RESUMEN
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.
Asunto(s)
Lepra Lepromatosa/inmunología , Lepra Tuberculoide/inmunología , Mycobacterium leprae/inmunología , Piel/inmunología , Adolescente , Adulto , Anciano , Femenino , Fibroblastos/inmunología , Fibroblastos/microbiología , Fibroblastos/patología , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Queratinocitos/inmunología , Queratinocitos/microbiología , Queratinocitos/patología , Lepra Lepromatosa/genética , Lepra Lepromatosa/microbiología , Lepra Lepromatosa/patología , Lepra Tuberculoide/genética , Lepra Tuberculoide/microbiología , Lepra Tuberculoide/patología , Macrófagos/inmunología , Macrófagos/microbiología , Macrófagos/patología , Masculino , Persona de Mediana Edad , Mycobacterium leprae/patogenicidad , RNA-Seq , Análisis de la Célula Individual , Piel/microbiología , Piel/patología , Linfocitos T/inmunología , Linfocitos T/microbiología , Linfocitos T/patología , TranscriptomaRESUMEN
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.