Biology subjects

Miller-Jensen, K.

Publications and source records attributed to Miller-Jensen, K..

11 recordsLinked to original sources

bioRxiv2025

Interferon-β paracrine signaling mediates synergy between TLR3 and TRIF-independent TLR pathways

Cytokine dysregulation during microbial infections can dangerously increase the severity of associated diseases. Non-additive responses to simultaneous activation of toll-like receptor (TLR) signaling pathways are one potential contributor. Here we explored mechanisms underlying the TLR2/MyD88 and TLR3/TRIF-mediated responses in macrophages for canonical pro-inflammatory and antiviral targets including TNF, IL-6, IL-12p40, IFN-{beta} and CXCL10. We found that all targets exhibited characteristic levels of synergistic cytokine activation that varied with TLR-ligand concentration and exposure time. These trends were conserved when the TLR3-ligand polyI:C (PIC) was combined with either of the TLR2-mediated pathogens S. aureus or L. pneumophila. Using pharmacological inhibitors, genetic knockouts, and recombinant cytokines, we explored how TLR2-TLR3-induced paracrine signaling via TNF and IFN-{beta} affected synergy. While TNF synergy is paracrine-independent, IFN-{beta} contributes to synergistic activation of IL-6 and IL-12p40. When IFN-{beta} was directly combined with P3C or S. aureus infection, low concentrations produced modest synergy for both, while high concentrations increased IL-6 but antagonized IL-12p40. Thus, multiple mechanisms regulate TLR2/TLR3-mediated synergistic cytokine activation in macrophages, including type I interferon modulation of TRIF-independent pathogen stimulation that increases inflammatory signaling. These findings have important implications for therapeutic modification of cytokine-driven inflammation, including cytokine storms and the development of vaccine adjuvants.

immunology↗

bioRxiv2025

Interplay between cytokine and FGF2 signaling in induction of entosis and vasculogenic mimicry response in glioblastoma

Tumor vascularization is critical to survival of cancer cells, but is frequently perturbed leading to disorganized angiogenesis and emergence of alternative means of delivery of oxygen and nutrients, such as vasculogenic mimicry (VM). Understanding of VM and its relationship to endothelial vascularization has been hampered by the lack of comprehensive combination of in vivo clinical data and relevant in vitro models. We address this challenge by analyzing glioblastoma (GBM) tumors and clinically isolated cancer cells. This analysis strongly suggests a key role of macrophage-induced controlled cell death in emergence of VM. The results further point to entosis of cancer cells as a critical intermediate state in this process, enabled by mechano-chemical cell heterogeneity. We find evidence that macrophages can regulate endothelial angiogenesis and VM as two alternative vascularization mechanisms. These results reveal mechanistic underpinnings of VM and pave the way to predictive analysis of tumor progression.

cancer biology↗

bioRxiv2024

Mapping intratumoral myeloid-T cell interactomes at single-cell resolution reveals targets for overcoming checkpoint inhibitor resistance

Effective cancer immunotherapies restore anti-tumor immunity by rewiring cell-cell communication. Treatment-induced changes in communication can be inferred from single-cell RNA-sequencing (scRNA-seq) data, but current methods do not effectively manage heterogeneity within cell types. Here we developed a computational approach to efficiently analyze scRNA-seq-derived, single-cell-resolved cell-cell interactomes, which we applied to determine how agonistic CD40 (CD40ag) alters immune cell crosstalk alone, across tumor models, and in combination with immune checkpoint blockade (ICB). Our analyses suggested that CD40ag improves responses to ICB by targeting both immuno-stimulatory and immunosuppressive macrophage subsets communicating with T cells, and we experimentally validated a spatial basis for these subsets with immunofluorescence and spatial transcriptomics. Moreover, treatment with CD40ag and ICB established coordinated myeloid-T cell interaction hubs that are critical for reestablishing antitumor immunity. Our work advances the biological significance of hypotheses generated from scRNA-seq-derived cell-cell interactomes and supports the clinical translation of myeloid-targeted therapies for ICB-resistant tumors.

systems biology↗

bioRxiv2023

Functionally and metabolically divergent melanoma-associated macrophages originate from common bone-marrow precursors

Melanomas display high numbers of tumor-associated macrophages (TAMs), which correlate with worse prognosis. Harnessing macrophages for therapeutic purposes has been particularly challenging due to their heterogeneity, based on their ontogeny and function and driven by the tissue-specific niche. In the present study, we used the YUMM1.7 model to better understand melanoma TAM origin and dynamics during tumor progression, with potential therapeutic implications. We identified distinct TAM subsets based on F4/80 expression, with the F4/80high fraction increasing over time and displaying tissue-resident-like phenotype. While skin-resident macrophages showed mixed on-togeny, F4/80+ TAM subsets in i.d. YUMM1.7 tumors originated almost exclusively from bone-marrow precursors. Mul-tiparametric analysis of macrophage phenotype showed a temporal divergence of F4/80+ TAM subpopulations, which also differed from skin-resident subsets, and from their monocytic precursors. Overall, F4/80+ TAMs displayed co-ex-pression of M1- and M2-like canonical markers, while RNA-seq and pathway analysis showed differential immunosup-pressive and metabolic profiles. GSEA showed F4/80high TAMs to rely on oxidative phosphorylation, with increased proliferation and protein secretion while F4/80low cells had high pro-inflammatory and intracellular signaling pathways, with lipid and polyamine metabolism. Overall, the present in-depth characterization provides further evidence of the ontogeny of the evolving melanoma TAMs, whose gene expression profiles matched recently-identified TAM clusters in other tumor models and human cancers. These findings provide evidence for potentially targeting specific immunosup-pressive TAMs in advanced tumor stages.

immunology↗

bioRxiv2023

Apoptosis recognition receptors regulate skin tissue repair in mice

Apoptosis and clearance of apoptotic cells via efferocytosis are evolutionarily conserved processes that drive tissue repair. However, the mechanisms by which recognition and clearance of apoptotic cells regulate repair are not fully understood. Here, we use single-cell RNA sequencing to provide a map of the cellular dynamics during early inflammation in mouse skin wounds. We find that apoptotic pathways and efferocytosis receptors are elevated in fibroblasts and immune cells, including resident Lyve1+ macrophages, during inflammation. Interestingly, human diabetic foot wounds upregulate mRNAs for apoptotic genes and display increased and altered efferocytosis signaling via the receptor Axl. During early inflammation in mouse wounds, we detect upregulation of Axl in dendritic cells and fibroblasts via TLR3-independent mechanisms. Inhibition studies in vivo in mice reveal that Axl signaling is required for wound repair but is dispensable for efferocytosis. By contrast, inhibition of another efferocytosis receptor, Timd4, in mouse wounds decreases efferocytosis and abrogates wound repair. These data highlight the distinct mechanisms by which apoptotic cell detection coordinates tissue repair and provides potential therapeutic targets for chronic wounds in diabetic patients.

cell biology↗

bioRxiv2022

A transcriptional cycling model recapitulates chromatin-dependent features of noisy inducible transcription

Activation of gene expression in response to environmental cues results in substantial phenotypic heterogeneity between cells that can impact a wide range of outcomes including differentiation, viral activation, and drug resistance. An important source of gene expression noise is transcriptional bursting, or the observation that transcripts are produced during infrequent bursts of promoter activity. Chromatin accessibility, which regulates assembly of polymerase complexes on promoters, impacts transcriptional bursting, suggesting that how an activating signal affects transcriptional noise will depend on the initial chromatin state at the promoter. To explore this possibility, we simulated transcriptional activation using a transcriptional cycling model with three promoter states that represent chromatin remodeling, polymerase binding and pause release. We initiated this model over a large parameter range representing target genes with different chromatin environments, and found that, upon increasing the polymerase pause release rate to activate transcription, changes in gene expression noise varied significantly across initial promoter states. This model captured phenotypic differences in activation of latent HIV viruses integrated at different chromatin locations and mediated by the transcription factor NF-{kappa}B. Activating transcription in the model via increasing one or more of the transcript production rates, as occurs following NF-{kappa}B activation, reproduced experimentally measured transcript distributions for four different latent HIV viruses, as well as the bimodal pattern of HIV protein expression that leads to a subset of reactivated virus. Importantly, the parameter activation path differentially affected gene expression noise, and ultimately viral activation, in line with experimental observations. This work demonstrates how upstream signaling pathways can be connected to biological processes that underlie transcriptional bursting, resulting in target gene-specific noise profiles following stimulation of a single upstream pathway. Author SummaryMany genes are transcribed in infrequent bursts of mRNA production through a process called transcriptional bursting, which contributes to variability in responses between cells. Heterogeneity in cell responses can have important biological impacts, such as whether a cell supports viral replication or responds to a drug, and thus there is an effort to describe this process with mathematical models to predict biological outcomes. Previous models described bursting as a transition between an "OFF" state or an "ON" state, an elegant and simple mathematical representation of complex molecular mechanisms, but one which failed to capture how upstream activation signals affected bursting. To address this, we added an additional promoter state to better reflect biological mechanisms underlying bursting. By fitting this model to variable activation of quiescent HIV infections in T cells, we showed that our model more accurately described viral expression variability across cells in response to an upstream stimulus. Our work highlights how mathematical models can be further developed to understand complex biological mechanisms and suggests ways to connect transcriptional bursting to upstream activation pathways.

systems biology↗

bioRxiv2020

TNF stimulation primarily modulates transcriptional burst size of NF-κB-regulated genes

Cell-to-cell heterogeneity is a characteristic feature of the tumor necrosis factor (TNF)-stimulated inflammatory response mediated by the transcription factor NF-{kappa}B, motivating an exploration of the underlying sources of this noise. Here we combined single-transcript measurements with computational models to study transcriptional noise at six NF-{kappa}B-regulated inflammatory genes. In the basal state, NF-{kappa}B-target genes displayed an inverse correlation between mean and noise. TNF stimulation increased transcription while maintaining noise, except for the most repressed genes. By fitting transcript distributions to a two-state model of promoter activity, we found that TNF primarily stimulated transcription by increasing burst size while maintaining burst frequency. Burst size increases were associated with enrichment of initiated-but-paused RNA polymerase II at the promoter, and blocking the release of paused RNAPII with a small molecule inhibitor decreased TNF-stimulated burst size. Finally, we used a mathematical model to show that TNF positive feedback further amplified gene expression noise resulting from burst-size mediated transcription, leading to diverse TNF functional outputs. Our results reveal potential sources of noise underlying intercellular heterogeneity in the TNF-mediated inflammatory response.

systems biology↗

bioRxiv2020

Single-cell secretion analysis reveals a dual role for IL-10 in restraining and resolving the TLR4-induced inflammatory response

Following TLR4 stimulation of macrophages, negative feedback mediated by the anti-inflammatory cytokine IL-10 limits the inflammatory response. However, extensive cell-to-cell variability in TLR4-stimulated cytokine secretion raises questions about how negative feedback is robustly implemented. To explore this, we characterized the TLR4-stimulated secretion program in primary murine macrophages using a single-cell microwell assay that enabled evaluation of functional autocrine IL-10 signaling. High-dimensional analysis of single-cell data revealed three distinct tiers of TLR4-induced proinflammatory activation based on levels of cytokine secretion. Surprisingly, while IL-10 inhibits TLR4-induced activation in the highest tier, it also contributes to the TLR4-induced activation threshold by regulating which cells transition from non-secreting to secreting states. This role for IL-10 in restraining TLR4 inflammatory activation is largely mediated by intermediate IFN-{beta} signaling, while TNF-a likely mediates response resolution by IL-10. Thus, cell-to-cell variability in cytokine regulatory motifs provides a means to tailor the TLR4-induced inflammatory response.

immunology↗

bioRxiv2020

Co-stimulation with opposing macrophage polarization cues leads to orthogonal secretion programs in individual cells

Macrophages are innate immune cells that contribute to fighting infections, tissue repair, and maintaining tissue homeostasis. To enable such functional diversity, macrophages resolve potentially conflicting cues in the microenvironment via mechanisms that remain unclear. Here, we used single-cell RNA sequencing to explore how individual macrophages respond when co-stimulated with the inflammatory stimuli, LPS+IFN-{gamma}, and the resolving cytokine, IL-4. We found that co-stimulated macrophages displayed a distinct global transcriptional program. However, variable negative cross-regulation between some LPS+IFN-{gamma}- and IL-4-specific genes resulted in significant cell-to-cell heterogeneity in transcription. Interestingly, negative cross-regulation led to mutually exclusive expression of the T-cell-polarizing cytokines Il6 and Il12b versus the IL-4-associated factors Arg1 and Chil3 in single co-stimulated macrophages, and single-cell secretion measurements showed that these specialized functions were maintained for at least 48 hours. Overall, our study suggests that increasing functional diversity in the population is one strategy macrophages use to respond to conflicting environmental cues.

immunology↗

bioRxiv2018

Systems analysis by mass cytometry identifies susceptibility of latent HIV-infected T cells to targeting of p38 and mTOR pathways

Efforts to cure HIV are hindered by viral persistence in latently infected memory CD4+ T cells. Targeting T cell death pathways dysregulated by HIV infection offers a novel approach for eradication of the latent reservoir. To identify potential therapeutic targets, we compared signaling and apoptosis in uninfected and latently infected primary cultured CD4+ central memory T cells by mass cytometry following T cell receptor stimulation. We found that HIV-infected cells were sensitized to activation of pro-apoptotic p38 kinase signaling via p53, and to inhibition of anti-apoptotic mTOR kinase signaling, even without HIV protein expression. Simultaneous targeting of p38 and mTOR kinases in resting CD4+ T cells from virally-suppressed HIV+ patients ex vivo reduced cell-associated HIV RNA and DNA. Our results demonstrate how systems biology approaches are useful for identifying novel therapeutic approaches to treat HIV latency, and further suggest that it may be possible to deplete latent HIV-infected T cells without viral reactivation.

systems biology↗

bioRxiv2018

Fold-change detection of NF-κB at target genes with different transcript outputs

The transcription factor NF-{kappa}B promotes inflammatory and stress-responsive gene transcription across a range of cell types in response to the cytokine tumor necrosis factor- (TNF). Although NF-{kappa}B signaling exhibits significant variability across single cells, some target genes exhibit fold-change detection of NF-{kappa}B, which may buffer against stochastic variation in signaling molecules. However, this observation was made at target genes supporting high levels of TNF-inducible transcription. It is unknown if fold-change detection is maintained at NF-{kappa}B target genes with low levels of TNF-inducible transcription, for which stochastic promoter events may be more pronounced. Here we used a microfluidic cell-trapping device to measure how TNF-induced activation of NF-{kappa}B controls transcription in single Jurkat T cells at the promoters of integrated HIV and the endogenous cytokine gene IL6, which produce only a few transcripts per cell. We tracked TNF-stimulated NF-{kappa}B RelA nuclear translocation by live-cell imaging and then quantified transcript number by RNA FISH in the same cell. We found that TNF-induced transcription correlates with fold change in nuclear NF-{kappa}B with similar strength at low versus high abundance target genes. A computational model of TNF-NF-{kappa}B signaling, which implements fold-change detection from competition for binding to {kappa}B motifs, was sufficient to reproduce fold-change detection across the experimentally measured range of transcript outputs. Nevertheless, we found that gene-specific trends in transcriptional noise and levels of promoter-bound NF-{kappa}B predicted by the model were inconsistent with our experimental observations at low abundance gene targets. Our results reveal a gap in our understanding of RelA-mediated transcription for low abundance transcripts and suggest that cells use additional biological mechanisms to maintain robustness of NF-{kappa}B fold-change detection while tuning transcriptional output.

systems biology↗