Keratinocyte-derived small extracellular vesicles supply antigens for CD1a-resticted T cells and promote their type 2 bias in the context of filaggrin insufficiency

IntroductionExosome-enriched small extracellular vesicles (sEVs) are nanosized organelles known to participate in long distance communication between cells, including in the skin. Atopic dermatitis (AD) is a chronic inflammatory skin disease for which filaggrin (FLG) gene mutations are the strongest genetic risk factor. Filaggrin insufficiency affects multiple cellular function, but it is unclear if sEV-mediated cellular communication originating from the affected keratinocytes is also altered, and if this influences peptide and lipid antigen presentation to T cells in the skin.MethodsAvailable mRNA and protein expression datasets from filaggrin-insufficient keratinocytes (shFLG), organotypic models and AD skin were used for gene ontology analysis with FunRich tool. sEVs secreted by shFLG and control shC cells were isolated from conditioned media by differential centrifugation. Mass spectrometry was carried out for lipidomic and proteomic profiling of the cells and sEVs. T cell responses to protein, peptide, CD1a lipid antigens, as well as phospholipase A2-digested or intact sEVs were measured by ELISpot and ELISA.ResultsData analysis revealed extensive remodeling of the sEV compartment in filaggrin insufficient keratinocytes, 3D models and the AD skin. Lipidomic profiles of shFLGsEV showed a reduction in the long chain (LCFAs) and polyunsaturated fatty acids (PUFAs; permissive CD1a ligands) and increased content of the bulky headgroup sphingolipids (non-permissive ligands). This resulted in a reduction of CD1a-mediated interferon-γ T cell responses to the lipids liberated from shFLG-generated sEVs in comparison to those induced by sEVs from control cells, and an increase in interleukin 13 secretion. The altered sEV lipidome reflected a generalized alteration in the cellular lipidome in filaggrin-insufficient cells and the skin of AD patients, resulting from a downregulation of key enzymes implicated in fatty acid elongation and desaturation, i.e., enzymes of the ACSL, ELOVL and FADS family.DiscussionWe determined that sEVs constitute a source of antigens suitable for CD1a-mediated presentation to T cells. Lipids enclosed within the sEVs secreted on the background of filaggrin insufficiency contribute to allergic inflammation by reducing type 1 responses and inducing a type 2 bias from CD1a-restricted T cells, thus likely perpetuating allergic inflammation in the skin

Increased μ-Calpain Activity in Blasts of Common B-Precursor Childhood Acute Lymphoblastic Leukemia Correlates with Their Lower Susceptibility to Apoptosis.

Childhood acute lymphoblastic leukemia (ALL) blasts are characterized by inhibited apoptosis promoting fast disease progress. It is known that in chronic lymphocytic and acute myeloid leukemias the reduced apoptosis is strongly related with the activity of calpain-calpastatin system (CCS) composed of cytoplasmic proteases--calpains--performing the modulatory proteolysis of key proteins involved in cell proliferation and apoptosis, and of their endogenous inhibitor--calpastatin. Here, the CCS protein abundance and activity was for the first time studied in childhood ALL blasts and in control bone marrow CD19+ B cells by semi-quantitative flow cytometry and western blotting of calpastatin fragments resulting from endogenous calpain activity. Significantly higher μ-calpain (CAPN1) gene transcription, protein amounts and activity (but not those of m-calpain), with calpastatin amount and transcription of its gene (CAST) greatly varying were observed in CD19(+) ALL blasts compared to control cells. Significant inverse relation between the amount/activity of calpain and spontaneous apoptosis was noted. Patients older than 10 years (considered at higher risk) displayed increased amounts and activities of blast calpain. Finally, treatment of blasts with the tripeptide calpain inhibitors II and IV significantly and in dose-dependent fashion increased the percentage of blasts entering apoptosis. Together, these findings make the CCS a potential new predictive tool and therapeutic target in childhood ALL

Endogenous calpain activity is present in ALL blasts.

<p><b>A</b>. Representative result of western blot determinantion of calpastatin and its immunoreactive fragments resulting from calpain activity in non-malignant BM CD19⁺ cells (lane 1), ALL blasts from a 12-year old patient (lane 2) and blasts from the same patient as in lane 2, but incubated in vitro for 24 hours with 4 μM calpain inhibitor IV (lane 3). Actin was used as a reference protein. For further details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136615#sec002" target="_blank">Materials and Methods</a>. B. Endogenous calpain activity in ALL blasts measured by degree of calpastatin degradation (loss of the native form) is significantly higher in the children more than 10 years old. Box-and-whisker plots depict the medians, 25^th and 75^th percentile and range respectively. Asterisk signifies p = 0.01; N (1–10 years old ALL patients) = 27, N(>10 years old ALL patients) = 10.</p

The amounts of calpastatin differ between CD19⁺ ALL blasts and non-malignant B cells.

<p>A. Representative two-parameter plots (dot plots) resulting from simultaneous staining of BM samples (left panel–ALL, right panel–control) with anti-CD19 and anti-calpastatin antibodies. Actual corrected MFI values for calpastatin signal in CD19⁺ cells are shown. B. No significant difference between the proportions of calpastatin-positive cells among ALL blasts and nonmalignant BM B cells. C. Significantly lower calpastatin amount (MFI) in the blasts (C). Box-and-whisker plots depict the medians, 25^th and 75^th percentile and range respectively. N(ALL) = 30, N(control) = 17. For the details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136615#sec002" target="_blank">Materials and Methods</a>.</p

Proportions of μ-calpain-positive cells and relative amounts of μ-calpain are elevated among ALL blasts.

<p>A. Representative two-parameter plots (dot plots) resulting from simultaneous staining of BM samples (left panel–ALL, right panel–control) with anti-CD19, anti-CD34 and anti-μ-calpain antibodies. Actual corrected MFI values for calpain signal in CD19⁺ cells are shown. B. Significant difference between the proportion of μ-calpain-positive cells among ALL blasts and nonmalignant BM B cells. Box-and-whisker plots depict the medians, 25^th and 75^th percentile and range respectively. Asterisk signifies p = 0.02; N(ALL) = 20, N(control) = 9. C. Amount of μ-calpain is significantly higher in ALL blasts than in nonmalignant B lymphocytes. Comparison of relative intensities (MFI) of μ-calpain–bound antibody in CD19⁺ ALL blasts (ALL) and non-malignant B cells (control). Asterisk denotes p = 0.03; N(ALL) = 16, N(control) = 9). For the details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136615#sec002" target="_blank">Materials and Methods</a>.</p

Levels of spontaneous apoptosis of ALL blasts depend on patient age and μ–calpain amount.

<p>Apoptosis was determined as the proportion of AnnexinV+ blasts and plotted against the amount of μ–calpain in the blasts (A, N = 17, r = -0.54, p< 0.05) and against patients’ age (B, N = 39, r = -0.31, p< 0.05). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136615#sec002" target="_blank">Materials and Methods</a> for details. When the patients were subdivided into below and above 10 years of age subgroups, the latter were characterized by significantly lower apoptosis (C, asterisk signifies p = 0.01) and significantly higher proportion of the μ–calpain positive blasts (D, asterisk signifies p = 0.04). N (1–10 years old ALL patients) = 29, N(>10 years old ALL patients) = 10.</p

Expression of μ-calpain but not other CCS genes is different in ALL blasts and nonmalignant BM B cells.

<p>A. Significantly higher expression of <i>CAPN1</i> (μ-calpain) gene in ALL blasts compared to control B cells. B,C. No differences between expression of <i>CAPN2</i> (m-calpain) and <i>CAST</i> (calpastatin) genes in ALL blasts vs non-malignant B cells. Please mark huge variability of expression of both the <i>CAPN1</i> and especially <i>CAST</i> genes. CCS gene expression is shown as proportion of the expression of <i>GAPDH</i> housekeeping gene considered 1. Box-and-whisker plots depict the means, SEM and SD respectively. P values (Kruskall-Wallis test) are given in the graphs; N(ALL) = 6, N(control) = 6. For the details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136615#sec002" target="_blank">Materials and Methods</a>.</p

Similar proportions of m-calpain-positive cells and amounts of m-calpain in ALL blasts and control cells.

<p>A. Representative two-parameter plots (dot plots) resulting from simultaneous staining of BM samples (left panel–ALL, right panel–control) with anti-CD19 and anti-m-calpain antibodies. Actual corrected MFI values for calpain signal in CD19⁺ cells are shown. Details in Materials and Methods. B, C. No significant difference between the proportion of m-calpain-positive cells among ALL blasts and nonmalignant BM B cells (B) and between amount (MFI) (C). Box-and-whisker plots depict the medians, 25^th and 75^th percentile and range respectively. N(ALL) = 6, N(control) = 6. For the details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136615#sec002" target="_blank">Materials and Methods</a>.</p

Threats to feminist identity and reactions to gender based discrimination

The aim of this research was to examine conditions that modify feminists’support for women as targets of gender discrimination. In an experimental study we tested a hypothesis that threatened feminist identity will lead to greater differentiation between feminists and conservative women as victims of discrimination and, in turn, a decrease in support for non-feminist victims. The study was conducted among 96 young Polish female professionals and graduate students from Gender Studies programs in Warsaw who self-identified as feminists (Mage=22.23). Participants were presented with a case of workplace gender discrimination. Threat to feminist identity and worldview of the discrimination victim (feminist vs. conservative) were varied between research conditions. Results indicate that identity threat caused feminists to show conditional reactions to discrimination. Under identity threat, feminists perceived the situation as less discriminatory when the target held conservative views on gender relations than when the target was presented as feminist. This effect was not observed under conditions of no threat. Moreover, feminists showed an increase in compassion for the victim when she was portrayed as a feminist compared to when she was portrayed as conservative. Implications for the feminist movement are discussed