathophysiology of FM.

Decreased soluble dipeptidyl peptidase IV activity as a potential serum biomarker for COPD.

OBJECTIVES The objective of this study was to measure soluble dipeptidyl peptidase IV (sDPPIV) activity in sera of patients with stable chronic obstructive pulmonary disease (COPD) in comparison to healthy controls. The main goal was to assess changes in the enzyme activity in relation to severity of the disease, age and smoking history and to evaluate diagnostic accuracy for prediction of COPD by level of serum sDPPIV activity. DESIGN AND METHODS The study included 106 patients with stable COPD (GOLD II-GOLD IV stages) and 38 healthy controls. Serum sDPPIV activity as well as some inflammatory markers (CRP, total and differential leukocyte counts) was measured. Multivariate logistic regression models were applied to analyze association of sDPPIV activity and inflammatory markers in risk estimation for COPD development. RESULTS sDPPIV activity in COPD patients was significantly reduced when compared to healthy controls. Decrease was observed already in GOLD II stage. Age and smoking history did not influence sDPPIV activity. Very good diagnostic accuracy (AUC=0.833; sensitivity and specificity of 85.7% and 78.9%, respectively) for GOLD II and good diagnostic accuracy (AUC=0.801; sensitivity and specificity of 65.1% and 86.8%, respectively) for total cohort of COPD patients were found. The multivariate logistic regression model showed that the use of sDPPIV in combination with CRP and lymphocyte proportion improved diagnostic strength and gave an AUC of 0.933. CONCLUSIONS sDPPIV activity is decreased in COPD patients as early as in GOLD II stage. Very good diagnostic accuracy of sDPPIV activity suggests it as a candidate biomarker for early diagnosis of COPD.

Association of hsp70-2 (+1267A/G), hsp70-hom (+2437T/C), HMOX-1 (number of GT repeats) and TNF-alpha (+489G/A) polymorphisms with COPD in Croatian population.

OBJECTIVE To test for possible association of hsp70-2 (+1267A/G), hsp70-hom (+2437T/C), HMOX-1 (number of GT repeats) and TNF-α (+489G/A) polymorphisms with chronic obstructive pulmonary disease (COPD) in Croatian population. METHODS Genotyping of DNA isolated from whole blood of 130 COPD patients (as defined by spirometry) and 95 healthy controls was performed. Fragment size analysis upon restriction enzyme digestion and/or sequencing was used for genotype/allele definition. Significance of findings was tested using χ(2) test. RESULTS hsp70-2 (+1267A/G) polymorphism was significantly associated with COPD. Results of genotyping analysis indicated that a genotype carrying G allele was preferentially associated with COPD; odds ratio (OR)=1.50, 95% confidence interval (CI)=1.00-2.24 and P=0.061. OR for the GG genotype was 3.47 with CI=1.26-9.56 and P=0.04. No association for hsp70-hom (+2437T/C), TNF-α (+489G/A) and HMOX-1 (number of GT repeats) polymorphisms were found. In addition, comparison of genotype frequencies among different stages of disease severity (GOLD II-IV) revealed no discrimination for any of the tested polymorphisms. CONCLUSION This study is supporting the association of hsp70-2 (+1267A/G) polymorphism and COPD. Higher frequency of G allele and GG genotype in Croatian COPD patients was observed. There was no evidence for the association of hsp70-hom (+2437T/C), TNF-α (+489G/A) SNPs and HMOX-1 (number of GT repeats) polymorphism with COPD. Allele and genotype frequencies for all of the tested polymorphisms show no association with disease severity (GOLD II-IV).

Mutation in cytochrome b gene of mitochondrial DNA in a family with fibromyalgia is associated with NLRP3-inflammasome activation

Background Fibromyalgia (FM) is a worldwide diffuse musculoskeletal chronic pain condition that affects up to 5% of the general population. Many symptoms associated with mitochondrial diseases are reported in patients with FM such as exercise intolerance, fatigue, myopathy and mitochondrial dysfunction. In this study, we report a mutation in cytochrome b gene of mitochondrial DNA (mtDNA) in a family with FM with inflammasome complex activation. Methods mtDNA from blood cells of five patients with FM were sequenced. We clinically and genetically characterised a patient with FM and family with a new mutation in mtCYB. Mitochondrial mutation phenotypes were determined in skin fibroblasts and transmitochondrial cybrids. Results After mtDNA sequence in patients with FM, we found a mitochondrial homoplasmic mutation m.15804T>C in the mtCYB gene in a patient and family, which was maternally transmitted. Mutation was observed in several tissues and skin fibroblasts showed a very significant mitochondrial dysfunction and oxidative stress. Increased NLRP3-inflammasome complex activation was observed in blood cells from patient and family. Conclusions We propose further studies on mtDNA sequence analysis in patients with FM with evidences for maternal inheritance. The presence of similar symptoms in mitochondrial myopathies could unmask mitochondrial diseases among patients with FM. On the other hand, the inflammasome complex activation by mitochondrial dysfunction could be implicated in the pathophysiology of mitochondrial diseases.

Porphyrins as new endogenous anti-inflammatory agents.

A series of porphyrins, tetrapyrrole natural organic compounds, are evaluated here as endogenous anti-inflammatory agents. They directly inhibit the activity of Fyn, a non-receptor Src-family tyrosine kinase, triggering anti-inflammatory events associated with down-regulation of T-cell receptor signal transduction, leading to inhibition of tumor necrosis factor alpha (TNF-α) production. This is one of the major pro-inflammatory cytokines, associated with diseases such as diabetes, tumorigenesis, rheumatoid arthritis, and inflammatory bowel disease. Porphyrins, as a chemical class, inhibited Fyn kinase activity in a non-competitive, linear-mixed fashion. In cell-based in vitro experiments on polymorphonuclear cells, porphyrins inhibited TNF-α cytokine production, T-cell proliferation, and the generation of free radicals in the oxidative burst, in a concentration-related manner. In vivo, lipopolysaccharide-induced TNF-α production in mice was inhibited by several of the porphyrins. These findings may be very important for the overall understanding of the role(s) of porphyrins in inflammation and their possible application as new anti-inflammatory agents.

Effect of coenzyme Q10 on psychopathological symptoms in fibromyalgia patients

This work has been supported by Grupo de Investigacion Junta de Andalucia CTS113, Consejeria de Salud of the Junta de Andalucia (PI-0036-2014).