Royal jelly extracellular vesicles promote wound healing by modulating underlying cellular responses.

Apis mellifera royal jelly (RJ) is a well-known remedy in traditional medicine around the world and its versatile effects range from antibacterial to anti-inflammatory properties and pro-regenerative properties. As a glandular product, RJ has been shown to contain a substantial number of extracellular vesicles (EVs), and, in this study, we aimed to investigate the extent of involvement of RJEVs in wound healing-associated effects. Molecular analysis of RJEVs verified the presence of exosomal markers such as CD63 and syntenin, and cargo molecules MRJP1, defensin-1, and jellein-3. Furthermore, RJEVs were demonstrated to modulate mesenchymal stem cell (MSC) differentiation and secretome, as well as decrease LPS-induced inflammation in macrophages by blocking the mitogen-activated protein kinase (MAPK) pathway. In vivo studies confirmed antibacterial effects of RJEVs and demonstrated an acceleration of wound healing in a splinted mouse model. This study suggests that RJEVs play a crucial role in the known effects of RJ by modulating the inflammatory phase and cellular response in wound healing. Transfer of RJ into the clinics has been impeded by the high complexity of the raw material. Isolating EVs from the raw RJ decreases the complexity while allowing standardization and quality control, bringing a natural nano-therapy one step closer to the clinics.

CD49b Targeting Inhibits Tumor Growth and Boosts Anti-tumor Immunity.

The suppressive function of T-regulatory cells (Tregs) can have a detrimental effect on immune responses against tumor cells. Within the Treg cells subset, a new non-classical population has been reported, which expresses high levels of CD49b molecule and, depending on their activation status, can also express the canonical Tregs transcription factor Foxp3. In this report, we sought to characterize Tregs subsets in a murine melanoma model and disrupt the CD49b/CD29 axis by administering an anti-CD29 antibody in tumor-bearing mice. Our data shows that whereas in the draining lymph nodes, the Tr1 cells subset composes <5% of CD4+ T cells, in the tumor, they reach ∼30% of CD4+ T cells. Furthermore, Tr1 cells share the expression of suppressive molecules, such as Nrp-1, PD-1, and CD73, which are highly expressed on Tr1 cells found in tumor-infiltrating leukocytes (TILs). Regardless of the phenotypic similarities with cTreg cells, Tr1 cells display a low proliferative activity, as shown in the kinetics and the incorporation of 5-bromodeoxyuridine (BrdU) experiments. With the intent to impact on Tr1 cells, we administered anti-CD29 antibody into tumor mice, observing that the treatment effectively inhibits tumor growth. This effect is at least mediated by the enrichment of pro-inflammatory T cells, including IFN-γ+ cTreg and IFN-γ+ Tr1 cells (with reduced expression of IL-10), plus Th1 and Tc cells. In this study, we present Tr1 cell characterization in tumor-bearing animals and introduce CD29 as a target for tumor therapy, supported by a meta-analysis indicating that CD29 is present in human biopsies.

Type I collagen hydrogels as a delivery matrix for royal jelly derived extracellular vesicles.

Throughout the last decade, extracellular vesicles (EVs) have become increasingly popular in several areas of regenerative medicine. Recently, Apis mellifera royal jelly EVs (RJ EVs) were shown to display favorable wound healing properties such as stimulation of mesenchymal stem cell migration and inhibition of staphylococcal biofilms. However, the sustained and effective local delivery of EVs in non-systemic approaches - such as patches for chronic cutaneous wounds - remains an important challenge for the development of novel EV-based wound healing therapies. Therefore, the present study aimed to assess the suitability of type I collagen -a well-established biomaterial for wound healing - as a continuous delivery matrix. RJ EVs were integrated into collagen gels at different concentrations, where gels containing 2 mg/ml collagen were found to display the most stable release kinetics. Functionality of released RJ EVs was confirmed by assessing fibroblast EV uptake and migration in a wound healing assay. We could demonstrate reliable EV uptake into fibroblasts with a sustained pro-migratory effect for up to 7 d. Integrating fibroblasts into the RJ EV-containing collagen gel increased the contractile capacity of these cells, confirming availability of RJ EVs to fibroblasts within the collagen gel. Furthermore, EVs released from collagen gels were found to inhibit Staphylococcus aureus ATCC 29213 biofilm formation. Overall, our results suggest that type I collagen could be utilized as a reliable, reproducible release system to deliver functional RJ EVs for wound healing therapies.

Complex Interaction between Resident Microbiota and Misfolded Proteins: Role in Neuroinflammation and Neurodegeneration.

Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) and Creutzfeldt-Jakob disease (CJD) are brain conditions affecting millions of people worldwide. These diseases are associated with the presence of amyloid-ß (Aß), alpha synuclein (α-Syn) and prion protein (PrP) depositions in the brain, respectively, which lead to synaptic disconnection and subsequent progressive neuronal death. Although considerable progress has been made in elucidating the pathogenesis of these diseases, the specific mechanisms of their origins remain largely unknown. A body of research suggests a potential association between host microbiota, neuroinflammation and dementia, either directly due to bacterial brain invasion because of barrier leakage and production of toxins and inflammation, or indirectly by modulating the immune response. In the present review, we focus on the emerging topics of neuroinflammation and the association between components of the human microbiota and the deposition of Aß, α-Syn and PrP in the brain. Special focus is given to gut and oral bacteria and biofilms and to the potential mechanisms associating microbiome dysbiosis and toxin production with neurodegeneration. The roles of neuroinflammation, protein misfolding and cellular mediators in membrane damage and increased permeability are also discussed.

T regulatory cells-derived extracellular vesicles and their contribution to the generation of immune tolerance.

T regulatory (Treg) cells have a major role in the maintenance of immune tolerance against self and foreign antigens through the control of harmful inflammation. Treg cells exert immunosuppressive function by several mechanisms, which can be distinguished as contact dependent or independent. Recently, the secretion of extracellular vesicles (EVs) by Treg cells has been reported as a novel suppressive mechanism capable of modulating immunity in a cell-contact independent and targeted manner, which has been identified in different pathologic scenarios. EVs are cell-derived membranous structures involved in physiologic and pathologic processes through protein, lipid, and genetic material exchange, which allow intercellular communication. In this review, we revise and discuss current knowledge on Treg cells-mediated immune tolerance giving special attention to the production and release of EVs. Multiple studies support that Treg cells-derived EVs represent a refined intercellular exchange device with the capacity of modulating immune responses, thus creating a tolerogenic microenvironment in a cell-free manner. The mechanisms proposed encompass miRNAs-induced gene silencing, the action of surface proteins and the transmission of enzymes. These observations gain relevance by the fact that Treg cells are susceptible to converting into effector T cells after exposition to inflammatory environments. Yet, in contrast to their cells of origin, EVs are unlikely to be modified under inflammatory conditions, highlighting the advantage of their use. Moreover, we speculate in the possibility that Treg cells may contribute to infectious tolerance via vesicle secretion, intervening with CD4+ T cells differentiation and/or stability.

CD4+Foxp3+T Regulatory Cells Promote Transplantation Tolerance by Modulating Effector CD4+ T Cells in a Neuropilin-1-Dependent Manner.

Several mechanisms of immune suppression have been attributed to Foxp3+ T regulatory cells (Treg) including modulation of target cells via inhibition of cell proliferation, alteration of cytokine secretion, and modification of cell phenotype, among others. Neuropilin-1 (Nrp1), a co-receptor protein highly expressed on Treg cells has been involved in tolerance-mediated responses, driving tumor growth and transplant acceptance. Here, we extend our previous findings showing that, despite expressing Foxp3, Nrp1KO Treg cells have deficient suppressive function in vitro in a contact-independent manner. In vivo, the presence of Nrp1 on Treg cells is required for driving long-term transplant tolerance. Interestingly, Nrp1 expression on Treg cells was also necessary for conventional CD4+ T cells (convT) to become Nrp1+Eos+ T cells in vivo. Furthermore, adoptive transfer experiments showed that the disruption of Nrp1 expression on Treg cells not only reduced IL-10 production on Treg cells, but also increased the frequency of IFNγ+ Treg cells. Similarly, the presence of Nrp1KO Treg cells facilitated the occurrence of IFNγ+CD4+ T cells. Interestingly, we proved that Nrp1KO Treg cells are also defective in IL-10 production, which correlates with deficient Nrp1 upregulation by convT cells. Altogether, these findings demonstrate the direct role of Nrp1 on Treg cells during the induction of transplantation tolerance, impacting indirectly the phenotype and function of conventional CD4+ T cells.

Mesenchymal stem cells and their immunosuppressive role in transplantation tolerance.

Since they were first described, mesenchymal stem cells (MSCs) have been shown to have important effector mechanisms and the potential for use in cell therapy. A great deal of research has been focused on unveiling how MSCs contribute to anti-inflammatory responses, including describing several cell populations involved and identifying soluble and other effector molecules. In this review, we discuss some of the contemporary evidence for use of MSCs in the field of immune tolerance, with a special emphasis on transplantation. Although considerable effort has been devoted to understanding the biological function of MSCs, additional resources are required to clarify the mechanisms of their induction of immune tolerance, which will undoubtedly lead to improved clinical outcomes for MSC-based therapies.