AMs are the primary immune cell type in the alveolar space and serve as the first line of cellular defense against inhaled pathogens (6
). Defects in AM phagocytosis/killing and production of inflammatory signals hinder the ability of the host to clear pathogens. In the case of HSCT, donor-derived AMs reconstituting the lung airspaces have a significantly impaired ability to ingest and kill bacterial pathogens (9
), leaving the host susceptible to organisms such as P. aeruginosa
Our previous work has shown that overproduction of PGE2
BMT directly impairs AM host defense (12
). One effector in the PGE2
signaling cascade, PTEN, has recently been shown to inhibit AM phagocytosis and killing of IgG-opsonized particles by negatively regulating the FcγR signaling pathway in control mice (21
). In this work, we show a similar mechanism after BMT, where PGE2
is overproduced by AMs and increases the lipid phosphatase activity of PTEN (see
). PTEN activity inhibits BMT AM phagocytosis of serum- and non–serum-opsonized P. aeruginosa
), although it appears that PTEN may play a more critical role in the negative regulation of opsonized phagocytosis. In addition, we show that myeloid-specific ablation of PTEN restores bacterial host defense functions in AMs (see
) and neutrophils () after BMT.
Our data demonstrate that BMT AMs have increased PTEN activity relative to nontransplant control AMs (see
). Because PGE2 has been shown to increase PTEN activity via activation of the cAMP signaling cascade, we examined whether BMT AM overproduction of PGE2 mediated this increase in PTEN activity. Indomethacin treatment, which inhibits COX-2 synthesis of prostaglandins, effectively abrogated the increase we observed in PTEN activity (see
). Enhanced PTEN activity translated into the functional consequence of suppressed AKT activity after FcγR stimulation in BMT AMs, but we observed that AKT function could be restored with indomethacin pretreatment (see
). Thus, these data suggest that PGE2 may increase BMT AM PTEN activity and diminish AKT activation after FcγR activation.
During acute bacterial lung infections, AM phagocytosis likely involves uptake of bacteria that are nonopsonized or opsonized by serum-derived complement (6
). It has previously been shown that PI3K activity, which is tightly regulated by PTEN, is involved in complement-mediated and nonopsonized phagocytosis (27
) in nontransplant settings. Therefore, we examined whether inhibiting PTEN activity could restore BMT AM phagocytosis of serum and non–serum-opsonized FITC–P. aeruginosa
. Pharmacologic PTEN inhibition increased phagocytosis of nonopsonized P. aeruginosa
but did not restore levels to those seen in control AMs. In contrast, pharmacologic inhibition of PTEN fully restored serum-opsonized phagocytosis to control levels (see
). It is likely that the presence of IgG (in addition to complement) in our immune serum contributed to the greater magnitude of improvement we observed in phagocytosis of serum-opsonized bacteria in bpV(pic)-treated BMT AMs compared with nonopsonized bacteria. As discussed below, these pharmacologic results were corroborated by studies using genetic deletion of PTEN.
It is interesting to speculate on the role that PTEN activation may play during acute infection in control mice. Our data demonstrate that PTEN activity is low in control AMs, allowing AKT to be phosphorylated in response to FcγR signaling (see
). It is likely, however, that during the course of infection, inflammatory mediators (including PGE2) may up-regulate PTEN activity at later time points after infection. Up-regulation of PTEN activity at later time points may serve as a negative-feedback control mechanism to limit further macrophage activation via IgG-opsonized targets. This may prevent potentially harmful and prolonged activation of the innate immune response.
Previous studies have shown that in murine models of neutropenia, myeloid-specific ablation of PTEN can enhance opsonized bacterial clearance and proinflammatory cytokine production in AMs (38
). Our data demonstrate a similar enhancement of host defense after transplantation of myeloid-specific PTEN KO bone marrow into WT recipients. Despite overproduction of PGE2
), we found that AM phagocytosis and killing of serum-opsonized P. aeruginosa
is restored (see
) and that AM TNF-α production is improved in the absence of PTEN expression after BMT (see
). Phagocytosis of nonopsonized bacteria is not fully restored in PTEN CKO BMT AMs (see
). Furthermore, bacterial clearance in PTEN CKO BMT mice challenged with P. aeruginosa
lung infection is only partially recovered (see
), despite the fact that lung TNF-α levels are restored (see
). Taken together, these data suggest that PTEN may be necessary for PGE2
to mediate its suppressive effects on BMT AM function against opsonized targets; however, BMT AMs retain PTEN-independent defects in nonopsonized phagocytosis.
After infection, total lung leukocyte numbers and neutrophil recruitment are similar in WT BMT and PTEN CKO BMT mice (). However, uninfected PTEN CKO BMT mice display an increase in total leukocyte number relative to WT BMT mice (). We found significantly more neutrophils and fewer macrophages in the lungs of uninfected PTEN CKO BMT mice compared with uninfected control and WT BMT mice (). In , we demonstrate that neutrophil killing of serum-opsonized P. aeruginosa is restored to control levels in PTEN CKO BMT mice. Taken together, our results indicate that PTEN CKO BMT mice have fully restored AM phagocytosis of serum-opsonized bacteria, more neutrophils at baseline, and normal neutrophil killing ability.
Despite these improvements in host defense, in vivo
clearance of P. aeruginosa
in PTEN CKO BMT mice is not fully restored to control levels. We believe this is best explained by the observation that nonopsonized bacterial phagocytosis is still impaired in PTEN CKO BMT AMs relative to control AMs (see
). This is likely because IRAK-M is elevated in the AMs from PTEN CKO BMT mice (see
). This elevation in IRAK-M in the PTEN CKO BMT AMs may also explain why TNF-α production is not fully restored to control levels (see
), assuming the signal to up-regulate TNF-α is mediated via TLR signaling. We have previously shown that increased PGE2
production after BMT elevates IRAK-M expression in AMs (20
). Transplantation of IRAK-M−/− bone marrow into WT mice fully restores AM phagocytosis of nonopsonized P. aeruginosa
and in vivo
clearance of P. aeruginosa
after an acute (24-h) infection. Putting our two studies together, we conclude that nonopsonized phagocytosis of P. aeruginosa
by AMs plays a critical role in clearance of acute infection and is negatively regulated by PGE2
–induced IRAK-M. Although we believe the partial restoration of bacterial clearance in our PTEN CKO BMT mice is best explained by our observation that nonopsonized phagocytosis by AMs is not fully recovered, another possibility is that neutrophils in the PTEN CKO BMT may display prolonged survival (30
), resulting in increased neutrophil damage to the lung, delayed immune resolution, and diminished tissue repair. Because PTEN is a critical regulator of cellular apoptosis, this may well be occurring.
IRAK-M protein expression is elevated in WT BMT and PTEN CKO BMT AMs relative to control AMs (see
). These data suggest that PGE2–mediated up-regulation of IRAK-M may occur independently of PTEN to regulate AM host defense functions. Thus, our data support a model where PGE2 signaling induces two unique pathways that regulate host defense. The up-regulation of PTEN by PGE2 primarily causes inhibition of opsonized AM phagocytosis and neutrophil killing after BMT. Simultaneously, PGE2–mediated up-regulation of IRAK-M inhibits nonopsonized phagocytosis in AMs after BMT.
Susceptibility to P. aeruginosa
is widely considered to be a consequence of neutropenia (44
). However, our data demonstrate that isolated defects in AM phagocytosis of nonopsonized bacteria can render the host susceptible to this infection. Our findings are supported by previous observations demonstrating that clodronate liposome depletion of AMs negatively affects lung host defense against P. aeruginosa in vivo
Overall, our data suggest that PGE2
may inhibit multiple mechanisms of phagocytosis in BMT AMs via the up-regulation of PTEN activity and the induction of IRAK-M. Blocking PTEN activity mitigates the suppressive effects of PGE2
on opsonized bacterial phagocytosis in AMs and restores neutrophil killing after BMT. However, ablation of PTEN may not be sufficient to fully restore host defense against acute P. aeruginosa
infection, given that nonopsonized phagocytosis by PTEN CKO BMT AMs appears to be inhibited by elevated IRAK-M expression. As such, our results suggest that a better strategy to restore host defense after BMT may be to target the production of PGE2
or EP2 receptor signaling. In fact, pharmacologic blockade of PGE2
production by indomethacin after BMT can fully restore host defense (12
). An important future goal will be to determine whether these same PGE2
–induced alterations are present in human AMs after HSCT.