Mast cells are efficient innate immune responders located at mucosal surfaces and within tissues37,38
. We previously reported that mast cells migrate to the lungs during F. tularensis
LVS pulmonary infection and that mast cell secreted IL-4 significantly reduces intramacrophage replication8
. In this study, we further analyzed mast cell/IL-4 mediated inhibition of bacterial replication and host cell death using mice deficient in IL-4 signaling. These mice (IL4R−/ −
) exhibited significantly higher levels of active caspase-3, a known cysteine protease associated with cell death39
, compared to wild-type mice, following LVS or human virulent SCHU S4 pulmonary challenge. However, there was higher macrophage recruitment to the lungs and increased cell death within this population following LVS infection compared to SCHU S4. This increase in active caspase-3 expression within lung macrophages of LVS or SCHU S4-infected IL-4R−/ −
mice suggests a physiological contribution of IL-4 in modulating host cell death following pulmonary infection. In this regard, Wickstrum and colleagues28
have recently reported that mice infected via aerosolization of SCHU S4 exhibit active caspase-3 and cell death, but minimal cleaved caspase-1 associated with the inflammasome, within the lungs.
Notably, our study demonstrates a plausible mechanism by which IL-4-mediated inhibition of F. tularensis
replication is associated with increased ATP production and improved acidification of organelles containing bacteria. Specifically, IL-4 increased ATP production, and MR expression and uptake with increased co-localization of MR within acidified regions in macrophages, as well as localization of Lucifer labeled F. tularensis
to the acidified organelles. Interestingly, addition of rIL-4 resulted in bacteria well encompassed by acidified regions in contrast to untreated macrophages. Furthermore, when IL4R−/ −
macrophages were co-cultured with mast cells, there was reduced acidification and increased Lucifer labeled bacteria compared to wild-type macrophages in co-culture with mast cells (Supplementary Fig. S4
). Also, at 24 h, IL4R−/ −
macrophages in co-culture with mast cells exhibited greater cell lysis than wild-type macrophages, which may be directly related to lack of IL4 uptake and resulting cell death. These results provide a foundation by which mast cells and IL-4 production mediate reduction of bacterial replication and host cell death associated with the modulation of ATP expression.
To analyze parameters associated with cell death following bacterial infection, we evaluated the effect of mast cells on LVS and SCHU S4 intramacrophage replication and induction of apoptosis. Under similar co-culture conditions, mast cells were shown to inhibit the intramacrophage replication of SCHU S4 and LVS, and to diminish active caspase-3 induction. Additionally, infected macrophages treated with rIL-4 demonstrated decreased Annexin V and propidium iodide staining. To determine if the inhibition of bacterial burden was dependent upon the reduction of host cell death, we utilized a pancaspase inhibitor, zVAD. Although active caspase-3 was significantly diminished in the presence of the inhibitor, bacterial replication was not altered, suggesting that the effect of IL-4 was upstream and independent of active caspase-3 inhibition. Interleukin-4 up-regulation of ATP by enhanced glucose uptake has been shown to inhibit cell death30
Additionally, IL-4 up-regulates Bcl-xL, which functions in maintenance of mitochondrial integrity30,40
, and prevention of cell death upstream of cytochrome c release, thereby reducing the progression to necrosis20
. Interestingly, ATP also has been shown to promote phagosome-lysosome fusion and intracellular killing of bacterial pathogens31,32
. Our recent findings8
and current published reports30–32
, suggest that the association between IL-4 inhibition of Francisella
bacterial replication and host cell death may result from increased ATP production. Enhancement of ATP, not only supports cell survival, but also promotes acidification and killing of invading pathogens.
Additionally, to determine if the inhibitory effects observed with mast cells are mast cells specific, primary hepatocytes were co-cultured with macrophages, and then infected with LVS as conducted in previous experiments of co-culture with mast cells. Bacterial replication and macrophage active caspase-3 expression were not altered (data not shown), supporting the findings that inhibition is dependent upon mast cell secreted IL-4. While mast cell spent supernatant or rIL-4 treatment reduced LVS intramacrophage replication, the levels of nitric oxide were not increased (Supplemental Fig. 5S
). Therefore, nitric oxide is ruled out as a possible factor influencing bacterial reduction. Additionally, STAT6−/ −
deficient mast cells which do not product detectable levels of IL-4, did not reduce bacterial replication or active caspase-3 expression in macrophages during co-culture. Considering that TNF-α has been shown to amplify IL-4 receptor signaling via inactivation of protein tyrosine phosphatase 1B41
, we also examined the effects of neutralizing antibody against TNF-α. This neutralizing antibody reduced the levels of IL4 (data not shown) in the system and the inhibitory effects of mast cells in co-culture. The highest level of abrogation of mast cell effects resulted with addition of anti-IL-4 and anti-TNF-α to the co-culture system during LVS infection (Supplemental Fig. S6
). In view of the fact that Francisella
infected macrophages produce TNF-α, this cytokine alone is not responsible for the decrease of bacterial replication or macrophage cell death observed with mast cells/IL4. The IL-4–TNF-α feedback mechanism during Francisella
infection is currently under further investigation.
Interleukin-4 has been shown to enhance MR expression and increase recycling of these receptors into the lysosomes33,42
. Additionally, up-regulation of MR has been reported to be essential to control of pulmonary pathogens such as Pseudomonas43
. To this end, Schulert and Allen33
demonstrated that IL-4 treatment promotes phagocytosis of LVS and up-regulation of MR expression within infected macrophages. However, the degree of expression of IL-4 and the origin (e.g., peritoneal or bone marrow derived) of macrophages may lead to phenotypic differences in cellular activity. MR expression induced by extended (48 h) rIL-4 pretreatment compared to the short term (2 h) pretreatment used in this study was associated with decreased phagocytosis and inefficient bacterial killing45
. The inefficient bacterial killing during extended rIL-4 treatment may promote excessive stimulation and reduced recycling of the MR. This is supported by a recent finding that sustained receptor stimulation results in sequestration of recycling endosomes46
. Given the plasticity of mononuclear cells and the stimuli of the microenvironment (i.e., peritoneal, lung, systemic or tissue), there may be several facets to the response of macrophages to cytokines such as IL-4 in vivo
. Collectively, our recent findings provide new insights to the contribution of mast cells and secreted IL-4 in mediating anti-bacterial innate immunity.