ECM Substrates Impact RNAi Localization at Adherens Junctions of Colon Epithelial Cells

The extracellular matrix (ECM) plays crucial roles in tissue homeostasis. Abnormalities in ECM composition are associated with pathological conditions, such as fibrosis and cancer. These ECM alterations are sensed by the epithelium and can influence its behavior through crosstalk with other mechanosensitive complexes, including the adherens junctions (AJs). We have previously shown that the AJs, through their component PLEKHA7, recruit the RNAi machinery to regulate miRNA levels and function. We have particularly shown that the junctional localization of RNAi components is critical for their function. Here, we investigated whether different ECM substrates can influence the junctional localization of RNAi complexes. To do this, we plated colon epithelial Caco2 cells on four key ECM substrates found in the colon under normal or pathogenic conditions, namely laminin, fibronectin, collagen I, and collagen IV, and we examined the subcellular distribution of PLEKHA7, and of the key RNAi components AGO2 and DROSHA. Fibronectin and collagen I negatively impacted the junctional localization of PLEKHA7, AGO2, and DROSHA when compared to laminin. Furthermore, fibronectin, collagen I, and collagen IV disrupted interactions of AGO2 and DROSHA with their essential partners GW182 and DGCR8, respectively, both at AJs and throughout the cell. Combinations of all substrates with fibronectin also negatively impacted junctional localization of PLEKHA7 and AGO2. Additionally, collagen I triggered accumulation of DROSHA at tri-cellular junctions, while both collagen I and collagen IV resulted in DROSHA accumulation at basal areas of cell–cell contact. Altogether, fibronectin and collagens I and IV, which are elevated in the stroma of fibrotic and cancerous tissues, altered localization patterns and disrupted complex formation of PLEKHA7 and RNAi components. Combined with our prior studies showing that apical junctional localization of the PLEKHA7-RNAi complex is critical for regulating tumor-suppressing miRNAs, this work points to a yet unstudied mechanism that could contribute to epithelial cell transformation

Predominant Distribution of the RNAi Machinery at Apical Adherens Junctions in Colonic Epithelia Is Disrupted in Cancer

The RNA interference (RNAi) machinery is an essential component of the cell, regulating miRNA biogenesis and function. RNAi complexes were thought to localize either in the nucleus, such as the microprocessor, or in the cytoplasm, such as the RNA-induced silencing complex (RISC). We recently revealed that the core microprocessor components DROSHA and DGCR8, as well as the main components of RISC, including Ago2, also associate with the apical adherens junctions of well-differentiated cultured epithelial cells. Here, we demonstrate that the localization of the core RNAi components is specific and predominant at apical areas of cell-cell contact of human normal colon epithelial tissues and normal primary colon epithelial cells. Importantly, the apical junctional localization of RNAi proteins is disrupted or lost in human colon tumors and in poorly differentiated colon cancer cell lines, correlating with the dysregulation of the adherens junction component PLEKHA7. We show that the restoration of PLEKHA7 expression at adherens junctions of aggressively tumorigenic colon cancer cells restores the junctional localization of RNAi components and suppresses cancer cell growth in vitro and in vivo. In summary, this work identifies the apical junctional localization of the RNAi machinery as a key feature of the differentiated colonic epithelium, with a putative tumor suppressing function

Imprinted Anti-Hemagglutinin and Anti-Neuraminidase Antibody Responses after Childhood Infections of A(H1N1) and A(H1N1)pdm09 Influenza Viruses

ABSTRACT Immune imprinting is a driver known to shape the anti-hemagglutinin (HA) antibody landscape of individuals born within the same birth cohort. With the HA and neuraminidase (NA) proteins evolving at different rates under immune selection pressures, anti-HA and anti-NA antibody responses since childhood influenza virus infections have not been evaluated in parallel at the individual level. This is partly due to the limited knowledge of changes in NA antigenicity, as seasonal influenza vaccines have focused on generating neutralizing anti-HA antibodies against HA antigenic variants. Here, we systematically characterized the NA antigenic variants of seasonal A(H1N1) viruses from 1977 to 1991 and completed the antigenic profile of N1 NAs from 1977 to 2015. We identified that NA proteins of A/USSR/90/77, A/Singapore/06/86, and A/Texas/36/91 were antigenically distinct and mapped N386K as a key determinant of the NA antigenic change from A/USSR/90/77 to A/Singapore/06/86. With comprehensive panels of HA and NA antigenic variants of A(H1N1) and A(H1N1)pdm09 viruses, we determined hemagglutinin inhibition (HI) and neuraminidase inhibition (NI) antibodies from 130 subjects born between 1950 and 2015. Age-dependent imprinting was observed for both anti-HA and anti-NA antibodies, with the peak HI and NI titers predominantly detected from subjects at 4 to 12 years old during the year of initial virus isolation, except the age-independent anti-HA antibody response against A(H1N1)pdm09 viruses. More participants possessed antibodies that reacted to multiple antigenically distinct NA proteins than those with antibodies that reacted to multiple antigenically distinct HA proteins. Our results support the need to include NA proteins in seasonal influenza vaccine preparations. IMPORTANCE Seasonal influenza vaccines have aimed to generate neutralizing anti-HA antibodies for protection since licensure. More recently, anti-NA antibodies have been established as an additional correlate of protection. While HA and NA antigenic changes occurred discordantly, the anti-HA and anti-NA antibody profiles have rarely been analyzed in parallel at the individual level, due to the limited knowledge on NA antigenic changes. By characterizing NA antigenic changes of A(H1N1) viruses, we determined the anti-HA and anti-NA antibody landscape against antigenically distinct A(H1N1) and A(H1N1)pdm09 viruses using sera of 130 subjects born between 1950 and 2015. We observed age-dependent imprinting of both anti-HA and anti-NA antibodies against strains circulated during the first decade of life. A total of 67.7% (88/130) and 90% (117/130) of participants developed cross-reactive antibodies to multiple HA and NA antigens at titers ≥1:40. With slower NA antigenic changes and cross-reactive anti-NA antibody responses, including NA protein in influenza vaccine preparation may enhance vaccine efficacy