Vitamin A in the interplay between ROS, autophagy and immunoglobulin production in normal B cells and B cells from patients with the immunodeficiency disorder CVID

Background and aims: Common variable immune deficiency (CVID) is an immune disorder characterized by recurrent infections, defective B cell functions and low production of immunoglobulins (Igs). CVID-derived B cells frequently have a low response to stimulation via toll like receptors such as TLR9 and RP105, and the patients often have low levels of serum vitamin A levels. TLR9/RP105-mediated IgG production in normal B cells requires autophagy, and the vitamin A metabolite retinoic acid (RA) can augment IgG production by enhancing this autophagy. Recent research has highlighted the importance of NOX2-induced ROS as a signal transducer in B cells, and ROS has also been implicated in autophagy in various cell systems. The aims of the present thesis were: 1) Investigate a possible interplay between ROS, autophagy and Ig secretion in normal B cell stimulated via TLR9 and RP105; 2) Explore whether dysregulation of ROS and/or autophagy might contribute to the low production of IgG in B cells from CVID patients; 3) Reveal the role of RA in the interplay between ROS, autophagy and Ig production both in normal- and CVID-derived B cells. Methods: CD19+ B cells were isolated from buffy coats or whole blood collected from CVID patients and healthy controls. The B cells were stimulated via TLR9 and RP105 in the presence or absence of RA. The levels of ROS and autophagy in the stimulated B cells were measured by flow cytometry. ELISA assays were performed to quantify Ig secretion. The results from these assays were combined to analyze for co-variations in ROS levels, autophagy and Ig secretion in normal- and CVID-derived B cells. Results: TLR9/RP105-mediated stimulation of normal B cells increased the levels of ROS, autophagy and Ig secretion. We found a significant positive correlation between ROS and autophagy in stimulated B cells from healthy donors (r=0.472), but not between Ig secretion and levels of either ROS or autophagy. However, NOX2-induced ROS was found to be essential for both autophagy and Ig secretion in the normal B cells, as the inhibitor significantly reduced these levels. There was a general tendency of lowered levels of autophagy in stimulated B cells derived from CVID patients compared to normal B cells (n=13, p=0.064). In a subgroup of CVID patients, the autophagy levels were significantly reduced. The levels of ROS in CVID-derived B cells did not differ from the levels in normal B cells, but we found aberrant responses to NOX2 inhibition on ROS levels or autophagy in B cells from three of the CVID patients. RA enhanced the levels of autophagy and Ig secretion in TLR9/RP105-stimulated B cells from both CVID patients and healthy controls, but the effects of RA was generally lower in the CVID B cells. RA did not affect the TLR9/RP105-induced ROS generation in neither normal- nor CVID-derived B cells. Conclusion: We observed a positive correlation between ROS levels and autophagy in normal TLR9/ RP105-stimulated B cells and revealed that NOX2-induced ROS is important for both autophagy and Ig secretion in these cells. The ROS levels were not dysregulated in CVID-derived B cells. However, in TLR9/RP105-stimulated B cells from a subgroup of CVID patients characterized by more adverse disease, we revealed that reduced levels of autophagy was associated with low IgG production. RA did not enhance the ROS levels in TLR9/RP105-stimulated B cells, but enhanced autophagy and Ig secretion in B cells from both CVID patients and healthy controls