Allergen-specific cytokine polarization protects Shetland ponies against Culicoides obsoletus-induced insect bite hypersensitivity

The immunological mechanisms explaining development of an allergy in some individuals and not in others remain incompletely understood. Insect bite hypersensitivity (IBH) is a common, seasonal, IgE-mediated, pruritic skin disorder that affects considerable proportions of horses of different breeds, which is caused by bites of the insect Culicoides obsoletus (C. obsoletus). We investigated the allergen-specific immune status of individual horses that had either been diagnosed to be healthy or to suffer of IBH. Following intradermal allergen injection, skin biopsies were taken of IBH-affected and healthy ponies and cytokine expression was determined by RT-PCR. In addition, allergen-specific antibody titers were measured and cytokine expression of in vitro stimulated, allergen-specific CD4 T-cells was determined. 24 hrs after allergen injection, a significant increase in mRNA expression of the type-2 cytokine IL-4 was observed in the skin of IBH-affected Shetland ponies. In the skin of healthy ponies, however, an increase in IFN¿ mRNA expression was found. Analysis of allergen-specific antibody titers revealed that all animals produced allergen-specific antibodies, and allergen-specific stimulation of CD4 T-cells revealed a significant higher percentage of IFN¿-expressing CD4 T-cells in healthy ponies compared to IBH-affected ponies. These data indicate that horses not affected by IBH, in contrast to the so far established dogma, are not immunologically ignorant but have a Th1-skewed allergen-specific immune response that appears to protect against IBH-associated symptoms. To our knowledge this is the first demonstration of a natural situation, in which an allergen-specific immune skewing is protective in an allergic disorder

Basophil-derived Amphiregulin is essential for UVB irradiation-induced immune suppression

UVB irradiation (290–320nm) is used to treat skin diseases like psoriasis and atopic dermatitis, and is known to suppress contact hypersensitivity (CHS) reactions in mouse models. Regulatory T cells (Treg cells) have been shown to be responsible for this UVB-induced suppression of CHS. The epidermal growth factor (EGF)-like growth factor amphiregulin (AREG) engages EGFR on Treg cells and, in different disease models, it was shown that mast cell–derived AREG is essential for optimal Treg cell function in vivo. Here we determined whether AREG has a role in UVB-induced, Treg cell–mediated suppression of CHS reactions in the skin. Our data show that AREG is essential for UVB-induced CHS suppression. In contrast to the general assumption, however, mast cells were dispensable for UVB-induced immune suppression, whereas basophil-derived AREG was essential. These data reveal, to our knowledge, a previously unreported function for basophils in the homeostasis of immune responses in the skin. Basophils thus fulfill a dual function: they contribute to the initiation of effective type 2 immune responses and, by enhancing the suppressive capacity of local Treg cell populations, also to local immune regulation in the skin.A stimulation grant from the University of Utrecht and a grant from the Dutch Technology Foundation (STW-NWO).http://www.nature.com/jid/hb201

Enhanced Inflammatory Potential of CD4(+) T-Cells That Lack Proteasome Immunosubunit Expression, in a T-Cell Transfer-Based Colitis Model

Proteasomes play a fundamental role in intracellular protein degradation and therewith regulate a variety of cellular processes. Exposure of cells to (pro)inflammatory cytokines upregulates the expression of three inducible catalytic proteasome subunits, the immunosubunits, which incorporate into newly assembled proteasome complexes and alter the catalytic activity of the cellular proteasome population. Single gene-deficient mice lacking one of the three immunosubunits are resistant to dextran sulfate sodium (DSS)-induced colitis development and, likewise, inhibition of one single immunosubunit protects mice against the development of DSS-induced colitis. The observed diminished disease susceptibility has been attributed to altered cytokine production and CD4+ T-cell differentiation in the absence of immunosubunits. To further test whether the catalytic activity conferred by immunosubunits plays an essential role in CD4+ T-cell function and to distinguish between the role of immunosubunits in effector T-cells versus inflamed tissue, we used a T-cell transfer-induced colitis model. Naïve wt or immunosubunit-deficient CD4+ T-cells were adoptively transferred into RAG1-/- and immunosubunit-deficient RAG1-/- mice and colitis development was determined six weeks later. While immunosubunit expression in recipient mice had no effect on colitis development, transferred immunosubunit-deficient T- cells were more potent in inducing colitis and produced more proinflammatory IL17 than wt T-cells. Taken together, our data show that modifications in proteasome-mediated proteolysis in T-cells, conferred by lack of immunosubunit incorporation, do not attenuate but enhance CD4+ T-cell-induced inflammation

CD4 T cells and iTreg cells in mice with T-cell transfer-induced colitis.

<p>Splenocytes of mice with T-cell transfer-induced colitis, harvested six weeks after T-cell transfer, were stained for CD4 and FoxP3 expression and analyzed by flow cytometry. (A) absolute numbers of splenocytes; (B, D) percentages, and (C, E) numbers of CD4+ T-cells and CD4+FoxP3+ T-cells in the spleen. Results for individual mice and means+SEM (n = 3–6 per group) are depicted. *p<0.05.</p

Recombinant Culicoides obsoletus complex allergens stimulate antigen-specific T cells of insect bite hypersensitive Shetland ponies in vitro

BACKGROUND: Ponies may suffer from Insect bite hypersensitivity (IBH), an allergic IgE-mediated pruritic skin disorder, induced by allergens from biting midges of the Culicoides spp. HYPOTHESIS/OBJECTIVES: To determine whether recombinant Culicoides obsoletus allergens are able to activate T cells of ponies exposed to C. obsoletus and whether these allergen-specific responses differ between IBH-affected and healthy ponies. ANIMALS: Ten IBH-affected Shetland ponies and 10 age-matched healthy controls taken from the same stables, to ensure similar exposure to midges. METHOD: Peripheral blood mononuclear cells (PBMC) were cultured with two different pools of recombinant C. obsoletus complex allergens to expand the allergen-specific T cells. These PBMC cultures were subsequently co-cultured with mature dendritic cells (DCs) loaded with the same antigens. Induction of Th1, Th2 and regulatory T (Treg) cells in these DC/PBMC co-cultures was assessed by analysis of IFN-γ, IL-4, IL-10 and FoxP3 expression levels using quantitative RT-PCR and phenotyping by flow cytometry. RESULTS: Recombinant C. obsoletus allergens increased IFN-γ mRNA expression levels, percentages of IFN-γ expressing (Th1) cells and CD25(high) FoxP3(+) IL-10(+) Tregs compared to unstimulated DC/PBMC co-cultures. Stimulation of IL-4 expressing Th2 cells by the recombinant allergens was far less pronounced. The DC/PBMC co-cultures did not reveal significant differences between healthy and IBH-affected ponies for any of the analysed parameters, except for higher IL-4 mRNA levels in IBH affected ponies after stimulation with one of the two allergen pools. CONCLUSION AND CLINICAL IMPORTANCE: The recombinant C. obsoletus complex allergens can stimulate antigen-specific Th1 and IL10 producing Treg cells and are therefore promising candidates for the immunotherapy of IBH

Colitis development in T-cell transferred mice.

<p>Flow cytometry-sorted naïve CD4⁺ T-cells of B6 (WT) or β2i/MECL-1^−/−β5i/LMP7^−/−(IS−/−) mice were transferred into RAG1^−/− or RAG1^−/−β2i/MECL-1^−/−β5i/LMP7^−/− (IS−/− x RAG1−/−) mice and colitis development was determined 6 weeks by histological scoring of H&E stained tissue samples (see M&M). (A) overall colitis scores, (B) colitis scores in the proximal −, (C) mid −, and (D) distal colon sections. Scores for individual mice and means+SEM (n = 3–6 per group) are depicted. *p<0.05. Data are representative of two independent experiments.</p

Cytokine expression in mice with T-cell transfer-induced colitis.

<p>Splenocytes of mice with T -cell transfer-induced colitis were stimulated with anti-CD3 mAb for 4 hrs. mRNA was extracted and cytokine expression was quantified by RT-PCR. Expression of (A) IL17a, (B) IFNy, (C) IL10, and (D) TNFα relative to 18S rRNA is shown for individual mice per experimental group. Bars represent means+SEM (n = 3–6). *p<0.05. Data are representative of two independent experiments.</p

Histological scoring of T-cell transfer colitis.

<p>Histological scoring of T-cell transfer colitis.</p

Basophil-Derived Amphiregulin Is Essential for UVB Irradiation-Induced Immune Suppression

UVB irradiation (290-320 nm) is used to treat skin diseases like psoriasis and atopic dermatitis, and is known to suppress contact hypersensitivity (CHS) reactions in mouse models. Regulatory T cells (Treg cells) have been shown to be responsible for this UVB-induced suppression of CHS. The epidermal growth factor (EGF)-like growth factor amphiregulin (AREG) engages EGFR on Treg cells and, in different disease models, it was shown that mast cell-derived AREG is essential for optimal Treg cell function in vivo. Here we determined whether AREG has a role in UVB-induced, Treg cell-mediated suppression of CHS reactions in the skin. Our data show that AREG is essential for UVB-induced CHS suppression. In contrast to the general assumption, however, mast cells were dispensable for UVB-induced immune suppression, whereas basophil-derived AREG was essential. These data reveal, to our knowledge, a previously unreported function for basophils in the homeostasis of immune responses in the skin. Basophils thus fulfill a dual function: they contribute to the initiation of effective type 2 immune responses and, by enhancing the suppressive capacity of local Treg cell populations, also to local immune regulation in the skin.Journal of Investigative Dermatology advance online publication, 11 September 2014; (2014) 0, 000-000. doi:10.1038/jid.2014.329

Increased frequencies of allergen-specific Th1 cells in healthy compared to IBH-affected ponies.

<p>Allergen-specific CD4 T-cells were <i>in vitro</i> expanded in summer from PBMC of 11 healthy and 8 IBH-affected animals, by stimulation with WBE. Cytokine expression of expanded CD4 T-cells was then determined by exposure to monocyte-derived DCs in the presence or absence of allergen. <b>A</b>) representative FACS blot; <b>B</b>) IFNγ- and <b>C</b>) IL4 CD4 T-cells; <b>D</b>) ratio IFNγ/ IL4 CD4 T-cells. Bars represent average +SEM; dots represent individual animals.</p