Extracellular vesicles derived from antigen-presenting cells pulsed with foot and mouth virus vaccine-antigens act as carriers of viral proteins and stimulate B cell response.

Foot and mouth disease (FMD) is a highly contagious infection caused by FMD-virus (FMDV) that affects livestock worldwide with significant economic impact. The main strategy for the control is vaccination with FMDV chemically inactivated with binary ethylenimine (FMDVi). In FMDV infection and vaccination, B cell response plays a major role by providing neutralizing/protective antibodies in animal models and natural hosts. Extracellular vesicles (EVs) and small EVs (sEVs) such as exosomes are important in cellular communication. EVs secreted by antigen-presenting cells (APC) like dendritic cells (DCs) participate in the activation of B and T cells through the presentation of native antigen membrane-associated to B cells or by transferring MHC-peptide complexes to T cells and even complete antigens from DCs. In this study, we demonstrate for the first time that APC activated with the FMDVi O1 Campos vaccine-antigens secrete EVs expressing viral proteins/peptides that could stimulate FMDV-specific immune response. The secretion of EVs-FMDVi is a time-dependent process and can only be isolated within the first 24 h post-activation. These vesicles express classical EVs markers (CD9, CD81, and CD63), along with immunoregulatory molecules (MHC-II and CD86). With an average size of 155 nm, they belong to the category of EVs. Studies conducted in vitro have demonstrated that EVs-FMDVi express antigens that can stimulate a specific B cell response against FMDV, including both marginal zone B cells (MZB) and follicular B cells (FoB). These vesicles can also indirectly or directly affect T cells, indicating that they express both B and T epitopes. Additionally, lymphocyte expansion induced by EVs-FMDVi is greater in splenocytes that have previously encountered viral antigens in vivo. The present study sheds light on the role of EVs derived from APC in regulating the adaptive immunity against FMDV. This novel insight contributes to our current understanding of the immune mechanisms triggered by APC during the antiviral immune response. Furthermore, these findings may have practical implications for the development of new vaccine platforms, providing a rational basis for the design of more effective vaccines against FMDV and other viral diseases.

Modulation of B cell activation by extracellular vesicles and potential alteration of this pathway in patients with rheumatoid arthritis.

BACKGROUND: Extracellular vesicles are involved in the intercellular communication of the immune system. In rheumatoid arthritis (RA), these structures are considered a source of autoantigens that drive proinflammatory responses of innate immune cells. A high concentration of circulating medium/large size extracellular vesicles (m/lEVs) and m/lEVs forming immune complexes (m/lEV-ICs) have been associated with disease activity and systemic inflammation in patients with RA. B cells are central components of RA immunopathology because of their involvement in the production of autoantibodies, antigen presentation, and cytokine production. However, the effect of m/lEVs on B cell function in the context of RA and other autoimmune diseases remains unknown. METHODS: We evaluated the effect of m/lEVs obtained from healthy donors (HD) and patients with RA on B cell responses in vitro. In addition, we evaluated the effect of pre-exposition of monocyte-derived macrophages (MDM) to m/lEVs on activation of autologous B cells from HD and patients. RESULTS: The presence of m/lEVs reduced the frequency of CD69+ and CD86+ B cells from HD activated by an agonist of antigen receptor. This regulation of the B cell activation markers by m/lEVs was partially dependent on phosphatidylserine binging. These m/lEVs also reduced the proliferation, calcium mobilization, and global phosphorylation of tyrosine. Similar responses were observed in B cells from patients with RA. However, the presence of m/lEVs promoted high antibody levels in B cells cultured with T cell-dependent stimuli by 7 days. In addition, despite the direct inhibitory effect of m/lEVs on early B cell responses, when B cells were cocultured with autologous MDM previously exposed to m/lEVs or m/lEV-ICs, an increased frequency of CD69+ B cells from patients with RA was observed, albeit not with cells from HD. CONCLUSIONS: These data together suggest that m/lEVs have a direct modulatory effect in early responses of B cells through B cell receptor that can potentially fail in patients with RA because of the impact of these vesicles over cells of the innate immune system. This phenomenon can potentially contribute to the loss of tolerance and disease activity in patients with RA.

Alterations in Lysosome Homeostasis in Lipid-Related Disorders: Impact on Metabolic Tissues and Immune Cells.

Lipid-related disorders, which primarily affect metabolic tissues, including adipose tissue and the liver are associated with alterations in lysosome homeostasis. Obesity is one of the more prevalent diseases, which results in energy imbalance within metabolic tissues and lysosome dysfunction. Less frequent diseases include Niemann-Pick type C (NPC) and Gaucher diseases, both of which are known as Lysosomal Storage Diseases (LSDs), where lysosomal dysfunction within metabolic tissues remains to be fully characterized. Adipocytes and hepatocytes share common pathways involved in the lysosome-autophagic axis, which are regulated by the function of cathepsins and CD36, an immuno-metabolic receptor and display alterations in lipid diseases, and thereby impacting metabolic functions. In addition to intrinsic defects observed in metabolic tissues, cells of the immune system, such as B cells can infiltrate adipose and liver tissues, during metabolic imbalance favoring inflammation. Moreover, B cells rely on lysosomes to promote the processing and presentation of extracellular antigens and thus could also present lysosome dysfunction, consequently affecting such functions. On the other hand, growing evidence suggests that cells accumulating lipids display defective inter-organelle membrane contact sites (MCSs) established by lysosomes and other compartments, which contribute to metabolic dysfunctions at the cellular level. Overall, in this review we will discuss recent findings addressing common mechanisms that are involved in lysosome dysregulation in adipocytes and hepatocytes during obesity, NPC, and Gaucher diseases. We will discuss whether these mechanisms may modulate the function of B cells and how inter-organelle contacts, emerging as relevant cellular mechanisms in the control of lipid homeostasis, have an impact on these diseases.

Differential capability of Bacillus thuringiensis Cry1Ac protoxin and toxin to induce in vivo activation of dendritic cells and B lymphocytes.

The insecticidal Bacillus thuringiensis protein Cry1Ac is produced as a protoxin and becomes activated to a toxin when ingested by larvae. Both proteins are immunogenic and able to activate macrophages. The proposed mechanism of immunostimulation by Cry1Ac protoxin has been related to its capacity to activate antigen-presenting cells (APC), but its ability to activate dendritic cells (DC) has not been explored. Here we evaluated, in the popliteal lymph nodes (PLN), spleen and peritoneum, the activation of DC CD11c+ MHC-II+ following injection with single doses (50 µg) of Cry1Ac toxin or protoxin via the intradermal (i.d.) and intraperitoneal (i.p.) routes in C57BL/6 mice. In vivo stimulation with both Cry1Ac proteins induced activation of DC via upregulation of CD86, primarily in PLN 24 h after i. d. injection. Moreover, this activation was detected in DC, displaying CD103+, a typical marker of migratory DC, while upregulation of CD80 was uniquely induced by toxin. Tracking experiments showed that Cy5-labeled Cry1Ac proteins could rapidly reach the PLN and localize near DC, but some label remained in the footpad. When the capacity of Cry1Ac-activated DC to induce antigen presentation was examined, significant proliferation of naïve T lymphocytes was induced exclusively by the protoxin. The protoxin elicited a Th17-biased cytokine profile. Moreover, only the Cry1Ac toxin induced a pronounced proliferation of B cells from both untreated and Cry1Ac-injected mice, suggesting that it acts as a polyclonal activator. In conclusion, Cry1Ac protoxin and toxin show a distinctive capacity to activate APCs.

Atypical phenotype and response of B cells in patients with seropositive rheumatoid arthritis.

Patients with rheumatoid arthritis (RA) may be classified as seropositive or seronegative according to the presence of autoantibodies. An abnormal B cell phenotype and function could be one of the main components of the immunopathology of seropositive patients; however, there is little information regarding B cell defects in these patients. This study shows a broad characterization of the B cell phenotype and function in patients with seropositive RA. We focused mainly on the evaluation of subsets, the expression of modulatory molecules of cell activation (CD22, FcÉ£RIIb and FcµR), calcium mobilization, global tyrosine phosphorylation, expression of activation markers, cytokine and immunoglobulin (Ig) production, proliferation and the in-vitro generation of plasma cells. Increased frequency of CD27- IgM- IgD- and CD21- B cells was observed in patients with seropositive RA compared with healthy donors (HD). Decreased expression of CD22 was primarily found in memory B cells of patients with RA regardless of seropositivity. B cells from seropositive patients exhibited normal proliferation, calcium mobilization kinetics and global tyrosine phosphorylation, but showed an increased frequency of CD86+ B cells compared with HD. B cells of seropositive patients secrete less interleukin-10 after in-vitro activation and showed a decreased frequency of plasma cell differentiation and IgM production compared with HD. Our data indicate that patients with seropositive RA have an increased frequency of atypical B cell populations previously associated with chronic activation and antigen exposure. This may result in the observed low responsiveness of these cells in vitro.

Overexpression of Fc receptor-like 1 associated with B-cell activation during hepatitis B virus infection

The role of B cells in the pathogenesis of hepatitis B virus (HBV) infection has not been explored in depth. In the present study, the activation status of B cells from peripheral blood of healthy controls (N = 20) and patients with acute hepatitis B (AHB, N = 15) or chronic hepatitis B (CHB, N = 30) was evaluated by measuring the expression levels of B-cell activation markers CD69 and CD86, using quantitative real-time PCR and flow cytometry. Moreover, the potential mechanism underlying B-cell activation during HBV infection was further investigated by analyzing the expression profile of FCRL1, an intrinsic activation molecule of B cells. An elevation in the levels of B-cell activation markers including CD69 and CD86 was observed in the AHB patients (44.31 ± 9.27, 27.64 ± 9.26%) compared to CHB patients (30.35 ± 11.27, 18.41 ± 6.56%, P < 0.05), which was still higher than healthy controls (12.23 ± 7.84, 8.22 ± 3.43%, P < 0.05). Furthermore, the expression of FCRL1 was found to be similar to B-cell activation markers, which was highest in AHB patients (70.15 ± 17.11%), lowest in healthy donors (36.32 ± 9.98%, P < 0.05) and half-way between these levels in patients with CHB (55.17 ± 12.03%, P < 0.05). The results were positively associated with aberrant B-cell activation. These data suggest that B cells can play a role in HBV infection, and therefore more effort should be devoted to exploring their functions.