Neutralization mechanism of human monoclonal antibodies against type B botulinum neurotoxin.

Botulism is a deadly neuroparalytic condition caused by the botulinum neurotoxin (BoNT) produced by Clostridium botulinum and related species. Toxin-neutralizing antibodies are the most effective treatments for BoNT intoxication. We generated human monoclonal antibodies neutralizing type B botulinum neurotoxin (BoNT/B), designated M2 and M4. The combination of these antibodies exhibited a strong neutralizing effect against BoNT/B toxicity. In this study, we analyzed the mechanisms of action of these antibodies in vitro. M4 binds to the C-terminus of the heavy chain (the receptor-binding domain) and inhibits BoNT/B binding to neuronal PC12 cells. Although M2 recognized the light (L) chain (the metalloprotease domain), it did not inhibit substrate (VAMP2) cleavage in the cleavage assay. M2 increased the surface localization of BoNT/B in PC12 cells at a later time point, suggesting that M2 inhibits the translocation of the L chain from synaptic vesicles to the cytosol. These results indicate that M2 and M4 inhibit the different processes of BoNT/B individually and that multistep inhibition is important for the synergistic effect of the combination of monoclonal antibodies. Our findings may facilitate the development of effective therapeutic antibodies against BoNTs.

Membrane Vesicles Derived From Clostridium botulinum and Related Clostridial Species Induce Innate Immune Responses via MyD88/TRIF Signaling in vitro.

Clostridium botulinum produces botulinum neurotoxin complexes that cause botulism. Previous studies elucidated the molecular pathogenesis of botulinum neurotoxin complexes; however, it currently remains unclear whether other components of the bacterium affect host cells. Recent studies provided insights into the role of bacterial membrane vesicles (MVs) produced by some bacterial species in host immunity and pathology. We herein examined and compared the cellular effects of MVs isolated from four strains of C. botulinum with those of closely related Clostridium sporogenes and two strains of the symbiont Clostridium scindens. MVs derived from all strains induced inflammatory cytokine expression in intestinal epithelial and macrophage cell lines. Cytokine expression was dependent on myeloid differentiation primary response (MyD) 88 and TIR-domain-containing adapter-inducing interferon-ß (TRIF), essential adaptors for toll-like receptors (TLRs), and TLR1/2/4. The inhibition of actin polymerization impeded the uptake of MVs in RAW264.7 cells, however, did not reduce the induction of cytokine expression. On the other hand, the inhibition of dynamin or phosphatidylinositol-3 kinase (PI3K) suppressed the induction of cytokine expression by MVs, suggesting the importance of these factors downstream of TLR signaling. MVs also induced expression of Reg3 family antimicrobial peptides via MyD88/TRIF signaling in primary cultured mouse small intestinal epithelial cells (IECs). The present results indicate that MVs from C. botulinum and related clostridial species induce host innate immune responses.

Osteoprotegerin-dependent M cell self-regulation balances gut infection and immunity.

Microfold cells (M cells) are responsible for antigen uptake to initiate immune responses in the gut-associated lymphoid tissue (GALT). Receptor activator of nuclear factor-κB ligand (RANKL) is essential for M cell differentiation. Follicle-associated epithelium (FAE) covers the GALT and is continuously exposed to RANKL from stromal cells underneath the FAE, yet only a subset of FAE cells undergoes differentiation into M cells. Here, we show that M cells express osteoprotegerin (OPG), a soluble inhibitor of RANKL, which suppresses the differentiation of adjacent FAE cells into M cells. Notably, OPG deficiency increases M cell number in the GALT and enhances commensal bacterium-specific immunoglobulin production, resulting in the amelioration of disease symptoms in mice with experimental colitis. By contrast, OPG-deficient mice are highly susceptible to Salmonella infection. Thus, OPG-dependent self-regulation of M cell differentiation is essential for the balance between the infectious risk and the ability to perform immunosurveillance at the mucosal surface.

The Roles of Peyer's Patches and Microfold Cells in the Gut Immune System: Relevance to Autoimmune Diseases.

Microfold (M) cells are located in the epithelium covering mucosa-associated lymphoid tissues, such as the Peyer's patches (PPs) of the small intestine. M cells actively transport luminal antigens to the underlying lymphoid follicles to initiate an immune response. The molecular machinery of M-cell differentiation and function has been vigorously investigated over the last decade. Studies have shed light on the role of M cells in the mucosal immune system and have revealed that antigen uptake by M cells contributes to not only mucosal but also systemic immune responses. However, M-cell studies usually focus on infectious diseases; the contribution of M cells to autoimmune diseases has remained largely unexplored. Accumulating evidence suggests that dysbiosis of the intestinal microbiota is implicated in multiple systemic diseases, including autoimmune diseases. This implies that the uptake of microorganisms by M cells in PPs may play a role in the pathogenesis of autoimmune diseases. We provide an outline of the current understanding of M-cell biology and subsequently discuss the potential contribution of M cells and PPs to the induction of systemic autoimmunity, beyond the mucosal immune response.

Fasting-Refeeding Impacts Immune Cell Dynamics and Mucosal Immune Responses.

Nutritional status potentially influences immune responses; however, how nutritional signals regulate cellular dynamics and functionality remains obscure. Herein, we report that temporary fasting drastically reduces the number of lymphocytes by ∼50% in Peyer's patches (PPs), the inductive site of the gut immune response. Subsequent refeeding seemingly restored the number of lymphocytes, but whose cellular composition was conspicuously altered. A large portion of germinal center and IgA+ B cells were lost via apoptosis during fasting. Meanwhile, naive B cells migrated from PPs to the bone marrow during fasting and then back to PPs during refeeding when stromal cells sensed nutritional signals and upregulated CXCL13 expression to recruit naive B cells. Furthermore, temporal fasting before oral immunization with ovalbumin abolished the induction of antigen-specific IgA, failed to induce oral tolerance, and eventually exacerbated food antigen-induced diarrhea. Thus, nutritional signals are critical in maintaining gut immune homeostasis.

Sox8 is essential for M cell maturation to accelerate IgA response at the early stage after weaning in mice.

Microfold (M) cells residing in the follicle-associated epithelium (FAE) of the gut-associated lymphoid tissue are specialized for antigen uptake to initiate mucosal immune responses. The molecular machinery and biological significance of M cell differentiation, however, remain to be fully elucidated. Here, we demonstrate that Sox8, a member of the SRY-related HMG box transcription factor family, is specifically expressed by M cells in the intestinal epithelium. The expression of Sox8 requires activation of RANKL-RelB signaling. Chromatin immunoprecipitation and luciferase assays revealed that Sox8 directly binds the promoter region of Gp2 to increase Gp2 expression, which is the hallmark of functionally mature M cells. Furthermore, genetic deletion of Sox8 causes a marked decrease in the number of mature M cells, resulting in reduced antigen uptake in Peyer's patches. Consequently, juvenile Sox8-deficient mice showed attenuated germinal center reactions and antigen-specific IgA responses. These findings indicate that Sox8 plays an essential role in the development of M cells to establish mucosal immune responses.

The Arf GTPase-activating protein SMAP1 promotes transferrin receptor endocytosis and interacts with SMAP2.

Arf GTPase-activating proteins (Arf GAP) play important roles in the formation of the membrane vesicles that traffic between subcellular membranous organelles. The small Arf GTPase-activating protein (SMAP) subfamily of Arf GAPs has two members, SMAP1 and SMAP2, in mammals. The present study investigated whether these two proteins may have an overlapping function in addition to their previously reported distinct functions. Results showed that the presence of either SMAP1 or SMAP2 was sufficient for endocytosis of the transferrin receptor, and that transferrin incorporation was impaired only by the absence of both SMAP1 and SMAP2. This suggests the involvement of both SMAP1 and SMAP2 in transferrin endocytosis. Results also demonstrated a physical association between SMAP1 and SMAP2, which might serve as a basis for a functional interaction, and identified the intramolecular domains responsible for this association.

Altered trafficking of mutated growth factor receptors and their associated molecules: implication for human cancers.

Ligand-stimulated receptor tyrosine kinases (RTKs) are phosphorylated/ubiquitinated, endocytosed and transported to the lysosomes via endosomes/multivesicular bodies, resulting in the attenuation of signal transmission. If this physiological mechanism of RTK signal downregulation is perturbed, signal transduction persists and may contribute to cellular transformation. This article presents several such examples. In some cases, endocytosis is impaired, and the activated RTK remains on the plasma membrane. In other cases, the activated RTK is endocytosed into endosomes/multivesicular bodies, but not subsequently sorted to the lysosomes for degradation. The latter cases indicate that even endocytosed RTKs can transmit signals. Transport of RTKs is accomplished via the formation and movement of membrane vesicles. Blockage or delay of endocytosis/trafficking can be caused by genetic alterations in the RTK itself or by mutations in CBL, Arf GAPs, or other components involved in internalization and vesicle transport. A survey of the literature indicates that, in some cases, even RTKs synthesized de novo can initiate signaling at the endoplasmic reticulum/Golgi before reaching the plasma membrane. The spectrum of molecules targeted by the signal is likely to be different between cell surface- and endoplasmic reticulum/Golgi-localized RTKs.