BackgroundUpon stimulation with different cytokines, macrophages can undergo classical or alternative activation to become M1 or M2 macrophages. Alternatively activated (or M2) macrophages are defined by their expression of specific gene products and play an important role in containing inflammation, removing apoptotic cells and repairing tissue damage. Whereas it is well-established that IL-4 can drive alternative activation, if lack of TGFβ signaling at physiological levels affects M2 polarization has not been addressed.ResultsVav1-Cre x TβRIIfx/fx mice, lacking TβRII function in hematopoietic cells, exhibited uncontrolled pulmonary inflammation and developed a lethal autoimmune syndrome at young age. This was accompanied by significantly increased numbers of splenic neutrophils and T cells as well as elevated hepatic macrophage infiltration and bone marrow monocyte counts. TβRII-/- CD4+ and CD8+ T-cells in the lymph nodes and spleen expressed increased cell surface CD44, and CD69 was also higher on CD4+ lymph node T-cells. Loss of TβRII in bone marrow-derived macrophages (BMDMs) did not affect the ability of these cells to perform efferocytosis. However, these cells were defective in basal and IL-4-induced arg1 mRNA and Arginase-1 protein production. Moreover, the transcription of genes that are typically upregulated in M2-polarized macrophages, such as ym1, mcr2 and mgl2, was also decreased in peritoneal macrophages and IL-4-stimulated TβRII-/- BMDMs. We found that cell surface and mRNA expression of Galectin-3, which also regulates M2 macrophage polarization, was lower in TβRII-/- BMDMs. Very interestingly, the impaired ability of these null mutant BMDMs to differentiate into IL-4 polarized macrophages was Stat6- and Smad3-independent, but correlated with reduced levels of phospho-Akt and β-catenin.ConclusionsOur results establish a novel biological role for TGFβ signaling in controlling expression of genes characteristic for alternatively activated macrophages. We speculate that lack of TβRII signaling reduces the anti-inflammatory M2 phenotype of macrophages because of reduced expression of these products. This would cause defects in the ability of the M2 macrophages to negatively regulate other immune cells such as T-cells in the lung, possibly explaining the systemic inflammation observed in Vav1-Cre x TβRIIfx/fx mice.

After Ag activation of naive T cells in vitro, extensive growth and differentiation into effector cells depend upon IL-2. DNA microarray analysis was used to identify IL-2-dependent molecules regulating this process. In this study, we show that the transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp-1) is expressed by a cytokine-dependent pathway in activated T lymphocytes. IL-2 production by activated CD4+ and CD8+ T cells inversely correlated with Blimp-1 levels as higher IL-2 production was associated with lower Blimp-1 expression. Furthermore, ectopic expression of Blimp-1 by activated T cells inhibited IL-2 production but enhanced granzyme B and CD25 expression. Collectively, these findings indicate that there is a negative feedback regulatory loop in activated T cells such that IL-2 inhibits its own production through induction of Blimp-1 while promoting an effector cell phenotype.

Because viral clearance was unimpaired, the study highlights the critical role of serpinB1 in mitigating inflammation and restricting pro-inflammatory cytokine production in influenza infection.

We have demonstrated previously that DNA binding and protein expression of the E2A-encoded transcription factor E47 are lower in nuclear extracts of activated splenic B cells from old mice. In the present study, we address how E47 protein expression is regulated in aging. Results herein show that E2A mRNA levels were decreased in stimulated splenic B cells from old as compared with young mice. RNA stability assays showed that the rate of E2A mRNA decay was accelerated in stimulated splenic B cells from old mice, but E47 protein degradation rates were comparable in young vs aged B cells, indicating that the regulation of E47 expression in activated splenic B cells occurs primarily by mRNA stability. The rates of decay of other mRNAs showed that the increased mRNA degradation in aged splenic activated B cells is not a general phenomenon but restricted to a subset of mRNAs. We next investigated the signal transduction pathways controlling E2A mRNA expression and stability and found that p38 MAPK regulates E2A mRNA expression through increased mRNA stability and is down-regulated in aged activated B cells. Results show that inhibition of p38 MAPK significantly reduces E2A mRNA stability in both young and old B cells, further stressing the role of p38 MAPK in E2A RNA stabilization. These studies demonstrate that the transcription factor E2A, critical for many aspects of B cell function, is regulated by a novel mechanism in aging.

Abr deactivates Rac, a master molecular switch that positively regulates many immune cell functions, by converting it to its GDP-bound conformation. Here we report that in the absence of Abr function, cockroach allergen (CRA)-immunized mice experienced a fatal asthma attack when challenged with CRA. The asthma in abr−/− mice was characterized by increased pulmonary mucus production, elevated serum IgE and leukocyte airway infiltration. Decreased pulmonary compliance was further documented by increased airway resistance upon methacholine challenge. Peribronchial and bronchio-alveolar lavage eosinophils, key cells associated with allergic asthma, were increased in abr−/− mice, but adoptive transfer of this cell type from immunized mice to naïve controls followed by CRA challenge showed that eosinophils are not primarily responsible for differences in airway resistance between controls and abr null mutants. CD4+ T cell numbers in the airways of CRA-challenged abr−/− mice were also significantly increased compared to controls, as were the Th2 T cell-secreted cytokines IL-4 and IL-5 in total lung. Interestingly, when control and abr−/− CD4+ T cells from CRA-immunized mice were transferred to wild type animals, airway resistance upon challenge with CRA was significantly higher in mice transplanted with T cells lacking Abr function. CD4+ T cells from CRA-immunized and challenged abr−/− mice contained elevated levels of activated Rac-GTP, compared to wild type controls. Functionally, abr−/− CD4+ T cells from CRA-exposed mice showed significantly enhanced chemotaxis towards CCL21. These results identify Abr-regulated CD4+ T cell migration as an important component of severe cockroach allergen evoked allergic asthma in mice.

IL-2- and IL-2R-deficient mice readily develop T cell-dependent immune responses in vivo, but the relevance of this finding is complicated by severe concurrent autoimmunity. Furthermore, the detection of such responses does not address whether under normal circumstances IL-2 dominates T cell immunity. In the present report, we investigated the extent IL-2-independent T cell growth is mediated by other cytokines in the IL-2 family and compared such responses to those generated by IL-2/IL-2R-sufficient T cells. T cell expansion and contraction to the superantigen staphylococcal enterotoxin A (SEA) by autoimmune-free IL-2Rbeta-/- CD4 and CD8 T cells were comparable to normal control mice, although their CD8+ T cells did not optimally develop into IFNgamma-producing effector cells. The proliferation by these IL-2Rbeta-deficient T cells in vivo was independent of IL-2, IL-4 and IL-15 and not blocked by mAbs to the common gamma chain. However, in co-adoptive transfer experiments, wild-type T cells exhibited somewhat more extensive proliferation than IL-2Rbeta-deficient T cells to SEA and this difference was almost entirely accounted for by CD8+ T cells. Collectively, these data indicate that substantial T cell proliferation occurs in the absence of responsiveness to cytokines in the IL-2 family, although maximal T cell proliferation and development of IFNgamma-producing effector CD8+ T cells depend upon IL-2Rbeta.

BackgroundBcr and Abr are GTPase activating proteins that specifically downregulate activity of the small GTPase Rac in restricted cell types in vivo. Rac1 is expressed in smooth muscle cells, a critical cell type involved in the pathogenesis of pulmonary hypertension. The molecular mechanisms that underlie hypoxia-associated pulmonary hypertension are not well-defined.Methodology/Principal Findings Bcr and abr null mutant mice were compared to wild type controls for the development of pulmonary hypertension after exposure to hypoxia. Also, pulmonary arterial smooth muscle cells from those mice were cultured in hypoxia and examined for proliferation, p38 activation and IL-6 production. Mice lacking Bcr or Abr exposed to hypoxia developed increased right ventricular pressure, hypertrophy and pulmonary vascular remodeling. Perivascular leukocyte infiltration in the lungs was increased, and under hypoxia bcr−/− and abr−/− macrophages generated more reactive oxygen species. Consistent with a contribution of inflammation and oxidative stress in pulmonary hypertension-associated vascular damage, Bcr and Abr-deficient animals showed elevated endothelial leakage after hypoxia exposure. Hypoxia-treated pulmonary arterial smooth muscle cells from Bcr- or Abr-deficient mice also proliferated faster than those of wild type mice. Moreover, activated Rac1, phosphorylated p38 and interleukin 6 were increased in these cells in the absence of Bcr or Abr. Inhibition of Rac1 activation with Z62954982, a novel Rac inhibitor, decreased proliferation, p38 phosphorylation and IL-6 levels in pulmonary arterial smooth muscle cells exposed to hypoxia.ConclusionsBcr and Abr play a critical role in down-regulating hypoxia-induced pulmonary hypertension by deactivating Rac1 and, through this, reducing both oxidative stress generated by leukocytes as well as p38 phosphorylation, IL-6 production and proliferation of pulmonary arterial smooth muscle cells.

Previously, we described the protective role of the neutrophil serine protease inhibitor serpinB1 in preventing early mortality of Pseudomonas aeruginosa lung infection by fostering bacterial clearance and limiting inflammatory cytokines and proteolytic damage. Surfactant protein D (SP-D), which maintains the antiinflammatory pulmonary environment and mediates bacterial removal, was degraded in infected serpinB1-deficient mice. Based on the hypothesis that increased SP-D would rescue or mitigate the pathological effects of serpinB1 deletion, we generated two serpinB1(-/-) lines overexpressing lung-specific rat SP-D and inoculated the mice with P. aeruginosa. Contrary to predictions, bacterial counts in the lungs of SP-D(low)serpinB1(-/-) and SP-D(high) serpinB1(-/-) mice were 4 logs higher than wild-type and not different from serpinB1(-/-) mice. SP-D overexpression also failed to mitigate inflammation (TNF-α), lung injury (free protein, albumin), or excess neutrophil death (free myeloperoxidase, elastase). These pathological markers were higher for infected SP-D(high)serpinB1(-/-) mice than for serpinB1(-/-) mice, although the differences were not significant after controlling for multiple comparisons. The failure of transgenic SP-D to rescue antibacterial defense of serpinB1-deficient mice occurred despite 5-fold or 20-fold increased expression levels, largely normal structure, and dose-dependent bacteria-aggregating activity. SP-D of infected wild-type mice was intact in 43-kD monomers by reducing SDS-PAGE. By contrast, proteolytic fragments of 35, 17, and 8 kD were found in infected SP-D(low)serpinB1(-/-), SP-D(high) serpinB1(-/-) mice, and serpinB1(-/-) mice. Thus, although therapies to increase lung concentration of SP-D may have beneficial applications, the findings suggest that therapy with SP-D may not be beneficial for lung inflammation or infection if the underlying clinical condition includes excess proteolysis.