Circadian Clock Programming of Anticipatory Antiviral Immunity Gates Enteric Virus Infection Susceptibility

Background

Vulnerability to viral infections can vary widely across individuals, and several factors could contribute to these differences. The circadian clock is a powerful biological system that governs the expression of certain genes in an oscillatory rhythmic fashion following the light-dark cycle, ultimately impacting physiological processes, metabolism and disease manifestations (1). Past studies have explored the impact of the circadian clock on respiratory and systemic viral-induced infections. These revealed that circadian rhythms can influence the intensity and duration of post-infection responses (2,3). In contrast, the impact of circadian rhythm on antiviral programs, and how this translates to infection outcome, remains poorly understood.

Using a model of murine coxsackievirus infection, the authors of this preprint sought to determine whether the circadian clock is involved in the local, temporal regulation of host antiviral defense within the gut.

Key Findings

The circadian clock governs the replication and transmission efficiencies of enteric virus

First, the authors established that the circadian clock contributes profoundly to the replication and transmission capacities of enteric viral infection. Using a murine coxsackievirus B3 (CVB3) infection model, the authors observed that mice had significantly higher viral loads especially when infections in the gut occurred during the dark phase of the Zeitgeber Time. The mice were, however, greatly protected if the infections occurred during the early light phase instead (Figure 1A-B). This diurnal oscillation of viral load required an intact circadian clock, as the absence of core circadian transcription factor, CLOCK, completely abolished the phenomenon.

The circadian clock controls the expression of antiviral regulator IRF1

The authors showed for the first time that the circadian clock-regulated Interferon Regulatory Factor 1 (IRF1) was directly responsible for enteric antiviral protection. Since early light phase infection with the CVB3 virus resulted in significant antiviral protection in these mice, the authors investigated if the expression of genes involved in antiviral immunity was altered. Interestingly, IRF1 expression uniquely exhibited a diurnal oscillation, similar to BMAL1, a second vital circadian clock transcription factor.

IRF1 is a transcription factor that regulates the expression of host sensors and restriction factors that collectively serve as early antiviral defences (4). The authors elegantly showed through a series of in vitro luciferase reporter and immunoprecipitation assays, that IRF1 expression was directly regulated by CLOCK and BMAL1 (Figure 2H-J), and that high expression of these genes corresponded with antiviral protections conferred during the early light phase. Furthermore, genetic deletion of IRF1 in mice completely abolished temporal variations of CVB3 replication, and reduced expression of antiviral genes without affecting the circadian system (Figure 3A-B).

Enteric antiviral protection is driven by a myeloid cell-intrinsic IRF1 program

The authors also showed that gut myeloid cells were the key mediators of IRF1-driven antiviral immunity against enteric viral infection. The Irf1 gene was more highly expressed within the lamina propria, which is enriched in myeloid cells, and transient deletion of colony-stimulating factor 1 receptor – positive (CSF1r⁺) myeloid cells greatly reduced Irf1 expression. Furthermore, myeloid cell–specific loss of Irf1 abolished the observed antiviral effect against CVB3 infection, confirming the role of gut myeloid cells in the lamina propria acting as a barrier against CVB3 replication and infection via IRF1.

Why is this preprint important?

This preprint elegantly demonstrates that the timing of infection is a critical determinant of viral pathogenesis, influencing not only host responses following infection but also the effectiveness of antiviral immunity. Notably, the authors are the first to provide evidence, in a mammalian system, for the direct role of the circadian clock in regulating some intrinsic antiviral defense programs. Circadian rhythms shape the host’s susceptibility to early viral infection and replication even before pathogen exposure, positioning the circadian system as a temporal guardian providing antiviral immunity. These results significantly advance our understanding of host–virus interactions and underscore the importance of considering biological time as a key factor in assessing infectious disease susceptibility and outcome.

References

1. Bell-Pedersen, V. M. Cassone, D. J. Earnest, S. S. Golden, P. E. Hardin, T. L. Thomas, M. J. Zoran, Circadian rhythms from multiple oscillators: lessons from diverse organisms. Nature Reviews Genetics 6, 544-556 (2005).
2. A. Ehlers, W. Xie, E. Agapov, S. Brown, D. Steinberg, R. Tidwell, G. Sajol, R. Schutz, R. Weaver, H. Yu, BMAL1 links the circadian clock to viral airway pathology and asthma phenotypes. Mucosal immunology 11, 97-111 (2018).
3. R. S. Edgar, A. Stangherlin, A. D. Nagy, M. P. Nicoll, S. Efstathiou, J. S. O’Neill, A. B. Reddy, Cell autonomous regulation of herpes and influenza virus infection by the circadian clock. Proceedings of the National Academy of Sciences 113, 10085-10090 (2016).
4. Zhou, H., Tang, Y.-D., & Zheng, C. (2022). Revisiting IRF1-mediated antiviral innate immunity. Cytokine & Growth Factor Reviews, 64, 1–6. https://doi.org/10.1016/j.cytogfr.2022.01.004

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