Mast cells as rapid innate sensors of cytomegalovirus by TLR3/TRIF signaling-dependent and -independent mechanisms

Abstract

The succinct metaphor, 'the immune system's loaded gun', has been used to describe the role of mast cells (MCs) due to their storage of a wide range of potent pro-inflammatory and antimicrobial mediators in secretory granules that can be released almost instantly on demand to fight invaders. Located at host-environment boundaries and equipped with an arsenal of pattern recognition receptors, MCs are destined to be rapid innate sensors of pathogens penetrating endothelial and epithelial surfaces. Although the importance of MCs in antimicrobial and antiparasitic defense has long been appreciated, their role in raising the alarm against viral infections has been noted only recently. Work on cytomegalovirus (CMV) infection in the murine model has revealed MCs as players in a novel cross-talk axis between innate and adaptive immune surveillance of CMV, in that infection of MCs, which is associated with MC degranulation and release of the chemokine CCL5, enhances the recruitment of protective CD8 T cells to extravascular sites of virus replication, specifically to lung interstitium and alveolar epithelium. Here, we have expanded on these studies by investigating the conditions for MC activation and the consequent degranulation in response to host infection. Surprisingly, the data revealed two temporally and mechanistically distinct waves of MC activation: an almost instant indirect activation that depended on TLR3/TRIF signaling and delayed activation by direct infection of MCs that did not involve TLR3/TRIF signaling. Cell type-specific Cre-recombination that yielded eGFP-expressing reporter virus selectively originating from MCs identified MC as a new in vivo, first-hit target cell of productive murine CMV infection.

Figure 1

Activation of peritoneal MCs. (a, b) WT C57BL/6 mice were either infected intraperitoneally with purified mCMV (10⁶ PFU in 100 µl PBS) or received PBS as a control. PECs were recovered 4 h later by lavage with PBS. An electronic gate was set on cells expressing FcεRI, and activation of peritoneal CD117⁺FcεRI⁺ MCs was evaluated from the cell-surface expression of CD107a, indicating MC degranulation. (a) Representative density plots for each group. (b) Quantitation and statistical analysis of MC degranulation. (c) Analysis of mCMV-induced MC activation in WT C57BL/6 mice and in the indicated, B6-congenic mouse strains deficient in TLR signaling pathways by lacking the adapter proteins MyD88 or TRIF or the receptor TLR3. Data were compiled from 4 independent experiments, not including the experiment of a and b. Throughout the graphs, the symbols represent data for individual mice, and horizontal bars indicate the median values. P values<0.05 indicate significant differences between bracketed groups. MC, mast cell; mCMV, murine cytomegalovirus; PEC, peritoneal exudate cell; TLR, Toll-like receptor; WT, wild-type.

Figure 2

Degranulation of MCs does not involve intrinsic TLR3 signaling. MC-deficient Kit^W-sh/W-sh mice were reconstituted with MCs by intraperitoneal transfer of BMMCs derived from either WT C57BL/6 (TLR3^+/+) or B6-congenic TLR3^−/− donors and were infected 5 weeks later with mCMV. Similar to the preceding experiments, the 4-h activation of peritoneal CD117⁺FcεRI⁺ MCs was assessed by analyzing the expression of CD107a. (a) Representative density plots. (b) Quantitation and statistical analysis. Symbols represent data for individual mice, and horizontal bars indicate the median values. BMMC, bone marrow-derived mast cell; MC, mast cell; mCMV, murine cytomegalovirus; TLR, Toll-like receptor; WT, wild-type.

Figure 3

Degranulation of MCs does not involve signals from NK cells. The 4-h in vivo MC degranulation assay was performed in C57BL/6 mice that were either undepleted or pan-NK cell-depleted prior to infection with mCMV (WT). Alternatively, undepleted C57BL/6 mice were infected with the mCMV-Δm157 virus lacking the ligand of the activating NK cell receptor Ly49H. The symbols represent data for individual mice, and horizontal bars indicate the median values. MC, mast cell; mCMV, murine cytomegalovirus; NK, natural killer; WT, wild-type.

Figure 4

TLR3/TRIF signaling-independent MC degranulation triggered by direct in vivo infection of MCs. PECs were recovered from the peritoneal cavity 24 h after intraperitoneal infection of WT C57BL/6 mice and B6-congenic TLR3^−/− or TRIF^−/− mice with reporter virus mCMV-gfp constitutively expressing GFP. In the cytofluorometric analysis, PECs were pre-gated for the expression of FcεRI and analyzed for the expression of CD117 and of the degranulation marker CD107a. GFP-expression in FcεRI⁺CD117⁺CD107a⁺ MCs identifies infected and degranulating MCs. The green-colored areas represent GFP⁺ cells within the gate of CD107a⁺ MC, and the dark-shaded area represents the ‘reverse gate' of MCs not expressing CD107a at the cell surface. GFP, green fluorescent protein; MC, mast cell; mCMV, murine cytomegalovirus; NK, natural killer; PEC, peritoneal exudate cell; TLR, Toll-like receptor; WT, wild-type.

Figure 5

MC-specific Cre-recombination reveals selective degranulation of infected MCs. PECs were recovered from the peritoneal cavity 24 h after intraperitoneal infection of B6-congenic Mcpt5-cre transgenic mice with the conditional reporter virus mCMV-Δm157-flox-egfp. (a) Map sketch explaining the principle of Cre-mediated recombination generating mCMV-Δm157-rec-egfp that expresses green fluorescent eGFP. Virion pictograms symbolize stop (red) and go (green). (b) Cytofluorometric analysis identifying degranulating FcεRI⁺CD117⁺CD107a⁺ MCs as those in which eGFP is expressed after Cre-recombination. For more detail, see the legend of Figure 4. GFP, green fluorescent protein; MC, mast cell; mCMV, murine cytomegalovirus; PEC, peritoneal exudate cell.

Figure 6

MCs support the productive viral replication cycle. (a) Flow chart of the experiment. (b) Quantitation of the recombined reporter virus mCMV-Δm157-rec-egfp present in the indicated organs of Mcpt5-cre transgenic mice on day 8 after intraperitoneal infection with the conditional reporter virus mCMV-Δm157-flox-egfp. Symbols represent data for individual mice. The arrow points to data for the liver for which the assay results are photodocumented. (c) Green fluorescent foci in a MEF monolayer on day 4 after infection with liver homogenate from the mouse that is indicated by an arrow in b. Top, fluorescence and phase contrast; bottom, fluorescence. mCMV, murine cytomegalovirus; MEF, mouse embryo fibroblast.