Subunit-Specific Reactivity of Autoantibodies Against Laminin-332 Reveals Direct Inflammatory Mechanisms on Keratinocytes

Laminin-332 pemphigoid is a rare and severe autoimmune blistering disease, caused by IgG autoantibodies targeting laminin-332 in the dermal-epidermal basement zone. Laminin-332 pemphigoid is characterized by variable inflammatory infiltrate and the predominance of non-complement-fixing antibodies. Given these findings, we hypothesized that IgG autoantibodies to laminin-332 directly resulted in keratinocyte expression of inflammatory factors. We performed RNA-seq on primary human keratinocytes treated with IgG from patients with laminin-332 pemphigoid. Genes for numerous cytokines and chemokines were upregulated, including CSF2, CSF3, CXCL1, CXCL5, CXCL3, CXCL8, CXCL10, CXCL1, IL6, IL7, IL15, IL23, IL32, IL37, TGFB2 as well as metalloproteases. Considering the pro-inflammatory and proteolytic effect of autoantibodies from patients with laminin-332 pemphigoid identified in our initial experiment, we next questioned whether the reactivity against specific laminin subunits dictates the inflammatory and proteolytic keratinocyte response. Then, we treated keratinocytes with IgG from a separate cohort of patients with reactivity against individual subunits of laminin-332. We identified upregulation of IL-1α, IL-6, IL-8, CXCL1, MMP9, TSLP, and GM-CSF at the protein level, most notably in keratinocytes treated with IgG from laminin β3-reactive patients. We for the first time demonstrated a pro-inflammatory response, similar to that described in keratinocytes treated with IgG autoantibodies from patients with bullous pemphigoid, providing novel insight into the pathogenesis of laminin-332 pemphigoid and laminin-332 biology.

Download Full-text

HaCaT Cells as a Reliable In Vitro Differentiation Model to Dissect the Inflammatory/Repair Response of Human Keratinocytes

Mediators of Inflammation ◽

10.1155/2017/7435621 ◽

2017 ◽

Vol 2017 ◽

pp. 1-12 ◽

Cited By ~ 43

Author(s):

Irma Colombo ◽

Enrico Sangiovanni ◽

Roberta Maggio ◽

Carlo Mattozzi ◽

Stefania Zava ◽

...

Keyword(s):

Cell Density ◽

Skin Diseases ◽

Inflammatory Responses ◽

Human Keratinocytes ◽

Suitable Model ◽

Hacat Cells ◽

Primary Human Keratinocytes ◽

Proinflammatory Mediators ◽

Cytokines And Chemokines

Cultured primary human keratinocytes are frequently employed for studies of immunological and inflammatory responses; however, interpretation of experimental data may be complicated by donor to donor variability, the relatively short culture lifetime, and variations between passages. To standardize the in vitro studies on keratinocytes, we investigated the use of HaCaT cells, a long-lived, spontaneously immortalized human keratinocyte line which is able to differentiate in vitro, as a suitable model to follow the release of inflammatory and repair mediators in response to TNFα or IL-1β. Different treatment conditions (presence or absence of serum) and differentiation stimuli (increase in cell density as a function of time in culture and elevation of extracellular calcium) were considered. ELISA and Multiplex measurement technologies were used to monitor the production of cytokines and chemokines. Taken together, the results highlight that Ca2+ concentration in the medium, cell density, and presence of serum influences at different levels the release of proinflammatory mediators by HaCaT cells. Moreover, HaCaT cells maintained in low Ca2+ medium and 80% confluent are similar to normal keratinocytes in terms of cytokine production suggesting that HaCaT cells may be a useful model to investigate anti-inflammatory interventions/therapies on skin diseases.

Download Full-text

Astragaloside Iv Suppresses Histamine-induced Inflammatory Factors and MUC5AC Overproduction in Nasal Epithelial Cells via Regulation of Inflammation-related Genes

10.21203/rs.3.rs-520631/v1 ◽

2021 ◽

Author(s):

Jie Guo ◽

Shuai Xu

Keyword(s):

Epithelial Cells ◽

Inflammatory Cytokines ◽

Underlying Mechanism ◽

Inflammatory Factors ◽

Rna Seq ◽

Astragaloside Iv ◽

Nasal Epithelial Cells ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony

Abstract Allergic rhinitis (AR) is a symptomatic allergic disease that leads to severe inflammation. Astragaloside IV (AS-IV) is a primary active component of Astragalus membranaceus and exerts immune-regulation and anti-inflammatory effects. However, the pharmacological effect of AS-IV in the nasal epithelial cells (NECs) has not been reported. The present study aimed to assess the effect of AS-IV on inflammatory cytokines and mucin 5 subtype AC (MUC5AC) overproduction in histamine (His)-stimulated NECs and its underlying mechanism. NECs were stimulated with or without His for 24 h in the absence or presence of AS-IV. The levels of inflammatory cytokines including IL-6, IL-8, MCP-1, IL-1β, granulocyte-macrophage colony-stimulating factor (GM-CSF), eotaxin, and MUC5AC were assayed. Our findings indicated that AS-IV inhibited His-evoked release and expression of inflammatory cytokines and MUC5AC in NECs. RNA-SEQ analyses indicated the significant changes in expression levels involved in inflammation genes upon treatment of His-induced NECs with AS-IV. Our findings indicated that AS-IV inhibited His-evoked inflammatory cytokines secretion and MUC5AC overproduction in NECs, which were partly mediated by regulation of inflammation-related genes. AS-IV might be a therapeutic agent for the treatment of AR.

Download Full-text

Comparison of Two Diagnostic Assays for Anti-Laminin 332 Mucous Membrane Pemphigoid

Frontiers in Immunology ◽

10.3389/fimmu.2021.773720 ◽

2021 ◽

Vol 12 ◽

Author(s):

Stephanie Goletz ◽

Federica Giurdanella ◽

Maike M. Holtsche ◽

Miranda Nijenhuis ◽

Barbara Horvath ◽

...

Keyword(s):

Mucous Membrane ◽

Blood Donors ◽

Pemphigus Vulgaris ◽

Mucous Membrane Pemphigoid ◽

Diagnostic Assays ◽

Autoimmune Blistering Disease ◽

Highly Sensitive ◽

Blistering Disease ◽

Mucosal Lesions ◽

Laminin 332

Anti-laminin 332 mucous membrane pemphigoid (MMP) is an autoimmune blistering disease characterized by predominant mucosal lesions and autoantibodies against laminin 332. The exact diagnosis of anti-laminin 332 MMP is important since nearly 30% of patients develop solid cancers. This study compared two independently developed diagnostic indirect immunofluorescence (IF) tests based on recombinant laminin 332 expressed in HEK239 cells (biochip mosaic assay) and the migration trails of cultured keratinocytes rich in laminin 332 (footprint assay). The sera of 54 anti-laminin 332 MMP, 35 non-anti-laminin 332 MMP, and 30 pemphigus vulgaris patients as well as 20 healthy blood donors were analyzed blindly and independently. Fifty-two of 54 and 54/54 anti-laminin 332 MMP sera were positive in the biochip mosaic and the footprint assay, respectively. In the 35 non-anti-laminin 332 MMP sera, 3 were positive in both tests and 4 others showed weak reactivity in the footprint assay. In conclusion, both assays are easy to perform, highly sensitive, and specific, which will further facilitate the diagnosis of anti-laminin 332 MMP.

Download Full-text

Cryptotanshinone from Salvia miltiorrhiza Bunge lowers the Cytokeratin 1/10 expression in primary human Keratinocytes in vitro

10.1055/s-0037-1608052 ◽

2017 ◽

Author(s):

S Esch ◽

A Hensel ◽

S Brandt ◽

S König

Keyword(s):

Salvia Miltiorrhiza ◽

Human Keratinocytes ◽

Salvia Miltiorrhiza Bunge ◽

Primary Human Keratinocytes

Download Full-text

Autoimmune Blistering Disease with Concurrent IgG Antibodies to Desmoglein 1 and BP180 : Two Cases

Nishi Nihon Hifuka ◽

10.2336/nishinihonhifu.75.415 ◽

2013 ◽

Vol 75 (5) ◽

pp. 415-418

Author(s):

Miki ICHIKAWA ◽

Maya TANAKA ◽

Kazunori URABE ◽

Masutaka FURUE

Keyword(s):

Igg Antibodies ◽

Autoimmune Blistering Disease ◽

Blistering Disease

Download Full-text

Faculty Opinions recommendation of Robust production and passaging of infectious HPV in squamous epithelium of primary human keratinocytes.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1144927.602030 ◽

2009 ◽

Author(s):

Cheng-Ming Chiang

Keyword(s):

Squamous Epithelium ◽

Human Keratinocytes ◽

Primary Human Keratinocytes

Download Full-text

lncRNA CASC19 Contributes to Radioresistance of Nasopharyngeal Carcinoma by Promoting Autophagy via AMPK-mTOR Pathway

International Journal of Molecular Sciences ◽

10.3390/ijms22031407 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1407

Author(s):

Hongxia Liu ◽

Wang Zheng ◽

Qianping Chen ◽

Yuchuan Zhou ◽

Yan Pan ◽

...

Keyword(s):

Nasopharyngeal Carcinoma ◽

Cell Line ◽

Malignant Tumors ◽

Underlying Mechanism ◽

Mtor Pathway ◽

Rna Seq ◽

Cellular Autophagy ◽

First Time

Nasopharyngeal carcinoma (NPC) is one of the most frequent head and neck malignant tumors and is majorly treated by radiotherapy. However, radiation resistance remains a serious obstacle to the successful treatment of NPC. The aim of this study was to discover the underlying mechanism of radioresistance and to elucidate novel genes that may play important roles in the regulation of NPC radiosensitivity. By using RNA-seq analysis of NPC cell line CNE2 and its radioresistant cell line CNE2R, lncRNA CASC19 was screened out as a candidate radioresistance marker. Both in vitro and in vivo data demonstrated that a high expression level of CASC19 was positively correlated with the radioresistance of NPC, and the radiosensitivity of NPC cells was considerably enhanced by knockdown of CASC19. The incidence of autophagy was enhanced in CNE2R in comparison with CNE2 and another NPC cell line HONE1, and silencing autophagy with LC3 siRNA (siLC3) sensitized NPC cells to irradiation. Furthermore, CASC19 siRNA (siCASC19) suppressed cellular autophagy by inhibiting the AMPK/mTOR pathway and promoted apoptosis through the PARP1 pathway. Our results revealed for the first time that lncRNA CASC19 contributed to the radioresistance of NPC by regulating autophagy. In significance, CASC19 might be a potential molecular biomarker and a new therapeutic target in NPC.

Download Full-text