Decoding spatiotemporal transcriptional dynamics and epithelial fibroblast crosstalk during gastroesophageal junction development through single cell analysis.

The gastroesophageal squamocolumnar junction (GE-SCJ) is a critical tissue interface between the esophagus and stomach, with significant relevance in the pathophysiology of gastrointestinal diseases. Despite this, the molecular mechanisms underlying GE-SCJ development remain unclear. Using single-cell transcriptomics, organoids, and spatial analysis, we examine the cellular heterogeneity and spatiotemporal dynamics of GE-SCJ development from embryonic to adult mice. We identify distinct transcriptional states and signaling pathways in the epithelial and mesenchymal compartments of the esophagus and stomach during development. Fibroblast-epithelial interactions are mediated by various signaling pathways, including WNT, BMP, TGF-ß, FGF, EGF, and PDGF. Our results suggest that fibroblasts predominantly send FGF and TGF-ß signals to the epithelia, while epithelial cells mainly send PDGF and EGF signals to fibroblasts. We observe differences in the ligands and receptors involved in cell-cell communication between the esophagus and stomach. Our findings provide insights into the molecular mechanisms underlying GE-SCJ development and fibroblast-epithelial crosstalk involved, paving the way to elucidate mechanisms during adaptive metaplasia development and carcinogenesis.

Human cytomegalovirus exploits STING signaling and counteracts IFN/ISG induction to facilitate infection of dendritic cells.

Human cytomegalovirus (HCMV) is a widespread pathogen that in immunocompromised hosts can cause life-threatening disease. Studying HCMV-exposed monocyte-derived dendritic cells by single-cell RNA sequencing, we observe that most cells are entered by the virus, whereas less than 30% of them initiate viral gene expression. Increased viral gene expression is associated with activation of the stimulator of interferon genes (STING) that usually induces anti-viral interferon responses, and with the induction of several pro- (RHOB, HSP1A1, DNAJB1) and anti-viral (RNF213, TNFSF10, IFI16) genes. Upon progression of infection, interferon-beta but not interferon-lambda transcription is inhibited. Similarly, interferon-stimulated gene expression is initially induced and then shut off, thus further promoting productive infection. Monocyte-derived dendritic cells are composed of 3 subsets, with one being especially susceptible to HCMV. In conclusion, HCMV permissiveness of monocyte-derived dendritic cells depends on complex interactions between virus sensing, regulation of the interferon response, and viral gene expression.

Sequential antigen loss and branching evolution in lymphoma after CD19- and CD20-targeted T-cell-redirecting therapy.

ABSTRACT: CD19 chimeric antigen receptor (CAR) T cells and CD20 targeting T-cell-engaging bispecific antibodies (bispecs) have been approved in B-cell non-Hodgkin lymphoma lately, heralding a new clinical setting in which patients are treated with both approaches, sequentially. The aim of our study was to investigate the selective pressure of CD19- and CD20-directed therapy on the clonal architecture in lymphoma. Using a broad analytical pipeline on 28 longitudinally collected specimen from 7 patients, we identified truncating mutations in the gene encoding CD20 conferring antigen loss in 80% of patients relapsing from CD20 bispecs. Pronounced T-cell exhaustion was identified in cases with progressive disease and retained CD20 expression. We also confirmed CD19 loss after CAR T-cell therapy and reported the case of sequential CD19 and CD20 loss. We observed branching evolution with re-emergence of CD20+ subclones at later time points and spatial heterogeneity for CD20 expression in response to targeted therapy. Our results highlight immunotherapy as not only an evolutionary bottleneck selecting for antigen loss variants but also complex evolutionary pathways underlying disease progression from these novel therapies.

Brain-to-gut trafficking of alpha-synuclein by CD11c+ cells in a mouse model of Parkinson's disease.

Inflammation in the brain and gut is a critical component of several neurological diseases, such as Parkinson's disease (PD). One trigger of the immune system in PD is aggregation of the pre-synaptic protein, α-synuclein (αSyn). Understanding the mechanism of propagation of αSyn aggregates is essential to developing disease-modifying therapeutics. Using a brain-first mouse model of PD, we demonstrate αSyn trafficking from the brain to the ileum of male mice. Immunohistochemistry revealed that the ileal αSyn aggregations are contained within CD11c+ cells. Using single-cell RNA sequencing, we demonstrate that ileal CD11c+ cells are microglia-like and the same subtype of cells is activated in the brain and ileum of PD mice. Moreover, by utilizing mice expressing the photo-convertible protein, Dendra2, we show that CD11c+ cells traffic from the brain to the ileum. Together these data provide a mechanism of αSyn trafficking between the brain and gut.

Vector-borne Trypanosoma brucei parasites develop in artificial human skin and persist as skin tissue forms.

Transmission of Trypanosoma brucei by tsetse flies involves the deposition of the cell cycle-arrested metacyclic life cycle stage into mammalian skin at the site of the fly's bite. We introduce an advanced human skin equivalent and use tsetse flies to naturally infect the skin with trypanosomes. We detail the chronological order of the parasites' development in the skin by single-cell RNA sequencing and find a rapid activation of metacyclic trypanosomes and differentiation to proliferative parasites. Here we show that after the establishment of a proliferative population, the parasites enter a reversible quiescent state characterized by slow replication and a strongly reduced metabolism. We term these quiescent trypanosomes skin tissue forms, a parasite population that may play an important role in maintaining the infection over long time periods and in asymptomatic infected individuals.

Microglia-mediated demyelination protects against CD8+ T cell-driven axon degeneration in mice carrying PLP defects.

Axon degeneration and functional decline in myelin diseases are often attributed to loss of myelin but their relation is not fully understood. Perturbed myelinating glia can instigate chronic neuroinflammation and contribute to demyelination and axonal damage. Here we study mice with distinct defects in the proteolipid protein 1 gene that develop axonal damage which is driven by cytotoxic T cells targeting myelinating oligodendrocytes. We show that persistent ensheathment with perturbed myelin poses a risk for axon degeneration, neuron loss, and behavioral decline. We demonstrate that CD8+ T cell-driven axonal damage is less likely to progress towards degeneration when axons are efficiently demyelinated by activated microglia. Mechanistically, we show that cytotoxic T cell effector molecules induce cytoskeletal alterations within myelinating glia and aberrant actomyosin constriction of axons at paranodal domains. Our study identifies detrimental axon-glia-immune interactions which promote neurodegeneration and possible therapeutic targets for disorders associated with myelin defects and neuroinflammation.

Genetic inhibition of CARD9 accelerates the development of atherosclerosis in mice through CD36 dependent-defective autophagy.

Caspase recruitment-domain containing protein 9 (CARD9) is a key signaling pathway in macrophages but its role in atherosclerosis is still poorly understood. Global deletion of Card9 in Apoe-/- mice as well as hematopoietic deletion in Ldlr-/- mice increases atherosclerosis. The acceleration of atherosclerosis is also observed in Apoe-/-Rag2-/-Card9-/- mice, ruling out a role for the adaptive immune system in the vascular phenotype of Card9 deficient mice. Card9 deficiency alters macrophage phenotype through CD36 overexpression with increased IL-1ß production, increased lipid uptake, higher cell death susceptibility and defective autophagy. Rapamycin or metformin, two autophagy inducers, abolish intracellular lipid overload, restore macrophage survival and autophagy flux in vitro and finally abolish the pro-atherogenic effects of Card9 deficiency in vivo. Transcriptomic analysis of human CARD9-deficient monocytes confirms the pathogenic signature identified in murine models. In summary, CARD9 is a key protective pathway in atherosclerosis, modulating macrophage CD36-dependent inflammatory responses, lipid uptake and autophagy.

Short-range interactions between fibrocytes and CD8+ T cells in COPD bronchial inflammatory response.

Bronchi of chronic obstructive pulmonary disease (COPD) are the site of extensive cell infiltration, allowing persistent contact between resident cells and immune cells. Tissue fibrocytes interaction with CD8+ T cells and its consequences were investigated using a combination of in situ, in vitro experiments and mathematical modeling. We show that fibrocytes and CD8+ T cells are found in the vicinity of distal airways and that potential interactions are more frequent in tissues from COPD patients compared to those of control subjects. Increased proximity and clusterization between CD8+ T cells and fibrocytes are associated with altered lung function. Tissular CD8+ T cells from COPD patients promote fibrocyte chemotaxis via the CXCL8-CXCR1/2 axis. Live imaging shows that CD8+ T cells establish short-term interactions with fibrocytes, that trigger CD8+ T cell proliferation in a CD54- and CD86-dependent manner, pro-inflammatory cytokines production, CD8+ T cell cytotoxic activity against bronchial epithelial cells and fibrocyte immunomodulatory properties. We defined a computational model describing these intercellular interactions and calibrated the parameters based on our experimental measurements. We show the model's ability to reproduce histological ex vivo characteristics, and observe an important contribution of fibrocyte-mediated CD8+ T cell proliferation in COPD development. Using the model to test therapeutic scenarios, we predict a recovery time of several years, and the failure of targeting chemotaxis or interacting processes. Altogether, our study reveals that local interactions between fibrocytes and CD8+ T cells could jeopardize the balance between protective immunity and chronic inflammation in the bronchi of COPD patients.

Lymph node medulla regulates the spatiotemporal unfolding of resident dendritic cell networks.

Unlike macrophage networks composed of long-lived tissue-resident cells within specific niches, conventional dendritic cells (cDCs) that generate a 3D network in lymph nodes (LNs) are short lived and continuously replaced by DC precursors (preDCs) from the bone marrow (BM). Here, we examined whether specific anatomical niches exist within which preDCs differentiate toward immature cDCs. In situ photoconversion and Prtn3-based fate-tracking revealed that the LN medullary cords are preferential entry sites for preDCs, serving as specific differentiation niches. Repopulation and fate-tracking approaches demonstrated that the cDC1 network unfolded from the medulla along the vascular tree toward the paracortex. During inflammation, collective maturation and migration of resident cDC1s to the paracortex created discontinuity in the medullary cDC1 network and temporarily impaired responsiveness. The decrease in local cDC1 density resulted in higher Flt3L availability in the medullary niche, which accelerated cDC1 development to restore the network. Thus, the spatiotemporal development of the cDC1 network is locally regulated in dedicated LN niches via sensing of cDC1 densities.

An interferon gamma response signature links myocardial aging and immunosenescence.

AIMS: Aging entails profound immunological transformations that can impact myocardial homeostasis and predispose to heart failure. However, preclinical research in the immune-cardiology field is mostly conducted in young healthy animals, which potentially weakens its translational relevance. Herein, we sought to investigate how the aging T-cell compartment associates with changes in myocardial cell biology in aged mice. METHODS AND RESULTS: We phenotyped the antigen-experienced effector/memory T cells purified from heart-draining lymph nodes of 2-, 6-, 12-, and 18-month-old C57BL/6J mice using single-cell RNA/T cell receptor sequencing. Simultaneously, we profiled all non-cardiomyocyte cell subsets purified from 2- to 18-month-old hearts and integrated our data with publicly available cardiomyocyte single-cell sequencing datasets. Some of these findings were confirmed at the protein level by flow cytometry. With aging, the heart-draining lymph node and myocardial T cells underwent clonal expansion and exhibited an up-regulated pro-inflammatory transcription signature, marked by an increased interferon-γ (IFN-γ) production. In parallel, all major myocardial cell populations showed increased IFN-γ responsive signature with aging. In the aged cardiomyocytes, a stronger IFN-γ response signature was paralleled by the dampening of expression levels of transcripts related to most metabolic pathways, especially oxidative phosphorylation. Likewise, induced pluripotent stem cells-derived cardiomyocytes exposed to chronic, low grade IFN-γ treatment showed a similar inhibition of metabolic activity. CONCLUSIONS: By investigating the paired age-related alterations in the T cells found in the heart and its draining lymph nodes, we provide evidence for increased myocardial IFN-γ signaling with age, which is associated with inflammatory and metabolic shifts typically seen in heart failure.

Cytotoxic CNS-associated T cells drive axon degeneration by targeting perturbed oligodendrocytes in PLP1 mutant mice.

Myelin defects lead to neurological dysfunction in various diseases and in normal aging. Chronic neuroinflammation often contributes to axon-myelin damage in these conditions and can be initiated and/or sustained by perturbed myelinating glia. We have previously shown that distinct PLP1 mutations result in neurodegeneration that is largely driven by adaptive immune cells. Here we characterize CD8+ CNS-associated T cells in myelin mutants using single-cell transcriptomics and identify population heterogeneity and disease-associated changes. We demonstrate that early sphingosine-1-phosphate receptor modulation attenuates T cell recruitment and neural damage, while later targeting of CNS-associated T cell populations is inefficient. Applying bone marrow chimerism and utilizing random X chromosome inactivation, we provide evidence that axonal damage is driven by cytotoxic, antigen specific CD8+ T cells that target mutant myelinating oligodendrocytes. These findings offer insights into neural-immune interactions and are of translational relevance for neurological conditions associated with myelin defects and neuroinflammation.

Advancing massive transcriptional profiling of single bacteria.

Single-bacteria RNA-seq is becoming a transformative technology for elucidating bacterial diversity in ecology, microbial communities, and pathogenicity. In a recent report by Ma et al. published in Cell, the authors present BacDrop, a versatile, high-throughput method for capturing the transcriptomes of millions of bacteria.

A primary cell-based in vitro model of the human small intestine reveals host olfactomedin 4 induction in response to Salmonella Typhimurium infection.

Infection research largely relies on classical cell culture or mouse models. Despite having delivered invaluable insights into host-pathogen interactions, both have limitations in translating mechanistic principles to human pathologies. Alternatives can be derived from modern Tissue Engineering approaches, allowing the reconstruction of functional tissue models in vitro. Here, we combined a biological extracellular matrix with primary tissue-derived enteroids to establish an in vitro model of the human small intestinal epithelium exhibiting in vivo-like characteristics. Using the foodborne pathogen Salmonella enterica serovar Typhimurium, we demonstrated the applicability of our model to enteric infection research in the human context. Infection assays coupled to spatio-temporal readouts recapitulated the established key steps of epithelial infection by this pathogen in our model. Besides, we detected the upregulation of olfactomedin 4 in infected cells, a hitherto unrecognized aspect of the host response to Salmonella infection. Together, this primary human small intestinal tissue model fills the gap between simplistic cell culture and animal models of infection, and shall prove valuable in uncovering human-specific features of host-pathogen interplay.

Myocardial Milieu Favors Local Differentiation of Regulatory T Cells.

BACKGROUND: In the past years, several studies investigated how distinct immune cell subsets affects post-myocardial infarction repair. However, whether and how the tissue environment controls these local immune responses has remained poorly understood. We sought to investigate how antigen-specific T-helper cells differentiate under myocardial milieu's influence. METHODS: We used a transgenic T cell receptor (TCR-M) model and major histocompatibility complex-II tetramers, both myosin-specific, combined with single-cell transcriptomics (single-cell RNA sequencing [scRNA-seq]) and functional phenotyping to elucidate how the antigen-specific CD4+ T cells differentiate in the murine infarcted myocardium and influence tissue repair. Additionally, we transferred proinflammatory versus regulatory predifferentiated TCR-M-cells to dissect how they specially contribute to post-myocardial infarction inflammation. RESULTS: Flow cytometry and scRNA-/TCR-seq analyses revealed that transferred TCR-M cells acquired an induced regulatory phenotype (induced regulatory T cell) in the infarcted myocardium and blunted local inflammation. Myocardial TCR-M cells differentiated into 2 main lineages enriched with either cell activation and profibrotic transcripts (eg, Tgfb1) or suppressor immune checkpoints (eg, Pdcd1), which we also found in human myocardial tissue. These cells produced high levels of LAP (latency-associated peptide) and inhibited IL-17 (interleukin-17) responses. Endogenous myosin-specific T-helper cells, identified using genetically barcoded tetramers, also accumulated in infarcted hearts and exhibited a regulatory phenotype. Notably, TCR-M cells that were predifferentiated toward a regulatory phenotype in vitro maintained stable in vivo FOXP3 (Forkhead box P3) expression and anti-inflammatory activity whereas TH17 partially converted toward a regulatory phenotype in the injured myocardium. Overall, the myosin-specific Tregs dampened post-myocardial infarction inflammation, suppressed neighboring T cells, and were associated with improved cardiac function. CONCLUSIONS: These findings provide novel evidence that the heart and its draining lymph nodes actively shape local immune responses by promoting the differentiation of antigen-specific Tregs poised with suppressive function.

Th17.1 cell driven sarcoidosis-like inflammation after anti-BCMA CAR T cells in multiple myeloma.

Pseudo-progression and flare-up phenomena constitute a novel diagnostic challenge in the follow-up of patients treated with immune-oncology drugs. We present a case study on pulmonary flare-up after Idecabtagen Vicleucel (Ide-cel), a BCMA targeting CAR T-cell therapy, and used single-cell RNA-seq (scRNA-seq) to identify a Th17.1 driven autoimmune mechanism as the biological underpinning of this phenomenon. By integrating datasets of various lung pathological conditions, we revealed transcriptomic similarities between post CAR T pulmonary lesions and sarcoidosis. Furthermore, we explored a noninvasive PET based diagnostic approach and showed that tracers binding to CXCR4 complement FDG PET imaging in this setting, allowing discrimination between immune-mediated changes and true relapse after CAR T-cell treatment. In conclusion, our study highlights a Th17.1 driven autoimmune phenomenon after CAR T, which may be misinterpreted as disease relapse, and that imaging with multiple PET tracers and scRNA-seq could help in this diagnostic dilemma.

Rapid neutrophil mobilization by VCAM-1+ endothelial cell-derived extracellular vesicles.

AIMS: Acute myocardial infarction rapidly increases blood neutrophils (<2 h). Release from bone marrow, in response to chemokine elevation, has been considered their source, but chemokine levels peak up to 24 h after injury, and after neutrophil elevation. This suggests that additional non-chemokine-dependent processes may be involved. Endothelial cell (EC) activation promotes the rapid (<30 min) release of extracellular vesicles (EVs), which have emerged as an important means of cell-cell signalling and are thus a potential mechanism for communicating with remote tissues. METHODS AND RESULTS: Here, we show that injury to the myocardium rapidly mobilizes neutrophils from the spleen to peripheral blood and induces their transcriptional activation prior to arrival at the injured tissue. Time course analysis of plasma-EV composition revealed a rapid and selective increase in EVs bearing VCAM-1. These EVs, which were also enriched for miRNA-126, accumulated preferentially in the spleen where they induced local inflammatory gene and chemokine protein expression, and mobilized splenic-neutrophils to peripheral blood. Using CRISPR/Cas9 genome editing, we generated VCAM-1-deficient EC-EVs and showed that its deletion removed the ability of EC-EVs to provoke the mobilization of neutrophils. Furthermore, inhibition of miRNA-126 in vivo reduced myocardial infarction size in a mouse model. CONCLUSIONS: Our findings show a novel EV-dependent mechanism for the rapid mobilization of neutrophils to peripheral blood from a splenic reserve and establish a proof of concept for functional manipulation of EV-communications through genetic alteration of parent cells.

Dynamics of monocyte-derived macrophage diversity in experimental myocardial infarction.

AIMS: Macrophages have a critical and dual role in post-ischaemic cardiac repair, as they can foster both tissue healing and damage. Multiple subsets of tissue resident and monocyte-derived macrophages coexist in the infarcted heart, but their precise identity, temporal dynamics, and the mechanisms regulating their acquisition of discrete states are not fully understood. To address this, we used multi-modal single-cell immune profiling, combined with targeted cell depletion and macrophage fate mapping, to precisely map monocyte/macrophage transitions after experimental myocardial infarction. METHODS AND RESULTS: We performed single-cell transcriptomic and cell-surface marker profiling of circulating and cardiac immune cells in mice challenged with acute myocardial infarction, and integrated single-cell transcriptomes obtained before and at 1, 3, 5, 7, and 11 days after infarction. Using complementary strategies of CCR2+ monocyte depletion and fate mapping of tissue resident macrophages, we determined the origin of cardiac macrophage populations. The macrophage landscape of the infarcted heart was dominated by monocyte-derived cells comprising two pro-inflammatory populations defined as Isg15hi and MHCII+Il1b+, alongside non-inflammatory Trem2hi cells. Trem2hi macrophages were observed in the ischaemic area, but not in the remote viable myocardium, and comprised two subpopulations sequentially populating the heart defined as Trem2hiSpp1hi monocyte-to-macrophage intermediates, and fully differentiated Trem2hiGdf15hi macrophages. Cardiac Trem2hi macrophages showed similarities to 'lipid-associated macrophages' found in mouse models of metabolic diseases and were observed in the human heart, indicating conserved features of this macrophage state across diseases and species. Ischaemic injury induced a shift of circulating Ly6Chi monocytes towards a Chil3hi state with granulocyte-like features, but the acquisition of the Trem2hi macrophage signature occurred in the ischaemic tissue. In vitro, macrophages acquired features of the Trem2hi signature following apoptotic-cell efferocytosis. CONCLUSION: Our work provides a comprehensive map of monocyte/macrophage transitions in the ischaemic heart, constituting a valuable resource for further investigating how these cells may be harnessed and modulated to promote post-ischaemic heart repair.

Integrated single-cell analysis-based classification of vascular mononuclear phagocytes in mouse and human atherosclerosis.

AIMS: Accumulation of mononuclear phagocytes [monocytes, macrophages, and dendritic cells (DCs)] in the vessel wall is a hallmark of atherosclerosis. Using integrated single-cell analysis of mouse and human atherosclerosis, we here aimed to refine the nomenclature of mononuclear phagocytes in atherosclerotic vessels and to compare their transcriptomic profiles in mouse and human disease. METHODS AND RESULTS: We integrated 12 single-cell RNA-sequencing (scRNA-seq) datasets of immune cells isolated from healthy or atherosclerotic mouse aortas, and data from 11 patients (n = 4 coronary vessels, n = 7 carotid endarterectomy specimens) from two studies. Integration of mouse data identified subpopulations with discrete transcriptomic signatures within previously described populations of aortic resident (Lyve1), inflammatory (Il1b), as well as foamy (Trem2hi) macrophages. We identified unique transcriptomic features distinguishing aortic intimal resident macrophages from atherosclerosis-associated Trem2hi macrophages. Also, populations of Xcr1+ Type 1 classical DCs (cDC1), Cd209a+ cDC2, and mature DCs (Ccr7, Fscn1) with a 'mreg-DC' signature were detected. In humans, we uncovered macrophage and DC populations with gene expression patterns similar to those observed in mice. In particular, core transcripts of the foamy/Trem2hi signature (TREM2, SPP1, GPNMB, CD9) mapped to a specific population of macrophages in human lesions. Comparison of mouse and human data and direct cross-species data integration suggested transcriptionally similar macrophage and DC populations in mice and humans. CONCLUSIONS: We refined the nomenclature of mononuclear phagocytes in mouse atherosclerotic vessels, and show conserved transcriptomic features of macrophages and DCs in atherosclerosis in mice and humans, emphasizing the relevance of mouse models to study mononuclear phagocytes in atherosclerosis.

Altered and allele-specific open chromatin landscape reveals epigenetic and genetic regulators of innate immunity in COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes severe COVID-19 in some patients and mild COVID-19 in others. Dysfunctional innate immune responses have been identified to contribute to COVID-19 severity, but the key regulators are still unknown. Here, we present an integrative single-cell multi-omics analysis of peripheral blood mononuclear cells from hospitalized and convalescent COVID-19 patients. In classical monocytes, we identified genes that were potentially regulated by differential chromatin accessibility. Then, sub-clustering and motif-enrichment analyses revealed disease condition-specific regulation by transcription factors and their targets, including an interaction between C/EBPs and a long-noncoding RNA LUCAT1, which we validated through loss-of-function experiments. Finally, we investigated genetic risk variants that exhibit allele-specific open chromatin (ASoC) in COVID-19 patients and identified a SNP rs6800484-C, which is associated with lower expression of CCR2 and may contribute to higher viral loads and higher risk of COVID-19 hospitalization. Altogether, our study highlights the diverse genetic and epigenetic regulators that contribute to COVID-19.

A MATQ-seq-Based Protocol for Single-Cell RNA-seq in Bacteria.

Microbes exhibit an extraordinary capacity to adapt their physiology to different environments using phenotypic heterogeneity. However, the majority of gene regulation studies are conducted in bulk reflecting only averaged gene expression levels across millions of cells. Bacterial single-cell RNA-seq (scRNA-seq) can overcome this by enabling whole transcriptome and unbiased analysis of microbes at the single-cell level. Here, we describe a detailed workflow of single-cell RNA-seq based on the multiple annealing and dC-tailing-based quantitative single-cell RNA-seq (MATQ-seq) protocol. Following adjustments to the original eukaryotic protocol, the workflow was applied to two major human pathogens Salmonella enterica serovar Typhimurium (henceforth Salmonella) and Pseudomonas aeruginosa (henceforth Pseudomonas). The development of bacterial scRNA-seq protocols offers promising avenues to explore the molecular programs underlying phenotypic heterogeneity on the transcriptome level in different settings such as infection, persistence, ecology, and biofilms.