1. Introduction
Macrophages are a cell population that comprises a variety of cell subpopulations, each one displaying specific phenotypic and functional characteristics. Macrophages are necessary to maintain an optimal immune response against pathogens. Likewise, they play a crucial role in maintaining tissue homeostasis under sterile conditions [
1].
Classically, macrophages are sub-grouped into M1, a more proinflammatory subpopulation, and M2, which are known as anti-inflammatory [
2]. However, in 2011, an even smaller subpopulation of monocytes/macrophages was described, which expresses the CD3–T cell receptor (TCR) complex, hereafter called CD3+ macrophages [
3]. Although there have been significant advances in the description of this subpopulation, currently, several questions regarding their origin, signaling, and function are still open. It was considered a dogma that the functional expression of the CD3–TCR complex is exclusive to T cells. Structurally, it is composed of eight chains, two of the TCR that could be αβ or γδ, and the CD3 that is constituted by six chains that form two heterodimers CD3Σγ and CD3Σδ, and the CD3ζζ homodimers [
4,
5]. CD3–TCR complex signaling in T cells is well known. First, the TCR is responsible for identifying the antigen; posteriorly, through phosphorylation of the CD3 chains, several molecules with different functions are recruited, such as protein tyrosine kinases (PTK), including Lck and Fyn (Src family), ZAP-70 (Syk family), linker for the activation of T cells (LAT), and mitogen-associated protein kinase (MAPK), among many others. Subsequently, this signaling activates nuclear factors, including nuclear factor-kappa B (NF-κB), nuclear factor of activated T cells (NFAT), and activator protein 1 (AP-1), to induce the T cell response [
6,
7,
8,
9].
It has been described that CD3+ macrophages express TCR that requires the RAG1/RAG2 system to be generated; they were identified for the first time in the granuloma of tuberculosis patients [
3]. More recently, evidence with murine models of mycobacterial infections, specifically with the
Mycobacterium bovis Bacillus Calmette–Guérin (BCG), demonstrated that the transmembrane form of tumor necrosis factor (tmTNF) and its receptor 1 (TNFR1) are indispensable to maintain the presence of this myeloid subpopulation [
10,
11]. Interestingly, around 15% of human monocyte-derived macrophages (MDMs) are CD3+, and in response to anti-CD3 and anti-TNF stimuli, these CD3+ macrophages exhibit a specific proinflammatory profile [
11].
Although several reports have demonstrated the relevance of this subpopulation in the context of mycobacterial infections [
3,
10,
11,
12,
13]; it is important to note that the presence of CD3+ myeloid cells has been described in several other inflammatory pathologies such as atherosclerosis, malaria, and hypersensitivity pneumonitis, among others [
14,
15,
16,
17,
18], strengthening the hypothesis that this subpopulation is essential to mediate the inflammatory process.
It is currently unclear whether the CD3 expression on macrophages is constant or cyclic; similarly, it is still unclear which molecules could participate in the CD3 downstream signaling to favor proinflammatory processes. The main issue for the study of this pathway is the low frequency of CD3+ monocytes/macrophages, representing a critical limitation to the design of experimental strategies. Previously, we demonstrated that a fraction of murine RAW 264.7 macrophages (hereafter called RAW) are CD3+ after a BCG infection [
10]. This study aimed to clarify if this murine macrophage cell line could be a suitable cell model to study the expression and signaling of the CD3 molecule in myeloid cells.
2. Materials and Methods
2.1. Raw Cell Culture
RAW cell line was acquired from American Type Culture Collection (ATCC). These cells were unfrozen and were cultured at 1 × 106 cells in a TC-25 flask (Costar, ON, Canada) with RPMI 1640 medium supplemented with 10% FBS (GIBCO BRL, Grand Island, NY, USA), 100 unit/mL penicillin, 10 μg/mL streptomycin and 250 ng/mL amphotericin B (Sigma-Aldrich; Saint Louis, MO, USA).
After a 2-day incubation period (37 °C, 5% CO
2), cells were collected, and viability was determined by trypan blue exclusion using a TC-20 cell counter system (Bio-Rad, Hercules, CA, USA). The cells in culture were routinely 90 to 95% live cells. The cells were used in passages 10–15, according to previous reports [
19].
2.2. BCG Preparation and CFU
M. bovis BCG Pasteur strain 1172 P2 (Pasteur Institute, Paris, France) was cultured and grown to the log phase in 7H9 Middlebrook medium. Bacteria were stored at −80 °C in PBS plus 10% glycerol until use. The mycobacterial load was determined by plating serial 10-fold dilutions on agar 7H10 Middlebrook, supplemented with Oleic Albumin Dextrose Catalase (OADC, BD, Franklin Lakes, NJ, USA). BCG colonies were counted after incubation for at least 3 weeks.
2.3. BCG In Vitro Infection
RAW cells (1 × 106) were cultured in 12-well flat-bottomed cell culture plates (Sarsted, Nümbrecht, Germany) and infected with BCG Pasteur strain at MOI 0.1 and 1 for 2 h. At this time, the culture medium was replaced to eliminate non-phagocytosed bacteria. Infected cells were maintained in antibiotic-free RPMI 1640 (10% FBS, 37 °C, 5% CO2). Supernatants and cells were collected at 5 and 24 h. Cells were stained for flow cytometry or prepared for protein or nucleic acid analysis, and supernatants were stored at −20 °C until use.
2.4. Flow Cytometry
CD3 and mTNF evaluation were performed by multiparametric flow cytometry in unstimulated RAW cells, stimulated with anti CD3 (24 h) or infected with BCG (5 and 24 h) as described in each section.
For this evaluation, cells were collected and stained for 20 min at 4 °C with mAb to CD3, TNF, CD11b, and F4/80 (Biolegend, San Diego, CA, USA). Fixation procedures were performed to maintain TNF expression, followed by flow cytometric analysis. For intracellular evaluation of CD3, fixation and permeabilization were performed using the citofix/citoperm kit (BD, Franklin Lakes, NJ, USA; Cytofix/Cytoperm™ Plus) according to the manufacturer’s instructions.
Acquisition of cells was performed using a FACS Aria II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA, USA) and compensated with a single fluorochrome. Data were analyzed with FlowJo software (Tree Star, San Carlos, CA, USA). The cells used for FMO conditions were stained and acquired in parallel. Dead cells were omitted by the side scatter/forward scatter gating strategy, and isotype-matched control antibodies were used to identify background levels of staining. Typically, 50,000 events were recorded. More details of the antibodies used in this study are shown in
Table A1 (
Appendix A).
2.5. Enrichment of Human T Cells, CD14+ and Obtention of MDM
Blood samples were obtained from donors attending the blood bank at the Instituto Nacional de Enfermedades Respiratorias “Ismael Cosio Villegas”. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats using a density gradient as the standard Lymphoprep. In PBMCs, a positive selection for CD14+ cells was performed using magnetic microbeads coated with an anti-CD14 monoclonal antibody (Miltenyi Biotech, Bergisch Gladbach, Germany). The purity of the CD14+ fraction was analyzed by flow cytometry using anti-human CD14, CD2, and CD19 monoclonal antibodies from BioLegend. The efficiency of this process was regularly >90%. To promote the differentiation from monocytes to macrophages (MDMs), 2 × 10
6 CD14+ cells were cultured in Costar 6-well plates in RPMI-1640 culture medium (GIBCO, Grand Island, NY, USA), supplemented with 2 mM L-glutamine (GIBCO, Grand Island, NY, USA), 100μg/mL streptomycin, 100 IU/mL penicillin, and 10% fetal bovine serum (FBS, GIBCO, Grand Island, NY, USA) for 7 days at 37 °C, 5% CO
2. Then, MDMs were lysed with 200 µL of lysis buffer (Pierce RIPA buffer; Thermo Scientific, Waltham, MA, USA) to evaluate proteins or suspended in DNA/RNA Shield solution (Zymo Research, Irvine, CA, USA) and stored at −70 °C until use for RNA extraction. More details of the antibodies used in this study are shown in
Table A1 (
Appendix A).
2.6. Splenocytes and Bone Marrow Cells Obtained from C57BL/6 Mice
In this study, we used C57BL/6 wild-type male mice, 8–12 weeks old, housed in animal facility of the Instituto Nacional de Enfermedades Respiratorias “Ismael Cosio Villegas”. All animal experiments were carried out in accordance with institutional guidelines and were approved by the ethical committee on animal experimentation. Bone marrow cells were isolated from the femur of euthanized mice; meanwhile, splenocytes were collected from digested spleen tissue. Bone marrow cells and splenocytes were used as negative and positive controls, respectively, for CD3.
2.7. Sorting of CD3+ RAW Cells
To obtain 1.2 × 107 RAW CD3+ cells, we usually sorted 4 × 107 total RAW cells. RAW cells were washed with sterile PBS at 300 g, 10 min, resuspended in 400 µL of staining buffer and labeled with anti-CD3 and anti-F4/80 as previously described. After labeling, cells were resuspended in 5 mL of PBS plus 1% FBS to be acquired and sorted in the FACS-Aria-II cytometer. Instrument configuration was performed by the staff of Flow Cytometry Core Facility of the Instituto Nacional de Enfermedades Respiratorias Ismael Cosío Villegas in Mexico City. The purity of the CD3+ fraction was confirmed by analyzing a sample of 1 × 105 cells by flow cytometry. The efficiency of this process was routinely >90%.
2.8. RAW Cells Stimulated with Anti-CD3 mAb
Indicated wells were coated with the anti-CD3 antibody or isotype control at 1 µg/mL (Biolegend, San Diego, CA, USA) in sterile PBS and incubated at 5% CO2 at 37 °C overnight a day before the experiment. Unbound anti-CD3 was removed on the day of the experiment, then 5 × 105 of total or sorted RAW cells were plated and incubated for 24 h in RPMI-1640 culture medium (GIBCO, Grand Island, NY, USA), supplemented with 2 mM L-glutamine (GIBCO, Grand Island, NY, USA), 100 μg/mL streptomycin, 100 IU/mL penicillin, and 10% fetal bovine serum (FBS, GIBCO, Grand Island, NY, USA) for 24 h at 37 °C, 5% CO2. Supernatant and cells were collected for ELISA and WB analysis.
2.9. RNA Extraction and q-PCR
Uninfected and infected RAW cells at 0, 5, and 24 h post-infection were suspended in DNA/RNA Shield solution (Zymo Research, Irvine, CA, USA) and stored at −70 °C until use for RNA extraction. Total RNA was obtained with the RNeasy Micro Kit (Qiagen, Hilden, Germany), and genomic DNA contamination was eliminated using RNA-Free DNAse Set (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Nucleic acid quantification was performed with the Qubit™ assay kit and Qubit 2.0 Fluorometer (Life Technologies, Waltham, USA). RNA in amounts of 500 ng and 100 ng (from RAW cells) or 100 ng (from human MDM and T cells) was used for cDNA conversion using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, USA) in a reaction volume of 20 µL following the manufacturer’s guidelines. Relative gene expression was evaluated by quantitative real-time PCR (qPCR) using the following TaqMan probes for mouse: TNF (Mm00443258_m1), CD3 (CD3e gene) (Mm01179194_m1); 18S (Rn18s, Mm03928990_g1), and ACTB (β-actin) (Mm00607939_s1). Furthermore, TaqMan probes used for human genes were TNF (Hs00174128_m1), CD3 (CD3e gene) (Hs01062241_m1), 18S (18S ribosomal RNA gene) (Hs03928990_g1), and ACTB (β-actin) (Hs01060665_g1). Single reactions were prepared with the Maxima Probe/ROX qPCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), diluting 3 to 1 of cDNA. Amplifications were performed in duplicate under the following thermal conditions: 95 °C for 10 min, followed by 40 cycles of 60 °C for 1 min and 95 °C for 15 s in the Step One Plus Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA).
Relative gene expression (R.E.) was determined using the ΔΔCT method to calculate the n-fold change for each target gene under each experimental condition. Results were normalized to their corresponding endogenous controls depending on origin cells (murine or human); thus, ACTB and 18S were specific for murine or human. Furthermore, gene expression was calculated relative to the corresponding reference group; for assessment of the anti-CD3 effect at different times, we used unstained RAW cells as the reference group, and for evaluation after BCG infection, uninfected RAW cells, where 2−ΔΔCT = 1. To compare human and mouse CD3 gene expression, first, the ΔCT was calculated considering normalizing to their corresponding housekeeping gene for mouse or human origin as described above. Then, relative expression was determined using uninfected-MDM.
2.10. Immunoprecipitation
After removing the medium, RAW cells were washed in PBS and lysed in ice-cold Pierce IP lysis buffer (Thermo Scientific, Waltham, MA, USA) containing Protease Inhibitor Cocktail P8340 (Sigma-Aldrich, Fluka, MO, USA) for 15 min at 4 °C. Lysates were harvested and centrifuged at 13,000× g/10 min to eliminate nuclei. Then, IP was performed using Dynabeads Protein G Kit and monoclonal antibodies for mouse CD3 (BioLegend, San Diego, CA, USA). IP was realized following the manufacturer’s guidelines, and precipitated proteins were then analyzed by electrophoresis and posteriorly by immunoblot.
2.11. Western Blot (Immunoblot)
Cells were lysed on ice for 10 min using Pierce RIPA buffer (Thermo Scientific, Waltham, MA, USA) containing phosphatases and protease inhibitor (Roche, Basel, Switzerland). Protein quantification was performed in lysates with the Pierce BCA assay protein kit (Thermo Scientific, Waltham, MA, USA), following the manufacturer’s guidelines. Then, lysates were boiled for 10 min in 2× SDS Laemmli sample buffer. Usually, 20–25 micrograms of protein extract were separated by SDS–PAGE, transferred onto nitrocellulose membranes, and analyzed by immunoblotting with mAbs for NFAT, IKK-α, pC-Jun, NF-kB, and pCD3ζchain. β-Actin, β-Tubulin, or GAPDH were used as load control. Western blot analysis was performed using ImageLab software V.6.1. (Bio-Rad, Hercules, CA, USA). Volume boxes were used to define protein bands in samples and controls. Data were normalized using housekeeping proteins as loading controls. This analysis was performed to obtain relative comparisons between different culture conditions. More details on the antibodies used in this study are shown in
Table A1 (
Appendix A).
2.12. Cytokine Measurement in Cell Culture Supernatants
Collected supernatants were thawed and analyzed by enzyme-linked immunosorbent assays (ELISA) to quantify cytokines such as IL-6 (Mouse IL-6 ELISA MAX Standard set Cat. No. 431301) and TNF (Mouse TNF-α ELISA Max Deluxe set Cat. No. 430904). The assays were performed following the manufacturer’s protocols (BioLegend, San Diego, CA, USA).
2.13. Statistical Analysis
Data analysis was performed using GraphPad Prism 9 (GraphPad Software, La Jolla, CA, USA). Data are presented as mean ± standard deviation (SD) and considered non-parametric. Multiple comparisons were performed with Kruskal–Wallis and corrected using Dunn’s test. When data are the means of means, they are presented as mean ± standard error of the mean (SEM), and the multiple comparisons were performed with one-way ANOVA followed by Bonferroni test. p-values < 0.05 were considered statistically significant.
4. Discussion
CD3+ myeloid cells are a small subpopulation, which can be found in peripheral blood (CD3+ monocytes) and tissue (CD3+ macrophages); however, under nonpathological conditions, only 12–15% of monocytes can be differentiated into CD3+ macrophages [
3,
12,
16]. Reports indicated that these cells can be activated by a CD3-dependent pathway to deliver proinflammatory cytokines by an—up till now—unknown pathway.
Although the primary cell culture is used as a model to study the cell signaling because it can provide information closer to what happens in an in vivo system, the low frequency of CD3+ myeloid cells is a significant limitation to design deep studies that allow us to clarify the CD3-dependent signaling. Thus, using a myeloid cell line to identify cell signaling is an alternative to screening molecules involved in the CD3-dependent cell activation; this information could be posteriorly translated to primary cultures of human cells to identify key molecules. In this study, we demonstrated the use of RAW cells as a new approach to studying the CD3 signaling in myeloid cells. Here, it is essential to note that a human monocyte cell line was also used; however, we could not identify the CD3 expression (data not shown).
Reports have described that CD3+ macrophages secrete proinflammatory cytokines such as TNF, IFN-γ, IP-10, CCL-2, and IL-1β, among others, by a CD3-dependent pathway [
11]. However, it is still unclear how the signal after CD3 stimulus is transduced. This knowledge is imperative to identify mechanisms to regulate its function under the different pathologies where this cell subpopulation has been identified. Probably, it also can provide highlights regarding its origin.
The role of macrophages is not limited to pathological conditions; they are necessary to repair tissue even under sterile conditions. We demonstrated that naïve mice have CD3+ myeloid cells in the lung tissue [
11]; in this regard, new questions are opened. For instance, it is necessary to know if a proinflammatory stimulus is required to induce CD3+ macrophage proliferation in situ, or if the monocytes’ recruitment is needed to increase the frequency of these cells during an inflammatory process.
CD3 expression has been almost exclusively associated with lymphoid cells, and the CD3-dependent signaling to favor T cell activation is well understood. Following the recent evidence reviewed by Ashouri JF [
22], it is summarized in
Figure 6 (left). Upon recognition by the TCR, intracellular downstream signals are initiated, and Lck kinase induces the phosphorylation of the CD3-ζ chain to favor the ZAP70 recruitment. Subsequently, it interacts with LAT to actively recruit LAT to the activated ZAP70. To regulate the effector functions of T cells, such as the delivery of cytokines, other molecules play an essential role; for example, the activated T cell increases its calcium level, increasing the function of calcineurin to dephosphorylate NFAT; then, it is translocated into the nucleus to regulate the expression of cytokines [
23]. Similarly, studies have shown that the c-Jun/c-Fos heterodimer functions downstream of the TCR to induce the synthesis of cytokines [
24].
In contrast to T cells, information on the CD3-signaling pathways in myeloid cells is limited. This work identified that RAW murine macrophages express CD3 at transcriptional and protein levels. Like lymphocytes, the CD3 expression on the cell surface is cycling even though it remains stable at the transcriptional level. Thus, our main aim was to support the use of this cell line as an option to design experimental strategies that allow us to identify the CD3-signaling pathway in myeloid cells. Although it is possible that the CD3-signaling in myeloid cells is not the same between humans and mice, this study can provide valuable information to facilitate future studies in human cells.
In this model, the anti-CD3 stimulus induces the delivery of the proinflammatory cytokines TNF and IL-6, but it is important to note that two different profiles could be observed, and that they were dependent on whether the RAW cells were mixed (CD3+/CD3−) or enriched. At baseline purified CD3+ showed a higher level of NFAT and pC-jun than did CD3-cells, but simultaneously, they delivered lower TNF and IL-6 levels, suggesting that the CD3 expression provides a different profile of activation. On the other hand, when all RAW cells were together, we did not identify changes in the involved molecules in the CD3-dependent pathway; we speculate that this was because the CD3 cell surface expression in RAW cells was not constant. So, the specific moment when the cell is activated by a CD3-dependent pathway is variable; consequently, these changes can be masked by the effect of CD3. Moreover, we cannot exclude the possibility that 24 h was too late to see differences in protein levels downstream of CD3 activation when heterogeneous RAW cultures were used because although we did not observe differences in the downstream molecules, we confirmed that at 24 h with the anti-CD3 stimulus, TNF and IL-6 were still very abundant in the medium supernatant.
In
Figure 6 (right), we summarized the current evidence for CD3 signaling in myeloid cells; the names of the new molecules identified in this work are indicated in red, and the names of molecules provided by previous reports are indicated in black. Thus, at present, it is still unclear if the TCRs expressed by some CD3+ myeloid cells are antigen-specific and if they can initiate phosphorylation of the CD3 chains, but we identified that this CD3 expresses both Σ and ζ chains. Moreover, it has been described that human macrophages constitutively express Lck, ZAP70, and LAT. Due to anti-CD3 stimulus, these cells produce proinflammatory cytokines, probably by a SOCS3-dependent pathway [
3,
11]. Our current results also showed that the CD3+ RAW cells display a high level of NFAT, pC-Jun, and IKK-a, suggesting that they could be participating in the CD3 downstream signaling, at least in mouse cells; future studies with human cells are necessary.
Figure 6.
Current knowledge of the CD3 downstream signaling in T cells and macrophages. T cells express the CD3/TCR complex, and these cells are activated after the TCR recognizes a specific antigen. Lck, ZAP70 and LAT are recruited to stimulate the nuclear factors NFAT and AP-1, to finally favor functions such as activation, proliferation, and cytokine production (left). CD3+ macrophages also express CD3, and some of them are TCR+. At present, it is still unclear if this TCR is antigen-specific, but these cells can be activated by anti-CD3 stimulus. Lck, ZAP70, LAT, IKK-a, NFAT and C-Jun play a role in activating the CD3+ macrophages and favor the delivery of pro-inflammatory cytokines such as TNF and IL-6 (right). The figure was created in BioRender.
Figure 6.
Current knowledge of the CD3 downstream signaling in T cells and macrophages. T cells express the CD3/TCR complex, and these cells are activated after the TCR recognizes a specific antigen. Lck, ZAP70 and LAT are recruited to stimulate the nuclear factors NFAT and AP-1, to finally favor functions such as activation, proliferation, and cytokine production (left). CD3+ macrophages also express CD3, and some of them are TCR+. At present, it is still unclear if this TCR is antigen-specific, but these cells can be activated by anti-CD3 stimulus. Lck, ZAP70, LAT, IKK-a, NFAT and C-Jun play a role in activating the CD3+ macrophages and favor the delivery of pro-inflammatory cytokines such as TNF and IL-6 (right). The figure was created in BioRender.
Previous reports indicated that in response to BCG infection, the frequency of CD3+ myeloid cells is increased in vivo. This was observed for cells of both human and murine origin [
3,
11,
12]. In this study, we did not identify an increased frequency of CD3+ RAW cells after BCG infection, but we observed that CD3 is increased at the transcriptional level 5 h post-infection (MOI 1). Therefore, the discrepancy in CD3 expression on the cell surface could be due to these cells being activated (because of the same infection). Consequently, the CD3 recycling is constantly happening, and it is a limitation to observation of the frequency of CD3+ RAW cells under this condition.
This study provides evidence that RAW cells are suitable as a model to study the CD3 signaling in myeloid cells. Identifying molecules involved in this pathway is necessary to modulate the function of the CD3+ myeloid cells observed in diverse pathologies.
This study is not free of limitations; perhaps the most important is that we used a cell line with a murine origin. However, we provided a suitable alternative to study CD3+ myeloid cell activation signaling in more detail. This model can be helpful to clarify questions such as the optimal timing for activation of CD3+ macrophages if there are co-stimulation mechanisms homologous to CD28 in macrophages, how the signal is amplified, and if these macrophages display a type of hybrid signaling in which innate receptors participate, among others. Finding the answer to these questions will highlight if there are pre-requisites for the complete activation of human CD3+ macrophages and which molecules may be involved in this pathway.