Project description:Class-switching to IgG2a/c in mice is a hallmark response to intracellular pathogens. T cells can promote class-switching and the predominant pathway for induction of IgG2a/c antibody responses has been suggested to be via stimulation from Th1 cells. We previously formulated CAF®01 (cationic liposomes containing dimethyldioctadecylammonium bromide (DDA) and Trehalose-6,6-dibehenate (TDB)) with the lipidated TLR7/8 agonist 3M-052 (DDA/TDB/3M-052), which promoted robust Th1 immunity in newborn mice. When testing this adjuvant in adult mice using the recombinant Chlamydia trachomatis (C.t.) vaccine antigen CTH522, it similarly enhanced IgG2a/c responses compared to DDA/TDB, but surprisingly reduced the magnitude of the IFN-g+ Th1 response in a TLR7 agonist dose-dependent manner. Single cell RNA-sequencing revealed that DDA/TDB/3M-052 liposomes initiated early transcription of class-switch regulating genes directly in pre-germinal center B cells. Mixed bone marrow chimeras further demonstrated that this adjuvant did not require Th1 cells for IgG2a/c switching, but rather facilitated TLR7-dependent T-bet programming directly in B cells. This study underlines that adjuvant-directed IgG2a/c class-switching in vivo can occur in the absence of T cell help, via direct activation of TLR7 on B cells and positions DDA/TDB/3M-052 as a powerful adjuvant capable of eliciting type I-like immunity in B cells without strong induction of Th1 responses.
Project description:Salinization poses a significant challenge in agriculture, exacerbated by anthropogenic global warming. Biostimulants, derived from living microorganisms or natural extracts, have emerged as valuable tools for conventional and organic agriculture. However, our understanding of the molecular mechanisms underlying the effects of biostimulants is very limited, especially in crops under real cultivation conditions. In this study, we adopted an integrative approach to investigate the effectiveness of the combined application of plant growth-promoting bacterium (Bacillus megaterium strain BM08) and a non-microbial biostimulant under control conditions (normal watering) and salt stress. After confirming the yield increase under both conditions, we investigated the molecular mechanisms underlying the observed effect by measuring a number of physiological parameters (i.e., lipid peroxidation, antioxidants, chlorophylls, total phenolics and phytohormone content), as well as RNA sequencing and primary metabolite analyses. Our findings reveal that the combined effect of the microbial and non-microbial biostimulants led to a decrease in the antioxidant response and an up-regulation of genes involved in cytokinin biosynthesis under salt stress conditions. This, in turn, resulted in a higher concentration of the bioactive cytokinin, isopentenyladenosine, in roots and leaves and an increase in γ-aminobutyric acid, a non-proteic amino acid related to abiotic stress responses. In addition, we observed a decrease in malic acid, along with an abscisic acid (ABA)-independent up-regulation of SR-kinases, a family of protein kinases associated with abiotic stress responses. Furthermore, we observed that the single application of the non-microbial biostimulant triggers an ABA-dependent response under salt stress; however, when combined with the microbial biostimulant, it potentiated the mechanisms triggered by the BM08 bacterial strain. This comprehensive investigation shows that the combination of two biostimulants is able to elicit a cytokinin-dependent response that may explain the observed yield increase under salt stress conditions.