Utilization of a photoactivatable antigen system to examine B-cell probing termination and the B-cell receptor sorting mechanisms during B-cell activation.

Antigen binding to the B-cell receptor (BCR) induces several responses, resulting in B-cell activation, proliferation, and differentiation. However, it has been difficult to study these responses due to their dynamic, fast, and transient nature. Here, we attempted to solve this problem by developing a controllable trigger point for BCR and antigen recognition through the construction of a photoactivatable antigen, caged 4-hydroxy-3-nitrophenyl acetyl (caged-NP). This photoactivatable antigen system in combination with live cell and single molecule imaging techniques enabled us to illuminate the previously unidentified B-cell probing termination behaviors and the precise BCR sorting mechanisms during B-cell activation. B cells in contact with caged-NP exhibited probing behaviors as defined by the unceasing extension of membrane pseudopods in random directions. Further analyses showed that such probing behaviors are cell intrinsic with strict dependence on F-actin remodeling but not on tonic BCR signaling. B-cell probing behaviors were terminated within 4 s after photoactivation, suggesting that this response was sensitive and specific to BCR engagement. The termination of B-cell probing was concomitant with the accumulation response of the BCRs into the BCR microclusters. We also determined the Brownian diffusion coefficient of BCRs from the same B cells before and after BCR engagement. The analysis of temporally segregated single molecule images of both BCR and major histocompatibility complex class I (MHC-I) demonstrated that antigen binding induced trapping of BCRs into the BCR microclusters is a fundamental mechanism for B cells to acquire antigens.

Substrate stiffness regulates B-cell activation, proliferation, class switch, and T-cell-independent antibody responses in vivo.

B cells use B-cell receptors (BCRs) to sense antigens that are usually presented on substrates with different stiffness. However, it is not known how substrate stiffness affects B-cell proliferation, class switch, and in vivo antibody responses. We addressed these questions using polydimethylsiloxane (PDMS) substrates with different stiffness (20 or 1100 kPa). Live cell imaging experiments suggested that antigens on stiffer substrates more efficiently trigger the synaptic accumulation of BCR and phospho-Syk molecules compared with antigens on softer substrates. In vitro expansion of mouse primary B cells shows different preferences for substrate stiffness when stimulated by different expansion stimuli. LPS equally drives B-cell proliferation on stiffer or softer substrates. Anti-CD40 antibodies enhance B-cell proliferation on stiffer substrates, while antigens enhance B-cell proliferation on softer substrates through a mechanism involving the enhanced phosphorylation of PI3K, Akt, and FoxO1. In vitro class switch differentiation of B cells prefers softer substrates. Lastly, NP67-Ficoll on softer substrates accounted for an enhanced antibody response in vivo. Thus, substrate stiffness regulates B-cell activation, proliferation, class switch, and T cell independent antibody responses in vivo, suggesting its broad application in manipulating the fate of B cells in vitro and in vivo.

Evodiamine activates AMPK and promotes adiponectin multimerization in 3T3-L1 adipocytes.

Adiponectin, an adipokine with insulin-sensitizing effect, is secreted from adipocytes into circulation as high, medium, and low molecular weight (HMW, MMW, and LMW) forms. The HMW adiponectin is more metabolically active and the ratio of HMW adiponectin to total adiponectin directly correlates with insulin sensitivity. Evodiamine is an indole alkaloid found in the traditional Chinese medicinal plant Evodia rutaecarpa. In this study, evodiamine was found to activate AMP-activated protein kinase (AMPK) in both 3T3-L1 adipocytes and 293T cells. Activation of AMPK by evodiamine promoted the assembly of HMW adiponectin and increased the HMW/total ratio of adiponectin in 3T3-L1 adipocytes. The Ca(2+)-dependent PI3K/Akt/CaMKII-signaling pathway was demonstrated to be involved in evodiamine-induced AMPK activation. This study revealed a novel role of this Ca(2+)-mediated signaling pathway in promoting the multimerization of adiponectin.

Promotion of adiponectin multimerization by emodin: a novel AMPK activator with PPARγ-agonist activity.

Adiponectin is an important insulin-sensitizing adipokine with multiple beneficial effects on obesity-associated medical complications. It is secreted from adipocytes into circulation as high, medium, and low molecular weight forms (HMW, MMW, and LMW). Each oligomeric form of adiponectin exerts non-overlapping biological functions, with the HMW oligomer possessing the most potent insulin-sensitizing activity. In this study, we reported that emodin, a natural product and active ingredient of various Chinese herbs, activates AMPK in both 3T3-L1 adipocytes and 293T cells. Activation of AMPK by emodin promotes the assembly of HMW adiponectin and increases the ratio of HMW adiponectin to total adiponectin in 3T1-L1 adipocytes. Emodin might activate AMPK by an indirect mechanism similar to berberine. We also found that emodin activates PPARγ and promotes differentiation and adiponectin expression during differentiation of 3T3-L1 preadipocytes. Therefore, emodin is a novel AMPK activator with PPARγ-agonist activity. Our results demonstrate that the effects of emodin on adiponectin expression and multimerization are the ultimate effects resulting from both AMPK activation and PPARγ activation. The dual-activity makes emodin or the derivatives potential drug candidates for the treatment of type 2 diabetes and other obesity-related metabolic diseases.

Fc receptor-like 1 intrinsically recruits c-Abl to enhance B cell activation and function.

B cell activation is regulated by the stimulatory or inhibitory co-receptors of B cell receptors (BCRs). Here, we investigated the signaling mechanism of Fc receptor-like 1 (FcRL1), a newly identified BCR co-receptor. FcRL1 was passively recruited into B cell immunological synapses upon BCR engagement in the absence of FcRL1 cross-linking, suggesting that FcRL1 may intrinsically regulate B cell activation and function. BCR cross-linking alone led to the phosphorylation of the intracellular Y281ENV motif of FcRL1 to provide a docking site for c-Abl, an SH2 domain-containing kinase. The FcRL1 and c-Abl signaling module, in turn, potently augmented B cell activation and proliferation. FcRL1-deficient mice exhibited markedly impaired formation of extrafollicular plasmablasts and germinal centers, along with decreased antibody production upon antigen stimulation. These findings reveal a critical BCR signal-enhancing function of FcRL1 through its intrinsic recruitment to B cell immunological synapses and subsequent recruitment of c-Abl upon BCR cross-linking.

Substrate stiffness governs the initiation of B cell activation by the concerted signaling of PKCß and focal adhesion kinase.

The mechanosensing ability of lymphocytes regulates their activation in response to antigen stimulation, but the underlying mechanism remains unexplored. Here, we report that B cell mechanosensing-governed activation requires BCR signaling molecules. PMA-induced activation of PKCß can bypass the Btk and PLC-γ2 signaling molecules that are usually required for B cells to discriminate substrate stiffness. Instead, PKCß-dependent activation of FAK is required, leading to FAK-mediated potentiation of B cell spreading and adhesion responses. FAK inactivation or deficiency impaired B cell discrimination of substrate stiffness. Conversely, adhesion molecules greatly enhanced this capability of B cells. Lastly, B cells derived from rheumatoid arthritis (RA) patients exhibited an altered BCR response to substrate stiffness in comparison with healthy controls. These results provide a molecular explanation of how initiation of B cell activation discriminates substrate stiffness through a PKCß-mediated FAK activation dependent manner.

Impairment on the lateral mobility induced by structural changes underlies the functional deficiency of the lupus-associated polymorphism FcγRIIB-T232.

FcγRIIB functions to suppress the activation of immune cells. A single-nucleotide polymorphism in the transmembrane (TM) domain of FcγRIIB, FcγRIIB-T232, is associated with lupus. In this study, we investigated the pathogenic mechanism of FcγRIIB-T232 at both functional and structural levels. Our results showed that FcγRIIB-T232 exhibited significantly reduced lateral mobility compared with FcγRIIB-I232 and was significantly less enriched into the microclusters of immune complexes (ICs) after stimulation. However, if sufficient responding time is given for FcγRIIB-T232 to diffuse and interact with the ICs, FcγRIIB-T232 can restore its inhibitory function. Moreover, substituting the FcγRIIB-T232 TM domain with that of a fast floating CD86 molecule restored both the rapid mobility and the inhibitory function, which further corroborated the importance of fast mobility for FcγRIIB to function. Mechanistically, the crippled lateral mobility of FcγRIIB-T232 can be explained by the structural changes of the TM domain. Both atomistic simulations and nuclear magnetic resonance measurement indicated that the TM helix of FcγRIIB-T232 exhibited a more inclined orientation than that of FcγRIIB-I232, thus resulting in a longer region embedded in the membrane. Therefore, we conclude that the single-residue polymorphism T232 enforces the inclination of the TM domain and thereby reduces the lateral mobility and inhibitory functions of FcγRIIB.

A negative-feedback function of PKCß in the formation and accumulation of signaling-active B cell receptor microclusters within B cell immunological synapse.

Advanced live cell imaging studies suggested that B cell activation is initiated by the formation of BCR microclusters and subsequent B cell IS upon BCR and antigen recognition. PKC family member PKCß is highly expressed in B cells and plays an important role in the initiation of B cell activation. Here, we reported an inhibitory function of PKCß through a negative-feedback manner in B cell activation. Compared with WT (PKCß-WT) or the constitutively active (PKCß-ΔNPS) form of PKCß, DN PKCß (PKCß-DN) unexpectedly enhanced the accumulation of BCR microclusters into the B cell IS, leading to the recruitment of an excessive amount of pSyk, pPLC-γ2, and pBLNK signaling molecules into the membrane-proximal BCR signalosome. Enhanced calcium mobilization responses in the decay phase were also observed in B cells expressing PKCß-DN. Mechanistic studies showed that this negative-feedback function of PKCß works through the induction of an inhibitory form of pBtk at S180 (pBtk-S180). Indeed, the capability of inducing the formation of an inhibitory pBtk-S180 is in the order of PKCß-ΔNPS > PKCß-WT > PKCß-DN. Thus, these results improve our comprehensive understanding on the positive and negative function of PKCß in the fine tune of B cell activation.

Pseudoginsenoside F11, a Novel Partial PPAR γ Agonist, Promotes Adiponectin Oligomerization and Secretion in 3T3-L1 Adipocytes.

PPAR γ is a nuclear hormone receptor that functions as a master regulator of adipocyte differentiation and development. Full PPAR γ agonists, such as the thiazolidinediones (TZDs), have been widely used to treat type 2 diabetes. However, they are characterized by undesirable side effects due to their strong agonist activities. Pseudoginsenoside F11 (p-F11) is an ocotillol-type ginsenoside isolated from Panax quinquefolium L. (American ginseng). In this study, we found that p-F11 activates PPAR γ with modest adipogenic activity. In addition, p-F11 promotes adiponectin oligomerization and secretion in 3T3-L1 adipocytes. We also found that p-F11 inhibits obesity-linked phosphorylation of PPAR γ at Ser-273 by Cdk5. Therefore, p-F11 is a novel partial PPAR γ agonist, which might have the potential to be developed as a new PPAR γ -targeted therapeutics for type 2 diabetes.