A gene silencing pipeline to interrogate human cDC1 and pDC development and functions

Type 1 conventional dendritic cells (cDC1s) and plasmacytoid dendritic cells (pDCs) are thought to be critical for anti-tumor or antiviral immunity. In vitro differentiation systems have unlocked the ability to produce large numbers of these cells. However, a method is lacking to systematically identify the cell-intrinsic factors controlling their differentiation and functions that remain therefore poorly understood, in contrast to the situation in mice. Here, we developed a workflow for efficient gene silencing and its tracing in human cDC1s/pDCs generated in vitro. As proof of concept, we confirmed the key role of IRF8 in their development, and of IRF7/MyD88 in human pDC production of interferons-α/λ. We found that SAMHD1 and RAB7B promote human cDC1 differentiation, while SEPT3 promotes human pDC differentiation. We also found that PPT1 and RAB5 are required for optimal differentiation of pDCs and cDC1s. Finally, we identified BCL11A, PPT1 and RAB7 as novel HIV-1 restriction factors in cDC1s/pDCs. This approach will enable broader genetic screens to advance our understanding of human cDC1s/pDCs and harness them against viral infections or cancer

Adhesion of <em>Neisseria meningitidis</em> to Dermal Vessels Leads to Local Vascular Damage and <em>Purpura</em> in a Humanized Mouse Model

<div><p>Septic shock caused by <em>Neisseria meningitidis</em> is typically rapidly evolving and often fatal despite antibiotic therapy. Further understanding of the mechanisms underlying the disease is necessary to reduce fatality rates. <em>Postmortem</em> samples from the characteristic <em>purpuric</em> rashes of the infection show bacterial aggregates in close association with microvessel endothelium but the species specificity of <em>N. meningitidis</em> has previously hindered the development of an <em>in vivo</em> model to study the role of adhesion on disease progression. Here we introduced human dermal microvessels into SCID/Beige mice by xenografting human skin. Bacteria injected intravenously exclusively associated with the human vessel endothelium in the skin graft. Infection was accompanied by a potent inflammatory response with the secretion of human inflammatory cytokines and recruitment of inflammatory cells. Importantly, infection also led to local vascular damage with hemostasis, thrombosis, vascular leakage and finally <em>purpura</em> in the grafted skin, replicating the clinical presentation for the first time in an animal model. The adhesive properties of the type IV pili of <em>N. meningitidis</em> were found to be the main mediator of association with the dermal microvessels <em>in vivo</em>. Bacterial mutants with altered type IV pili function also did not trigger inflammation or lead to vascular damage. This work demonstrates that local type IV pili mediated adhesion of <em>N. meningitidis</em> to the vascular wall, as opposed to circulating bacteria, determines vascular dysfunction in meningococcemia.</p> </div

Human skin graft.

<p>(A) 21 days post graft of human skin onto SCID/Beige mouse. (B) Histology of grafted human skin showing dermal/epidermal border (arrow); (H&E). (C) Graft interface (dotted line) between human (Hu) and mouse (Ms) skin (H&E). (D) Human endothelium in the grafted skin labeled with <i>Ulex europaeus agglutinin</i> lectin (UEA, red). Cell nuclei are labeled with DAPI (blue). (E) Junction between human (UEA lectin, red) and mouse (msCD31, green) vessels at the graft border. (F) Frame from Movie S1. Intravital microscopy showing perfusion of human vessels labeled with UEA lectin (red). Blood plasma is labeled with 150 kDa FITC-dextran (green). Black silhouettes within the flow are red blood cells.</p

Infection with non-adherent mutants of <i>N. meningitidis</i>.

<p>(A) Bacterial CFU counts from the blood of mice infected with wild type <i>N. meningitidis</i> (WT), an isogenic non-piliated <i>pilD</i> mutant strain (<i>pilD</i>), and a piliated but non-adherent <i>pilC1</i> mutant strain (<i>pilC1</i>). (B) Bacterial counts of biopsies from grafted human skin and contralateral mouse skin of mice infected for 24 h as in (A). (C) 3D reconstruction of human vessel from a grafted mouse infected with the <i>pilD</i> mutant strain (UEA lectin, red). Small amounts of tissue auto-fluorescence and collagen can be seen in the green channel. (D) H&E staining of grafted human skin in a mouse infected with the <i>pilD</i> mutant strain. Arrow highlights very mild inflammation around a vessel. (E) H&E staining of grafted human skin in a mouse infected with the <i>pilC1</i> mutant strain. Arrow highlights signs of congestion in a vessel. (F) Cytokine analysis of human IL-6 in serum of mice grafted with human skin and infected with the WT or mutant strains. (G) Cytokine analysis of human IL-8 in the same mice as (F).</p

Bacterial association with vessels.

<p>(A) Bacterial colony forming unit (CFU) counts from the blood of mice grafted with human skin and infected with <i>N. meningitidis</i>. These counts represent the number of bacteria circulating in the blood at the given time-point. (B) Bacterial CFU counts from skin biopsies taken from either grafted human skin (Hu Skin) or contralateral mouse skin (Ms Skin). (C) Bacterial CFU counts in biopsies taken from organs of mice grafted with human skin. (D) Confocal microscopy showing <i>N. meningitidis</i> (green) associated with human vessels (UEA lectin, red) in the skin, 2 h post infection. (E) 3D reconstruction showing bacteria (green) in a human vessel (UEA lectin, red) in the skin, 6 h post infection (F) Bacteria (green) in a human vessel (HuCD31, red), 24 h post infection. (G) Bacteria (green) expressing tfp (red) 24 h post infection. (H) Bacteria (green) expressing capsule (red), 24 h post infection. (I) CFU counts from skin biopsies of mice grafted with mouse skin (MOM graft, mouse on mouse), and contralateral mouse skin (Ms), after 24 h of infection with <i>N. meningitidis</i>.</p

Histology scoring.

<p>0 = none detected.</p><p>+ = very mild/mild – occasional events detected, 1–2 per field of view.</p><p>++ = moderate – more than occasional, multiple events per field of view.</p><p>+++ = severe – dominant phenotype, most/all of field of view affected.</p>1<p>Mice grafted with human skin and injected with PBS 24h prior to sacrifice.</p>2<p>Mice grafted with mouse skin and infected with WT bacteria for 24 h.</p

Inflammatory signaling following infection.

<p>(A) Human IL-6 concentrations in serum of mice grafted with human skin and injected with either PBS or <i>N. meningitidis</i>. MOM, control mice grafted with mouse skin and infected for 24 h. (B) Human IL-8 concentrations from serum of mice as in (A). (C) Neutrophil infiltration (Ly6G/Ly6C, red) at 24 h post infection, a few bacteria can be seen (green) in the inflamed area. Cell nuclei stained with DAPI (blue). (D) Neutrophil infiltration (red) at 6 h post infection. (E) <i>N. meningitidis</i> (green) phagocytosed by a neutrophil (red), 6 h post infection.</p

Tissue morphology following infection with <i>N. meningitidis</i>.

<p>(A) Tissue morphology 6 h post infection with <i>N. meningitidis</i> (H&E). Thrombosis and mild inflammation can be seen in a vessel (arrow). (B) Bacteria (green) at 6 h infection, human endothelium labeling (huCD31; red), indicates loss of continuous staining (arrow). Cell nuclei are labeled with DAPI (blue). (C) Platelet aggregation (red) in close proximity to bacteria (green), 6 h post infection. (D) Inflammation, vascular leak (arrow) and congestion (arrowheads) at 24 h post infection (H&E). (E) Bacteria (green) filling a vessel in close proximity to the basement membrane (Collagen IV, red). (F) Disruption of Collagen IV (red) in a heavily infected vessel at 24 h. (G) Thrombosed vessel (arrow) 24 h post infection (H&E). (H) Small vascular hemorrhage (arrow) in a congested vessel also showing neutrophil infiltration (*). (I) Extensive vascular leak, red/brown color shows large lake of RBCs at the dermal/epidermal border at 24 h (H&E). (J) RBC (red) leakage in the dermis (d). Leakage occurs at the epidermal (e) border. (K) Human skin graft prior to infection. (L) The same mouse as (K) 24 h post infection showing <i>purpura</i>, seen as dark areas (arrow).</p