Reduced response to TLR ligation in leucocytes of Type1 Diabetics

Objectives: This study aims to investigate the activation of Toll-like receptors (TLR), using a specific ligands, to discover whether there is an altered inflammatory response in type 1 diabetic(T1DM) white cells compared with those of non-diabetic(ND) control subjects. Methods: Peripheral blood leukocytes from 14 T1DM patients and 9 ND controls were maintained in the presence or absence of TLR2, -3, -4, -7 & -9 ligands; consisting of: Porphryomonas gingivalis lipopolysaccharide (Glasgow Dental School), polyI/C (Sigma), purified Esherishia coli lipopolysaccharide(Sigma), R848 (InvivoGen) and CpG ODN 2336 (Coley Pharmaceuticals). After incubating for 24 hours the supernatants were collected from replicate wells and stored at –70oC for IL-8 and IL-6 ELISA. Results: Since un-stimulated ND leukocytes synthesized statistically significantly greater amounts of IL-6 and IL-8 than cells from T1DM the data were normalized and the amounts of cytokine expressed after stimulation were compared. Similar levels of IL-8 and IL-6 were synthesized by ND and T1DM cells following ligation of TLR7 and TLR9. However, statistically significantly lower levels of IL-8 were detected in the T1DM group compared with the ND group following ligation of TLR2 and TLR3 (p=0.031, p=0.047, respectively). A similar trend was noted in IL-6 expression, but statistically significant differences were not observed. When the T1DM group was subdivided into two subsets (above the median and below the median of 9.05) based on their HbA1c levels, the cells of the higher HbA1c group were statistically significantly less responsive to TLR2 and TLR3 ligands (p= 0.0113, p= 0.0167, respectively) than ND cells. The group with the lower HbA1c profile was only statistically significant regarding TLR2 ligation. Conclusion: The results show that the response of T1DM patients to ligands specific for TLR 2 and 3 are reduced in relation to IL8 production. This may indicate a reduced effective response to viral and bacterial infection.