Activated T cells secrete an alternatively spliced form of common γ-chain that inhibits cytokine signaling and exacerbates inflammation.

The common γ-chain (γc) plays a central role in signaling by IL-2 and other γc-dependent cytokines. Here we report that activated T cells produce an alternatively spliced form of γc mRNA that results in protein expression and secretion of the γc extracellular domain. The soluble form of γc (sγc) is present in serum and directly binds to IL-2Rß and IL-7Rα proteins on T cells to inhibit cytokine signaling and promote inflammation. sγc suppressed IL-7 signaling to impair naive T cell survival during homeostasis and exacerbated Th17-cell-mediated inflammation by inhibiting IL-2 signaling upon T cell activation. Reciprocally, the severity of Th17-cell-mediated inflammatory diseases was markedly diminished in mice lacking sγc. Thus, sγc expression is a naturally occurring immunomodulator that regulates γc cytokine signaling and controls T cell activation and differentiation.

Mepolizumab targets multiple immune cells in aspirin-exacerbated respiratory disease.

BACKGROUND: Eosinophilic asthma and nasal polyposis are hallmarks of aspirin-exacerbated respiratory disease (AERD), and IL-5 inhibition has been shown to provide therapeutic benefit. However, IL-5Rα is expressed on many cells in addition to eosinophils, and the mechanisms by which IL-5 inhibition leads to clinical benefit in eosinophilic asthma and nasal polyposis are unlikely to be due exclusively to antieosinophil effects. OBJECTIVE: We sought to identify the mechanisms by which anti-IL-5 treatment with mepolizumab improves respiratory inflammation in AERD. METHODS: The clinical characteristics, circulating granulocytes, nasal scraping transcripts, eosinophilic cationic protein, tryptase, and antibody levels, and urinary and nasal eicosanoid levels were measured for 18 subjects with AERD who were taking mepolizumab and compared with those of 18 matched subjects with AERD who were not taking mepolizumab. RESULTS: Subjects taking mepolizumab had significantly fewer peripheral blood eosinophils and basophils, and those cells that remained had higher surface CRTH2 expression than did the cells from subjects not taking mepolizumab. Nasal prostaglandin F2α, prostaglandin D2 metabolites, leukotriene B4, and thromboxane levels were lower in subjects taking mepolizumab, as were urinary levels of tetranor-prostaglandin D2 and leukotriene E4. The nasal epithelial cell transcripts that were overexpressed among subjects with AERD who were taking mepolizumab were enriched for genes involved in tight junction formation and cilium organization. Nasal and urinary prostaglandin E2, tryptase, and antibody levels were not different between the 2 groups. CONCLUSION: IL-5 inhibition in AERD decreases production of inflammatory eicosanoids and upregulates tight junction-associated nasal epithelial cell transcripts, likely due to decreased IL-5 signaling on tissue mast cells, eosinophils, and epithelial cells. These direct effects on multiple relevant immune cells contribute to the mechanism of benefit afforded by mepolizumab.

Immune Profile of the Nasal Mucosa in Patients with Cutaneous Leishmaniasis.

Localized skin lesions are characteristic of cutaneous leishmaniasis (CL); however, Leishmania (Viannia) species, which are responsible for most CL cases in the Americas, can spread systemically, sometimes resulting in mucosal disease. Detection of Leishmania has been documented in healthy mucosal tissues (conjunctiva, tonsils, and nasal mucosa) and healthy skin of CL patients and in individuals with asymptomatic infection in areas of endemicity of L (V) panamensis and L (V) braziliensis transmission. However, the conditions and mechanisms that favor parasite persistence in healthy mucosal tissues are unknown. In this descriptive study, we compared the cell populations of the nasal mucosa (NM) of healthy donors and patients with active CL and explored the immune gene expression signatures related to molecular detection of Leishmania in this tissue in the absence of clinical signs or symptoms of mucosal disease. The cellular composition and gene expression profiles of NM samples from active CL patients were similar to those of healthy volunteers, with a predominance of epithelial over immune cells, and within the CD45+ cell population, a higher frequency of CD66b+ followed by CD14+ and CD3+ cells. In CL patients with molecular evidence of Leishmania persistence in the NM, genes characteristic of an anti-inflammatory and tissue repair responses (IL4R, IL5RA, POSTN, and SATB1) were overexpressed relative to NM samples from CL patients in which Leishmania was not detected. Here, we report the first immunological description of subclinically infected NM tissues of CL patients and provide evidence of a local anti-inflammatory environment favoring parasite persistence in the NM.

NLS-Cholic Acid Conjugation to IL-5Rα-Specific Antibody Improves Cellular Accumulation and In Vivo Tumor-Targeting Properties in a Bladder Cancer Model.

Receptor-mediated internalization followed by trafficking and degradation of antibody-conjugates (ACs) via the endosomal-lysosomal pathway is the major mechanism for delivering molecular payloads inside target tumor cells. Although a mainstay for delivering payloads with clinically approved ACs in cancer treatment and imaging, tumor cells are often able to decrease intracellular payload concentrations and thereby reduce the effectiveness of the desired application. Thus, increasing payload intracellular accumulation has become a focus of attention for designing next-generation ACs. We developed a composite compound (ChAcNLS) that enables ACs to escape endosome entrapment and route to the nucleus resulting in the increased intracellular accumulation as an interleukin-5 receptor α-subunit (IL-5Rα)-targeted agent for muscle invasive bladder cancer (MIBC). We constructed 64Cu-A14-ChAcNLS, 64Cu-A14-NLS, and 64Cu-A14 and evaluated their performance by employing mechanistic studies for endosome escape coupled to nuclear routing and determining whether this delivery system results in improved 64Cu cellular accumulation. ACs consisting of ∼20 ChAcNLS or NLS moieties per 64Cu-A14 were prepared in good yield, high monomer content, and maintaining high affinity for IL-5Rα. Confocal microscopy analysis demonstrated ChAcNLS mediated efficient endosome escape and nuclear localization. 64Cu-A14-ChAcNLS increased 64Cu cellular accumulation in HT-1376 and HT-B9 cells relative to 64Cu-A14 and 64Cu-A14-NLS. In addition, we tested 64Cu-A14-ChAcNLS in vivo to evaluate its tissue distribution properties and, ultimately, tumor uptake and targeting. A model of human IL-5Rα MIBC was developed by implanting NOD/SCID mice with subcutaneous HT-1376 or HT-B9MIBC tumors, which grow containing high and low IL-5Rα-positive tumor cell densities, respectively. ACs were intravenously injected, and daily blood sampling, biodistribution at 48 and 96 h, and positron emission tomography (PET) at 24 and 48 h were performed. Region of interest (ROI) analysis was also performed on reconstructed PET images. Pharmacokinetic analysis and biodistribution studies showed that 64Cu-A14-ChAcNLS had faster clearance rates from the blood and healthy organs relative to 64Cu-A14. However, 64Cu-A14-ChAcNLS maintained comparable tumor accumulation relative to 64Cu-A14. This resulted in 64Cu-A14-ChAcNLS having superior tumor/normal tissue ratios at both 48 and 96 h biodistribution time points. Visualization of AC distribution by PET and ROI analysis confirmed that 64Cu-A14-ChAcNLS had improved targeting of MIBC tumor relative to 64Cu-A14. In addition, 64Cu-A14 modified with only NLS had poor tumor targeting. This was a result of poor tumor uptake due to extremely rapid clearance. Thus, the overall findings in this model of human IL-5Rα-positive MIBC describe an endosome escape-nuclear localization cholic-acid-linked peptide that substantially enhances AC cellular accumulation and tumor targeting.

SLy2 controls the antibody response to pneumococcal vaccine through an IL-5Rα-dependent mechanism in B-1 cells.

The adaptor protein SLy2 (Src homology domain 3 lymphocyte protein 2) is located on human chromosome 21 and was reported to be among a group of genes amplified in Down's syndrome (DS) patients. DS patients characteristically show an impaired immunity to pneumococcal infections. However, molecular mechanisms linking gene amplifications with specific DS phenotypes remain elusive. To investigate the effect of SLy2 gene amplification on the mammalian immune system, we studied SLy2 overexpressing transgenic-SLy2 (TG) mice. We found that baseline immunoglobulin M (IgM) levels as well as IgM responses following Pneumovax immunizations were reduced in TG mice. Moreover, B-1 cells, the major natural IgM-producing population in mice, were reduced in the peritoneal cavity of TG mice, while other immune cell compartments were unaltered. Mechanistically, SLy2 overexpression attenuated the expression of the IL-5 receptor α chain on B-1 cells, resulting in decreased B-1 cell numbers and decreased differentiation into Ab-secreting cells. Since B-1 cells essentially contribute to immunity against Streptococcus pneumoniae, the present study provides a novel molecular link between SLy2 expression and pneumococcal-specific IgM responses in vivo. These studies suggest that the adaptor protein SLy2 is a potential future target for immunomodulatory strategies for pneumococcal infections.

IL-5 triggers a cooperative cytokine network that promotes eosinophil precursor maturation.

Eosinophils originate in the bone marrow from an eosinophil lineage-committed, IL-5Rα-positive, hematopoietic progenitor (eosinophil progenitor). Indeed, IL-5 is recognized as a critical regulator of eosinophilia and has effects on eosinophil progenitors, eosinophil precursors, and mature eosinophils. However, substantial levels of eosinophils remain after IL-5 neutralization or genetic deletion, suggesting that there are alternative pathways for promoting eosinophilia. In this study, we investigated the contributory role of IL-5 accessory cytokines on the final stages of eosinophil differentiation. IL-5 stimulation of low-density bone marrow cells resulted in expression of a panel of cytokines and cytokine receptors, including several ligand-receptor pairs. Notably, IL-4 and IL-4Rα were expressed by eosinophil precursors and mature eosinophils. Signaling through IL-4Rα promoted eosinophil maturation when IL-5 was present, but IL-4 stimulation in the absence of IL-5 resulted in impaired eosinophil survival, suggesting that IL-4 cooperates with IL-5 to promote eosinophil differentiation. In contrast, CCL3, an eosinophil precursor-produced chemokine that signals through CCR1, promotes terminal differentiation of CCR1-positive eosinophil precursors in the absence of IL-5, highlighting an autocrine loop capable of sustaining eosinophil differentiation. These findings suggest that brief exposure to IL-5 is sufficient to initiate a cytokine cooperative network that promotes eosinophil differentiation of low-density bone marrow cells independent of further IL-5 stimulation.

IL-5 receptor α levels in patients with marked eosinophilia or mastocytosis.

BACKGROUND: IL-5 plays a central role in the development and maintenance of eosinophilia (EO) and eosinophil activation in a wide variety of eosinophilic disorders. Although IL-5, IL-3, and GM-CSF can modulate the expression of IL-5 receptor α (IL-5Rα) on eosinophils in vitro, little is known about soluble and surface IL-5Rα levels in vivo. OBJECTIVE: To assess soluble and surface IL-5Rα levels in patients with EO and/or mastocytosis. METHODS: Surface IL-5Rα expression was assessed by flow cytometry in blood and/or bone marrow from subjects with EO (n = 39) and systemic mastocytosis (n = 8) and from normal volunteers (n = 28). Soluble IL-5Rα (sIL-5Rα) level was measured in a cohort of 177 untreated subjects and correlated with EO, eosinophil activation, and serum tryptase and cytokine levels. RESULTS: IL-5Rα expression on eosinophils inversely correlated with EO (r = -0.48; P < .0001), whereas serum levels of sIL-5Rα increased with the eosinophil count (r = 0.56; P < .0001) and serum IL-5 (r = 0.40; P < .0001) and IL-13 (r = 0.29; P = .004) levels. Of interest, sIL-5Rα level was significantly elevated in patients with systemic mastocytosis without EO. Although sIL-5Rα levels correlated with serum tryptase levels in these patients, eosinophil activation, assessed by CD69 expression on eosinophils and serum eosinophil-derived neurotoxin levels, was increased compared with that in normal subjects. CONCLUSIONS: These data are consistent with an in vivo IL-5Rα regulatory pathway in human eosinophils similar to that described in vitro and involving a balance between soluble and surface receptor levels. This may have implications with respect to the use of novel therapeutic agents targeting IL-5 and its receptor in patients with EO and/or mastocytosis.

Are serum immunoglobulin concentrations a predictive biomarker of response to anti-IL5/IL5Rα therapies?

BACKGROUND: Approval of biologics has recently revolutionized T2 severe asthma management. However, predictive biomarkers remain highly needed to improve patient's selection. OBJECTIVE: This study aims to determine whether serum immunoglobulins (Igs) levels might be predictive biomarkers of response to anti-interleukin-5 (IL5)/IL5Rα therapies. METHODS: Severe asthma patients eligible for mepolizumab or benralizumab were included herein. Serum immunoglobulin quantification was performed at baseline before mepolizumab or benralizumab initiation. After a 6-month treatment of mepolizumab or benralizumab, patients presented a second serum immunoglobulin quantification. The treatment response was evaluated by the GETE (Global Evaluation of Treatment Effectiveness) score at 6 months. RESULTS: A total of 50 patients were included. Median age was 56 [IQR 48.8-65.3] and 50% were females. Compared to baseline, a significant increase in IgG was observed at 6 months (9.2 [7.8-10.2] g/l vs 10.1 [8.8-11.1] g/l, p = 0.04). The area under the ROC curve was 0.58 [95%IC 0.40-0.77] for blood eosinophil count (p = 0.37), 0.75 [95%IC: 0.58-0.92] for serum IgG concentration (p = 0.009) for predicting the treatment response. According to the Youden index, serum IgG concentration ≥ 9.2 g/l predicts the response to anti-IL5 therapies with a sensitivity of 76.9% and a specificity of 75.7%. CONCLUSION: Baseline serum IgG concentrations may be a useful tool to predict the response to anti-IL5/IL5Rα therapies but should be confirmed in larger clinical trials. Interestingly, anti-IL5/IL5Rα therapies are associated with a significant increase in serum IgG concentrations at 6 months.

Local proliferation and mobilization of CCR3(+) CD34(+) eosinophil-lineage-committed cells in the lung.

Emerging evidence suggests that haematopoietic CD34(+) progenitor cells migrate from bone marrow (BM) to sites of allergen exposure where they can undergo further proliferation and final maturation, potentially augmenting the degree of tissue inflammation. In the current study we used a well-characterized mouse model of allergen-induced airway inflammation to determine the role of CCR3 receptor-ligand interactions in the migration and function of CD34(+) cells. Allergen exposure significantly increased BM, blood and airway CD34(+) CCR3(+) cells as well as airway CD34(+) CCR3(+) stem cell antigen-1-positive (Sca-1(+) ) and CD34(+) CD45(+) interleukin-5 receptor-α-positive (IL-5Rα(+) ) cells. A portion of the newly produced CD34(+) CCR3(+), Sca-1(+) CCR3(+) and IL-5Ralpha(+) lung cells showed a significant proliferative capacity in response to allergen when compared with saline-treated animals. In addition, in vitro colony formation of lung CD34(+) cells was increased by IL-5 or eotaxin-2 whereas eotaxin-2 had no effect on BM CD34(+) cells. Furthermore, both eotaxin-1 and eotaxin-2 induced migration of BM and blood CD34(+) CCR3(+) cells in vitro. These data suggest that the CCR3/eotaxin pathway is involved in the regulation of allergen-driven in situ haematopoiesis and the accumulation/mobilization of eosinophil-lineage-committed progenitor cells in the lung. Hence, targeting both IL-5 and CCR3-mediated signalling pathways may be required to control the inflammation associated with allergen-induced asthma.

Interleukin 5 in the link between the innate and acquired immune response.

Interleukin-5 (IL-5) is an interdigitating homodimeric glycoprotein that is initially identified by its ability to support the in vitro growth and differentiation of mouse B cells and eosinophils. IL-5 transgenic mouse shows two predominant features, remarkable increase in B-1 cells resulting in enhanced serum antibody levels, predominantly IgM, IgA, and IgE classes and in expansion of eosinophil numbers in the blood and eosinophil infiltration into various tissues. Conversely, mice lacking a functional gene for IL-5 or IL-5 receptor alpha chain (IL-5Ralpha) display a number of developmental and functional impairments in B cells and eosinophils. IL-5 receptor (IL-5R) comprises alpha and betac chains. IL-5 specifically binds to IL-5Ralpha and induces the recruitment of betac to IL-5R. Although precise mechanisms on cell-lineage-specific IL-5Ralpha expression remain elusive, several transcription factors including Sp1, E12/E47, Oct-2, and c/EBPbeta have been shown to regulate its expression in B cells and eosinophils. JAK2 and JAK1 tyrosine kinase are constitutively associated with IL-5Ralpha and betac, respectively, and are activated by IL-5 stimulation. IL-5 activates at least three different signaling pathways including JAK2/STAT5 pathway, Btk pathway, and Ras/ERK pathway. IL-5 is one of key cytokines for mouse B cell differentiation in general, particularly for fate-determination of terminal B cell differentiation to antibody-secreting plasma cells. IL-5 critically regulates homeostatic proliferation and survival of and natural antibody production by B-1 cells, and enhances the AID and Blimp-1 expression in activated B-2 cells leading to induce mu to gamma1 class switch recombination and terminal differentiation to IgM- and IgG1-secreting plasma cells, respectively. In humans, major target cells of IL-5 are eosinophils. IL-5 appears to play important roles in pathogenesis of asthma, hypereosinophilic syndromes, and eosinophil-dependent inflammatory diseases. Clinical studies will provide a strong impetus for investigating the means of modulating IL-5 effects. We will discuss the role of IL-5 in the link between innate and acquired immune response, particularly emphasis of the molecular basis of IL-5-dependent B cell activation, allergen-induced chronic inflammation and hypereosinophilic syndromes on a novel target for therapy.

MEDI-563, a humanized anti-IL-5 receptor alpha mAb with enhanced antibody-dependent cell-mediated cytotoxicity function.

BACKGROUND: Peripheral blood eosinophilia and lung mucosal eosinophil infiltration are hallmarks of bronchial asthma. IL-5 is a critical cytokine for eosinophil maturation, survival, and mobilization. Attempts to target eosinophils for the treatment of asthma by means of IL-5 neutralization have only resulted in partial removal of airway eosinophils, and this warrants the development of more effective interventions to further explore the role of eosinophils in the clinical expression of asthma. OBJECTIVE: We sought to develop a novel humanized anti-IL-5 receptor alpha (IL-5Ralpha) mAb with enhanced effector function (MEDI-563) that potently depletes circulating and tissue-resident eosinophils and basophils for the treatment of asthma. METHODS: We used surface plasmon resonance to determine the binding affinity of MEDI-563 to FcgammaRIIIa. Primary human eosinophils and basophils were used to demonstrate antibody-dependent cell-mediated cytotoxicity. The binding epitope of MEDI-563 on IL-5Ralpha was determined by using site-directed mutagenesis. The consequences of MEDI-563 administration on peripheral blood and bone marrow eosinophil depletion was investigated in nonhuman primates. RESULTS: MEDI-563 binds to an epitope on IL-5Ralpha that is in close proximity to the IL-5 binding site, and it inhibits IL-5-mediated cell proliferation. MEDI-563 potently induces antibody-dependent cell-mediated cytotoxicity of both eosinophils (half-maximal effective concentration = 0.9 pmol/L) and basophils (half-maximal effective concentration = 0.5 pmol/L) in vitro. In nonhuman primates MEDI-563 depletes blood eosinophils and eosinophil precursors in the bone marrow. CONCLUSIONS: MEDI-563 might provide a novel approach for the treatment of asthma through active antibody-dependent cell-mediated depletion of eosinophils and basophils rather than through passive removal of IL-5.

Safety profile, pharmacokinetics, and biologic activity of MEDI-563, an anti-IL-5 receptor alpha antibody, in a phase I study of subjects with mild asthma.

BACKGROUND: Increased eosinophil levels have been linked to airway inflammation and asthma exacerbations. IL-5 is responsible for eosinophil differentiation, proliferation, and activation; IL-5 receptors are expressed on eosinophils and their progenitors, and targeting such receptors induces eosinophil apoptosis. OBJECTIVE: To evaluate the safety profile, pharmacokinetics, and pharmacodynamics of MEDI-563, a humanized mAb targeting the IL-5 receptor alpha chain. METHODS: Single, escalating, intravenous doses (0.0003-3 mg/kg) of MEDI-563 were administered to subjects with mild atopic asthma (n = 44) over approximately 3 to 30 minutes in this open-label study. Pulmonary function, symptom scores, adverse events, MEDI-563 pharmacokinetics, and levels of C-reactive protein (CRP), IL-6, eosinophil cationic protein (ECP), and eosinophils were evaluated. RESULTS: Mean peripheral blood (PB) eosinophil levels decreased in a dose-dependent fashion (baseline +/- SD, 0.27 +/- 0.2 x 10(3)/microL; 24 hours postdose, 0.01 +/- 0.0 x 10(3)/microL); 94.0% of subjects receiving >or=0.03 mg/kg exhibited levels between 0.00 x 10(3)/microL and 0.01 x 10(3)/microL. Eosinopenia lasted at least 8 or 12 weeks with doses of 0.03 to 0.1 and 0.3 to 3 mg/kg, respectively. ECP levels were reduced from 21.4 +/- 17.2 microg/L (baseline) to 10.3 +/- 7.0 microg/L (24 hours postdose). The most frequently reported adverse events were reduced white blood cell counts (34.1%), nasopharyngitis (27.3%), and increased blood creatine phosphokinase (25.0%). Mean C-reactive protein levels increased approximately 5.5-fold at 24 hours postdose but returned to baseline by study end; mean IL-6 levels increased approximately 3.9-fold to 4.7-fold at 6 to 12 hours postdose, respectively. Pharmacokinetic activity was dose proportional at doses of 0.03 to 3 mg/kg. CONCLUSION: Single escalating doses of MEDI-563 had an acceptable safety profile and resulted in marked reduction of PB eosinophil counts within 24 hours after dosing.

Molecular mechanism of asthma and its novel molecular target therapeutic agent.

Asthma is a chronic disease with major public health ramifications owing to its high morbidity and mortality rates, especially in severe and recurrent cases. Conventional therapeutic options could partially alleviate the burden of asthma, yet a novel approach is needed to completely control this condition. To do so, a comprehensive understanding of the molecular mechanism underlying asthma is essential to recognize and treat the major pathways that drive its pathophysiology. In this review, we will discuss the molecular mechanism of asthma, in particular focusing on the type of inflammatory responses it elicits, namely type 2 and non-type 2 asthma. Furthermore, we will discuss the novel therapeutic options that target the aberrant molecules found in asthma pathophysiology. We will specifically focus on the role of novel monoclonal antibody therapies recently developed, such as the anti-IgE, IL-5, IL-5Rα, and IL-4Rα antibodies, drugs that have been extensively studied preclinically and clinically.