Analysis of phagocytosis induced TNF-α production of murine macrophage using traditional flow cytometry in a mixed population

Introduction
Phagocytosis is one of the most ancient immune defenses and is vastly conserved across evolution. It is critical not only in pathogen defence, but also in the clearance of dead or dying cells. Traditionally, phagocytosis was studied using light or fluorescence microscopy to quantify particle internalization, but this technique is meticulous, slow, and usually generates statistically weak data. By utilizing flow cytometry, fluorescent target particles associated (both surface bound and internalized) with tagged effector cells can be analyzed at high speed to generate large statistically robust datasets. In addition, other downstream effects of phagocytosis can be analyzed in a multi-parametric manner using other fluorescent markers and channels. Here in we demonstrate the use of traditional flow cytometry to analyze the production of TNF-α by murine macrophages in relation to bacterial internalization.

Materials and Methods

Intracellular staining of RAW macrophages following phagocytosis of Eschericheri coli.
Cultured murine macrophage were incubated with GFP-E. coli according to MMI Lab 3 protocol. Briefly, 1x10⁶ RAW murine macrophage and Jurkat T-cells were seeded into complete DMEM with GFP expressing DH5α E. coli at a 21.1:1 target to effector ratio. Tubes were incubated with Golgiplug (BD) at 37°C and 5% CO₂. Samples were then spun down at 200 x g and washed twice with PBS before blocking with FACS buffer (2% serum) and incubated with rat anti-mouse αCD45-PE IgG (Biolegend) for 30 minutes, washed twice and resuspended in Cytofix buffer and held at room temperature for 20 minutes. Following PBS wash, cells were resuspended in permeablization buffer (0.01% Triton X-100) and held at room temp for 10 minutes before incubation with rat anti mouse αTNFα IgG-APC (Biolegend) for 30 minutes at 4°C. Samples were washed with permeablization buffer and spun at 326 x g twice before being stored overnight at 4°C in PBS then being run on a BD FACS Canto II. Small debris and potential clumps were gated out using gating techniques described in MMI 590 Lab 1.

Results and Discussion

Antibody staining allows for high resolution of gating on desired cell populations.
Although forward and side scatter can sometimes be used to separate out morphologically distinct cells, side scatter profiles often overlap and prevent analysis of a population of interest. In this experiment, RAW macrophage and Jurkat T-cells are seen to overlap quite heavily in SSC-FSC plots (Figure 1a). The use of fluorescently tagged αCD-45 antibodies allows a much higher level of resolution when separating these two populations. Although CD-45 is a pan- leukocyte marker, this particular antibody is against murine CD-45 and does not cross-react with human CD-45. Thus the antibody stains RAW cells, which are of murine origin, but not human Jurkat cells and allows for downstream analysis of each population respectively (Figure 1b).

Figure 1. Analysis of distinct cell types in a mixed population using surface markers. Co-incubated RAW murine macrophage and Jurkat human T-cells were acquired on a BD FACS Canto II. a) Forward and side scatter profile of the mixed population P1 gate (RAW cells orange, Jurkat cells Green based on P4/P5 gates) b) Total single cell events were gated based on fluorescence of anti-mouse CD45 antibody conjugated to a PE fluorochrome.

Raw macrophage exposed to E. coli produce TNFα regardless of bacterial internalization.
Macrophage are professional phagocytes, and tend to be some of the first responders in infection which recruit other cells to the site and activate them via chemokine/cytokine production. Thus, as expected, our RAW macrophages responded quickly when exposed to E. coli, being roughly 15% and 22% phagocytic by 30 and 60 minutes respectively (Figure 2, GFP+). This rate seems low in relation to total TNFα producing, APC+ RAW cells (85% and 95% at 30 and 60 minutes respectively), but may be due to paracrine action of cytokine production by phagocytic cells. Though this scenario seems unlikely since the addition of golgiplug should prevent the secretion of any produced cytokines. Most likely, these non phagocytic TNF producing cells are being activated through bacterial PAMPs present in the culture media binding through TLRs. A non-stimulated control could also be used to see if TNF production is constitutive in this cell line.

 Since Jurkat cells are lymphocytes, which are not known to be phagocytic except for in specific species, we expect them to not internalize any bacteria.  As expected, the vast majority of the Jurkat cells were negative for bacterial internalization. The very small proportions of GFP+ Jurkat cells (1.7% and 2.5% at 30 and 60 minutes respectively) could be due to a bacterial cell stuck to the outside of a Jurkat cell, which would likely not change the area enough to be removed as a clump using a height to surface area gate. This is where traditional flow cytometry falters, as it cannot distinguish associated fluorescence from true internalization. The use of imaging flow cytometry could eliminate these events and greatly increase confidence in the data.

 CD4+ T helper cells have been known to produce TNFα in macrophage co-cultures. Since Jurkat cell lines are CD4+ derived, we expected some level of TNFα production, but virtually no TNF staining was seen in these cell types. This could be explained by murine macrophages not being able to stimulate human T cells. Alternatively, there may be TNF production, but since we used an anti-mouse TNF antibody, there may simply be no cross-reactivity and thus the cells appear to be TNF(-). A repeated experiment using anti-human antibodies may be useful in deciphering these results.

Figure 2. Macrophage exposed to bacteria produce TNFα regardless of bacterial internalization. Mixed RAW macrophage and Jurkat T cells were incubated for 30 or 60 minutes with 22.1:1 GFP E. coli. Populations gated based on anti-mouse CD-45 staining. Samples were also stained with anti-mouse anti-TNFα antibody conjugated to APC. Population proportions are noted in the corner of each respective gate.