Investigation of il-27 secreting regulatory B cells(i27breg) and i27breg derived exosomes

Abstract

B cells are one of the key players of adaptive immunity with their ability to produce antibodies and antigen presentation to T lymphocytes. They can also differentiate into memory cells which protect the organism from the re-encounter of the same pathogen contributing to the immune response. B cells are divided into two major groups: B1 and B2 cells. B1 cells are tissue-resident cells, they possess innate-like features, they can self-renew and produce natural IgM antibodies which are secreted non-specifically to the antigen. B1 cells are originated in yolk salk and fetal liver, after a short amount of time from neonatal phase, they migrate towards the pleural and peritoneal cavities, their major residing region. B1 cells are maturated into B-1a and B-1b subsets, which are discriminated by the specific surface marker expression of CD5 on B-1a subset. B2 cells have been known as the 'conventional' B cells which are able to present antigens and generate antibodies.Immunological tolerance is an important mechanism of the immune system. It protects the organism from its own immune system and prevent the formation excessive immune responses. Recent studies have enlightened the presence of a new B cell subpopulation which possess immune suppressive characteristics. Bhan and colleagues have identified this subpopulation of B cells as regulatory B cells (Breg). Bregs have been divided into subgroups based on their surface marker expressions such as Interleukin-10 (IL-10) producing CD1dhiCD5+ B10 cells, CD19+CD21hiCD23hiCD24hi transitional 2 marginal-zone precursor cells (T2-MZP), CD19+CD5+ B-1a cells, CD138+ plasma B cells have been defined in murine models. Bregs exert their immune suppressive functions by secreting anti-inflammatory cytokines such as IL-10, TGF- and IL-35. Additionally, they can also utilize membrane associated molecules like programmed death-ligand 1 (PD-L1), CD73, CD39 to maintain functionality. Upon different stimuli, Bregs differentiation is triggered. It was shown that B cells stimulated with H.felis whole antigen triggers the IL-10 secreting Breg differentiation through TLR2 and MyD88 signaling. Similarly, TLR9 ligand CpG(K3) also induce the B cell differentiation into IL-10 secreting plasma blasts.IL-27 is a heterodimeric cytokine which is composed of p28 and EBI-3 subunits. It belongs to the IL-6/IL-12 family. IL-27 can act as a pro-inflammatory by inducing Th1 differentiation and IFN- production. IL-27 can also act as an anti-inflammatory cytokine by modulating T cell responses modulation as it can inhibit the differentiation of Th17 cells, thereby altering the balance between Th1 and Th17 cell responses. IL-27 is a dual functioning cytokine that vary depending on the cellular context and stimuli. Therefore, it gained a great potential to be used in therapy. IL-27 has been investigated in different types of autoimmune diseases including Multiple Sclerosis (MS) which is a chronic autoimmune disorder of the central nervous system, characterized with the disrupted myelin sheath. In a recent study by Choi et al., IL-27 secreting peritoneal cavity (PeC) derived B-1a cells have been discovered and named as i27Bregs. These cells are not disease or antigen specific. Their data showed that stimulation with LPS and BCR co-stimulation (CD40/IgM) increased IL-27 production. Furthermore, these i27Bregs were found to alleviate uveitis and encephalomyelitis by upregulating PD-1 and Lag3 and promoting the differentiation of regulatory B cells that secrete IL-35 and IL-10, suggesting potential therapeutic use in various autoimmune CNS disorders.Exosomes are type of extracellular vesicles secreted by all living cells. They can carry proteins, metabolites and nucleic acids. Due to their small size, they hold great therapeutic potential for treating various diseases. It was shown that, i27Breg derived exosomes ameliorate the symptoms of Uveitis and can be used in treating other central nervous system disorders. Immunometabolism has become an increasingly important area of study, as immune cells rely on metabolic signals and ATP for activation. Mitochondrial metabolism is the key energy producing mechanism which is important for immune cell functions. It was shown that, B cells activated with B cell receptor (BCR) and Toll-like receptor (TLR) have altered mitochondrial dynamics. The primary aim of our study is to investigate the IL-27 secreting regulatory B cells upon different TLR stimulation and characterize exosomes secreted from these cells. To achieve our goal, we have isolated PeC cells and collected spleens from VertX IL10egfp, IL-10 GFP reporter mice. PeC cells were cultured with LPS (5µg/ml), H.felis (10µg/ml), CpG(K3) (10µg/ml) and PAM3CSK4 (2.5µg/ml) for 72 hours. Splenocytes were cultured with LPS (5µg/ml), H.felis (10µg/ml) and CpG(K3) (10µg/ml) for 72 hours. Then, cells were collected and stained with CD19 to detect B cells and intracellular IL-27p28 staining was performed to assess IL-27p28 production by flow cytometry. Moreover, proteins were extracted from LPS, H.felis and CpG(K3) stimulated PeC cells and splenocytes to evaluate IL-27p28 protein expression by western blot. In addition, exosomes from unstimulated and CpG(K3) stimulated PeC cells were isolated by ultracentrifugation. Our results demonstrated that, IL-27p28 production levels in PeC B cells were significantly increased following stimulation with H.felis, PAM3CSK4 and CpG(K3) compared to unstimulated control. Interestingly, LPS stimulation did not induce a significant change which contrasts with previous literature. Western blot analysis confirmed increased IL-27p28 expression in total PeC cells stimulated with H. felis and CpG (K3), while LPS-stimulated cells showed no detectable protein levels compared to controls. In the splenic B cells, IL-27p28 production levels were significantly increased in all stimulated groups compared to unstimulated control, as shown by flow cytometry. Consistently, western blot analysis revealed increased IL-27p28 expression in all stimulated splenocyte groups compared to unstimulated controls. Furthermore, stimulation with LPS, H.felis, CpG(K3) and PAM3CSK4 led to a significant increase in the percentage of IL-27p28+IL-10+ cells within PeC B cells. Similarly, B cells stimulated with LPS, H. felis, and CpG (K3) also showed a significant rise in the proportion of IL-27p28⁺IL-10⁺ cells, suggesting that a substantial fraction of IL-10-producing PeC and splenic B cells concurrently produce IL-27p28. Additionally, we also succesfully isolated and start to characterize exosomes from both unstimulated and CpG (K3) stimulated PeC cells.Previously, our group demonstrated that H.felis LPS and PAM3CSK4 stimulation of splenic B cells increased their mitochondrial membrane potential (MMP) and IL-10 production. However, for PeC derived B cells, while LPS-induced IL-10 secretion had been reported, the effects of other TLR ligands on MMP and IL-10 production remained unexplored. Our second aim is to investigate MMP and IL-10 levels in PeC derived B and B1 cells. To do this, total PeC cells were isolated from VertX IL10egfp, IL-10 GFP reporter mice and stimulated with H.felis (10µg/ml), LPS (5µg/ml) and PAM3CSK4 (2.5µg/ml) for 72 hours. Afterward, cells were stained for the B cell marker CD19 and CD23 to identify B1 cells (CD19+CD23-), and MMP was assessed by Mitoview633 via flow cytometry. Our results have demonstrated that, IL-10 production were significantly increased in both PeC B and B1 cells upon H.felis, LPS and PAM3CSK4 stimulationMMP levels were significantly elevated in B1 cells with all three stimuli, while in PeC B cells, MMP increased significantly only following H. felis stimulation; LPS and PAM3CSK4 did not induce significant changes. In addition, in all stimulated groups the proportion of Mitoview633+IL-10+ cells have significantly increased, suggesting that cells acquiring suppressive functions possess functional mitochondria. In summary, this study demonstrates that, IL-27p28 secretion of peritoneal cavity derived B cells and B-1a cells and splenic B cells is significantly enhanced upon various TLR stimulations. We also successfully isolated and characterized IL-27p28-containing exosomes from PeC cells Moreover, IL-27 protein levels from PeC cells increased after LPS, PAM3CSK4, CpG (K3), and H. felis stimulation, and IL-10 production by PeC B and B1 cells appears to be linked to mitochondrial metabolism.