This protocol describes a technique using mouse splenocytes to discover pathogen-associated molecular patterns that alter molecular clock gene expression.
From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse splenocytes with the pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS), ODN1826, and heat-killed Listeria monocytogenes and examine their effect on the molecular circadian clock. Previously, studies have focused on examining the influence of LPS on the molecular clock using a variety of in vivo and ex vivo approaches from an assortment of models (e.g., mouse, rat, and human). This protocol describes the isolation and challenge of splenocytes, as well as the methodology to assess clock gene expression post-challenge via quantitative PCR. This approach can be used to assess not only the influence of microbial components on the molecular clock but other molecules as well that may alter expression of the clock. This approach could be utilized to tease apart the molecular mechanism of how PAMP-Toll-like receptor interaction influences clock expression.
The master clock in mammals, which orchestrates 24 h oscillations for nearly all aspects of physiology and behavior, is located within the suprachiasmatic nucleus (SCN) of the hypothalamus1,2. In addition to regulating biological processes on an organismal level, the master clock also synchronizes peripheral cellular clocks throughout the body3,4,5. While the molecular clock machinery consists of at least three interlocking transcriptional-translational feedback loops, the core is comprised of the Period (Per1-3), Cryptochrome (Cry1-2), Bmal1, and Clock genes6,7. Besides maintaining the accurate timing of the core molecular clock, some ancillary clock gene products (e.g., Rev-erbα and Dbp) also regulate expression of non-clock genes, i.e., clock controlled genes (CCGs)6,7.
Functional molecular clocks have been described in various immune tissues (e.g., spleen and lymph nodes)8 and cells (e.g., B cells, dendritic cells, macrophages)8,9. These cells detect and respond to pathogen-associated molecular patterns (PAMPs), conserved microbial components, via innate immune recognition receptors such as Toll-like receptors (TLRs)10. To date, 13 functional TLRs have been described, which recognize microbial constituents such as bacterial cell wall components, flagellar protein, and microbial nucleic acids10. The PAMP, lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria recognized by TLR4, has been shown to alter circadian rhythms at the both organismal and molecular levels. For example, in vivo challenge of LPS induced photic-like phase delays as measured by activity in mice11 and led to reduced clock gene expression in the SCN and liver as determined by in situ hybridization and quantitative PCR, respectively, in rats12. After an in vivo challenge with LPS, analysis of human peripheral blood leukocytes13 and subcutaneous adipose tissue14 revealed altered expression of several clock genes as measured via qPCR. Lastly, ex vivo LPS challenges of human macrophages and mouse peritoneal macrophages, also led to altered clock expression as measured by qPCR14.
Here, we describe a protocol to assess the influence of the PAMPs LPS, ODN1826 (synthetic oligonucleotides containing unmethylated CpG motifs), and heat-killed Listeria monocytogenes (HKLM), recognized by TLR4, TLR9, and TLR2, respectively, on molecular clock gene expression in mouse splenocytes. The protocol includes mouse splenectomy, splenocyte isolation and challenge, RNA extraction, cDNA synthesis, and qPCR to assess expression of several clock genes. This protocol allows for the timely acquisition of a large number of immune cells with very little animal or cellular manipulation, which can then be challenged ex vivo with various PAMPs. The molecular clock has been shown to modulate various aspects of the immune response8,15,16, therefore, disruption of the molecular clock would most likely impair the proper time-dependent variation of the immune response. In addition, since disruptions of circadian rhythms can lead to serious pathologies17,18,19,20, it may be of interest for researchers to challenge splenocytes with a wide range of molecules and assess their influence on the clock.
During the study, animal care and treatment complied with National Institutes of Health policy, were in accordance with institutional guidelines, and were approved by the University of Hartford Animal Institutional Animal Care and Use Committee.
1. Entrainment of Animals
NOTE: Twenty week-old male B6129SF2/J mice are used in the study.
2. Preparation of Instruments, Culture Medium, and Challenge Medium
3. Splenocyte Isolation and Challenge
4. RNA Isolation and cDNA Synthesis
5. Quantitative Polymerase Chain Reaction (qPCR)
6. Statistical Analysis
Mice were sacrificed at ZT13, splenocytes were isolated and challenged ex vivo with the PAMPs LPS, ODN1826, or HKLM. After 3 h, RNA was isolated, and qPCR was used to assess relative expression levels of the molecular clock genes Clock, Per2, Dbp, and Rev-erbα compared to unchallenged control cells. After PAMP challenge, Clock expression levels were not significantly different than expression in the control cells (Figure 1A). Per2 expression levels were significantly elevated in cells challenged with LPS and ODN1826 when compared to unchallenged controls (Figure 1B). LPS was the only PAMP to alter Rev-erbα expression, as mRNA levels were significantly lower than in the unchallenged controls (Figure 1C). Lastly, significantly lower mRNA levels were observed for Dbp after challenge with each of the PAMPs when compared to the controls (Figure 1D). Consistent with what has been previously shown, out of all the clock associated genes examined, Dbp expression tends to be most affected by PAMP challenge23.
Figure 1: Altered clock gene expression in mouse splenocytes after ex vivo PAMP challenge. Splenocytes were isolated at ZT13 and challenged with LPS, ODN1826, or HKLM. Relative clock mRNA levels (normalized to β-actin) were determined by qPCR 3 h after challenge. Each data point represents expression level for 1 animal. Experimental mean + SEM are given. *p <0.05, **p <0.01, ***p <0.001. The indicated challenges were significantly different from the control (unchallenged cells) as per one-way ANOVA with the Dunnett's post hoc test. Please click here to view a larger version of this figure.
Within this protocol, a microvolume spectrophotometer can be used to quantify and assess the purity of the RNA being used in determining gene expression. Nucleic acids absorb UV light at 260 nm, proteins typically absorb light at 280 nm, while other potential contaminants used during an RNA extraction procedure (e.g., phenol) are detectable at 230 nm. Therefore, by assessing the absorbance (A) ratio at 260/280 nm (RNA to protein) and 260/230 nm (RNA to non-protein contaminants) the quality of the RNA can be assessed. High quality RNA has an A260/280 ratio between 1.8–2.1, as lower ratios indicate protein contamination. A pure RNA sample will have an A260/230 ratio of 2.0.
When determining the relative expression of a target gene (i.e., Per2, Clock, Rev-erbα, and Dbp), an endogenous control gene (a gene in which expression levels do not differ between samples) must also be selected. Relative expression of the target gene is then normalized to the expression of the endogenous control gene. Differences in starting material (number of splenocytes), variation in reverse-transcriptase efficiency, varying rates of RNA degradation, etc., will be corrected for by the endogenous control gene (β-actin in this protocol). However, it is wise to verify that the treatment being examined does not alter expression of the endogenous control gene. This can be accomplished by assessing β-actin levels from several replicates of an equal number of cells (treatment vs. non-treatment). In theory, their β-actin levels should be identical. Another approach to guard against endogenous control variation would be to use a panel of endogenous controls (e.g., β-actin, Gapdh, and 18S rRNA gene).
When examining the impact of PAMPs on the molecular clock, the time of day when mice are euthanized and splenocytes are subsequently challenged must be taken into consideration. Tlr expression and responsiveness has previously been shown to demonstrate time-of-day dependent variation8,15, therefore, a time of day when TLR responsiveness is at its peak, could result in a greater influence on the clock. Furthermore, expression of molecular clock genes will also fluctuate throughout the day in splenocytes, therefore, a reduction of clock gene expression due to PAMP challenge would be most significant if examined during the time of peak expression9. Since Dbp and Rev-erbα have been shown to demonstrate significant expression peaks in splenocytes and splenic immune cells around the light-dark interphase 8,9,23,24 (ZT12), in the current method, cells were isolated and challenged at ZT13 in order to have a greater chance at detecting a reduction in these genes. Conversely, a PAMP that could increase clock expression, would most likely be observed if looking at a time of day when clock expression is at its lowest.
Since mice are nocturnal animals, their rest phase is during the light period, which corresponds to human activity. Therefore, ideally, mice will be housed in a room with minimal traffic and one that contains a white-noise machine in order to reduce daytime disturbances as this could disrupt the circadian rhythms of the mice. Furthermore, cage changes should be done well in advance of the experiment date. Any work in the animal room (including euthanasia of the animals) during the dark period, should be conducted under red light in order to avoid disrupting the rhythms of the mice.
Diurnal rhythms are subjected to environmental stimuli (e.g., light or food), which are termed zeitgebers. In the case of a 12 h light/12 h dark cycle, the zeitgeber (i.e., light) resets the clock to a 24 h period. While most diurnal rhythms are circadian (i.e., daily rhythms that occur in the absence of an external cue), they are not true circadian rhythms until they have been shown to oscillate with an approximate 24 h period under constant environmental conditions. Therefore, this procedure could be performed using mice under constant conditions, which would entail entraining mice to the light-dark cycle as described above, but then holding the animals in constant darkness for 3 days prior to sampling. This type of experiment is referred to as a dark-dark (DD) experiment and the time point of sampling would be referred to as CT (circadian time), not ZT.
While this method can identify PAMPs that alter clock gene expression within splenocytes, it does not take into account how these PAMPs affect the master clock or peripheral clocks throughout the body. Since the spleen is composed of a heterogeneous population of cells, individual PAMPs could impact each cell type differently. For example, Tlr9 expression rhythms in the mouse spleen differ between splenocytes, macrophages, B cells, and DCs15. Additionally, Tlr1, Tlr3, Tlr4, Tlr6, Tlr7,and Tlr8 displayed significant daily oscillations in an adherent splenocyte population but only Tlr2 and Tlr6 experience daily oscillations in enriched splenic macrophages24. Therefore, in order to investigate the outcome of a challenge on individual cell types, cells could be isolated via magnetic cell sorting, as previously described9,15 and then subsequently challenged. Additionally, the splenic cell population fluctuates over the daily cycle, which could also play a role in sensitivity to a particular PAMP and subsequent impact on the clock8.
This method allows for the isolation of a large number of immune cells that consist predominately of B cells (~58%), T cells (~21%), dendritic cells (~5%), and macrophages (~4%)25. The large number of cells provides the opportunity to challenge splenocytes with a variety of PAMPs in a single experiment. The splenocyte isolation procedure is very easy to perform, can be completed within minutes (depending on the number of animals), and with minimal animal or cellular manipulation, which is essential when examining the molecular clock because as mentioned above, these actions can disrupt the timing of the clock as well as clock-controlled genes. The results for this procedure were highly reproducible, as significance between challenged and unchallenged cells was achieved with just three animals and the results were consistent with previously published work23 (Figure 1). It should be noted that increasing the number of animals per group might have revealed statistically significant differences between a challenge group and control (e.g., ODN 1826 and Rev-erbα).
Moving forward, while this protocol only addresses the acute effects on clock gene expression after PAMP challenge, it could provide proof of principle for further investigation. For example, this assay could be used as a model to decipher the molecular mechanisms regarding TLR – PAMP interaction and how it influences the molecular clock. It could also be used to determine the length of time it takes for the molecular clock to recover after a PAMP challenge, which could be determined by conducting a time-course experiment (i.e., assessing expression after varying times post challenge). As mentioned above, subsequent experiments could be performed to examine PAMP challenge on specific splenocyte cell populations. Since several pathogens stimulate multiple TLRs upon infection, it would be interesting to use this protocol to investigate if challenging with multiple PAMPs have a synergistic effect on clock gene expression.
The authors have nothing to disclose.
This work was supported by Faculty Research grants from the College of Arts and Sciences Dean’s Office at the University of Hartford.
Frosted slides | Fisher | 12-550-343 | |
Cell strainers | Fisher | 22363547 | |
Lipopolysaccharide | InvivoGen | ltrl-eklps | |
ODN1826 | InvivoGen | Tlrl-1826-1 | |
HKLM | InvivoGen | Tlrl-hklm | |
RPMI 1640 | Gibco | 11875-093 | |
PBS | Gibco | 20012-043 | |
RNeasy Mini Kit | Qiagen | 74104 or 74106 | |
RNase-Free DNase Set | Qiagen | 79254 | |
6-well cell culture plate | Denville | T1006 | |
50 ml tubes | Corning | 352070 | |
15 ml tubes | Corning | 352097 | |
High Capacity cDNA Reverse Transcription Kit | ThermoFisher | 4368814 | |
TaqMan Gene Expression Assays b-actin | ThermoFisher | Mm00607939_s1 | |
TaqMan Gene Expression Assays Per2 | ThermoFisher | Mm00478113_m1 | |
TaqMan Gene Expression Assays Rev-erba | ThermoFisher | Mm00520708_m1 | |
TaqMan Gene Expression Assays Bmal1 | ThermoFisher | Mm00500226_m1 | |
TaqMan Gene Expression Assays Dbp | ThermoFisher | Mm00497539_m1 | |
qPCR machine StepOnePlus | ThermoFisher | ||
TaqMan Gene Expression Master Mix | ThermoFisher | 4369016 | |
MicroAmp Fast 96-well reaction plate (0.1 ml) | ThermoFisher | 4346907 | |
Statistical Analysis Software | Prism 7.0a |