Neutrophil extracellular traps in pediatric inflammatory bowel disease.

Neutrophil extracellular traps (NETs) are fibers composed of chromatin and neutrophil proteins released by activated neutrophils. NETs trap and kill microbes, activate dendritic and T cells, and are implicated in autoimmune and vascular diseases. The pathogenesis of inflammatory bowel disease (IBD) is multifactorial and characterized by chronic active mucosal inflammation with controversial contribution of neutrophils. Our aim is to describe the involvement of NETs in pediatric IBD. We retrospectively examined biopsies from the small bowel and colon of children at diagnosis of Crohn's disease (CD) or ulcerative colitis (UC). The biopsies were labeled for neutrophil elastase, myeloperoxidase, DNA, chromatin and histones in order to identify NETs. Samples of two children with normal colonoscopy served as controls. Twelve patients (5 boys) were included, 6 with CD and 6 with UC. Their average age was 12.2 years (range 5-16). NETs were found in all samples from patients and not in the samples from the two controls. This is the first demonstration of the presence of NETs in biopsies taken from the small bowel and colon of pediatric patients with IBD. More studies are needed in order to identify the role of NETs in CD/UC pathogenesis.

Calpain 12 Function Revealed through the Study of an Atypical Case of Autosomal Recessive Congenital Ichthyosis.

Congenital erythroderma is a rare and often life-threatening condition, which has been shown to result from mutations in several genes encoding important components of the epidermal differentiation program. Using whole exome sequencing, we identified in a child with congenital exfoliative erythroderma, hypotrichosis, severe nail dystrophy and failure to thrive, two heterozygous mutations in ABCA12 (c.2956C>T, p.R986W; c.5778+2T>C, p. G1900Mfs*16), a gene known to be associated with two forms of ichthyosis, autosomal recessive congenital ichthyosis, and harlequin ichthyosis. Because the patient displayed an atypical phenotype, including severe hair and nail manifestations, we scrutinized the exome sequencing data for additional potentially deleterious genetic variations in genes of relevance to the cornification process. Two mutations were identified in CAPN12, encoding a member of the calpain proteases: a paternal missense mutation (c.1511C>A; p.P504Q) and a maternal deletion due to activation of a cryptic splice site in exon 9 of the gene (c.1090_1129del; p.Val364Lysfs*11). The calpain 12 protein was found to be expressed in both the epidermis and hair follicle of normal skin, but its expression was dramatically reduced in the patient's skin. The downregulation of capn12 expression in zebrafish was associated with abnormal epidermal morphogenesis. Small interfering RNA knockdown of CAPN12 in three-dimensional human skin models was associated with acanthosis, disorganized epidermal architecture, and downregulation of several differentiation markers, including filaggrin. Accordingly, filaggrin expression was almost absent in the patient skin. Using ex vivo live imaging, small interfering RNA knockdown of calpain 12 in skin from K14-H2B GFP mice led to significant hair follicle catagen transformation compared with controls. In summary, our results indicate that calpain 12 plays an essential role during epidermal ontogenesis and normal hair follicle cycling and that its absence may aggravate the clinical manifestations of ABCA12 mutations.

Endoplasmic reticulum anchored heme-oxygenase 1 faces the cytosol.

Heme-oxygenase 1 is an endoplasmic reticulum-anchored enzyme that breaks down heme into iron, carbon monoxide and biliverdin. Heme is a hydrophobic co-factor in many proteins, including hemoglobin. Free heme is highly cytotoxic and, therefore, both heme synthesis and breakdown are tightly regulated. During turnover of heme proteins, heme is released in the phago-lysosomal compartment or the cytosol. The subcellular location of the heme-oxygenase 1 active site has not been clarified. Using constructs of heme-oxygenase 1 with fluorescent proteins, and the endogenous heme-oxygenase 1 in two variations of protease protection assays, we determined that heme-oxygenase 1 is membrane-bound and faces the cytosol in non-activated macrophages in vivo. These findings imply that in quiescent macrophages, heme breakdown products are generated in the cytosol. This facilitates iron recycling to ferroportin for iron export and to ferritin for iron storage.

Physiologically aged red blood cells undergo erythrophagocytosis in vivo but not in vitro.

BACKGROUND: The lifespan of red blood cells is terminated when macrophages remove senescent red blood cells by erythrophagocytosis. This puts macrophages at the center of systemic iron recycling in addition to their functions in tissue remodeling and innate immunity. Thus far, erythrophagocytosis has been studied by evaluating phagocytosis of erythrocytes that were damaged to mimic senescence. These studies have demonstrated that acquisition of some specific individual senescence markers can trigger erythrophagocytosis by macrophages, but we hypothesized that the mechanism of erythrophagocytosis of such damaged erythrocytes might differ from erythrophagocytosis of physiologically aged erythrocytes. DESIGN AND METHODS: To test this hypothesis we generated an erythrocyte population highly enriched in senescent erythrocytes by a hypertransfusion procedure in mice. Various erythrocyte-aging signals were analyzed and erythrophagocytosis was evaluated in vivo and in vitro. RESULTS: The large cohort of senescent erythrocytes from hypertransfused mice carried numerous aging signals identical to those of senescent erythrocytes from control mice. Phagocytosis of fluorescently-labeled erythrocytes from hypertransfused mice injected into untreated mice was much higher than phagocytosis of labeled erythrocytes from control mice. However, neither erythrocytes from hypertransfused mice, nor those from control mice were phagocytosed in vitro by primary macrophage cultures, even though these cultures were able to phagocytose oxidatively damaged erythrocytes. CONCLUSIONS: The large senescent erythrocyte population found in hypertransfused mice mimics physiologically aged erythrocytes. For effective erythrophagocytosis of these senescent erythrocytes, macrophages depend on some features of the intact phagocytosing tissue for support.