In JoVE (1)

Other Publications (40)

Articles by Donald N. Cook in JoVE

Other articles by Donald N. Cook on PubMed

Impaired T Cell Function in RANTES-deficient Mice

Clinical Immunology (Orlando, Fla.). Mar, 2002  |  Pubmed ID: 11890717

The chemokine RANTES is a chemoattractant for monocytes and T cells and is postulated to participate in many aspects of the immune response. To evaluate the biological roles of RANTES in vivo, we generated RANTES-deficient (-/-) mice and characterized their T cell function. In cutaneous delayed-type hypersensitivity assays, a 50% reduction in ear and footpad swelling was seen in -/- mice compared to +/+ mice. In vitro, polyclonal and antigen-specific T cell proliferation was decreased. Quantitative analysis using the fluorescent dye carboxy-fluorescein succinimidyl ester revealed that this proliferative defect was due both to fewer antigen-reactive T cells and to a reduction in the capacity of these cells to proliferate. In addition, IFN-gamma and IL-2 production by the -/- T cells was dramatically decreased. Together, these data suggest that RANTES is required for normal T cell functions as well as for recruiting monocytes and T cells to sites of inflammation.

A Matrix for New Ideas in Pulmonary Fibrosis

American Journal of Respiratory Cell and Molecular Biology. Aug, 2002  |  Pubmed ID: 12151302

The Genetics of Innate Immunity in the Lung

Chest. Mar, 2003  |  Pubmed ID: 12628980

Toll-like Receptors and the Genetics of Innate Immunity

Current Opinion in Allergy and Clinical Immunology. Dec, 2003  |  Pubmed ID: 14612679

The discovery that mammalian Toll-like receptors recognize microbial products and initiate innate immune responses to them has spawned a new field of biology, namely the study of molecular interactions linking microbial recognition to innate and adaptive immune responses. This field has grown very rapidly in recent years, due largely to recent advances in genetic technology. This review summarizes recent work in which genetic approaches have been used to identify novel and important facets of Toll-like receptor function.

The Role of Toll-like Receptor 4 in Environmental Airway Injury in Mice

American Journal of Respiratory and Critical Care Medicine. Jul, 2004  |  Pubmed ID: 15020293

Inhalation of toxins commonly found in air pollution contributes to the development and progression of asthma and environmental airway injury. In this study, we investigated the requirement of toll-like receptor 4 (TLR4) in mice for pulmonary responses to three environmental toxins: aerosolized lipopolysaccharide, particulate matter (residual oil fly ash), and ozone. The physiologic and biologic responses to these toxins were evaluated by the extent of airway responsiveness, neutrophil recruitment to the lower respiratory tract, changes in inflammatory cytokines, and the concentration of protein in the lavage fluid. Genetically engineered, TLR4-deficient mice (C57BL/6(TLR4-/-)) were unresponsive to inhaled lipopolysaccharide, except for minimal increases in some inflammatory cytokines. In contrast, C57BL/6(TLR4-/-) mice did not differ from wild-type mice in their airway response to instilled residual oil fly ash or acute ozone exposure; however, we found that, despite a robust inflammatory response, C57BL/6(TLR4-/-) mice are protected against the development of airway hyperresponsiveness after subchronic ozone exposure. These data demonstrate in the mouse that the requirement of TLR4 for pulmonary inflammation depends on the nature of the toxin and appears specific to toxin and exposure conditions.

Depletion of Host Langerhans Cells Before Transplantation of Donor Alloreactive T Cells Prevents Skin Graft-versus-host Disease

Nature Medicine. May, 2004  |  Pubmed ID: 15098028

Skin is the most commonly affected organ in graft-versus-host disease (GVHD). To explore the role of Langerhans cells in GVHD, the principal dendritic cells of the skin, we studied the fate of these cells in mice transplanted with allogeneic bone marrow. In contrast to other dendritic cells, host Langerhans cells were replaced by donor Langerhans cells only when donor T cells were administered along with bone marrow, and the extent of Langerhans cell chimerism correlated with the dose of donor T cells injected. Donor T cells depleted host Langerhans cells through a Fas-dependent pathway and induced the production in skin of CCL20, which was required for the recruitment of donor Langerhans cells. Administration of donor T cells to bone marrow-chimeric mice with persistent host Langerhans cells, but not to mice whose Langerhans cells had been replaced, resulted in marked skin GVHD. These findings indicate a crucial role for donor T cells in host Langerhans cell replacement, and show that host dendritic cells can persist in nonlymphoid tissue for the duration of an animal's life and can trigger GVHD despite complete blood chimerism.

Genetic Regulation of Endotoxin-induced Airway Disease

Genomics. Jun, 2004  |  Pubmed ID: 15177550

To identify novel genes regulating the biologic response to lipopolysaccharide (LPS), we used a combination of quantitative trait locus (QTL) analysis and microarray-based gene expression studies of C57BL/6J x DBA/2J(BXD) F2 and recombinant inbred (RI) mice. A QTL affecting pulmonary TNF-alpha production was identified on chromosome 2, and a region affecting both polymorphonuclear leukocyte recruitment and TNF-alpha levels was identified on chromosome 11. Microarray analyses of unchallenged and LPS-challenged BXD RI strains identified approximately 500 genes whose expression was significantly changed by inhalation of LPS. Of these genes, 28 reside within the chromosomal regions identified by the QTL analyses, implicating these genes as high priority candidates for functional studies. Additional high priority candidate genes were identified based on their differential expression in mice having high and low responses to LPS. Functional studies of these genes are expected to reveal important molecular mechanisms regulating the magnitude of biologic responses to LPS.

Toll-like Receptors in the Pathogenesis of Human Disease

Nature Immunology. Oct, 2004  |  Pubmed ID: 15454920

Members of the Toll-like receptor (TLR) family are key regulators of both innate and adaptive immune responses. The function of TLRs in various human diseases has been investigated by comparison of the incidence of disease among people having different polymorphisms in genes that participate in TLR signaling. These studies have shown that TLR function affects several diseases, including sepsis, immunodeficiencies, atherosclerosis and asthma. As this body of data grows, it will provide new insights into disease pathogenesis as well as valuable information on the merits of various therapeutic options.

The Critical Role of Hematopoietic Cells in Lipopolysaccharide-induced Airway Inflammation

American Journal of Respiratory and Critical Care Medicine. Apr, 2005  |  Pubmed ID: 15618460

Rapid and selective recruitment of neutrophils into the airspace in response to LPS facilitates the clearance of bacterial pathogens. However, neutrophil infiltration can also participate in the development and progression of environmental airway disease. Previous data have revealed that Toll-like receptor 4 (tlr4) is required for neutrophil recruitment to the lung after either inhaled or systemically administrated LPS from Escherichia coli. Although many cell types express tlr4, endothelial cell expression of tlr4 is specifically required to sequester neutrophils in the lung in response to systemic endotoxin. To identify the cell types requiring trl4 expression for neutrophil recruitment after inhaled LPS, we generated chimeric mice separately expressing tlr4 on either hematopoietic cells or on structural lung cells. Neutrophil recruitment into the airspace was completely restored in tlr4-deficient mice receiving wild-type bone marrow. By contrast, wild-type animals receiving tlr4-deficient marrow had dramatically reduced neutrophil recruitment. Moreover, adoptive transfer of wild-type alveolar macrophages also restored the ability of tlr4-deficient recipient mice to recruit neutrophils to the lung. These data demonstrate the critical role of hematopoietic cells and alveolar macrophages in initiating LPS-induced neutrophil recruitment from the vascular space to the airspace.

Attenuation of Allergen-induced Responses in CCR6-/- Mice is Dependent Upon Altered Pulmonary T Lymphocyte Activation

Journal of Immunology (Baltimore, Md. : 1950). Feb, 2005  |  Pubmed ID: 15699135

We have established a defect in CCR6-/- mice in response to a cockroach allergen airway challenge characterized by decreased IL-5 production, reduced CD4+ T and B cells as well as decreased eosinophil accumulation. To determine the nature of the defect in CCR6-/- mice T lymphocyte populations from allergen-sensitized wild-type mice were transferred into sensitized CCR6-/- mice. The reconstituted response was characterized by an increase in IL-5 levels, eosinophil accumulation, and serum IgE levels in recipient CCR6-/- mice. Analysis of lymphocytes from draining lymph nodes of CCR6+/+ and CCR6-/- sensitized or challenged mice demonstrated a significant decrease in IL-5 and IL-13 production in CCR6-/- mice. In contrast, the systemic response in allergen-rechallenged spleen cells demonstrated no significant alteration in allergen-induced cytokine production. Transfer of isolated splenic T lymphocytes from sensitized CCR6+/+ mice induced airway hyperresponsiveness in wild-type but not CCR6-/- naive mice, suggesting that T cells alone were not sufficient to induce airway hyperresponsiveness in CCR6-/- mice. Additional analysis demonstrated decreased CD11c+, CD11b+ and CD11c, and B220 subsets of dendritic cells in the lungs of CCR6-/- mice after allergen challenge. Using in vitro cell mixing studies with isolated pulmonary CD4+ T cells and CD11c+ cells from CCR6+/+ or CCR6-/- mice, we demonstrate alterations in both CCR6-/- T cells and CCR6-/- pulmonary APCs to elicit IL-5 responses. Altogether, the defect in CCR6-/- mice appears to be primarily due to an alteration in T cell activation, but also appears to include local pulmonary APC defects.

The Chemokine Receptor D6 Limits the Inflammatory Response in Vivo

Nature Immunology. Apr, 2005  |  Pubmed ID: 15750596

How the inflammatory response is initiated has been well defined but relatively little is known about how such responses are resolved. Here we show that the D6 chemokine receptor is involved in the post-inflammatory clearance of beta-chemokines from cutaneous sites. After induction of inflammation by phorbol esters, wild-type mice showed a transient inflammatory response. However, in D6-deficient mice, an excess concentration of residual chemokines caused a notable inflammatory pathology with similarities to human psoriasis. These results suggest that D6 is involved in the resolution of the cutaneous inflammatory response.

Increased Inflammation in Mice Deficient for the Chemokine Decoy Receptor D6

European Journal of Immunology. May, 2005  |  Pubmed ID: 15789340

Chemokines are chemotactic cytokines with a key role in the control of cell trafficking and positioning under homeostatic and inflammatory conditions. D6 is a promiscuous 7-transmembrane-domain receptor expressed on lymphatic vessels which recognizes most inflammatory, but not homeostatic, CC chemokines. In vitro experiments demonstrated that D6 is unable to signal after ligand engagement, and it is structurally adapted to sustain rapid and efficient ligand internalization and degradation. These unique functional properties lead to the hypothesis that D6 may be involved in the control of inflammation by acting as a decoy and scavenger receptor for inflammatory chemokines. Consistent with this hypothesis, here we report that D6(-/-) mice showed an anticipated and exacerbated inflammatory response in a model of skin inflammation. Moreover, the absence of D6 resulted in increase cellularity and inflammatory-chemokine levels in draining lymph nodes. Thus, D6 is a decoy receptor structurally adapted and strategically located to tune tissue inflammation and control transfer of inflammatory chemokines to draining lymph nodes.

CCL5-CCR5 Interaction Provides Antiapoptotic Signals for Macrophage Survival During Viral Infection

Nature Medicine. Nov, 2005  |  Pubmed ID: 16208318

Host defense against viruses probably depends on targeted death of infected host cells and then clearance of cellular corpses by macrophages. For this process to be effective, the macrophage must presumably avoid its own virus-induced death. Here we identify one such mechanism. We show that mice lacking the chemokine Ccl5 are immune compromised to the point of delayed viral clearance, excessive airway inflammation and respiratory death after mouse parainfluenza or human influenza virus infection. Virus-inducible levels of Ccl5 are required to prevent apoptosis of virus-infected mouse macrophages in vivo and mouse and human macrophages ex vivo. The protective effect of Ccl5 requires activation of the Ccr5 chemokine receptor and consequent bilateral activation of G(alphai)-PI3K-AKT and G(alphai)-MEK-ERK signaling pathways. The antiapoptotic action of chemokine signaling may therefore allow scavengers to finally stop the host cell-to-cell infectious process.

Polymorphisms of the Toll-like Receptors and Human Disease

Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America. Nov, 2005  |  Pubmed ID: 16237638

The Toll-like receptor (TLR) family regulates both innate and adaptive immune responses. Given its broad effect on immunity, the function of TLRs in various human diseases has been investigated largely by comparing the incidence of disease among persons with different polymorphisms in the genes that participate in TLR signaling. These studies demonstrate that TLR function affects several diseases, including sepsis, immunodeficiencies, atherosclerosis, and asthma. These findings have resulted in new opportunities to study the pathogenesis of disease, identify subpopulations at greater risk of disease, and, potentially, identify novel therapeutic approaches.

Spontaneous Mutations in Recombinant Inbred Mice: Mutant Toll-like Receptor 4 (Tlr4) in BXD29 Mice

Genetics. Mar, 2006  |  Pubmed ID: 16322526

Recombinant inbred (RI) mice are frequently used to identify QTL that underlie differences in measurable phenotypes between two inbred strains of mice. Here we show that one RI strain, C57BL/6J x DBA/2J (BXD29), does not develop an inflammatory response following inhalation of LPS. Approximately 25% of F2 mice [F1(BXD29 x DBA/2J) x F1] are also unresponsive to inhaled LPS, suggesting the presence of a recessive mutation in the BXD29 strain. A genomic scan of these F2 mice revealed that unresponsive animals, but not responsive animals, are homozygous for C57BL/6J DNA at a single locus on chromosome 4 close to the genomic location of Tlr4. All progeny between BXD29 and gene-targeted Tlr4-deficient mice are unresponsive to inhaled LPS, suggesting that the mutation in the BXD29 strain is allelic with Tlr4. Moreover, the intact Tlr4 receptor is not displayed on the cell surface of BXD29 macrophages. Finally, a molecular analysis of the Tlr4 gene in BXD29 mice revealed that it is interrupted by a large insertion of repetitive DNA. These findings explain the unresponsiveness of BXD29 mice to LPS and suggest that data from BXD29 mice should not be included when using BXD mice to study phenotypes affected by Tlr4 function. Our results also suggest that the frequency of such unidentified, spontaneously occurring mutations is an issue that should be considered when RI strains are used to identify QTL.

TLR4 Signaling Attenuates Ongoing Allergic Inflammation

Journal of Immunology (Baltimore, Md. : 1950). May, 2006  |  Pubmed ID: 16670292

The relationship between LPS exposure and allergic asthma is poorly understood. Epidemiologic studies in humans have found that exposure to LPS can protect, have no effect, or exacerbate allergic asthma. Similarly, LPS has had variable effects on allergic pulmonary inflammation in the mouse, depending on the model used. In the present study, we studied the effect of very low doses of LPS in models of both short-term and long-term allergen challenge. When challenged with allergen for short periods, wild-type and tlr4-deficient mice had similar responses. However, when challenged for periods of 1 wk or longer, tlr4-deficient mice developed dramatically increased airway eosinophils, serum IgE, and Th2 cytokines compared with similarly challenged, genetically matched C57BL/6 mice. The relative attenuation of allergic responses seen in C57BL/6 mice was dependent on bone marrow-derived cell-specific expression of tlr4, and was not associated with an increase in Th1 responses. The number of dendritic cells in lungs of challenged tlr4-deficient mice was significantly increased compared with those in challenged C57BL/6 mice. No differences were seen in the abilities of naive C57BL/6 and tlr4-deficient mice to develop allergen-specific tolerance after exposure to similar preparations of OVA, suggesting that tolerance and regulation of existing inflammation develop through different mechanisms. The attenuation of eosinophilic inflammation in C57BL/6 mice was abolished when these mice were challenged with OVA supplemented with additional LPS. Together, these findings show that low doses of endotoxin can have regulatory effects on allergic inflammation, particularly in the setting of ongoing allergen exposure.

Control of Microglial Neurotoxicity by the Fractalkine Receptor

Nature Neuroscience. Jul, 2006  |  Pubmed ID: 16732273

Microglia, the resident inflammatory cells of the CNS, are the only CNS cells that express the fractalkine receptor (CX3CR1). Using three different in vivo models, we show that CX3CR1 deficiency dysregulates microglial responses, resulting in neurotoxicity. Following peripheral lipopolysaccharide injections, Cx3cr1-/- mice showed cell-autonomous microglial neurotoxicity. In a toxic model of Parkinson disease and a transgenic model of amyotrophic lateral sclerosis, Cx3cr1-/- mice showed more extensive neuronal cell loss than Cx3cr1+ littermate controls. Augmenting CX3CR1 signaling may protect against microglial neurotoxicity, whereas CNS penetration by pharmaceutical CX3CR1 antagonists could increase neuronal vulnerability.

Cutting Edge: the Silent Chemokine Receptor D6 is Required for Generating T Cell Responses That Mediate Experimental Autoimmune Encephalomyelitis

Journal of Immunology (Baltimore, Md. : 1950). Jul, 2006  |  Pubmed ID: 16785491

D6, a promiscuous nonsignaling chemokine binding molecule expressed on the lymphatic endothelium, internalizes and degrades CC chemokines, and D6(-/-) mice demonstrated increased cutaneous inflammation following topical phorbol ester or CFA injection. We report that D6(-/-) mice were unexpectedly resistant to the induction of experimental autoimmune encephalomyelitis due to impaired encephalitogenic responses. Following induction with myelin oligodendroglial glycoprotein (MOG) peptide 35-55 in CFA, D6(-/-) mice showed reduced spinal cord inflammation and demyelination with lower incidence and severity of experimental autoimmune encephalomyelitis attacks as compared with D6(+/+) littermates. In adoptive transfer studies, MOG-primed D6(+/-) T cells equally mediated disease in D6(+/+) or D6(-/-) mice, whereas cells from D6(-/-) mice transferred disease poorly to D6(+/-) recipients. Lymph node cells from MOG-primed D6(-/-) mice showed weak proliferative responses and made reduced IFN-gamma but normal IL-5. CD11c(+) dendritic cells accumulated abnormally in cutaneous immunization sites of D6(-/-) mice. Surprisingly, D6, a "silent" chemokine receptor, supports immune response generation.

The Chemokine Receptor D6 Has Opposing Effects on Allergic Inflammation and Airway Reactivity

American Journal of Respiratory and Critical Care Medicine. Feb, 2007  |  Pubmed ID: 17095748

The D6 chemokine receptor can bind and scavenge several chemokines, including the T-helper 2 (Th2)-associated chemokines CCL17 and CCL22. Although D6 is constitutively expressed in the lung, its pulmonary function is unknown.

Protection Against Inflammation- and Autoantibody-caused Fetal Loss by the Chemokine Decoy Receptor D6

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2007  |  Pubmed ID: 17283337

Fetal loss in animals and humans is frequently associated with inflammatory conditions. D6 is a promiscuous chemokine receptor with decoy function, expressed in lymphatic endothelium, that recognizes and targets to degradation most inflammatory CC chemokines. Here, we report that D6 is expressed in placenta on invading extravillous trophoblasts and on the apical side of syncytiotrophoblast cells, at the very interface between maternal blood and fetus. Exposure of D6-/- pregnant mice to LPS or antiphospholipid autoantibodies results in higher levels of inflammatory CC chemokines and increased leukocyte infiltrate in placenta, causing an increased rate of fetal loss, which is prevented by blocking inflammatory chemokines. Thus, the promiscuous decoy receptor for inflammatory CC chemokines D6 plays a nonredundant role in the protection against fetal loss caused by systemic inflammation and antiphospholipid antibodies.

Innate Immune Control of Pulmonary Dendritic Cell Trafficking

Proceedings of the American Thoracic Society. Jul, 2007  |  Pubmed ID: 17607005

Dendritic cells (DC) are potent antigen-presenting cells that are essential for initiating adaptive immune responses. Residing within the airway mucosa, pulmonary DC continually sample the antigenic content of inhaled air and migrate to draining lymph nodes, where they present these antigens to naive T cells. The migratory patterns of pulmonary DC are highly dependent upon inflammatory conditions in the lung. Under steady-state, or non-inflammatory, conditions, pulmonary DC undergo slow but constitutive migration to draining lymph nodes, where they remain for several days and confer antigen-specific tolerance. With the onset of pulmonary inflammation, airway DC trafficking increases dramatically, and these cells rapidly accumulate within draining lymph nodes. However, within a few days, the number of airway-derived DC in lymph nodes stabilizes or declines, even in the face of ongoing pulmonary inflammation. Here, we summarize current understanding of the molecular and cellular mechanisms underlying pulmonary DC trafficking to the lymph node and the recruitment of DC precurors to the lung. It is hoped that an improved understanding of these mechanisms will lead to novel DC-mediated therapeutic strategies to treat immune-related pulmonary disease.

The Chemokine Receptor CCR6 is an Important Component of the Innate Immune Response

European Journal of Immunology. Sep, 2007  |  Pubmed ID: 17694574

In our initial studies we found that naïve CCR6-deficient (CCR6(-/-)) C57BL/6 mice possessed significantly lower number of both F4/80(+) macrophages and dendritic cells (DC), but higher number of B cells in the peritoneal cavity, as compared to naïve wild type (WT) controls. Furthermore, peritoneal macrophages isolated from CCR6(-/-) mice expressed significantly lower levels of inflammatory cytokines and nitric oxide following lipopolysaccharide (LPS)stimulation, as compared to WT macrophages. In a severe experimental peritonitis model induced by cecal ligation and puncture (CLP), CCR6(-/-) mice were protected when compared with WT controls. At 24 h following the induction of peritonitis, CCR6(-/-) mice exhibited significantly lower levels of inflammatory cytokines/chemokines in both the peritoneal cavity and blood. Interestingly, DC recruitment into the peritoneal cavity was impaired in CCR6(-/-) mice during the evolution of CLP-induced peritonitis. Peritoneal macrophages isolated from surviving CCR6(-/-) mice 3 days after CLP-induced peritonitis exhibited an enhanced LPS response compared with similarly treated WT peritoneal macrophages. These data illustrate that CCR6 deficiency alters the innate response via attenuating the hyperactive local and systemic inflammatory response during CLP-induced peritonitis.

Alloimmune Lung Injury Induced by Local Innate Immune Activation Through Inhaled Lipopolysaccharide

Transplantation. Oct, 2007  |  Pubmed ID: 17989607

Alloimmune lung injury, characterized by perivascular lymphocytic inflammation, lymphocytic bronchiolitis (LB), and obliterative bronchiolitis (OB), causes substantial morbidity and mortality after lung transplantation and bone marrow transplantation (BMT), but little is known regarding its pathogenesis. We have developed and pursued the hypothesis that local activation of pulmonary innate immunity through toll-like receptor (TLR)-4 is critical to the development of posttransplant alloimmune lung injury.

Blood-derived Inflammatory Dendritic Cells in Lymph Nodes Stimulate Acute T Helper Type 1 Immune Responses

Nature Immunology. Apr, 2009  |  Pubmed ID: 19252492

T helper type 1 (T(H)1)-polarized immune responses, which confer protection against intracellular pathogens, are thought to be initiated by dendritic cells (DCs) that enter lymph nodes from peripheral tissues. Here we found after viral infection or immunization, inflammatory monocytes were recruited into lymph nodes directly from the blood to become CD11c(+)CD11b(hi)Gr-1(+) inflammatory DCs, which produced abundant interleukin 12p70 and potently stimulated T(H)1 responses. This monocyte extravasation required the chemokine receptor CCR2 but not the chemokine CCL2 or receptor CCR7. Thus, the accumulation of inflammatory DCs and T(H)1 responses were much lower in Ccr2(-/-) mice, were preserved in Ccl2(-/-) mice and were relatively higher in CCL19-CCL21-Ser-deficient plt mutant mice, in which all other lymph node DC types were fewer in number. We conclude that blood-derived inflammatory DCs are important in the development of T(H)1 immune responses.

Allergic Sensitization Through the Airway Primes Th17-dependent Neutrophilia and Airway Hyperresponsiveness

American Journal of Respiratory and Critical Care Medicine. Oct, 2009  |  Pubmed ID: 19661246

In humans, immune responses to inhaled aeroallergens develop in the lung and draining lymph nodes. Many animal models of asthma bypass this route and instead use intraperitoneal injections of allergen using aluminum hydroxide as an adjuvant.

Ozone Activates Pulmonary Dendritic Cells and Promotes Allergic Sensitization Through a Toll-like Receptor 4-dependent Mechanism

The Journal of Allergy and Clinical Immunology. May, 2010  |  Pubmed ID: 20394980

The Chemokine, CCL3, and Its Receptor, CCR1, Mediate Thoracic Radiation-induced Pulmonary Fibrosis

American Journal of Respiratory Cell and Molecular Biology. Jul, 2011  |  Pubmed ID: 20870892

Patients receiving thoracic radiation often develop pulmonary injury and fibrosis. Currently, there are no effective measures to prevent or treat these conditions. We tested whether blockade of the chemokine, CC chemokine ligand (CCL) 3, and its receptors, CC chemokine receptor (CCR) 1 and CCR5, can prevent radiation-induced lung inflammation and fibrosis. C57BL/6J mice received thoracic radiation, and the interaction of CCL3 with CCR1 or CCR5 was blocked using genetic techniques, or by pharmacologic intervention. Lung inflammation was assessed by histochemical staining of lung tissue and by flow cytometry. Fibrosis was measured by hydroxyproline assays and collagen staining, and lung function was studied by invasive procedures. Irradiated mice lacking CCL3 or its receptor, CCR1, did not develop the lung inflammation, fibrosis, and decline in lung function seen in irradiated wild-type mice. Pharmacologic treatment of wild-type mice with a small molecule inhibitor of CCR1 also prevented lung inflammation and fibrosis. By contrast, mice lacking CCR5 were not protected from radiation-induced injury and fibrosis. The selective interaction of CCL3 with its receptor, CCR1, is critical for radiation-induced lung inflammation and fibrosis, and these conditions can be largely prevented by a small molecule inhibitor of CCR1.

Strain-dependent Genomic Factors Affect Allergen-induced Airway Hyperresponsiveness in Mice

American Journal of Respiratory Cell and Molecular Biology. Oct, 2011  |  Pubmed ID: 21378263

Asthma is etiologically and clinically heterogeneous, making the genomic basis of asthma difficult to identify. We exploited the strain-dependence of a murine model of allergic airway disease to identify different genomic responses in the lung. BALB/cJ and C57BL/6J mice were sensitized with the immunodominant allergen from the Dermatophagoides pteronyssinus species of house dust mite (Der p 1), without exogenous adjuvant, and the mice then underwent a single challenge with Der p 1. Allergic inflammation, serum antibody titers, mucous metaplasia, and airway hyperresponsiveness were evaluated 72 hours after airway challenge. Whole-lung gene expression analyses were conducted to identify genomic responses to allergen challenge. Der p 1-challenged BALB/cJ mice produced all the key features of allergic airway disease. In comparison, C57BL/6J mice produced exaggerated Th2-biased responses and inflammation, but exhibited an unexpected decrease in airway hyperresponsiveness compared with control mice. Lung gene expression analysis revealed genes that were shared by both strains and a set of down-regulated genes unique to C57BL/6J mice, including several G-protein-coupled receptors involved in airway smooth muscle contraction, most notably the M2 muscarinic receptor, which we show is expressed in airway smooth muscle and was decreased at the protein level after challenge with Der p 1. Murine strain-dependent genomic responses in the lung offer insights into the different biological pathways that develop after allergen challenge. This study of two different murine strains demonstrates that inflammation and airway hyperresponsiveness can be decoupled, and suggests that the down-modulation of expression of G-protein-coupled receptors involved in regulating airway smooth muscle contraction may contribute to this dissociation.

Cyclooxygenase-2 Regulates Th17 Cell Differentiation During Allergic Lung Inflammation

American Journal of Respiratory and Critical Care Medicine. Jul, 2011  |  Pubmed ID: 21474648

Th17 cells comprise a distinct lineage of proinflammatory T helper cells that are major contributors to allergic responses. It is unknown whether cyclooxygenase (COX)-derived eicosanoids regulate Th17 cells during allergic lung inflammation.

IL-35 Production by Inducible Costimulator (ICOS)-positive Regulatory T Cells Reverses Established IL-17-dependent Allergic Airways Disease

The Journal of Allergy and Clinical Immunology. Jan, 2012  |  Pubmed ID: 21906794

Recent evidence suggests that IL-17 contributes to airway hyperresponsiveness (AHR); however, the mechanisms that suppress the production of this cytokine remain poorly defined.

ATP Binding Cassette Transporter G1 Deletion Induces IL-17-dependent Dysregulation of Pulmonary Adaptive Immunity

Journal of Immunology (Baltimore, Md. : 1950). Jun, 2012  |  Pubmed ID: 22539789

Mice with genetic deletion of the cholesterol transporter ATP binding cassette G1 (ABCG1) have pulmonary lipidosis and enhanced innate immune responses in the airway. Whether ABCG1 regulates adaptive immune responses to the environment is unknown. To this end, Abcg1(+/+) and Abcg1(-/-) mice were sensitized to OVA via the airway using low-dose LPS as an adjuvant, and then challenged with OVA aerosol. Naive Abcg1(-/-) mice displayed increased B cells, CD4(+) T cells, CD8(+) T cells, and dendritic cells (DCs) in lung and lung-draining mediastinal lymph nodes, with lung CD11b(+) DCs displaying increased CD80 and CD86. Upon allergen sensitization and challenge, the Abcg1(-/-) airway, compared with Abcg1(+/+), displayed reduced Th2 responses (IL-4, IL-5, eosinophils), increased neutrophils and IL-17, but equivalent airway hyperresponsiveness. Reduced Th2 responses were also found using standard i.p. OVA sensitization with aluminum hydroxide adjuvant. Mediastinal lymph nodes from airway-sensitized Abcg1(-/-) mice produced reduced IL-5 upon ex vivo OVA challenge. Abcg1(-/-) CD4(+) T cells displayed normal ex vivo differentiation, whereas Abcg1(-/-) DCs were found paradoxically to promote Th2 polarization. Th17 cells, IL-17(+) γδT cells, and IL-17(+) neutrophils were all increased in Abcg1(-/-) lungs, suggesting Th17 and non-Th17 sources of IL-17 excess. Neutralization of IL-17 prior to challenge normalized eosinophils and reduced neutrophilia in the Abcg1(-/-) airway. We conclude that Abcg1(-/-) mice display IL-17-mediated suppression of eosinophilia and enhancement of neutrophilia in the airway following allergen sensitization and challenge. These findings identify ABCG1 as a novel integrator of cholesterol homeostasis and adaptive immune programs.

The Toll-like Receptor 5 Ligand Flagellin Promotes Asthma by Priming Allergic Responses to Indoor Allergens

Nature Medicine. Nov, 2012  |  Pubmed ID: 23064463

Allergic asthma is a complex disease characterized by eosinophilic pulmonary inflammation, mucus production and reversible airway obstruction. Exposure to indoor allergens is a risk factor for asthma, but this disease is also associated with high household levels of total and particularly Gram-negative bacteria. The ability of bacterial products to act as adjuvants suggests they might promote asthma by priming allergic sensitization to inhaled allergens. In support of this idea, house dust extracts (HDEs) can activate antigen-presenting dendritic cells (DCs) in vitro and promote allergic sensitization to inhaled innocuous proteins in vivo. It is unknown which microbial products provide most of the adjuvant activity in HDEs. A screen for adjuvant activity of microbial products revealed that the bacterial protein flagellin (FLA) stimulated strong allergic airway responses to an innocuous inhaled protein, ovalbumin (OVA). Moreover, Toll-like receptor 5 (TLR5), the mammalian receptor for FLA, was required for priming strong allergic responses to natural indoor allergens present in HDEs. In addition, individuals with asthma have higher serum levels of FLA-specific antibodies as compared to nonasthmatic individuals. Together, these findings suggest that household FLA promotes the development of allergic asthma by TLR5-dependent priming of allergic responses to indoor allergens.

CC Chemokine Receptor 8 Potentiates Donor Treg Survival and is Critical for the Prevention of Murine Graft-versus-host Disease

Blood. Aug, 2013  |  Pubmed ID: 23798714

The infusion of donor regulatory T cells (Tregs) has been used to prevent acute graft-versus-host disease (GVHD) in mice and has shown promise in phase 1 clinical trials. Previous work suggested that early Treg migration into lymphoid tissue was important for GVHD prevention. However, it is unclear how and where Tregs function longitudinally to affect GVHD. To better understand their mechanism of action, we studied 2 Treg-associated chemokine receptors in murine stem cell transplant models. CC chemokine receptor (CCR) 4 was dispensable for donor Treg function in the transplant setting. Donor Tregs lacking CCR8 (CCR8(-/-)), however, were severely impaired in their ability to prevent lethal GVHD because of increased cell death. By itself, CCR8 stimulation was unable to rescue Tregs from apoptosis. Instead, CCR8 potentiated Treg survival by promoting critical interactions with dendritic cells. In vivo, donor bone marrow-derived CD11c(+) antigen-presenting cells (APCs) were important for promoting donor Treg maintenance after transplant. In contrast, host CD11c(+) APCs appeared to be dispensable for early activation and expansion of donor Tregs. Collectively, our data indicate that a sustained donor Treg presence is critical for their beneficial properties, and that their survival depends on CCR8 and donor but not host CD11c(+) APCs.

Pulmonary Antigen Presenting Cells: Isolation, Purification, and Culture

Methods in Molecular Biology (Clifton, N.J.). 2013  |  Pubmed ID: 23943441

Antigen presenting cells (APCs) such as dendritic cells (DCs) and macrophages comprise a relatively small fraction of leukocytes residing in lymphoid and non-lymphoid tissues. Accordingly, functional analyses of these cells have been hampered by low cell yields. Also, alveolar macrophages share several physical properties with DCs, and this has complicated efforts to prepare pure populations of lung APCs. To overcome these difficulties, we have developed improved flow cytometry-based methods to analyze and purify APCs from the lung and its draining lymph nodes (LNs). In this chapter, we describe these methods in detail, as well as methods for culturing APCs and characterizing their interactions with T cells.

Modulation of Distinct Asthmatic Phenotypes in Mice by Dose-dependent Inhalation of Microbial Products

Environmental Health Perspectives. Jan, 2014  |  Pubmed ID: 24168764

Humans with asthma display considerable heterogeneity with regard to T helper (Th) 2-associated eosinophilic and Th17-associated neutrophilic inflammation, but the impact of the environment on these different forms of asthma is poorly understood.

Epigenetic Control of Ccr7 Expression in Distinct Lineages of Lung Dendritic Cells

Journal of Immunology (Baltimore, Md. : 1950). Nov, 2014  |  Pubmed ID: 25297875

Adaptive immune responses to inhaled allergens are induced following CCR7-dependent migration of precursor of dendritic cell (pre-DC)-derived conventional DCs (cDCs) from the lung to regional lymph nodes. However, monocyte-derived (moDCs) in the lung express very low levels of Ccr7 and consequently do not migrate efficiently to LN. To investigate the molecular mechanisms that underlie this dichotomy, we studied epigenetic modifications at the Ccr7 locus of murine cDCs and moDCs. When expanded from bone marrow precursors, moDCs were enriched at the Ccr7 locus for trimethylation of histone 3 lysine 27 (H3K27me3), a modification associated with transcriptional repression. Similarly, moDCs prepared from the lung also displayed increased levels of H3K27me3 at the Ccr7 promoter compared with migratory cDCs from that organ. Analysis of DC progenitors revealed that epigenetic modification of Ccr7 does not occur early during DC lineage commitment because monocytes and pre-DCs both had low levels of Ccr7-associated H3K27me3. Rather, Ccr7 is gradually silenced during the differentiation of monocytes to moDCs. Thus, epigenetic modifications of the Ccr7 locus control the migration and therefore the function of DCs in vivo. These findings suggest that manipulating epigenetic mechanisms might be a novel approach to control DC migration and thereby improve DC-based vaccines and treat inflammatory diseases of the lung.

Complement Receptor C5aR1/CD88 and Dipeptidyl Peptidase-4/CD26 Define Distinct Hematopoietic Lineages of Dendritic Cells

Journal of Immunology (Baltimore, Md. : 1950). Apr, 2015  |  Pubmed ID: 25769922

Differential display of the integrins CD103 and CD11b are widely used to distinguish two major dendritic cell (DC) subsets in nonlymphoid tissues. CD103(+) DCs arise from FLT3-dependent DC precursors (preDCs), whereas CD11b(hi) DCs can arise either from preDCs or FLT3-independent monocytes. Functional characterization of these two lineages of CD11b(hi) DCs has been hindered by the lack of a widely applicable method to distinguish between them. We performed gene expression analysis of fractionated lung DCs from C57BL/6 mice and found that monocyte-derived DCs (moDCs), including CD11b(hi)Ly-6C(lo) tissue-resident and CD11b(hi)Ly-6C(hi) inflammatory moDCs, express the complement 5a receptor 1/CD88, whereas preDC-derived conventional DCs (cDCs), including CD103(+) and CD11b(hi) cDCs, express dipeptidyl peptidase-4/CD26. Flow cytometric analysis of multiple organs, including the kidney, liver, lung, lymph nodes, small intestine, and spleen, confirmed that reciprocal display of CD88 and CD26 can reliably distinguish FLT3-independent moDCs from FLT3-dependent cDCs in C57BL/6 mice. Similar results were obtained when DCs from BALB/c mice were analyzed. Using this novel approach to study DCs in mediastinal lymph nodes, we observed that most blood-derived lymph node-resident DCs, as well as tissue-derived migratory DCs, are cDCs. Furthermore, cDCs, but not moDCs, stimulated naive T cell proliferation. We anticipate that the use of Abs against CD88 and CD26 to distinguish moDCs and cDCs in multiple organs and mouse strains will facilitate studies aimed at assigning specific functions to distinct DC lineages in immune responses.

Inhaled House Dust Programs Pulmonary Dendritic Cells to Promote Type 2 T-cell Responses by an Indirect Mechanism

American Journal of Physiology. Lung Cellular and Molecular Physiology. Nov, 2015  |  Pubmed ID: 26386119

The induction of allergen-specific T helper 2 (Th2) cells by lung dendritic cells (DCs) is a critical step in allergic asthma development. Airway delivery of purified allergens or microbial products can promote Th2 priming by lung DCs, but how environmentally relevant quantities and combinations of these factors affect lung DC function is unclear. Here, we investigated the ability of house dust extract (HDE), which contains a mixture of environmental adjuvants, to prime Th2 responses against an innocuous inhaled antigen. Inhalational exposure to HDE conditioned lung conventional DCs, but not monocyte-derived DCs, to induce antigen-specific Th2 differentiation. Conditioning of DCs by HDE was independent of Toll-like receptor 4 signaling, indicating that environmental endotoxin is dispensable for programming DCs to induce Th2 responses. DCs directly treated with HDE underwent maturation but were poor stimulators of Th2 differentiation. In contrast, DCs treated with bronchoalveolar lavage fluid (BALF) from HDE-exposed mice induced robust Th2 differentiation. DC conditioning by BALF was independent of the proallergic cytokines IL-25, IL-33, and thymic stromal lymphopoietin. BALF treatment of DCs resulted in upregulation of CD80 but low expression of CD40, CD86, and IL-12p40, which was associated with Th2 induction. These findings support a model whereby environmental adjuvants in house dust indirectly program DCs to prime Th2 responses by triggering the release of endogenous soluble factor(s) by airway cells. Identifying these factors could lead to novel therapeutic targets for allergic asthma.

Retinoic Acid-Related Orphan Receptors (RORs): Regulatory Functions in Immunity, Development, Circadian Rhythm, and Metabolism

Nuclear Receptor Research. 2015  |  Pubmed ID: 26878025

In this overview, we provide an update on recent progress made in understanding the mechanisms of action, physiological functions, and roles in disease of retinoic acid related orphan receptors (RORs). We are particularly focusing on their roles in the regulation of adaptive and innate immunity, brain function, retinal development, cancer, glucose and lipid metabolism, circadian rhythm, metabolic and inflammatory diseases and neuropsychiatric disorders. We also summarize the current status of ROR agonists and inverse agonists, including their regulation of ROR activity and their therapeutic potential for management of various diseases in which RORs have been implicated.

Distinct Functions of CXCR4, CCR2, and CX3CR1 Direct Dendritic Cell Precursors from the Bone Marrow to the Lung

Journal of Leukocyte Biology. Feb, 2017  |  Pubmed ID: 28148720

Precursors of dendritic cells (pre-DCs) arise in the bone marrow (BM), egress to the blood, and finally migrate to peripheral tissue, where they differentiate to conventional dendritic cells (cDCs). Upon their activation, antigen-bearing cDCs migrate from peripheral tissue to regional lymph nodes (LNs) in a manner dependent on the chemokine receptor, CCR7. To maintain immune homeostasis, these departing cDCs must be replenished by new cDCs that develop from pre-DCs, but the molecular signals that direct pre-DC trafficking from the BM to the blood and peripheral tissues remain poorly understood. In the present study, we found that pre-DCs express the chemokine receptors CXCR4, CCR2, and CX3CR1, and that each of these receptors has a distinct role in pre-DC trafficking. Flow cytometric analysis of pre-DCs lacking CXCR4 revealed that this receptor is required for the retention of pre-DCs in the BM. Analyses of mice lacking CCR2 or CX3CR1, or both, revealed that they promote pre-DC migration to the lung at steady state. CCR2, but not CX3CR1, was required for pre-DC migration to the inflamed lung. Thus, these multiple chemokine receptors cooperate in a step-wise fashion to coordinate the trafficking of pre-DCs from the BM to the circulation and peripheral tissues.

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