L.m strain 1778+H 1b (ATCC 43249, USA, Manassas, VA, USA) was grown in brain heart infusion broth (BHI, BD-Difco, USA) for 18 h at 37°C with constant shaking at 112 xg. Bacterial cultures were washed with Hanks Balanced Saline Solution (HBSS) (Life Technologies, USA) and bacterial pellets resuspended in RPMI-1640 Glutamax (Life Technologies, USA) supplemented with 40% Fetal Bovine Serum (Life Technologies, USA) and frozen at -70 °C until use. Aliquots of L.m were serially diluted and plated in BHI agar at 37°C for 18-24 h. Bacterial numbers were determined by counting Colony-Forming Units (CFU).

Bone Marrow-derived Mast Cell (BMMC) was obtained following the protocol described by (30). Briefly, bone marrow cells were obtained from femurs and tibias of 6-8-week-old C57BL/6 female mice. Cells were maintained in RPMI-1640 supplemented with 10% FBS, 5 μM β-mercaptoethanol (Life Technologies, USA) and 2% antibiotic and antimycotic (Sigma, USA) (complete RPMI 1640 medium) plus 10 ng/mL of murine recombinant IL-3 (Peprotech, USA) and 10 ng/mL of murine recombinant stem cell factor (Peprotech, USA)). Non-adherent cells were transferred to fresh culture medium twice a week for 6−9 weeks. The purity of BMMC was ≥90% assessed by flow cytometry after staining of CD117 (clone: 2B8, BioLegend, USA; 0.25 μg/mL) and FcϵRI (clone: MAR-1, BioLegend, USA; 0.16 μg/mL) ( Supplementary Figure 1A ).

Peritoneum-derived mast cells (PMC), cells were obtained from peritoneal cavity of mice and cultured in complete RPMI-1640 medium plus IL-3 (30 ng/mL) and SCF (20 ng/mL). Non-adherent cells were transferred to fresh culture medium twice a week for 3−4 weeks. The purity of PMC was ≥90% assessed by flow cytometry ( Supplementary Figure 1C ).

All experiments followed institutional biosecurity and safety procedures. All animal experiments were approved by the Research Ethics Committee of the ENCB, IPN (ZOO-016-2019).

2x10⁵ BMMC or 2x10⁵ PMC/0.25 mL of RPMI-1640 supplemented with 10% FBS and 5μM β-mercaptoethanol (complete medium) were plated in cytospin chambers, and then stained with toluidine blue for 10 minutes. Finally, slides were air-dried and mounted with Entellan resin (Merck Millipore, USA) under a coverslip. Images were captured with a digital camera attached to a brightfield microscope (Zeiss Primo Star, Germany), and analyzed with Micro capture v7.9 software ( Supplementary Figure 1B, D ).

2.5X10⁵ BMMC/0.25 mL of complete medium were stimulated with L.m at different MOI (1:1, 10:1, 100:1) or stimulated with LLO (125, 250, 500, 1000 ng/mL) for 24 h. Then cells were washed with 1 mL of Annexin V binding Buffer 1X (Invitrogen, USA) and stained with 1 μg/mL Annexin V (BioLegend, USA) and 0.5 μg/mL propidium iodide (eBioscience, USA). After staining, cell viability was measured by flow cytometry ( Supplementary Figure 2 ).

The degranulation assay was carried out as described previously (31). Briefly, 2x10⁵ BMMC/0.25 mL of complete medium were incubated with L.m at different MOI for 90 minutes. Then were washed and stained with anti-CD107a (clone: 1D4B Biolegend, USA; 0.25 μg/mL) and anti-FcϵRI. Staining was measured by flow cytometry.

β-Hexosaminidase release was performed as follows: 2x10⁵ BMMC/0.25 mL of HEPES-Tyrode Buffer (HBT) (130 mM NaCl, 5.5 mM glucose, 2.7 mM KCl, 1.0 mM CaCl₂ 2 H₂O, 0.1% [wt/vol] Bovine Serum Albumin (BSA), 12 mM HEPES, 0.45 mM NaH₂PO₄ 1H₂O, pH 7.2) were stimulated with L.m at different MOI or stimulated with PMA (125 nM) plus Ionomicin (10 μM) for 90 minutes at 37°C. The supernatants were then recovered, and the cell pellet was lysed with 200 μL of 0.2% Triton X-100 in HBT. Both supernatants and cell lysates were incubated with 4-methylumbelliferyl N-acetyl-β-D-glucosaminide (Sigma-Aldrich, USA; 1 mM in 200 mM Na Citrate Buffer pH 4.5) for 2 h at 37°C. The enzyme reaction was stopped by the addition of 100 μL of 200 mM Tris base, pH 10.7. The samples were analyzed in a fluorescence plate reader (SpectraMax M, USA) using excitation 356 nm and emission 450 nm. The percentage of release of β-Hexosaminidase is calculated using the formula: %   R e l e a s e = [ s u p e r n a t a n ( s u p e r n a t a n + c e l l p e l l e t ) ] × 100 .

2x10⁵ BMMC were cultured in 0.25 mL of RPMI-1640 without phenol red (Life Technologies, USA) and then, stimulated with L.m at different MOI for 120 minutes. In the last 15 minutes of incubation 25 μL of a solution of p-nitro blue tetrazolium (NBT) (Sigma-Aldrich, USA) at 1 mg/mL were added. Then, cells were washed with PBS 1X (Life Technologies, USA) and fixed with absolute methanol. The formazan precipitates were dissolved by adding 54 μL of potassium hydroxide (KOH) 2mM and 46 μL of Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, USA). The samples were read at a wavelength of 620 nm in a plate reader (Multiskan EX, Thermo Scientific, USA).

2x10⁵ BMMC/0.25 mL of complete medium were incubated with L.m at different MOI for 2 h. At the end of incubation, HBSS supplemented with ampicillin at 200 ng/mL was added for 1 h. Afterwards, cells were washed and then transferred to complete medium supplemented with ampicillin at 200 ng/mL for 0 and 24 h. Then, cells were washed with HBSS and then lysed with sterile distilled water. Cell suspension were homogenized, and serial decimal dilutions were prepared in saline solution. Afterwards, 20μL of each dilution were plated on BHI agar at 37°C for 48 h, bacterial numbers were determined by counting CFU.

2x10⁵ BMMC/0.25 mL of complete medium were stimulated with L.m at different MOI or stimulated with Recombinant Listeriolysin-O (LLO; RayBiotech, USA) at different concentration (125, 250, 500 and 1000 ng/mL) for 24h. Then, supernatants were collected for the detection of TNF-α, IL-1β, IL-6, MCP-1 (Biolegend, San Diego, CA., USA) and IL-13 (eBioscience, USA) by ELISA according to manufacturer’s instructions.

2x10⁵ BMMC/0.25 mL of complete medium were stimulated with L.m MOI 100:1 for 15 minutes to evaluate p-ERK 1/2, 30 minutes for p-p65 and 60 minutes for p-p38. Then, the cells were preserved with 250 μL of Fixation buffer (BD-Bioscience, USA) for 10 min/37°C. Subsequently, were washed with 1 mL of Stain Buffer (BD-Bioscience, USA) and permeabilized with 1mL of 0.5x Perm buffer IV (BD-Bioscience, USA) for 15 min at room temperature (RT) and protected from light. Then, the cells were washed with 1 mL of Stain Buffer. After blocking with 0.015 μg of anti-CD16/32 (Mouse BD Fc Block™, clone: 2.4G2. BD-Biosciences, USA) cells were stained with antibodies to p-ERK 1/2-PE (clone: 20A; 4 μL per tube), p-p65-PE (clone: K10-895.12.50; 4 μL per tube), p-p38-PE (Clone: 36/p38; 4 μL per tube) or isotype controls (Clone: MOPC-21; Mouse IgG₁, κ-PE or Clone: MCP-11; Mouse IgG_2b, κ-PE). (All from BD-Biosciences, USA) for 60 min at RT and protected from light. Finally, the cells were washed and resuspended in 0.15 mL of Stain Buffer and analyzed by flow cytometry. The times chosen for the detection of each phosphorylated protein were selected based on kinetic assay ( Supplementary Figure 4 ).

2x10⁵ BMMC/0.1 mL of PBS 1X were marked with anti-TLR2-biotin (clone: 6C2, eBioscience, USA; 1 μg/0.1 mL) or isotype control-biotin (clone: eB149/10H5, eBioscience, USA; Rat IgG_2b, κ-biotin; 1 μg/0.1 mL) and stained with streptavidin-APC (BD-Bioscience, USA; 0.02 μg/mL), prior blocking with anti-CD16/32. Staining was measured by flow cytometry.

2x10⁵ BMMC/0.25 mL of complete medium were pre-incubated 30 minutes with 200 ng/mL of anti-TLR2 (clone: C9A12; IgG2a) or 200 ng/mL of isotype control (clone: T9C6; 1gG2a), both from Invivogen, USA. Afterwards, cells were stimulated with L.m MOI 100:1 and the bacterial load, O 2 − production, degranulation, cytokines production and evaluation of phosphorylated proteins assays were performed as indicated above. The bacterial load was analyzed as endocytic activity and bactericidal activity, according to the following formula: endocytic activity = (CFU at 0 h/MOI added) *100 (%). Bactericidal activity = (100-(CFU at 24 h/CFU at 0 h) *100) (%) (32). To evaluate the efficiency of antibody-mediated TLR2 inhibition, 2x10⁵ BMMC/0.25 mL were pre-incubated with different concentrations of anti-TLR2 (12.5, 25, 50, 100 y 200 ng/mL) for 30 min and then stimulated with the TLR2 agonist: Staphylococcus aureus peptidoglycan (PGN) (Sigma-Aldrich, USA) at 10 μg/mL for 24 h. Then, supernatants were collected for the detection of IL-6 by ELISA ( Supplementary Figure 5 ).

All cell samples stained with fluorochrome-conjugated antibodies were acquired using FACSAria Fusion (BD Biosciences, USA) and analyzed with FlowJo software version 6.0 (FlowJo, LLC). Cell debris and doublets were excluded from the analysis.

All statistical analyses were performed with SigmaPlot software version 14.0, from Systat Software, Inc., San Jose California USA, www.systatsoftware.com. Data normality was assessed by Kolmogorov-Smirnov with Lilliefors correction. Variables that followed normal distribution were plotted as mean ± standard error mean (s.e.m), represented as bars and analyzed with one way-analysis of variance (ANOVA) with Student-Newman-Keuls (SNK) post-hoc. While variables that did not follow normal distribution, semi-quantitative variables, normalized variables or percentages, were plotted as median + range, represented as boxes and analyzed with the Mann-Whitney test (comparisons between two groups) or Kruskal-Wallis test (comparisons between more than two groups). A value of p < 0.05 was considered to be significant.

Previous studies have noticed that MC can respond to L.m infection through degranulation, ROS production, internalization, and clearance of this bacterium (16–18). To corroborate the presence of these MC activation mechanisms during L.m infection, we incubated BMMC with different MOI of this bacterium to determine degranulation either through the surface expression of CD107a ( Figure 1A ) or β-hexosaminidase release ( Figure 1B ), ROS production with detection of O 2 − ( Figure 1C ) and internalization and clearance of L.m by evaluating intracellular CFU ( Figure 1D ). We found that degranulation and O 2 − production from BMMC occurred using L.m MOI 100:1 ( Figures 1A–C ). Also, we detected viable bacteria within BMMC at 0-hour post-infection (hpi) with the different MOI of L.m tested; however, we recovered small amounts of this bacterium at 24h, corresponding to a reduction of more than 3 logarithms for any MOI tested ( Figure 1D ). These results indicate that BMMC degranulate and produce ROS with high amounts of L.m and that BMMC are capable of internalizing and controlling L.m infection.

Previous reports have shown that L.m induces the de novo synthesis of different cytokines and chemokines (16, 33). To further corroborate the MC response to L.m we determined the production of TNF-α, IL-1β, IL-6, IL-13 and MCP-1. We stimulated BMMC with different MOI of L.m and at 24 h we determined the levels of each mediator. We found that BMMC produced TNF-α with MOI 1:1, 10:1 and 100:1 of L.m ( Figure 2A ). While IL-1β production was only detected in BMMC incubated with MOI 100:1 of L.m ( Figure 2B ). Similarly, we found that this bacterium induced IL-6 ( Figure 2C ) and IL-13 ( Figure 2D ) only with MOI 100:1 of L.m in BMMC. While MCP-1 was induced with MOI 10:1 and 100:1 of L.m in BMMC ( Figure 2E ). Together, these results indicate that L.m induces de novo synthesis of cytokines and chemokines.

Since L.m promoted the activation of BMMC, we decided to evaluate if this was associated with the induction of some intracellular signals that are associated with TLR receptors (22). To this end, we incubated BMMC with L.m (MOI 100:1) and determined the phosphorylation of some signaling proteins by flow cytometry. Initially we evaluated the phosphorylation of ERK 1/2 ( Figure 3A ) and p38 ( Figure 3B ), molecules belonging to the MAPK signaling pathway and which have been related to the production of pro-inflammatory cytokines in macrophages infected with L.m (34, 35). Interestingly, we found that L.m induced phosphorylation of both signaling proteins in BMMC ( Figures 3A, B ). An important transcription factor in the production of pro-inflammatory cytokines and chemokines by macrophages infected with L.m is NF-κB (34). Therefore, we determined whether this transcription factor was activated in BMMC in response to L.m, by evaluating the phosphorylation of p65 subunit. Interestingly, we found that L.m induced phosphorylation of p65 in BMMC ( Figure 3C ). Together, these results indicate that L.m induces the activation in BMMC of cell signaling molecules that are associated with TLR activation.

One of the main receptors found in various cells of the innate immune response that recognizes different components of the cell wall of Gram-positive bacteria, such as L.m, is TLR2 (20). The importance of macrophage TLR2 for phagocytosis and ROS production during infection with L.m has been demonstrated previously (36). Furthermore, BMMC TLR2 is known to participate in degranulation upon stimulation with peptidoglycan (PGN) (26). Based on these findings, we decided to evaluate whether TLR2 is involved in recognizing L.m and promoting the activation mechanisms in MC. Because some studies have suggested that human MCs do not express TLR (37), we evaluated the expression of this receptor on the surface of BMMC by flow cytometry. As reported previously (18, 22), this receptor was present in BMMC ( Figure 4A ). Afterwards, we pre-incubated BMMC with anti-TLR2 or isotype control for 30 minutes and then added to L.m (MOI 100:1). Then again, we evaluated cell degranulation through CD107a expression, ROS production with O 2 − detection, endocytic and bactericidal activity with intracellular CFU. We found that TLR2 blockade did not affect degranulation ( Figure 4B ), the levels of O 2 − produced ( Figure 4C ). We did not observe any change in the endocytic or bactericidal activity of BMMC incubated with L.m when TLR2 was blocked ( Figures 4D, E ). Altogether, our results indicate that MC TLR2 is not involved in the development of degranulation, ROS production, endocytosis or bacteria clearance during L.m infection.

Previous studies have shown the importance of macrophage TLR2 activation for the secretion of IL-1β, IL-6, TNF-α, IFN-β during the infection with L.m (38, 39). Furthermore, MyD88-deficient BMMC are reduced in their ability to produce IL-6 and MCP-1 in response to L.m implying a role of TLR in MC response (16). Therefore, we decided to investigate whether this MC TLR2 could mediate de novo synthesis of the cytokines TNF-α, IL-1β, IL-6, IL-13 and MCP-1 in two different sources of MCs: peritoneal MCs (PMC) and bone-marrow-derived MCs (BMMC). To this end, we pre-incubated MCs with 200 ng/mL anti-TLR2, a dose that efficiently blocks the interaction of TLR-2 and its natural ligand ( Supplementary Figure 5 ), or its respective isotype control for 30 minutes and then stimulated with L.m MOI 100:1 for 24 h. We found that blocking TLR2 in both PMC and BMMC did not affect the levels of TNF-α ( Figure 5A ), IL-1β ( Figure 5B ) or MCP-1 ( Figure 5E ). However, we observed that blocking this receptor significantly reduced the levels of IL-6 ( Figure 5C ) and IL-13 ( Figure 5D ) in both PMC and BMMC. These results show that MC TLR2 does not participate in the production of TNF-α, IL-1β; and MCP-1; but regulates IL-6 and IL-13 production during L.m infection.

To discern which cell signaling pathway was regulated in MC by TLR2 during L.m infection, p38, ERK and p65 activation was evaluated after incubating MCs with anti-TLR2. Interestingly, we noticed that p38 phosphorylation was inhibited when TLR-2 was blocked, while ERK and p65 phosphorylation were not affected ( Figures 6A–C ). This result showed that p38 cell signaling is regulated by TLR2 in MCs during L.m infection.

Finally, previous reports indicate that L.m soluble products, like listeriolysin O, can activate MCs through increasing intracellular Ca²⁺ by altering endoplasmic reticulum independently of MCs receptors (17). To evaluate whether MC TLR2 was involved in LLO mediated activation, BMMCs were incubated with blocking antibodies to TLR-2 and stimulated with 1000 ng/mL LLO, a dose that did not induced MC apoptosis nor necrosis, but induced cytokine production ( Supplementary Figure 2 and 3 ). We noticed that MC released similar amounts of TNF-α, IL-6, IL-13 and MCP-1 in response to LLO independently of TLR2 ( Figures 7A–D ). These results indicated that TLR2 was not necessary for MC activation by LLO.

Collectively, our data show that MC activation by TLR2 during L.m infection regulates IL-6 and IL-13 production and suggest that other receptors could be involved in mediating other mechanisms of MC activation ( Figure 8 ).