The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

Summary

This protocol describes a technique using mouse splenocytes to discover pathogen-associated molecular patterns that alter molecular clock gene expression.

Abstract

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.

Introduction

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 hypothalamus^1,2. In addition to regulating biological processes on an organismal level, the master clock also synchronizes peripheral cellular clocks throughout the body^3,4,5. 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 genes^6,7. 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 response^8,15,16, 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 pathologies^17,18,19,20, it may be of interest for researchers to challenge splenocytes with a wide range of molecules and assess their influence on the clock.

Protocol

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.

1. Entrain mice to a 12 h light (standard overhead white light)/12 h dark cycle for 2 weeks prior to the experiment.
   NOTE: Here zeitgeber time (ZT) 0 corresponds to lights on and ZT12 to lights off, while keeping all other environmental factors (i.e., food, water, and room temperature) constant ²¹.

2. Preparation of Instruments, Culture Medium, and Challenge Medium

1. Autoclave forceps and dissecting scissors. Wrap pairs of frosted microscope slides in aluminum foil and autoclave.
2. Prepare culture medium by adding fetal bovine serum (FBS) to RPMI 1640 to a final concentration of 10%. Prepare 10 mL of challenge medium in 50 mL tubes by adding the following PAMPs to the culture medium (RPMI with 10% FBS); LPS (5 µg/mL), ODN1826 (5 µg/mL), heat-killed Listeria monocytogenes (10⁸ HKLM/mL), or another PAMP in its suggested concentration.
3. Warm culture medium and challenge medium to 37 °C in a water bath, and keep approximately 70 mL of sterile phosphate buffered saline (PBS, pH 7.2) at room temperature.
4. Add 10 mL of culture medium using a 10 mL pipette and pipette aid to a 50 mL tube and place on ice.
5. Add approximately 30 mL of 70% ethanol to a 100 mL beaker and place ends of dissecting scissors and forceps into the beaker to prevent microbial contamination.
6. Prepare lysis buffer for RNA isolation by adding 10 µL β-Mercaptoethanol (under a fume hood) to every 1 mL of Buffer RLT in a 50 mL tube. Make only the amount that will be needed (600 µL per sample, which consists of approximately 1 x 10⁶ cells).
   NOTE: The Buffer RLT is a component of the RNA extraction kit that supports the binding of RNA to the silica membrane.

3. Splenocyte Isolation and Challenge

1. Euthanize mice at a particular zeitgeber time via narcosis by keeping them in their original cage and adding CO₂ to the cage at a flow rate of 3 L/min. Continue supplying CO₂ for 1 min after breathing stops.
2. Confirm death via cervical dislocation by placing the thumb and index finger on either side of the neck at the base of the skull. Alternatively, press a rod at the base of the skull while quickly pulling (using the other hand) the base of the tail or the hind limbs to cause separation of the cervical vertebrae from the skull²².
3. Spray the mouse trunk with 70% ethanol and wipe with a paper towel. Place the mouse on its back and slightly tilted onto its right side. Cut away the fur, using dissecting scissors, along the mouse's left side, about halfway between the front and back legs.
4. Using forceps, grab the peritoneum and carefully make an incision so as not to damage the spleen. Remove spleen with forceps and place into a sterile 50 mL tube containing approximately 10 mL of culture media on ice. Repeat for the remaining animals.
   ​NOTE: The spleen is the color of a kidney bean, and it is longer and flatter than the kidney.
5. Transfer one spleen with 2 mL of culture medium to a small sterile Petri dish.
   1. Homogenize the spleen by grinding it between the frosted portion of two sterile frosted slides. If possible, keep the issue and cells in the medium during the homogenization process.
   2. Once thoroughly homogenized, pipet the 2 mL of culture medium containing the splenocytes through a 40 µm nylon cell strainer into a 50 mL tube.
   3. Repeat the above steps for the remaining spleens using new pairs of frosted slides, 50 mL tubes with culture medium, and cell strainers.
6. Add 8 mL of cold culture medium to each of the 50 mL tubes containing the splenocytes for a total volume of 10 mL/tube. Determine the number of cells per milliliter using a hemocytometer.
7. Add approximately 1 x 10⁶ cells/well to 6-well culture plates. Add cells to the number of wells that correspond to the number of different PAMPs being used for the experiment and include a control well. Add 3 mL of culture medium or 3 mL of challenge medium to the respective wells.
8. Incubate the plates at 37 °C in 5% CO₂ for 3 h.
9. Scrape the cells from the bottom of the well using a 1,000 µL pipet tip attached to a P1000 micropipette. Transfer the medium containing the cells using the same 1,000 µL pipet tip to a 15 mL tube.
10. Pellet the cells via centrifugation at 167 x g for 5 min at room temperature. Remove the supernatant and wash the cell pellet with 5 mL of PBS.
11. Pellet the cells a second time at 167 x g for 5 min, remove the supernatant, and add 600 µL of lysis buffer to the cell pellet in order to lyse the cells. Then proceed to RNA isolation.

4. RNA Isolation and cDNA Synthesis

1. Isolate RNA from splenocytes using the RNA extraction kit according to manufacturer's instructions and perform the 'optional' on-column DNA digestion.
2. Prior to cDNA synthesis, determine RNA concentration using a microvolume spectrophotometer to verify that the concentration is within the optimal RNA range (up to 2 µg) of the cDNA synthesis kit.
3. Synthesize cDNA for each of the samples using the reverse transcription kit according to manufacturer's instructions. Use 10 µL of RNA for each of the samples in a 20 µL total reaction volume. At the completion of the reverse transcription (thermocycler run) add 20 μL H₂O to each reaction.
4. Construct the standard curving using a P10 or P20 micropipette to pool mRNA from a few control samples (e.g., 5 µL from 2 samples for a total of 10 µL) into a 0.5 mL tube to prepare the cDNA.
   1. Add 10 µL 2x reverse transcriptase master mix.
   2. At the completion of the reverse transcription, add 10 µL of H₂O to the reaction tube, which will serve as the starting concentration ("1") in the dilution series for the standard curve.
   3. Perform a 10-fold dilution series (1 to 10^-4) by adding 45 µL of water using a P100 or P200 micropipette into four 0.5 mL tubes designated 10^-1, 10^-2, 10^-3, and 10^-4.
      1. Add 5 µL of the starting concentration "1" into the first tube (10^-1) using a P20 micropipettor, mix by pipetting up and down several times, then transfer 5 µL from the 10^-1 tube into the tube designated 10^-2 and mix.
      2. Transfer 5 µL using a P20 micropipette from the 10^-2 tube into the tube designated 10^-3 and mix. Transfer 5 µL using a P20 micropipettor from the 10^-3 tube into the tube designated 10^-4 and mix.

5. Quantitative Polymerase Chain Reaction (qPCR)

1. Determine relative quantitation of mRNA levels by qPCR. Within the experimental set up, select "Quantitation - Relative Standard Curve" and "TaqMan" chemistry. Change the reaction volume to 10 µL. Enter the relevant information into the plate layout (e.g., target gene, standard curve, reporter, etc.).
2. Prepare the reaction so that it contains 0.5 µL of the primer/probe assay, 5 µL of gene expression master mix, 2 µL of H₂O and 2.5 µL of cDNA (10–100 ng).
   1. Prepare a master mix by multiplying each of the preceding reagents (except the cDNA) by the number of reactions, making sure to include those from the standard curve, negative controls, performing each reaction in duplicate, and 2 extra reactions that will account for pipetting variation.
   2. Pipet 7.5 µL of the prepared master mix into pre-determined wells in a 96-well reaction plate with a micropipette or a multichannel pipette. Pipet 2.5 µL of cDNA into the appropriate well for the unknowns and standards, and 2.5 µL of H₂O into the negative control wells.
   3. Include primer/probe assays for various molecular clock genes, and also include an assay for an endogenous control (e.g., β-actin).
3. In order to determine relative expression values, calculate the mean relative quantity for each replicate, then, for each sample, divide the mean relative quantity for the target gene by the mean relative quantity for β-actin.

6. Statistical Analysis

1. Using statistical analysis software, enter data into the program under the column statistics option. Select a one-way ANOVA with the Dunnett's post hoc test to assess differences between mean values of clock gene expression after PAMP challenge versus the control.

Results

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 (...

Discussion

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 asse...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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