Yarrowia lipolytica yeast is a model species of the group of oleaginous microorganisms capable of intracellular lipids accumulation in an amount exceeding 20% of the dry mass. Single cell oil biosynthesis can follow one of two biochemical pathways-de novo accumulation of cellular lipids in medium containing non-lipid carbon sources (including saccharides, glycerol) and ex novo microbial oil synthesis which involves fatty acids uptake from the environment. The mRNA expression of selected genes of de novo and ex novo lipid synthesis pathways was analyzed and correlated with the phenotypically observed features. It was proved that the accumulation yield of storage lipids via ex novo pathway was to some extent dependent on the limitation of the nitrogen source in the medium. It was also proposed that the synthesis of intracellular lipids in lipid-rich medium proceeded mainly via ex novo pathway, although the activity of genes encoding the enzymes of the de novo pathway were not completely inhibited at the stage of transcription by fatty acids present in the medium (e.g., ATP-citrate lyase). Molecular markers of two biosynthesis routes has been outlined and a hypothetical connection point between de novo and ex novo route were indicated.
Rationale: Chitotriosidase (CHIT1) and acidic mammalian chitinase (AMCase) are the enzymatically active chitinases in humans. CHIT1 has been implicated in the pathogenesis of interstitial lung diseases (ILDs) including IPF. Methods: Chitinolytic activity was assessed in induced sputum (IS) and serum from IPF patients and controls (n=20-28) and CHIT1 expression was evaluated in bronchoalveolar lavage (BAL) cells from IPF patients. The therapeutic efficacy of OATD-01 – a selective chitinase inhibitor was evaluated in the mouse model of bleomycin-induced pulmonary fibrosis. Mice were dosed with OATD-01 or nintedanib (100 mg/kg, po, qd) in a therapeutic scheme. Results: The chitinolytic activity in serum and IS from IPF patients was significantly elevated when compared to controls. Additionally, CHIT1 was shown to be highly expressed in BAL macrophages from IPF patients. OATD-01 - a potent chitinase inhibitor (hAMCase IC50=9nM, hCHIT1 IC50=26nM) significantly reduced lung fibrosis in the bleomycin-induced fibrosis model, comparably to nintedanib, as assessed by modified Ashcroft scoring. The anti-fibrotic activity of OATD-01 was associated with pharmacodynamic effect: plasma chitinolytic activity was significantly reduced in mice dosed with OATD-01 as compared to control group. Conclusion: We have demonstrated that expression of CHIT1 is increased in IPF and that inhibition of chitinases with OATD-01 provided significant therapeutic efficacy, comparable to nintedanib, in the bleomycin-induced pulmonary fibrosis model. These data support further development of OATD-01 as a first-in-class therapy for IPF.
Introduction: sarcoidosis and IPF are diffuse parenchymal lung diseases with a complex immune background. Bronchoalveolar lavage fluid (BALf) is used to investigate inflammation and immune response at the local level. Aim: evaluation mRNA expression of selected mediators of inflammation (CCL26, ECP, IL10, IL12p40, IL13, IL17, IL2, IL25, IL33, IL6, IL8, MCP1, MMP9, PCNA, TGF, TSLP, mast cell tryptase) in BALf cells from patients with sarcoidosis and IPF and its correlation with clinical data. Methods: total RNA isolated from cell suspensions obtained from BALf was used to reverse transcription. Expression of selected mediators was measured using quantitative RT- PCR. Results: 38 patients (26 - sarcoidosis, 12 – IPF) were enrolled. Patients with IPF were older and had a lower TLC and DLCO - 93.6 (IQR 75.6-102.8)% vs 115.9 (IQR 102.8-120), p=0.01 and 74.0 (IQR 57.4-86.4)%. vs 96.4 (IQR 88.8-104.8)%, p=0.009, respectively. We found no differences between both diseases in the mRNA expression of the investigated cytokines on BALf cells. The expression of all but one mediator (IL25) correlated inversely with FEV1 (%) in patients with sarcoidosis. There were significant positive correlations between the expression of ECP, IL13, IL17, IL25, IL33 and MCP1 and DLCO in patients with IPF; no correlations between the expression of the investigated mediators and DLCO in patients with sarcoidosis were found. Conclusions: the inflammatory and immune pathways in sarcoidosis and IPF show some similarities. However, the clinical implication of the expression of inflammatory markers is different in both diseases.
Introduction: Body composition is an important prognostic factor in patients with COPD. The decrease in fat free mass (FFM), muscle mass (MM) and increase in visceral fat is associated with an elevated secretion of cytokines which promote systemic inflammation. The aim of the study was to evaluate body composition and the cytokine profile in patients with COPD in relation with the presence of hyperinflation. Material and Methods: The study group consisted of 149 patients (61F, 88M) with stable COPD in all stages of severity aged 68 ± 8.8 yrs. All the patients underwent spirometry and bodypletysmography with bronchial reversibility testing. Hyperinflation was defined as RV%TLC > 48% and > 126% predicted. Body composition was analyzed by bioimpedance. The following serum inflammatory markers were evaluated: C-reactive protein, IL-6, IL-8, TNF-a, CC16, adiponectin and resistin. Results: Hyperinflation was found in 96 patients (group A) and it was more frequent in women than men (49/61 vs. 47/88, p < 0.001). BMI and age in this group were comparable to those in patients without hyperinflation (group B). Patients with hyperinflation have lover FFM, FFM index, MM and MM index and total body water and higher fat mass and fat mass index. We found significantly higher serum concentrations of inflammatory markers in group A: IL-6 – 6.4 ± 10.9 vs. 3.6 ± 4.2 pg/ml, resistin – 9.3 ± 4.2 vs. 7.6 ± 2.4 ng/ml, CRP 4.1 ± 2.3 vs. 2.9 ± 2.1 mg/l, respectively. Conclusions: Patients with hyperinflation have a lower FFMI, TBW and MMI and a higher proportion of fat tissue. Hyperinflation is associated with elevated concentrations of inflammatory markers what may be associated with more severe disease. Body compositions abnormality and higher activity of systemic inflammation could therefore be a negative prognostic factor in COPD patients.
Macrophages are important immune cells involved in airway inflammation in obstructive lung diseases. Depending on local milieu macrophages can undergo distinct polarization with different functional capacities. Expression of CCR3 receptor on macrophages is associated with Th2 response but its role in Th1 response remains unclear. The aim was to evaluate the expression of CCR3 receptor on M1- and M2-like sputum macrophages in obstructive lung diseases and healthy controls. Study involved 19 asthmatics, 21 COPD patients, 9 healthy non-smokers, and 9 healthy smokers who underwent sputum induction. In immunofluorescence staining, CCR3 expression was assessed on CD80/CD86 positive (M1) and CD163/CD206 positive (M2) macrophages in sputum smears. Non-smokers and smokers were comparators for asthma and COPD, respectively. We noted increased number of M1+CCR3+ and M2+CCR3+ sputum macrophages in asthma vs non-smoking controls, in smoking control vs non-smoking control and in smoking control vs COPD. Conclusion: Our study demonstrated an enhanced expression of CCR3 on M1 and M2 macrophages in asthma, COPD patients and healthy smokers. It suggests that expression of CCR3 on macrophages may be associated not only with Th2 response but also with Th1 response and be influenced by cigarette smoke exposure.
Exhaled breath condenstae (EBC) is a non-invasive, easily performed method for obtaining samples from respiratory tract. The use of EBC may be of great importance in patients with severe asthma, because of the difficulty in sputum induction (IS) in these patients. The aim of our experiments was to determine the concentrations of selected cytokines in IS and EBC and verify the hypothesis that in patients with severe asthma sputum induction may be omitted for the EBC. The study enrolled 17 patients with severe asthma who underwent sputum induction and EBC. Patients were free from acute exacerbations, but they received steroids therapy. The respiratory specimens were stored at -80 °C, and EBC was not densified. The concentration of IL-6, IL-13, TNF-α, TSLP, CCL-24 and IFNγ was determined in all samples with ELISA tests . The obtained results are presented in the table. The concentration of IL-13, TNF-α and TSLP were comparable in IS and EBC. Unexpectedly high levels of IFNγ in asthma patients in both IS and EBC may due to therapy, because steroid treatment may result in elevation of these markers. High significantly greater concentration of IL-6 and CCL-24 in IS vs. EBC was probably depended on the recovery of mucosal deposits of these markers during extraction of sputum. The results of our study have confirmed the usefulness of EBC for analyze IL-13, and TNF α. The sputum samples seems to be better in analyze of IL-6 and definitely only one a good enough source of CCL-24.
IL-6 is strongly implicated in the development of chronic obstructive pulmonary disease (COPD). IL-13 is the well-documented central mediator in allergic asthma. IL-6 is attributed to the proinflammatory activities in COPD as well as asthma. In COPD patients exacerbation is increased by serum IL-6. The association of IL-13 as well as IL-6 with the impaired respiratory function of asthma patients remains controversial.The aim of this study was to compare the concentration of IL-6 and IL-13 in the induced sputum of asthma and COPD patients, and to assess the possible association of these cytokines with the impairment of lung function.Twenty-six subjects with COPD and 18 subjects with asthma were enrolled in this study. IL-6 and IL-13 levels were measured in induced sputum by ELISA and correlated with the results of respiratory tests.The induced sputum of COPD patients had a significantly higher IL-6 level than the sputum of asthma subjects while no significant differences were found in the levels of IL-13. There was a statistically significant negative correlation between IL-6 level and FEV(1) or FEV(1)/FVC in asthma patients (r = -0.59 and -0.54, respectively) and a negative correlation that did not reach statistical significance between IL-6 level and FEV(1), FEV(1)% or FVC in COPD subjects (r = -0.30, -0.30 and -0.38, respectively). There was no relationship between concentrations of IL-13 and impaired respiratory function.Our results confirmed that IL-6, but not of IL-13, is associated with respiratory disorders in both asthma and COPD patients.
Background: IL-17A and IL-25 (IL-17 cytokines family) play an important role in the development of asthma, and allergy. Both cytokines act by binding to heterodimeric receptors, whose common subunit is IL17RA. This receptor is found on macrophages and some other cell types. The aim of the study was to determine the expression of IL17RA on induced sputum (IS) CD206+ macrophages (M2 type) from asthmatics and controls with regard to IL-17/IL-25 background and relation to clinical features of the disease. Methods: The study enrolled 34 asthma patients and 34 controls. The expression of CD206 and IL17RA on IS macrophages was assessed by the immunofluorescence method. In the IS supernatant, IL-17A and IL-25 levels were evaluated (ELISA method). Results: An elevated expression of IL17RA on IS macrophages in asthmatics vs controls was found. In asthmatics, CD206+IL17RA+ macrophage percentage was significantly higher than in healthy subjects and correlated positively with total IgE level. A characteristic sputum profile of atopic asthmatic was as follows: high CD260+IL17RA+ macrophage percentage, elevated IL-25 level and low CD206+IL17RA- macrophage percentage. Conclusion: It seems that M2 rather than M1 type macrophages are the effector cells expressed common subunit of the receptor for IL-17A and IL-25 in asthma. This may be related to the Th2-environment in asthma and increased concentrations of IL-25 and IL-13 as well as eosinophils. To our knowledge, our study provides the first data on a possible link between immunological reaction orchestrating M2 macrophages and IL-25 via IL-17RA pathway in the asthmatic airways.
