Differences in allergen-specific basophil activation and T cell proliferation in atopic dermatitis patients with comorbid allergic rhinoconjunctivitis treated with a monoclonal anti-IL-4Rα antibody or allergen-specific immunotherapy.

BACKGROUND: Atopic dermatitis (AD), a chronic inflammatory disorder, is often accompanied by allergic rhinoconjunctivitis (ARC) as a co-morbidity. The use of a monoclonal anti-IL-4Rα antibody has been effective in controlling moderate to severe AD symptoms. Allergen-specific immunotherapy (AIT) is widely used for the treatment of ARC and asthma. The effects of AIT on basophil reactivity/effector functions have already been examined and used as indicators of the treatment efficacy. However, it is unclear, how an anti-IL-4Rα antibody can influence allergen-specific immune responses of basophils and T cells of AD patients with comorbid ARC. OBJECTIVE: To investigate the effect of a monoclonal anti-IL-4Rα antibody on the in vitro allergic responses of basophils and T cells deriving from AD patients with comorbid ARC. METHODS: Blood samples of 32 AD patients were obtained before, after 4 and 16 weeks of an anti-IL-4Rα antibody therapy (300 mg subcutaneously/2 weeks; n = 21) or AIT (daily sublingual application; n = 11). Patients treated with an anti-IL-4Rα antibody were grouped according to their serum specific immunoglobulin E levels and ARC symptoms, while patients receiving an AIT were additionally grouped according to the allergen specificity of their AIT. Basophil activation test and T cell proliferation assays were undertaken after an in vitro allergen stimulation. RESULTS: A significant reduction of the immunoglobulin E levels and the allergen-specific T cell proliferation was observed in AD patients treated with an anti-IL-4Rα -antibody, while the allergen-specific basophil activation/sensitivity were found to be significantly increased. In patients receiving an AIT, the in vitro allergen-specific basophil activation and the T cell proliferation were found to be significantly decreased in response to seasonal allergens. CONCLUSIONS: An IL-4Rα blockade induced by a monoclonal anti-IL-4Rα antibody leads to an increased activity/sensitivity of early effector cells (such as basophils), in contrast to a decreasing reactivity observed under an AIT. The late-phase T cell reaction to allergens did not differ between the herein assessed treatments.

Elarekibep (PRS-060/AZD1402), a new class of inhaled Anticalin medicine targeting IL-4Ra for type 2 endotype asthma.

BACKGROUND: Type 2 endotype asthma is driven by IL-4 and IL-13 signaling via IL-4Ra, which is highly expressed on airway epithelium, airway smooth muscle, and immunocytes in the respiratory mucosa, suggesting potential advantages of an inhalable antagonist. Lipocalin 1 (Lcn1), a 16 kDa protein abundant in human periciliary fluid, has a robust drug-like structure well suited to protein engineering, but it has never been used to make an inhaled Anticalin protein therapeutic. OBJECTIVES: We sought to reengineer Lcn1 into an inhalable IL-4Ra antagonist and assess its pharmacodynamic/kinetic profile. METHODS: Lcn1 was systematically modified by directed protein mutagenesis yielding a high-affinity, slowly dissociating, long-acting full antagonist of IL-4Ra designated PRS-060 with properties analogous to dupilumab, competitively antagonizing IL-4Ra-dependent cell proliferation, mucus induction, and eotaxin expression in vitro. Because PRS-060 displayed exquisite specificity for human IL-4Ra, with no cross-reactivity to rodents or higher primates, we created a new triple-humanized mouse model substituting human IL-4Ra, IL-4, and IL-13 at their correct syntenic murine loci to model clinical dosing. RESULTS: Inhaled PRS-060 strongly suppressed acute allergic inflammation indexes in triple-humanized mice with a duration of action longer than its bulk clearance, suggesting that it may act locally in the lung. CONCLUSION: Lcn1 can be reengineered into the Anticalin antagonist PRS-060 (elarekibep), exemplifying a new class of inhaled topical, long-acting therapeutic drugs with the potential to treat type 2 endotype asthma.

IL-4 expressing cells are recruited to nerve after injury and promote regeneration.

Interleukin-4 (IL-4) has garnered interest as a cytokine that mediates regeneration across multiple tissues including peripheral nerve. Within nerve, we previously showed endogenous IL-4 was critical to regeneration across nerve gaps. Here, we determined a generalizable role of IL-4 in nerve injury and regeneration. In wild-type (WT) mice receiving a sciatic nerve crush, IL-4 expressing cells preferentially accumulated within the injured nerve compared to affected sites proximal, such as dorsal root ganglia (DRGs), or distal muscle. Immunohistochemistry and flow cytometry confirmed that eosinophils (CD45+, CD11b+, CD64-, Siglec-F+) were sources of IL-4 expression. Examination of targets for IL-4 within nerve revealed macrophages, as well as subsets of neurons expressed IL-4R, while Schwann cells expressed limited IL-4R. Dorsal root ganglia cultures were exposed to IL-4 and demonstrated an increased proportion of neurons that extended axons compared to cultures without IL-4 (control), as well as longer myelinated axons compared to cultures without IL-4. The role of endogenous IL-4 during nerve injury and regeneration in vivo was assessed following a sciatic nerve crush using IL-4 knockout (KO) mice. Loss of IL-4 affected macrophage accumulation within injured nerve compared to WT mice, as well as shifted macrophage phenotype towards a CD206- phenotype with altered gene expression. Furthermore, this loss of IL-4 delayed initial axon regeneration from the injury crush site and subsequently delayed functional recovery and re-innervation of neuromuscular junctions compared to wild-type mice. Given the role of endogenous IL-4 in nerve regeneration, exogenous IL-4 was administered daily to WT mice following a nerve crush to examine regeneration. Daily IL-4 administration increased early axonal extension and CD206+ macrophage accumulation but did not alter functional recovery compared to untreated mice. Our data demonstrate IL-4 promotes nerve regeneration and recovery after injury.

A New Era with the Development of Cytokine-Based Therapy for Pruritus.

Pruritus is a common dermatological condition and negatively impacts QOL. Persistent pruritus and excessive scratching behavior can lead to the itch-scratch cycle that exacerbates inflammatory skin diseases. Conventional antipruritic drugs, such as antihistamines, corticosteroids, or anticonvulsants, are sometimes insufficient. Recently, however, molecularly targeted drugs, such as IL-31 or IL-4 receptor-targeting antibodies, have become available or are under clinical trials, dramatically changing the clinical situation. In fact, some of these drugs can improve pruritus without the need for topical steroids. Taken together, these observations point to the importance of cytokine-mediated pruritus, further understanding of which may guide improved therapies.

Sex differences in M2 polarization, chemokine and IL-4 receptors in monocytes and macrophages from asthmatics.

Allergic asthma affects more women than men. It is mediated partially by IL-4/IL-13-driven polarization of monocyte-derived macrophages in the lung. We tested whether sex differences in asthma are due to differential IL-4 responsiveness and/or chemokine receptor expression in monocytes and monocyte-derived macrophages from healthy and allergic asthmatic men and women. We found female cells expressed M2 genes more robustly following IL-4 stimulation than male cells, as did cells from asthmatics than those from healthy controls. This likely resulted from increased expression ofγC, part of the type I IL-4 receptor, and reduced IL-4-induced SOCS1, a negative regulator of IL-4 signaling, in asthmatic compared to healthy macrophages. Monocytes from asthmatic women expressed more CX3CR1, which enhances macrophage survival. Our findings highlight how sex differences in IL-4 responsiveness and chemokine receptor expression may affect monocyte recruitment and macrophage polarization in asthma, potentially leading to new sex-specific therapies to manage the disease.

Tetracyclines improve experimental lymphatic filariasis pathology by disrupting interleukin-4 receptor-mediated lymphangiogenesis.

Lymphatic filariasis is the major global cause of nonhereditary lymphedema. We demonstrate that the filarial nematode Brugia malayi induced lymphatic remodeling and impaired lymphatic drainage following parasitism of limb lymphatics in a mouse model. Lymphatic insufficiency was associated with elevated circulating lymphangiogenic mediators, including vascular endothelial growth factor C. Lymphatic insufficiency was dependent on type 2 adaptive immunity, the interleukin-4 receptor, and recruitment of C-C chemokine receptor-2-positive monocytes and alternatively activated macrophages with a prolymphangiogenic phenotype. Oral treatments with second-generation tetracyclines improved lymphatic function, while other classes of antibiotic had no significant effect. Second-generation tetracyclines directly targeted lymphatic endothelial cell proliferation and modified type 2 prolymphangiogenic macrophage development. Doxycycline treatment impeded monocyte recruitment, inhibited polarization of alternatively activated macrophages, and suppressed T cell adaptive immune responses following infection. Our results determine a mechanism of action for the antimorbidity effects of doxycycline in filariasis and support clinical evaluation of second-generation tetracyclines as affordable, safe therapeutics for lymphedemas of chronic inflammatory origin.

Interrupting reactivation of immunologic memory diverts the allergic response and prevents anaphylaxis.

BACKGROUND: IgE production against innocuous food antigens can result in anaphylaxis, a severe life-threatening consequence of allergic reactions. The maintenance of IgE immunity is primarily facilitated by IgG+ memory B cells, as IgE+ memory B cells and IgE+ plasma cells are extremely scarce and short-lived, respectively. OBJECTIVE: Our aim was to investigate the critical requirements for an IgE recall response in peanut allergy. METHODS: We used a novel human PBMC culture platform, a mouse model of peanut allergy, and various experimental readouts to assess the IgE recall response in the presence and absence of IL-4Rα blockade. RESULTS: In human PBMCs, we have demonstrated that blockade of IL-4/IL-13 signaling aborted IgE production after activation of a recall response and skewed the cytokine response away from a dominant type 2 signature. TH2A cells, identified by single-cell RNA sequencing, expanded with peanut stimulation and maintained their pathogenic phenotype in spite of IL-4Rα blockade. In mice with allergy, anti-IL-4Rα provided long-lasting suppression of the IgE recall response beyond antibody treatment and fully protected against anaphylaxis. CONCLUSION: The findings reported here advance our understanding of events mediating the regeneration of IgE in food allergy.

Interleukin-4 Responsive Dendritic Cells Are Dispensable to Host Resistance Against Leishmania mexicana Infection.

IL-4 and IL-13 cytokines have been associated with a non-healing phenotype in murine leishmaniasis in L. mexicana -infected BALB/c mice as demonstrated in IL-4-/-, IL-13-/- and IL-4Rα-/- global knockout mouse studies. However, it is unclear from the studies which cell-type-specific IL-4/IL-13 signaling mediates protection to L. mexicana. Previous studies have ruled out a role for IL-4-mediated protection on CD4+ T cells during L. mexicana infections. A candidate for this role may be non-lymphocyte cells, particularly DCs, as was previously shown in L. major infections, where IL-4 production drives dendritic cell-IL-12 production thereby mediating a type 1 immune response. However, it is unclear if this IL-4-instruction of type 1 immunity also occurs in CL caused by L. mexicana, since the outcome of cutaneous leishmaniasis often depends on the infecting Leishmania species. Thus, BALB/c mice with cell-specific deletion of the IL-4Rα on CD11c+ DCs (CD11ccreIL-4Rα-/lox) were infected with L. mexicana promastigotes in the footpad and the clinical phenotype, humoral and cellular immune responses were investigated, compared to the littermate control. Our results show that CL disease progression in BALB/c mice is independent of IL-4Rα signaling on DCs as CD11ccreIL-4Rα-/lox mice had similar footpad lesion progression, parasite loads, humoral responses (IgE, IgG1, IgG 2a/b), and IFN-γ cytokine secretion in comparison to littermate controls. Despite this comparable phenotype, surprisingly, IL-4 production in CD11ccreIL-4Rα-/lox mice was significantly increased with an increasing trend of IL-13 when compared to littermate controls. Moreover, the absence of IL-4Rα signaling did not significantly alter the frequency of CD4 and CD8 lymphocytes nor their activation, or memory phenotype compared to littermate controls. However, these populations were significantly increased in CD11ccreIL-4Rα-/lox mice due to greater total cell infiltration into the lymph node. A similar trend was observed for B cells whereas the recruitment of myeloid populations (macrophages, DCs, neutrophils, and Mo-DCs) into LN was comparable to littermate IL-4Rα-/lox mice. Interestingly, IL-4Rα-deficient bone marrow-derived dendritic cells (BMDCs), stimulated with LPS or L. mexicana promastigotes in presence of IL-4, showed similar levels of IL-12p70 and IL-10 to littermate controls highlighting that IL-4-mediated DC instruction was not impaired in response to L. mexicana. Similarly, IL-4 stimulation did not affect the maturation or activation of IL-4Rα-deficient BMDCs during L. mexicana infection nor their effector functions in production of nitrite and arginine-derived metabolite (urea). Together, this study suggests that IL-4 Rα signaling on DCs is not key in the regulation of immune-mediated protection in mice against L. mexicana infection.

Evolution and function of interleukin-4 receptor signaling in adaptive immunity and neutrophils.

The cytokines interleukin (IL)-4 and IL-13, signaling via the IL-4 receptor (IL-4R), orchestrate type 2 immunity to helminth infections and toxins. Activation of epithelial and myeloid cells, and a transient neutrophils influx initiates type 2 immune responses, which are dominated by basophils, eosinophils, mast cells, B cell immunoglobulin E production, and type 2 T helper and T follicular helper cells. Interestingly, IL-4 and IL-13 can curtail chemotaxis and several effector functions of neutrophils in mice and humans. This inhibitory role of IL-4 and IL-13 probably developed to limit tissue damage by neutrophils during type 2 immunity where a "weep and sweep" response aims at expulsion and decreased fecundity, instead of killing, of macroparasites. Here, we review when IL-4R signaling cytokines appeared during evolution relative to neutrophils and adaptive immunity. Neutrophil-like granular phagocytes were present in invertebrates throughout the bilaterian clade, but we were unable to find data on IL-4, IL-13, or their receptors in invertebrates. Conversely, vertebrates had both adaptive immunity and IL-4, IL-13, and IL-4Rs, suggesting that type 2 cytokines evolved together with adaptive immunity. Further studies are necessary to determine whether IL-4R signaling in neutrophils was established simultaneously with the appearance of adaptive immunity or later.

Dupilumab: Basic aspects and applications to allergic diseases.

Interleukin (IL)-4 and IL-13, signature type 2 cytokines, exert their actions by binding to two types of receptors sharing the IL-4R α chain (IL-4Rα). Since IL-4 and IL-13 play important and redundant roles in the pathogenesis of allergic diseases, blocking both the IL-4 and IL-13 signals would be a powerful and effective strategy for treating allergic diseases. Dupilumab (Dupixent®) is a fully human monoclonal antibody recognizing IL-4Rα and blocking both the IL-4 and IL-13 signals. Dupilumab was first prescribed for atopic dermatitis (AD) patients and has been widely approved for adult patients with moderate to severe AD since 2018. Dupilumab has since been used for asthma, receiving approval for uncontrolled asthma in 2019. A phase 3 study using dupilumab for chronic rhinosinusitis with nasal polyps (CRSwNP) has been just completed, with positive results. Several clinical trials of dupilumab for other diseases in which type 2 inflammation is dominant are now underway. It is hoped that dupilumab will open the door to a new era for treating allergic patients with AD, asthma, and CRSwNP, and for more patients with type 2 inflammations.

Kit activates interleukin-4 receptor and effector signal transducer and activator of transcription 6 independent of its cognate ligand in mouse mast cells.

Signaling by Kit has been extensively studied in hematopoietic cells and is essential for the survival, proliferation and maintenance of hematopoietic stem and progenitor cells. In addition to the activation of intrinsic signaling pathways, Kit has been shown to interact with lineage-restricted type I cytokine receptors and produce cross signals, e.g. erythropoietin receptor, interleukin-7 receptor (IL-7R), IL-3R. Based on the earlier studies, we hypothesize that Kit activate other type I cytokine receptors in a cell-specific manner and execute cell-specific function. To investigate other Kit-activated receptors, we tested Kit and IL-4R cross-receptor activation in murine bone-marrow-derived mast cells, which express both Kit and IL-4R at the surface level. Kit upon activation by Kit ligand (KL), activated IL-4Rα, γC , and signal transducer and activator of transcription 6 independent of its cognate ligand IL-4. Though KL and IL-4 are individually mitogenic, combinations of KL and IL-4 synergistically promoted mast cell proliferation. Furthermore, inhibition of lipid raft formation by methyl-ß-cyclodextrin resulted in loss of synergistic proliferation. Together the data suggest IL-4R as a novel Kit-activated receptor. Such cross-receptor activations are likely to be a universal mechanism of Kit signaling in hematopoiesis.

Anti-IL-4/IL-13 for the treatment of asthma: the story so far.

Introduction: Severe asthma is a global health concern with high morbidity and mortality. Understanding of its complex pathophysiology continues to increase, providing specific immune targets for therapeutic intervention.Areas covered: In this review, we focus on the role of IL-4 and IL-13 in severe asthma and on the biologic therapies developed to target them, particularly dupilumab, a monoclonal antibody against the IL-4 receptor α subunit and IL-4/IL-13 receptor complex. A literature search was undertaken for all studies of monoclonal antibodies against IL-4 and IL-13.Expert Opinion: Dupilumab decreases the rate of severe asthma exacerbations and improves symptoms, lung function, and quality of life. Importantly, these effects are also observed during reduction of maintenance oral corticosteroid doses. Those with the highest T2 biomarkers derive the greatest benefit and the presence of atopic dermatitis or chronic rhinosinusitis with or without nasal polyposis may recommend dupilumab as the preferred biologic treatment for a patient.

An IL-4/21 Inverted Cytokine Receptor Improving CAR-T Cell Potency in Immunosuppressive Solid-Tumor Microenvironment.

Incorporation of inverted cytokine receptor (ICR) such as interleukin (IL)-4 vs. IL-7 (4/7) ICR is one strategy to improve the antitumor activities of chimeric antigen receptor (CAR) modified T (CAR-T) cells facing immunosuppressive cytokines. Here we report a novel interleukin (IL)-4 vs. IL-21 ICR (4/21 ICR) that enhanced CAR-T cell potency in IL-4+ tumor milieu via a different working-mechanism from 4/7 ICR. Upon IL-4 stimulation, 4/21 ICR activated the STAT3 pathway and promoted Th17-like polarization and tumor-targeted cytotoxicity in CAR-T cells in vitro. Furthermore, 4/21 ICR-CAR T cells persisted and eradicated established IL-4+ tumors in vivo. Thus, 4/21 ICR is a promising clinical CAR-T cell therapeutics for solid tumors rich in IL-4.

Pathogenesis of Atopic Dermatitis: Current Paradigm.

Atopic dermatitis (AD) is characterized by skin inflammation, barrier dysfunction and chronic pruritus. In this review, recent advances in the pathogenesis of AD are summarized. Clinical efficacy of the anti-IL-4 receptor antibody dupilumab implies that type 2 cytokines IL-4 and IL-13 have pivotal roles in atopic inflammation. The expression of IL-4 and IL-13 as well as type 2 chemokines such as CCL17, CCL22 and CCL26 is increased in the lesional skin of AD. In addition, IL-4 and IL-13 down-regulate the expression of filaggrin in keratinocytes and exacerbate epidermal barrier dysfunction. Keratinocytes in barrier-disrupted epidermis produce large amounts of thymic stromal lymphopoietin, IL-25 and IL-33, conducing to type 2 immune deviation via OX40L/OX40 signaling. IL-31, produced by type 2 T cells, is a cardinal pruritogenic cytokine. IL-4 and IL-13 also amplify the IL-31-mediated sensory nerve signal. These molecules are particularly important targets for future drug development for AD.

Systemic Treatment for Severe Atopic Dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory, relapsing disease of the skin, characterized by intense pruritus, maculopapular or vesicular erythematous lesions and scaling, sometimes accompanied by oozing, crusts and/or lichenification that has a negative impact on patients' quality of life. Prevalence is higher in children, around 15%, and approximately 5% in adults. Before introducing systemic therapy, it is mandatory to review patients' adherence to the correct use of topical treatments (corticosteroids, calcineurin inhibitors or cresoborole) and/or phototherapy. Ensure that environmental measures are being taken care of, irritant or proven allergic substances are not in use and even if the diagnostic is correct. If all is being done and topical treatment with corticosteroid, emollients and phototherapy have not been sufficient to achieve a good control in AD of adults or children patients, it is time to consider systemic agents. Up to now, most of systemic treatments were based on immunosuppressive therapies, being cyclosporine A, the usually first choice for moderate-to-severe AD. Recently, biologic drugs have been developed and approved for AD, as dupilumab, and a whole new group of drugs is giving much hope for patients to have a better control of the disease with less side effects.

Interleukin-4/interleukin-13 versus interleukin-5: a comparison of molecular targets in biologic therapy for the treatment of severe asthma.

PURPOSE OF REVIEW: Asthma is a chronic, inflammatory disorder of the airways caused by a complex interplay of various biologic mechanisms. Several monoclonal antibody therapies targeting interleukin (IL)-4/IL-13 and IL-5 cytokine pathways have been developed for the treatment of severe eosinophilic asthma. As individuals can display biomarkers and clinical features characteristic of several asthma phenotypes, selection of anoptimal biologic can be difficult. RECENT FINDINGS: Dupilumab, a monoclonal antibody that binds to the α subunit of the IL-4 receptor (IL-4Rα) and has been approved for the treatment of adults with severe atopic dermatitis, has been shown in recent phase 3 trials to also have significant clinical benefits in the asthmatic population irrespective of baseline eosinophil counts. SUMMARY: As monoclonal antibodies targeting either IL-4 or IL-13 cytokines individually have failed to demonstrate significant clinical benefits, biologics that target cytokine receptors may be more efficacious compared to those that target cytokines. Furthermore, inhibition of the IL-4/IL-13 signaling cascades may disrupt a broader Th2 inflammatory response compared to a more selective impairment of eosinophil proliferation and activity via blockage of the IL-5 pathway. Future research with independently funded, head-to-head trials of approved biologics is needed to elucidate a favorable therapeutic option.