Muldoon, M. | bioRxiv Science

bioRxiv2026

Heritable single-cell gene expression states shape functional variability in innate immune responses

Activation of innate immunity at the single-cell level is inherently heterogeneous, yet the mechanisms underlying this variability remain incompletely understood. Here, we integrate transcriptomics, high-content imaging and mathematical modelling to quantify transcriptional heritability within the evolutionarily conserved Toll-like receptor (TLR) system. RNA-seq-based fluctuation tests identified a subset of TLR4-dependent genes, including cytokines and immune effectors, that retain transcriptional heritability for more than 25 cell divisions in clonal macrophage populations. High-content microscopy confirmed gene-specific propagation of heritable states and revealed that environmental context shapes their persistence and expression. CD36, a scavenger receptor involved in bacterial recognition and lipid uptake, exhibited a stable, cell density-reinforced heritable state, whereas the inflammatory programme exemplified by IL1{beta} was transient, with heritability decaying upon clonal expansion. The interplay between heritable transcriptional states and population context generated emergent spatial organisation in high-density populations, with CD36-high cells forming discrete pockets and IL1{beta}-high cells enriched in surrounding regions. Functionally, CD36 expression determined clonal susceptibility to Listeria monocytogenes infection, linking transcriptional heritability to heterogeneous infection outcomes. Together, these findings identify transcriptional heritability as a key determinant of innate immune heterogeneity and demonstrate how heritable cellular states interact with population context to generate complex immune behaviours. Key point summaryO_LIMemorySeq and scRNA-seq identify long-term heritable gene expression states within TLR4-induced macrophage populations. C_LIO_LIHigh-content imaging across thousands of clonal populations reveals gene-specific dynamics of heritable states at the protein level. C_LIO_LIIl1{beta} and Cd36 define mutually exclusive heritable states that are differently regulated by population context and drive spatial organisation in cellular monolayers. C_LIO_LIHeritable CD36 protein expression shapes heterogeneous outcomes during Listeria monocytogenes infection. C_LI

immunology↗

bioRxiv2023

Variability of the innate immune response is globally constrained by transcriptional bursting

Transcription of almost all mammalian genes occurs in stochastic bursts, however the fundamental control mechanisms that allow appropriate single-cell responses remain unresolved. Here we utilise single cell genomics data and stochastic models of transcription to perform global analysis of the toll-like receptor (TLR)-induced gene expression variability. Based on analysis of more than 2000 TLR-response genes across multiple experimental conditions we demonstrate that the single-cell, gene-by-gene expression variability can be empirically described by a linear function of the population mean. We show that response heterogeneity of individual genes can be characterised by the slope of the mean-variance line, which captures how cells respond to stimulus and provides insight into evolutionary differences between species. We further demonstrate that linear relationships theoretically determine the underlying transcriptional bursting kinetics, revealing different regulatory modes of TLR response heterogeneity. Stochastic modelling of temporal scRNA-seq count distributions demonstrates that increased response variability is associated with larger and more frequent transcriptional bursts, which emerge via increased complexity of transcriptional regulatory networks between genes and different species. Overall, we provide a methodology relying on inference of empirical mean-variance relationships from single cell data and new insights into control of innate immune response variability.

immunology↗