Objectives: Monocytes and dendritic cells (DCs) are essential players in the immune response to infections, involved in shaping innate and adaptive immunity. However, a complete understanding of their specific roles in respiratory infections, including SARS-CoV-2, remains elusive.

Methods: To investigate the dynamics of monocytes and DCs in blood as well as the upper and lower airways, we sampled 147 patients with varying degree of COVID-19 severity longitudinally during the spring of 2020.

Results: Using flow cytometry, proteomics and in vitro TLR stimulation, we found differences in the distribution and function of monocytes and DCs in patients compared with controls, and importantly, reduced levels of DCs in both blood and airways. In fact, lower frequencies of cDC2s (Lin⁻ HLA-DR⁺ CD1c⁺) early after symptom onset predicted subsequent severe disease, and depletion of DC subsets lasted longer in patients with more severe disease. In contrast, severe COVID-19 was associated with increased frequencies of activated monocytes in the lower, but not the upper, airways. Proteomic analysis showed that monocyte and DC-related cytokines in plasma and airways associated with disease severity. During convalescence, cell frequencies and responses to TLR ligands normalised in blood, except for persistently low plasmacytoid DCs.

Conclusion: Our study reveals a distinct pattern of recruitment of monocytes but not DCs to the airways during severe COVID-19. Instead, decreased levels of DCs in both blood and airways were found, possibly contributing to more severe COVID-19. The connection between low blood DCs early in disease course and more severe outcomes provides insight into COVID-19 immunopathology, with possible therapeutic implications.

Uncontrolled regeneration leads to neoplastic transformation1–3. The intestinal epithelium requires precise regulation during continuous homeostatic and damage-induced tissue renewal to prevent neoplastic transformation, suggesting that pathways unlinking tumour growth from regenerative processes must exist. Here, by mining RNA-sequencing datasets from two intestinal damage models4,5 and using pharmacological, transcriptomics and genetic tools, we identified liver X receptor (LXR) pathway activation as a tissue adaptation to damage that reciprocally regulates intestinal regeneration and tumorigenesis. Using single-cell RNA sequencing, intestinal organoids, and gain- and loss-of-function experiments, we demonstrate that LXR activation in intestinal epithelial cells induces amphiregulin (Areg), enhancing regenerative responses. This response is coordinated by the LXR-ligand-producing enzyme CYP27A1, which was upregulated in damaged intestinal crypt niches. Deletion of Cyp27a1 impaired intestinal regeneration, which was rescued by exogenous LXR agonists. Notably, in tumour models, Cyp27a1 deficiency led to increased tumour growth, whereas LXR activation elicited anti-tumour responses dependent on adaptive immunity. Consistently, human colorectal cancer specimens exhibited reduced levels of CYP27A1, LXR target genes, and B and CD8 T cell gene signatures. We therefore identify an epithelial adaptation mechanism to damage, whereby LXR functions as a rheostat, promoting tissue repair while limiting tumorigenesis.

The Xenium In Situ platform is a new spatial transcriptomics product commercialized by 10x Genomics, capable of mapping hundreds of genes in situ at subcellular resolution. Given the multitude of commercially available spatial transcriptomics technologies, recommendations in choice of platform and analysis guidelines are increasingly important. Herein, we explore 25 Xenium datasets generated from multiple tissues and species, comparing scalability, resolution, data quality, capacities and limitations with eight other spatially resolved transcriptomics technologies and commercial platforms. In addition, we benchmark the performance of multiple open-source computational tools, when applied to Xenium datasets, in tasks including preprocessing, cell segmentation, selection of spatially variable features and domain identification. This study serves as an independent analysis of the performance of Xenium, and provides best practices and recommendations for analysis of such datasets.

The injectable contraceptive, depot medroxyprogesterone acetate (DMPA), is associated with compromised cervical mucosal barriers. High-resolution spatial transcriptomics is applied here to reveal the spatial localization of these altered molecular markers. Ectocervical tissue samples from Kenyan sex workers using DMPA, or non-hormonal contraceptives, underwent spatial transcriptomics and gene set enrichment analyses. Integrated systemic estradiol levels and bulk tissue gene expression data from a larger cohort enhanced the study’s scope. Unsupervised clustering unveiled four epithelial and seven submucosal layers, showcasing spatially restricted and diverse functional epithelial responses, and a less structured submucosal spatial ordering. DMPA associated with mucosal-wide immunoglobulin gene upregulation, verified by CD20⁺ B-cell immunostaining, and upregulated immune markers adjacent to the basal membrane. Downregulated genes represented spatially restricted disrupted epithelial barrier integrity and submucosal extracellular matrix dysfunction. The transcriptional profile was associated with markers of estrogen regulation. Collectively, our findings reveal estrogen-modulated distinct ectocervical transcriptional profiles associated with DMPA usage. While upregulation of immunoglobulin genes occurs throughout the mucosa, activation of innate immune responses and dysregulation of barrier integrity markers are spatially restricted. These results extend previous analyses using bulk transcriptomics and provide insights into the molecular landscape influenced by DMPA, shedding light on contraceptive effects and health implications.

BACKGROUND: Separation of the pulmonic and systemic circulation is essential for terrestrial life, and mammals have evolved distinct cardiac chambers with specialized structures and functions. Transcriptomics profiling revealed cellular heterogeneity between heart chambers. However, the mechanisms underlying chamber-specific transcriptomic and metabolic differences—and their functional significance—remain poorly understood. The Hippo/YAP (yes-associated protein) pathway is a conserved signaling network that regulates diverse cellular processes. The Hippo kinases inhibit YAP in cardiac fibroblasts (CF) to restrict fibrosis and inflammation. Nonetheless, how YAP regulates the metabolic microenvironment during homeostasis and fibroinflammation remains unclear.

METHODS: We investigated YAP and glycolysis activity in the 4 cardiac chambers by scoring the expression of YAP target genes and glycolysis genes in human single-nucleus RNA sequencing data. To compare glucose uptake between the left and right atria, we measured isotope-labeled glucose uptake in isolated mouse atria. To study the role of YAP in CFs, we inactivated the Hippo kinases, Lats1 and Lats2, in mouse CFs and performed metabolic studies, snRNA-seq, single-nucleus assay for transposase-accessible chromatin with sequencing, and spatial transcriptomics.

RESULTS: Metabolic and sequencing approaches revealed that Hippo-deficient CFs activated glycolysis to promote fibroinflammation. Inhibition of glycolysis or lactate production suppressed Hippo-deficient CF-induced fibrosis. Elevated YAP activity disrupted fibroblast lineage fidelity by inducing an osteochondroprogenitor cell state. Blocking macrophage expansion pharmacologically reduced Hippo-deficient CF proliferation and fibrosis. Sequencing and functional studies showed that macrophages secreted IGF1 (insulin-like growth factor 1) to activate IGF1 signaling in Hippo-deficient CFs to increase cell proliferation and fibrosis.

CONCLUSIONS: We discovered that right atrial CFs are more glycolytic and have higher YAP activity than CFs in other heart chambers. YAP activation in CFs induces glycolysis to drive fibrosis. YAP disrupts fibroblast lineage fidelity, driving them to a SOX9 (SRY-box transcription factor 9)-expressing osteochondroprogenitor cell state. Mechanistically, YAP activates the secretion of CSF1 (colony-stimulating factor 1) to promote macrophage expansion. Blocking macrophage expansion reduces Hippo-deficient CF proliferation, osteochondroprogenitor differentiation, and fibrosis, revealing that macrophages signal reciprocally to regulate CF cell states. Genomic and functional studies revealed that the upregulated IGF1 receptor in Hippo-deficient CFs enables them to receive macrophage-secreted IGF1, thereby further enhancing CF proliferation and fibrosis.

Stromal cells support epithelial cell and immune cell homeostasis and play an important role in inflammatory bowel disease (IBD) pathogenesis. Here, we quantify the stromal response to inflammation in pediatric IBD and reveal subset-specific inflammatory responses across colon segments and intestinal layers. Using data from a murine dynamic gut injury model and human ex vivo transcriptomic, protein and spatial analyses, we report that PDGFRA⁺CD142^−/low fibroblasts and monocytes/macrophages co-localize in the intestine. In primary human fibroblast-monocyte co-cultures, intestinal PDGFRA⁺CD142^−/low fibroblasts foster monocyte transition to CCR2⁺CD206⁺ macrophages through granulocyte-macrophage colony-stimulating factor (GM-CSF). Monocyte-derived CCR2⁺CD206⁺ cells from co-cultures have a phenotype similar to intestinal CCR2⁺CD206⁺ macrophages from newly diagnosed pediatric IBD patients, with high levels of PD-L1 and low levels of GM-CSF receptor. The study describes subset-specific changes in stromal responses to inflammation and suggests that the intestinal stroma guides intestinal macrophage differentiation.

Objective. B cells are important in the pathogenesis of primary Sjögren's syndrome (pSS). Patients positive for Sjögren's syndrome antigen A/Sjögren syndrome antigen B (SSA/SSB) autoantibodies are more prone to systemic disease manifestations and adverse outcomes. We aimed to determine the role of B cell composition, gene expression, and B cell receptor usage in pSS subgroups stratified for SSA/SSB antibodies.

Methods. Over 230,000 B cells were isolated from peripheral blood of patients with pSS (n = 6 SSA−, n = 8 SSA+ single positive and n = 10 SSA/SSB+ double positive) and four healthy controls and processed for single-cell RNA sequencing (scRNA-seq) and single-cell variable, diversity, and joining (VDJ) gene sequencing (scVDJ-seq).

Results. We show that SSA/SSB+ patients present the highest and lowest proportion of naïve and memory B cells, respectively, and the highest up-regulation of interferon-induced genes across all B cell subtypes. Differential usage of IGHV showed that IGHV1-69 and IGHV4-30-4 were more often used in all pSS subgroups compared with controls. Memory B cells from SSA/SSB+ patients displayed a higher proportion of cells with unmutated VDJ transcripts compared with other pSS patient groups and controls, indicating altered somatic hypermutation processes. Comparison with previous studies revealed heterogeneous clonotype pools, with little overlap in CDR3 sequences. Joint analysis using scRNA-seq and scVDJ-seq data allowed unsupervised stratification of patients with pSS and identified novel parameters that correlated to disease manifestations and antibody status.

Conclusion. We describe heterogeneity and molecular characteristics in B cells from patients with pSS, providing clues to intrinsic differences in B cells that affect the phenotype and outcome and allowing stratification of patients with pSS at improved resolution.

Chronic systemic immune activation significantly influences human immunodeficiency virus (HIV) disease progression. Despite evidence of a pro-inflammatory environment in the genital tract of HIV-infected women, comprehensive investigations into cervical tissue from this region remain limited. Similarly, the consequences of chronic HIV infection on the integrity of the female genital epithelium are poorly understood, despite its importance in HIV transmission and replication. Ectocervical biopsies were obtained from HIV-seropositive (n = 14) and HIV-seronegative (n = 47) female Kenyan sex workers. RNA sequencing and bioimage analysis of epithelial junction proteins (E-cadherin, desmoglein-1, claudin-1, and zonula occludens-1) were conducted, along with CD4 staining. RNA sequencing revealed upregulation of immunoregulatory genes in HIV-seropositive women, primarily associated with heightened T cell activity and interferon signaling, which further correlated with plasma viral load. Transcription factor analysis confirmed the upregulation of pro-inflammatory transcription factors, such as RELA, NFKB1, and IKZF3, which facilitates HIV persistence in T cells. Conversely, genes and pathways associated with epithelial barrier function and structure were downregulated in the context of HIV. Digital bioimage analysis corroborated these findings, revealing significant disruption of various epithelial junction proteins in ectocervical tissues of the HIV-seropositive women. Thus, chronic HIV infection associated with ectocervical inflammation, characterized by induced T cell responses and interferon signaling, coupled with epithelial disruption. These alterations may influence HIV transmission and heighten susceptibility to other sexually transmitted infections. These findings prompt exploration of therapeutic interventions to address HIV-related complications and mitigate the risk of sexually transmitted infection transmission.

Problem: Overgrowth of candida species in the human vaginal mucosa causes inflammation, which could render the mucosal barrier more susceptible to HIV infection. Here, we investigated whether this condition also affects the ectocervical mucosa, a potential site of HIV entry, in women at high risk of HIV infection.

Method of study: Retrospective medical data and ectocervical tissue samples were obtained from a cohort of Kenyan sex workers. Among 108 women, seven had signs of vaginal candida infection by wet smear microscopy and/or the presence of characteristic discharge. Women lacking these two criteria served as controls. Host transcriptomic profiling and quantitative in situ image analysis of epithelial barrier markers and CD4⁺ cell distribution were performed.

Results: The candida group had 162 differentially expressed genes out of 15 435 genes as compared with the control group. Among these 162 genes, 147 were upregulated and 15 were downregulated. Gene expression pathway analysis indicated associations with an upregulated inflammatory response, defined primarily by markers of neutrophil activation. Transcription factor analysis revealed upregulation of pathways related to RELA/REL/NFKB1, JUN and STAT1 in the candida group. In situ image analysis of ectocervical tissue samples showed no differences between groups in terms of epithelial height, expression of epithelial junction proteins (E-cadherin, claudin-1, zonula occludens 1, and desmoglein-1), or epithelial CD4⁺ cell distribution.

Conclusions: Vaginal candida infection was associated with inflammation and neutrophil infiltration, but not with severe epithelial disruption or CD4+ cell infiltration, in the ectocervical mucosa.

Effective humoral immune responses require well-orchestrated B and T follicular helper (Tfh) cell interactions. Whether these interactions are impaired and associated with COVID-19 disease severity is unclear. Here, longitudinal blood samples across COVID-19 disease severity are analysed. We find that during acute infection SARS-CoV-2-specific circulating Tfh (cTfh) cells expand with disease severity. SARS-CoV-2-specific cTfh cell frequencies correlate with plasmablast frequencies and SARS-CoV-2 antibody titers, avidity and neutralization. Furthermore, cTfh cells but not other memory CD4 T cells, from severe patients better induce plasmablast differentiation and antibody production compared to cTfh cells from mild patients. However, virus-specific cTfh cell development is delayed in patients that display or later develop severe disease compared to those with mild disease, which correlates with delayed induction of high-avidity neutralizing antibodies. Our study suggests that impaired generation of functional virus-specific cTfh cells delays high-quality antibody production at an early stage, potentially enabling progression to severe disease.