Conjunctival Goblet Cell Responses to TLR5 Engagement Promote Activation of Local Antigen-Presenting Cells.

Conjunctival epithelium forms a barrier between the ocular surface microbial flora and the ocular mucosa. In addition to secreting gel-forming mucins, goblet cells, located in the conjunctival epithelium, help maintain local immune homeostasis by secreting active TGFß2 and promoting tolerogenic phenotype of dendritic cells in the vicinity. Although dendritic cell subsets, characteristic of mucosal tissues, are found in the conjunctiva, previous studies provided limited information about their location within the tissue. In this study, we examine immunostained conjunctiva explants to determine the location of CD11c-positive dendritic cells in the context of MUC5AC-positive goblet cells. Considering that conjunctival goblet cells are responsive to signaling induced by pathogen recognition receptors, we also assess if their responses to microbial product, flagellin, can contribute to the disruption of ocular mucosal homeostasis that promotes activation of dendritic cells and results in chronic ocular surface inflammation. We find that dendritic cells in the conjunctiva with an increased microbial colonization are located adjacent to goblet cells. While their cell bodies in the stromal layer are immediately below the epithelial layer, several extensions of dendritic cells are projected across the epithelium towards the ocular surface. Such trans-epithelial dendrites are not detectable in healthy ocular mucosa. In response to topically applied flagellin, increased proportion of CD11c-positive cells in the conjunctiva strongly express MHC class II relative to the untreated conjunctiva. This change is accompanied by reduced immunoreactivity to TGFß-activating Thrombospondin-1 in the conjunctival epithelium. These findings are supported by in vitro observations in primary cultures of goblet cells that respond to the TLR5 stimulation with an increased expression of IL-6 and reduced level of active TGFß. The observed changes in the conjunctiva after flagellin application correspond with the development of clinical signs of chronic ocular mucosal inflammation including corneal epitheliopathy. Collectively, these findings demonstrate the ability of ocular mucosal dendritic cells to extend trans-epithelial dendrites in response to increased microbial colonization at the ocular surface. Moreover, this study provides key insight into how goblet cell responses to microbial stimuli may contribute to the disruption of ocular mucosal homeostasis and chronic ocular mucosal inflammation.

Challenges for Super-Resolution Localization Microscopy and Biomolecular Fluorescent Nano-Probing in Cancer Research.

Understanding molecular interactions and regulatory mechanisms in tumor initiation, progression, and treatment response are key requirements towards advanced cancer diagnosis and novel treatment procedures in personalized medicine. Beyond decoding the gene expression, malfunctioning and cancer-related epigenetic pathways, investigations of the spatial receptor arrangements in membranes and genome organization in cell nuclei, on the nano-scale, contribute to elucidating complex molecular mechanisms in cells and tissues. By these means, the correlation between cell function and spatial organization of molecules or molecular complexes can be studied, with respect to carcinogenesis, tumor sensitivity or tumor resistance to anticancer therapies, like radiation or antibody treatment. Here, we present several new applications for bio-molecular nano-probes and super-resolution, laser fluorescence localization microscopy and their potential in life sciences, especially in biomedical and cancer research. By means of a tool-box of fluorescent antibodies, green fluorescent protein (GFP) tagging, or specific oligonucleotides, we present tumor relevant re-arrangements of Erb-receptors in membranes, spatial organization of Smad specific ubiquitin protein ligase 2 (Smurf2) in the cytosol, tumor cell characteristic heterochromatin organization, and molecular re-arrangements induced by radiation or antibody treatment. The main purpose of this article is to demonstrate how nano-scaled distance measurements between bio-molecules, tagged by appropriate nano-probes, can be applied to elucidate structures and conformations of molecular complexes which are characteristic of tumorigenesis and treatment responses. These applications open new avenues towards a better interpretation of the spatial organization and treatment responses of functionally relevant molecules, at the single cell level, in normal and cancer cells, offering new potentials for individualized medicine.