Dashlkhumbe Byamba

Corticotropin-releasing hormone downregulates IL-10 production by adaptive forkhead box protein 3–negative regulatory T cells in patients with atopic dermatitis

BACKGROUND Corticotropin-releasing hormone (CRH) is the central regulating hormone of the hypothalamic-pituitary-adrenal axis. CRH also has diverse functional effects in the periphery and is related to the aggravation of several cutaneous diseases; however, the effect of CRH on T cells in patients with atopic dermatitis (AD) has not been well evaluated. OBJECTIVE We investigated whether CRH directly affects peripheral T(H)1, T(H)2, and regulatory T (Treg) cells in patients with AD. METHODS We assessed whether T cells express the CRH receptor protein and mRNA by using flow cytometry, Western blotting, immunofluorescence, immunohistochemistry, and RT-PCR. We evaluated cytokine expression using ELISA after treating the T cells extracted from patients with AD and healthy control subjects (HCs) with CRH. Flow cytometry was then used to evaluate any direct effects of CRH on T(H)1, T(H)2, and Treg cells from patients with AD and HCs. RESULTS T cells from patients with AD expressed significantly lower CRH receptor 1/2 mRNA levels than T cells from HCs. T cells from HCs reacted with different IL-4 and IFN-γ secretions to CRH treatment, whereas T cells from patients with AD did not. IL-10 production was significantly decreased in the supernatants from both the HCs and patients with AD after CRH treatment. CRH upregulated IL-4 production by T(H)2 cells and downregulated IFN-γ production by T(H)1 cells in HCs. CRH also suppressed the production of IL-10 by forkhead box protein 3-negative Treg cells in both groups, but the difference was only significant in patients with AD. CONCLUSIONS CRH-mediated suppression of IL-10 secretion from Treg cells might explain stress-related exacerbations in patients with AD.

Different characteristics of reactive oxygen species production by human keratinocyte cell line cells in response to allergens and irritants

Abstract:  Keratinocytes mount immune responses through the secretion of a variety of inflammatory cytokines, soluble proteins and reactive oxygen species (ROS). However, the role of ROS in keratinocytes in response to allergens and irritants has not yet been elucidated. In this study, we investigated the (i) ROS production; (ii) potential sites of ROS production; (iii) expression of cell surface molecules; (iv) secretion of cytokines; and (v) ROS‐dependent protein carbonylation in chemical‐treated human keratinocyte cell line (HaCaT) cells. Treatment of HaCaT cells with 2,4‐dinitrochlorobenzene (DNCB) and benzalkonium chloride (BKC) increased ROS levels in a time‐ and dose‐dependent manner, as determined with dichlorodihydrofluorescein diacetate (CM‐H2DCFDA), without reducing cell viability. Potential sources of ROS production were evaluated with pretreatment of diphenylene iodonium (DPI), an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase; rotenone, an inhibitor of the mitochondrial electron transport chain complex or allopurinol, a xanthine oxidase inhibitor. The DNCB‐induced ROS was related to both NADPH oxidase and mitochondrial electron transport chain complex. Conversely, BKC‐induced ROS was related to NADPH oxidase only. Western blotting using an anti‐DNP antibody revealed ROS‐dependent protein carbonylation in response to DNCB but not BKC. Both DNCB and BKC increased the secretion of IL‐1α from HaCaT cells; however, ROS production as well as other changes, except DNCB‐induced secretion of IL‐1α, was not inhibited by antioxidants. Although the role of ROS in keratinocytes in response to chemicals was inconclusive, our results suggest that the characteristics of ROS produced by keratinocytes in response to chemicals might differ.

Skin-penetrating methotrexate alleviates imiquimod-induced psoriasiform dermatitis via decreasing IL-17-producing gamma delta T cells.

Accumulating evidence has shown that the Toll‐like receptor 7 agonist imiquimod (IMQ) induces psoriasiform skin inflammation in mice and that this inflammation is dependent on the IL‐23/IL‐17 axis. Moreover, it has been demonstrated that the main source of IL‐17 is not Th17 but is dermal gamma delta (γδ) T cells in mouse psoriasiform skin. Recent advances in the understanding of immunopathogenesis of psoriasis led to an alteration in the treatment paradigm to the use of highly efficacious biologics. However, their high cost impedes the extensive use of these agents. Thus, inexpensive and safe medications are still considered valuable. In this study, we introduce the therapeutic efficacy of a newly formulated methotrexate (MTX), a chemical conjugate of MTX with cell permeable peptide, for the treatment of psoriasis. Topically applied skin‐penetrating (SP)‐MTX reduced the psoriasiform skin phenomenon, epidermal thickness and infiltrating immune cells into the dermis. IL‐17A‐producing dermal γδ T cells in the cellular infiltrate that contribute IL‐23/IL‐17 axis were well abrogated by SP‐MTX. Furthermore, SP‐MTX had no toxic effects on liver, kidney or myeloid cells, unlike systemic administration of MTX. In conclusion, topically applied SP‐MTX ameliorated psoriasiform skin inflammation in mice with the criteria of clinical phenomenon, histopathology and immunology, without inducing systemic toxic effects.

Statins inhibit chemotactic interaction between CCL20 and CCR6 in vitro: possible relevance to psoriasis treatment

Abstract:  Psoriasis is a chronic IL‐23/Th17 pathway‐associated skin disease. An increased expression of lesional CCL20 can recruit CCR6+ Th17, and lesional cytokine milieu persistently activates keratinocytes to produce CCL20. Lipid‐lowering drugs, statins, are known to possess immune‐modulating functions. In this study, we explored an inhibitory effect of statins on CCL20/CCR6 interaction. We demonstrated that IL‐1β, TNF‐α, and IL‐17A significantly increased CCL20 production from HaCaT cells. However, these increments were markedly inhibited by fluvastatin and simvastatin, but not by pravastatin. In the chemotaxis migration assay, pretreatment with fluvastatin and simvastatin inhibited the migration of human CD4+ T cells towards CCL20. However, the level of CCR6 surface expression in memory CD4+ T cells was not affected. Our results suggest that not all, but specific types of statins may be of benefit in alleviating psoriasis partially via interrupting CCL20/CCR6 chemotactic interaction, the mechanism which may eventually lessen the infiltration of Th17 cells.

Immunotherapy of Malignant Melanoma with Tumor Lysate-Pulsed Autologous Monocyte-Derived Dendritic Cells

Purpose Dendritic cell (DC) vaccination for melanoma was introduced because melanoma carries distinct tumor-associated antigens. The purpose of this study was to investigate the efficacy and safety of DC vaccination for melanoma in Korea. Materials and Methods Five patients with stage IV and one with stage II were enrolled. Autologous monocyte-derived DCs (MoDCs) were cultured and pulsed with tumor-lysate, keyhole limpet hemocyanin, and cytokine cocktail for mature antigen-loaded DC. DC vaccination was repeated four times at 2-week intervals and 2-4×107 DC were injected each time. Results Reduced tumor volume was observed by PET-CT in three patients after DC vaccination. Delayed type hypersensitivity responses against tumor antigen were induced in five patients. Tumor antigen-specific IFN-γ-producing peripheral blood mononuclear cells were detected with enzyme-linked immunosorbent spot in two patients. However, the overall clinical outcome showed disease progression in all patients. Conclusion In this study, DC vaccination using tumor antigen-loaded, mature MoDCs led to tumor regression in individual melanoma patients. Further standardization of DC vaccination protocol is required to determine which parameters lead to better anti-tumor responses and clinical outcomes.

The Antioxidative Effect of Heat‐Shock Protein 70 in Dendritic Cells

Reactive oxygen species (ROS) are produced by dendritic cells (DCs) during antigen presentation in contact hypersensitivity (CHS). ROS cause a number of non‐enzymatic protein modifications, such as carbonylation. Carbonylated proteins in DCs in response to hapten have not been fully identified yet. To identify the proteins carbonylated by ROS, murine epidermis‐derived DC line XS106 was challenged with a hapten, 2,4,6‐trinitrobenzene sulphonic acid (TNBS). MALDI‐TOF analysis revealed that heat‐shock protein 70 (HSP70) was one of the carbonylated proteins induced by TNBS. To verify the role of HSP70 in TNBS‐treated XS106 cell, we fused protein transduction domain (PTD) with HSP70 to facilitate protein delivery into the cell. The transfected fusion protein HSP70 within the cell caused transient increase of the cellular level of HSP70. Transient increase of HSP70 level in XS‐106 DCs resulted in inhibition of ROS production, carbonylation of HSP70, p38 MAPK activation and subsequently IL‐12 secretion. To investigate the effects of PTD–HSP70 in vivo, ear‐swelling experiments with 2,4,6‐trinitro‐1‐chlorobenzene (TNCB) were performed in BALB/c mice. Pretreatment of PTD–HSP70 reduced the CHS response to TNCB in vivo. We report here that carbonylation of HSP70 by ROS is associated with the pathogenesis of CHS, suggesting possibility of HSP70‐targeting therapy in CHS.