RNA-based therapeutics hold great promise for treating diseases and lipid nanoparticles (LNPs) represent the most advanced platform for RNA delivery. However, the fate of the LNP-mRNA after endosome-engulfing and escape from the autophagy-lysosomal pathway remains unclear. To investigate this, mRNA (encoding human erythropoietin) was delivered to cells using LNPs, which shows, for the first time, a link between LNP-mRNA endocytosis and its packaging into extracellular vesicles (endo-EVs: secreted after the endocytosis of LNP-mRNA). Endosomal escape of LNP-mRNA is dependent on the molar ratio between ionizable lipids and mRNA nucleotides. Our results show that fractions of ionizable lipids and mRNA (1:1 molar ratio of hEPO mRNA nucleotides:ionizable lipids) of endocytosed LNPs were detected in endo-EVs. Importantly, these EVs can protect the exogenous mRNA during in vivo delivery to produce human protein in mice, detected in plasma and organs. Compared to LNPs, endo-EVs cause lower expression of inflammatory cytokines.

Regulatory T cells (Treg) are crucial for the maintenance of tolerance to auto-antigens and harmless exogenous antigens. Here, we studied the role of the commensal microbiota for the development and function of Treg. CD4 + CD25 + T cells were obtained from peripheral lymph nodes (PLN) and mesenteric lymph nodes (MLN) of germ-free (GF) and conventional (conv) NMRI mice and tested for phenotype and functional suppressive capacity. CD4 + CD25 + T cells from GF mice showed a lower relative gene expression of fork head box p3 gene (Foxp3) and were not as potent suppressors in vitro as CD4 + CD25 + T cells from conv animals. Intracellular staining for Foxp3 and CTLA-4 revealed proportional and regional differences in putative Treg subsets between conv and GF mice. Fewer of the CD4 + CD25 + T cells in GF MLN expressed Foxp3 and CTLA-4, while the expression of these markers was similar amongst the CD4 + CD25 + T cells in PLN of conv and GF mice. The largest difference between conv and GF Treg was observed in the liver draining celiac lymph node, where GF mice had fewer putative Treg as compared to conv mice. We propose that the presence of a microbial flora favors the development of a fully functional Treg population. IntroductionRegulatory T cells down-regulate unwanted and exaggerated immune reactions to auto-antigens as well as innocuous environmental antigens. Different subsets of Treg have been defined. Natural CD4 + CD25 + Treg are positively selected for tissue-specific self-Ag in the thymus and exert their suppressive effect by cell-cell contact-dependent mechanisms [1]. Deficiency in the Treg population or neonatal thymectomy result in multiorgan autoimmune diseases [2][3][4][5][6][7][8]. Treg are characterized by a dense surface expression of CD25 (the a-chain of the IL-2-receptor), glucocorticoidinduced TNF receptor (GITR) and intracellular CTLA-4, however, these markers may be present on activated T cells. CD4 + CD25 + Tregs appear to be unique in transcribing the fork head box p3 gene (Foxp3) transcription factor [6,9], which enable their identification either with PCR are or newly developed mAb to the Foxp3 protein. Lack of a functional Foxp3 gene result in the severe autoimmune syndrome IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome), characterized by organ-specific autoimmunity, colitis and high IgE levels [10,11]. Mice with spontaneous or induced mutations in the Foxp3 gene that impair Foxp3 expression fail to develop CD4 + CD25 + Treg and exhibit a syndrome similar to IPEX [5,6].Other subsets of Treg are induced in the periphery and are referred to as Th3 [12][13][14] and Tr1 cells [15][16][17][18][19] can also generate Treg. This implies that the state of the APC and the local cytokine milieu, e.g. in the normal gut where both IL-10 and TGF-b are abundant, can determine the outcome of an immune reaction, and that Treg are induced under these circumstances.The aim of this study was to investigate the phenotype and functionality of CD4 + CD25 + Treg from germ-free (GF) ...

SummaryOral administration of a protein antigen generates a serum factor that induces tolerance when transferred into naïve recipients. This serum factor has been described in rats as consisting of exosome-like structures or tolerosomes, which express major histocompatibility complex class II molecules (MHCII) and mediate antigen-specific tolerance. In this study, we investigated the functions of serum-derived tolerosomes both in vivo and in vitro. Tolerosomes were purified from the 100 000 g pellet fraction of serum from ovalbumin (OVA)-fed mice. When transferred into naïve recipient mice, the tolerosomes mediated OVA-specific tolerance. We also found that tolerosomes from OVA-fed mice induced the activation of OVA-specific T cells both in vivo and in vitro. The inoculation of severe combined immunodeficiency (SCID) mice with an interferon-c-producing cell line normalized the expression of MHCII in the intestinal epithelial cells and restored their ability to generate tolerosomes. Syngeneic but not allogeneic transfer of tolerosomes from OVA-fed donors induced tolerance in the recipients. Our results show that tolerosomes can be isolated from mouse serum, that tolerosome-induced oral tolerance requires MHCII expression in intestinal epithelial cells, and that tolerosomes are functional only in syngeneic recipients.

Background: Specific pathogenic bacteria have been implicated in recurrent aphthous stomatitis (RAS), a chronic inflammatory condition characterised by ulcerations in the oral mucosa. However, the aetiology behind this condition still remains unclear. Objective: The buccal microbiota of patients with RAS was compared to that of control subjects to investigate its potential role for this condition. Design: Buccal swabs were obtained from non-ulcerative areas of 60 patients, of whom 42 patients had lesions at the time of sampling, and 60 healthy age-and gender-matched controls. Bacterial DNA was extracted and analysed by Terminal-Restriction Fragment Length Polymorphism, using enzymatic digestion of the polymerase chain reactionamplified 16S rRNA gene, yielding a series of peaks, each representing a bacterial taxon. Results: Two peaks, 60 and 489, were more prevalent in patients with RAS than controls. Conversely, peaks 58 and 490 were less common in patients than controls. When the patients were divided into subgroups, we found that the observed differences in peak-pattern were related to the presence of lesions during sampling. Conclusions: The microbiota of the non-inflamed buccal mucosa differed between patients and controls. The differences were most pronounced in patients who presented with lesions during sampling, suggesting that a disturbance in the normal buccal microbiota triggers the presence of lesions or that presence of lesions alters the microbiota.