IFN-γ-mediated control of SARS-CoV-2 infection through nitric oxide.

Introduction: The COVID-19 pandemic has highlighted the need to identify mechanisms of antiviral host defense against SARS-CoV-2. One such mediator is interferon-g (IFN-γ), which, when administered to infected patients, is reported to result in viral clearance and resolution of pulmonary symptoms. IFN-γ treatment of a human lung epithelial cell line triggered an antiviral activity against SARS-CoV-2, yet the mechanism for this antiviral response was not identified. Methods: Given that IFN-γ has been shown to trigger antiviral activity via the generation of nitric oxide (NO), we investigated whether IFN-γ induction of antiviral activity against SARS-CoV-2 infection is dependent upon the generation of NO in human pulmonary epithelial cells. We treated the simian epithelial cell line Vero E6 and human pulmonary epithelial cell lines, including A549-ACE2, and Calu-3, with IFN-γ and observed the resulting induction of NO and its effects on SARS-CoV-2 replication. Pharmacological inhibition of inducible nitric oxide synthase (iNOS) was employed to assess the dependency on NO production. Additionally, the study examined the effect of interleukin-1b (IL-1ß) on the IFN-g-induced NO production and its antiviral efficacy. Results: Treatment of Vero E6 cells with IFN-γ resulted in a dose-responsive induction of NO and an inhibitory effect on SARS-CoV-2 replication. This antiviral activity was blocked by pharmacologic inhibition of iNOS. IFN-γ also triggered a NO-mediated antiviral activity in SARS-CoV-2 infected human lung epithelial cell lines A549-ACE2 and Calu-3. IL-1ß enhanced IFN-γ induction of NO, but it had little effect on antiviral activity. Discussion: Given that IFN-g has been shown to be produced by CD8+ T cells in the early response to SARS-CoV-2, our findings in human lung epithelial cell lines, of an IFN-γ-triggered, NO-dependent, links the adaptive immune response to an innate antiviral pathway in host defense against SARS-CoV-2. These results underscore the importance of IFN-γ and NO in the antiviral response and provide insights into potential therapeutic strategies for COVID-19.

TREM2 macrophages induced by human lipids drive inflammation in acne lesions.

Acne affects 1 in 10 people globally, often resulting in disfigurement. The disease involves excess production of lipids, particularly squalene, increased growth of Cutibacterium acnes, and a host inflammatory response with foamy macrophages. By combining single-cell and spatial RNA sequencing as well as ultrahigh-resolution Seq-Scope analyses of early acne lesions on back skin, we identified TREM2 macrophages expressing lipid metabolism and proinflammatory gene programs in proximity to hair follicle epithelium expressing squalene epoxidase. We established that the addition of squalene induced differentiation of TREM2 macrophages in vitro, which were unable to kill C. acnes. The addition of squalene to macrophages inhibited induction of oxidative enzymes and scavenged oxygen free radicals, providing an explanation for the efficacy of topical benzoyl peroxide in the clinical treatment of acne. The present work has elucidated the mechanisms by which TREM2 macrophages and unsaturated lipids, similar to their involvement in atherosclerosis, may contribute to the pathogenesis of acne.

The cellular architecture of the antimicrobial response network in human leprosy granulomas.

Granulomas are complex cellular structures composed predominantly of macrophages and lymphocytes that function to contain and kill invading pathogens. Here, we investigated the single-cell phenotypes associated with antimicrobial responses in human leprosy granulomas by applying single-cell and spatial sequencing to leprosy biopsy specimens. We focused on reversal reactions (RRs), a dynamic process whereby some patients with disseminated lepromatous leprosy (L-lep) transition toward self-limiting tuberculoid leprosy (T-lep), mounting effective antimicrobial responses. We identified a set of genes encoding proteins involved in antimicrobial responses that are differentially expressed in RR versus L-lep lesions and regulated by interferon-γ and interleukin-1ß. By integrating the spatial coordinates of the key cell types and antimicrobial gene expression in RR and T-lep lesions, we constructed a map revealing the organized architecture of granulomas depicting compositional and functional layers by which macrophages, T cells, keratinocytes and fibroblasts can each contribute to the antimicrobial response.

Extracellular traps released by antimicrobial TH17 cells contribute to host defense.

TH17 cell subpopulations have been defined that contribute to inflammation and homeostasis, yet the characteristics of TH17 cells that contribute to host defense against infection are not clear. To elucidate the antimicrobial machinery of the TH17 subset, we studied the response to Cutibacterium acnes, a skin commensal that is resistant to IL-26, the only known TH17-secreted protein with direct antimicrobial activity. We generated C. acnes-specific antimicrobial TH17 clones (AMTH17) with varying antimicrobial activity against C. acnes, which we correlated by RNA sequencing to the expression of transcripts encoding proteins that contribute to antimicrobial activity. Additionally, we validated that AMTH17-mediated killing of C. acnes and bacterial pathogens was dependent on the secretion of granulysin, granzyme B, perforin, and histone H2B. We found that AMTH17 cells can release fibrous structures composed of DNA decorated with histone H2B that entangle C. acnes that we call T cell extracellular traps (TETs). Within acne lesions, H2B and IL-17 colocalized in CD4+ T cells, in proximity to TETs in the extracellular space composed of DNA decorated with H2B. This study identifies a functionally distinct subpopulation of TH17 cells with an ability to form TETs containing secreted antimicrobial proteins that capture and kill bacteria.

Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies.

High-throughput single-cell RNA-sequencing (scRNA-seq) methodologies enable characterization of complex biological samples by increasing the number of cells that can be profiled contemporaneously. Nevertheless, these approaches recover less information per cell than low-throughput strategies. To accurately report the expression of key phenotypic features of cells, scRNA-seq platforms are needed that are both high fidelity and high throughput. To address this need, we created Seq-Well S3 ("Second-Strand Synthesis"), a massively parallel scRNA-seq protocol that uses a randomly primed second-strand synthesis to recover complementary DNA (cDNA) molecules that were successfully reverse transcribed but to which a second oligonucleotide handle, necessary for subsequent whole transcriptome amplification, was not appended due to inefficient template switching. Seq-Well S3 increased the efficiency of transcript capture and gene detection compared with that of previous iterations by up to 10- and 5-fold, respectively. We used Seq-Well S3 to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation.

Cell-type deconvolution with immune pathways identifies gene networks of host defense and immunopathology in leprosy.

Transcriptome profiles derived from the site of human disease have led to the identification of genes that contribute to pathogenesis, yet the complex mixture of cell types in these lesions has been an obstacle for defining specific mechanisms. Leprosy provides an outstanding model to study host defense and pathogenesis in a human infectious disease, given its clinical spectrum, which interrelates with the host immunologic and pathologic responses. Here, we investigated gene expression profiles derived from skin lesions for each clinical subtype of leprosy, analyzing gene coexpression modules by cell-type deconvolution. In lesions from tuberculoid leprosy patients, those with the self-limited form of the disease, dendritic cells were linked with MMP12 as part of a tissue remodeling network that contributes to granuloma formation. In lesions from lepromatous leprosy patients, those with disseminated disease, macrophages were linked with a gene network that programs phagocytosis. In erythema nodosum leprosum, neutrophil and endothelial cell gene networks were identified as part of the vasculitis that results in tissue injury. The present integrated computational approach provides a systems approach toward identifying cell-defined functional networks that contribute to host defense and immunopathology at the site of human infectious disease.

CD163 favors Mycobacterium leprae survival and persistence by promoting anti-inflammatory pathways in lepromatous macrophages.

Lepromatous macrophages possess a regulatory phenotype that contributes to the immunosuppression observed in leprosy. CD163, a scavenger receptor that recognizes hemoglobin-haptoglobin complexes, is expressed at higher levels in lepromatous cells, although its functional role in leprosy is not yet established. We herein demonstrate that human lepromatous lesions are microenvironments rich in IDO⁺CD163⁺. Cells isolated from these lesions were CD68⁺IDO⁺CD163⁺ while higher levels of sCD163 in lepromatous sera positively correlated with IL-10 levels and IDO activity. Different Myco-bacterium leprae (ML) concentrations in healthy monocytes likewise revealed a positive correlation between increased concentrations of the mycobacteria and IDO, CD209, and CD163 expression. The regulatory phenotype in ML-stimulated monocytes was accompanied by increased TNF, IL-10, and TGF-ß levels whereas IL-10 blockade reduced ML-induced CD163 expression. The CD163 blockade reduced ML uptake in human monocytes. ML uptake was higher in HEK293 cells transfected with the cDNA for CD163 than in untransfected cells. Simultaneously, increased CD163 expression in lepromatous cells seemed to be dependent on ML uptake, and contributed to augmented iron storage in lepromatous macrophages. Altogether, these results suggest that ML-induced CD163 expression modulates the host cell phenotype to create a favorable environment for myco-bacterial entry and survival.