IL-4Rα deletion disrupts psychomotor performance and reference memory in mice while sparing behavioural phenotype associated with spatial learning.

Contribution of immune mediators, interleukin-4 and interferon gamma to cognitive functioning is receiving increasing attention. However, the fundamental question about how heterodimeric interleukin-4 receptor alpha- and interferon gamma- producing myeloid cells converge to influence hippocampal-dependent spatial memory tasks through immunomodulation of multisensory inputs from other brain areas remains unexplored. Here, we show that mice lacking interleukin-4 receptor alpha are able to successfully learn spatial tasks, while reference memory is impaired. Moreover, the absence of interleukin-4 receptor alpha leads to simultaneous increase in proportions of CD11b + myeloid cells in the hippocampus and thalamus, but not the brainstem during acquisition. Interleukin-4 receptor alpha deletion significantly decreased expression of myeloid cell-derived interferon gamma in the thalamus during the acquisition phase and simultaneously increased brain-derived neurotrophic factor production in the thalamus and brainstem of trained mice. We provide evidence that interleukin-4 receptor alpha is essential for cognitive performance while training-induced alterations in interferon gamma activity and brain-derived neurotrophic factor signalling may contribute to neuromodulation of learned tasks and consequently affect systems-level memory encoding and consolidation.

Role of IL-4Rα during acute schistosomiasis in mice.

Schistosomiasis is an important parasitic disease that causes major host morbidity and mortality in endemic areas. Research conducted in mouse models of schistosomiasis has provided great insights and understanding of how host protective immunity is orchestrated and key cellular populations involved in this process. Earlier studies using cytokine-deficient mice demonstrated the importance of IL-4 and IL-10 in mediating host survival during acute schistosomiasis. Subsequent studies employing transgenic mice carrying cell-specific deletion of IL-4Rα generated using the Cre/LoxP recombination system have been instrumental in providing more in-depth understanding of the mechanisms conferring host resistance to Schistosoma mansoni infection. In this review, we will summarize the contributions of IL-4/IL-13-responsive cellular populations in host resistance during acute schistosomiasis and their role in limiting tissue pathology.

A recombinant Der p 1-specific allergen-toxin demonstrates superior killing of allergen-reactive IgG+ hybridomas in comparison to its recombinant allergen-drug conjugate.

Introduction: Current treatments for asthma help to alleviate clinical symptoms but do not cure the disease. In this study, we explored a novel therapeutic approach for the treatment of house dust mite allergen Der p 1induced asthma by aiming to eliminate specific population of B-cells involved in memory IgE response to Der p 1. Materials and Methods: To achieve this aim, we developed and evaluated two different proDer p 1-based fusion proteins; an allergen-toxin (proDer p 1-ETA) and an allergen-drug conjugate (ADC) (proDer p 1-SNAP-AURIF) against Der p 1 reactive hybridomas as an in vitro model for Der p 1 reactive human B-cells. The strategy involved the use of proDer p 1 allergen as a cell-specific ligand to selectively deliver the bacterial protein toxin Pseudomonas exotoxin A (ETA) or the synthetic small molecule toxin Auristatin F (AURIF) into the cytosol of Der p 1 reactive cells for highly efficient cell killing. Results: As such, we demonstrated recombinant proDer p 1 fusion proteins were selectively bound by Der p 1 reactive hybridomas as well as primary IgG1+ B-cells from HDM-sensitized mice. The therapeutic potential of proDer p 1-ETA' and proDer p 1-SNAP-AURIF was confirmed by their selective cytotoxic activities on Der p 1 reactive hybridoma cells. The allergen-toxin demonstrated superior cytotoxic activity, with IC50 values in the single digit nanomolar value, compared to the ADC. Discussions: Altogether, the proof-of-concept experiments in this study provide a promising approach for the treatment of patients with house dust mite-driven allergic asthma.

Interleukin-33 amplifies IgE synthesis and triggers mast cell degranulation via interleukin-4 in naïve mice.

BACKGROUND: The regulation and function of IgE in healthy individuals and in antigen-naïve animals is not well understood. IL-33 administration increases serum IgE in mice with unknown mechanism. We tested the hypothesis that IL-33 provides an antigen-independent stimulus for IgE production and mast cell degranulation. METHODS: IL-33 was administered to naïve wild-type (WT), nude and ST2(-/-) , IL-4(-/-) , IL4Rα(-/-) and T-or B-cell-specific IL-4Rα(-/-) mice. IgE and cytokines were quantified by ELISA. T- and B-lymphocyte numbers and CD40L expression were determined by flow cytometry. Anaphylaxis was measured by temperature, mast cell degranulation and histamine release. RESULTS: IL-33 enhanced IgE production in naïve WT, T-IL-4Rα(-/-) but not in ST2(-/-) , IL-4(-/-) , IL-4Rα(-/-) or B-cell-specific IL-4Rα(-/-) mice, demonstrating IL-33 specificity and IL-4 dependency. Moreover, IL-4 was required for IL-33-induced B-cell proliferation and T-cell CD40L expression, which promotes IgE production. IL-33-induced IL-4 production was mainly from innate cells including mast cells and eosinophils. IL-33 increased mast cell surface IgE and triggered degranulation and systemic anaphylaxis in allergen-naïve WT but not in IL-4Rα(-/-) mice. CONCLUSION: IL-33 amplifies IgE synthesis and triggers anaphylaxis in naïve mice via IL-4, independent of allergen. IL-33 may play an important role in nonatopic allergy and idiopathic anaphylaxis.

B cell tolerance in mice transgenic for anti-CD8 immunoglobulin mu chain.

To analyze in vivo the induction of B cell tolerance against a T cell surface antigen, we generated transgenic mice expressing an anti-CD8.2 mu heavy chain gene. We show that self-specific B cells are efficiently tolerized if they express the membrane-bound form of the transgenic mu chain on their surface but that they can escape tolerization if they express only the secreted form. In the latter, we find an enhanced expression of anti-CD8.2 antibodies after polyclonal B cell activation. As a result, transgenic anti-CD8.2 antibodies bind to the CD8+ T cells but they did not induce their elimination. Furthermore, we observed the preferential expression of a limited subset of endogenous light chains with the transgenic mu chain. This suggests a positive or negative selection for particular heavy and light chain combinations in B lymphocytes.

Interleukin 9-induced in vivo expansion of the B-1 lymphocyte population.

The activity of interleukin (IL)-9 on B cells was analyzed in vivo using transgenic mice that constitutively express this cytokine. These mice show an increase in both baseline and antigen-specific immunoglobulin concentrations for all isotypes tested. Analysis of B cell populations showed a specific expansion of Mac-1(+) B-1 cells in the peritoneal and pleuropericardial cavities, and in the blood of IL-9 transgenic mice. In normal mice, the IL-9 receptor was found to be expressed by CD5(+) as well as CD5(-) B-1 cells, and repeated injections of IL-9 resulted in accumulation of B-1 cells in the peritoneal cavity, as observed in transgenic animals. Unlike other mouse models, such as IL-5 transgenic mice, in which expansion of the B-1 population is associated with high levels of autoantibodies, IL-9 did not stimulate the production of autoantibodies in vivo, and most of the expanded cells were found to belong to the B-1b subset (IgM+Mac-1(+)CD5(-)). In addition, we found that these IL-9-expanded B-1b cells do not share the well-documented antibromelain-treated red blood cell specificity of CD5(+) B-1a cells. The increase of antigen-specific antibody concentration in immunized mice suggests that these B-1 cells are directly or indirectly involved in antibody responses in IL-9 transgenic mice.

IL-4-deficient Balb/c mice resist infection with Leishmania major.

Mice with a genetically engineered deficiency for either IL-4 or IFN-gamma R1 (single mutants), and IL-4/IFN-gamma R1 (double mutants) on the Balb/c and 129Sv background were used to study the course of infection with Leishmania major. In contrast to genetically resistant 129Sv wildtype mice, IL-4/IFN-gamma R1 double mutant mice developed fetal disease with parasite dissemination to visceral organs similar to mice lacking IFN-gamma R1 only. Balb/c mice, which are exquisitely susceptible to L. major, were rendered resistant to infection by disruption of the IL-4 gene. As compared to homozygous IL-4+/- mice, heterozygous IL-4+/- mice, heterozygous IL-4+/- animals consistently developed smaller lesions with less ulceration and necrosis, indicating the likelihood of gene-dosage effects. This implicates that the magnitude of the IL-4 response determines the severity of disease. CD4+ T cells of IL-4-deficient mice showed impaired Th2 cell development, as assessed by quantitative RT-PCR of characteristic cytokines. Development of resistance is not explained by default Th1 development, because this was observed only at very late stages of infection. Moreover, the induction of inflammatory cytokines (e.g., IL-1 alpha, IL-1 beta, TNF-alpha, IL-12) together with iNOS in the lesion and draining lymph nodes was not altered in the absence of IL-4.

IL-4R alpha deficiency influences hippocampal-BDNF signaling pathway to impair reference memory.

Like pro-inflammatory cytokines, the role of anti-inflammatory cytokines in both learning and memory has been investigated, revealing beneficial effects for both interleukin-4 and interleukin-13 via the common interleukin-4 receptor alpha chain complex. In this study, using the Morris water maze spatial task for cognition, we compared interleukin-4 receptor alpha- deficient mice and their ligands interleukin-4/ interleukin-13 double deficient mice, on a Balb/c background. We demonstrate that while interleukin-4/ interleukin-13 double deficient mice are significantly impaired in both learning and reference memory, interleukin-4 receptor alpha-deficiency impairs only reference memory, compared to the wild-type control mice. In order to better understand how interleukin-4 receptor alpha- deficient mice are able to learn but not remember, we investigated the BDNF/TrkB- and the ARC-signaling pathways. We show that interleukin-4 receptor alpha-deficiency disrupts activation of BDNF/TrkB- and ARC-signaling pathways during reference memory, while the pathway for spatial learning is spared.

Differential requirements for interleukin (IL)-4 and IL-13 in protein contact dermatitis induced by Anisakis.

BACKGROUND: Exposure to antigens of the fish parasite Anisakis is associated with the development of protein contact dermatitis in seafood-processing workers. Understanding the basic mechanisms controlling allergic sensitization through the skin is critical for designing therapies that will prevent the progression of allergic disease. OBJECTIVE: To investigate the roles of interleukin (IL)-4, IL-13 and the IL-4Ralpha in both local skin pathology and systemic sensitization following epicutaneous exposure to Anisakis proteins. METHODS: BALB/c wild-type (WT) mice and mice deficient in IL-4, IL-13 or IL-4 and IL-13, as well as mice with cell-specific impairment of IL-4Ralpha expression, were sensitized to Anisakis antigen by repeated epicutaneous application of Anisakis extract. Following this sensitization, skin pathology was recorded and systemic responses were investigated. Intravenous challenge with Anisakis extract was performed to test for the development of biologically relevant systemic sensitization. RESULTS: In WT mice, epicutaneous sensitization with Anisakis larval antigens induced localized inflammation, epidermal hyperplasia, production of T(H)2 cytokines, antigen-specific IgE and IgG1. Intravenous challenge of sensitized mice resulted in anaphylactic shock. Interestingly, IL-13 deficient mice failed to develop epidermal hyperplasia and inflammation, whilst anaphylaxis was reduced only in strains deficient either in IL-4 only, or deficient in IL-4 and IL-13 concurrently, as well as in mice deficient in IL-4Ralpha or with impaired IL-4Ralpha expression on CD4(+) T cells. CONCLUSIONS: Interleukin-13 plays a central role in protein contact dermatitis associated with repeated epicutaneous exposure to Anisakis extract, whereas IL-4 drives systemic sensitization and resultant anaphylactic shock.

Control of early viral and bacterial distribution and disease by natural antibodies.

Natural antibodies are often dismissed from immunological analysis as "background," but they may play an important role in conferring immunity against infections. In antibody-free mice infected with various viruses or with Listeria monocytogenes, viral or bacterial titers in peripheral organs, including the kidney and brain, were 10 to 100 times greater than in antibody-competent mice (and enhanced their susceptibility to some infections), and titers in secondary lymphoid organs were 10 to 100 times lower than in antibody-competent mice. Thus, natural antibodies play a crucial role by preventing pathogen dissemination to vital organs and by improving immunogenicity through enhanced antigen-trapping in secondary lymphoid organs.

Requirement for IL-13 independently of IL-4 in experimental asthma.

The pathogenesis of asthma reflects, in part, the activity of T cell cytokines. Murine models support participation of interleukin-4 (IL-4) and the IL-4 receptor in asthma. Selective neutralization of IL-13, a cytokine related to IL-4 that also binds to the alpha chain of the IL-4 receptor, ameliorated the asthma phenotype, including airway hyperresponsiveness, eosinophil recruitment, and mucus overproduction. Administration of either IL-13 or IL-4 conferred an asthma-like phenotype to nonimmunized T cell-deficient mice by an IL-4 receptor alpha chain-dependent pathway. This pathway may underlie the genetic associations of asthma with both the human 5q31 locus and the IL-4 receptor.

Nippostrongylus brasiliensis infection leads to impaired reference memory and myeloid cell interference.

Hookworm infection is endemic in developing countries, leading to poor cognitive function-among other disruptions. In this study, the effects of Nippostrongylus brasiliensis infection (a murine model of Necator Americanus) on cognitive function were investigated. Though impaired cognition has been extensively reported, the exact domain of cognition affected is still unknown, hence requiring investigation. The objective of this study was to identify possible cognitive changes during Nippostrongylus brasiliensis infection in mice, using the Morris water maze. Here, we show for the first time that mice infected with Nippostrongylus brasiliensis were able to learn the Morris water maze task, but demonstrated impaired reference memory. Anxiety measured by thigmotaxis in the maze, did not play a role for the observed cognitive impairment. Of further interest, an increase in the number of hippocampal macrophages and microglia with training and/or infection suggested a significant role of these cell types during spatial learning. Together, these experimental mouse studies suggest that helminth infections do have an impact on cognition. Further experimental animal studies on cognition and infection might open new approaches for a better understanding and impact of pathogen infections.

Protein kinase C-delta (PKCδ), a marker of inflammation and tuberculosis disease progression in humans, is important for optimal macrophage killing effector functions and survival in mice.

We previously demonstrated that protein kinase C-δ (PKCδ) is critical for immunity against Listeria monocytogenes, Leishmania major, and Candida albicans infection in mice. However, the functional relevance of PKCδ during Mycobacterium tuberculosis (Mtb) infection is unknown. PKCδ was significantly upregulated in whole blood of patients with active tuberculosis (TB) disease. Lung proteomics further revealed that PKCδ was highly abundant in the necrotic and cavitory regions of TB granulomas in multidrug-resistant human participants. In murine Mtb infection studies, PKCδ-/- mice were highly susceptible to tuberculosis with increased mortality, weight loss, exacerbated lung pathology, uncontrolled proinflammatory cytokine responses, and increased mycobacterial burdens. Moreover, these mice displayed a significant reduction in alveolar macrophages, dendritic cells, and decreased accumulation of lipid bodies (lungs and macrophages) and serum fatty acids. Furthermore, a peptide inhibitor of PKCδ in wild-type mice mirrored lung inflammation identical to infected PKCδ-/- mice. Mechanistically, increased bacterial growth in macrophages from PKCδ-/- mice was associated with a decline in killing effector functions independent of phagosome maturation and autophagy. Taken together, these data suggest that PKCδ is a marker of inflammation during active TB disease in humans and required for optimal macrophage killing effector functions and host protection during Mtb infection in mice.

Protein kinase C-delta (PKCδ), a marker of inflammation and tuberculosis disease progression in humans, is important for optimal macrophage killing effector functions and survival in mice.

This corrects the article DOI: 10.1038/mi.2017.68.

Differences between IL-4R alpha-deficient and IL-4-deficient mice reveal a role for IL-13 in the regulation of Th2 responses.

Allergens and infections with parasitic helminths preferentially induced Th2 immune responses associated with elevated levels of serum immunoglobulin E (IgE) and expansion of eosinophils and mast cells. Interleukin-4 (IL-4) is a key cytokine in the differentiation of naive CD4+ T cells into Th2 cells, which produce a panel of cytokines including IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 [1] and have been shown to trigger recovery from gastrointestinal nematodes [2]. Nonetheless, mice deficient for IL-4 have been shown to develop residual Th2 responses [3-5] and can expel the nematode Nippostrongylus brasiliensis [6], suggesting that there is a functional equivalent of IL-4 in these processes. IL-13 is a cytokine that shares some, but not all, biological activities with IL-4 [7,8]. There is now compelling evidence that IL-4 and IL-13 share receptor components, including IL-4R alpha and IL-13R alpha 1 [9]. In order to dissect the roles of IL-4 and IL-13 in the regulation of Th2 cells and in the response to nematode infections, we looked for differences between mice deficient for either the IL-4 gene or the IL-4R alpha gene. Unlike IL-4, IL-4R alpha was required for control of N. brasiliensis, and Th2 development during infection--as characterized by cytokine production, GATA-3 and surface CD30 expression--was more severely affected in IL-4R alpha-/- mice than in IL-4-/- mice. Injection of recombinant IL-13 induced worm expulsion in otherwise incompetent RAG2-/- mice. Our results suggest that IL-13 regulates Th2 responses to nematode infection and requires IL-4R alpha.

Cloning of a carcinoembryonic antigen gene family member expressed in leukocytes of chronic myeloid leukemia patients and bone marrow.

The carcinoembryonic antigen (CEA) gene family belongs to the immunoglobulin superfamily and can be subdivided into the CEA and pregnancy-specific glycoprotein subgroups. The basic structure of the encoded proteins consists of, in addition to a leader, one IgV-like and 2, 3, or 6 IgC-like domains. These domains are followed by varying COOH-terminal regions responsible for secretion, transmembrane anchoring, or insertion into the membrane by a glycosyl phosphatidylinositol tail. Here we report on the characterization of CGM6, a new member of the CEA gene subgroup, by complementary DNA cloning. The deduced coding region comprises 349 amino acids and consists of a leader, one IgV-like, two IgC-like domains, and a hydrophobic region, which is replaced by a glycosyl phosphatidylinositol moiety in the mature protein. CGM6 transcripts were only found thus far in leukocytes of chronic myeloid leukemia patients, in normal bone marrow, and in marginal amounts in normal granulocytes. The CGM6 gene product might, therefore, represent a myeloid marker. Analyses of CGM6 protein-expressing HeLa transfectants with monoclonal antibodies strongly indicate that the CGM6 gene codes for the CEA family member NCA-95.

The IL-33 receptor (ST2) regulates early IL-13 production in fungus-induced allergic airway inflammation.

Allergic airway inflammation (AAI) in response to environmental antigens is an increasing medical problem, especially in the Western world. Type 2 interleukins (IL) are central in the pathological response but their importance and cellular source(s) often rely on the particular allergen. Here, we highlight the cellular sources and regulation of the prototypic type 2 cytokine, IL-13, during the establishment of AAI in a fungal infection model using Cryptococcus neoformans. IL-13 reporter mice revealed a rapid onset of IL-13 competence within innate lymphoid cells type 2 (ILC2) and IL-33R(+) T helper (Th) cells. ILC2 showed IL-33-dependent proliferation upon infection and significant IL-13 production. Th cells essentially required IL-33 to become either GATA3(+) or GATA3(+)/Foxp3(+) hybrids. GATA3(+) Th cells almost exclusively contributed to IL-13 production but hybrid GATA3(+)/Foxp3(+) Th cells did not. In addition, alveolar macrophages upregulated the IL-33R and subsequently acquired a phenotype of alternative activation (Ym1(+), FIZZ1(+), and arginase-1(+)) linked to type 2 immunity. Absence of adaptive immunity in rag2(-/-) mice resulted in attenuated AAI, revealing the need for Th2 cells for full AAI development. Taken together, in pulmonary cryptococcosis ILC2 and GATA3(+) Th2 cells produce early IL-13 largely IL-33R-dependent, thereby promoting goblet cell metaplasia, pulmonary eosinophilia, and alternative activation of alveolar macrophages.

The role of IL-4 in adult acquired and congenital toxoplasmosis.

The course of Toxoplasma gondii infection was studied in IL-4-deficient mice from three genetic backgrounds and their wild-type counterparts following peroral inoculation of tissue cysts. Survival rates were significantly reduced in disease-susceptible C57 BL/6 mice and F1 (C57BL/6 x 129Sv) mice deficient in IL-4 compared with wild-type controls. In contrast, this difference was not observed in T. gondii-resistant BALB/c mice. However, brain tissue cyst burdens in IL-4-deficient mice were either equivalent to (C57BL/6 and BALB/c mice) or significantly less (B6/129 mice) than similarly infected wild-type mice. Thus strain-specific differences in the course of T. gondii were demonstrated in the absence of IL-4. The course of T. gondii infection was also compared between B6/129 IL-4-deficient mice and their wild-type counterparts following peroral challenge with 20 tissue cysts on day 12 of pregnancy. Age-matched non-pregnant IL-4-/- and IL-4+/+ mice were also infected to assess the role of IL-4 on T. gondii infection during pregnancy. Disease phenotypes, as measured by mortality, were reversed if infections were initiated during pregnancy compared with non-pregnant infection. Thus significant mortality occurred immediately post partum in IL-4+/+ mothers, while all IL-4-/- mothers survived. Cyst burdens 28 days p.i. were significantly lower in IL-4-/- mothers than IL-4+/+ mothers and both IL-4-/- and IL-4+/+ non-pregnant mice. Congenital disease transmission as measured by foetal death or vertical disease transmission was independent of the presence or absence of IL-4. These studies demonstrate a role for IL-4 in pregnancy-induced immunosuppression and the associated increased susceptibility to T. gondii infection.

Lung-resident CD4⁺ T cells are sufficient for IL-4Rα-dependent recall immunity to Nippostrongylus brasiliensis infection.

Immunity to Nippostrongylus brasiliensis reinfection requires pulmonary CD4⁺ T-cell responses. We examined whether secondary lymphoid recruited or pre-existing lung CD4⁺ T-cell populations coordinated this immunity. To do this, we blocked T-cell egress from lymph nodes using Fingolimod (FTY720). This impaired host ability to resolve a primary infection but did not change effectiveness of recall immunity. Associated with this effective recall immunity was the expansion and T helper type 2 polarization of a pre-existing pulmonary CD4⁺ T-cell population. LTßR-Ig (lymphotoxin beta-receptor fusion protein)-mediated disruption of stromal cell organization of immune cells did not disrupt this recall immunity, suggesting that protection was mediated by a pulmonary interstitial residing CD4⁺ T-cell population. Adoptive transfer of N. brasiliensis-experienced pulmonary CD4⁺ T cells from FTY720-treated wild-type or T-cell interleukin (IL)-4Rα-deficient mice demonstrated protection to be IL-4Rα dependent. These results show that pre-existing CD4⁺ T cells can drive effective recall immunity to N. brasiliensis infection independently of T-cell recruitment from secondary lymphoid organs.

Acute helminth infection enhances early macrophage mediated control of mycobacterial infection.

Co-infection with mycobacteria and helminths is widespread in developing countries, but how this alters host immunological control of each pathogen is not comprehensively understood. In this study, we demonstrate that acute Nippostrongylus brasiliensis (Nb) murine infection reduce early pulmonary mycobacterial colonization. This Nb-associated reduction in pulmonary Mycobacterium tuberculosis colony-forming units was associated with early and increased activation of pulmonary CD4 T cells and increased T helper type 1 (Th1) and Th2 cytokine secretion. An accelerated and transient augmentation of neutrophils and alveolar macrophages (AMs) was also observed in co-infected animals. AMs displayed markers of both classical and alternative activation. Intranasal transfer of pulmonary macrophages obtained from donor mice 5 days after Nb infection significantly reduced pulmonary Mycobacterium bovis Bacille Calmette-Guérin clearance in recipient mice. These data demonstrate that early stage Nb infection elicits a macrophage response, which is protective during the early stages of subsequent mycobacterial infection.