Aude Thiriot

In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight

Dysfunctional endothelium contributes to more diseases than any other tissue in the body. Small interfering RNAs (siRNAs) can help in the study and treatment of endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here, we show that polymeric nanoparticles made of low-molecular-weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. These nanoparticles mediate the most durable non-liver silencing reported so far and facilitate the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth and metastasis.

Nociceptive sensory neurons drive interleukin-23-mediated psoriasiform skin inflammation

The skin has a dual function as a barrier and a sensory interface between the body and the environment. To protect against invading pathogens, the skin harbours specialized immune cells, including dermal dendritic cells (DDCs) and interleukin (IL)-17-producing γδ T (γδT17) cells, the aberrant activation of which by IL-23 can provoke psoriasis-like inflammation. The skin is also innervated by a meshwork of peripheral nerves consisting of relatively sparse autonomic and abundant sensory fibres. Interactions between the autonomic nervous system and immune cells in lymphoid organs are known to contribute to systemic immunity, but how peripheral nerves regulate cutaneous immune responses remains unclear. We exposed the skin of mice to imiquimod, which induces IL-23-dependent psoriasis-like inflammation. Here we show that a subset of sensory neurons expressing the ion channels TRPV1 and Nav1.8 is essential to drive this inflammatory response. Imaging of intact skin revealed that a large fraction of DDCs, the principal source of IL-23, is in close contact with these nociceptors. Upon selective pharmacological or genetic ablation of nociceptors, DDCs failed to produce IL-23 in imiquimod-exposed skin. Consequently, the local production of IL-23-dependent inflammatory cytokines by dermal γδT17 cells and the subsequent recruitment of inflammatory cells to the skin were markedly reduced. Intradermal injection of IL-23 bypassed the requirement for nociceptor communication with DDCs and restored the inflammatory response. These findings indicate that TRPV1+Nav1.8+ nociceptors, by interacting with DDCs, regulate the IL-23/IL-17 pathway and control cutaneous immune responses.

Endothelial Heparan Sulfate Controls Chemokine Presentation in Recruitment of Lymphocytes and Dendritic Cells to Lymph Nodes

Heparan sulfate can bind several adhesion molecules involved in lymphocyte trafficking. However, the in vivo function of endothelial heparan sulfate in lymphocyte homing and stimulation of the immune response has not been elucidated. Here, we generated mutant mice deficient in the enzyme Ext1, which is required for heparan sulfate synthesis, in a Tek-dependent and inducible manner. Chemokine presentation was diminished in the mutant mice, causing the lack of appropriate integrin-mediated adhesion, and resulted in a marked decrease in lymphocyte sticking to high endothelial venules and in recruitment of resident dendritic cells through lymphatic vessels to the lymph nodes. As a consequence, mutant mice displayed a severe impairment in lymphocyte homing and a compromised contact hypersensitivity response. By contrast, lymphocyte rolling was increased because of loss of electrostatic repulsion by heparan sulfate. These results demonstrate critical roles of endothelial heparan sulfate in immune surveillance and immune response generation.

The Bw Cells, a Novel B Cell Population Conserved in the Whole Genus Mus

In common laboratory mouse strains, which are derived from the crossing between three subspecies, peritoneal B cells are enriched in B-1a cells characterized by the CD5+Mac-1+B220lowIgMhighIgDlowCD43+CD9+ phenotype. Intriguingly in other vertebrates, CD5+Mac-1+ cells have never been found in a specific anatomic site. To ascertain the peculiarity of the CD5+ peritoneal B cells in laboratory mice, we analyzed the peritoneal B cell subsets in 9 inbred and 39 outbred wild-derived mouse strains belonging to 13 different species/subspecies. We found that most of these strains do not have the CD5+ B-1a cell population. However, all of these strains including classical laboratory mouse strains, have variable proportions of a novel B cell population: Bw, which is characterized by a unique phenotype (CD5−Mac-1+B220highIgMhighIgDhighCD43−CD9−) and is not restricted to the peritoneal cavity. Bw cells are also distinct from both B-1 and B-2 cells from a functional point of view both by proliferative responses, cytokine secretion and Ab synthesis. Moreover, transfer experiments show that bone marrow and fetal liver cells from wild mice can give rise to Bw cells in alymphoid mice. The conservation of this B cell population, but not of the CD5+ B-1a, during evolution of the genus Mus, its readiness to respond to TLR ligands and to produce high concentration of autoantibodies suggest that Bw cells play a key role in innate immunity.

Atypical chemokine receptor 1 on nucleated erythroid cells regulates hematopoiesis

Healthy individuals of African ancestry have neutropenia that has been linked with the variant rs2814778(G) of the gene encoding atypical chemokine receptor 1 (ACKR1). This polymorphism selectively abolishes the expression of ACKR1 in erythroid cells, causing a Duffy-negative phenotype. Here we describe an unexpected fundamental role for ACKR1 in hematopoiesis and provide the mechanism that links its absence with neutropenia. Nucleated erythroid cells had high expression of ACKR1, which facilitated their direct contact with hematopoietic stem cells. The absence of erythroid ACKR1 altered mouse hematopoiesis including stem and progenitor cells, which ultimately gave rise to phenotypically distinct neutrophils that readily left the circulation, causing neutropenia. Individuals with a Duffy-negative phenotype developed a distinct profile of neutrophil effector molecules that closely reflected the one observed in the ACKR1-deficient mice. Thus, alternative physiological patterns of hematopoiesis and bone marrow cell outputs depend on the expression of ACKR1 in the erythroid lineage, findings with major implications for the selection advantages that have resulted in the paramount fixation of the ACKR1 rs2814778(G) polymorphism in Africa.

Spinal cord injury-induced immunodeficiency is mediated by a sympathetic-neuroendocrine adrenal reflex

Acute spinal cord injury (SCI) causes systemic immunosuppression and life-threatening infections, thought to result from noradrenergic overactivation and excess glucocorticoid release via hypothalamus–pituitary–adrenal axis stimulation. Instead of consecutive hypothalamus–pituitary–adrenal axis activation, we report that acute SCI in mice induced suppression of serum norepinephrine and concomitant increase in cortisol, despite suppressed adrenocorticotropic hormone, indicating primary (adrenal) hypercortisolism. This neurogenic effect was more pronounced after high-thoracic level (Th1) SCI disconnecting adrenal gland innervation, compared with low-thoracic level (Th9) SCI. Prophylactic adrenalectomy completely prevented SCI-induced glucocorticoid excess and lymphocyte depletion but did not prevent pneumonia. When adrenalectomized mice were transplanted with denervated adrenal glands to restore physiologic glucocorticoid levels, the animals were completely protected from pneumonia. These findings identify a maladaptive sympathetic-neuroendocrine adrenal reflex mediating immunosuppression after SCI, implying that therapeutic normalization of the glucocorticoid and catecholamine imbalance in SCI patients could be a strategy to prevent detrimental infections.

Wild-derived mouse strains, a valuable model to study B cell responses.

In the present report, we revisited the B cell responsiveness of 7 wild-derived mouse strains to various toll-like receptor ligands (TLR-L). We found that 2 of them, namely PWK and STF presented profound defects in B cell proliferative responses to most of the TLR-L. Yet, their macrophage responses were largely unaffected, suggesting that regulation of TLR pathways are distinct in B cells and macrophages. We also showed that, anti-CD40 mAbs rescued the low proliferative responses to CpG in both PWK and STF B cells. In the other hand, CpG synergized with LPS to induce high levels of proliferation in STF B cells, which did not respond to LPS alone. Cytokine or immunoglobulin (Ig) productions, in vitro, were less impaired than the proliferative responses to LPS or CpG alone. In STF B cells, both ERK, P38 and JNK pathways were affected following in vitro TLR4 or TLR9 signaling. Moreover, while the basal levels of Ig secreting cells and of serum Igs were similar to that of control mice, antibody responses to both TI and TD antigens were severely affected, mainly in STF mice. Our findings therefore highlight the relevance of wild-derived mouse strains and TLR-L to study B cell physiology.

Differential DARC/ACKR1 expression distinguishes venular from non-venular endothelial cells in murine tissues

BackgroundIntravascular leukocyte recruitment in most vertebrate tissues is restricted to postcapillary and collecting venules, whereas capillaries and arterioles usually support little or no leukocyte adhesion. This segmental restriction is thought to be mediated by endothelial, rather than hemodynamic, differences. The underlying mechanisms are largely unknown, in part because effective tools to distinguish, isolate, and analyze venular endothelial cells (V-ECs) and non-venular endothelial cells (NV-ECs) have been unavailable. We hypothesized that the atypical chemokine receptor DARC (Duffy Antigen Receptor for Chemokines, a.k.a. ACKR1 or CD234) may distinguish V-ECs versus NV-ECs in mice.MethodsWe generated a rat-anti-mouse monoclonal antibody (MAb) that specifically recognizes the erythroid and endothelial forms of native, surface-expressed DARC. Using this reagent, we characterized DARC expression and distribution in the microvasculature of murine tissues. ResultsDARC was exquisitely restricted to post-capillary and small collecting venules and completely absent from arteries, arterioles, capillaries, veins, and most lymphatics in every tissue analyzed. Accordingly, intravital microscopy showed that adhesive leukocyte-endothelial interactions were restricted to DARC+ venules. DARC was detectable over the entire circumference of V-ECs, but was more concentrated at cell-cell junctions. Analysis of single-cell suspensions suggested that the frequency of V-ECs among the total microvascular EC pool varies considerably between different tissues.ConclusionsImmunostaining of endothelial DARC allows the identification and isolation of intact V-ECs from multiple murine tissues. This strategy may be useful to dissect the mechanisms underlying segmental microvascular specialization in healthy and diseased tissues and to characterize the role of EC subsets in tissue-homeostasis, immune surveillance, infection, inflammation, and malignancies.

Of Origins and Pedigrees: Lineage Tracing of Dendritic Cells

Understanding the ontogeny of distinct hematopoietic cell types remains a challenge. In this issue, Schraml et al. contribute to unraveling the complexity of a central component of the mononuclear phagocyte system by using a new in vivo approach to trace the progeny of common dendritic cell precursors.