Orchestrated control of filaggrin-actin scaffolds underpins cornification

Epidermal stratification critically depends on keratinocyte differentiation and programmed death by cornification, leading to formation of a protective skin barrier. Cornification is dynamically controlled by the protein filaggrin, rapidly released from keratohyalin granules (KHGs). However, the mechanisms of cornification largely remain elusive, partly due to limitations of the observation techniques employed to study filaggrin organization in keratinocytes. Moreover, while the abundance of keratins within KHGs has been well described, it is not clear whether actin also contributes to their formation or fate. We employed advanced (super-resolution) microscopy to examine filaggrin organization and dynamics in skin and human keratinocytes during differentiation. We found that filaggrin organization depends on the cytoplasmic actin cytoskeleton, including the role for α- and β-actin scaffolds. Filaggrin-containing KHGs displayed high mobility and migrated toward the nucleus during differentiation. Pharmacological disruption targeting actin networks resulted in granule disintegration and accelerated cornification. We identified the role of AKT serine/threonine kinase 1 (AKT1), which controls binding preference and function of heat shock protein B1 (HspB1), facilitating the switch from actin stabilization to filaggrin processing. Our results suggest an extended model of cornification in which filaggrin utilizes actins to effectively control keratinocyte differentiation and death, promoting epidermal stratification and formation of a fully functional skin barrier

Filaggrin inhibits generation of CD1a neolipid antigens by house dust mite-derived phospholipase.

Atopic dermatitis is a common pruritic skin disease in which barrier dysfunction and cutaneous inflammation play a role in pathogenesis. Mechanisms underlying the associated inflammation are not fully understood, and while CD1a-expressing Langerhans cells are known to be enriched within lesions, their role in clinical disease pathogenesis has not been studied. Here we observed that house dust mite (HDM) generates neolipid antigens for presentation by CD1a to T cells in the blood and skin lesions of affected individuals. HDM-responsive CD1a-reactive T cells increased in frequency after birth and showed rapid effector function, consistent with antigen-driven maturation. To define the underlying mechanisms, we analyzed HDM-challenged human skin and observed allergen-derived phospholipase (PLA2) activity in vivo. CD1a-reactive T cell activation was dependent on HDM-derived PLA2 and such cells infiltrated the skin after allergen challenge. Filaggrin insufficiency is associated with atopic dermatitis, and we observed that filaggrin inhibits PLA2 activity and inhibits CD1a-reactive PLA2-generated neolipid-specific T cell activity from skin and blood. The most widely used classification schemes of hypersensitivity, such as Gell and Coombs are predicated on the idea that non-peptide stimulants of T cells act as haptens that modify peptides or proteins. However our results point to a broader model that does not posit haptenation, but instead shows that HDM proteins generate neolipid antigens which directly activate T cells. Specifically, the data identify a pathway of atopic skin inflammation, in which house dust mite-derived phospholipase A2 generates antigenic neolipids for presentation to CD1a-reactive T cells, and define PLA2 inhibition as a function of filaggrin, supporting PLA2 inhibition as a therapeutic approach

NKT Cells in Rheumatoid Arthritis

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Ni2+-Assisted Hydrolysis May Affect the Human Proteome; Filaggrin Degradation Ex Vivo as an Example of Possible Consequences.

Peer reviewed: TrueAcknowledgements: We thank MSc Małgorzata Orłowska for her help with the iTOL visualization.Deficiency in a principal epidermal barrier protein, filaggrin (FLG), is associated with multiple allergic manifestations, including atopic dermatitis and contact allergy to nickel. Toxicity caused by dermal and respiratory exposures of the general population to nickel-containing objects and particles is a deleterious side effect of modern technologies. Its molecular mechanism may include the peptide bond hydrolysis in X1-S/T-c/p-H-c-X2 motifs by released Ni2+ ions. The goal of the study was to analyse the distribution of such cleavable motifs in the human proteome and examine FLG vulnerability of nickel hydrolysis. We performed a general bioinformatic study followed by biochemical and biological analysis of a single case, the FLG protein. FLG model peptides, the recombinant monomer domain human keratinocytes in vitro and human epidermis ex vivo were used. We also investigated if the products of filaggrin Ni2+-hydrolysis affect the activation profile of Langerhans cells. We found X1-S/T-c/p-H-c-X2 motifs in 40% of human proteins, with the highest abundance in those involved in the epidermal barrier function, including FLG. We confirmed the hydrolytic vulnerability and pH-dependent Ni2+-assisted cleavage of FLG-derived peptides and FLG monomer, using in vitro cell culture and ex-vivo epidermal sheets; the hydrolysis contributed to the pronounced reduction in FLG in all of the models studied. We also postulated that Ni-hydrolysis might dysregulate important immune responses. Ni2+-assisted cleavage of barrier proteins, including FLG, may contribute to clinical disease associated with nickel exposure

Corrigendum: Ni 2+ -assisted hydrolysis may affect the human proteome; filaggrin degradation ex vivo as an example of possible consequences

Peer reviewed: Tru

Ni2+-Assisted Hydrolysis May Affect the Human Proteome; Filaggrin Degradation Ex Vivo as an Example of Possible Consequences

Deficiency in a principal epidermal barrier protein, filaggrin (FLG), is associated with multiple allergic manifestations, including atopic dermatitis and contact allergy to nickel. Toxicity caused by dermal and respiratory exposures of the general population to nickel-containing objects and particles is a deleterious side effect of modern technologies. Its molecular mechanism may include the peptide bond hydrolysis in X1-S/T-c/p-H-c-X2 motifs by released Ni2+ ions. The goal of the study was to analyse the distribution of such cleavable motifs in the human proteome and examine FLG vulnerability of nickel hydrolysis. We performed a general bioinformatic study followed by biochemical and biological analysis of a single case, the FLG protein. FLG model peptides, the recombinant monomer domain human keratinocytes in vitro and human epidermis ex vivo were used. We also investigated if the products of filaggrin Ni2+-hydrolysis affect the activation profile of Langerhans cells. We found X1-S/T-c/p-H-c-X2 motifs in 40% of human proteins, with the highest abundance in those involved in the epidermal barrier function, including FLG. We confirmed the hydrolytic vulnerability and pH-dependent Ni2+-assisted cleavage of FLG-derived peptides and FLG monomer, using in vitro cell culture and ex-vivo epidermal sheets; the hydrolysis contributed to the pronounced reduction in FLG in all of the models studied. We also postulated that Ni-hydrolysis might dysregulate important immune responses. Ni2+-assisted cleavage of barrier proteins, including FLG, may contribute to clinical disease associated with nickel exposure

The Mucosal Immune Response to Laryngopharyngeal Reflux

Rationale: Laryngopharyngeal reflux (LPR) affects up to 20% of Western populations. Although individual morbidity is usually moderate, treatment costs are high and there are associations with other diseases, including laryngeal cancer. To date, there have been no studies of the mucosal immune response to this common inflammatory disease