Peritoneal B cells play a crucial role in the immune system and are divided into two categories based on their expression of the CD11b molecule: B1 (CD11b+CD19+) and B2 (CD11b-CD19+) cells (Hastings et al., 2006; Suchanek and Clatworthy, 2023). B1 cells are further classified based on the CD5 molecule into B1a (CD5+CD11b+CD19+) and B1b (CD5-CD11b+CD19+), which are long-lasting, self-renewing innate B cells residing in both the peritoneal and pleural cavities (Hastings et al., 2006; Wong et al., 2019). Peritoneal B1 cells play a key role in the body's innate immune response by producing natural antibodies (Ehrenstein and Notley, 2010; Rodriguez-Zhurbenko et al., 2022; Uehre et al., 2023). These cells are also vital in regulating immune reactions. Within this group, a notable subset, known as B10 cells, can produce IL-10. This cytokine is known for its ability to reduce inflammation (Maseda et al., 2013). These B10 cells play a critical role in regulating immune responses and minimizing the severity of autoimmune disorders, highlighting their importance in maintaining the balance of both the gastrointestinal and immune systems (Maseda et al., 2013). B2 cells are present in minimal amounts in the peritoneal cavity, they share similar surface markers to their splenic counterparts (Hastings et al., 2006). However, influenced by the peritoneal environment, peritoneal B2 cells exhibit phenotypic and functional changes, such as the expression of the anti-inflammatory cytokine IL-10 (Maseda et al., 2013). Nonetheless, it is unclear if the B1 and B2 cells within the peritoneum respond similarly to the same stimulus.

Lipopolysaccharide (LPS), an essential constituent of the outer membrane of Gram-negative bacteria, has vital regulatory impacts on peritoneal B cells, adjusting their migration, activation, proliferation, differentiation, and cytokine and antibody secretion capabilities (Chishima et al., 2000). Upon binding to Toll-Like Receptor 4 (TLR4) on peritoneal B cells, LPS stimulation triggers a sequence of intracellular signaling cascades, prominently involving the MAPK and JAK/STAT pathways. This results in the phosphorylation of key mediators such as MEK1, ERK1/2, and STAT3. Activation of both the MAPK and STAT3 pathways subsequently triggers production of pro-inflammatory and anti-inflammatory cytokines, including IL-6 and IL-10, which possess immunoregulatory functions under inflammatory conditions (Venkataraman et al., 1999; Mishima et al., 2019). Current data indicates that LPS provokes higher secretion of IL-6 and IL-10 from peritoneal B cells than their splenic equivalents (Margry et al., 2014). It is yet to be determined whether LPS selectively stimulates IL-10 secretion from peritoneal B1 cells but not B2 cells. Interestingly, LPS exposure causes a short-term decrease in all peritoneal B-cell subpopulations, including B1a, B1b, and B2 cells, with a notable impact on B1a cells that endures up to 48 hours post-stimulation (Moon et al., 2012).These findings suggest that LPS may affect the homeostatic equilibrium of peritoneal B cells, but the mechanism needs to be further elucidated.

IL-21, classified as a Type I cytokine, is primarily secreted by Th1, TFH cells and NK cells (Mehta et al., 2004; Lüthje et al., 2012). It crucially shapes immune responses by promoting the differentiation, proliferation, and activation of effector cells, including B cells, T cells, and NK cells (Mehta et al., 2004). In addition to its immunostimulatory functions, IL-21 exerts immunomodulatory effects through mechanisms like B10 cell-mediated suppression (Wu et al., 2016). IL-21 specifically promotes the differentiation of regulatory B cells capable of producing IL-10, thereby enhancing their immunosuppressive capacity (Yoshizaki et al., 2012). For instance, IL-21 can induce precursor B10 cells’ maturation into effector B10 cells, leading to an increased population of IL-10-producing B cells (Yoshizaki et al., 2012). Furthermore, IL-21 can inhibit B-cell proliferation caused by inflammatory stimuli by suppressing LPS-induced Bcl-xL expression (Wu et al., 2016). Although there have been many studies on B cells in the spleen and peripheral compartments, further investigation is needed to understand the responsiveness of peritoneal B1 and B2 cells to IL-21.

IL-21 and LPS can both modulate the potential secretory IL-10 of peritoneal B cells (Yoshizaki et al., 2012; Maseda et al., 2013). However, the mechanisms underlying how these agents stimulate IL-10 production in peritoneal B1 and B2 subtypes are still unknown. It is also unclear whether B1 and B2 cells exhibit different reactions when exposed to identical stimuli in the peritoneal environment. The primary objective of this study is to analyze the varying reactivity patterns of peritoneal B1 and B2 cells in response to IL-21 and LPS. Our results demonstrate that these cell subtypes exhibit diverse reactions to IL-21 and LPS, reflected in IL-10 production, apoptosis induction, surface marker expression, and intracellular signaling molecule activation. There is heterogeneity across these metrics. This suggests that although B1 and B2 cells live in the same peritoneal environment, they demonstrate unique functional characteristics due to their differing cellular lineages.