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Jul 2021

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A Mouse Infection Model with a Wildtype Salmonella enterica Serovar Typhimurium Strain for the Analysis of Inflammatory Innate Immune Cells
用于分析炎性先天免疫细胞的野生型肠沙门氏菌鼠伤寒血清型小鼠感染模型   

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Abstract

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative, facultative intracellular bacterium, which causes gastrointestinal disorders in humans, and systemic, typhoid fever-like infections in mice. Our current knowledge regarding the involvement of cellular and humoral immunity in the defense from S. Typhimurium infections is largely based on animal models with attenuated strains. Cells of the innate immune system act as one of the first barriers in the defense from bacteria. We established a robust experimental model for the characterization of these cell types and their response during host-pathogen interactions. Therefore, this protocol focuses on the characterization of macrophages, monocytes, and neutrophils in the spleens of infected animals by employing multi-color flow cytometry.

Keywords: Macrophages (巨噬细胞), Intracellular bacteria (细胞内细菌), Gram-negative bacteria (革兰氏阴性菌), Infection control (感染控制)

Background

Salmonella enterica is a highly iron-dependent siderophilic intracellular Gram-negative pathogen, which can cause local intestinal disease, or severe systemic infection and septicemia. Hence, it was included in the WHO list of the most serious infectious disease threats to human health (Keestra-Gounder et al., 2015 ; Bumann and Schothorst, 2017). Salmonella invades and multiplies within mononuclear phagocytic cells in the liver, spleen, lymph nodes, and Peyer’s patches, in so-called Salmonella-containing vacuoles (SCV) (Steele-Mortimer, 2008). However, Salmonella exhibits different mechanisms to evade antimicrobial activities, for example by inhibiting phagolysosomal fusion (Baumler and Fang, 2013; Bhutta et al., 2018).


Several immune cells are important in the defense against bacterial infections. While some act as the first barrier, by taking up and killing bacteria, others are responsible for long-term immunity or for coordinating efficient anti-microbial immune responses. Macrophages, dendritic cells (DCs), and neutrophils represent one of the first lines of defense against invading pathogens like Salmonella, by recognizing specific pathogen-associated molecular patterns (PAMPs), and danger-associated-molecular patterns (DAMPs) (de Jong et al., 2012). Recognition of Gram-negative bacteria in macrophages involves the binding of LPS to a receptor complex, leading to the secretion of pro- and anti-inflammatory cytokines (Nagai et al., 2002; Bode et al., 2012). Macrophages can polarize dependending on local factors within the microenvironment, microbial stimuli, and cytokines secreted by other cells. Pro-inflammatory macrophages are essential components of anti-microbial host defense mechanisms, and can be characterized by the synthesis of inducible nitric oxide synthase (iNOS). Anti-inflammatory macrophages dampen the inflammatory state, by producing anti-inflammatory cytokines and Arginase-1 (ARG1). ARG1 cleaves L-arginine, the substrate of iNOS, and thereby impairs the control of various intracellular pathogens (Mosser and Edwards, 2008; Murray, 2017).


Several studies have shown that Salmonella is also phagocytosed by dendritic cells, which serve as an important link between innate and adaptive immunity (Steinman and Hemmi, 2006). Upon phagocytic internalization of bacteria, DCs mature and migrate to defined lymphoid tissues. Thereby, they increase the expression of the major histocompatibility complex II (MHCII) to present specific antigens to T cells, initiating adaptive immune response.


Due to the widespread development of multidrug resistances against antibiotics, current antibiotic treatments of invasive salmonellosis is often not successful. In addition, it is uncertain whether standard antibiotics can penetrate into the Salmonella-containing vacuole. Thus, it is necessary to better understand the host-pathogen interplay in salmonellosis, to develop novel effective antimicrobial therapies targeting intracellular pathogens (Lahiri et al., 2010; Navarre et al., 2010; Mastroeni and Grant, 2011).

Materials and Reagents

  1. 10 cm bacteriological Petri dish (Falcon, catalog number: 351029)

  2. Cell strainer 100 µm (Falcon, catalog number: 352360)

  3. 5 ml syringe (BD, DiscarditTM II 309050)

  4. Sterile wedge shaped spreader (Microspec, catalog number: 211738)

  5. Syringes for intraperitoneal injection (30G × ½’’ 0.3 mm × 12 mm, Braun, Omnican F 9161502S)

  6. 1.5 mL Eppendorf Tubes (Eppendorf, catalog number: T9661)

  7. 96 well round-bottom plates (BRAND, catalog number: 781600)

  8. Cryovials 2 mL round bottom (Simport, catalog number: T311-3)

  9. Salmonella enterica serovar Typhimurium ATCC14028 (ATCC)

  10. LB Broth Lennox (Roth, catalog number: X964.2)

  11. Phosphate buffer saline (PBS) (Lonza, catalog number: 17-515 F)

  12. Agar-Agar Kobe I (Roth, catalog number: 5210.3)

  13. Paraformaldehyde (Sigma-Aldrich, Formalin-solution, neutral buffered 10%, HT501128)

  14. APC anti-mouse CD11b (BioLegend, catalog number: 101212)

  15. FITC anti-mouse CD45 (BioLegend, catalog number: 103108)

  16. BV421 rat anti-mouse F4/80 (BD Biosciences, catalog number: 565411)

  17. BV510 anti-mouse Ly6C (BioLegend, catalog number: 128033)

  18. PerCP-eFluorTM 710 anti-mouse Ly6G (Invitrogen, catalog number: 46-9668-82)

  19. PerCP/Cyanine5.5 anti-mouse MHCII (BioLegend, catalog number: 116416)

  20. BV421 anti-mouse CD11c (BioLegend, catalog number: 117330)

  21. PE-Cyanine7 anti-mouse iNOS (Invitrogen, catalog number: 25-5920-80)

  22. anti-mouse ARG1 (Invitrogen, catalog number: 17-3697-82)

  23. Glycerol (Sigma-Aldrich, catalog number: G5516)

  24. Fetal Bovine Serum heat inactivated (FBS) (PAN Biotech, catalog number: P30-3031)

  25. Ethylenedinitrilotetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884)

  26. Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: 254134)

  27. Potassium hydrogen carbonate (KHCO3) (Sigma-Aldrich, catalog number: 237205)

  28. Ethylenediaminetetraacetic acid, disodium salt (Na2EDTA) (Sigma-Aldrich, catalog number: E5134)

  29. Triton X-100 (Roth, catalog number: 3051.3)

  30. LB-Agar plates (see Recipes)

  31. ACK lysis buffer (see Recipes)

  32. FACS buffer (see Recipes)

  33. Triton buffer (see Recipes)

  34. LB agar plates (see Recipes)

  35. LB medium (see Recipes)

  36. 4% PFA (see Recipes)

  37. LB-medium with 30% glycerol (see Recipes)

Equipment

  1. Incubator 37°C (ELOS Breed, catalog number: B110N)

  2. Shaking incubator (VWR, GFL, catalog number: 3031)

  3. Photometer (Eppendorf, BioPhotometer D30, catalog number: 6133000001)

  4. Centrifuge (Hettich Micro 200R, catalog number: Z652113)

  5. CytoFLEX S V4-B4-R2-I2 Flow Cytometer (13 detectors, 4 lasers, Beckman Coulter, catalog number: C01161)

  6. CASY TT counting system (OMNI Life Science, catalog number: TT-20A-2571)

Software

  1. FlowJo v10.7.0 (BD Biosciences, https://www.flowjo.com/solutions/flowjo)

  2. GraphPad Prism V8.4.1 (https://www.graphpad.com/scientific-software/prism)

Procedure

  1. Grow Salmonella enterica serovar Typhimurium ATCC14028 in an Erlenmeyer flask, until they reach the logarithmic growth phase

    1. Open the original vial and rehydrate the entire pellet in 1 mL of LB-medium.

    2. Mix well by pipetting up and down.

    3. Pipette 10 µL in 10 mL of LB-medium as pre-culture.

      Note: Centrifuge the remaining 950 µL at 1920 × g at room temperature (RT) for 5 min, resuspend the pellet in 1 mL of LB-medium containing 30% Glycerol, aliquot as 10 × 100 µL in cryovials, and freeze the aliquots at -80°C. This will be the stock for the pre-culture in further experiments.

    4. Incubate the pre-culture in a rotating incubator at 200 rpm at 37°C overnight.

    5. Next day, pipette 50 µL of this overnight pre-culture in 10 mL of LB-medium in an Erlenmeyer flask.

    6. Incubate in an orbital shaker at 200 rpm at 37°C for 1–2 h, until an OD600 of 0.5 (measured in a photometer using LB-medium as blank).


  2. Counting of viable Salmonella enterica serovar Typhimurium using for example a CASY TT counting system

    1. Use the 45 µm capillary.

    2. Measure the background, by placing a new Casy cup with fresh Casy ton buffer under the measuring unit.

    3. Select program for background measurement (Table 1).

    4. Measure background. This should be below 30 counts and 1 µm size. Otherwise, wash the system.

    5. Prepare a new Casy cup with 10 mL of Casy ton buffer, and add 5 µL of S. typhimurium solution.

    6. Shake gently.

    7. Place sample under the measuring unit.

    8. Select program for measuring between 1–3 µm (Table 1).

    9. Measure.

    10. Click next to get the number of viable counts per milliliter = viable S.tm/mL.

    11. After the measurement is completed, remove the sample cup, and add a fresh Casy cup with 10 mL Casy ton buffer.

    12. Perform Casy Clean up to five times.

    13. Select Program for Washing (Table 1).

    14. After washing is completed, check the background.

    15. If the background is below 30, the Casy counting system can be turned off; otherwise, continue washing.


      Table 1. Programs CASY TT Counting system

      Background Measurement Measurement of S. Typhimurium

      Washing Program

      Capillary 45 µm   X-Axis: 5 µm 45 µm X-Axis: 3 µm 45 µm   X-Axis: 5 µm
      Sample Volume 200 µL Cycles: 1 200 µL Cycles: 3 200 µL Cycles: 10
      Dilution 1.00 × 100 2.00 × 103 1.00 × 100
      Y-Axis Auto Auto Auto
      Eval.Cursor 1.00–4.89 µm 0.75–2.93 µm 0.00–5.00 µm
      Norm. Cursor 0.5–4.89 µm 0.3–2.93 µm 0.00–5.00 µm
      %Calculation %ViaDebris: On %Via  Debris: On %Via  Debris: On
      Aggregation Correction Auto  Auto  Auto 
      Interface Par   P.Feed: On Par   P.Feed: On Par   P.Feed: On
      Print Mode Manual Graphic: On Manual   Graphic: On Manual   Graphic: On


  3. Intraperitoneal injection of mice

    Using Omnican F syringes (30G × ½’’ (0.3 mm × 12 mm), intraperitoneally (i.p.) inject mice with 1000 bacteria in 200 µL of PBS, according to the measurement in the Casy counting system.

    Negative control mice are injected with 200 µL of PBS.

    A video demonstrating the procedure can be found at: Intraperitonelal Injection in the Mouse: https://researchanimaltraining.com/articles/intraperitoneal-injection-in-the-mouse/.

    Notes:

    1. The injection of mice is performed in a bio-safety level 2 animal facility, where mice are housed in individually ventilated cages (IVC). After infection, all cages, litter, and used material must be autoclaved.

    2. Weight and body surface temperature are important markers for monitoring the mice during the infection. Therefore, mice should be familiarized to daily weighing and temperature measurements 3–5 days prior to the experiment. This will avoid stress-induced weight loss and changes in body temperature.

    3. All substances used should be warmed to RT. The injection of cold substances influences the wellbeing of the mice, and can lead to a sudden drop in body temperature.


  4. Monitoring of mice during the infection

    Note: Mice on a C57BL/6 background express a non-functional NRAMP1 (natural resistance associated macrophage protein 1) protein. NRAMP is a phagolysosomal membrane transporter for iron, protons, and divalent cations, and is associated with host resistance to various intracellular pathogens. Non-functional NRAMP leads to intracellular persistence of S. typhimurium within macrophages, and early death after 5 days at the latest. Therefore, it is crucial to monitor the mice in the most careful way.

    1. Body weight:

      The body weight should be measured at the same time each day, to avoid behavioral changes due to food intake. It is important to use a lockable box and a precision scale, which can be disinfected.

    2. Body temperature:

      The body temperature is measured with an infrared thermometer at the same time each day.

      Pick the mouse up by the middle of the tail and expose its abdomen. Allow the mouse to hold on to the grid of a cage with its forepaws, which allows the mouse to stretch its upper body and expose the abdomen. Hold the trigger to measure the temperature, taking care to aways measure at the same location of the abdomen.

    3. General appearance of the mice:

      The general appearance of the mice should be documented twice a day, for example according to the M-CASS scoring sheet in Lilley et al. (2015) , and your experimental animal license. Severe pain, suffering, and distress must be prevented.


  5. Isolation of splenocytes

    1. After 72 h of infection, isolate the spleens, and press the organs through a 100 µm cell strainer with the plunger of a 5 mL syringe, onto a Petri dish with 5 mL of cold PBS.

    2. Centrifuge at 300 × g and 4°C for 5 min, and wash once with 5 mL of PBS (300 × g at 4°C for 5 min), discard the supernatant.

    3. Subject the cell pellet to erythrocyte lysis, by resuspending in 2 mL of ACK lysis buffer.

    4. Incubate at RT for 2 min.

    5. Wash once with 5 mL of FACS buffer (300 × g at 4°C for 5 min).


  6. Flow Cytometry stain of innate immune cells

    All procedures are performed in 1.5 mL Eppendorf tubes.

    1. Extracellular stain (Table 2)

      1. Prepare an appropriate mix of antibodies (1:200) in 100 µL of FACS buffer per sample (Table 2).


        Table 2. Antibodies for flow cytometry – extracellular stain

        Antibody Clone Company Catalog number expressed on
        CD11b APC M1/70 BioLegend 101212 neutrophils, monocytes
        CD45 FITC 30-F11 BioLegend 103108 leukocytes
        F4/80 BV421 T45-2342 BD Biosciences 565411 macrophages
        Ly6C BV510 HK1.4 BioLegend 128033 monocytes
        Ly6G PerCP-eFluorTM710 1A8-Ly6g Invitrogen 46-9668-82 neutrophils
        MHCII PerCP/Cyanine5.5 AF6-120.1 BioLegend 116416 dendritic cells
        CD11c BV421 N418 BioLegend 117330 dendritic cells


      1. Resuspend the cell pellet in this mix.

      2. Incubate in the dark at 4°C for 15 min.

      3. Wash once with 1,000 µL of FACS buffer (300 × g at 4°C for 5 min).

      4. Resuspend the pellets in 4% paraformaldehyde (PFA) to fix the cells for intracellular staining.

      5. Transfer into 96-well round-bottom plates.

      6. Analyze directly in a flow cytometer.

    2. Intracellular stain (Table 3)

      1. Prepare extracellular stain as described in steps 1a–d.

      2. Resuspend the pellet in 4% PFA.

      3. Incubate at 4°C for 15 min in the dark.

      4. Centrifuge and resolve the pellet in Triton buffer.

      5. Mix well and incubate at room temperature for 15 min in the dark.

      6. Prepare appropriate mix of antibodies (1:100) in 50 µL of Triton buffer per sample (Table 3).


        Table 3. Antibodies for flow cytometry – intracellular stain

        Antibody Clone Company Catalog number expressed on
        iNOS PE-Cyanine7 CXNFT Invitrogen 25-5920-80 pro-inflammatory macrophages
        ARG1 APC A1exF5 Invitrogen 17-3697-82 anti-inflammatory macrophages


      7. Resuspend the cell pellet in this mix.

      8. Incubate at 4°C for 45 min.

      9. Wash once with 1,000 µL Triton buffer (300 × g at 4°C for 5 min).

      10. Resuspend the pellets in 4% PFA.

      11. Transfer into 96-well round-bottom plates.

      12. Analyze directly in a flow cytometer.

Data analysis

FlowJo software can be used to analyze data. Plot a linear FSC (forward scatter) versus SSC (side scatter) dot plot, and create a gate to select all cells. Using these cells, select CD45+ cells (pan-leucocytes), and follow the gating strategy described in Figure 1.

Statistical analysis and graphs are performed and generated in GraphPad Prism. When data are normally distributed (assess by using histograms and statistical tools, for example the Shapiro-Wilk test), analyze by the unpaired Student's t-test for samples with equal variances, or by two-way ANOVA (F test). When data are not normally distributed, the Mann-Whitney U, or an ANOVA on ranks tests can be performed.



Figure 1. Gating strategy for the analysis of innate immune cells (infected mouse - upper panel, and uninfected mouse – lower panel).

(A) forward scatter (FSC) against side scatter (SSC), (B) exclusion of doublets, (C) leukocytes: CD45+, (D) neutrophils: CD45+Ly6G+CD11b+, (E) macrophages: CD45+CD11b-Ly6G-F4/80+, (F) proinflammatory macrophages express iNOS, (G) antiinflammatory macrophages express Arginase, (H) dendritic cells: CD45+Ly6G-CD11c+MHCII+, (I) + (J) monocytes: CD45+Ly6G-F4/80+CD11b+, (K) inflammatory and resident monocytes: Ly6Chigh or Ly6Clow, respectively.

Recipes

  1. FACS buffer

    PBS with 0.5% heat-inactivated FBS

    2 mM EDTA

  2. ACK (Ammonium-Chloride-Potassium) lysis buffer

    150 mM NH4Cl

    10 mM KHCO3

    0.1 mM Na2EDTA

    pH 7.2–7.4

  3. Triton buffer

    PBS with 0.05% Triton X-100

  4. LB agar plates

    2% LB-Broth

    1.5% Agar-Agar

    Autoclave (121°C for 20 min), cool down, and pipette 15 mL in 10 cm bacteriological Petri dishes.

    Store at 4°C.

  5. LB medium

    2% LB-Broth

    Autoclave (121°C for 20 min).

  6. 4% PFA

    Add 40 µL of PFA to 960 µL of PBS.

  7. LB-medium with 30% glycerol

    Add 300 µL of glycerol to 700 µL of LB-medium.

Acknowledgments

Author G.W. is supported by grants from the Christian Doppler Society and an ERA-NET grant by the FWF (EPICROSS, I-3321), N.B. was supported by the FWF doctoral college project W1253 HOROS. This protocol was adapted and modified after Nairz et al. (2009).

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.

Ethics

Animal experiments were approved by the Austrian Federal Ministry of Science and Research according to the directive 2010/63/EU.

References

  1. Baumler, A. and Fang, F. C. (2013). Host specificity of bacterial pathogens. Cold Spring Harb Perspect Med 3(12): a010041.
  2. Bhutta, Z. A., Gaffey, M. F., Crump, J. A., Steele, D., Breiman, R. F., Mintz, E. D., Black, R. E., Luby, S. P. and Levine, M. M. (2018). Typhoid Fever: Way Forward. Am J Trop Med Hyg 99(3_Suppl): 89-96.
  3. Lahiri, A., Lahiri, A., Iyer, N., Das, P. and Chakravortty, D. (2010). Visiting the cell biology of Salmonella infection. Microbes Infect 12(11): 809-818.
  4. Lilley, E., Armstrong, R., Clark, N., Gray, P., Hawkins, P., Mason, K., Lopez-Salesansky, N., Stark, A. K., Jackson, S. K., Thiemermann, C., et al. (2015). Refinement of animal models of sepsis and septic shock. Shock 43(4): 304-316.
  5. Mastroeni, P. and Grant, A. J. (2011). Spread of Salmonella enterica in the body during systemic infection: unravelling host and pathogen determinants. Expert Rev Mol Med 13: e12.
  6. Navarre, W. W., Zou, S. B., Roy, H., Xie, J. L., Savchenko, A., Singer, A., Edvokimova, E., Prost, L. R., Kumar, R., Ibba, M., et al. (2010). PoxA, yjeK, and elongation factor P coordinately modulate virulence and drug resistance in Salmonella enterica. Mol Cell 39(2): 209-221.
  7. Bode, J. G., Ehlting, C. and Haussinger, D. (2012). The macrophage response towards LPS and its control through the p38(MAPK)-STAT3 axis. Cell Signal 24(6): 1185-1194.
  8. Bumann, D. and Schothorst, J. (2017). Intracellular Salmonella metabolism. Cell Microbiol 19(10).
  9. de Jong, H. K., Parry, C. M., van der Poll, T. and Wiersinga, W. J. (2012). Host-pathogen interaction in invasive Salmonellosis. PLoS Pathog 8(10): e1002933.
  10. Keestra-Gounder, A. M., Tsolis, R. M. and Baumler, A. J. (2015). Now you see me, now you don't: the interaction of Salmonella with innate immune receptors. Nat Rev Microbiol 13(4): 206-216.
  11. Mosser, D. M. and Edwards, J. P. (2008). Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8(12): 958-969.
  12. Murray, P. J. (2017). Macrophage Polarization. Annu Rev Physiol 79: 541-566.
  13. Nagai, Y., Akashi, S., Nagafuku, M., Ogata, M., Iwakura, Y., Akira, S., Kitamura, T., Kosugi, A., Kimoto, M. and Miyake, K. (2002). Essential role of MD-2 in LPS responsiveness and TLR4 distribution. Nat Immunol 3(7): 667-672.
  14. Nairz, M., Theurl, I., Schroll, A., Theurl, M., Fritsche, G., Lindner, E., Seifert, M., Crouch, M. L., Hantke, K., Akira, S., et al. (2009). Absence of functional Hfe protects mice from invasive Salmonella enterica serovar Typhimurium infection via induction of lipocalin-2. Blood 114(17): 3642-3651.
  15. Steele-Mortimer, O. (2008). The Salmonella-containing vacuole: moving with the times. Curr Opin Microbiol 11(1): 38-45.
  16. Steinman, R. M. and Hemmi, H. (2006). Dendritic cells: translating innate to adaptive immunity. Curr Top Microbiol Immunol 311: 17-58.

简介

鼠伤寒沙门氏菌 (S. Typhimurium) 是一种革兰氏阴性、兼性细胞内细菌,可导致人类胃肠道疾病和小鼠全身性伤寒样感染。 我们目前关于细胞和体液免疫参与防御鼠伤寒沙门氏菌感染的知识主要基于减毒菌株的动物模型。 先天免疫系统的细胞是抵御细菌的第一道屏障。 我们建立了一个强大的实验模型来表征这些细胞类型及其在宿主-病原体相互作用期间的反应。 因此,该协议侧重于通过采用多色流式细胞术来表征受感染动物脾脏中的巨噬细胞、单核细胞和中性粒细胞。

背景

肠沙门氏菌是一种高度铁依赖的嗜铁细胞内革兰氏阴性病原体,可引起局部肠道疾病,或严重的全身感染和败血症。因此,它被列入世界卫生组织对人类健康最严重的传染病威胁清单(Keestra-Gounder等人,2015 年;Bumann 和 Schothorst,2017 年) 。沙门氏菌在所谓的含沙门氏菌空泡 (SCV)中侵入并在肝脏、脾脏、淋巴结和派尔斑块中的单核吞噬细胞内增殖(Steele-Mortimer, 2008) 。然而,沙门氏菌表现出不同的机制来逃避抗菌活性,例如通过抑制吞噬溶酶体融合(Baumler 和 Fang,2013;Bhutta等人,2018) 。
几种免疫细胞在防御细菌感染方面很重要。虽然一些作为第一道屏障,通过吸收和杀死细菌,但另一些则负责长期免疫或协调有效的抗微生物免疫反应。巨噬细胞、树突状细胞(DC)和中性粒细胞通过识别特定的病原体相关分子模式 (PAMP) 和危险相关分子模式 (DAMP),代表了抵御沙门氏菌等入侵病原体的第一道防线(de Jong等人)等人,2012) 。巨噬细胞中革兰氏阴性菌的识别涉及 LPS 与受体复合物的结合,导致促炎和抗炎细胞因子的分泌(Nagai等人,2002;Bode等人,2012) 。巨噬细胞的极化取决于微环境中的局部因素、微生物刺激和其他细胞分泌的细胞因子。促炎巨噬细胞是抗微生物宿主防御机制的重要组成部分,其特征在于诱导型一氧化氮合酶 (iNOS) 的合成。抗炎巨噬细胞通过产生抗炎细胞因子和精氨酸酶-1 (ARG1) 来抑制炎症状态。 ARG1 裂解 iNOS 的底物 L-精氨酸,从而削弱对各种细胞内病原体的控制(Mosser 和 Edwards,2008;Murray,2017) 。
几项研究表明,沙门氏菌也被树突状细胞吞噬,树突状细胞是先天免疫和适应性免疫之间的重要联系(Steinman 和 Hemmi,2006) 。在细菌吞噬内化后,DC 成熟并迁移到确定的淋巴组织。因此,它们增加主要组织相容性复合物 II (MHCII) 的表达,以将特定抗原呈递给 T 细胞,从而启动适应性免疫反应。
由于对抗生素的多药耐药性的广泛发展,目前对侵袭性沙门氏菌病的抗生素治疗通常并不成功。此外,尚不确定标准抗生素是否可以渗透到含沙门氏菌的液泡中。因此,有必要更好地了解沙门氏菌病中的宿主-病原体相互作用,以开发针对细胞内病原体的新型有效抗菌疗法(Lahiri等人,2010;Navarre等人,2010;Mastroeni 和 Grant,2011) 。

关键字:巨噬细胞, 细胞内细菌, 革兰氏阴性菌, 感染控制

材料和试剂
1.10厘米细菌培养皿(Falcon,目录号: 351029)
2.细胞过滤器100 µm(Falcon,目录号:352360)
3.5 毫升注射器(BD,Discardit TM II 309050)
4.无菌楔形吊具(Microspec,目录号: 211738)
5.注射器(30G × ½'' 0.3 mm × 12 mm,Braun,Omnican F 9161502S)
6.1.5 mL Eppendorf Tubes(Eppendorf,目录号:T9661)
7.96孔圆底板(BRAND,目录号:781600)
8.冷冻管2 mL圆底(Simport,目录号:T311-3)
9.沙门氏菌鼠伤寒血清型 ATCC14028 (ATCC)
10.LB Broth Lennox(Roth,目录号:X964.2)
11.磷酸盐缓冲盐水(PBS)(Lonza,目录号: 17-515 F)
12.Agar-Agar Kobe I(Roth,目录号: 5210.3)
13.多聚甲醛(Sigma-Aldrich,福尔马林溶液,中性缓冲 10%,HT501128)
14.APC 抗小鼠 CD11b(BioLegend,目录号: 101212)
15.FITC 抗小鼠 CD45(BioLegend,目录号: 103108)
16.BV421 大鼠抗小鼠 F4/80(BD Biosciences,目录号: 565411)
17.BV510 抗小鼠 Ly6C(BioLegend,目录号: 128033)
18.PerCP-eFluor TM 710 抗小鼠 Ly6G(Invitrogen,目录号: 46-9668-82)
19.PerCP/Cyanine5.5 抗小鼠 MHCII(BioLegend,目录号: 116416)
20.BV421抗小鼠CD11c(BioLegend,目录号: 117330)
21.PE-Cyanine7抗小鼠iNOS(Invitrogen,目录号: 25-5920-80)
22.抗小鼠 ARG1(Invitrogen,目录号:17-3697-82)
23.甘油(Sigma-Aldrich,目录号:G5516)
24.胎牛血清热灭活(FBS)(PAN Biotech,目录号:P30-3031)
25.乙酸(EDTA) ( Sigma-Aldrich,目录号: E9884)
26.氯化铵( NH 4 Cl)( Sigma-Aldrich,目录号: 254134)
27.碳酸氢钾 ( KHCO 3 ) ( Sigma-Aldrich,目录号:237205)
28.乙二胺四乙酸二钠盐( Na 2 EDTA)( Sigma-Aldrich,目录号:E5134)
29.Triton X-100(Roth,目录号:3051.3)
30.LB-琼脂板(见食谱)
31.ACK 裂解缓冲液(参见配方)
32.FACS 缓冲液(见配方)
33.Triton 缓冲液(见配方)
34.LB琼脂板(见食谱)
35.LB培养基(见食谱)
36.4% PFA(见食谱)
37.含有 30% 甘油的 LB 培养基(参见食谱)




设备


1.培养箱37°C(ELOS Breed,目录号:B110N)
2.摇动培养箱(VWR,GFL,目录号:3031)
3.光度计(Eppendorf,BioPhotometer D30,目录号:6133000001)
4.离心机(Hettich Micro 200R,目录号:Z652113)
5.CytoFLEX S V4-B4-R2-I2 流式细胞仪(13 个检测器,4 个激光器,Beckman Coulter,目录号:C01161)
6.CASY TT 计数系统(OMNI Life Science,目录号:TT-20A-2571)




软件


1.FlowJo v10.7.0(BD Biosciences, https: //www.flowjo.com/solutions/flowjo )
2.GraphPad Prism V8.4.1 ( https://www.graphpad.com/scientific-software/prism )




程序


A.沙门氏菌鼠伤寒血清型ATCC14028 在锥形瓶中,直到它们达到对数生长期


1.打开原始小瓶并在 1 mL 的 LB 培养基中重新水合整个颗粒。
2.通过上下移液充分混合。
3.移液器 10 μL 在 10 mL 的 LB 培养基中作为预培养。


 


4.将预培养物在旋转培养箱中以 200 rpm 在 37°C 下孵育过夜。
5.第二天,在锥形瓶中的 10 mL LB 培养基中吸取 50 μL 的这种过夜预培养物。
6.在 37°C 下以 200 rpm 在n 轨道摇床上孵育1-2 小时,直到 OD 600为0.5(在光度计中使用 LB 介质作为空白测量) 。


B.计数活的肠沙门氏菌鼠伤寒血清型 使用例如 CASY TT 计数系统


1.使用 45 µm 毛细管。
2.通过在测量单元下方放置一个装有新鲜 Casy ton 缓冲液的新 Casy 杯来测量背景。
3.选择背景测量程序(表 1)。
4.测量背景。这应该低于 30 个计数和 1 µm 大小。否则,清洗系统。
5.用 10 mL 的 Casy ton 缓冲液准备一个新的 Casy 杯,并添加 5 μL 的S。 鼠伤寒溶液。
6.轻轻摇晃。
7.将样品放在测量单元下方。
8.选择测量 1 – 3 µm 的程序(表 1)。
9.措施。
10.单击下一步以获取每毫升的存活计数 = 存活S.tm /mL。
11.测量完成后,取出样品杯,加入一个新的装有 10 mL Casy ton 缓冲液的 Casy 杯。
12.最多执行五次 Casy Clean。
13.选择洗涤程序(表 1)。
14.洗涤完成后,检查背景。
15.如果背景低于30,可以关闭Casy计数系统;否则,继续洗涤。


表 1. CASY TT 计数系统程序


背景测量测量 鼠伤寒沙门氏菌洗涤程序


毛细管45 µm X 轴:5 µm45 µm X 轴:3 µm45 µm X 轴:5 µm
样品量200 µL 循环:1200 µL 循环:3200 µL 循环:10
稀释1.00 × 10 02.00 × 10 31.00 × 10 0
Y轴汽车汽车汽车
评估光标1.00–4.89 µm0.75–2.93 µm0.00–5.00 µm
规范。光标0.5–4.89 µm0.3–2.93 µm0.00–5.00 µm
%计算%通过碎片:开%通过碎片:开%通过碎片:开
聚合校正汽车汽车汽车
界面Par P.Feed:开Par P.Feed:开Par P.Feed:开
打印模式手动图形:开手动图形:开手动图形:开


C.小鼠腹腔注射


使用Omnican F 注射器 (30G × ½'' (0.3 mm × 12 mm),在 200 µL PBS 中腹膜内 (ip) 向小鼠注射 1000 个细菌。
阴性对照小鼠注射 200 µL PBS。
可在以下位置找到演示该过程的视频:小鼠腹腔内注射: https ://researchanimaltraining.com/articles/intraperitoneal-injection-in-the-mouse/ 。


 


D.在感染期间监测小鼠


 


1.体重:
应每天在同一时间测量体重,以避免因食物摄入而导致行为改变。使用可上锁的盒子和可以消毒的精密秤很重要。
2.体温:
每天在同一时间用红外线温度计测量体温。
拿起鼠标的尾巴中间,露出它的腹部。让老鼠用前爪抓住笼子的网格,这样老鼠就可以伸展上半身并露出腹部。 握住扳机测量温度,注意在腹部的同一位置进行测量。
3.小鼠的一般外观:
小鼠的一般外观应每天记录两次,例如根据Lilley等人的 M-CASS 评分表。 (2015),以及您的实验动物许可证。必须防止严重的疼痛、痛苦和痛苦。


E.脾细胞的分离


1.感染 72 小时后,分离脾脏,并用 5 mL 注射器的柱塞将器官通过 100 μm 细胞过滤器压到带有 5 mL 冷 PBS 的培养皿上。
2.以300 × g离心 4°C 5 分钟,然后用 5 mL PBS(300 × g 4°C 5 分钟),弃去上清液。
3.通过在 2 mL 的 ACK 裂解缓冲液中重新悬浮,使细胞颗粒进行红细胞裂解。
4.在 RT 孵育 2 分钟。
5.用 5 mL 的 FACS 缓冲液(300 × g 4°C 下 5 分钟)。


F.先天免疫细胞的流式细胞仪染色


所有程序均在 1.5 mL Eppendorf 管中进行。
1.细胞外染色(表 2)
a.在每个样品的 100 μL FACS 缓冲液中制备适当的抗体混合物(1:200)(表 2)。


表 2. 流式细胞术抗体——细胞外染色


抗体克隆公司目录编号表达于
CD11b APCM1/70生物传奇101212中性粒细胞、单核细胞
CD45 FITC30-F11生物传奇103108白细胞
F4/80 BV421T45-2342BD 生物科学565411巨噬细胞
Ly6C BV510HK1.4生物传奇128033单核细胞
Ly6G PerCP-eFluor TM 7101A8-Ly6g英杰公司46-9668-82中性粒细胞
MHCII PerCP/Cyanine5.5AF6-120.1生物传奇116416树突状细胞
CD11c BV421N418生物传奇117330树突状细胞


a.重悬此混合物中的细胞沉淀。
b.4°C 避光孵育 15 分钟。
c.用 1,000 μL 的 FACS 缓冲液(300 × g 4°C 下 5 分钟)。
d.在 4% 多聚甲醛 (PFA) 中重新悬浮颗粒,以固定细胞进行细胞内染色。
e.转移到 96 孔圆底板中。
f.直接在流式细胞仪中分析。
2.细胞内染色(表 3)
a.- d中的说明制备细胞外染色剂。
b.在 4% PFA 中重新悬浮颗粒。
c.在黑暗中在 4°C 下孵育 15 分钟。
d.离心并在 Triton 缓冲液中溶解沉淀。
e.充分混合并在黑暗中在室温下孵育 15 分钟。
f.在每个样品的 50 μL Triton 缓冲液中制备适当的抗体混合物(1:100)(表 3)。


表 3. 流式细胞术抗体 - 细胞内染色


抗体克隆公司目录编号表达于
iNOS PE-花青7长鑫通英杰公司25-5920-80促炎巨噬细胞
ARG1 装甲运兵车A1exF5英杰公司17-3697-82抗炎巨噬细胞


g.重悬此混合物中的细胞沉淀。
h.在 4°C 下孵育 45 分钟。
i.用 1,000 µL Triton 缓冲液(300 × g 4°C 下 5 分钟)。
j.在 4% PFA 中重新悬浮颗粒。
k.转移到 96 孔圆底板中。
l.直接在流式细胞仪中分析。




数据分析


FlowJo 软件可用于分析数据。绘制线性 FSC(前向散射)与 SSC(侧向散射)点图,并创建一个门以选择所有单元格。使用这些细胞,选择 CD45 +细胞(泛白细胞),并遵循图 1 中描述的门控策略。
在 GraphPad Prism 中执行和生成统计分析和图表。当数据呈正态分布时(使用直方图和统计工具进行评估,例如 Shapiro-Wilk 检验),通过未配对的学生 t 检验分析具有相等方差s 的样本,或通过双向 ANOVA( F检验)进行分析。当数据不是正态分布时,可以执行 Mann-Whitney U 或秩检验的 ANOVA。
 


图 1. 先天免疫细胞分析的门控策略(感染小鼠 - 上图,未感染小鼠 - 下图)。 
(A) 前向散射 (FSC) 对抗侧向散射 (SSC),(B) 排除双峰,(C) 白细胞:CD45 + ,(D) 中性粒细胞:CD45 + Ly6G + CD11b + ,(E) 巨噬细胞:CD45 + CD11b - Ly6G - F4/80 + ,(F) 促炎巨噬细胞表达 iNOS,(G) 抗炎巨噬细胞表达精氨酸酶,(H) 树突状细胞:CD45 + Ly6G - CD11c + MHCII + ,(I) + (J) 单核细胞:CD45 + Ly6G - F4/80 + CD11b + ,(K) 炎症和驻留单核细胞:分别为 Ly6C高或 Ly6C低。




食谱


1.流式细胞仪缓冲器
PBS 含0.5% 热灭活 FBS 
2 毫米乙二胺四乙酸


2.ACK(氯化铵-钾)裂解缓冲液
150 毫米NH 4氯
10 毫米碳酸氢钾3
0.1 mM Na 2 EDTA
pH 7.2–7.4


3.海卫一缓冲液
PBS 和 0.05%海卫 X-100


4.LB琼脂板
2% LB-肉汤
1.5% 琼脂
高压灭菌器(121°C 20 分钟),冷却,移取 15 mL 移液器到 10 cm 细菌培养皿中。
储存于 4°C。


5.LB培养基
2% LB-肉汤
高压灭菌器(121°C 20 分钟)。


6.4% PFA
将 40 μL 的 PFA 添加到 960 μL 的 PBS 中。


7.含 30% 甘油的 LB 培养基
将 300 μL 的甘油添加到 700 μL 的 LB 培养基中。




致谢


作者 GW 得到了基督教多普勒协会的资助和 FWF(EPICROSS,I-3321)的 ERA-NET 资助,NB 得到了 FWF 博士学院项目 W1253 HOROS 的支持。该协议在Nairz等人之后进行了调整和修改。 (2009 年) 。




利益争夺


作者声明,该研究是在没有任何可能构成潜在利益冲突的商业或财务关系的情况下进行的。




伦理


动物实验由奥地利联邦科学与研究部根据指令 2010/63/EU 批准。




参考


Baumler, A. 和 Fang, FC (2013)。细菌病原体的宿主特异性。 Cold Spring Harb Perspect Med 3(12):a010041。
Bhutta, ZA, Gaffey, MF, Crump, JA, Steele, D., Breiman, RF, Mintz, ED, Black, RE, Luby, SP 和 Levine, MM (2018)。伤寒:前进之路。 Am J Trop Med Hyg 99(3_Suppl):89-96。
Lahiri, A.、Lahiri, A.、Iyer, N.、Das, P. 和 Chakravortty, D. (2010)。参观沙门氏菌感染的细胞生物学。 微生物感染12(11):809-818。
Lilley, E., Armstrong, R., Clark, N., Gray, P., Hawkins, P., Mason, K., Lopez-Salesansky, N., Stark, AK, Jackson, SK, Thiemermann, C.,等人。 (2015 年)。优化脓毒症和脓毒性休克的动物模型。 冲击43(4):304-316。
Mastroeni, P. 和 Grant, AJ (2011)。全身感染期间肠沙门氏菌在体内的传播:解开宿主和病原体的决定因素。 专家 Rev Mol Med 13:e12。
Navarre, WW, Zou, SB, Roy, H., Xie, JL, Savchenko, A., Singer, A., Edvokimova, E., Prost, LR, Kumar, R., Ibba, M.等。 (2010)。 PoxA、yjeK 和延伸因子 P 协同调节肠道沙门氏菌的毒力和耐药性。 摩尔细胞39(2):209-221。
Bode, JG, Ehlting, C. 和 Haussinger, D. (2012)。巨噬细胞对 LPS 的反应及其通过 p38(MAPK)-STAT3 轴的控制。 细胞信号24(6):1185-1194。
Bumann, D. 和 Schothorst, J. (2017)。细胞内沙门氏菌代谢。 细胞微生物学19(10)。
de Jong, HK, Parry, CM, van der Poll, T. 和 Wiersinga, WJ (2012)。侵袭性沙门氏菌病中的宿主-病原体相互作用。 PLoS Pathog 8(10):e1002933。
Keestra-Gounder, AM, Tsolis, RM 和 Baumler, AJ (2015)。现在你看到我了,现在你没有看到:沙门氏菌与先天免疫受体的相互作用。 Nat Rev 微生物13(4):206-216。
Mosser, DM 和 Edwards, JP (2008)。探索巨噬细胞活化的全谱。 Nat Rev Immunol 8(12):958-969。
默里,PJ(2017)。巨噬细胞极化。 Annu Rev Physiol 79:541-566。
Nagai, Y.、Akashi, S.、Nagafuku, M.、Ogata, M.、Iwakura, Y.、Akira, S.、Kitamura, T.、Kosugi, A.、Kimoto, M. 和 Miyake, K.( 2002)。 MD-2 在 LPS 反应性和 TLR4 分布中的重要作用。 Nat Immunol 3(7):667-672。
Nairz, M.、Theurl, I.、Schroll, A.、Theurl, M.、Fritsche, G.、Lindner, E.、Seifert, M.、Crouch, ML、Hantke, K.、Akira, S.等人_ (2009 年)。功能性 Hfe 的缺乏通过诱导 lipocalin-2 保护小鼠免受侵袭性肠沙门氏菌鼠伤寒血清型感染。 血液114(17):3642-3651。
斯蒂尔-莫蒂默,O.(2008 年)。含沙门氏菌的液泡:与时俱进。 Curr Opin Microbiol 11(1):38-45。
Steinman, RM 和 Hemmi, H. (2006)。树突状细胞:将先天免疫转化为适应性免疫。 Curr Top Microbiol Immunol 311:17-58。


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引用:Pfeifhofer-Obermair, C., Brigo, N., Tymoszuk, P. and Weiss, G. (2022). A Mouse Infection Model with a Wildtype Salmonella enterica Serovar Typhimurium Strain for the Analysis of Inflammatory Innate Immune Cells. Bio-protocol 12(7): e4378. DOI: 10.21769/BioProtoc.4378.
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