Staphylococcus aureus is a significant human pathogen which can colonize the skin. Neutrophils are well known to be involved in clearance of the bacterium. This study focused on exploring the role human keratinocytes play as first responders to bacterial challenges. IL-1α and IL-1β increased mRNA production and protein secretion of the neutrophil chemotactic CXCL1, CXCL2 and IL-8 in keratinocytes. S. aureus and the bacterial cell wall components lipoteichoic acid (LTA) and peptidoglycan (PGN) induced similar expression profiles in a Toll-like receptor (TLR)-2 dependent manner. Interestingly, the S. aureus induced mRNA levels peaked at later time-points than those induced by IL-1. The S. aureus activated chemokine production was preceded by significant IL-1α and IL-1β secretion. Expression of IL-1α was significantly higher than that of IL-1β. Inhibition of IL-1RI using neutralizing antibodies revealed that S. aureus derived LTA and PGN induced chemokine expression requires IL-1RI engagement. Surprisingly, we further found that chemokine secretion is dependent upon endocrine IL-1α, but not IL-1β, signaling. Our data demonstrate that the innate immune response of keratinocytes is regulated differently than those of other cell types. This may represent a fail-safe system, which protects the host against genetic variation and immune evasion mechanisms developed by pathogens.
Epidermolysis bullosa acquisita (EBA) is an autoimmune blistering disease caused by autoantibodies against type VII collagen. The neonatal Fc receptor (FcRn) regulates immunoglobulin G (IgG) homeostasis and thus controls serum levels of antibodies. In this study, we investigated the effects of FcRn deficiency on the levels of autoantibodies against type VII collagen and blistering in EBA. For this purpose, rabbit IgG against murine type VII collagen was injected into FcRn-deficient and wild-type (n = 10 per group) mice. Enzyme-linked immunosorbent assay levels of serum IgG against type VII collagen were significantly lower in mutant compared with wild-type mice. Analysis of serum levels of specific autoantibodies induced in FcRn-deficient and wild-type mice (n = 10 per group) by immunization with type VII collagen showed significantly lower serum levels of IgG against type VII collagen in FcRn-deficient mice compared with wild-type animals. Importantly, the extent of blistering disease after injection of IgG against type VII collagen was significantly reduced in FcRn-deficient mice compared to wild-type controls. Our data demonstrate that FcRn maintains levels of pathogenic autoantibodies and thereby promotes tissue injury in experimental EBA. Therefore, modulation of FcRn function using inhibitors may reduce pathogenic IgG levels, offering therapeutic benefit in patients with antibody-mediated diseases.
IL-1 is a potent pro-inflammatory cytokine that activates intracellular signaling cascades some of which may involve IL-1 receptor associated kinase-1 (IRAK1). Psoriasis is a T cell dependent chronic inflammatory condition of the skin of unknown cause. IL-1 has been implicated in psoriasis pathology, but the mechanism has not been elucidated. Interestingly, expression of IRAK1 is elevated in psoriatic skin. To identify a potential link between IL-1, keratinocytes and T cells in skin inflammation we employed pathway-focused microarrays to evaluate IL-1 dependent gene expression in keratinocytes. Several candidate mRNAs encoding known T cell chemoattractants were identified in primary keratinocytes and the stable keratinocyte cell line HaCaT. CCL5 and CCL20 mRNA and protein levels were confirmed up-regulated by IL-1 in concentration and time-dependent manners. Furthermore IL-1 synergized with IFN-γ and TNF-α. Expression of CXCL9, CXCL10 and CXCL11 mRNAs was also increased in response to IL-1, but protein could only be detected in medium from cells treated with IFN-γ alone or in combination with IL-1. Over-expression of IRAK1 led to increased constitutive and cytokine induced production of CCL5 and CCL20. Inhibition of IRAK1 activity through RNAi or expression of a dominant negative mutant blocked production of CCL5 and CCL20 but had no effect upon the IL-1 enhancement of IFN-γ induced CXCL9, CXCL10 and CXCL11 production. In conclusion IL-1 regulates T cell targeting chemokine production in keratinocytes through IRAK1 dependent and independent pathways. These pathways may contribute to acute and chronic skin inflammation.
Membranous nephropathy is an immune kidney disease caused by IgG antibodies that form glomerular subepithelial immune complexes. Proteinuria is mediated by complement activation, as a result of podocyte injury by C5b-9, but the role of specific complement pathways is not known. Autoantibodies-mediating primary membranous nephropathy are predominantly of IgG4 subclass, which cannot activate the classical pathway. Histologic evidence from kidney biopsies suggests that the lectin and the alternative pathways may be activated in membranous nephropathy, but the pathogenic relevance of these pathways remains unclear. In this study, we evaluated the role of the alternative pathway in a mouse model of membranous nephropathy. After inducing the formation of subepithelial immune complexes, we found similar glomerular IgG deposition in wild-type mice and in factor B-null mice, which lack a functional alternative pathway. Unlike wild-type mice, mice lacking factor B did not develop albuminuria nor exhibit glomerular deposition of C3c and C5b-9. Albuminuria was also reduced but not completely abolished in C5-deficient mice. Our results provide the first direct evidence that the alternative pathway is necessary for pathogenic complement activation by glomerular subepithelial immune complexes and is, therefore, a key mediator of proteinuria in experimental membranous nephropathy. This knowledge is important for the rational design of new therapies for membranous nephropathy.
Keratinocytes in the skin play an important role in innate immune responses by secreting chemokines. This study aimed to determine if keratinocyte cell lines can be used for studies of innate immune mechanisms. Human primary keratinocytes and the HaCaT, CCD 1106 KERTr (KERTr) and HEK001 cell lines were treated with a panel of Toll-like receptor (TLR)-ligands. Expression of IL-8, CCL20, CXCL9 and CXCL10 was determined. All three cell lines expressed TLR1-6 and TLR9. KERTr cells responded to the same TLR-ligands as primary keratinocytes. Overall HEK001 responded similarly, but appeared to be relatively more sensitive to flagellin. This was in agreement with increased expression of TLR5. The expression profiles were most distinct in HaCaT cells. Furthermore, our data confirm and extend previously reported TLR7 and TLR8 independent IL-8 secretion by keratinocytes after Imiquimod treatment. The different cell lines represent complementary tools for molecular studies of innate immunity of the skin.
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