FASCIA Method in the Assessment of Lymphocyte Mitogen Responses in the Laboratory Diagnostics of Primary Immunodeficiencies.

Lymphocyte responses to mitogens constitute a key part of the diagnostics of combined immunodeficiency (CID). Currently, mostly radioactive thymidine incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution methods are used. Flow-cytometric assay for specific cell-mediated immune-response in activated whole blood (FASCIA) has been put forth as an easy-to-perform option for the measurement of lymphocyte responses with the advantage of recognizing different lymphocyte subtypes and avoiding the use of radioactive reagents. Our aim was to analyze retrospectively the usefulness of FASCIA in the diagnostics of CID. We included all lymphocyte stimulation tests done with FASCIA in HUSLAB (Helsinki, Finland) between February 2015 and September 2018 in our analysis. The cohort was divided into two groups according to the patients' final diagnoses: CID (n = 30) or non-CID (n = 159). We evaluated the stimulation responses with a combined FASCIA score (the average of all mitogen responses). The FASCIA score was significantly lower among the CID group compared to the other patients (p = 0.002), and in the ROC analysis, the AUC was 0.75 (p < 0.001) for the FASCIA score. When the three mitogens were analyzed separately, phytohemagglutinin (PHA) was best in separating patients with CID from non-CID (in the ROC analysis AUC 0.71, p = 0.001). Immunosuppressive medication affected the FASCIA result significantly and needs to be considered when evaluating the results. In conclusion, FASCIA can reliably detect the CID patients in the absence of immunosuppressive medication. It emerges as a method with many benefits compared to tests requiring radioactive reagents or the complicated CFSE staining.

Immune-microbiota interaction in Finnish and Russian Karelia young people with high and low allergy prevalence.

BACKGROUND: After the Second World War, the population living in the Karelian region was strictly divided by the "iron curtain" between Finland and Russia. This resulted in different lifestyle, standard of living, and exposure to the environment. Allergic manifestations and sensitization to common allergens have been much more common on the Finnish compared to the Russian side. OBJECTIVE: The remarkable allergy disparity in the Finnish and Russian Karelia calls for immunological explanations. METHODS: Young people, aged 15-20 years, in the Finnish (n = 69) and Russian (n = 75) Karelia were studied. The impact of genetic variation on the phenotype was studied by a genome-wide association analysis. Differences in gene expression (transcriptome) were explored from the blood mononuclear cells (PBMC) and related to skin and nasal epithelium microbiota and sensitization. RESULTS: The genotype differences between the Finnish and Russian populations did not explain the allergy gap. The network of gene expression and skin and nasal microbiota was richer and more diverse in the Russian subjects. When the function of 261 differentially expressed genes was explored, innate immunity pathways were suppressed among Russians compared to Finns. Differences in the gene expression paralleled the microbiota disparity. High Acinetobacter abundance in Russians correlated with suppression of innate immune response. High-total IgE was associated with enhanced anti-viral response in the Finnish but not in the Russian subjects. CONCLUSIONS AND CLINICAL RELEVANCE: Young populations living in the Finnish and Russian Karelia show marked differences in genome-wide gene expression and host contrasting skin and nasal epithelium microbiota. The rich gene-microbe network in Russians seems to result in a better-balanced innate immunity and associates with low allergy prevalence.

Acinetobacter species in the skin microbiota protect against allergic sensitization and inflammation.

BACKGROUND: The human commensal microbiota interacts in a complex manner with the immune system, and the outcome of these interactions might depend on the immune status of the subject. OBJECTIVE: Previous studies have suggested a strong allergy-protective effect for Gammaproteobacteria. Here we analyze the skin microbiota, allergic sensitization (atopy), and immune function in a cohort of adolescents, as well as the influence of Acinetobacter species on immune responses in vitro and in vivo. METHODS: The skin microbiota of the study subjects was identified by using 16S rRNA sequencing. PBMCs were analyzed for baseline and allergen-stimulated mRNA expression. In in vitro assays human monocyte-derived dendritic cells and primary keratinocytes were incubated with Acinetobacter lwoffii. Finally, in in vivo experiments mice were injected intradermally with A lwoffii during the sensitization phase of the asthma protocol, followed by readout of inflammatory parameters. RESULTS: In healthy subjects, but not in atopic ones, the relative abundance of Acinetobacter species was associated with the expression of anti-inflammatory molecules by PBMCs. Moreover, healthy subjects exhibited a robust balance between anti-inflammatory and TH1/TH2 gene expression, which was related to the composition of the skin microbiota. In cell assays and in a mouse model, Acinetobacter species induced strong TH1 and anti-inflammatory responses by immune cells and skin cells and protected against allergic sensitization and lung inflammation through the skin. CONCLUSION: These results support the hypothesis that skin commensals play an important role in tuning the balance of TH1, TH2, and anti-inflammatory responses to environmental allergens.

Smad3 regulates dermal cytokine and chemokine expression and specific antibody production in murine responses to a respiratory chemical sensitizer.

BACKGROUND: The cytokine transforming growth factor-beta(TGF-beta) has important regulatory roles in the immune system. To investigate the role of intact TGF-beta signaling during the contact hypersensitivity (CHS) response to a respiratory allergen, we exposed Smad3-/- mice to topical trimellitic anhydride (TMA). METHODS: CHS was induced by topical application of TMA. The swelling of the TMA-exposed ears was analyzed, and lymph node, ear tissue and skin biopsies were collected for RNA isolation, histology and histochemical analyses. Lymph node cell proliferation was measured and blood samples were collected for analysis of TMA-specific immunoglobulin. RESULTS: Topical TMA exposure resulted in increased mRNA expression of proinflammatory and suppressive cytokines (IL-1beta, TNF-alpha, IL-6, IFN-gamma, IL-4, IL-10, IL-17, IL-23, TGF-beta), chemokines (CXCL9, CXCL10, CCL24) and chemokine receptors (CCR7, CCR8, CXCR2), increased numbers of CD3+ T cells in ear tissue, and lymphadenopathy in the Smad3-/- mice. The IL-10 result was confirmed at the protein level by immunohistochemistry. However, the ear-swelling response and infiltration of eosinophils, F4/80+ cells, CD11c+ cells and mast cells were similar in the Smad3-/- mice compared to their wild-type (WT) siblings. While TMA-specific IgE was induced equally in the WT and Smad3-/- mice, the concentration of TMA-specific IgG2a was significantly lower in the Smad3-/- mice. CONCLUSIONS: The Smad3 molecule contributes significantly to the regulation of the cytokine and chemokine network during the CHS response to TMA. The lack of Smad3 resulted in a potent Th2 shift, confirmed by strongly impaired IgG2a levels.

Transcriptional Repressor HIC1 Contributes to Suppressive Function of Human Induced Regulatory T Cells.

Regulatory T (Treg) cells are critical in regulating the immune response. In vitro induced Treg (iTreg) cells have significant potential in clinical medicine. However, applying iTreg cells as therapeutics is complicated by the poor stability of human iTreg cells and their variable suppressive activity. Therefore, it is important to understand the molecular mechanisms of human iTreg cell specification. We identified hypermethylated in cancer 1 (HIC1) as a transcription factor upregulated early during the differentiation of human iTreg cells. Although FOXP3 expression was unaffected, HIC1 deficiency led to a considerable loss of suppression by iTreg cells with a concomitant increase in the expression of effector T cell associated genes. SNPs linked to several immune-mediated disorders were enriched around HIC1 binding sites, and in vitro binding assays indicated that these SNPs may alter the binding of HIC1. Our results suggest that HIC1 is an important contributor to iTreg cell development and function.

Complex 2B4 regulation of mast cells and eosinophils in murine allergic inflammation.

The cell surface molecule 2B4 (CD244) is an important regulator of lymphocyte activation, and its role in antiviral immunity and lymphoproliferative disorders is well established. Although it is also expressed on mast cells (MCs) and eosinophils (Eos), the functions of 2B4 on these allergy-orchestrating cells remain unclear. We therefore investigated the role of 2B4 on murine MCs and Eos, particularly how this molecule affects allergic and nonallergic inflammatory processes involving these effector cells. Experiments in bone marrow-derived cultures revealed an inhibitory effect for 2B4 in MC degranulation, but also an opposing stimulatory effect in eosinophil migration and delayed activation. Murine disease models supported the dual 2B4 function: In 2B4-/- mice with nonallergic peritonitis and mild atopic dermatitis (AD), modest infiltrates of Eos into the peritoneum and skin (respectively) confirmed that 2B4 boosts eosinophil trafficking. In a chronic AD model, 2B4-/- mice showed overdegranulated MCs, confirming the inhibiting 2B4 effect on MC activation. This multifunctional 2B4 profile unfolded in inflammation resembles a similar mixed effect of 2B4 in natural killer cells. Taken together, our findings provide evidence for physiological 2B4 stimulatory/inhibitory effects in MCs and Eos, pointing to a complex role for 2B4 in allergy.

IL-17/Th17 pathway is activated in acne lesions.

The mechanisms of inflammation in acne are currently subject of intense investigation. This study focused on the activation of adaptive and innate immunity in clinically early visible inflamed acne lesions and was performed in two independent patient populations. Biopsies were collected from lesional and non-lesional skin of acne patients. Using Affymetrix Genechips, we observed significant elevation of the signature cytokines of the Th17 lineage in acne lesions compared to non-lesional skin. The increased expression of IL-17 was confirmed at the RNA and also protein level with real-time PCR (RT-PCR) and Luminex technology. Cytokines involved in Th17 lineage differentiation (IL-1ß, IL-6, TGF-ß, IL23p19) were remarkably induced at the RNA level. In addition, proinflammatory cytokines and chemokines (TNF-α, IL-8, CSF2 and CCL20), Th1 markers (IL12p40, CXCR3, T-bet, IFN-γ), T regulatory cell markers (Foxp3, IL-10, TGF-ß) and IL-17 related antimicrobial peptides (S100A7, S100A9, lipocalin, hBD2, hBD3, hCAP18) were induced. Importantly, immunohistochemistry revealed significantly increased numbers of IL-17A positive T cells and CD83 dendritic cells in the acne lesions. In summary our results demonstrate the presence of IL-17A positive T cells and the activation of Th17-related cytokines in acne lesions, indicating that the Th17 pathway is activated and may play a pivotal role in the disease process, possibly offering new targets of therapy.

Regulatory T Cells Control Immune Responses through Their Non-Redundant Tissue Specific Features.

Regulatory T cells (Treg) are needed in the control of immune responses and to maintain immune homeostasis. Of this subtype of regulatory lymphocytes, the most potent are Foxp3 expressing CD4+ T cells, which can be roughly divided into two main groups; natural Treg cells (nTreg), developing in the thymus, and induced or adaptive Treg cells (iTreg), developing in the periphery from naïve, conventional T cells. Both nTreg cells and iTreg cells have their own, non-redundant roles in the immune system, with nTreg cells mainly maintaining tolerance toward self-structures, and iTreg developing in response to externally delivered antigens or commensal microbes. In addition, Treg cells acquire tissue specific features and are adapted to function in the tissue they reside. This review will focus on some specific features of Treg cells in different compartments of the body.

Toll-like receptor activation during cutaneous allergen sensitization blocks development of asthma through IFN-gamma-dependent mechanisms.

Toll-like receptors (TLRs) are pattern-recognition receptors that have a pivotal role as primary sensors of microbial products and as initiators of innate and adaptive immune responses. We investigated the role of TLR2, TLR3, and TLR4 activation during cutaneous allergen sensitization in the modulation of allergic asthma. The results show that dermal exposure to TLR4 ligand lipopolysaccharide (LPS) or TLR2 ligand Pam3Cys suppresses asthmatic responses by reducing airway hyperreactivity, mucus production, Th2-type inflammation in the lungs, and IgE antibodies in serum in a dose-dependent manner. In contrast, TLR3 ligand Poly(I:C) did not protect the mice from asthmatic symptoms but reduced IgE and induced IgG2a in serum. LPS (especially) and Pam3Cys enhanced the activation of dermal dendritic cell (DCs) by increasing the expression of CD80 and CD86 but decreased DC numbers in draining lymph nodes at early time points. Later, these changes in DCs led to an increased number of CD8(+) T cells and enhanced the production of IFN-γ in bronchoalveolar lavage fluid. In conclusion, dermal exposure to LPS during sensitization modulates the asthmatic response by skewing the Th1/Th2 balance toward Th1 by stimulating the production of IFN-γ. These findings support the hygiene hypothesis and pinpoint the importance of dermal microbiome in the development of allergy and asthma.

ST2 regulates allergic airway inflammation and T-cell polarization in epicutaneously sensitized mice.

IL-33 is an inducer of proinflammatory and T-helper type 2 (Th2) cytokines, which have an important role in atopic dermatitis (AD) and allergic asthma. ST2 is a specific receptor for IL-33 and is expressed on Th2 cells, eosinophils and mast cells. A murine model of AD was used to characterize the role of ST2 in allergen-induced skin inflammation and allergic asthma. ST2-/- and wild-type (WT) mice were epicutaneously sensitized with ovalbumin (OVA) and staphylococcal enterotoxin B, and intranasally challenged with OVA. ST2-/- mice exhibited increased production of IFNγ and increased number of CD8(+) T cells in the sensitized skin and in the airways compared with WT mice. The number of eosinophils was decreased, and Th2 cytokines were downregulated in the airways of epicutaneously sensitized ST2-/- mice compared with WT controls. However, dermal eosinophil numbers were as in WT, and the levels of Th2 cytokines were even elevated in the sensitized skin of ST2-/- mice. ST2-/- mice had elevated numbers of neutrophils and macrophages and increased levels of proinflammatory cytokines in the sensitized skin. The role of ST2 differs between different target tissues: ST2 is dispensable for the development of Th2 response in the sensitized skin, whereas it is a main inducer of Th2 cytokines in asthmatic airways.

Foxp3+ cells control Th2 responses in a murine model of atopic dermatitis.

The role of Foxp3+ regulatory T (Treg) cells in atopic dermatitis (AD) is still unclear. In a murine AD model, the number of Foxp3+ cells increased in the allergen-exposed skin area and in the secondary lymphoid organs. Both Foxp3+ and Foxp3- IL-10+ T cells accumulated at the site of allergen exposure, and CD103+ effector/memory Foxp3+ Treg cells expanded gradually in the lymph nodes throughout the sensitization protocol. The depletion of Foxp3+ Treg cells led to significantly exacerbated skin inflammation, including increased recruitment of inflammatory cells and expression of T helper type 2 cytokines, as well as elevated serum IgE levels. The effect of depleting Treg cells during epicutaneous sensitization was mirrored off by a stronger inflammatory response also in the lungs following airway challenge. Thus, Treg cells have an important role in controlling AD-like inflammation and the transfer of allergic skin inflammation to the lungs.

The temporal and spatial dynamics of Foxp3+ Treg cell-mediated suppression during contact hypersensitivity responses in a murine model.

Regulatory T (Treg) cells suppress contact hypersensitivity (CHS) responses, but the dynamics, mode, and site of their action is not well characterized. We studied forkhead box P3+ (Foxp3+) Treg cells during the CHS response in conditional Foxp3 knockout depletion of regulatory T cell (DEREG) mice, where Foxp3+ cells can be transiently deleted by diphtheria toxin. The mice were sensitized and challenged with oxazolone, and Foxp3+ cells were depleted either during sensitization or elicitation. Treg cell depletion before sensitization led to significantly exacerbated and prolonged CHS responses. In contrast, depleting Treg cells during elicitation had no effect on the 24-hour response, but the response was significantly prolonged. In wild-type mice, the gradual resolution of the CHS response was accompanied by a similarly gradual accumulation of Foxp3+ Treg cells relative to T effector cells in the skin. This effect was not as marked in the Treg cell-depleted mice, suggesting that the skin is an important site of Treg cell activities during the resolution phase. Together, our results show that endogenous Foxp3+ Treg cell function is important during the sensitization and resolution phases, but their depletion just before elicitation does not have an effect on the CHS response during the first 24 hours after elicitation.

IL-33 and ST2 in atopic dermatitis: expression profiles and modulation by triggering factors.

In the acute phase of atopic dermatitis (AD), T-helper type 2 (Th2) cytokines characterize the inflammatory response in the skin. IL-33 is a new tissue-derived cytokine, which is mainly expressed by cells of barrier tissues, and is known to activate Th2 lymphocytes, mast cells, and eosinophils. IL-33 signals through a receptor complex consisting of IL-33-specific receptor ST2 and a co-receptor IL-1RAcP. As IL-33 is known to promote Th2-type immunity, we examined expression profiles of IL-33 and its receptor components in human AD skin, in the murine model of AD, and in various cell models. We found increased expression of IL-33 and ST2 in AD skin after allergen or staphylococcal enterotoxin B (SEB) exposure, as well as in the skin of 22-week-old filaggrin-deficient mice. In addition, skin fibroblasts, HaCaT keratinocytes, primary macrophages, and HUVEC endothelial cells efficiently produced IL-33 in response to the combined stimulation of tumor necrosis factor-α and IFN-γ, which was further enhanced by a mimetic of double-stranded RNA. Finally, the increased expression of IL-33 and ST2 caused by irritant, allergen, or SEB challenge was suppressed by topical tacrolimus treatment. These results suggest an important role for IL-33-ST2 interaction in AD and highlight the fact that bacterial and viral infections may increase the production of IL-33.

Absence of CCR4 exacerbates skin inflammation in an oxazolone-induced contact hypersensitivity model.

Chemokine receptor CCR4 is expressed by Th2 cells and is involved in the recruitment of inflammatory cells into the skin. We studied the effects of CCR4 deficiency in the murine model of oxazolone-induced contact hypersensitivity in CCR4-/- and wild-type (WT) mice. The inflammatory response in the skin at 24 hours post-elicitation was stronger in CCR4-/- mice compared with WT, evidenced by increased ear swelling and inflammatory cell infiltration. In addition, the mRNA expression levels of several cytokines, chemokines, chemokine receptors, and selectins in the skin of CCR4-/- mice were significantly elevated compared with WT mice. Time kinetic experiments during the sensitization and elicitation phases revealed that the number of CD3+CD4+ cells in CCR4-/- mice remained high longer during the sensitization phase and increased more rapidly during the elicitation phase compared with WT mice. These data demonstrate that the absence of CCR4 results in enhanced secondary immune response during allergic skin inflammation.

Topical superantigen exposure induces epidermal accumulation of CD8+ T cells, a mixed Th1/Th2-type dermatitis and vigorous production of IgE antibodies in the murine model of atopic dermatitis.

Patients with atopic dermatitis (AD) have repeated cutaneous exposure to both environmental allergens and superantigen-producing strains of Staphylococcus aureus. We used a murine model of AD to investigate the role of staphylococcal enterotoxin B (SEB) in the modulation of allergen-induced skin inflammation. Mice were topically exposed to SEB, OVA, a combination of OVA and SEB (OVA/SEB), or PBS. Topical SEB and OVA/SEB exposure induced epidermal accumulation of CD8+ T cells and TCRVbeta8+ cells in contrast to OVA application, which induced a mainly dermal infiltration of CD4+ cells. SEB and OVA/SEB exposure elicited a mixed Th1/Th2-associated cytokine and chemokine expression profile within the skin. Restimulation of lymph node cells from OVA- and OVA/SEB-exposed mice with OVA elicited strong production of IL-13 protein, whereas substantial amounts of IFN-gamma protein were detected after SEB stimulation of cells derived from SEB- or OVA/SEB-exposed mice. Topical SEB treatment elicited vigorous production of SEB-specific IgE and IgG2a Abs and significantly increased the production of OVA-specific IgE and IgG2a Abs. The present study shows that topical exposure to SEB provokes epidermal accumulation of CD8+ T cells, a mixed Th2/Th1 type dermatitis and vigorous production of specific IgE and IgG2a Abs, which can be related to the chronic phase of atopic skin inflammation.

CCL1-CCR8 interactions: an axis mediating the recruitment of T cells and Langerhans-type dendritic cells to sites of atopic skin inflammation.

Atopic dermatitis represents a chronically relapsing skin disease with a steadily increasing prevalence of 10-20% in children. Skin-infiltrating T cells, dendritic cells (DC), and mast cells are thought to play a crucial role in its pathogenesis. We report that the expression of the CC chemokine CCL1 (I-309) is significantly and selectively up-regulated in atopic dermatitis in comparison to psoriasis, cutaneous lupus erythematosus, or normal skin. CCL1 serum levels of atopic dermatitis patients are significantly higher than levels in healthy individuals. DC, mast cells, and dermal endothelial cells are abundant sources of CCL1 during atopic skin inflammation and allergen challenge, and Staphylococcus aureus-derived products induce its production. In vitro, binding and cross-linking of IgE on mast cells resulted in a significant up-regulation of this inflammatory chemokine. Its specific receptor, CCR8, is expressed on a small subset of circulating T cells and is abundantly expressed on interstitial DC, Langerhans cells generated in vitro, and their monocytic precursors. Although DC maintain their CCR8+ status during maturation, brief activation of circulating T cells recruits CCR8 from intracytoplamic stores to the cell surface. Moreover, the inflammatory and atopy-associated chemokine CCL1 synergizes with the homeostatic chemokine CXCL12 (SDF-1alpha) resulting in the recruitment of T cell and Langerhans cell-like DC. Taken together, these findings suggest that the axis CCL1-CCR8 links adaptive and innate immune functions that play a role in the initiation and amplification of atopic skin inflammation.