Frontline Science: Rev-Erbα links blue light with enhanced bacterial clearance and improved survival in murine Klebsiella pneumoniae pneumonia

Abstract

The wavelength of light is a critical determinant of light's capacity to entrain adaptive biological mechanisms, such as enhanced immune surveillance, that precede and prepare us for the active circadian day, a time when the risk of encountering pathogen is highest. Light rich in the shorter wavelength visible blue spectrum maximally entrains these circadian rhythms. We hypothesized that exposure to blue light during sepsis will augment immunity and improve outcome. Using a clinically relevant Klebsiella pneumoniae acute lower respiratory tract infection model, we show that blue spectrum light shifts autonomic tone toward parasympathetic predominance and enhances immune competence, as characterized by accelerated pathogen clearance that is accompanied by reduced alveolar neutrophil influx, inflammation, and improved survival. Blue light functioned through an optic-cholinergic pathway and expansion of splenic Ccr2⁺ monocytes to increase control of the infection and improve survival. The "keystone" mediating these effects is the circadian clock protein Rev-Erbα, and biochemical activation with Rev-Erbα agonist SR9009 enhanced mononuclear cell phagocytosis in vitro and recapitulated the enhanced pathogen elimination in vivo observed with blue light. These findings underscore the potential therapeutic value of blue light and modulating Rev-Erbα to enhance host immunity against infection.

Keywords: circadian; innate immunity; sepsis.

FIGURE 1. The lower wavelength of light reduced the heart rate and the LF/HF parameter of heart rate variability.

Mice underwent laparotomy and implantation of a DSI HD-X11 telemetry device. Each day for the next 5 days, each mouse was exposed to a 12-h photoperiod (12h light:12h dark) of each of the following spectrums of light that were of equal illuminance (∼1400 lux): peak 442, 486, 510, 617, and 720 nm. After 5 days of continuous physiologic monitoring, the data for each spectrum were combined and analyzed. A, Heart rate; B, LF/HF ratio (a parameter of heart rate variability that qualifies the balance between parasympathetic and sympathetic tone).^– Dark horizontal bar indicates circadian night (ZT12 to ZT24). White horizontal bar indicates circadian day (ZT0 to ZT12). Lines represent a fractional polynomial estimate of the mean of each parameter for the entire cohort of mice exposed to each wavelength (n = 5 mice for all experiments combined)

FIGURE 2. Blue light enhances bacterial clearance, reduces bacterial dissemination, and improves survival during Klebsiella pneumoniae pneumonia.

Mice underwent intratracheal inoculation with K. pneumoniae (∼4 × 10³ CFU) and then were exposed to ambient, blue or red light for either a 24-h photoperiod or 36 h followed by a 12-h photoperiod daily for 72 h. Organs and blood were harvested at the times indicated, and total tissue CFUs were quantified. A, lung tissue CFU/mL tissue homogenate; B, blood CFU/mL tissue homogenate; C, liver tissue CFU/mL tissue homogenate; and D, spleen tissue CFU/mL tissue homogenate. (10 to 16 total mice per group for all three experiments combined). Data were analyzed by Wilcoxon rank sum, bar = median. E, In separate experiments, mice underwent intratracheal inoculation with K. pneumoniae (4 × 10³ CFU) and then were exposed to ambient, blue or red light for 36 h followed by a 12-h photoperiod daily for 72 h and then observed for 7 days (n = 12 mice per group). Comparison (blue vs. red) of survival at 7 days was analyzed by log rank test

FIGURE 3. Blue light reduces neutrophilic infiltration and regional inflammation during Klebsiella pneumoniae pneumonia.

A, Mice underwent intratracheal inoculation with K. pneumoniae (∼4 × 10³ CFU) and then were exposed to ambient, blue or red light for 36 h followed by a 12-h photoperiod daily for 72 h. Lungs were harvested at 72 h and histology (H&E) performed. Scale bar, 125 µm. B-J, Mice underwent intratracheal inoculation with K. pneumoniae (∼4 × 10³ CFU) and then were exposed to ambient, blue or red light for either a 24-h photoperiod or 36 h followed by a 12-h photoperiod daily and then observed for 72 h. BAL was performed at the times indicated, and BAL fluid analyzed for B, total cell count; C, neutrophil count; D, myeloperoxidase (MPO) activity; E, mononuclear cell count; F-H, chemokine concentrations; I, J, cytokine concentrations (12–16 total mice per group for all three experiments combined). Data were analyzed by Wilcoxon rank sum, bar = median

FIGURE 4. Blue light functions through an optic pathway to enhance control of the infection and reduce neutrophil influx.

129S1/SvlmJ (WT) or 129S1/Sv-Vsx2^or-J/J optic cup and nerve degeneration (Vsx2^−/−) mice underwent intratracheal inoculation with K. pneumoniae (∼4 × 10³ CFU) and then were exposed to red or blue light for 36 h followed by a 12-h photoperiod daily for 72 h. Organs, blood, and BAL fluid were harvested at 72 h. A-D, Tissue and blood bacterial CFUs per mL of tissue homogenate; E, total cell count; F, neutrophil count; G, MPO activity; H, mononuclear cell count; I-K, chemokine concentrations (seven to eight total mice per group for all two experiments combined). Data were analyzed by Wilcoxon rank sum, bar = median

FIGURE 5. Blue light functions through a cholinergic pathway to enhance control of the infection and reduce neutrophil influx and inflammation.

Mice underwent intratracheal inoculation with K. pneumoniae (∼4 × 10³ CFU), were administered either α-bungarotoxin (1 µg/kg; i.p.) or equivolume normal (0.9%) saline vehicle control, and then were exposed to red or blue light for 36 h followed by a 12-h photoperiod daily for 72 h. Organs, blood, and BAL fluid were harvested at 72 h. A-D, Tissue and blood bacterial CFUs per mL of tissue homogenate; E, total cell count; F, neutrophil count; G, MPO activity; H, mononuclear cell count; I-K, chemokine concentrations (seven to eight total mice per group for all two experiments combined). Data were analyzed by Wilcoxon rank sum, bar = median

FIGURE 6. Blue light functions through the spleen to enhance control of the infection, reduce neutrophil influx and inflammation, and improve survival.

Mice underwent splenectomy (Spl-X) or sham operation (Sham), were intratracheally inoculated with K. pneumoniae (∼4 × 10³ CFU) and then exposed to red or blue light for 36 h followed by a 12-h photoperiod daily for 72 h. Organs, blood, and BAL fluid were harvested at 72 h. A-C, Tissue and blood bacterial CFUs per mL of tissue homogenate; D, total cell count; E, neutrophil count; F, MPO activity; G, mononuclear cell count; H-J, chemokine concentrations (seven to eight total mice per group for all two experiments combined). Data were analyzed by Wilcoxon rank sum, bar = median. K, 7-day survival curves (12 total mice per group for all three experiments combined). Comparison of survival at 7 days analyzed by log rank test

FIGURE 7. Blue light requires CCR2 to enhance bacterial clearance and reduce neutrophil influx during K. pneumoniae pneumonia.

C57Bl/6J (WT) or B6.129S4-Ccr2^tm1lfc/J (Ccr2^−/−), mice underwent intratracheal inoculation with K. pneumoniae (∼4 × 10³ CFU) and then were exposed to red or blue light for 36 h followed by a 12-h photoperiod daily for 72 h. Organs, blood, and BAL fluid were harvested at 72 h. A-D, Tissue and blood bacterial CFUs per mL of tissue homogenate; E, total cell count; F, neutrophil count; G, MPO activity; H, macrophage count (five to eight total mice per group for all two experiments combined). I-K, Splenic Ly-6C⁺ and CCR2⁺ monocyte concentrations at 24- and 72-h inoculation with K. pneumoniae and exposure to each light spectrum (four total mice per group). Data were analyzed by Wilcoxon rank sum, bar = median. Gating flow strategy shown in Supplementary Fig. 1

FIGURE 8. Blue light induces Rev-Erbα in lung and spleen tissue and the alveolar macrophage.

A,B Mice underwent intratracheal inoculation with K. pneumoniae (∼4 × 10³ CFU) and then were exposed to blue or red light. At the time points denoted, mice were euthanized and A, lung and B, spleen tissue were harvested. Total cell lysate was analyzed for Rev-Erbα and tubulin expression by immunoblot. Densitometry of Rev-Erbα expression adjusted for tubulin expression was performed (two mice per group for all experiments combined). C, Mice underwent intratracheal inoculation with K. pneumoniae (∼4 × 10³ CFU) and then were exposed to ambient, blue or red light for 36 h followed by a 12-h photoperiod daily for 72 h. After 72 h, the left lung was harvested and fixed with 4% paraformaldehyde and analyzed by immunofluorescence. Rev-Erbα, white. F4/80 macrophage, red. DAPI nuclei, blue. Scale bar, 125 µm (12 mice per group)

FIGURE 9. Augmenting Rev-Erbα enhances macrophage phagocytosis in vitro and enhances bacterial clearance and reduces neutrophil influx and inflammation in vivo.

A, RAW 264 cells were incubated with either SR9009 (10 mM) or equivolume DMSO vehicle and then administered heat killed E. coli labeled with pHrhodo. Phagocytosis was then quantified using the pHrodo Phagocytosis Assay. B-O, Mice were intratracheally inoculated with K. pneumoniae (∼4 × 10³ CFU). Four hours later they were administered either SR9009 (100 mg/kg; i.p.) or equivolume 15% Kollifor vehicle and then daily for 3 days. Organs, blood, and BAL fluid were harvested at 72 h. B-E, Tissue and blood bacterial CFUs per mL of tissue homogenate; F, BAL total cell count; G, BAL neutrophil count; H, BAL MPO activity; I, BAL mononuclear cell count; J-L, BAL chemokine concentrations; M-O, BAL cytokine concentrations (13 total mice per group for all four experiments combined). Data analyzed by Wilcoxon rank sum, bar = median