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Method for Studying the Effect of Gene Silencing on Bacterial Infection-induced ERK1/2 Signaling in Bone-marrow Derived Macrophages

基因沉默对于细菌感染诱导骨髓巨噬细胞中ERK1/2信号通路的影响研究方法

Gaurav Kumar Gaurav Kumar *
SA Subhash B. Arya *
Amit Tuli Amit Tuli

 (*contributed equally to this work) 发布: 2018年12月20日第8卷第24期 DOI: 10.21769/BioProtoc.3123 浏览次数: 8022

评审: Shantibhusan SenapatiHaixia XuAnonymous reviewer(s)

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Abstract

Macrophages are highly phagocytic cells that utilize various pathogen recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs). These PAMPs can be present within the microbe, such as bacterial CpG DNA, and are recognized by Toll-like receptor 9 (TLR9), a PRR present on the endosomal membrane of macrophages. PAMPs can also be present on the surface of microbes, such as Lipopolysaccharide (LPS), which decorates the outer membrane of gram-negative bacteria like Salmonella typhimurium and Escherichia coli. LPS is recognized by TLR4 present on the plasma membrane of macrophages, and LPS-TLR4 association leads to activation of signaling cascades including MAPK phosphorylation, which in turn promotes macrophage activation and microbial killing. This protocol describes the method for studying the role of a gene of interest in Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) signaling, induced by bacterial infection in primary bone-marrow derived macrophages (BMDMs).

Keywords: Bone-marrow derived macrophages (骨髓巨噬细胞) search Salmonella (沙门氏菌) search Infection (感染) search ERK signaling (ERK信号通路) search Western blotting (蛋白质印迹) search

Background

Macrophages are phagocytic cells which can either be resident to specific tissues as Kupffer cells (in the liver) and peritoneal macrophages (in peritoneal cavity) or, can enter tissues in response to an infection. The primary function of macrophages involves phagocytosis and clearance of old damaged cells, and recycling of nutrients in the serum (recently reviewed in Shapouri-Moghaddam et al., 2018). However, for macrophages to clear microbes, there exists a need for their activation. Macrophages obtained from many tissues such as alveoli and peritoneal cavity includes high numbers of pre-activated macrophage population. However, macrophages derived from myeloid progenitor cells present in the bone marrow are comparatively naive and more responsive to activating stimulus (Epelman et al., 2014). Upon encounter of gram-negative bacteria by macrophages, LPS present on either the surface of a bacterium or shed by bacteria in the blood flow is captured by LBP (LPS-binding protein) and presented as a ligand to TLR4, a type I transmembrane protein present on the plasma membrane of macrophage that mediate the recognition of PAMPS such as LPS (Shimazu et al., 1999). The engagement of LPS with TLR4 (along with other co-stimulatory molecules) leads to recruitment of several adaptor proteins at the cytoplasmic tail of TLR4 followed by a cascade of intracellular events leading to activation of the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling cascades downstream to TLR4 including ERK1/2 (Buscher et al., 1995; Schaeffer and Weber, 1999; Gay et al., 2014; Platko et al., 2018; Arya et al., 2018). These signaling pathways directly or indirectly phosphorylate and activate various transcription factors that lead to the expression of genes involved in production and release of pro-inflammatory cytokines, reactive oxygen species, nitrosative burst and promotes macrophage activation (Satoh and Akira, 2016). Thus, ERK1/2 signaling plays an important role in augmenting macrophage response against intracellular pathogens. This protocol describes a method to investigate the role of a host factor of interest in modulating ERK1/2 activation in primary BMDMs in response to Salmonella typhimurium infection (see schematic shown in Figure 1 and Arya et al., 2018).

Materials and Reagents

  1. 5 ml syringe (Dispovan)
  2. 60-mm tissue culture dish (BD Falcon®, catalog number: 353002)
  3. 10-cm tissue culture dish (BD Falcon®, catalog number: 353003)
  4. 6-well tissue culture plate (BD Falcon®, catalog number: 353046) 
  5. 50 ml Falcon tube (BD Falcon®, catalog number: 352070)
  6. 15 ml Falcon tube (BD Falcon®, catalog number: 352096)
  7. 14 ml round-bottom polypropylene tube (Corning, catalog number: 352006)
  8. 1.5 ml microcentrifuge tube (MCT) (Tarson, catalog number: 50010)
  9. 0.2 micron PVDF membrane (Bio-Rad, catalog number: 162-077)
  10. X-ray film (Carestream, catalog number: 6574958)
  11. Aluminum foil
  12. Cell scraper (HiMedia, catalog number: TCP104)
  13. Sterile 22 G needle (Dispovan)
  14. C57BL/6 male mice (typically 6-8 weeks old)
  15. Salmonella typhimurium strain SL1344
  16. RPMI medium 1640 (Lonza, catalog number: 12702F)
  17. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10082147)
  18. HEPES (1 M) (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080)
  19. MEM Non-Essential Amino Acids Solution (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 11140-050)
  20. Antibiotic-Antimycotic (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15240-062)
  21. Sodium Pyruvate (100 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11360-070)
  22. GlutaMAXTM Supplement (Thermo Fisher Scientific, GibcoTM, catalog number: 35050-061)
  23. DPBS (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-144) 
  24. PBS, pH 7.4 (Thermo Fisher Scientific, GibcoTM, catalog number: 10010-023) 
  25. Trypsin-EDTA (0.05%) (Thermo Fisher Scientific, GibcoTM, catalog number: 25300-054) 
  26. Trypan Blue Solution (Thermo Fisher Scientific, GibcoTM, catalog number: 15250-061) 
  27. Mouse M-CSF (macrophage-colony stimulating factor) (eBiosciences, catalog number: 34-8983)
  28. ACK (Ammonium-Chloride-Potassium) lysing buffer (Thermo Fisher Scientific, GibcoTM, catalog number: A10492-01)
  29. Protease inhibitor (Sigma-Aldrich, catalog number: P8340)
  30. Phosphatase inhibitor (Roche, catalog number: 4906837001)
  31. Bradford reagent (Sigma-Aldrich, catalog number: B6916)
  32. TWEEN® 20 (Sigma-Aldrich, catalog number: P9146) 
  33. TritonTM X-100 (Sigma-Aldrich, catalog number: T8787) 
  34. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771) 
  35. Sodium deoxycholate (DOC) (Sigma-Aldrich, catalog number: D6750)
  36. Trizma® base (Tris base) (Sigma-Aldrich, catalog number: T6066) 
  37. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 3014)
  38. Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: 3889)
  39. Ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA-Na2) (Sigma-Aldrich, catalog number: E5134)
  40. Gentamicin solution (Sigma-Aldrich, catalog number: G1272)
  41. Streptomycin sulfate (Sigma-Aldrich, catalog number: S6501)
  42. Ethanol (Merck, catalog number: 108543)
  43. Skim milk (BD Difco, catalog number: 232100)
  44. LB broth (BD Difco, catalog number: 244620)
  45. SS agar (HiMedia, catalog number: M108) 
  46. Laemmlisample buffer (Bio-Rad, catalog number: 161-0747)
  47. ON-TARGETplus Non-targeting Control siRNA Pool (Dharmacon, catalog number: D-001810-10) 
  48. ON-TARGETplus Mouse Arl11 siRNA (Dharmacon, catalog number: L-055672-01-0005)
  49. Polyclonal anti-total ERK1/2 antibody (Cell Signaling Technology, catalog number: 4695)
  50. Polyclonal anti-phospho-ERK1/2 antibody (Cell Signaling Technology, catalog number: 4370)
  51. Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch, catalog number: 111-035-144)
  52. ECL Plus Western Blotting Substrate (Pierce, catalog number: 32132)
  53. 70% ethanol (see Recipes)
  54. Complete RPMI medium (see Recipes)
  55. Mouse M-CSF stock solution (0.4 mg/ml) (see Recipes)
  56. SS agar plate (see Recipes)
  57. Streptomycin stock solution (50 mg/ml) (see Recipes)
  58. LB broth (see Recipes)
  59. Opsonization buffer (see Recipes)
  60. Growth medium (GM) for infection (see Recipes)
  61. RIPA lysis buffer (see Recipes)
  62. Blocking buffer (see Recipes)
  63. PBST (see Recipes)
  64. Antibody dilution buffer (see Recipes)

Equipment

  1. Beaker
  2. 0.2 micron polyethersulfone filter
  3. Scissors and forceps (Fisher Scientific)
  4. Cell culture CO2 incubator (Thermo Scientific, model: 371)
  5. Biosafety cabinet class II (ESCO, model: AC2-4S8-NS)
  6. Inverted tissue culture microscope (Nikon, model: TS2) 
  7. Hemocytometer (Sigma, catalog number: Z359629)
  8. Multimode reader (Tecan, model: Infinite M200)
  9. Incubator shaker (New Brunswick, model: Innova 42) 
  10. Automatic X-ray Film Processor (Protec, model: OPTIMAX 2010)
  11. Flatbed scanner (Microtek, model: Bio-5000)
  12. Refrigerated centrifuge with plate centrifuge bucket big enough for a 6-well plate (Eppendorf, model: 5810 R)
  13. Semi-dry horizontal blotting system (Atto, model: WSE-4020)
  14. SDS-PAGE gel apparatus (Bio-Rad, model: Mini Protean Tetra Cell)
  15. Dancing shaker (Tarson, model: MC-02)
  16. Rocking shaker (Tarson, model: 4080)
  17. Flow cytometry

Software

  1. ImageJ software (NIH, https://imagej.nih.gov/ij/)
  2. Microsoft Excel software (MS Office)

Procedure

English
中文翻译

文章信息

版权信息

© 2018 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Kumar, G., Arya, S. B. and Tuli, A. (2018). Method for Studying the Effect of Gene Silencing on Bacterial Infection-induced ERK1/2 Signaling in Bone-marrow Derived Macrophages. Bio-protocol 8(24): e3123. DOI: 10.21769/BioProtoc.3123.
  2. Arya, S. B., Kumar, G., Kaur, H., Kaur, A. and Tuli, A. (2018). ARL11 regulates lipopolysaccharide-stimulated macrophage activation by promoting mitogen-activated protein kinase (MAPK) signaling. J Biol Chem 293(25): 9892-9909.

    3. ris ris Download Citation in RIS Format

分类

免疫学 > 免疫细胞功能 > 巨噬细胞

生物化学 > 蛋白质 > 定量

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