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1.  A crucial role for β2 integrins in podosome formation, dynamics and Toll-like-receptor-signaled disassembly in dendritic cells 
Journal of Cell Science  2014;127(19):4213-4224.
ABSTRACT
The dynamic properties of podosomes, their ability to degrade the underlying matrix and their modulation by Toll-like receptor (TLR) signaling in dendritic cells (DCs) suggests they have an important role in migration. Integrins are thought to participate in formation and dynamics of podosomes but the multiplicity of integrins in podosomes has made this difficult to assess. We report that murine DCs that lack β2 integrins fail to form podosomes. Re-expression of β2 integrins restored podosomes but not when the membrane proximal or distal NPxF motifs, or when an intervening triplet of threonine residues were mutated. We show that β2 integrins are remarkably long-lived in podosome clusters and form a persistent framework that hosts multiple actin-core-formation events at the same or adjacent sites. When β2 integrin amino acid residues 745 or 756 were mutated from Ser to Ala, podosomes became resistant to dissolution mediated through TLR signaling. TLR signaling did not detectably modulate phosphorylation at these sites but mutation of either residue to phospho-mimetic Asp increased β2 integrin turnover in podosomes, indicating that phosphorylation at one or both sites establishes permissive conditions for TLR-signaled podosome disassembly.
doi:10.1242/jcs.151167
PMCID: PMC4179490  PMID: 25086067
Podosomes; Dendritic cells; Integrins
2.  Siglec-F-dependent negative regulation of allergen-induced eosinophilia depends critically on the experimental model 
Immunology Letters  2014;160(1):11-16.
Highlights
•Siglec-F-dependent negative regulation of eosinophilia depends on experimental model.•Siglec-F-dependent suppression of lung eosinophilia may not depend on ligand-induced apoptosis.•Implications for therapeutic approaches to treating human disease in which siglec-8, is targeted.
Siglec-8 and siglec-F are paralogous membrane proteins expressed on human and murine eosinophils respectively. They bind similar sialylated and sulphated glycans and mediate eosinophil apoptosis when cross-linked with antibodies or glycan ligands. In models of allergic eosinophilic airway inflammation, siglec-F was shown previously to be important for negatively regulating eosinophilia. It was proposed that this was due to siglec-F-dependent apoptosis, triggered via engagement with ligands that are upregulated on bronchial epithelium. Our aim was to further investigate the functions of siglec-F by comparing two commonly used models of ovalbumin-induced airway inflammation that differ in the dose and route of administration of ovalbumin. In confirmation of published results, siglec-F-deficient mice had enhanced lung tissue eosinophilia in response to intranasal ovalbumin delivered every other day. However, following aerosolised ovalbumin delivered daily, there was no influence of siglec-F deficiency on lung eosinophilia. Expression of siglec-F ligands in lung tissues was similar in both models of allergen induced inflammation. These data demonstrate that siglec-F-dependent regulation of eosinophilia is subtle and depends critically on the model used. The findings also indicate that mechanisms other than ligand-induced apoptosis may be important in siglec-F-dependent suppression of eosinophilia.
doi:10.1016/j.imlet.2014.03.008
PMCID: PMC4045373  PMID: 24698729
Siglec, sialic acid binding immunoglobulin like lectin; OVA, ovalbumin; WT, wild type; KO, knock out; KI, knock in; ITIM, immunoreceptor tyrosine based inhibitory motifs; Siglec-F; Siglec-8; Eosinophil; Allergic airway inflammation
3.  Siglec-E Promotes β2-Integrin-dependent NADPH Oxidase Activation to Suppress Neutrophil Recruitment to the Lung* 
The Journal of Biological Chemistry  2014;289(29):20370-20376.
Background: Siglec-E is a negative regulator of neutrophil recruitment to the lung in a mouse model of sepsis.
Results: Siglec-E promotes integrin-dependent production of reactive oxygen species (ROS) and Akt activation.
Conclusion: Siglec-E-promoted ROS plays a key role in its suppression of neutrophil recruitment to the lung.
Significance: Siglecs may be new therapeutic targets in inflammatory lung disease.
Siglec-E is a sialic acid-binding Ig-like lectin expressed on murine myeloid cells. It has recently been shown to function as a negative regulator of β2-integrin-dependent neutrophil recruitment to the lung following exposure to lipopolysaccharide (LPS). Here, we demonstrate that siglec-E promoted neutrophil production of reactive oxygen species (ROS) following CD11b β2-integrin ligation with fibrinogen in a sialic acid-dependent manner, but it had no effect on ROS triggered by a variety of other stimulants. Siglec-E promotion of ROS was likely mediated via Akt activation, because siglec-E-deficient neutrophils plated on fibrinogen exhibited reduced phosphorylation of Akt, and the Akt inhibitor, MK2206, blocked fibrinogen-induced ROS. In vivo imaging showed that siglec-E also promoted ROS in acutely inflamed lungs following exposure of mice to LPS. Importantly, siglec-E-promoted ROS were required for its inhibitory function, as the NADPH oxidase inhibitor, apocynin, reversed the siglec-E-mediated suppression of neutrophil recruitment and blocked neutrophil ROS production in vitro. Taken together, these results demonstrate that siglec-E functions as an inhibitory receptor of neutrophils via positive regulation of NADPH oxidase activation and ROS production. Our findings have implications for the inhibitory role of siglec-9 on human neutrophils in sepsis and acute lung injury.
doi:10.1074/jbc.M114.574624
PMCID: PMC4106349  PMID: 24895121
Lipopolysaccharide (LPS); Lung Injury; NADPH Oxidase; Neutrophil; Reactive Oxygen Species (ROS); Siglec-E
4.  Peptide immunotherapy in allergic asthma generates IL-10–dependent immunological tolerance associated with linked epitope suppression 
The Journal of Experimental Medicine  2009;206(7):1535-1547.
Treatment of patients with allergic asthma using low doses of peptides containing T cell epitopes from Fel d 1, the major cat allergen, reduces allergic sensitization and improves surrogate markers of disease. Here, we demonstrate a key immunological mechanism, linked epitope suppression, associated with this therapeutic effect. Treatment with selected epitopes from a single allergen resulted in suppression of responses to other (“linked”) epitopes within the same molecule. This phenomenon was induced after peptide immunotherapy in human asthmatic subjects and in a novel HLA-DR1 transgenic mouse model of asthma. Tracking of allergen-specific T cells using DR1 tetramers determined that suppression was associated with the induction of interleukin (IL)-10+ T cells that were more abundant than T cells specific for the single-treatment peptide and was reversed by anti–IL-10 receptor administration. Resolution of airway pathophysiology in this model was associated with reduced recruitment, proliferation, and effector function of allergen-specific Th2 cells. Our results provide, for the first time, in vivo evidence of linked epitope suppression and IL-10 induction in both human allergic disease and a mouse model designed to closely mimic peptide therapy in humans.
doi:10.1084/jem.20082901
PMCID: PMC2715096  PMID: 19528258
5.  Th2-driven, allergen-induced airway inflammation is reduced after treatment with anti–Tim-3 antibody in vivo 
The Journal of Experimental Medicine  2007;204(6):1289-1294.
T cell immunoglobulin and mucin domain–containing molecule-3 (Tim-3) is a surface molecule that is preferentially expressed on activated Th1 cells in comparison to Th2 cells. Blockade of Tim-3 has been shown to enhance Th1-driven pathology in vivo, suggesting that blockade of Tim-3 may improve the development of Th2-associated responses such as allergy. To examine the effects of Tim-3 blockade on the Th2 response in vivo, we administered anti–Tim-3 antibody during pulmonary inflammation induced by transfer of ovalbumin (OVA)-reactive Th2 cells, and subsequent aerosol challenge with OVA. In this model, anti–Tim-3 antibody treatment before each airway challenge significantly reduced airway hyperreactivity, with a concomitant decrease in eosinophils and Th2 cells in the lung. We examined Th1 and Th2 cytokine levels in the lung after allergen challenge and found that pulmonary expression of the Th2 cytokine IL-5 was significantly reduced, whereas IFN-γ levels were significantly increased by anti–Tim-3 antibody treatment. Thus, blocking Tim-3 function has a beneficial effect during pulmonary inflammation by skewing the Th2 response toward that of a Th1 type, suggesting an important role for Tim-3 in the regulation of allergic disease.
doi:10.1084/jem.20062093
PMCID: PMC2118608  PMID: 17517968
6.  The Absence of Interleukin 9 Does Not Affect the Development of Allergen-induced Pulmonary Inflammation nor Airway Hyperreactivity 
Interleukin (IL)-9 is a pleiotropic cytokine secreted by T helper (Th)2 cells and has been proposed as a candidate gene for asthma and allergy. We have used mice genetically deficient in IL-9 to determine the role of this cytokine in the pathophysiologic features of the allergic pulmonary response–airway hyperreactivity (AHR) and eosinophilia. We have demonstrated that IL-9 is not required for the development of a robust Th2 response to allergen in sensitized mice. IL-9 knockout mice developed a similar degree of eosinophilic inflammation and AHR to their wild-type littermates. Goblet cell hyperplasia and immunoglobulin (Ig) E production were also unaffected by the lack of IL-9. Moreover, levels of bronchoalveolar lavage (BAL) IL-4, IL-5, and IL-13 were comparable between wild-type and knockout mice. These findings indicate that IL-9 is not obligatory for the development of eosinophilia and AHR, and imply that other Th2 cytokines can act in a compensatory fashion.
doi:10.1084/jem.20011732
PMCID: PMC2196020  PMID: 11781365
Th2 cytokines; asthma; airway hyperreactivity; eosinophilia; mucus

Results 1-6 (6)