Effect of external cues on clock-driven protection from influenza A infection

Abstract

Influenza and other respiratory viral pathogens remain leading causes of mortality and morbidity. Circadian rhythms play a critical role in regulating immune responses and can confer temporal protection from influenza infection. Here, we investigated whether this protection requires rhythmic function after the initial infection by manipulating environmental cycles. We found that disrupting environmental lighting cues within a critical window of vulnerability abrogated the time-of-day-specific protection. This poor outcome was mediated by a dysregulated immune response, as evidenced by the accumulation of inflammatory monocytes and CD8+ T cells in the lungs and a transcriptomic profile indicative of an exaggerated inflammation. Disruption of the light cycle did not affect outcomes in a clock mutant, indicating that it acts through the host's circadian clock. Importantly, rhythmic meal timing mitigated the adverse effects of disrupted light cycles, supporting the idea that external cues acting through different body clocks can compensate for one another. Together, these findings underscore the critical interplay between environmental timing cues and endogenous circadian rhythms in determining influenza outcomes and offer translational insight into optimizing care for critically ill patients with respiratory viral infections.

Keywords: Immunology; Inflammation; Influenza; Innate immunity; Pulmonology; T cells.

Figure 1. Constant light exposure abrogated the specific time-of-day protection following influenza A infection.

(A) Experimental model. (B) Survival (**P < 0.01, log-rank test; *P < 0.05, log-rank test from 3 independent experiments). (C) Weight loss trajectory (*P < 0.001, ANOVA for repeated measures; ^#P < 0.001, ANOVA for repeated measures). (D) Average clinical score as a representation of disease progression (*P < 0.001, ANOVA for repeated measures; ^#P < 0.001, ANOVA for repeated measures) following IAV infection (n = 16–36 per group). All data were pooled from 3–5 independent experiments. ZT23 vs. ZT11 comparisons are indicated with an *; ZT23 vs. ZT23(LL) comparisons are indicated with a #.

Figure 2. Exposure to constant light following IAV infection caused exaggerated inflammation independent of viral burden.

(A) Quantification of viral titers (day 6, n = 8–14 per group; day 8, n = 6–10 per group; *P < 0.05, 1-way ANOVA; ***P < 0.0005, 2-way ANOVA with main effects only). (B) Total bronchoalveolar lavage (BAL) on 6 days p.i. (n = 13–15 per group; *P < 0.05, 1-way ANOVA, Kruskal Wallis test). (C) Differential of the total BAL cells (n = 6–13 per group; *P < 0.05, **P < 0.001 2-way ANOVA, mixed effects model). (D) Chemokine levels in BAL on day 6 p.i. (n = 3–8 per group; *P < 0.05, **P < 0.001, 1-way ANOVA). (E) Cytokine levels in BAL on day 6 p.i. (n = 3–7 per group; *P < 0.05, **P < 0.001, ****P < 0.0001, 1-way ANOVA). (F) Absolute number of CD45⁺ cells. (G) Absolute number of CD4⁺ T lymphocytes. (H) Absolute number of CD8⁺ T lymphocytes. (F–H) n = 9–15 per group; *P < 0.05, **P < 0.001, 1-way ANOVA. (I) Absolute number of Ly6C^hi inflammatory monocytes and (J) absolute number of Neutrophils. (K) Absolute number of B cells (n = 4–7 per group *P < 0.05, 1-way ANOVA; Kruskal-Wallis test). All data were pooled from 3–5 independent experiments.

Figure 3. Immunopathology from IAV is worsened by environmental light cycling disruption.

(A) Representative micrographs of H&E-stained lung sections on day 8 p.i. Left: Scale bar: 5 mm (left); 200 μm (right). Original magnification, ×20 (right). (B) Quantification of acute lung injury (n = 12–14 per group; ****P < 0.0001, 1-way ANOVA). (C) Representative micrographs of CD3-stained lung sections on day 8 p.i. Original magnification, ×20; scale bar: 200 μm. (D) Quantification of CD3⁺ cells per HPF (n = 5–7 per group; **P < 0.001, ****P < 0.0001, 1-way ANOVA). (E) Representative micrographs of F4/80-stained lung sections on day 8 p.i. Original magnification, ×20; Scale bar: 200 μm. (F) Quantification of F4/80⁺ cells per HPF (n = 5–7 per group; ****P < 0.0001, 1-way ANOVA). (G) Representative images of pro-SPC-stained lung sections on day 8 p.i. Original magnification, ×40. (H) Quantification of SFTPC⁺ (SPC⁺) alveolar type 2 cells per HPF (n = 5–11 per group; ****P < 0.0001 1-way ANOVA). Scale bar: 100 μm. All data were pooled from 5 independent experiments.

Figure 4. A specific window of vulnerability to light disruption following influenza A infection existed.

(A) Experimental model. (B) Survival (n = 10–21 per group, **P < 0.001 log-rank test). (C) Weight loss trajectory (n = 10–21 per group, *P < 0.05 ANOVA for repeated measures) following IAV infection. (D) Average clinical score (n = 10–21 per group, *P < 0.05 ANOVA for repeated measures). ZT23 vs. ZT11 comparisons are indicated with an *; ZT23(LL) day 7 vs. ZT11 comparisons are indicated with a &. (E) Experimental model. (F) Survival (n = 10–18 per group, **P < 0.001 log-rank test). All data were pooled from 3 independent experiments.

Figure 5. Transcriptomic analyses showed global immune activation in the ZT23 and ZT23(LL) groups.

(A) Schematic of bulk RNA-Seq processing on day 8 p.i. lungs. (B) Heatmaps of all differentially regulated genes. (C) Volcano plots showing upregulated and downregulated genes. In each volcano plot, the horizontal dotted line represents a P_adj = 0.05, and the vertical dotted line represents a log (fold change) >2 or <–2. (D) Plot of log-adjusted fold change for ZT23 vs. ZT23(LL) shows the directionality of the most differentially expressed genes. n = 5 per group, all females. All data were pooled from 3 independent experiments.

Figure 6. Transcriptomic analyses showed global immune activation in the ZT23(LL) and ZT11 LD groups.

(A) Venn diagram (sizes not to scale) depicting the number of differentially expressed genes. (B) Volcano plots showing upregulated and downregulated genes. In each volcano plot, the horizontal dotted line represents a P_adj = 0.05, and the vertical dotted line represents a log (fold change) >2 or <–2. (C) Plot of log-adjusted fold change for overlapping pathways between ZT23 LD vs. ZT23(LL) and ZT23 LD vs. ZT11 LD. (D–H) Relative gene expression of selected genes as a confirmation for bulk RNA-Seq. n = 4–8 per group, *P < 0.05, **P < 0.001, ***P < 0.0005, ****P < 0.0001; 1-way ANOVA. All data were pooled from 3 independent experiments.

Figure 7. Food cycling rescued the loss of time-of-day protection from influenza A following environmental light disruption.

(A) Experimental model. (B) Survival (n = 21–50 per group; **P < 0.01 log-rank test from 3 independent experiments, *P < 0.05 log-rank test from 3 independent experiments). (C) Weight loss trajectory (n = 21–50 per group, *P < 0.05, ANOVA for repeated measures; ^#P < 0.001, ANOVA for repeated measures) following IAV. (D) Average clinical score. (n = 21–50 per group, *P < 0.05 ANOVA for repeated measures) following IAV infection. All data were pooled from 3 independent experiments. ZT23 vs. ZT11 comparisons are indicated with an *; ZT23 vs. ZT23(LL-food cycling) comparisons are indicated with a #.