CIRCULATING NUCLEIC ACIDS AS DAMAGE- ASSOCIATED MOLECULAR PATTERNS (DAMPS) MODULATE IMMUNE RESPONSE
IN TYPE 1 DIABETES
G. Kocic, R. Kocic, T. Jevtovic, D. Sokolovic, T. Cvetkovic, P. Vlahovic, Lj. Saranac. Medical Faculty Nis, Serbia
Objectives: The immune response may be triggered by molecules derived from microorganisms, known as the pathogen-associated molecular-pattern molecules (PAMP) or by damage-associated molecular patterns (DAMPs), derived from damaged, dying or necrotic tissues. DAMPs initiate strong inflammatory response via activation of antigen presenting cells (APC), production of pro-inflammatory cytokines (IL-1, TNF-α, IL-6, IL-8), cell adhesion molecules (ICAM-1, VCAM-1) by activation of vascular endothelium and may accelerate atherosclerosis. DAMPs mediate immune responses through the TLRs, RAGE and NOD1-like receptors.Nucleic acids, single, double stranded and specific “danger motifs” (unmethylated CpG), represent strong TLR ligands but may also act via TLR-independent pathways (via MDA-5), when the central event of the host immune system is the rapid activation of multiple trancription factors, NF-kB, IRF-3 and modulation of apoptotic machinery. Extracellular ribonucleases (RNases) are involved in degradation of all circulating ribonucleic acids (RNAs) or oligonucleotide derivatives. In our previous reports the fall of extracellular nuclease activity toward TLR 3, 7 and 9 ligands was documented to occur in type 1 diabetes, followed by the retention of different-sized oligonucleotides in plasma. The in vitro RNase susceptibility to glycation in type 1 diabetes was confirmed. Circulating ribonucleic acids (CRNAs) were not considered as the potential DAMPs immunogenic molecules up to now.
Methods:Since the higher prevalence of various infectious diseases is reported to occur in type 1 diabetic patients, the aim of the present study is to evaluate modulation of immuno-inflammatory cascade and antiviral response by CRNAs in type 1 diabetes. For the isolation of nucleic acids, examined groups were: juvenile type 1 diabetic patients (n=37), adult type 1 diabetic patients (n=23), control children (n=27) and healthy adult subjects (n=30). Peripheral blood mononuclear cells (PBMCs) were isolated from 100mL blood of one donor volunteer. PBMC were treated with: RNA samples purified from plasma of: type 1 juvenile and adult diabetic patients, control healthy adults, healthy children and PolyI:C(5μg/ml). Corresponding intact PBMC contained equal aliquots of physiological saline. The Bcl2, Bax, NF-kB, MDA-5 and IRF-3 expression was estimated after 6 hours of incubation.
Results and Discussion: The ratio between two apoptosis regulators, Bcl-2 (apoptosis suppressor) and Bax (apoptosis inducer) was significantly increased in juvenile type 1 diabetes (0.93 vs 0.89 in control children p<0.05) more than in adult type 1 diabetes (0.89 vs 0.73 p<0.001). The expression of NF-κB was altered (10.5%) more with the CRNAs od adult diabetic patients (87.7%), potentiating age-dependent inflammatory effect. The CRNAs of both groups of diabetic patients were able to downregulate MDA-5 (-24.3% and -18.9%) and IRF-3 (-12.58% and -19.32%), supressing very important subjects of the surveillance andcellular innate immunity antiviral response. Obtained results may be of importance for future therapeutic immune anti-inflammatory and antiviral response modulation in type 1 diabetes according to the patient age.