Roger Peronet

Mast cell-derived exosomes induce phenotypic and functional maturation of dendritic cells and elicit specific immune responses in vivo

Mast cells (MCs) are considered major players in IgE-mediated allergic responses, but have also recently been recognized as active participants in innate as well as specific immune responses. Recent work provided evidence that MCs are able to activate B and T lymphocytes through the release of vesicles called exosomes. Here we demonstrate that exosomes, which are located in the endocytic pathway, harbor exogenous Ags that associate with other molecules endowed with immunomodulatory functions, including 60- and 70-kDa heat shock proteins. Administration to naive mice of Ag-containing exosomes in the absence of conventional adjuvants elicits specific Ab responses across the MHC II haplotype barrier. We demonstrate that MC-exosomes induce immature dendritic cells (DCs) to up-regulate MHC class II, CD80, CD86, and CD40 molecules and to acquire potent Ag-presenting capacity to T cells. Uptake and processing of Ag-associated exosomes by endogenous DCs were also demonstrated. Finally, exosome-associated heat shock proteins are critical for the acquisition by DCs of the Ag-presenting function. This work demonstrates a heretofore unrecognized collaborative interaction between MCs and DCs leading to the elicitation of specific immune responses.

Mast Cell-Dependent B and T Lymphocyte Activation Is Mediated by the Secretion of Immunologically Active Exosomes

Mitogenic activity of bone marrow-derived mouse mast cells and mast cell lines P815 and MC/9 on B and T lymphocytes is present in their culture supernatants. To identify this activity, mast cells were incubated in serum-free medium and the supernatant was subjected to differential centrifugation, which resulted in two fractions, the hypodense and dense fraction (pellet). When analyzed for their mitogenic activity on spleen cells, all activity was found to be associated with the dense fraction. Electron microscopy studies revealed the presence in this fraction of small vesicles called exosomes with a heterogeneous size from 60 to 100 nm of diameter. When cocultured with spleen cells, purified exosomes induced blast formation, proliferation, as well as IL-2 and IFN-γ production, but no detectable IL-4. Similar data were obtained by injecting exosomes into naive mice. In contrast to mast cell lines, a pretreatment with IL-4 is required for bone marrow-derived mast cells to secrete active exosomes. Structurally, exosomes were found to harbor immunologically relevant molecules such as MHC class II, CD86, LFA-1, and ICAM-1. These findings indicate that mast cells can represent a critical component of the immunoregulatory network through secreted exosomes that display mitogenic activity on B and T lymphocytes both in vitro and in vivo.

Anopheles mosquito bites activate cutaneous mast cells leading to a local inflammatory response and lymph node hyperplasia.

When Anopheles mosquitoes probe the skin for blood feeding, they inject saliva in dermal tissue. Mosquito saliva is known to exert various biological activities, but its perception by the immune system and its role in parasite transmission remain poorly understood. In the present study, we report on the cellular changes occurring in the mouse skin and draining lymph nodes after a Anopheles stephensi mosquito bite. We show that mosquito bites induce dermal mast cell degranulation, leading to fluid extravasation and neutrophil influx. This inflammatory response does not occur in mast cell-deficient W/Wv mice, unless these are reconstituted specifically with mast cells. Mast cell activation caused by A. stephensi mosquito bites is followed by hyperplasia of the draining lymph node due to the accumulation of CD3+, B220+, CD11b+, and CD11c+ leukocytes. The T cell enrichment of the draining lymph nodes results from their sequestration from the circulation rather than local proliferation. These data demonstrate that mosquito bites and very likely saliva rapidly trigger the immune system, emphasizing the critical contribution of peripheral mast cells in inducing T cell and dendritic cell recruitment within draining lymph nodes, a prerequisite for the elicitation of T and B lymphocyte priming.

Mast Cell-Dependent Down-Regulation of Antigen-Specific Immune Responses by Mosquito Bites

While probing host skin to search for blood vessels, the female Anopheles mosquito delivers Plasmodium parasites in the presence of saliva. Saliva from various blood-feeding vectors which contains several pharmacologically active components is believed to facilitate blood feeding as well as parasite transmission to the host. Recently, we found that mosquito saliva has the capacity to activate dermal mast cells and to induce local inflammatory cell influx. Our main objective in the present work is to investigate whether saliva, through mosquito bites, controls the magnitude of Ag-specific immune responses and whether this control is dependent on the mast cell-mediated inflammatory response. Using a mast cell knockin mouse model, we found that mosquito bites consistently induced MIP-2 in the skin and IL-10 in draining lymph nodes, and down-regulate Ag-specific T cell responses by a mechanism dependent on mast cells and mediated by IL-10. Our results provide evidence for new mechanisms which may operate during Plasmodium parasite transmission by mosquito bites.

Inhibition of histamine-mediated signaling confers significant protection against severe malaria in mouse models of disease

From the inoculation of Plasmodium sporozoites via Anopheles mosquito bites to the development of blood-stage parasites, a hallmark of the host response is an inflammatory reaction characterized by elevated histamine levels in the serum and tissues. Given the proinflammatory and immunosuppressive activities associated with histamine, we postulated that this vasoactive amine participates in malaria pathogenesis. Combined genetic and pharmacologic approaches demonstrated that histamine binding to H1R and H2R but not H3R and H4R increases the susceptibility of mice to infection with Plasmodium. To further understand the role of histamine in malaria pathogenesis, we used histidine decarboxylase–deficient (HDC−/−) mice, which are free of histamine. HDC−/− mice were highly resistant to severe malaria whether infected by mosquito bites or via injection of infected erythrocytes. HDC−/− mice displayed resistance to two lethal strains: Plasmodium berghei (Pb) ANKA, which triggers cerebral malaria (CM), and Pb NK65, which causes death without neurological symptoms. The resistance of HDC−/− mice to CM was associated with preserved blood–brain barrier integrity, the absence of infected erythrocyte aggregation in the brain vessels, and a lack of sequestration of CD4 and CD8 T cells. We demonstrate that histamine-mediated signaling contributes to malaria pathogenesis. Understanding the basis for these biological effects of histamine during infection may lead to novel therapeutic strategies to alleviate the severity of malaria.

Critical role of the neutrophil-associated high-affinity receptor for IgE in the pathogenesis of experimental cerebral malaria

FcεR1-expressing neutrophils accumulate in the brain of mice infected with Plasmodium berghei (PbANKA) and promote the development of experimental cerebral malaria.

Capacity of mouse mast cells to prime T cells and to induce specific antibody responses in vivo

Mouse, human and rat mast cells have been shown to express major histocompatibility complex II molecules and present antigens to specific T‐cell hybridomas in vitro. The purpose of our investigation was to determine whether mouse mast cells are able to initiate specific immune responses in vivo. Induction of anti‐dinitrophenyl (DNP) immunoglobulin G1 (IgG1) and IgG2a antibodies was performed by transferring ovalbumin (OVA)–DNP‐pulsed bone marrow‐derived mast cells (BMMC), B cells, or macrophages into naive mice which were boosted later with soluble antigen. Cultured spleen cells from immunized mice were tested for their cytokine content. Our data show that mast cells were by far better inducers of anti‐DNP IgG1 antibodies than were B cells and macrophages. In contrast, anti‐DNP IgG2a response induced by macrophages was much stronger than that obtained with mast cells whereas B cells were completely unable to elicit this response. In addition to a high index of cell proliferation, spleen cells from mast cell‐injected mice produced more interferon‐γ than those mice who received macrophages or B cells by two‐ to fivefold, and almost 10‐fold, respectively. Mast cell‐deficient Wf/Wf mice were compared with their normal +/+ littermates and with mast cell‐reconstituted Wf/Wf mice to develop delayed‐type hypersensitivity (DTH) reactions as well as humoral immune responses. Mast cell sufficient mice as well as mast cell‐reconstituted Wf/Wf mice developed significantly increased DTH reactions (P = 0·02, and 0·03, repectively) and higher anti‐OVA‐specific antibody responses as compared with Wf/Wf mice. Our data suggest that mast cells have the potential to up‐regulate both humoral and cellular immune responses in vivo.

Nonspecific B and T cell-stimulatory activity mediated by mast cells is associated with exosomes.

Bone marrow-derived mouse mast cells (BMMC) and mast cell lines P815 and MC9 have recently been shown to induce antigen-independent B and T lymphocyte activation. It has been demonstrated that a physical contact between mast cells and B and T lymphocytes is not necessary since mast cell supernatants contain full activity. Electron microscopy studies revealed the presence in mast cell supernatants of small vesicles called exosomes with a heterogeneous size from 60 to 100 nm of diameter. When cocultured with spleen cells, purified exosomes induce B and T cell blast formation, proliferation as well as IL-2 and IFN-γ production. In contrast to P815 and MC9 mast cell lines, a pretreatment with IL-4 is required for BMMC to produce active exosomes. Structurally, these exosomes were found to harbor immunologically relevant molecules such as MHC class II, CD86, LFA-1 and ICAM-1. Here we provide for the first time the evidence that mast cells use exosomes as sophisticated messengers to communicate with cells of the immune system.

FcεRI-mediated antigen endocytosis turns interferon-γ-treated mouse mast cells from inefficient into potent antigen-presenting cells

Previous studies in our laboratory have shown that bone‐marrow‐derived mast cells (BMMC) could present immunogenic peptides, from soluble antigens endocytosed through fluid phase, only if they were subjected to a 48‐hr treatment with interleukin‐4 (IL‐4) and granulocyte–macrophage colony‐stimulating factor (GM‐CSF). In contrast to GM‐CSF, interferon‐γ (IFN‐γ) which highly upregulates major histocompatibility complex (MHC) class II expression, completely inhibits the generation of immunogenic peptides. We have used this model to study the role of FcεRI‐mediated antigen internalization in the regulation of the antigen‐presenting function of IFN‐γ‐treated mast cells. Here, we report that FcεRI can reverse the IFN‐γ‐treated mast cells from inefficient to highly efficient antigen‐presenting cells. Inhibition of the antigen presenting capacity by piceatannol, a protein tyrosine kinase (PTK) syk inhibitor, indicates that this is an active process resulting from immunoglobulin E (IgE)–antigen–FcεRI engagement which involves tyrosines found in the immunoreceptor tyrosine‐based activation motif (ITAM) embedded in the cytoplasmic tail of the FcεRI β and γ chains. Antigen‐presenting function was also shown to require the activation of phosphatidyl inositol 3 (PI3) kinase, downstream of PTK syk phosphorylation, since this activity was completely blocked by wortmannin, a PI3 kinase inhibitor. These data suggest that signalling generated by FcεRI provides mast cells with IgE‐mediated enhanced antigen presentation to T cells and emphasize a so far unknown immunoregulatory mast‐cell function that might take place in inflammatory sites.

Histamine H3 Receptor-Mediated Signaling Protects Mice from Cerebral Malaria

Background Histamine is a biogenic amine that has been shown to contribute to several pathological conditions, such as allergic conditions, experimental encephalomyelitis, and malaria. In humans, as well as in murine models of malaria, increased plasma levels of histamine are associated with severity of infection. We reported recently that histamine plays a critical role in the pathogenesis of experimental cerebral malaria (CM) in mice infected with Plasmodium berghei ANKA. Histamine exerts its biological effects through four different receptors designated H1R, H2R, H3R, and H4R. Principal Findings In the present work, we explored the role of histamine signaling via the histamine H3 receptor (H3R) in the pathogenesis of murine CM. We observed that the lack of H3R expression (H3R−/− mice) accelerates the onset of CM and this was correlated with enhanced brain pathology and earlier and more pronounced loss of blood brain barrier integrity than in wild type mice. Additionally tele-methylhistamine, the major histamine metabolite in the brain, that was initially present at a higher level in the brain of H3R−/− mice was depleted more quickly post-infection in H3R−/− mice as compared to wild-type counterparts. Conclusions Our data suggest that histamine regulation through the H3R in the brain suppresses the development of CM. Thus modulating histamine signaling in the central nervous system, in combination with standard therapies, may represent a novel strategy to reduce the risk of progression to cerebral malaria.