Extracellular Hsp70 induces inflammation and modulates LPS/LTA-stimulated inflammatory response in THP-1 cells.

Extracellular Hsp70 (eHsp70) can act as damage-associated molecular pattern (DAMP) via Toll-like receptors TLR2 and TLR4, and stimulate immune and inflammatory responses leading to sterile inflammation and propagation of already existing inflammation. It was found elevated in the blood of patients with chronic obstructive pulmonary disease (COPD), who might suffer occasional bacterial colonizations and infections. We used a monocytic THP-1 cell line as a cellular model of systemic compartment of COPD to assess inflammatory effects of eHsp70 when present alone or together with bacterial products lypopolysaccharide (LPS) and lypoteichoic acid (LTA). THP-1 cells were differentiated into macrophage-like cells and treated with various concentrations of recombinant human Hsp70 protein (rhHsp70), LPS (TLR4 agonist), LTA (TLR2 agonist), and their combinations for 4, 12, 24, and 48 h. Concentrations of IL-1α, IL-6, IL-8, and TNF-α were determined by ELISA. Cell viability was assessed by MTS assay, and mode of cell death by luminometric measurements of caspases-3/7, -8, and -9 activities. rhHsp70 showed cell protecting effect by suppressing caspases-3/7 activation, while LPS provoked cytotoxicity through caspases-8 and -3/7 pathway. Regarding inflammatory processes, rhHsp70 alone induced secretion of IL-1α and IL-8, but had modulatory effects on release of all four cytokines when applied together with LPS or LTA. Combined effect with LPS was mainly synergistic, and with LTA mainly antagonistic, although it was cytokine- and time-dependent. Our results confirmed pro-inflammatory function of extracellular Hsp70, and suggest its possible implication in COPD exacerbations caused by bacterial infection through desensitization or inappropriate activation of TLR2 and TLR4 receptors.

Paraoxonase 1 activity and phenotype distribution in premenopausal and postmenopausal women.

INTRODUCTION: Postmenopausal women have higher risk of cardiovascular disease. One of the contributing factors could be reduced activity of anti-atherogenic enzyme paraoxonase 1 (PON1). The aim of this study was to examine differences in the lipid status, paraoxonase and arylesterase PON1 activities and PON1 phenotype in women with regular menstrual cycle and in postmenopausal women. MATERIALS AND METHODS: The study included 51 women in reproductive age (25 in follicular and 26 in luteal phase of the menstrual cycle) and 23 women in postmenopause. Lipid parameters in sera were determined using original reagents and according to manufacturer protocol. PON1 activity in serum was assessed by spectrophotometric method with substrates: paraoxon and phenylacetate. PON1 phenotype was determined by double substrate method. RESULTS: Compared to the women in follicular and luteal phase, postmenopausal women have significantly higher concentration of triglyceride [0.9 (0.7-1.3), 0.7 (0.6-1.0) vs. 1.5 (0.9-1.7) mmol/L; P = 0.002], cholesterol [5.10 (4.78-6.10), 5.05 (4.70-5.40) vs. 6.30 (5.73-7.23) mmol/L; P < 0.001], LDL [3.00 (2.56-3.63), 3.00 (2.70-3.70) vs. 3.90 (3.23-4.50) mmol/L; P < 0.001], and apolipoprotein B [0.88 (0.75-1.00), 0.79 (0.68-1.00) vs. 1.07 (0.90-1.24) mmol/L; P = 0.002]. PON1 basal [104 (66-260), 106 (63-250) vs. 93 (71-165) U/L; P = 0.847] and salt-stimulated paraoxonase activity [210 (131-462), 211 (120-442) vs. 180 (139-296) U/L; P = 0.857] as well as arylesterase activity [74 (63-82), 70 (54-91) vs. 70 (60-81) kU/L; P = 0.906] and PON1 phenotype (P = 0.810) were not different in the study groups. CONCLUSION: There are no differences in PON1 activity and PON1 phenotype between women with regular menstrual cycle and postmenopausal women.

Disturbed Hsp70 and Hsp27 expression and thiol redox status in porcine kidney PK15 cells provoked by individual and combined ochratoxin A and citrinin treatments.

The aim of this study was to explore the oxidative properties of ochratoxin A (OTA) and citrinin (CTN) as a possible underlying mechanism of their individual and/or combined cytotoxicity. Metabolic activity of PK15 porcine kidney cells was significantly reduced with OTA and CTN co-exposures, with synergistic cytotoxic interactions. Single CTN increased both reduced (GSH) and oxidized (GSSG) glutathione after 24 h. However, GSH was significantly lowered with all OTA and CTN combined applications in synergistic manner after 12 and 24 h. GSH/GSSG ratio was reduced in most single and dual treatments, which suggested the presence of oxidative stress. In addition, OTA and CTN exposures significantly decreased concentrations of total thiols, with mycotoxins interactions being synergistic or antagonistic. The expression levels of Hsps were differentially affected by single and dual mycotoxin(s) applications. Single OTA provoked significant down-regulation of Hsp70 and Hsp27 expressions, while CTN stimulated Hsps expressions. Hsps were also up-regulated by dual treatments, and this induction was much stronger then with single CTN. In conclusion, significant alterations in cellular redox status (glutathione, thiols) and protective mechanisms (Hsps) suggest that those disturbances might be involved in OTA and CTN individual and combined mechanisms of cytotoxicity.