Retinoic acid-induced IgG production in TLR-activated human primary B cells involves ULK1-mediated autophagy

Abstract

(2015) Retinoic acid-induced IgG production in TLR-activated human primary B cells involves ULK1-mediated autophagy, Autophagy, 11:3, 460-471, DOI: 10.1080/15548627.2015 Keywords: antibody secretion, autophagy, B lymphocytes, CD180, plasma cell differentiation, retinoic acid, RP105, TLR9, ULK1Abbreviations: ATG, autophagy-related; BDS, bright detail similarity; CD180, CD180 molecule; CVID, common variable immune deficiency; ELISA, enzyme-linked immunosorbent assay; Ig, immunoglobulin; IL, interleukin; MAP1LC3B/LC3B, microtubuleassociated protein 1 light chain 3 b; MTOR, mechanistic target of rapamycin (serine/threonine kinase); PAMP, pathogen-associated molecular pattern, PML/RARA, promyelocytic leukemia/ retinoic acid receptor a; RA, all-trans retinoic acid; RAR, retinoic acid receptor; SQSTM1, sequestosome 1; TLR, toll-like receptor; ULK1, unc-51 like autophagy activating kinase 1.In the present study we have established a vital role of autophagy in retinoic acid (RA)-induced differentiation of tolllike receptor (TLR)-stimulated human B cells into Ig-secreting cells. Thus, RA enhanced autophagy in TLR9-and CD180-stimulated peripheral blood B cells, as revealed by increased levels of the autophagosomal marker LC3B-II, enhanced colocalization between LC3B and the lysosomal marker Lyso-ID, by a larger percentage of cells with more than 5 characteristic LC3B puncta, and by the concomitant reduction in the level of SQSTM1/p62. Furthermore, RA induced expression of the autophagy-inducing protein ULK1 at the transcriptional level, in a process that required the retinoic acid receptor RAR. By inhibiting autophagy with specific inhibitors or by knocking down ULK1 by siRNA, the RAstimulated IgG production in TLR9-and CD180-mediated cells was markedly reduced. We propose that the identified prominent role of autophagy in RA-mediated IgG-production in normal human B cells provides a novel mechanism whereby vitamin A exerts its important functions in the immune system.

An Integrative Framework Reveals Signaling-to-Transcription Events in Toll-like Receptor Signaling

“…27 out of the 131 genes tested significantly affected TLR signature gene expression, which included known TLR signaling components such as TICAM1, TBK1, MAPK9, RIPK3 and IRAK2 (Figure 3B). Furthermore, several phosphoproteins were reported to function in TLR signaling by independent studies: TRAFD1 (Sanada et al, 2008), STK3 (Geng et al, 2015), ULK1 (Eriksen et al, 2015), and CORO1A (Tanigawa et al, 2009). Interestingly, known and candidate components had similar effects on the TLR gene signature upon knockdown.…”

Myeloid cell leukaemia 1 has a vital role in retinoic acid‐mediated protection of Toll‐like receptor 9‐stimulated B cells from spontaneous and DNA damage‐induced apoptosis

“…MCL1 siRNA or control siRNA was introduced into purified normal B cells using the Nucleofector device (Amaxa Biosciences, Basel, Switzerland) in accordance with the manufacturer's instructions and the program U‐15, as previously described . Typically, 3 × 10 6 B cells were resuspended in 100 μl Nucleofector solution V and mixed with 0·06 n m siRNA.…”

“…RA exerts its effects primarily as a ligand for retinoic acid receptors (RARs) and retinoic X receptors (RXRs) acting as transcription regulators, and numerous genes have been identified as direct or indirect targets of RA‐mediated transcription . We have previously shown that RA stimulates normal human T cells, as well as normal B cells when activated via Toll‐like receptors (TLR) such as TLR9 and RP105 (CD180) . The natural ligand for TLR9 is bacterial and viral DNA rich in unmethylated CpG, and these ligands can be mimicked in vitro by synthetic unmethylated CpG oligonucleotides (CpG‐ODNs) …”

“…Previous research from our laboratory has revealed the ability of RA to strengthen TLR9‐mediated B‐cell functions linked to proliferation and differentiation . The role of RA in regulating normal B‐cell homeostasis as well as DNA damage‐induced cell death has, however, not been explored.…”

“…11 We have previously shown that RA stimulates normal human T cells, 12,13 as well as normal B cells when activated via Toll-like receptors (TLR) such as TLR9 and RP105 (CD180). [14][15][16][17][18] The natural ligand for TLR9 is bacterial and viral DNA rich in unmethylated CpG, 19,20 and these ligands can be mimicked in vitro by synthetic unmethylated CpG oligonucleotides (CpG-ODNs). 21 Previous research from our laboratory has revealed the ability of RA to strengthen TLR9-mediated B-cell functions linked to proliferation and differentiation.…”