Human lung cDC1 drive increased perforin-mediated NK cytotoxicity in chronic obstructive pulmonary disease.

In chronic obstructive pulmonary disease (COPD), lung natural killer cells (NKs) lyse autologous lung epithelial cells in vitro, but underlying mechanisms and their relationship to epithelial cell apoptosis in vivo are undefined. Although this cytolytic capacity of lung NKs depends on priming by dendritic cells (DCs), whether priming correlates with DC maturation or is limited to a specific DC subset is also unknown. We recruited ever-smokers (≥10 pack-years; n = 96) undergoing clinically indicated lung resections. We analyzed lung NKs for cytotoxic molecule transcripts and for cytotoxicity, which we correlated with in situ detection of activated Caspase-3/7+ airway epithelial cells. To investigate DC priming, we measured lung DC expression of CCR2, CCR7, and CX3CR1 and cocultured peripheral blood NKs with autologous lung DCs, either matured using lipopolysaccharide (LPS) (nonobstructed smokers) or separated into conventional dendritic cell type-1 (cDC1) versus cDC type-2 (cDC2) (COPD). Lung NKs in COPD expressed more perforin (P < 0.02) and granzyme B (P < 0.03) transcripts; inhibiting perforin blocked in vitro killing by lung NKs. Cytotoxicity in vitro correlated significantly (Sr = 0.68, P = 0.0043) with numbers of apoptotic epithelial cells per airway. In nonobstructed smokers, LPS-induced maturation enhanced DC-mediated priming of blood NKs, reflected by greater epithelial cell death. Although CCR7 expression was greater in COPD in both cDC1 (P < 0.03) and cDC2 (P = 0.009), only lung cDC1 primed NK killing. Thus, rather than being intrinsic to those with COPD, NK priming is a capacity of human lung DCs that is inducible by recognition of bacterial (and possibly other) danger signals and restricted to the cDC1 subset.

Lung Dendritic Cells Drive Natural Killer Cytotoxicity in Chronic Obstructive Pulmonary Disease via IL-15Rα.

RATIONALE: Lung natural killer cells (NKs) kill a greater percentage of autologous lung parenchymal cells in chronic obstructive pulmonary disease (COPD) than in nonobstructed smokers. To become cytotoxic, NKs require priming, typically by dendritic cells (DCs), but whether priming occurs in the lungs in COPD is unknown. METHODS: We used lung tissue and in some cases peripheral blood from patients undergoing clinically indicated resections to determine in vitro killing of CD326+ lung epithelial cells by isolated lung CD56+ NKs. We also measured the cytotoxicity of unprimed blood NKs after preincubation with lung DCs. To investigate mechanisms of DC-mediated priming, we used murine models of COPD induced by cigarette smoke (CS) exposure or by polymeric immunoglobulin receptor (pIgR) deficiency, and blocked IL-15Rα (IL-15 receptor α subunit) trans-presentation by genetic and antibody approaches. RESULTS: Human lung NKs killed isolated autologous lung epithelial cells; cytotoxicity was increased (P = 0.0001) in COPD, relative to smokers without obstruction. Similarly, increased lung NK cytotoxicity compared with control subjects was observed in CS-exposed mice and pIgR-/- mice. Blood NKs both from smokers without obstruction and subjects with COPD showed minimal epithelial cell killing, but in COPD, preincubation with lung DCs increased cytotoxicity. NKs were primed by CS-exposed murine DCs in vitro and in vivo. Inhibiting IL-15Rα trans-presentation eliminated NK priming both by murine CS-exposed DCs and by lung DCs from subjects with COPD. CONCLUSIONS: Heightened NK cytotoxicity against lung epithelial cells in COPD results primarily from lung DC-mediated priming via IL-15 trans-presentation on IL-15Rα. Future studies are required to test whether increased NK cytotoxicity contributes to COPD pathogenesis.

Disruption of Early Tumor Necrosis Factor Alpha Signaling Prevents Classical Activation of Dendritic Cells in Lung-Associated Lymph Nodes and Development of Protective Immunity against Cryptococcal Infection.

UNLABELLED: Anti-tumor necrosis factor alpha (anti-TNF-α) therapies have been increasingly used to treat inflammatory diseases and are associated with increased risk of invasive fungal infections, including Cryptococcus neoformans infection. Using a mouse model of cryptococcal infection, we investigated the mechanism by which disruption of early TNF-α signaling results in the development of nonprotective immunity against C. neoformans We found that transient depletion of TNF-α inhibited pulmonary fungal clearance and enhanced extrapulmonary dissemination of C. neoformans during the adaptive phase of the immune response. Higher fungal burdens in TNF-α-depleted mice were accompanied by markedly impaired Th1 and Th17 responses in the infected lungs. Furthermore, early TNF-α depletion also resulted in disrupted transcriptional initiation of the Th17 polarization program and subsequent upregulation of Th1 genes in CD4(+) T cells in the lung-associated lymph nodes (LALN) of C. neoformans-infected mice. These defects in LALN T cell responses were preceded by a dramatic shift from a classical toward an alternative activation of dendritic cells (DC) in the LALN of TNF-α-depleted mice. Taken together, our results indicate that early TNF-α signaling is required for optimal DC activation, and the initial Th17 response followed by Th1 transcriptional prepolarization of T cells in the LALN, which further drives the development of protective immunity against cryptococcal infection in the lungs. Thus, administration of anti-TNF-α may introduce a particularly greater risk for newly acquired fungal infections that require generation of protective Th1/Th17 responses for their containment and clearance. IMPORTANCE: Increased susceptibility to invasive fungal infections in patients on anti-TNF-α therapies underlines the need for understanding the cellular effects of TNF-α signaling in promoting protective immunity to fungal pathogens. Here, we demonstrate that early TNF-α signaling is required for classical activation and accumulation of DC in LALN of C. neoformans-infected mice. Subsequent transcriptional initiation of Th17 followed by Th1 programming in LALN results in pulmonary accumulation of gamma interferon- and interleukin-17A-producing T cells and effective fungal clearance. All of these crucial steps are severely impaired in mice that undergo anti-TNF-α treatment, consistent with their inability to clear C. neoformans This study identified critical interactions between cells of the innate immune system (DC), the emerging T cell responses, and cytokine networks with a central role for TNF-α which orchestrate the development of the immune protection against cryptococcal infection. This information will be important in aiding development and understanding the potential side effects of immunotherapies.

MicroRNA-34a Negatively Regulates Efferocytosis by Tissue Macrophages in Part via SIRT1.

Apoptotic cell (AC) clearance (efferocytosis) is an evolutionarily conserved process essential for immune health, particularly to maintain self-tolerance. Despite identification of many recognition receptors and intracellular signaling components of efferocytosis, its negative regulation remains incompletely understood and has not previously been known to involve microRNAs (miRs). In this article, we show that miR-34a (gene ID 407040), well recognized as a p53-dependent tumor suppressor, mediates coordinated negative regulation of efferocytosis by resident murine and human tissue macrophages (Mø). The miR-34a expression varied greatly between Mø from different tissues, correlating inversely with their capacity for AC uptake. Transient or genetic knockdown of miR-34a increased efferocytosis, whereas miR-34a overexpression decreased efferocytosis, without altering recognition of live, necrotic, or Ig-opsonized cells. The inhibitory effect of miR-34a was mediated both by reduced expression of Axl, a receptor tyrosine kinase known to recognize AC, and of the deacetylase silent information regulator T1, which had not previously been linked to efferocytosis by tissue Mø. Exposure to AC downregulated Mø miR-34a expression, resulting in a positive feedback loop that increased subsequent capacity to engulf AC. These findings demonstrate that miR-34a both specifically regulates and is regulated by efferocytosis. Given the ability of efferocytosis to polarize ingesting Mø uniquely and to reduce their host-defense functions, dynamic negative regulation by miR-34a provides one means of fine-tuning Mø behavior toward AC in specific tissue environments with differing potentials for microbial exposure.

Glucocorticoid-Augmented Efferocytosis Inhibits Pulmonary Pneumococcal Clearance in Mice by Reducing Alveolar Macrophage Bactericidal Function.

Inhaled corticosteroids (ICS) increase community-acquired pneumonia (CAP) incidence in patients with chronic obstructive pulmonary disease (COPD) by unknown mechanisms. Apoptosis is increased in the lungs of COPD patients. Uptake of apoptotic cells (ACs) ("efferocytosis") by alveolar macrophages (AMøs) reduces their ability to combat microbes, including Streptococcus pneumoniae, the most common cause of CAP in COPD patients. Having shown that ICS significantly increase AMø efferocytosis, we hypothesized that this process, termed glucocorticoid-augmented efferocytosis, might explain the association of CAP with ICS therapy in COPD. To test this hypothesis, we studied the effects of fluticasone, AC, or both on AMøs of C57BL/6 mice in vitro and in an established model of pneumococcal pneumonia. Fluticasone plus AC significantly reduced TLR4-stimulated AMø IL-12 production, relative to either treatment alone, and decreased TNF-α, CCL3, CCL5, and keratinocyte-derived chemoattractant/CXCL1, relative to AC. Mice treated with fluticasone plus AC before infection with viable pneumococci developed significantly more lung CFUs at 48 h. However, none of the pretreatments altered inflammatory cell recruitment to the lungs at 48 h postinfection, and fluticasone plus AC less markedly reduced in vitro mediator production to heat-killed pneumococci. Fluticasone plus AC significantly reduced in vitro AMø killing of pneumococci, relative to other conditions, in part by delaying phagolysosome acidification without affecting production of reactive oxygen or nitrogen species. These results support glucocorticoid-augmented efferocytosis as a potential explanation for the epidemiological association of ICS therapy of COPD patients with increased risk for CAP, and establish murine experimental models to dissect underlying molecular mechanisms.

Design of a multi-center immunophenotyping analysis of peripheral blood, sputum and bronchoalveolar lavage fluid in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS).

BACKGROUND: Subpopulations and Intermediate Outcomes in COPD Study (SPIROMICS) is a multi-center longitudinal, observational study to identify novel phenotypes and biomarkers of chronic obstructive pulmonary disease (COPD). In a subset of 300 subjects enrolled at six clinical centers, we are performing flow cytometric analyses of leukocytes from induced sputum, bronchoalveolar lavage (BAL) and peripheral blood. To minimize several sources of variability, we use a "just-in-time" design that permits immediate staining without pre-fixation of samples, followed by centralized analysis on a single instrument. METHODS: The Immunophenotyping Core prepares 12-color antibody panels, which are shipped to the six Clinical Centers shortly before study visits. Sputum induction occurs at least two weeks before a bronchoscopy visit, at which time peripheral blood and bronchoalveolar lavage are collected. Immunostaining is performed at each clinical site on the day that the samples are collected. Samples are fixed and express shipped to the Immunophenotyping Core for data acquisition on a single modified LSR II flow cytometer. Results are analyzed using FACS Diva and FloJo software and cross-checked by Core scientists who are blinded to subject data. RESULTS: Thus far, a total of 152 sputum samples and 117 samples of blood and BAL have been returned to the Immunophenotyping Core. Initial quality checks indicate useable data from 126 sputum samples (83%), 106 blood samples (91%) and 91 BAL samples (78%). In all three sample types, we are able to identify and characterize the activation state or subset of multiple leukocyte cell populations (including CD4+ and CD8+ T cells, B cells, monocytes, macrophages, neutrophils and eosinophils), thereby demonstrating the validity of the antibody panel. CONCLUSIONS: Our study design, which relies on bi-directional communication between clinical centers and the Core according to a pre-specified protocol, appears to reduce several sources of variability often seen in flow cytometric studies involving multiple clinical sites. Because leukocytes contribute to lung pathology in COPD, these analyses will help achieve SPIROMICS aims of identifying subgroups of patients with specific COPD phenotypes. Future analyses will correlate cell-surface markers on a given cell type with smoking history, spirometry, airway measurements, and other parameters. TRIAL REGISTRATION: This study was registered with ClinicalTrials.gov as NCT01969344 .

Human CD56+ cytotoxic lung lymphocytes kill autologous lung cells in chronic obstructive pulmonary disease.

UNLABELLED: CD56+ natural killer (NK) and CD56+ T cells, from sputum or bronchoalveolar lavage of subjects with chronic obstructive pulmonary disease (COPD) are more cytotoxic to highly susceptible NK targets than those from control subjects. Whether the same is true in lung parenchyma, and if NK activity actually contributes to emphysema progression are unknown. To address these questions, we performed two types of experiments on lung tissue from clinically-indicated resections (n = 60). First, we used flow cytometry on fresh single-cell suspension to measure expression of cell-surface molecules (CD56, CD16, CD8, NKG2D and NKp44) on lung lymphocytes and of the 6D4 epitope common to MICA and MICB on lung epithelial (CD326+) cells. Second, we sequentially isolated CD56+, CD8+ and CD4+ lung lymphocytes, co-cultured each with autologous lung target cells, then determined apoptosis of individual target cells using Annexin-V and 7-AAD staining. Lung NK cells (CD56+ CD3-) and CD56+ T cells (CD56+ CD3+) were present in a range of frequencies that did not differ significantly between smokers without COPD and subjects with COPD. Lung NK cells had a predominantly "cytotoxic" CD56+ CD16+ phenotype; their co-expression of CD8 was common, but the percentage expressing CD8 fell as FEV1 % predicted decreased. Greater expression by autologous lung epithelial cells of the NKG2D ligands, MICA/MICB, but not expression by lung CD56+ cells of the activating receptor NKG2D, correlated inversely with FEV1 % predicted. Lung CD56+ lymphocytes, but not CD4+ or CD8+ conventional lung T cells, rapidly killed autologous lung cells without additional stimulation. Such natural cytotoxicity was increased in subjects with severe COPD and was unexplained in multiple regression analysis by age or cancer as indication for surgery. These data show that as spirometry worsens in COPD, CD56+ lung lymphocytes exhibit spontaneous cytotoxicity of autologous structural lung cells, supporting their potential role in emphysema progression. TRIAL REGISTRATION: ClinicalTrials.gov NCT00281229.

Role of CC chemokine receptor 4 in natural killer cell activation during acute cigarette smoke exposure.

Cigarette smoke (CS)-induced lung injury involves innate immune responses. The activation of innate effector cells is thought to require cross talk with dendritic cells (DCs) and macrophages, but the mediators of interaction are unknown. One candidate, CC chemokine receptor 4 (CCR4), is expressed by innate and adaptive effector cells, and its ligands are produced by DCs and macrophages. Using flow cytometry and confocal microscopy, we defined innate responses of lung myeloid DCs, macrophages, and conventional natural killer (NK) cells in mice exposed to CS over 4 days and examined the contribution of CCR4 using CCR4 knockout (CCR4(-/-)) mice. CS affected populations differently, causing an increase in F4/80(+) macrophages, a reduction in parenchymal CD11c(+)CD11b(+)CD103(-) DCs, but no effect on mucosal CD11c(+)CD11b(-)CD103(+) DCs. CS also induced a population of primed/activated CD69(+) NK cells and bronchoepithelial expression of the stress-related NKG2D receptor-activating protein, retinoic acid early transcript 1. CS-exposed CCR4(-/-) mice were similar to controls regarding effects on DCs and macrophages but displayed substantially impaired NK priming/activation and reduced expression of transcripts for interferon gamma, CXCL10, and retinoic acid early transcript 1. Quantitative confocal microscopy revealed that lungs of CS-exposed CCR4(-/-) mice had significantly reduced contacts of NK cells with CD11c(+) cells. These findings demonstrate that acute CS exposure elicits NK cell responses and suggest that CCR4 promotes NK cell priming/activation by mediating contacts with sentinel cells in the lung.

Cutting edge: Central memory CD8 T cells in aged mice are virtual memory cells.

The number of memory phenotype CD8 T cells increases dramatically with aging in both humans and mice. However, the mechanism for this is unknown. The prevailing hypothesis is that memory T cells accumulate with aging as a result of lifelong antigenic stimulation. However, data supporting this supposition are lacking. In this study, we demonstrate that central memory CD8 T cells, which represent a large majority of memory CD8 T cells in aged mice, are not memory cells that develop in response to antigenic stimulation but are virtual memory cells that develop without antigenic stimulation. In addition to phenotypic evidence, we show that accumulation of central memory CD8 T cells is independent of CD4 T cells, CCR5, and CXCR3, all of which are known to be essential for Ag-driven development of central memory CD8 T cells. Thus, this study reveals a novel mechanism for aging-related changes in CD8 T cells.

The host environment is responsible for aging-related functional NK cell deficiency.

NK cells play an important role in immunity against infection and tumors. Aging-related functional NK cell deficiency is well documented in humans and mice. However, the mechanism for this is poorly understood. Using an adoptive transfer approach in mice, we found that NK cells from both young and aged mice responded vigorously to priming by pathogen-derived products after being cotransferred into young mice. In contrast, NK cells from young mice responded poorly to priming by pathogen-derived products after being transferred to aged mice. In addition to defects in NK cell priming, maturation of NK cells under steady-state conditions is also impaired in aged mice, resulting in a decreased proportion of CD27(-) mature NK cells. We found that bone marrow from young and aged mice gave rise to CD27(-) mature NK cells similarly in young mixed bone marrow chimeric mice. Furthermore, by using a novel bone marrow transfer approach without irradiation, we found that after being transferred to aged mice, bone marrow from young mice gave rise to NK cells with maturation defects. Finally, we found that aging-related functional NK cell deficiency was completely reversed by injecting soluble IL-15/IL-15Rα complexes. In contrast, blockade of IL-10 signaling, which broadly augments inflammatory responses to pathogen-derived products, had little effect on aging-related defects in NK cell priming. These data demonstrate that the aged host environment is responsible for aging-related functional NK cell deficiency. Additionally, our data suggest that IL-15 receptor agonists may be useful tools in treating aging-related functional NK cell deficiency.

IL-4 acts as a potent stimulator of IFN-γ expression in CD8+ T cells through STAT6-dependent and independent induction of Eomesodermin and T-bet.

CD8+ T cell synthesis of IFN-γ is an important component of the CD8+ T cell immune response. In short-term cultures of murine pan-T cells, we found that IL-4 was the principal cytokine responsible for driving IFN-γ synthesis by CD3/CD28-activated CD8+ T cells. IL-4 was able to induce low levels of IFN-γ mRNA in CD8+ T cells even in the absence of CD3/CD28 engagement, although concomitant CD3/CD28 stimulation was necessary for IFN-γ secretion. IL-4 induction of IFN-γ was explained by its ability to induce Eomesodermin and T-bet transcription factors whose expression was further increased by CD3/CD28. Expression of Eomesodermin, T-bet and IFN-γ induced by IL-4 was partially dependent upon activation of MAPK and PI3K but independent of the canonical IL-4-activated transcription factor, STAT6. In contrast, expression of IFN-γ induced by IL-4/CD3/CD28 stimulation showed additional dependency upon STAT6 which functions to increase expression of Eomesodermin specifically. These novel findings point to a function for IL-4 as a direct regulator of IFN-γ expression in CD8+ T cells and reveal the molecular mechanisms involved.

CC chemokine receptor 4 contributes to innate NK and chronic stage T helper cell recall responses during Mycobacterium bovis infection.

Cysteine-cysteinyl chemokine receptor 4 (CCR4) is expressed by a variety of T-cell subsets and leukocytes. This study examined the participation of CCR4 in response to pulmonary infection with Mycobacterium bovis Bacille-Calmette-Guerin (BCG). Constitutive and induced CCR4 agonist expression was detected among large mononuclear cells. The course of infection and mobilization of effector cell populations were then analyzed in CCR4 knockout (CCR4(-/-)) mice. Compared with controls, CCR4(-/-) mice displayed delayed innate stage (<2 weeks) bacterial clearance and reduced late stage inflammation. Innate impairment was associated with reduced natural killer cell activation. In the adaptive phase, CCR4(-/-) mice generated effector T cells in draining lymph nodes and accumulated effector T cells in lungs, which resulted in normal adaptive stage bacterial elimination at 2 to 4 weeks. However, during the late stage, CCR4(-/-) mice had reduced interferonγ+CD4(+)α/ß+ (Th1) and interleukin (IL)-17+CD4(+)α/ß+ (Th17) T helper cells in lungs. In contrast, IL-17+ γ/δ T cells in lungs were unaffected. When challenged with mycobacterial antigen- (Ag-) Ag-coated beads to elicit a recall granulomatous response, CCR4(-/-) mice displayed abrogated recall granuloma formation and reduced interferon γ+ Th1 cells. These findings indicate that CCR4 supports innate natural killer cell activation and sustains later CD4(+) Th effector/memory antimycobacterial responses in the lung but is redundant in the early adaptive elimination phase.

Cysteine-cysteinyl chemokine receptor 6 mediates invariant natural killer T cell airway recruitment and innate stage resistance during mycobacterial infection.

This study examined the contribution of cysteine-cysteinyl chemokine receptor 6 (CCR6) to the innate pulmonary antimycobacterial immune response. Using a mouse model of Mycobacterium bovis BCG airway infection, we detected maximal induction of the CCR6 agonist CCL20 in lungs at 1 week after infection. Infected CCR6 knockout (CCR6-/-) mice displayed an early impairment of bacterial clearance, but ultimately eliminated the attenuated organisms with the onset of adaptive immunity. Flow-cytometric analyses of bronchoalveolar lavages and dispersed lungs revealed a 60% reduction in TCR-α/ß+ T cells in airways but no compromise of TCR-γ/δ+ T cells. The subset of CD1d-restricted, CD8-TCR-α/ß+ natural killer cells, which mediate innate mycobacterial resistance, was profoundly reduced (90%). Analysis of the adaptive response using ovalbumin-specific transgenic TCR T cell (OT-II) transfer combined with infection with recombinant M. bovis BCG producing ovalbumin peptide indicated no impairment of adaptive T cell activation in CCR6-/- mice. There was also no impairment of the induction of cytokine-producing cells in draining lymphoid tissue of CCR6-/- mice. Taken together, our findings indicate that CCR6 is not required for induction of the adaptive antimycobacterial response, but is likely critical to airway compartment mobilization of TCR-α/ß+CCR6+ innate and adaptive effector T cells.

Mononuclear phagocyte-derived IL-10 suppresses the innate IL-12/IFN-gamma axis in lung-challenged aged mice.

Previously, we reported that IL-10-producing mononuclear phagocytes increase in lungs of aged mice, causing impaired innate cytokine expression. Since dendritic cells (DCs) contribute to innate NK cell and adaptive T cell immunity, we tested the hypothesis that age-related IL-10 might influence DC function with effects on NK and T cell activation. The results showed that DC recruitment to sites of lung inflammation was normal in aged mice (>20 mo). However, IFN-gamma-producing NK cells in LPS-challenged lungs were decreased in aged as compared with young mice, which was associated with increased IL-10(+)CD11b(+)Gr-1(low)CD11c(-) cells consistent with mononuclear phagocytes. In vivo or in vitro blockade of IL-10 signaling restored IFN-gamma-producing NK cells. This restoration was reversed by IL-12 neutralization, indicating that IL-10 suppressed sources of IL-12 in aged mice. To probe DC function in adaptive immunity, we transferred young naive OVA-specific TCR transgenic T cells to old mice. Following challenge with OVA plus LPS, Ag presentation in the context of MHC-I and MHC-II occurred with similar kinetics and intensity in draining lymph nodes of young and old recipients as measured by proliferation. Despite this, aged hosts displayed impaired induction of IFN-gamma(+)CD4(+), but not IFN-gamma(+)CD8(+), effector T cells. Blockade of IL-10 signaling reversed age-associated defects. These studies indicate that the innate IL-12/IFN-gamma axis is not intrinsically defective in lungs of aged mice, but is rather suppressed by enhanced production of mononuclear phagocyte-derived IL-10. Our data identify a novel mechanism of age-associated immune deficiency.

Increased Foxp3(+) Treg cell activity reduces dendritic cell co-stimulatory molecule expression in aged mice.

As potent suppressors of immune responses to self- and foreign-antigens, Foxp3(+) Treg cells are suspected to be involved in immunosuppression leading to cancer, neurodegeneration and infection. Since ageing is associated with increased incidence of these diseases, we compared Treg activity in blood, lymphoid organs and lungs of young (5-6 months) and old (21-22 months) mice. Both the proportion and absolute number of Foxp3(+) CD4(+) Treg cells increased with age in secondary lymphoid organs but not in blood and lungs as compared to Foxp3(-) CD4(+) T cells. Although numbers of thymic and naïve conventional T and Treg cells decreased with age, Treg cells with memory/effector phenotype increased disproportionately in peripheral lymphoid tissues. In addition, CD40 and CD86 co-stimulatory molecule expression by lymph node dendritic cells was impaired in old mice and could be restored to levels of young mice by inactivating Treg cells with anti-CD25 monoclonal antibodies. These findings have important implications for the understanding of age-related immune dysfunction.

Mononuclear phagocyte-derived interleukin-10 suppresses the innate pulmonary granuloma cytokine response in aged mice.

Granulomas are sequestration responses observed in a wide variety of clinical conditions, including mycobacterial infection. We previously reported impaired adaptive, Th1 cell-mediated pulmonary granuloma formation in response to bead-immobilized Mycobacterium bovis-purified protein derivative in aged mice. To reveal determinants of age-related immune deficits, the present study examined the effect of aging on early innate stage pulmonary granuloma formation. Aged mice formed more neutrophil-rich innate granulomas with augmented CXCL2 expression followed by a pattern of rapid decay of tumor necrosis factor-alpha, interleukin (IL)-6, CCL3, and CXCL2. This was associated with enhanced IL-10 expression. Blockade of IL-10 signaling with anti-IL-10 receptor antibody reversed the age-related decay. Intracellular flow cytometric analysis revealed that CD11b(+)Gr-1(+/-) mononuclear phagocytes were the primary leukocyte sources of IL-10 in lungs, and their numbers were increased in aged mice. When exposed to purified protein derivative in vitro, young and old CD11b(+)Gr-1(+/-) mononuclear phagocytes from blood or lung had comparable IL-10 expression, suggesting in vivo signals in the aged environment enhanced the number of IL-10-producing cells in the aged lung. Our findings reveal a novel mechanism of age-associated IL-10 mediated pulmonary immune suppression with the potential to alter downstream adaptive immunity.

CCR4 participation in Th type 1 (mycobacterial) and Th type 2 (schistosomal) anamnestic pulmonary granulomatous responses.

CCR4 is purported to be a Th type 2 (Th2) cell-biased receptor but its functional role is unclear. Recent studies suggest that chemokine receptor expression and function are more complex in vivo and raise doubts regarding restricted CCR4 expression by Th2 cells. To address these issues, we analyzed the role of CCR4 in highly polarized models of Th type 1 (Th1) and Th2 cell-mediated pulmonary granulomas, respectively, elicited by i.v. challenge of primed mice with either mycobacterial purified protein derivative or schistosomal egg Ag-coated beads. CCR4 agonists were expressed during both responses, correlating with a shift of CCR4+ CD4+ T cells from blood to lungs. CCL22 dominated in draining nodes during the Th1 response. Analysis of CD4+ effector T cells revealed CCR4 expression and CCR4-mediated chemotaxis by both IFN-gamma and IL-4 producers. Studies of CCR4 knockout (CCR4(-/-)) mice showed partial impairment of the local type-2 cytokine response and surprisingly strong impairment of the Th1 response with abrogated IFN-gamma production during secondary but not primary challenge. Adoptive transfer indicated CCR4(-/-)CD4+ Th1 cell function was defective but this could not be reconstituted with wild-type (CCR4(+/+)) CD4+ T cells indicating involvement of another CCR4+ population. Coculture of CCR4(+/+)CD4+ T cells and CCR4(-/-) dendritic cells revealed intact IL-2 but impaired IFN-gamma production, pointing to a role for CCR4+ dendritic cells in effector cell expression. Therefore, CCR4 is not Th2-restricted and was required for sustenance and expression of the Th1 effector/memory response to mycobacterial Ags.

AMD3465, a novel CXCR4 receptor antagonist, abrogates schistosomal antigen-elicited (type-2) pulmonary granuloma formation.

CXCR4 is a major receptor for CXCL12 and is known to participate in multiple physiological systems. The present study tested a second generation CXCR4 antagonist, AMD3465, for effects on highly defined models of Th1- and Th2-cell-mediated hypersensitivity-type pulmonary granuloma formation. Type-1 and type-2 granulomas were induced, respectively, by intravenous challenge of sensitized CBA/J mice with Mycobacteria bovis purified protein derivative- or Schistosoma mansoni egg antigen-coated beads. Before challenge, mice were implanted with osmotic pumps releasing AMD3465 at 5 microg/hour (6 mg/kg/day). Compared to vehicle, AMD3465 had minimal effect on type-1 inflammation or cytokine responses in draining lymph nodes, but the type-2 inflammation was significantly abrogated with reductions in lesion size and eosinophil content as well as abrogated interleukin (IL)-5, IL-10, and IL-13 cytokine production in draining lymph nodes. The biased effect of AMD3465 correlated with greater CXCR4 ligand expression in the type-2 model. Treatment during a primary response impaired lymph node IL-2 production after both Mycobacteria bovis purified protein derivative and Schistosoma mansoni egg antigen challenge indicating an unbiased effect during immune induction. In summary, CXCR4 blockade inhibited eosinophil recruitment during type-2 granuloma formation and interfered with primary and secondary T-cell activation events in lymphoid tissue, suggesting potential therapeutic application for chronic hypersensitivity diseases.