Molecular clock gene regulates immune response in mice

Circadian rhythms – biological processes that oscillate regularly about every 24 hours – are widespread in animals, plants, fungi and cyanobacteria. They are regulated by clock genes, which, in turn, are highly influenced by daylight. They govern sleep patterns and daily fluctuations in core body temperature, brain wave activity, hormone production, cell division and other physiological and behavioral processes. Increasing evidence suggests that disrupting circadian rhythms compromises the immune response.

In 2012, a Yale University School of Medicine research team led by Dr. Erol Fikrig found that deficiency for the molecular clock period homolog 2 (Per2) gene in the B6.Cg-Per2^tm1Brd Tyr^c-Brd/J (003819) mouse disrupts the expression and function of Toll-like receptor 9 (Tlr9), an immune cell surface receptor that mediates immune responses (Silver et al. 2012). Their findings highlight the importance of regular sleep patterns and imply that exploiting the circadian clock's influences on Tlr9 may improve the efficacy of immunomodulatory therapies.

The discovery of evolutionarily conserved proteins that mobilize the immune system when they detect conserved pathogen-associated molecular patterns (PAMPs) is one of the most important in the field of immunology. One of these proteins, Tlr9, is highly expressed in antigen presenting cells – macrophages, B cells, and dendritic cells. It is unique in that it can detect not only PAMPs from bacteria and viruses but also synthetic CpG oligodeoxynucleotides (CpG ODNs). The Fikrig team found that, in mice, circadian rhythms influence Tlr9 expression and Tlr9-mediated innate and adaptive immune responses, and can impact vaccine efficacy:

Circadian rhythms modulate Tlr9 expression

• Splenic Tlr9 mRNA expression in wild-type C57BL/6J (B6J, 000664) mice peaks at night.
• Peritoneal macrophages in B6J mice express Tlr9 and key clock genes Per2 and Nr1d1 in a strong circadian pattern following a serum shock which triggers circadian oscillations in gene expression. In contrast, Tlr9 expression levels in Per2-deficient mice are significantly lower and are less responsive to serum shock.
• Even in complete darkness, Tlr9 expression in B6J spleens, macrophages and B cells oscillates in a daily rhythm.
• The CLOCK:BMAL1 heterodimer, the core circadian transcription factor, binds to the Tlr9 promoter, indicating that it regulates Tlr9 expression.
• B6J (but not Per2-deficient) mice challenged with CpG ODNs respond by expressing splenic Tnfa, Mcp1, Cd80, and Cd86 in a strong circadian pattern.

Circadian rhythms modulate Tlr9-dependent immune response

• TLR9-mediated responses are restricted to specific immune cell types – macrophages and B cells, but not dendritic cells.
• When challenged with TLR9-specific CpG ODNs, Per2-deficient peritoneal macrophages produce significantly less Tnfa and Il12 than wild-type B6J macrophages.
• When challenged with PAMPs other than CpG ODNs, Per2-deficient and B6J peritoneal macrophages produce comparable amounts of cytokines, indicating that Per2-deficiency affects only TLR9-mediated immune responses.
• B6J mice subjected to cecal ligation and puncture (CLP) – a sepsis model – when Tlr9 expression peaks develop a more severe sepsis than mice subjected to CLP when Tlr9 expression is at its lowest, suggesting that daily variations in Tlr9 expression and function affect the progression and severity of sepsis.

Circadian rhythms influence vaccine efficacy

• Lymphocytes from B6J mice immunized with ovalbumin (OVA) and a Tlr9-adjuvant at peak Tlr9 expression produce substantially more immune response cells and interferon-g than those from mice immunized when Tlr9 expression is at its lowest, suggesting that a Tlr9-adjuvanted vaccine is more efficacious if administered when Tlr9 expression peaks.

In summary, the Fikrig team demonstrated that, in mice, circadian rhythms affect expression of the pattern recognition receptor Tlr9 and Tlr9-dependent immune responses. The team also found that the timing of immunization using Tlr9-adjuvanted vaccines can affect efficacy. These findings imply that any disruptions in a person's circadian rhythms can compromise the immune response. Examples of such disturbances include working night shifts, irregular sleep patterns, and noises, lights, and medication regimens that interrupt a hospitalized person's sleep. The findings also suggest that the time at which immunomodulatory therapies are administered may influence the efficacy of those therapies for a number of diseases, including sepsis and cancer.

Molecular clock strains available from The Jackson Laboratory:

• B6.Cg-Per2^tm1Brd Tyrc-^Brd/J (003819), the albino congenic Per2 knockout on a B6J background used in this study
• B6.129S6-Per2^tm1Jt/J (006852), a congenic Per2:: Luciferase reporter on a B6J background
• 129S-Per2^tm1Drw/J (010832), a Per2 targeted mutant on the 129 background
• B6.129-Per2^tm1Drw/J (010492), a congenic Per2 targeted mutant on the B6J background
• B6.129S4-Clock^tm1.1Rep/J (010925), a congenic Clock gene knockout on a B6J background 
• C.B6-Clock^m1Jt/J (016175), a congenic Clock gene ENU mutant on the BALB/cJ (000651) background
• B6.Cg-Nr1d1^tm1Ven/LazJ (018447), a congenic Nr1d1 knockout/reporter on a B6J background (under development, accepting orders)