Altered circadian expression of cytokines and cytolytic factors in splenic natural killer cells of Per1(-/-) mutant mice

Abstract

Circadian systems regulate the immune system by various molecular and physiological pathways. Disruption to the circadian temporality of these pathways is associated with disease formation and progression. Circadian clock genes have been shown to regulate pathways involved in cellular proliferation, apoptosis, and DNA damage response, as aberrant rhythms in these genes are associated with various diseases. However, there is growing evidence that specific circadian genes differentially regulate functional pathways of immunocompetent cells. To extend our previous findings of the role of Period 2 in regulating splenocyte rhythms, we report that mice carrying a mutation in the Period 1 gene (Per1(-/-) mice), involved in the negative limb of the molecular clock, display significantly altered rhythms of cytokine (eg, interferon-γ) and cytolytic factors (eg, perforin and granzyme B) in splenic natural killer (NK) cells. Altered rhythms of NK cell immune factors were accompanied by changes in circadian expression of circadian clock genes, Bmal1 and Per2. In addition, Per1(-/-) circadian running-wheel activity rhythms remained rhythmic during constant darkness, although with a shortened free-running circadian period, suggesting primary involvement of peripheral molecular clocks. These findings indicate that the Per1 gene through NK cellular clocks modulates immune pathways.

FIG. 1.

Circadian wheel-running activity under entrained and free-running conditions. Both wild-type (WT) (A) and Per1^−/− mice (B) display typical entrainment under a standard 12:12 LD cycle. Per1^−/− mice remain rhythmic under free-running darkness (DD) conditions (B), while displaying a shortened circadian period (B, C). ****P<0.0001, significant difference between groups.

FIG. 2.

A mutation in the Per1 gene alters circadian rhythm gene and protein expression of core circadian genes Per2 (A, B) and Bmal1 (C, D) in natural killer (NK) cells. Male WT and Per1^−/− mice were sacrificed at 4-h intervals across the circadian day in DD. Protein levels were quantified using densitometric analyses by Image J software. Representative immunoblots are shown for each protein across each circadian time (CT). Sine wave fits using linear harmonic regression assumed a 24 h period for both WT and mutant mice (solid and dotted lines, respectively). Lines are superimposed on group means±standard error of the means (SEMs) for each CT. All curve fits are significant (P<0.01). Significant differences determined by Bonferroni post hoc tests between WT and mutant mice at given CT: *P<0.05, **P<0.01, and ***P<0.001.

FIG. 3.

Circadian rhythms of interferon-γ (IFN-γ) gene and protein expression are altered in Per1^−/− mice. Gene rhythms of IFN-γ appear phase delayed (A), whereas protein rhythms are reversed (B) in NK cells due to the Per1 mutation. Protein levels were quantified using densitometric analyses by Image J software. Representative immunoblots are shown for each protein across each CT. Sine wave fits using linear harmonic regression assumed a 24 h period for WT and mutant mice (solid and dotted lines, respectively). Lines are superimposed on group means±SEMs for each CT. All curve fits are significant (P<0.01). Significant differences determined by Bonferroni post hoc tests between WT and mutant mice at given CT: *P<0.05 and **P<0.01.

FIG. 4.

Circadian rhythms of perforin and granzyme B gene and protein expression are altered in Per1^−/− mice. Gene rhythms of perforin appear reversed (A) and perforin protein rhythms are dampened (B) in NK cells due to Per1 mutation. Granzyme B gene rhythms appear phase delayed (C) while protein rhythms are attenuated (D) in NK cells of Per1^−/− mice. Protein levels were quantified using densitometric analyses by Image J software. Representative immunoblots are shown for each protein across each CT. Sine wave fits using linear harmonic regression assumed a 24 h period for WT and mutant mice (solid and dotted lines, respectively). Lines are superimposed on group means±SEMs for each CT. All curve fits are significant (P<0.01). Significant differences determined by Bonferroni post hoc tests between WT and mutant mice at given CT: *P<0.05, **P<0.01, and ***P<0.001.