Interstitial macrophages are a focus of viral takeover and inflammation in COVID-19 initiation in human lung.

Early stages of deadly respiratory diseases including COVID-19 are challenging to elucidate in humans. Here, we define cellular tropism and transcriptomic effects of SARS-CoV-2 virus by productively infecting healthy human lung tissue and using scRNA-seq to reconstruct the transcriptional program in "infection pseudotime" for individual lung cell types. SARS-CoV-2 predominantly infected activated interstitial macrophages (IMs), which can accumulate thousands of viral RNA molecules, taking over 60% of the cell transcriptome and forming dense viral RNA bodies while inducing host profibrotic (TGFB1, SPP1) and inflammatory (early interferon response, CCL2/7/8/13, CXCL10, and IL6/10) programs and destroying host cell architecture. Infected alveolar macrophages (AMs) showed none of these extreme responses. Spike-dependent viral entry into AMs used ACE2 and Sialoadhesin/CD169, whereas IM entry used DC-SIGN/CD209. These results identify activated IMs as a prominent site of viral takeover, the focus of inflammation and fibrosis, and suggest targeting CD209 to prevent early pathology in COVID-19 pneumonia. This approach can be generalized to any human lung infection and to evaluate therapeutics.

Abnormal Lymphatic Sphingosine-1-Phosphate Signaling Aggravates Lymphatic Dysfunction and Tissue Inflammation.

BACKGROUND: Lymphedema is a global health problem with no effective drug treatment. Enhanced T-cell immunity and abnormal lymphatic endothelial cell (LEC) signaling are promising therapeutic targets for this condition. Sphingosine-1-phosphate (S1P) mediates a key signaling pathway required for normal LEC function, and altered S1P signaling in LECs could lead to lymphatic disease and pathogenic T-cell activation. Characterizing this biology is relevant for developing much needed therapies. METHODS: Human and mouse lymphedema was studied. Lymphedema was induced in mice by surgically ligating the tail lymphatics. Lymphedematous dermal tissue was assessed for S1P signaling. To verify the role of altered S1P signaling effects in lymphatic cells, LEC-specific S1pr1-deficient (S1pr1LECKO) mice were generated. Disease progression was quantified by tail-volumetric and -histopathologic measurements over time. LECs from mice and humans, with S1P signaling inhibition, were then cocultured with CD4 T cells, followed by an analysis of CD4 T-cell activation and pathway signaling. Last, animals were treated with a monoclonal antibody specific to P-selectin to assess its efficacy in reducing lymphedema and T-cell activation. RESULTS: Human and experimental lymphedema tissues exhibited decreased LEC S1P signaling through S1P receptor 1 (S1PR1). LEC S1pr1 loss-of-function exacerbated lymphatic vascular insufficiency, tail swelling, and increased CD4 T-cell infiltration in mouse lymphedema. LECs, isolated from S1pr1LECKO mice and cocultured with CD4 T cells, resulted in augmented lymphocyte differentiation. Inhibiting S1PR1 signaling in human dermal LECs promoted T-helper type 1 and 2 (Th1 and Th2) cell differentiation through direct cell contact with lymphocytes. Human dermal LECs with dampened S1P signaling exhibited enhanced P-selectin, an important cell adhesion molecule expressed on activated vascular cells. In vitro, P-selectin blockade reduced the activation and differentiation of Th cells cocultured with shS1PR1-treated human dermal LECs. P-selectin-directed antibody treatment improved tail swelling and reduced Th1/Th2 immune responses in mouse lymphedema. CONCLUSIONS: This study suggests that reduction of the LEC S1P signaling aggravates lymphedema by enhancing LEC adhesion and amplifying pathogenic CD4 T-cell responses. P-selectin inhibitors are suggested as a possible treatment for this pervasive condition.

Abnormal lymphatic S1P signaling aggravates lymphatic dysfunction and tissue inflammation.

BACKGROUND: Lymphedema is a global health problem with no effective drug treatment. Enhanced T cell immunity and abnormal lymphatic endothelial cell (LEC) signaling are promising therapeutic targets for this condition. Sphingosine-1-phosphate (S1P) mediates a key signaling pathway required for normal LEC function, and altered S1P signaling in LECs could lead to lymphatic disease and pathogenic T cell activation. Characterizing this biology is relevant for developing much-needed therapies. METHODS: Human and mouse lymphedema was studied. Lymphedema was induced in mice by surgically ligating the tail lymphatics. Lymphedematous dermal tissue was assessed for S1P signaling. To verify the role of altered S1P signaling effects in lymphatic cells, LEC-specific S1pr1 -deficient ( S1pr1 LECKO ) mice were generated. Disease progression was quantified by tail-volumetric and -histopathological measurements over time. LECs from mice and humans, with S1P signaling inhibition, were then co-cultured with CD4 T cells, followed by an analysis of CD4 T cell activation and pathway signaling. Finally, animals were treated with a monoclonal antibody specific to P-selectin to assess its efficacy in reducing lymphedema and T cell activation. RESULTS: Human and experimental lymphedema tissues exhibited decreased LEC S1P signaling through S1PR1. LEC S1pr1 loss-of-function exacerbated lymphatic vascular insufficiency, tail swelling, and increased CD4 T cell infiltration in mouse lymphedema. LECs, isolated from S1pr1 LECKO mice and co-cultured with CD4 T cells, resulted in augmented lymphocyte differentiation. Inhibiting S1PR1 signaling in human dermal LECs (HDLECs) promoted T helper type 1 and 2 (Th1 and Th2) cell differentiation through direct cell contact with lymphocytes. HDLECs with dampened S1P signaling exhibited enhanced P-selectin, an important cell adhesion molecule expressed on activated vascular cells. In vitro , P-selectin blockade reduced the activation and differentiation of Th cells co-cultured with sh S1PR1 -treated HDLECs. P-selectin-directed antibody treatment improved tail swelling and reduced Th1/Th2 immune responses in mouse lymphedema. CONCLUSION: This study suggests that reduction of the LEC S1P signaling aggravates lymphedema by enhancing LEC adhesion and amplifying pathogenic CD4 T cell responses. P-selectin inhibitors are suggested as a possible treatment for this pervasive condition. Clinical Perspective: What is New?: Lymphatic-specific S1pr1 deletion exacerbates lymphatic vessel malfunction and Th1/Th2 immune responses during lymphedema pathogenesis. S1pr1 -deficient LECs directly induce Th1/Th2 cell differentiation and decrease anti-inflammatory Treg populations. Peripheral dermal LECs affect CD4 T cell immune responses through direct cell contact.LEC P-selectin, regulated by S1PR1 signaling, affects CD4 T cell activation and differentiation.P-selectin blockade improves lymphedema tail swelling and decreases Th1/Th2 population in the diseased skin.What Are the Clinical Implications?: S1P/S1PR1 signaling in LECs regulates inflammation in lymphedema tissue.S1PR1 expression levels on LECs may be a useful biomarker for assessing predisposition to lymphatic disease, such as at-risk women undergoing mastectomyP-selectin Inhibitors may be effective for certain forms of lymphedema.

Neuroendocrinology of the lung revealed by single-cell RNA sequencing.

Pulmonary neuroendocrine cells (PNECs) are sensory epithelial cells that transmit airway status to the brain via sensory neurons and locally via calcitonin gene-related peptide (CGRP) and γ- aminobutyric acid (GABA). Several other neuropeptides and neurotransmitters have been detected in various species, but the number, targets, functions, and conservation of PNEC signals are largely unknown. We used scRNAseq to profile hundreds of the rare mouse and human PNECs. This revealed over 40 PNEC neuropeptide and peptide hormone genes, most cells expressing unique combinations of 5-18 genes. Peptides are packaged in separate vesicles, their release presumably regulated by the distinct, multimodal combinations of sensors we show are expressed by each PNEC. Expression of the peptide receptors predicts an array of local cell targets, and we show the new PNEC signal angiotensin directly activates one subtype of innervating sensory neuron. Many signals lack lung targets so may have endocrine activity like those of PNEC-derived carcinoid tumors. PNECs are an extraordinarily rich and diverse signaling hub rivaling the enteroendocrine system.