Circadian clock regulates the host response to Salmonella

Abstract

Organisms adapt to day-night cycles through highly specialized circadian machinery, whose molecular components anticipate and drive changes in organism behavior and metabolism. Although many effectors of the immune system are known to follow daily oscillations, the role of the circadian clock in the immune response to acute infections is not understood. Here we show that the circadian clock modulates the inflammatory response during acute infection with the pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). Mice infected with S. Typhimurium were colonized to higher levels and developed a higher proinflammatory response during the early rest period for mice, compared with other times of the day. We also demonstrate that a functional clock is required for optimal S. Typhimurium colonization and maximal induction of several proinflammatory genes. These findings point to a clock-regulated mechanism of activation of the immune response against an enteric pathogen and may suggest potential therapeutic strategies for chronopharmacologic interventions.

Keywords: clock genes; gastroenteritis; inflammation; intestine; microbes.

Fig. 1.

The inflammatory response is time-of-day–dependent in the cecum of mice infected with S. Typhimurium. (A) Recovery of S. Typhimurium from colon content of WT mice at 48, 60, 72, and 78 h p.i. at different circadian times (day, 10:00 AM or ZT4; night, 10:00 PM or ZT16). Each circle represents an individual animal. Red bars indicate the geometric means (n ≥ 7). (B) Cecal histopathology score of mice from A. Each bar represent the combined score of at least seven mice. PMN, polymorphonuclear leukocytes. (C) Representative images (10× magnification) of cecal inflammation in WT mice infected (Salmonella) or not at day (ZT4) or night (ZT16). (D) mRNA expression of Tnfα, Cxcl-1, Lcn2, and Per2 in the cecum of mice from A–C (n ≥ 7). Data are represented as geometric means of fold increases compared with uninfected WT (48 h) day ± SEM. Significant day–night changes are shown. *P < 0.05; **P < 0.01; ***P < 0.001. (Left) Graphs with yellow and gray areas represent the progression of mRNA expression of Tnfα, Cxcl-1, and Lcn2 depending on the time of infection.

Fig. 2.

Reduced cytokine production from macrophages of Clock mutant mice. (A) Time course of mRNA expression of different cytokines after LPS stimulation of BMDMs. Time 0 (unstimulated cells) in both WT and Clock mutant cells was set to 1. Bars represent mean ± SEM (n = 3). (B) Supernatant protein level of TNF-α and IL-6 from BMDMs of WT and Clock mutant mice after 24 h of LPS stimulation. Bars represent means ± SEM (n = 4). (C) IL-6 production in BMDMs from WT and Clock mutant mice 24 h p.i. with S. Typhimurium WT or with the msbB mutant. MOI, multiplicity of infection. (D) BMDMs from WT and Clock mutant mice were infected with different strains of S. Typhimurium as indicated. After 24 h, supernatants were collected, and secretion of IL-1β was measured. Data represent means ± SEM (n = 3).

Fig. 3.

Altered inflammatory response in the intestine of Clock mutant mice after in vivo infection with S. Typhimurium. (A) Tissue colonization in WT and Clock mutant mice 72 h p.i. with S. Typhimurium. Day, 10:00 AM, ZT4; night, 10:00 PM, ZT16. Each circle represents an individual animal. Red bars indicate the geometric means (n ≥ 8). WT mice are as in Fig. 1A and Fig. S1A. (B) Histopathology of cecum of infected Clock mutant mice from A. Each bar represent the combined score of at least eight mice. PMN, polymorphonuclear leukocytes. (C) Representative images (10× magnification) of the ceca from Clock mutant mice at 72 h p.i. (Salmonella) or mock, at day or night.

Fig. 4.

Microarray analysis from cecum of mice infected with S. Typhimurium reveals a circadian mechanism modulating the response to acute bacterial infection. (A) Heat diagram showing changes in gene expression detected in the ceca of mice 72 h p.i. with S. Typhimurium at day (D, ZT4) or night (N, ZT16) in WT and Clock mutant mice, compared with uninfected controls (n ≥ 3). Representative results from two animals are shown. Relative increase (red) or decrease (green) of mRNA level is shown. A list of the most represented subcategories of genes from each cluster, the number of genes included in each subcategory, and the relative P value are shown. (B and C) Transcriptional profiles of selected proinflammatory/antimicrobial genes identified in cluster 1. Significant changes are shown. *P < 0.05; **P < 0.01; ***P < 0.001. (D) Network of transcription factors involved in regulation of subsets of genes included in cluster 1. Significant changes (P < 0.05) are shown as colored circles (blue, WT infected vs. uninfected day; green, WT infected vs. uninfected night; brown, Clock mutant infected vs. uninfected day; orange, Clock mutant infected vs. uninfected night). (E) Competitive infection with a mixture of S. Typhimurium WT and iroN mutant at two different times of the day (day, ZT4; night, ZT16). Bars indicate the average competitive index of bacteria recovered from colon contents. Data represent geometric means ± SEM (n = 10 each group).