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

Redondo Marin, J. A.; Nguyen, T. C.; Alachkar, N.; Moran, J.; Srivastav, A.; Kochanczyk, M.; Muldoon, M.; Roberts, I.; Krawczyk-Balska, A.; Singh, A.; Paszek, P.

doi:10.64898/2026.06.17.732820

bioRxiv · 10.64898/2026.06.17.732820

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

Redondo Marin, J. A.·Nguyen, T. C.·Alachkar, N.·Moran, J.·Srivastav, A.·Kochanczyk, M.·Muldoon, M.·Roberts, I.·Krawczyk-Balska, A.·Singh, A.·Paszek, P.

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Abstract

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

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Redondo Marin, J. A., Nguyen, T. C., Alachkar, N., Moran, J., Srivastav, A., Kochanczyk, M., Muldoon, M., Roberts, I., Krawczyk-Balska, A., Singh, A., Paszek, P.. 2026-06-19. Heritable single-cell gene expression states shape functional variability in innate immune responses. https://doi.org/10.64898/2026.06.17.732820

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