Céline Beauvillain

CCR7 is involved in the migration of neutrophils to lymph nodes

Increasing evidence suggests that neutrophils may participate in the regulation of adaptive immune responses, and can reach draining lymph nodes and cross-prime naive T cells. The aim of this study was to identify the mechanism(s) involved in the migration of neutrophils to the draining lymph nodes. We demonstrate that a subpopulation of human and mouse neutrophils express CCR7. CCR7 is rapidly expressed at the membrane upon stimulation. In vitro, stimulated human neutrophils migrate in response to the CCR7 ligands CCL19 and CCL21. In vivo, injection of complete Freund adjuvant induces a rapid recruitment of neutrophils to the lymph nodes in wild-type mice but not in Ccr7(-/-) mice. Moreover, intradermally injected interleukin-17-and granulocyte-macrophage colony-stimulating factor-stimulated neutrophils from wild-type mice, but not from Ccr7(-/-) mice, migrate to the draining lymph nodes. These results identify CCR7 as a chemokine receptor involved in the migration of neutrophils to the lymph nodes.

Neonatal and adult microglia cross-present exogenous antigens

Some observations have suggested that cells from the central nervous system (CNS) could present exogenous antigens on major histocompatibility complex (MHC) class I molecules to CD8+ T cells (a process called cross‐presentation). Microglia are the major myeloid immunocompetent cells of the CNS. When activated, following the injury of the nervous parenchyma, they become fully competent antigen‐presenting cells (APC) that prime CD4+ T lymphocytes. We therefore tested the cross‐presentation capacity of murine microglia. We report that a microglial cell line (C8‐B4), neonatal microglia, and interestingly adult microglia cross‐present soluble exogenous antigen (ovalbumin) to a OVA‐specific CD8+ T‐cell hybridoma and cross‐prime OVA‐specific naive OT‐1 CD8+ T cells. In both these cases, C8‐B4 and neonatal microglia cross‐present OVA as well as peritoneal macrophages. Although cross‐presentation by adult microglia is less efficient, it is increased by GM‐CSF and CpG oligodeoxynucleotide (ODN) stimulation. Using microglial cells either exposed to an inhibitor of proteasome, lactacystin, or purified from TAP−/− mice, we demonstrate that the microglia cross‐present antigen in proteasome‐ and TAP‐dependant pathways, respectively. Last, microglia purified from adult mice injected intracerebrally with OVA efficiently stimulate OVA‐specific CD8+ T cells, thereby showing that microglia take up and process exogenous antigen into MHC class I in vivo. This first demonstration of the cross‐presentation property of microglia offers novel therapeutic approaches to modulate CD8 T‐cell responses in the brain.

The scavenger receptors SRA-1 and SREC-I cooperate with TLR2 in the recognition of the hepatitis C virus non-structural protein 3 by dendritic cells

BACKGROUNDS & AIMS The hepatitis C virus NS3 protein is taken up by myeloid cells in a TLR2-independent manner and activates myeloid cells via TLR2. This study aimed to identify the endocytic receptor(s) involved in the uptake of NS3 by myeloid cells and its relation with TLR2. METHODS Inhibitors and transfected cells were used to identify the nature of the NS3-binding receptors expressed by myeloid cells. The cooperation between scavenger receptors (SRs) and TLR2 in the NS3-mediated activation of myeloid cells was evaluated using inhibitors, cells from TLR2(-/-) mice, and confocal microscopy. The involvement of SRs in NS3 cross-presentation was evaluated in vitro using an NS3-specific human T-cell clone. RESULTS We observed that SRs are the main binding structures for NS3 on myeloid cells and identified the SRs SRA-1 and SREC-I as endocytic receptors for NS3. Moreover, both SRs and TLR2 cooperate in NS3-induced myeloid cell activation. CONCLUSION This study highlights a central role for SRs in NS3 uptake and cross-presentation, and demonstrates a tightly orchestrated cooperation between signalling and endocytic innate receptors in NS3 recognition.

Prototypic Long Pentraxin PTX3 Is Present in Breast Milk, Spreads in Tissues, and Protects Neonate Mice from Pseudomonas aeruginosa Lung Infection

Newborns and infants present a higher susceptibility to infection than adults, a vulnerability associated with deficiencies in both the innate and adaptive immune systems. Innate immune receptors are sensors involved in the recognition and elimination of microbes that play a pivotal role at the interface between innate and adaptive immunity. Pentraxin 3 (PTX3), the prototypic long pentraxin, is a soluble pattern recognition receptor involved in the initiation of protective responses against selected pathogens. Because neonates are generally resistant to these pathogens, we suspected that PTX3 may be provided by a maternal source during the early life times. We observed that human colostrum contains high levels of PTX3, and that mammary epithelial cell and CD11b+ milk cells constitutively produce PTX3. Interestingly, PTX3 given orally to neonate mice was rapidly distributed in different organs, and PTX3 ingested during lactation was detected in neonates. Finally, we observed that orally administered PTX3 provided protection against Pseudomonas aeruginosa lung infection in neonate mice. Therefore, breastfeeding constitutes, during the early life times, an important source of PTX3, which actively participates in the protection of neonates against infections. In addition, these results suggest that PTX3 might represent a therapeutic tool for treating neonatal infections and support the view that breastfeeding has beneficial effects on the neonates’ health.

Clusterin facilitates apoptotic cell clearance and prevents apoptotic cell-induced autoimmune responses

Clusterin (Clu), an extracellular chaperone, exhibits characteristics of soluble innate immunity receptors, as assessed by its ability to bind some bacteria strains. In this study, we report that Clu also binds specifically to late apoptotic cells but not to live, early apoptotic, or necrotic cells. Histones, which accumulate on blebs during the apoptotic process, represent privileged Clu-binding motifs at the surface of late apoptotic cells. As a consequence, Clu potentiates, both in vitro and in vivo, the phagocytosis of late apoptotic cells by macrophages. Moreover, the increased phagocytosis of late apoptotic cells induced by Clu favors the presentation and cross-presentation of apoptotic cell-associated antigens. Finally, we observed that, in a model of apoptotic cell-induced autoimmunity, and relative to control mice, Clu−/− mice develop symptoms of autoimmunity, including the generation of anti-dsDNA antibodies, deposition of immunoglobulins and complement components within kidneys, and splenomegaly. These results identify Clu as a new molecule partner involved in apoptotic cell efferocytosis and suggest a protective role for Clu in inflammation and autoimmune diseases.

The angiotensin II type 2 receptor activates flow-mediated outward remodelling through T cells-dependent interleukin-17 production

AIMS The angiotensin II type 1 receptor (AT1R) through the activation of immune cells plays a key role in arterial inward remodelling and reduced blood flow in cardiovascular disorders. On the other side, flow (shear stress)-mediated outward remodelling (FMR), involved in collateral arteries growth in ischaemic diseases, allows revascularization. We hypothesized that the type 2 receptor (AT2R), described as opposing the effects of AT1R, could be involved in FMR. METHODS AND RESULTS We studied FMR using a model of ligation of feed arteries supplying collateral pathways in the mouse mesenteric arterial bed in vivo. Seven days after ligation, diameter increased by 30% in high flow (HF) arteries compared with normal flow vessels. FMR was absent in mice lacking AT2R. At Day 2, T lymphocytes expressing AT2R were present preferentially around HF arteries. FMR did not occur in athymic (nude) mice lacking T cells and in mice treated with anti-CD3ε antibodies. AT2R activation induced interleukin-17 production by memory T cells. Treatment of nude mice or AT2R-deficient mice with interleukin-17 restored diameter enlargement in HF arteries. Interleukin-17 increased NO-dependent relaxation and matrix metalloproteinases activity, both important in FMR. Remodelling of feeding arteries in the skin flap model of ischaemia was also absent in AT2R-deficient mice and in anti-interleukin-17-treated mice. Finally, remodelling, absent in 12-month-old mice, was restored by a treatment with the AT2R non-peptidic agonist C21. CONCLUSION AT2R-dependent interleukin-17 production by T lymphocyte is necessary for collateral artery growth and could represent a new therapeutic target in ischaemic disorders.

PPARα Regulates Endothelial Progenitor Cell Maturation and Myeloid Lineage Differentiation Through a NADPH Oxidase-Dependent Mechanism in Mice

Peroxisome proliferator‐activated receptor‐alpha (PPARα) is a key modulator of lipid metabolism. Here, we propose that PPARα regulates the maturation and function of bone marrow (BM) progenitor cells. Although PPARα deletion increased the number of BM‐resident cells and the differentiation of endothelial progenitor cells (EPCs) and monocytic progenitor cells, it impaired re‐endothelialization of injured carotid artery that was associated with reduced circulating EPCs. Also, PPARα deletion diminished the in vivo proangiogenic effect of PPARα agonist without affecting EPC differentiation markers. Macrophage colony‐stimulating factor treatment increased the population of monocytic progenitor cells as well as secretome of BM‐derived cells in PPARα wild‐type but not in knockout mice. In addition, PPARα‐null mice displayed reduced lymphocytes and increased monocytes and neutrophils in the blood. Furthermore, PPARα‐null mice exhibited increments in the number of total cells (as well as of phenotypically distinct subpopulations of lymph node cells) but also a significant alteration in the number of various subpopulations of splenocytes and thymocytes. Finally, PPARα negatively regulated reactive oxygen species derived by NADPH oxidase in BM‐resident progenitor cells. Taken together, our data provide evidence that PPARα is a critical regulator of recruitment, homing, and maturation of BM‐derived progenitor cells. Stem Cells 2015;33:1292–1303

Comparison of two enzymatic immunoassays, high resolution mass spectrometry method and radioimmunoassay for the quantification of human plasma histamine

Histamine (HA) is one of the main immediate mediators involved in allergic reactions. HA plasma concentration is well correlated with the severity of vascular and respiratory signs of anaphylaxis. Consequently, plasma quantification of HA is useful to comfort the diagnosis of anaphylaxis. Currently, radioimmunoassay (RIA) is the gold standard method to quantify HA due to its high sensitivity, but it is time consuming, implicates specific formations and cautions for technicians, and produces hazardous radioactive wastes. The aim of this study was to compare two enzymatic immunoassays (EIA) and one in-house liquid chromatography high-resolution mass spectrometry method (LC-HRMS) with the gold standard method for HA quantification in plasma samples of patients suspected of anaphylaxis reactions. Ninety-two plasma samples were tested with the 4 methods (RIA, 2 EIA and LC-HRMS) for HA quantification. Fifty-eight samples displayed HA concentrations above the positive cut-off of 10nM evaluated by RIA, including 18 highly positive samples (>100 nM). This study shows that Immunotech(®) EIA and LC-HRMS concentrations were highly correlated with RIA values, in particular for samples with a HA concentration around the positive cut-off. In our hands, plasma concentrations obtained with the Demeditec Diagnostics(®) EIA correlated less with results obtained by RIA, and an underestimation of plasma HA levels led to a lack of sensitivity. In conclusion, this study demonstrates that Immunotech(®) EIA and LC-HRMS method could be used instead of RIA to assess plasma HA in human diagnostic use.

Immunoglobulines monoclonales : méthodes diagnostiques en 2011

Resume Une immunoglobuline monoclonale se caracterise par l’augmentation selective d’une seule espece moleculaire d’immunoglobuline serique, causee par la proliferation incontrolee d’un clone unique de lymphocytes B, constituee soit d’une seule classe de chaine lourde et d’un seul type de chaine legere, soit de chaines legeres isolees d’un seul type, soit beaucoup plus rarement de fragments de chaines lourdes d’une seule classe. Sa presence n’est pas systematiquement synonyme de malignite. Le diagnostic biologique d’une immunoglobuline monoclonale repose sur la realisation d’une analyse conjointe du serum et des urines qui vise a affirmer son homogeneite de charge et d’isotypie. Le diagnostic biologique de gammapathie monoclonale ne se limite pas a un seul dosage, mais depend d’une strategie raisonnee, utilisant les differents outils diagnostiques que sont l’electrophorese, qu’elle soit sur gel ou capillaire, l’immunoelectrophorese ou immunofixation, l’immunoselection, les dosages d’immunoglobulines et de leurs chaines legeres libres, et enfin la recherche de cryoglobuline. L’interpretation des resultats menant au diagnostic d’immunoglobuline monoclonale reste encore source de difficultes, car certaines situations cliniques (myelome a chaines legeres, cryoglobulinemie, …) necessitent de combiner de maniere judicieuse l’ensemble des techniques mises a disposition. Une grande expertise technique et biologique est donc requise pour, au vu des renseignements cliniques indispensables, choisir au mieux la sequence des examens, et ainsi assurer la qualite et l’efficacite de la strategie mise en œuvre.

FVB/N Mice Spontaneously Heal Ulcerative Lesions Induced by Mycobacterium ulcerans and Switch M. ulcerans into a Low Mycolactone Producer

Buruli ulcer, a debilitating disease, is caused by Mycobacterium ulcerans. The incidence of this neglected tropical disease is steadily increasing. As a rule, without treatment, skin ulcers occur and a lengthy healing process may be observed associated with severe functional disabilities. Mouse models are already available to study establishment of lesions or evaluation of therapy but a lack of a suitable animal model, mimicking all clinical stages, in particular the healing process, remains an obstacle to understand the pathophysiology of M. ulcerans infection. M. ulcerans was s.c. inoculated in three consanguine mouse strains, that is, BALB/c and C57BL/6, classically used to study mycobacterial infection, and FVB/N. Strikingly, FVB/N mice, although as sensitive as all other mouse strains with respect to M. ulcerans infection, presented a spontaneous healing after the ulcerative phase despite stable bacterial load, and mycolactone toxin was not detected in the healed tissues. The spontaneous healing process was accompanied by an activation of the innate immune system. The adaptive response initiated by FVB/N mice was not involved in the healing process and did not confer protection against M. ulcerans. Our work highlights the importance of innate immune responses to control M. ulcerans infection. This in vivo model of M. ulcerans infection now paves the way for new avenues of research toward the elucidation of critical stages of this disease, such as the characterization of the regulation of mycolactone production, a better understanding of the pathophysiology of M. ulcerans infection, and the development of new therapeutic strategies.