Differential Effects of Reactive Oxygen Species on IgG versus IgM Levels in TLR-Stimulated B Cells.

It is becoming increasingly evident that reactive oxygen species (ROS) have critical roles as "second messengers" in cell signaling. In B cells, ROS can be generated either as a byproduct of mitochondrial respiration, as a result of the endoplasmic reticulum stress response induced by high production of Igs, or by the activation of NADPH oxidase (NOX) complexes. Having previously shown that costimulation of B cells via TLR 9 and the TLR-related receptor RP105 drives maturation of human peripheral blood B cells into Ig-producing cells, we aimed to study the role of ROS generated during this vital process. To this end, the ROS levels were either reduced by the NOX inhibitor VAS2870 or by the ROS scavenger N-acetyl cysteine (NAC). We revealed that TLR9/RP105-mediated stimulation of human B cells involved a rapid activation of NOX. Moreover, VAS2870 blocked the TLR9/RP105-induced B cell activation and thereby all Ig production. Importantly, we showed that ROS targeted by NAC was selectively required for IgG but not for IgM production. The endoplasmic reticulum stress response in the TLR9/RP105-stimulated cells was higher in IgG+ than in IgG- cells and was reduced by NAC in IgG+ cells only. Of note, we revealed that substantially higher levels of IgG than IgM were produced per cell and that IgG+ cells produced significantly higher ROS levels than IgG- cells. Taken together, our results imply that NAC-targeted ROS may be particularly important for sustaining the high Ig production in IgG+ B cells.

TLR9 stimulation of B-cells induces transcription of p53 and prevents spontaneous and irradiation-induced cell death independent of DNA damage responses. Implications for Common variable immunodeficiency.

In the present study, we address the important issue of whether B-cells protected from irradiation-induced cell death, may survive with elevated levels of DNA damage. If so, such cells would be at higher risk of gaining mutations and undergoing malignant transformation. We show that stimulation of B-cells with the TLR9 ligands CpG-oligodeoxynucleotides (CpG-ODN) prevents spontaneous and irradiation-induced death of normal peripheral blood B-cells, and of B-cells from patients diagnosed with Common variable immunodeficiency (CVID). The TLR9-mediated survival is enhanced by the vitamin A metabolite retinoic acid (RA). Importantly, neither stimulation of B-cells via TLR9 alone or with RA increases irradiation-induced DNA strand breaks and DNA damage responses such as activation of ATM and DNA-PKcs. We prove that elevated levels of γH2AX imposed by irradiation of stimulated B-cells is not due to induction of DNA double strand breaks, but merely reflects increased levels of total H2AX upon stimulation. Interestingly however, we unexpectedly find that TLR9 stimulation of B-cells induces low amounts of inactive p53, explained by transcriptional induction of TP53. Taken together, we show that enhanced survival of irradiated B-cells is not accompanied by elevated levels of DNA damage. Our results imply that TLR9-mediated activation of B-cells not only promotes cell survival, but may via p53 provide cells with a barrier against harmful consequences of enhanced activation and proliferation. As CVID-derived B-cells are more radiosensitive and prone to undergo apoptosis than normal B-cells, our data support treatment of CVID patients with CpG-ODN and RA.

Coffee inhibits nuclear factor-kappa B in prostate cancer cells and xenografts.

Chronic inflammation contributes to prostate cancer and the transcription factor Nuclear Factor-kappa B (NF-κB) is constitutively active in most such cancers. We examine the effects of coffee on NF-κB and on the regulation of selected genes in human-derived prostate cancer cells (PC3) and in PC3 xenografts in athymic nude mice. PC3 cells stably transduced with an NF-κB-luciferase reporter were used both in vitro and for xenografts. NF-κB activity was measured by reporter assays, DNA binding and in vivo imaging. Gene expression was measured in PC3 cells, xenografts and tumor microenvironment by low-density arrays. Western blotting of activated caspases was used to quantify apoptosis. Coffee inhibited TNFα-induced NF-κB activity and DNA-binding in PC3 cells. Furthermore, coffee increased apoptosis and modulated expression of a number of inflammation- and cancer-related genes in TNFα-treated PC3 cells. In vivo imaging revealed a 31% lower NF-κB-luciferase activation in the xenografts of the mice receiving 5% coffee compared to control mice. Interestingly, we observed major changes in gene expression in the PC3 cells in xenografts as compared to PC3 cells in vitro. In PC3 xenografts, genes related to inflammation, apoptosis and cytoprotection were down-regulated in mice receiving coffee, and coffee also affected the gene expression in the xenograft microenvironment. Our data demonstrate that coffee inhibits NF-κB activity in PC3 cells in vitro and in xenografts. Furthermore, coffee modulates transcription of genes related to prostate cancer and inflammation. Our results are the first to suggest mechanistic links between coffee consumption and prostate cancer in an experimental mouse model.

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

In the present study we have established a vital role of autophagy in retinoic acid (RA)-induced differentiation of toll-like 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 RA-stimulated 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.

TLR9-signaling is required for turning retinoic acid into a potent stimulator of RP105 (CD180)-mediated proliferation and IgG synthesis in human memory B cells.

The role of vitamin A in the various parts of the immune system remains elusive. Toll-like receptors (TLRs) are involved in innate polyclonal activation of B-cells, and as such they are important for maintaining long-lasting first line defense against pathogens. Here we explore the impact of all-trans retinoic acid (RA) on B cell responses mediated via the TLR homolog RP105 (CD180). We show that RA slightly reduces the proliferation and IgG production in CD27+ memory B cells stimulated by anti-RP105 alone. However, co-stimulation with the TLR9-ligand CpG results in turning RA into a potent stimulator of RP105-induced proliferation and IgG synthesis in memory B cells. The results emphasize the important role of RA in stimulating TLR-mediated polyclonal activation and differentiation of B cells, and reveal the complex interplay between various TLRs that may underlie the ability of RA to fight pathogens.