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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Bio Protoc. Author manuscript; available in PMC 2017 July 25.
Published in final edited form as:
PMCID: PMC5526106
NIHMSID: NIHMS878763

In vivo Analysis of Neutrophil Infiltration during LPS-induced Peritonitis

Abstract

Bacterial lipopolysaccharide (LPS) is present in the outer membrane of Gram-negative bacteria and functions as pathogen-associated molecular pattern (PAMP) (Whitfield and Trent, 2014). LPS therefore is a potent activator of inflammatory responses leading to cytokine release and neutrophils recruitment. The lipid A moiety of LPS activates the complex consisting of the LPS binding protein (LBP), CD14, MD-2 and Toll-like receptor 4 (TLR4) and the non-canonical inflammasome-linked caspases-4, 5 and 11, which in turn activate the canonical NLRP3 inflammasome (Shi et al., 2014; Hagar et al., 2013; Kayagaki et al., 2013; Hoshino et al., 1999; Poltorak, 1998; Nagai et al., 2002; Park et al., 2009; Ratsimandresy et al., 2013). In particular, the cytokine interleukin (IL)-1β produced in response to inflammasome activation has a crucial role in neutrophil recruitment through promoting neutrophil adhesion and migration (McDonald et al., 2010).This protocol allows studying of inflammatory response induced by LPS that affect neutrophil infiltration by tracking myeloperoxidase (MPO) activity in vivo (de Almeida et al., 2015).

Materials and Reagents

  1. Insulin syringes (Thermo Fisher Scientific, catalog number: 14-841-31)
  2. 0.22 μm filters
  3. C57BL/6 mice, typically of 8-12 weeks old mice (male or female)
  4. LPS E.coli 0111:B4 (Sigma-Aldrich, catalog number: L2630-100MG)
  5. Dulbecco's phosphate-buffered saline (DPBS) (Corning, catalog number: 21-030-CV)
  6. XenoLight RediJect inflammation probe (PerkinElmer, catalog number: 760535)
  7. Luminol sodium salt (Sigma-Aldrich, catalog number: A4685)
  8. Isofluorane (Henry Schein, Isothesia&trade, catalog number: 10014450)
  9. 5 mg/ml LPS (see Recipes)
  10. 20 mg/ml luminol sodium salt stock solution (see Recipes)

Equipment

  1. Anesthesia machine (VetEquip, model: 901808) or similar anesthesia equipment
  2. Rechargeable trimmer (Braintree Scientific, catalog number: VLP-323 75)
  3. Scale (Kent Scientific, catalog number: SCL66110)
  4. Biosafety cabinet
  5. IVIS spectrum (PerkinElmer, model: 124262) or a comparable luminescence imaging equipment

Software

  1. Living Image software (PerkinElmer)

Procedure

  1. Two days before the LPS intra peritoneal injection place mice in the anesthesia machine and once the mice are anesthetized shave abdominal area with a trimmer, as fur quenches the luminescence signal (Figure 1).
    Figure 1
    A representative picture of mice under anesthesia getting their abdominal area shaved with a trimmer
  2. Weigh mice.
  3. In the day of the experiment dilute LPS in DPBS and prepare syringes for injection.
  4. Intraperitoneally inject mice with 2.5 mg/kg of LPS or the same volume DPBS for the control group (injection volume approximately 200 μl).
  5. 3 h later intraperitoneally inject mice with 200 mg/kg of XenoLight Rediject inflammation probe or 200 mg/kg luminol sodium salt (injection volume approximately 200 μl).
  6. Place mice in the IMPAC6 anesthesia chamber attached to the IVIS spectrum.
  7. Transfer mice to the IVIS spectrum and place mice abdomen facing up into the chamber and position each nose inside the cone that delivers the isofluorane (Figure 2).
    Figure 2
    A representative example of in vivo imaging in 8 wk of age male C57BL/6 mice after i.p. injection of PBS (left) or LPS (2.5 mg/kg body weight) (right)
  8. Start imaging anesthetized mice 10 min post XenoLight Rediject inflammation probe injection with a 5 min exposure capturing in vivo bioluminescence generated by the activity of MPO as a marker for infiltration of neutrophils. In order to imagine 5 mice select field of view D and select 1.5 cm subject height (Figures 2 and and3)3) (Gross et al., 2009; Tseng and Kung, 2012).
    Figure 3
    Screen capture image of the IVIS acquisition control panel
  9. Quantify the MPO signal using the Living Image software. First select region of interest (ROI) using ROI tools and choose to automatically draw measurement ROIs and perform ROI analyses to measure photon radiance. Also measure background ROI and subtract from your ROI measurement. Use average radiance to plot your graph.

Recipes

  1. 5 mg/ml LPS
    Dilute LPS in DPBS.
    Filter sterilize with a 0.22 μm filter and aliquot stock solution at -80 °C.
  2. 20 mg/ml luminol sodium salt stock solution
    Prepare 20 mg/ml luminol sodium salt stock solution in DPBS.
    Note: Luminol sodium salt for injection has to be prepared fresh each time in DPBS (20 mg/ml), filter sterilize with a 0.22 μm filter and protected from light until use.

Acknowledgments

This protocol was adapted from a previously published study (de Almeida et al., 2015). This work was supported by grants from the National Institutes of Health (AI099009 and AR064349 to C.S., AR066739 to A.D., AI120625 and AI120618 to C.S. and A.D., T32AR007611 to L.d.A., and the American Heart Association 13GRNT17110117 to C.S.).

References

  • de Almeida L, Khare S, Misharin AV, Patel R, Ratsimandresy RA, Wallin MC, Perlman H, Greaves DR, Hoffman HM, Dorfleutner A, Stehlik C. The PYRIN domain-only protein POP1 inhibits inflammasome assembly and ameliorates inflammatory disease. Immunity. 2015;43(2):264–276. [PMC free article] [PubMed]
  • Gross S, Gammon ST, Moss BL, Rauch D, Harding J, Heinecke JW, Ratner L, Piwnica-Worms D. Bioluminescence imaging of myeloperoxidase activity in vivo. Nat Med. 2009;15(4):455–461. [PMC free article] [PubMed]
  • Hagar JA, Powell DA, Aachoui Y, Ernst RK, Miao EA. Cytoplasmic LPS activates caspase-11 : implications in TLR4-independent endotoxic shock. Science. 2013;341(6151):1250–1253. [PMC free article] [PubMed]
  • Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, Takeda K, Akira S. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol. 1999;162(7):3749–3752. [PubMed]
  • Kayagaki N, Wong MT, Stowe IB, Ramani SR, Gonzalez LC, Akashi-Takamura S, Miyake K, Zhang J, Lee WP, Muszynski A, Forsberg LS, Carlson RW, Dixit VM. Noncanonical inflammasome activation by intracellular LPS independent of TLR4. Science. 2013;341(6151):1246–1249. [PubMed]
  • McDonald B, Pittman K, Menezes GB, Hirota SA, Slaba I, Waterhouse CC, Beck PL, Muruve DA, Kubes P. Intravascular danger signals guide neutrophils to sites of sterile inflammation. Science. 2010;330(6002):362–366. [PubMed]
  • Nagai Y, Akashi S, Nagafuku M, Ogata M, Iwakura Y, Akira S, Kitamura T, Kosugi A, Kimoto M, Miyake K. Essential role of MD-2 in LPS responsiveness and TLR4 distribution. Nat Immunol. 2002;3(7):667–672. [PubMed]
  • Park BS, Song DH, Kim HM, Choi BS, Lee H, Lee JO. The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex. Nature. 2009;458(7242):1191–1195. [PubMed]
  • Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, Beutler B. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science. 1998;282(5396):2085–2088. [PubMed]
  • Ratsimandresy RA, Dorfleutner A, Stehlik C. An update on PYRIN domain-containing pattern recognition receptors: from immunity to pathology. Front Immunol. 2013;4:440. [PMC free article] [PubMed]
  • Shi J, Zhao Y, Wang Y, Gao W, Ding J, Li P, Hu L, Shao F. Inflammatory caspases are innate immune receptors for intracellular LPS. Nature. 2014;514(7521):187–192. [PubMed]
  • Tseng JC, Kung AL. In vivo imaging of inflammatory phagocytes. Chem Biol. 2012;19(9):1199–1209. [PubMed]
  • Whitfield C, Trent MS. Biosynthesis and export of bacterial lipopolysaccharides. Annu Rev Biochem. 2014;83:99–128. [PubMed]