Citations

Objective Perianal Crohn’s disease (pCD) occurs in up to 40% of patients with CD and is associated with poor quality of life, limited treatment responses and poorly understood aetiology. We performed a genetic association study comparing CD subjects with and without perianal disease and subsequently performed functional follow-up studies for a pCD associated SNP in Complement Factor B ( CFB ). Design Immunochip-based meta-analysis on 4056 pCD and 11 088 patients with CD from three independent cohorts was performed. Serological and clinical variables were analysed by regression analyses. Risk allele of rs4151651 was introduced into human CFB plasmid by site-directed mutagenesis. Binding of recombinant G252 or S252 CFB to C3b and its cleavage was determined in cell-free assays. Macrophage phagocytosis in presence of recombinant CFB or serum from CFB risk, or protective CD or healthy subjects was assessed by flow cytometry. Results Perianal complications were associated with colonic involvement, OmpC and ASCA serology, and serology quartile sum score. We identified a genetic association for pCD (rs4151651), a non-synonymous SNP (G252S) in CFB , in all three cohorts. Recombinant S252 CFB had reduced binding to C3b, its cleavage was impaired, and complement-driven phagocytosis and cytokine secretion were reduced compared with G252 CFB. Serine 252 generates a de novo glycosylation site in CFB. Serum from homozygous risk patients displayed significantly decreased macrophage phagocytosis compared with non-risk serum. Conclusion pCD-associated rs4151651 in CFB is a loss-of-function mutation that impairs its cleavage, activation of alternative complement pathway, and pathogen phagocytosis thus implicating the alternative complement pathway and CFB in pCD aetiology.

Abstract Eosinophils are a major pathological component of the inflammatory cell infiltrate found in lungs as a result of allergic disease. They migrate into lungs in response to a variety of chemokines. The development and effector phases of the allergic response are dependent on the actions of interleukin (IL)-4 and IL-13 on a wide variety of cell types. However, how these two cytokines directly affect eosinophil signaling, function and chemotaxis is largely unknown. Using mouse eosinophils purified from IL-5 transgenic mice and a transwell migration assay, eotaxin-1 (30nM) induced eosinophil migration 6–11x over background. IL-4 alone was able to induce eosinophil migration 2x over background. More eosinophils migrated in response to eotaxin when IL-14 or IL-13 was also placed in the bottom chamber. IL-4 appeared to be more potent than IL-13. This effect was dependent upon cytokine concentration. Interestingly, when eosinophils were pretreated with IL-4 for 30 minutes prior to chemotaxis, migration to eotaxin-1 was enhanced 4-fold. However, pretreatment with IL-13 did not enhance chemotaxis to eotaxin-1. These data suggest that IL-4 and IL-13 have direct and rapid effects on the chemotactic function of eosinophils with some differences. We are investigating the mechanisms of these effects, as well as other signaling and functional responses of eosinophils to these cytokines. (AI38985 and HLT32007698)

Abstract Type 2 immune cells and eosinophils are transiently present in lung tissue not only in pathology but also during normal postnatal development. However, the lung developmental processes underlying postnatal airway recruitment of eosinophils after birth remain unexplored. We determined that in mice, mature eosinophils are recruited to the lung during P3–14, which corresponds to the primary septation/alveolarization phase of lung development. Developmental eosinophils peaked during P10–14 and exhibited Siglec-Fmed/highCD11c−/low phenotypes, similar to allergic asthma models. By interrogating the lung transcriptome and proteome during peak eosinophil recruitment in postnatal development, we identified markers that functionally capture mesenchymal-epithelial interface establishment (Nes, Smo, Wnt5a, Nog) and provisional extracellular matrix (ECM) deposition (Tnc, Postn, Spon2, Thbs2) as key lung morphogenetic events associating with eosinophils. We identifiedTenascin-C (TNC) as one of the key ECM markers in the lung epithelial-mesenchymal interface at the RNA and protein levels, consistently associating with eosinophils in development and disease. By RNA-seq analysis, naïve murine eosinophils cultured with TNC-enriched ECM significantly induced expression of Siglec-F, CD11c, eosinophil peroxidase, and other markers typical for activated eosinophils in development and allergic inflammatory responses. TNC knockout mice had an altered eosinophil recruitment profile in development. Collectively, our results indicate that lung morphogenetic processes associated with heightened Type 2 immunity are not merely tissue “background” but specifically guide immune cells both in development and pathology.

Abstract Eosinophils comprise only 1-3% of circulating leukocytes but are often a diagnostic feature associated with a variety of inflammatory and disease states, including parasitic and fungal infections, allergic diseases (e.g., asthma, rhinitis and sensitivities to specific foods), cancer, and transplant rejection. The accurate evaluations of eosinophilia as well as the release of stored eosinophil granule proteins are important for the monitoring disease progression and/or assessing the effectiveness of a given treatment strategy(ies). Despite the availability of detection methods for each of the eosinophil granule proteins logistical difficulties limiting either the assays specificity and/or sensitivity have prevented their extensive use. We have generated unique eosinophil peroxidase specific monoclonal antibodies (EPX-mAb) and a high throughput sandwich ELISA assay as a means of overcoming these difficulties. Our EPX-mAb based ELISA is ~10 times more sensitive than traditional OPD based activity assays and allows detection of EPX in mouse BAL fluid after an acute OVA protocol as well as EPX release from mouse eosinophils stimulated ex vivo. The assay is highly specific and gives no signal in samples from EPX deficient mice even with massive eosinophilia (e.g., EPX deficient IL-5 transgenic mice). More significantly, this assay detects EPX in human tissue extracts and biological fluids and thus represents novel diagnostic assay previously unavailable in clinical settings.

Abstract Chronic asthma is characterized by pulmonary remodeling and sustained lung dysfunction. The expression of IL-13 has been demonstrated as sufficient and necessary to induce these pathologies. Despite the common assumption that T cells are the primary source of IL-13 in the lung following allergen provocation, the mechanistic events leading to IL-13 expression are unclear, including the potential importance of other cells (i.e., non-T cells). Methods: Co-expression of IL-5 and eotaxin-2 induces pulmonary histopathologies and lung dysfunction that are eosinophil-dependent (i.e., they are absent in triple transgenic I5/hE2/PHIL) animals. IL-13 knockout and CD4 knockout mice were crossed with I5/hE2 mice to assess the effects of these deficiencies on the induced pathologies occurring in this double transgenic model. Conclusions: The Th2 pulmonary pathologies associated with I5/hE2 mice included recruitment of pulmonary effector T cells and increased brochoalveolar Th2 cytokine levels. Conversely, I5/hE2/CD4-/- had only a ~50% reduction in IL-13 levels as compared to I5/hE2 mice, suggesting that a significant portion of IL-13 is non-T cell derived in these mice. I5/hE2/IL-13-/- mice are improved in lung dysfunction and mucus production as compared to I5/hE2 mice. All mice had similar levels of eosinophil degranulation. These results demonstrate a role for non-T cell derived IL-13 that contributes to mucus production and lung dysfunction in a mouse model of chronic severe asthma.

Abstract Eosinophil infiltration into tumors has been associated with positive and negative growth human cancers and in mouse models of tumorigenesis. Our previous studies have shown that eosinophils infiltrate tumors and differentially accumulate in the necrotic and capsule regions of tumors. Using various eosinophil-deficient mouse models, we demonstrate that in the absence of eosinophils tumor growth is attenuated. Methods: Tumor growth kinetics were assessed using a B16F10 melanoma subcutaneous cell injection model. Eosinophil chemotaxis was assessed using an ex vivo transwell insert assay system. We compared tumor growth in wild type animals vs. various eosinophil-deficient mouse models that are transgenic line of mice (PHIL) and MBP-/-/EPO-/- double knockout mice that are deficient in eosinophil-specific proteins (major basic protein (MBP) and eosinophil peroxide (EPO)) resulting in a blockade of eosinophilpoiesis. Conclusions: The migration of eosinophils was inhibited through blocking the sphingosine-1-phosphate pathway, suggesting recruitment and accumulation is primarily an inflammatory response. Tumor growth was decreased by ~40-60% in PHIL and in MBP-/-/EPO-/- double knockout mice relative to tumors in wild type animals. In addition, EPO-/- mice, and not MBP-/-mice, replicate this ~40% reduction in tumor size implicating a significant role for EPO in tumor growth kinetics. These data suggest eosinophils respond to inflammatory mediators and mediate augment tumor growth.

Abstract Products of the 5-lipoxygenase (5-LO) pathway (i.e., cys-leukotrienes and LTB4), are contributors to lung inflammation, airway remodeling, and hyperresponsiveness associated with allergic respiratory inflammation. Eosinophils are capable of leukotriene synthesis/secretion and chemotactically respond to these compounds. However, the in vivo role of eosinophils in leukotriene mediated events during asthmatic airway inflammation is not defined. To study the link between eosinophils and leukotrienes we used the I5/E2 double transgenic mouse model of severe asthma, over-expressing IL-5 systemically and eotaxin-2 in airways, in which lung pathologies are entirely dependent on the presence of eosinophils. We show that airway levels of cysteinyl leukotrienes and LTB4 are significantly elevated in I5/E2 mice. Crosses of I5E2 mice with 5-LO knockout animals abolished AHR in the I5/E2 parental model without significant effects on lung histopathology. Administration of a cys-leukotriene receptor antagonist (Montelukast®) to I5/E2 mice replicates the effects of the total leukotriene loss in I5/E2/5-LO-/- animals. In contrast, I5/E2 mice crossed with BLT-1-/- mice which are deficient of the LTB4 receptor fail to display effects on the pulmonary pathologies occurring in the I5/E2 parental model. These data demonstrate a unique role for cys-leukotrienes in the development of AHR independent of lung remodeling events occurring in this transgenic model of severe asthma.

Abstract Atopic dermatitis and allergic contact dermatitis are two of the most common skin diseases. In both diseases, eosinophils have been suggested as potential contributors of the pathologies linked with these skin conditions. Our goal was to establish a causative relationship between the inflammatory response of chemically induced contact hypersensitivity and eosinophil-mediated activities. Wild type and eosinophil-less PHIL mice were sensitized and challenged with 2,4-dinitrochlorobenzene (DNFB) or alternatively trimellitic anhydride (TMA). Time lapsed videography was used to capture the number of itching events associated with treatment. Immunohistochemistry for eosinophils and nerves was performed to correlate the effect of dermatitis on innervation and eosinophil accumulation in these mice. Our studies showed that in both the DNFB and TMA models a robust eosinophil infiltrate occurred that was accompanied by copious levels of eosinophil degranulation. More importantly, these studies demonstrated that metrics of inflammation (e.g., induced ear swelling (i.e., thickness)) was reduced in PHIL mice relative to wild type. Our observations were also directly correlative with a loss of nerve growth and branching in PHIL mice. These data implicate eosinophils as contributors to the pathologies linked with chemically-induced contact hypersensitivity, including the induced itch responses associated with these models.

Abstract Eosinophilia is a hallmark of worm infection and allergy. The parasitic nematode Trichinella spiralis establishes chronic infection in skeletal muscle. We have shown previously that in eosinophil-ablated, transgenic mice, T. spiralis larvae die in large numbers in the muscle. Death is promoted by inducible nitric oxide synthase (iNOS). We report here that transcription of iNOS was upregulated in the context of an established alternative activation phenotype in local leukocytes. Infiltrating macrophages, dendritic cells, and neutrophils produced iNOS. These changes were preceded by a reduced T cell recruitment to infected muscle that was coincident with a lag in larval growth, suggesting compromise of the parasite’s habitat. The results further define the cellular interactions that occur at a site of chronic nematode infection in which the eosinophil functions as a pivotal regulator of immunity.

Abstract Eosinophil effector functions have been hypothesized to be mediated in part by the release of proteins stored in the secondary granules of mature cells of this leukocyte lineage. Major basic protein-1 (MBP-1) and eosinophil peroxidase (EPO) comprise the majority of protein in these granules. No role has been speculated for these proteins in eosinophilopoiesis and/or the accumulation of mature cells in circulation. Methods: Eosinophilopoiesis and eosinophil accumulation in the periphery were assessed in single and double knockout (MBP-1-/- and/or EPO-/-) mice. Bone marrow and peripheral blood leukocytes were assessed by IHC, FACS and in vitro cell culture assays. Mice were subjected to an acute OVA protocol assessing induced pulmonary pathologies in each of the strains of granule protein knockout mice. Results: We demonstrate that, unlike the single deficiency of MBP-1 or EPO, the absence of both granule proteins resulted in the loss of peripheral blood eosinophils. IHC assessments of bone marrow and spleen demonstrate that eosinophil lineage commitment occurs in these mice. However, these assessments and FACS studies demonstrated a blockade in the maturation of eosinophil-lineage committed cells. This blockade is not rescued by ex vivo culture or by bone marrow engraftment into wild type recipient mice. Similar to other eosinophil-less mouse models, MBP-1-/-/EPO-/- mice also fail to develop pulmonary inflammation in an OVA protocol.