Deficiency of BMAL1 promotes ROS generation and enhances IgE-dependent degranulation in mast cells.

BACKGROUND: Bmal1 (Brain and muscle arnt-like, or Arntl) is a bHLH/PAS domain transcription factor central to the transcription/translation feedback loop of the circadian clock. Mast cells are crucial for effector functions in allergic reaction and their activity follows a circadian rhythm. However, the functional roles of Bmal1 in mast cells remain to be determined. PURPOSE: This study aimed to elucidate the specific roles of Bmal1 in IgE-dependent mast cell degranulation. RESULTS: IgE-dependent degranulation was enhanced in bone marrow-derived mast cells (BMMCs) derived from Bmal1-deficient mice (Bmal1-KO mice) compared to that in BMMCs derived from wild-type mice (WT mice) in the absence of 2-Mercaptoethanol (2-ME) in culture. Mast cell-deficient KitW-sh mice reconstituted with Bmal1-KO BMMCs showed more robust passive cutaneous anaphylactic (PCA) reactions, an in vivo model of IgE-dependent mast cell degranulation, than KitW-sh mice reconstituted with WT BMMCs. In the absence of 2-ME in culture, the mRNA expression of the anti-oxidative genes NF-E2-related factor 2 (Nrf2), superoxide dismutase 2 (SOD2), and heme oxygenase-1 (HO-1) was lower and reactive oxygen species (ROS) generation was higher in Bmal1-KO BMMCs than in WT BMMCs at steady state. The IgE-dependent ROS generation and degranulation were enhanced in Bmal1-KO BMMCs compared to WT BMMCs in the absence of 2-ME in culture. The addition of 2-ME into the culture abrogated or weakened the differences in anti-oxidative gene expression, ROS generation, and IgE-dependent degranulation between WT and Bmal1-KO BMMCs. CONCLUSION: The current findings suggest that Bmal1 controls the expression of anti-oxidative genes in mast cells and Bmal1 deficiency enhanced IgE-dependent degranulation associated with promotion of ROS generation. Thus, Bmal1 may function as a key molecule that integrates redox homeostasis and effector functions in mast cells.

Cha o 3, a cypress pollen allergen, does not activate basophils in Japanese cypress pollinosis.

Background: In Japan, pollinosis caused by the Japanese cypress (JCy) Chamaecyparis obtusa is among the very common seasonal allergies. In JCy pollinosis, Cha o 1 is the first major allergen, and Cha o 2 is the second major allergen. Recently, Cha o 3 was identified as a new JCy pollen allergen in JCy pollinosis. However, the relative contribution of Cha o 3 to JCy pollinosis compared with that of Cha o 1 and that of Cha o 2 has not been fully elucidated. Objective: This study aimed to clarify the allergenicity of Cha o 3 compared with that of Cha o 1 and Cha o 2 in JCy pollinosis. Methods: We recruited 27 patients with JCy pollinosis and performed the basophil activation test (BAT) with native (n) Cha o 1, Cha o 2, and Cha o 3 purified from JCy pollen. In addition, we a performed JCy-specific IgE suppression test. Results: In the BAT, 26 of 27 patients (96%) and 18 of 27 patients (67%) showed positive basophil activation in response to n Cha o 1 and n Cha o 2, respectively, as judged by CD203c expression. Little CD203c expression in response to n Cha o 3 was seen. The presence of n Cha o 3 marginally reduced the titer levels of JCy-specific IgE. Conclusion: Cha o 3 showed little ability to activate basophils and suppress JCy-specific IgE titers compared with Cha o 1 or Cha o 2 in patients with JCy pollinosis. Thus, Cha o 3 may not be a major allergen in JCy pollinosis.

Characterization of human epithelial resident memory regulatory T cells.

Human resident memory regulatory T cells (Tregs) exist in the normal, noninflamed skin. Except one, all previous studies analyzed skin Tregs using full-thickness human skin. Considering that thick dermis contains more Tregs than thin epidermis, the current understanding of skin Tregs might be biased toward dermal Tregs. Therefore, we sought to determine the phenotype and function of human epidermal and epithelial Tregs. Human epidermis and epithelium were allowed to float on a medium without adding any exogenous cytokines and stimulations for two days and then emigrants from the explants were analyzed. Foxp3 was selectively expressed in CD4+CD103- T cells in the various human epithelia, as it is highly demethylated. CD4+CD103-Foxp3+ cells suppressed proliferation of other resident memory T cells. The generation and maintenance of epithelial Tregs were independent of hair density and Langerhans cells. Collectively, immune-suppressive CD4+CD103-Foxp3+ Tregs are present in the normal, noninflamed human epidermis and mucosal epithelia.

Revisiting the Experimental Methods for Human Skin T-Cell Analysis.

Tissue-resident memory T cells exist in both the epidermis and the dermis in human skin. To analyze these cells, the skin needs to be incubated with dispase II to separate the two layers, that is, the epidermis and the dermis. The next step varies among researchers; the subsequent enzymatic digestion of the two layers is popular, whereas the spontaneous migration method can also be done. Scraping of these layers to yield skin T cells may reduce antigen modulation. This study aimed to determine each method's limitations. Dispase II incubation itself cleaves T-cell antigens. Therefore, further enzymatic digestion with collagenases strongly cleaves antigens. The scraping method yields skin T cells that are affected by dispase II as it is. However, skin T-cell yield is low. The spontaneous migration method recovers and/or upregulates antigens with T-cell activation and loses ∼20% of T cells in the floating sheets. However, there was no prominent bias regarding CD103 expression between emigrants and the remaining T cells in the sheets. There were 104 and 105 CD3+ T cells per 1 cm2 of the epidermis and upper dermis, respectively. Collectively, each method has strengths and limitations to analyze both the epidermal and dermal T cells.

Tissue Expander-associated T Cells: Relevance to Breast Implant-associated Anaplastic Large-cell Lymphoma.

The generation of breast implant-associated anaplastic large-cell lymphoma (BIA-ALCL) is closely associated with textured implants. The phenotype of BIA-ALCL cells is well examined, but its cell of origin remains unknown. Here we investigate what types of T cells are recruited and differentiated in the surrounding capsules and tissues as a consequence of continuous contact with a textured surface. Methods: Capsule and pericapsule tissues were recovered from patients who had textured or smooth tissue expanders (TEs). These samples were enzymatically digested, and T cells in the samples were analyzed using flow cytometry. Peripheral blood mononuclear cells from the same donors were utilized as a control. Results: Effector memory CD4+ T cells predominantly infiltrated capsules and tissues without apparent differences between textured and smooth TEs. In these effector memory CD4+ T cells, CD4+ resident memory T cells were generated by smooth TEs but not by textured TEs. However, TNFRSF8/CD30 mRNA expression is higher in the CD69- effector memory CD4+ T cells than in the CD69+ ones. Conclusion: Textured and smooth TEs differentially recruit and/or differentiate T cells in situ.