Influenza A virus (IAV) and SARS-CoV-2 are pandemic viruses causing millions of deaths, yet their clinical manifestations are distinctly different. With the hypothesis that upper airway immune and epithelial cell responses are also distinct, we performed single-cell RNAsequencing (scRNA-Seq) on nasal wash cells freshly collected from adults with either acute COVID-19 or influenza or from healthy controls. We focused on major cell types and subtypes in a subset of donor samples. Nasal wash cells are enriched for macrophages and neutrophils for both influenza and COVID-19 compared to healthy controls. Hillock-like epithelial cells, M2-like macrophages, and age-dependent B cells are enriched in COVID-19 samples. A global decrease in interferon (IFN)-associated transcripts in neutrophils, macrophages, and epithelial cells is apparent in COVID-19 compared to influenza. The innate immune response to SARS-CoV-2 appears to be maintained in macrophages, despite evidence for limited epithelial immune sensing. Cell-to-cell interaction analyses reveal a decrease in epithelial interactions in COVID-19 and highlight differences in macrophage-macrophage interactions for COVID-19 and influenza. Our study demonstrates that scRNA-Seq can define host and viral transcriptional activity at the site of infection and reveal distinct local epithelial and immune cell responses for COVID-19 and influenza that may contribute to their divergent disease courses.

The study of complex heterodimeric peptide ligands has been hampered by a paucity of pharmacological tools. To facilitate such investigations, we have explored the utility of membrane tethered ligands (MTLs). Feasibility of this recombinant approach was explored with a focus on Drosophila bursicon, a heterodimeric cystine-knot protein that activates the G proteincoupled receptor rickets (rk). Rk/bursicon signaling is an evolutionarily conserved pathway in insects required for wing expansion, cuticle hardening, and melanization during development. We initially engineered two distinct MTL constructs, each composed of a type II transmembrane domain, a peptide linker, and a C terminal extracellular ligand that corresponded to either the a or b bursicon subunit. Coexpression of the two complementary bursicon MTLs triggered rk-mediated signaling in vitro.We were then able to generate functionally active bursicon MTLs in which the two subunits were fused into a single heterodimeric peptide, oriented as either a-b or b-a. Carboxy-terminal deletion of 32 amino acids in the b-a MTL construct resulted in loss of agonist activity. Coexpression of this construct with rk inhibited receptor-mediated signaling by soluble bursicon. We have thus generated membrane-anchored bursicon constructs that can activate or inhibit rk signaling. These probes can be used in future studies to explore the tissue and/or developmental stage-dependent effects of bursicon in the genetically tractable Drosophila model organism. In addition, our success in generating functionally diverse bursicon MTLs offers promise that such technology can be broadly applied to other complex ligands, including the family of mammalian cystine-knot proteins.

Pediatric patients with constitutively active mutations in the cytosolic double-stranded-DNA-sensing adaptor STING develop an autoinflammatory syndrome known as STING-associated vasculopathy with onset in infancy (SAVI). SAVI patients have elevated interferon-stimulated gene expression and suffer from interstitial lung disease (ILD) with lymphocyte predominate bronchus-associated lymphoid tissue (BALT). Mice harboring SAVI mutations (STING V154M [VM]) that recapitulate human disease also develop lymphocyte-rich BALT. Ablation of either T or B lymphocytes prolongs the survival of SAVI mice, but lung immune aggregates persist, indicating that T cells and B cells can independently be recruited as BALT. VM T cells produced IFNγ, and IFNγR deficiency prolonged the survival of SAVI mice; however, T-cell-dependent recruitment of infiltrating myeloid cells to the lung was IFNγ independent. Lethally irradiated VM recipients fully reconstituted with wild type bone-marrow-derived cells still developed ILD, pointing to a critical role for VM-expressing radioresistant parenchymal and/or stromal cells in the recruitment and activation of pathogenic lymphocytes. We identified lung endothelial cells as radioresistant cells that express STING. Transcriptional analysis of VM endothelial cells revealed up-regulation of chemokines, proinflammatory cytokines, and genes associated with antigen presentation. Together, our data show that VM-expressing radioresistant cells play a key role in the initiation of lung disease in VM mice and provide insights for the treatment of SAVI patients, with implications for ILD associated with other connective tissue disorders.

Detection of DNA is an important determinant of host-defense but also a driver of autoinflammatory and autoimmune diseases. Failure to degrade self-DNA in DNAseII or III(TREX1)-deficient mice results in activation of the cGAS-STING pathway. Deficiency of cGAS or STING in these models ameliorates disease manifestations. However, the contribution of the cGAS-STING pathway, relative to endosomal TLRs, in systemic lupus erythematosus (SLE) is controversial. In fact, STING deficiency failed to rescue, and actually exacerbated, disease manifestations in Fas-deficient SLE-prone mice. We have now extended these observations to a chronic model of SLE induced by the i.p. injection of TMPD (pristane). We found that both cGAS- and STING-deficiency not only failed to rescue mice from TMPD-induced SLE, but resulted in increased autoantibody production and higher proteinuria levels compared to cGAS STING sufficient mice. Further, we generated cGASKOFaslpr mice on a pure MRL/Faslpr background using Crispr/Cas9 and found slightly exacerbated, and not attenuated, disease. We hypothesized that the cGAS-STING pathway constrains TLR activation, and thereby limits autoimmune manifestations in these two models. Consistent with this premise, mice lacking cGAS and Unc93B1 or STING and Unc93B1 developed minimal systemic autoimmunity as compared to cGAS or STING single knock out animals. Nevertheless, TMPD-driven lupus in B6 mice was abrogated upon AAV-delivery of DNAse I, implicating a DNA trigger. Overall, this study demonstrated that the cGAS-STING pathway does not promote systemic autoimmunity in murine models of SLE. These data have important implications for cGAS-STING-directed therapies being developed for the treatment of systemic autoimmunity.

The Cys-Cys chemokine receptor 6 (CCR6) is a well-established modulator of inflammation. Although several genetic associations have been identified between CCR6 polymorphisms and immune system disorders (e.g., rheumatoid arthritis and Crohn's disease), the pharmacological effects of naturally occurring missense mutations in this receptor have yet to be characterized. In this study, we initially assessed G protein-mediated signaling and observed that wild-type (WT) CCR6 exhibited ligand-independent activity. In addition, we found that the five most frequent CCR6 missense variants (A89T, A150V, R155W, G345S, and A369V) exhibited decreased basal and/or ligand induced Gαi protein signaling. To complement the study of these loss-of-function variants, we engineered a set of constitutively active CCR6 receptors. Selected mutations enhanced basal G protein-mediated signaling up to 3-fold relative to the WT value. Using a bioluminescence resonance energy transfer assay we investigated the ability of each naturally occurring and engineered CCR6 receptor mutant to recruit β-arrestin. In contrast to G protein-mediated signaling, β-arrestin mobilization was largely unperturbed by the naturally occurring loss-of-function CCR6 variants. Elevated recruitment of β-arrestin was observed in one of the engineered constitutively active mutants (T98P). Our results demonstrate that point mutations in CCR6 can result in either a gain or loss of receptor function. These observations underscore the need to explore how CCR6 natural variants may influence immune cell physiology and human disease.