Local GM-CSF-Dependent Differentiation and Activation of Pulmonary Dendritic Cells and Macrophages Protect against Progressive Cryptococcal Lung Infection in Mice.

Patients with acquired deficiency in GM-CSF are susceptible to infections with Cryptococcus neoformans and other opportunistic fungi. We previously showed that GM-CSF protects against progressive fungal disease using a murine model of cryptococcal lung infection. To better understand the cellular and molecular mechanisms through which GM-CSF enhances antifungal host defenses, we investigated temporal and spatial relationships between myeloid and lymphoid immune responses in wild-type C57BL/6 mice capable of producing GM-CSF and GM-CSF-deficient mice infected with a moderately virulent encapsulated strain of C. neoformans (strain 52D). Our data demonstrate that GM-CSF deficiency led to a reduction in: 1) total lung leukocyte recruitment; 2) Th2 and Th17 responses; 3) total numbers of CD11b(+) dendritic cells (DC) and CD11b(-) and CD11b(+) macrophages (Mϕ); 4) DC and Mϕ activation; and 5) localization of DC and Mϕ to the microanatomic sites of alveolar infection. In contrast, GM-CSF deficiency resulted in increased accumulation of DC and Mϕ precursors, namely Ly-6C(high) monocytes, in the blood and lungs of infected mice. Collectively, these results show that GM-CSF promotes the local differentiation, accumulation, activation, and alveolar localization of lung DC and Mϕ in mice with cryptococcal lung infection. These findings identify GM-CSF as central to the protective immune response that prevents progressive fungal disease and thus shed new light on the increased susceptibility to these infections observed in patients with acquired GM-CSF deficiency.

Early or late IL-10 blockade enhances Th1 and Th17 effector responses and promotes fungal clearance in mice with cryptococcal lung infection.

The potent immunoregulatory properties of IL-10 can counteract protective immune responses and, thereby, promote persistent infections, as evidenced by studies of cryptococcal lung infection in IL-10-deficient mice. To further investigate how IL-10 impairs fungal clearance, the current study used an established murine model of C57BL/6J mice infected with Cryptococcus neoformans strain 52D. Our results demonstrate that fungal persistence is associated with an early and sustained expression of IL-10 by lung leukocytes. To examine whether IL-10-mediated immune modulation occurs during the early or late phase of infection, assessments of fungal burden and immunophenotyping were performed on mice treated with anti-IL-10R-blocking Ab at 3, 6, and 9 d postinfection (dpi) (early phase) or at 15, 18, and 21 dpi (late phase). We found that both early and late IL-10 blockade significantly improved fungal clearance within the lung compared with isotype control treatment when assessed 35 dpi. Immunophenotyping identified that IL-10 blockade enhanced several critical effector mechanisms, including increased accumulation of CD4(+) T cells and B cells, but not CD8(+) T cells; specific increases in the total numbers of Th1 and Th17 cells; and increased accumulation and activation of CD11b(+) dendritic cells and exudate macrophages. Importantly, IL-10 blockade effectively abrogated dissemination of C. neoformans to the brain. Collectively, this study identifies early and late cellular and molecular mechanisms through which IL-10 impairs fungal clearance and highlights the therapeutic potential of IL-10 blockade in the treatment of fungal lung infections.

Implicating exudate macrophages and Ly-6C(high) monocytes in CCR2-dependent lung fibrosis following gene-targeted alveolar injury.

The alveolar epithelium is characteristically abnormal in fibrotic lung disease, and we recently established a direct link between injury to the type II alveolar epithelial cell (AEC) and the accumulation of interstitial collagen. The mechanisms by which damage to the epithelium induces lung scarring remain poorly understood. It is particularly controversial whether an insult to the type II AEC initiates an inflammatory response that is required for the development of fibrosis. To explore whether local inflammation occurs following a targeted epithelial insult and contributes to lung fibrosis, we administered diphtheria toxin to transgenic mice with type II AEC-restricted expression of the diphtheria toxin receptor. We used immunophenotyping techniques and diphtheria toxin receptor-expressing, chemokine receptor-2-deficient (CCR2(-/-)) mice to determine the participation of lung leukocyte subsets in pulmonary fibrogenesis. Our results demonstrate that targeted type II AEC injury induces an inflammatory response that is enriched for CD11b(+) nonresident exudate macrophages (ExM) and their precursors, Ly-6C(high) monocytes. CCR2 deficiency abrogates the accumulation of both cell populations and protects mice from fibrosis, weight loss, and death. Further analyses revealed that the ExM are alternatively activated and that ExM and Ly-6C(high) monocytes express mRNA for IL-13, TGF-ß, and the collagen genes, COL1A1 and COLIIIA1. Furthermore, the accumulated ExM and Ly-6C(high) monocytes contain intracellular collagen, as detected by immunostaining. Together, these results implicate CCR2 and the accumulation of ExM and Ly-6C(high) monocytes as critical determinants of pulmonary fibrosis induced by selective type II AEC injury.

Early induction of CCL7 downstream of TLR9 signaling promotes the development of robust immunity to cryptococcal infection.

We investigated mechanisms by which TLR9 signaling promoted the development of the protective response to Cryptococcus neoformans in mice with cryptococcal pneumonia. The afferent (week 1) and efferent (week 3) phase immune parameters were analyzed in the infected wild-type (TLR9(+/+)) and TLR-deficient (TLR9(-/-)) mice. TLR9 deletion diminished 1) accumulation and activation of CD11b(+) dendritic cells (DCs), 2) the induction of IFN-γ and CCR2 chemokines CCL7, CCL12, but not CCL2, at week 1, and 3) pulmonary accumulation and activation of the major effector cells CD4(+) and CD8(+) T cells, CD11b(+) lung DCs, and exudate macrophages at week 3. The significance of CCL7 induction downstream of TLR9 signaling was investigated by determining whether CCL7 reconstitution would improve immunological parameters in C. neoformans-infected TLR9(-/-) mice. Early reconstitution with CCL7 1) improved accumulation and activation of CD11b(+) DCs at week 1, 2) restored early IFN-γ production in the lungs, and 3) restored the accumulation of major effector cell subsets. CCL7 administration abolished the difference in lung fungal burdens between TLR9(+/+) and TLR9(-/-) mice at week 3; however, significant reduction of fungal burdens between PBS- and CCL7-treated mice has not been observed, suggesting that additional mechanism(s) apart from early CCL7 induction contribute to optimal fungal clearance in TLR9(+/+) mice. Collectively, we show that TLR9 signaling during the afferent phase contributes to the development of protective immunity by promoting the early induction of CCL7 and IFN-γ and the subsequent early recruitment and activation of DCs and additional effector cells in mice with cryptococcal pneumonia.

Chemokine receptor 2-mediated accumulation of fungicidal exudate macrophages in mice that clear cryptococcal lung infection.

Clearance of pulmonary infection with the fungal pathogen Cryptococcus neoformans is associated with the accumulation and activation of lung macrophages. However, the phenotype of these macrophages and the mechanisms contributing to their accumulation are not well-defined. In this study, we used an established murine model of cryptococcal lung infection and flow cytometric analysis to identify alveolar macrophages (AMs) and the recently described exudate macrophages (ExMs). Exudate macrophages are distinguished from AMs by their strong expression of CD11b and major histocompatibility complex class II and modest expression of costimulatory molecules. Exudate macrophages substantially outnumber AMs during the effector phase of the immune response; and accumulation of ExMs, but not AMs, was chemokine receptor 2 (CCR2) dependent and attributable to the recruitment and subsequent differentiation of Ly-6C(high) monocytes originating from the bone marrow and possibly the spleen. Peak ExM accumulation in wild-type (CCR2(+/+)) mice coincided with maximal lung expression of mRNA for inducible nitric oxide synthase and correlated with the known onset of cryptococcal clearance in this strain of mice. Exudate macrophages purified from infected lungs displayed a classically activated effector phenotype characterized by cryptococcal-enhanced production of inducible nitric oxide synthase and tumor necrosis factor α. Cryptococcal killing by bone marrow-derived ExMs was CCR2 independent and superior to that of AMs. We conclude that clearance of cryptococcal lung infection requires the CCR2-mediated massive accumulation of fungicidal ExMs derived from circulating Ly-6C(high) monocytes.

Dual roles of CD40 on microbial containment and the development of immunopathology in response to persistent fungal infection in the lung.

Persistent pulmonary infection with Cryptococcus neoformans in C57BL/6 mice results in chronic inflammation that is characterized by an injurious Th2 immune response. In this study, we performed a comparative analysis of cryptococcal infection in wild-type versus CD40-deficient mice (in a C57BL/6 genetic background) to define two important roles of CD40 in the modulation of fungal clearance as well as Th2-mediated immunopathology. First, CD40 promoted microanatomic containment of the organism within the lung tissue. This protective effect was associated with: i) a late reduction in fungal burden within the lung; ii) a late accumulation of lung leukocytes, including macrophages, CD4+ T cells, and CD8+ T cells; iii) both early and late production of tumor necrosis factor-α and interferon-γ by lung leukocytes; and iv) early IFN-γ production at the site of T cell priming in the regional lymph nodes. In the absence of CD40, systemic cryptococcal dissemination was increased, and mice died of central nervous system infection. Second, CD40 promoted pathological changes in the airways, including intraluminal mucus production and subepithelial collagen deposition, but did not alter eosinophil recruitment or the alternative activation of lung macrophages. Collectively, these results demonstrate that CD40 helps limit progressive cryptococcal growth in the lung and protects against lethal central nervous system dissemination. CD40 also promotes some, but not all, elements of Th2-mediated immunopathology in response to persistent fungal infection in the lung.

CD11c+ cells are required to prevent progression from local acute lung injury to multiple organ failure and death.

To investigate the role of CD11c(+) cells in endotoxin-induced acute lung injury, wild-type or CD11c-diphtheria toxin receptor transgenic mice were treated with intraperitoneal diphtheria toxin (5 ng/g b.wt.) in the presence or absence of intratracheal lipopolysaccharide (51 microg). Lipopolysaccharide treatment resulted in 100% mortality in CD11c-depleted animals but not in control animals. Analysis of local lung tissue revealed no differences in acute lung injury severity; however, analysis of distal tissues revealed severe damage and necrosis to multiple organs (liver, spleen, and kidneys) in CD11c-diphtheria toxin receptor mice but not in wild-type mice. In addition, dramatic increases in systemic levels of liver enzymes (alanine aminotransferase, 657 U/L, aspartate aminotransferase, 1401 U/L), blood urea (53 mg/dl), and 8-iso-prostaglandin F(2alpha), a marker of oxidative stress (350 pg/ml), were observed. These data demonstrate that CD11c(+) cells play a critical role in protecting the organs from systemic injury caused by a pulmonary endotoxin challenge.

Accumulation of CD11b+ lung dendritic cells in response to fungal infection results from the CCR2-mediated recruitment and differentiation of Ly-6Chigh monocytes.

Pulmonary clearance of the encapsulated yeast Cryptococcus neoformans is associated with the CCR2-mediated accumulation of lung dendritic cells (DC) and the development of a T1 adaptive immune response. The objective of this study was to identify the circulating DC precursor(s) responsible for this large increase in lung DC numbers. An established murine model was used to evaluate putative DC precursors in the blood, bone marrow, and lungs of CCR2(+/+) mice and CCR2(-/-) mice throughout a time course following infection with C. neoformans. Results demonstrate that numbers of Ly-6C(high) monocytes increased in parallel in the peripheral blood and lungs of CCR(+/+) mice, whereas CD11c(+) MHC class II(+) pre-DC were 10-fold less prevalent in the peripheral blood and did not differ between the two strains. Accumulation of Ly-6C(high) monocytes correlated with a substantial increase in the numbers of CD11b(+) DC in the lungs of infected CCR2(+/+) mice. Comparative phenotypic analysis of lung cells recovered in vivo suggests that Ly-6C(high) monocytes differentiate into CD11b(+) DC in the lung; differentiation is associated with up-regulation of costimulatory molecules and decreased Ly-6C expression. Furthermore, in vitro experiments confirmed that Ly-6C(high) monocytes differentiate into CD11b(+) DC. Accumulation of Ly-6C(high) monocytes and CD11b(+) DC was not attributable to their proliferation in situ. We conclude that the CCR2-mediated accumulation of CD11b(+) DC in the lungs of Cryptococcus-infected mice is primarily attributable to the continuous recruitment and differentiation of Ly-6C(high) monocytes.

Th2 but not Th1 immune bias results in altered lung functions in a murine model of pulmonary Cryptococcus neoformans infection.

Changes in airway dynamics have been reported in the rat model of pulmonary cryptococcosis. However, it is not known if Cryptococcus neoformans-induced changes in lung functions are related to the immunophenotype that develops in response to cryptococcal infection in the lungs. In this study we performed a parallel analysis of the immunophenotype and airway resistance (standard resistance of the airways [SRAW]) in BALB/c mice infected with highly virulent C. neoformans strain H99 and moderately virulent strain 52D. H99 infection evoked a Th2 response and was associated with increased SRAW, while the SRAW for 52D infection, which resulted in a predominantly Th1-skewed response, did not differ from the SRAW for uninfected mice. We found that an altered SRAW in mice did not positively or negatively correlate with the pulmonary fungal burden, the magnitude of inflammatory response, the numbers of T cells, eosinophils or eosinophil subsets, neutrophils, or monocytes/macrophages, or the levels of cytokines (interleukin-4 [IL-4], IL-10, gamma interferon, or IL-13) produced by lung leukocytes. However, the level of a systemic Th2 marker, serum immunoglobulin E (IgE), correlated significantly with SRAW, indicating that the changes in lung functions were proportional to the level of Th2 skewing in this model. These data also imply that IgE may contribute to the altered SRAW observed in H99-infected mice. Lung histological analysis revealed severe allergic bronchopulmonary mycosis pathology in H99-infected mice and evidence of protective responses in 52D-infected mice with well-marginalized lesions. Taken together, the data show that C. neoformans can significantly affect airflow physiology, particularly in the context of a Th2 immune response with possible involvement of IgE as an important factor.

Role of dendritic cells and alveolar macrophages in regulating early host defense against pulmonary infection with Cryptococcus neoformans.

Successful pulmonary clearance of the encapsulated yeast Cryptococcus neoformans requires a T1 adaptive immune response. This response takes up to 3 weeks to fully develop. The role of the initial, innate immune response against the organism is uncertain. In this study, an established model of diphtheria toxin-mediated depletion of resident pulmonary dendritic cells (DC) and alveolar macrophages (AM) was used to assess the contribution of these cells to the initial host response against cryptococcal infection. The results demonstrate that depletion of DC and AM one day prior to infection results in rapid clinical deterioration and death of mice within 6 days postinfection; this effect was not observed in infected groups of control mice not depleted of DC and AM. Depletion did not alter the microbial burden or total leukocyte recruitment in the lung. Mortality (in mice depleted of DC and AM) was associated with increased neutrophil and B-cell accumulation accompanied by histopathologic evidence of suppurative neutrophilic bronchopneumonia, cyst formation, and alveolar damage. Collectively, these data define an important role for DC and AM in regulating the initial innate immune response following pulmonary infection with C. neoformans. These findings provide important insight into the cellular mechanisms which coordinate early host defense against an invasive fungal pathogen in the lung.

Cryptococcal urease promotes the accumulation of immature dendritic cells and a non-protective T2 immune response within the lung.

Urease, a major virulence factor for Cryptococcus neoformans, promotes lethal meningitis/encephalitis in mice. The effect of urease within the lung, the primary site of most invasive fungal infections, is unknown. An established model of murine infection that utilizes either urease-producing (wt and ure1::URE1) or urease-deficient (ure1) strains (H99) of C. neoformans was used to characterize fungal clearance and the resultant immune response evoked by these strains within the lung. Results indicate that mice infected with urease-producing strains of C. neoformans demonstrate a 100-fold increase in fungal burden beginning 2 weeks post-infection (as compared with mice infected with urease-deficient organisms). Infection with urease-producing C. neoformans was associated with a highly polarized T2 immune response as evidenced by increases in the following: 1) pulmonary eosinophils, 2) serum IgE levels, 3) T2 cytokines (interleukin-4, -13, and -4 to interferon-gamma ratio), and 4) alternatively activated macrophages. Furthermore, the percentage and total numbers of immature dendritic cells within the lung-associated lymph nodes was markedly increased in mice infected with urease-producing C. neoformans. Collectively, these data define cryptococcal urease as a pulmonary virulence factor that promotes immature dendritic cell accumulation and a potent, yet non-protective, T2 immune response. These findings provide new insights into mechanisms by which microbial factors contribute to the immunopathology associated with invasive fungal disease.

Tumor necrosis factor-related apoptosis-inducing ligand inhibits experimental autoimmune thyroiditis by the expansion of CD4+CD25+ regulatory T cells.

There have been several reports that TNF-related apoptosis-inducing ligand (TRAIL) has the ability to suppress the development of experimental autoimmune diseases, including a mouse model of experimental autoimmune encephalomyelitis, a rabbit model of rheumatoid arthritis, type 1 diabetes mellitus, in mice and experimental autoimmune thyroiditis (EAT) in mice. However, the mechanism underlying TRAIL effect is not well defined. In the present study, we specifically examined TRAIL effects on CD4(+)CD25(+) regulatory T cells. CD4(+)CD25(+) T cells prepared from mouse thyroglobulin (mTg)-immunized CBA/J mice proliferate in the presence of TRAIL and dendritic cells in vitro. These CD4(+)CD25(+) T cells included both CD4(+)CD25(+)CD45RB(Low) (regulatory) and CD4(+)CD25(+)CD45RB(High) (effector) T cells. Our results demonstrated that mTg-immunized mice treated with TRAIL showed significant increases in the number of CD4(+)CD25(+)CD45RB(Low) T cells compared with mice immunized with mTg alone. CD4(+)CD25(+)CD45RB(Low) T cells expressed much higher levels of the forkhead family transcription factor, IL-10, and TGFbeta1 than CD4(+)CD25(+)CD45RB(High) T cells, and these cells can completely suppress the proliferation of the mTg-primed splenocytes in lower concentrations than the unfractionated CD4(+)CD25(+) T cells. Furthermore, transfer of these cells into CBA/J mice prior to mTg-primed splenocyte injection could markedly reduce the frequency and severity of EAT development. CD4(+)CD25(+)CD45RB(Low) T cells were more effective at suppressing histological thyroiditis than unfractionated cells. These results indicated that TRAIL can increase the number of mTg-specific CD4(+)CD25(+)CD45RB(Low) T cells, inhibiting autoimmune responses and preventing the progression of EAT. These findings reveal a novel mechanism by which TRAIL could inhibit autoimmune disease.

Loss of Na+ channel beta2 subunits is neuroprotective in a mouse model of multiple sclerosis.

Multiple sclerosis (MS) is a CNS disease that includes demyelination and axonal degeneration. Voltage-gated Na+ channels are abnormally expressed and distributed in MS and its animal model, Experimental Allergic Encephalomyelitis (EAE). Up-regulation of Na+ channels along demyelinated axons is proposed to lead to axonal loss in MS/EAE. We hypothesized that Na+ channel beta2 subunits (encoded by Scn2b) are involved in MS/EAE pathogenesis, as beta2 is responsible for regulating levels of channel cell surface expression in neurons. We induced non-relapsing EAE in Scn2b(+/+) and Scn2b(-/-) mice on the C57BL/6 background. Scn2b(-/-) mice display a dramatic reduction in EAE symptom severity and lethality as compared to wildtype, with significant decreases in axonal degeneration and axonal loss. Scn2b(-/-) mice show normal peripheral immune cell populations, T cell proliferation, cytokine release, and immune cell infiltration into the CNS in response to EAE, suggesting that Scn2b inactivation does not compromise immune function. Our data suggest that loss of beta2 is neuroprotective in EAE by prevention of Na+ channel up-regulation in response to demyelination.

Misoprostol impairs female reproductive tract innate immunity against Clostridium sordellii.

Fatal cases of acute shock complicating Clostridium sordellii endometritis following medical abortion with mifepristone (also known as RU-486) used with misoprostol were reported. The pathogenesis of this unexpected complication remains enigmatic. Misoprostol is a pharmacomimetic of PGE(2), an endogenous suppressor of innate immunity. Clinical C. sordellii infections were associated with intravaginal misoprostol administration, suggesting that high misoprostol concentrations within the uterus impair immune responses against C. sordellii. We modeled C. sordellii endometritis in rats to test this hypothesis. The intrauterine but not the intragastric delivery of misoprostol significantly worsened mortality from C. sordellii uterine infection, and impaired bacterial clearance in vivo. Misoprostol also reduced TNF-alpha production within the uterus during infection. The intrauterine injection of misoprostol did not enhance mortality from infection by the vaginal commensal bacterium Lactobacillus crispatus. In vitro, misoprostol suppressed macrophage TNF-alpha and chemokine generation following C. sordellii or peptidoglycan challenge, impaired leukocyte phagocytosis of C. sordellii, and inhibited uterine epithelial cell human beta-defensin expression. These immunosuppressive effects of misoprostol, which were not shared by mifepristone, correlated with the activation of the G(s) protein-coupled E prostanoid (EP) receptors EP2 and EP4 (macrophages) or EP4 alone (uterine epithelial cells). Our data provide a novel explanation for postabortion sepsis leading to death and also suggest that PGE(2), in which production is exaggerated within the reproductive tract during pregnancy, might be an important causal determinant in the pathogenesis of more common infections of the gravid uterus.

CCR2 mediates conventional dendritic cell recruitment and the formation of bronchovascular mononuclear cell infiltrates in the lungs of mice infected with Cryptococcus neoformans.

Pulmonary clearance of the encapsulated yeast Cryptococcus neoformans requires the development of T1-type immunity. CCR2-deficient mice infected with C. neoformans develop a non-protective T2 immune response and persistent infection. The mechanisms responsible for this aberrant response are unknown. The objective of this study was to define the number, phenotype, and microanatomic location of dendritic cells (DC) residing within the lung of CCR2+/+ or CCR2-/- mice throughout a time course following infection with C. neoformans. Results demonstrate the CCR2-mediated recruitment of conventional DC expressing modest amounts of costimulatory molecules. DC recruitment was preceded by the up-regulation in the lung of the CCR2 ligands CCL2 and CCL7. Colocalization of numerous DC and CD4+ T cells within bronchovascular infiltrates coincided with increased expression of IL-12 and IFN-gamma. By contrast, in the absence of CCR2, DC recruitment was markedly impaired, bronchovascular infiltrates were diminished, and mice developed features of T2 responses, including bronchovascular collagen deposition and IL-4 production. Our results demonstrate that CCR2 is required for the recruitment of large numbers of conventional DC to bronchovascular infiltrates in mice mounting a T1 immune response against a fungal pathogen. These findings shed new insight into the mechanism(s) by which DC recruitment alters T cell polarization in response to an infectious challenge within the lung.

Inheritance of immune polarization patterns is linked to resistance versus susceptibility to Cryptococcus neoformans in a mouse model.

Genetic background variation between inbred strains accounts for different levels of susceptibility to Cryptococcus neoformans in the mouse infection model. To elucidate the inheritance of immunophenotypic traits and their associations with clearance outcomes during cryptococcal infection, we compared C57BL/6, BALB/c, and their first-generation hybrid, CB6F1 (F1), mice. Mice from each group were infected with C. neoformans (10(4) CFU) and analyzed at weekly intervals over a 6-week period. BALB/c mice progressively cleared the cryptococcal infection in the lungs and showed a Th1-skewed immune response: a Th1-shifted cytokine profile, modest lung pathology, and no significant elevation in the systemic immunoglobulin E (IgE) level. In contrast, C57BL/6 mice developed a chronic infection with a Th2-skewed immune response: a Th2-shifted cytokine profile, pulmonary eosinophilia, severe lung pathology, elevated serum IgE, fungemia, and cryptococcal dissemination in the central nervous system. F1 mice demonstrated intermediate resistance to C. neoformans, with a stronger resemblance to the immunophenotype of the resistant (BALB/c) mice. F1 mice also demonstrated enhanced pulmonary recruitment of lymphocytes, especially CD8(+) T cells, in comparison to both parental strains, suggesting positive heterosis. We conclude that the inheritance of traits responsible for early cytokine induction in the infected lungs and dendritic-cell maturation/activation status in draining nodes is responsible for the intermediate immune response polarization and clearance outcome observed initially in the lungs of F1 mice. The enhanced pulmonary lymphocyte recruitment could be responsible for a gradual shutdown of the undesirable Th2 arm of the immune response and subsequently improved anticryptococcal resistance in F1 mice.

The role of dendritic cells in the development of acute dextran sulfate sodium colitis.

Dendritic cells (DCs) are essential mediators of the host immune response to surrounding microbes. In this study, we investigate the role of DCs in the pathogenesis of a widely used colitis model, dextran sulfate sodium-induced colitis. The effect of dextran sulfate sodium on the production of proinflammatory cytokines and chemokines by bone marrow-derived DCs (BM-DCs) was analyzed. BM-DCs were adoptively transferred into C57BL/6 mice or DCs were ablated using transgenic CD11c-DTR/GFP mice before treatment with 5% dextran sulfate sodium in drinking water. We found that dextran sulfate sodium induced production of proinflammatory cytokines (IL-12 and TNF-alpha) and chemokines (KC, MIP-1alpha, MIP-2, and MCP-1) by DCs. Adoptive transfer of BM-DCs exacerbated dextran sulfate sodium colitis while ablation of DCs attenuated the colitis. We conclude that DCs are critical in the development of acute dextran sulfate sodium colitis and may serve a key role in immune balance of the gut mucosa.

Role of granulocyte macrophage colony-stimulating factor in host defense against pulmonary Cryptococcus neoformans infection during murine allergic bronchopulmonary mycosis.

We investigated the role of granulocyte macrophage colony-stimulating factor (GM-CSF) in host defense in a murine model of pulmonary cryptococcosis induced by intratracheal inoculation of Cryptococcus neoformans. Pulmonary C. neoformans infection of C57BL/6 mice is an established model of an allergic bronchopulmonary mycosis. Our objective was to determine whether GM-CSF regulates the pulmonary Th2 immune response in C. neoformans-infected C57BL/6 mice. Long-term pulmonary fungistasis was lost in GM-CSF knockout (GM(-/-)) mice, resulting in increased pulmonary burden of fungi between weeks 3 and 5. GM-CSF was required for the early influx of macrophages and CD4 and CD8 T cells into the lungs but was not required later in the infection. Lack of GM-CSF also resulted in reduced eosinophil recruitment and delayed recruitment of mononuclear cells into the airspace. Macrophages from GM(+/+) mice showed numerous hallmarks of alternatively activated macrophages: higher numbers of intracellular cryptococci, YM1 crystals, and induction of CCL17. These hallmarks are absent in macrophages from GM(-/-) mice. Mucus-producing goblet cells were abundantly present within the bronchial epithelial layer in GM(+/+) mice but not in GM(-/-) mice at week 5 after infection. Production of both Th1 and Th2 cytokines was impaired in the absence of GM-CSF, consistent with both reduced C. neoformans clearance and absence of allergic lung pathology.

Short communication: differences between macrophages and dendritic cells in the cyclic AMP-dependent regulation of lipopolysaccharide-induced cytokine and chemokine synthesis.

Cyclic adenosine monophosphate (cAMP) is an intracellular signaling molecule responsible for directing cellular responses to extracellular signals. Once believed to signal exclusively through its ability to bind protein kinase A (PKA), recent research has revealed alternative cAMP-binding targets involved in PKA-independent processes. In this study we addressed the hypothesis that the guanine nucleotide exchange protein directly activated by cAMP (Epac-1) and PKA differentially regulate inflammatory mediator production in distinct phagocytic cell types. To accomplish this, we compared the release of cAMP-regulated polypeptide inflammatory mediators in both macrophages (obtained from the lung and peritoneum) and bone marrow-derived dendritic cells (DCs) stimulated with bacterial endotoxin. Using the highly selective Epac-1 and PKA activating cAMP analogs 8-pCPT-2 -O-Me-cAMP and 6-Bnz-cAMP, respectively, we found that macrophages differ from DCs in the involvement of these distinct cAMP pathways in modulating inflammatory mediator release in response to endotoxin. Whereas the regulation of cytokine and chemokine production in macrophages by cAMP was solely dependent on PKA, we found that both Epac-1 and PKA activation could regulate mediator production in DCs. This finding may be important in the pharmacologic regulation of immune responses through manipulation of cAMP signaling cascades and contributes to our understanding of the differences between these cell types.

Differential regulation by leukotrienes and calcium of Fc gamma receptor-induced phagocytosis and Syk activation in dendritic cells versus macrophages.

Macrophage (MØ) phagocytosis via the Fc receptor for immunoglobulin G (Fc gammaR) requires the spleen tyrosine kinase (Syk) and serves an important antimicrobial function. We have reported previously that Fc gammaR-mediated ingestion and Syk activation in MØ are amplified by and depend on the proinflammatory lipid mediator leukotriene B4 (LTB4). Although Fc gammaR-mediated ingestion is also important for antigen uptake, there is no information about LTB4 regulation of these processes in dendritic cells (DCs). In this study, we compared murine bone marrow (BM)-derived DCs to MØ from BM, peritoneum, and the pulmonary alveolar space. Neither phagocytosis nor Syk activation in DCs was influenced by exogenous LTB4. Unlike the various MØ populations, Syk activation in DCs was likewise unaffected by pharmacologic or genetic strategies to inhibit endogenous LTB4 synthesis or to block the high-affinity LTB4 receptor BLT1. DCs were refractory to regulation by LTB4 despite the fact that they expressed BLT1 and mobilized intracellular calcium in response to its ligation. This resistance to LTB4 in DCs instead reflected the fact that in contrast to MØ, Syk activation in DCs was itself entirely independent of calcium. These results identify a fundamental difference in Fc gammaR signaling between DCs and MØ, which may relate to the divergent, functional consequences of target ingestion in the two cell types.