LIGHT signaling through LTßR and HVEM in keratinocytes promotes psoriasis and atopic dermatitis-like skin inflammation.

Psoriasis (PS) and atopic dermatitis (AD) are common skin inflammatory diseases characterized by hyper-responsive keratinocytes. Although, some cytokines have been suggested to be specific for each disease, other cytokines might be central to both diseases. Here, we show that Tumor necrosis factor superfamily member 14 (TNFSF14), known as LIGHT, is required for experimental PS, similar to its requirement in experimental AD. Mice devoid of LIGHT, or deletion of either of its receptors, lymphotoxin ß receptor (LTßR) and herpesvirus entry mediator (HVEM), in keratinocytes, were protected from developing imiquimod-induced psoriatic features, including epidermal thickening and hyperplasia, and expression of PS-related genes. Correspondingly, in single cell RNA-seq analysis of PS patient biopsies, LTßR transcripts were found strongly expressed with HVEM in keratinocytes, and LIGHT was upregulated in T cells. Similar transcript expression profiles were also seen in AD biopsies, and LTßR deletion in keratinocytes also protected mice from allergen-induced AD features. Moreover, in vitro, LIGHT upregulated a broad spectrum of genes in human keratinocytes that are clinical features of both PS and AD skin lesions. Our data suggest that agents blocking LIGHT activity might be useful for therapeutic intervention in PS as well as in AD.

Tumor Necrosis Factor Superfamily 14 (LIGHT) Restricts Neovascularization by Decreasing Circulating Endothelial Progenitor Cells and Function.

Tumor necrosis factor superfamily 14 (TNFSF14) is also known as the LT-related inducible ligand (LIGHT). It can bind to the herpesvirus invasion mediator and lymphotoxin-ß receptor to perform its biological activity. LIGHT has multiple physiological functions, including strengthening the synthesis of nitric oxide, reactive oxygen species, and cytokines. LIGHT also stimulates angiogenesis in tumors and induces the synthesis of high endothelial venules; degrades the extracellular matrix in thoracic aortic dissection, and induces the expression of interleukin-8, cyclooxygenase-2, and cell adhesion molecules in endothelial cells. While LIGHT induces tissue inflammation, its effects on angiogenesis after tissue ischemia are unclear. Thus, we analyzed these effects in the current study. In this study, the animal model of hind limb ischemia surgery in C57BL/6 mice was performed. Doppler ultrasound, immunohistochemical staining, and Western blotting were employed to analyze the situation of angiogenesis. In addition, human endothelial progenitor cells (EPCs) were used for in vitro studies to analyze the possible mechanisms. The results in the animal study showed that LIGHT injection inhibited angiogenesis in ischemic limbs. For the in vitro studies, LIGHT inhibited the expression of integrins and E-selectin; decreased migration and tube formation capabilities, mitochondrial respiration, and succinate dehydrogenase activity; and promoted senescence in EPCs. Western blotting revealed that the impairment of EPC function by LIGHT may be due to its effects on the proper functioning of the intracellular Akt signaling pathway, endothelial nitrite oxide synthase (eNOS), and mitochondrial respiration. In conclusion, LIGHT inhibits angiogenesis after tissue ischemia. This may be related to the clamped EPC function.

LIGHT Amplification by NF-κB Contributes to TLR3 Signaling Pathway-Induced Acute Hepatitis.

LIGHT is a member of the TNF superfamily and a proinflammatory cytokine involved in liver pathogenesis. Many liver diseases involve activation of Toll-like receptor 3 (TLR3), which is activated by double-stranded RNA (dsRNA). However, the involvement of LIGHT in TLR3 implicated liver diseases is not clear. In this study, we investigated the role of LIGHT in TLR3 involved liver pathogenesis by using a mouse model of TLR3 agonist poly(I:C)-induced hepatitis. We found LIGHT expression at both protein and mRNA level in liver tissues is dramatically increased during the course of poly(I:C)-induced liver injury. This induction depends on NF-κB activation as pretreating the mice with a NF-κB inhibitor abrogates LIGHT upregulation. Importantly, blockade of the LIGHT signaling pathway with the recombinant LIGHT receptor HVEM protein ameliorates liver injury in poly(I:C)-induced hepatitis. Conclusions. These results indicate that LIGHT amplification by NF-κB plays a significant role in TLR3 involved hepatitis and points LIGHT to be a potential drug target for liver disease therapy.

Dual Roles of Tumor Necrosis Factor Superfamily 14 in Antiviral Immunity.

Tumor necrosis factor superfamily 14 (TNFSF14) (LIGHT) is an interesting costimulatory molecule associated with T lymphocyte activation, and it mainly exerts its biological effects by binding to its receptors herpesvirus invasion mediator (HVEM) and lymphotoxin-ß receptor. Research shows that TNFSF14 plays a critical regulatory role in immune responses to viral infection, but its role is different in different diseases. TNFSF14 can be a cytokine neutralization target during novel coronavirus infection, and anti-TNFSF14 monoclonal antibody treatment can reduce the risk of respiratory failure and mortality. When the host is infected with adenovirus, TNFSF14 can be used as an inflammatory biomarker to indicate whether there was an adenovirus infection in the host and the degree of disease caused by viral infection. When hosts suffer influenza virus infection, the TNFSF14-HVEM signaling pathway can stimulate the maturation and proliferation of memory CD8+ T cells, which helps the host immune system stimulate a second immune response against respiratory virus infection. TNFSF14 can act as an immune adjuvant and enhance the immunogenicity of the human papillomavirus (HPV) DNA vaccine when the host is infected with HPV. During hepatitis virus infection, TNFSF14 acts as a proinflammatory factor, participates in inflammation and causes tissue damage. In conclusion, TNFSF14 plays different and significant roles in diverse viral infections. This article reviews the current research on TNFSF14 in antiviral immunity.

Analysis of Slow-Cycling Variants of the Light-Inducible Nuclear Protein Export System LEXY in Mammalian Cells.

The optogenetic tool LEXY consists of the second light oxygen voltage (LOV) domain of Avena sativa phototropin 1 mutated to contain a nuclear export signal. It allows exporting from the nucleus with blue light proteins of interest (POIs) genetically fused to it. Mutations slowing the dark recovery rate of the LOV domain within LEXY were recently shown to allow for better depletion of some POIs from the nucleus in Drosophila embryos and for the usage of low light illumination regimes. We investigated these variants in mammalian cells and found they increase the cytoplasmic localization of the proteins we tested after illumination, but also during the dark phases, which corresponds to higher leakiness of the system. These data suggest that, when aiming to sequester into the nucleus a protein with a cytoplasmic function, the original LEXY is preferable. The iLEXY variants are, instead, advantageous when wanting to deplete the nucleus of the POI as much as possible.

Observation of conformational dynamics in single light-harvesting proteins from cryptophyte algae.

Photosynthetic organisms use pigment-protein complexes to capture the sunlight that powers most life on earth. Within these complexes, the position of the embedded pigments is all optimized for light harvesting. At the same time, the protein scaffold undergoes thermal fluctuations that vary the structure, and, thus, photophysics, of the complexes. While these variations are averaged out in ensemble measurements, single-molecule spectroscopy provides the ability to probe these conformational changes. We used single-molecule fluorescence spectroscopy to identify the photophysical substates reflective of distinct conformations and the associated conformational dynamics in phycoerythrin 545 (PE545), a pigment-protein complex from cryptophyte algae. Rapid switching between photophysical states was observed, indicating that ensemble measurements average over a conformational equilibrium. A highly quenched conformation was also identified, and its population increased under high light. This discovery establishes that PE545 has the characteristics to serve as a photoprotective site. Finally, unlike homologous proteins from the evolutionarily related cyanobacteria and red algae, quenching was not observed upon photobleaching, which may allow for robust photophysics without the need for rapid repair or replacement machinery. Collectively, these observations establish the presence of a rich and robust set of conformational states of PE545. Cryptophytes exhibit particularly diverse energetics owing to the variety of microenvironments in which they survive, and the conformational states and dynamics reported here may provide photophysical flexibility that contributes to their remarkable ability to flourish under diverse conditions.

Structural dynamics of light harvesting proteins, photosynthetic membranes, and cells observed by spectral editing solid-state NMR.

Photosynthetic light-harvesting complexes have a remarkable capacity to perform robust photo-physics at ambient temperatures and in fluctuating environments. Protein conformational dynamics and membrane mobility are processes that contribute to the light-harvesting efficiencies and control photoprotective responses. This short review describes the application of magic angle spinning nuclear magnetic resonance (NMR) spectroscopy for characterizing the structural dynamics of pigment, protein, and thylakoid membrane components related to light harvesting and photoprotection. I will discuss the use of dynamics-based spectral editing solid-state NMR for distinguishing rigid and mobile components and assessing protein, pigment, and lipid dynamics on sub-nanosecond to millisecond timescales. Dynamic spectral editing NMR has been applied to investigate light-harvesting complex II protein conformational dynamics inside lipid bilayers and in native membranes. Furthermore, we used the NMR approach to assess thylakoid membrane dynamics. Finally, it is shown that dynamics-based spectral editing NMR for reducing spectral complexity by filtering motion-dependent signals enabled us to follow processes in live photosynthetic cells.

Controlling synthetic membraneless organelles by a red-light-dependent singlet oxygen-generating protein.

Membraneless organelles (MLOs) formed via protein phase separation have great implications for both physiological and pathological processes. However, the inability to precisely control the bioactivities of MLOs has hindered our understanding of their roles in biology, not to mention their translational applications. Here, by combining intrinsically disordered domains such as RGG and mussel-foot proteins, we create an in cellulo protein phase separation system, of which various biological activities can be introduced via metal-mediated protein immobilization and further controlled by the water-soluble chlorophyll protein (WSCP)-a remarkably stable, red-light-responsive singlet oxygen generator. The WSCP-laden protein condensates undergo a liquid-to-solid phase transition on light exposure, due to oxidative crosslinking, providing a means to control catalysis within synthetic MLOs. Moreover, these photoresponsive condensates, which retain the light-induced phase-transition behavior in living cells, exhibit marked membrane localization, reminiscent of the semi-membrane-bound compartments like postsynaptic densities in nervous systems. Together, this engineered system provides an approach toward controllable synthetic MLOs and, alongside its light-induced phase transition, may well serve to emulate and explore the aging process at the subcellular or even molecular level.

Hepatic stellate cell mediates transcription of TNFSF14 in hepatocellular carcinoma cells via H2S/CSE-JNK/JunB signaling pathway.

Hepatic stellate cells (HSC) and hydrogen sulfide (H2S) both play important roles in the development of hepatocellar carcinoma (HCC). Whereas, in the microenvironment of HCC, whether HSC participate in regulating the biological process of HCC cells by releasing H2S remains elusive. In vitro, Flow cytometry (FCM), CCK-8, RNA-sequencing, Western blotting, RT-qPCR, immunofluorescence and ChIP assays were carried out in the HCC cells to investigate the effect of H2S on biological functions and JNK/JunB-TNFSF14 signaling pathway. Specimens from HCC patients were analyzed by RT-qPCR and Western blotting assays for evaluating the expression of TNFSF14 and CSE. Statistical analysis was used to analyze the correlation between TNFSF14 expression and clinical data of HCC patients. Based on the FCM and CCK-8 results, we found the LX-2 cells were able to induce HCC cells apoptosis through releasing H2S. RNA-sequencing, RT-qPCR, and Western blotting results showed that TNFSF14 gene was upregulated in both LX-2 and NaHS group. NaHS treated in HCC cells led to JNK/JunB signaling pathway activating and greater binding of p-JunB to its responsive elements on TNFSF14 promoter. Impairment of TNFSF14 induction alleviated LX-2 and NaHS induced apoptosis of HepG2 and PLC/PRF/5 cells. Furthermore, TNFSF14 expression in HCC tissues was lower than the adjacent tissue. HCC patients with low expression of TNFSF14 had higher malignant degree and poor prognosis. In summary, demonstration of the involvement of HSC-derived H2S in JNK/JunB mediated expression of TNFSF14 gene strongly indicates H2S palys an important role in the regulation of HCC apoptosis.

Psb34 protein modulates binding of high-light-inducible proteins to CP47-containing photosystem II assembly intermediates in the cyanobacterium Synechocystis sp. PCC 6803.

Assembly of photosystem II (PSII), a water-splitting catalyst in chloroplasts and cyanobacteria, requires numerous auxiliary proteins which promote individual steps of this sequential process and transiently associate with one or more assembly intermediate complexes. In this study, we focussed on the role of a PSII-associated protein encoded by the ssl1498 gene in the cyanobacterium Synechocystis sp. PCC 6803. The N-terminal domain of this protein, which is here called Psb34, is very similar to the N-terminus of HliA/B proteins belonging to a family of high-light-inducible proteins (Hlips). Psb34 was identified in both dimeric and monomeric PSII, as well as in a PSII monomer lacking CP43 and containing Psb28. When FLAG-tagged, the protein is co-purified with these three complexes and with the PSII auxiliary proteins Psb27 and Psb28. However, the preparation also contained the oxygen-evolving enhancers PsbO and PsbV and lacked HliA/B proteins even when isolated from high-light-treated cells. The data suggest that Psb34 competes with HliA/B for the same binding site and that it is one of the components involved in the final conversion of late PSII assembly intermediates into functional PSII complexes, possibly keeping them free of Hlips. Unlike HliA/B, Psb34 does bind to the CP47 assembly module before its incorporation into PSII. Analysis of strains lacking Psb34 indicates that Psb34 mediates the optimal equilibrium of HliA/B binding among individual PSII assembly intermediates containing CP47, allowing Hlip-mediated photoprotection at all stages of PSII assembly.

Photoprotective conformational dynamics of photosynthetic light-harvesting proteins.

Under high light conditions, excess energy can damage the machinery of oxygenic photosynthesis. Plants have evolved a series of photoprotective processes, including conformational changes of the light-harvesting complexes that activate dissipation of energy as heat. In this mini-review, we will summarize our recent work developing and applying single-molecule methods to investigate the conformational states of the light-harvesting complexes. Through these measurements, we identified dissipative conformations and how they depend on conditions that mimic high light. Our studies revealed an equilibrium between the light-harvesting and dissipative conformations, and that the nature of the equilibrium varies with cellular environment, between proteins, and between species. Finally, we conclude with an outlook on open questions and implications for photosynthetic yields.

Photosystem I light-harvesting proteins regulate photosynthetic electron transfer and hydrogen production.

Linear electron flow (LEF) and cyclic electron flow (CEF) compete for light-driven electrons transferred from the acceptor side of photosystem I (PSI). Under anoxic conditions, such highly reducing electrons also could be used for hydrogen (H2) production via electron transfer between ferredoxin and hydrogenase in the green alga Chlamydomonas reinhardtii. Partitioning between LEF and CEF is regulated through PROTON-GRADIENT REGULATION5 (PGR5). There is evidence that partitioning of electrons also could be mediated via PSI remodeling processes. This plasticity is linked to the dynamics of PSI-associated light-harvesting proteins (LHCAs) LHCA2 and LHCA9. These two unique light-harvesting proteins are distinct from all other LHCAs because they are loosely bound at the PSAL pole. Here, we investigated photosynthetic electron transfer and H2 production in single, double, and triple mutants deficient in PGR5, LHCA2, and LHCA9. Our data indicate that lhca2 and lhca9 mutants are efficient in photosynthetic electron transfer, that LHCA2 impacts the pgr5 phenotype, and that pgr5/lhca2 is a potent H2 photo-producer. In addition, pgr5/lhca2 and pgr5/lhca9 mutants displayed substantially different H2 photo-production kinetics. This indicates that the absence of LHCA2 or LHCA9 impacts H2 photo-production independently, despite both being attached at the PSAL pole, pointing to distinct regulatory capacities.

High-light-inducible proteins HliA and HliB: pigment binding and protein-protein interactions.

High-light-inducible proteins (Hlips) are single-helix transmembrane proteins that are essential for the survival of cyanobacteria under stress conditions. The model cyanobacterium Synechocystis sp. PCC 6803 contains four Hlip isoforms (HliA-D) that associate with Photosystem II (PSII) during its assembly. HliC and HliD are known to form pigmented (hetero)dimers that associate with the newly synthesized PSII reaction center protein D1 in a configuration that allows thermal dissipation of excitation energy. Thus, it is expected that they photoprotect the early steps of PSII biogenesis. HliA and HliB, on the other hand, bind the PSII inner antenna protein CP47, but the mode of interaction and pigment binding have not been resolved. Here, we isolated His-tagged HliA and HliB from Synechocystis and show that these two very similar Hlips do not interact with each other as anticipated, rather they form HliAC and HliBC heterodimers. Both dimers bind Chl and ß-carotene in a quenching conformation and associate with the CP47 assembly module as well as later PSII assembly intermediates containing CP47. In the absence of HliC, the cellular levels of HliA and HliB were reduced, and both bound atypically to HliD. We postulate a model in which HliAC-, HliBC-, and HliDC-dimers are the functional Hlip units in Synechocystis. The smallest Hlip, HliC, acts as a 'generalist' that prevents unspecific dimerization of PSII assembly intermediates, while the N-termini of 'specialists' (HliA, B or D) dictate interactions with proteins other than Hlips.

LIGHT controls distinct homeostatic and inflammatory gene expression profiles in esophageal fibroblasts via differential HVEM and LTßR-mediated mechanisms.

Fibroblasts mediate tissue remodeling in eosinophilic esophagitis (EoE), a chronic allergen-driven inflammatory pathology. Diverse fibroblast subtypes with homeostasis-regulating or inflammatory profiles have been recognized in various tissues, but which mediators induce these alternate differentiation states remain largely unknown. We recently identified that TNFSF14/LIGHT promotes an inflammatory esophageal fibroblast in vitro. Herein we used esophageal biopsies and primary fibroblasts to investigate the role of the LIGHT receptors, herpes virus entry mediator (HVEM) and lymphotoxin-beta receptor (LTßR), and their downstream activated pathways, in EoE. In addition to promoting inflammatory gene expression, LIGHT down-regulated homeostatic factors including WNTs, BMPs and type 3 semaphorins. In vivo, WNT2B+ fibroblasts were decreased while ICAM-1+ and IL-34+ fibroblasts were expanded in EoE, suggesting that a LIGHT-driven gene signature was imprinted in EoE versus normal esophageal fibroblasts. HVEM and LTßR overexpression and deficiency experiments demonstrated that HVEM regulates a limited subset of LIGHT targets, whereas LTßR controls all transcriptional effects. Pharmacologic blockade of the non-canonical NIK/p100/p52-mediated NF-κB pathway potently silenced LIGHT's transcriptional effects, with a lesser role found for p65 canonical NF-κB. Collectively, our results show that LIGHT promotes differentiation of esophageal fibroblasts toward an inflammatory phenotype and represses homeostatic gene expression via a LTßR-NIK-p52 NF-κB dominant pathway.

The Contribution of LIGHT (TNFSF14) to the Development of Systemic Sclerosis by Modulating IL-6 and T Helper Type 1 Chemokine Expression in Dermal Fibroblasts.

Systemic sclerosis (SSc) is an autoimmune and vascular disease resulting in multiple organ fibrosis, in which IL-6 and T helper (Th)2/Th17 cytokines serve as critical disease drivers. LIGHT is a proinflammatory cytokine promoting IL-6 production in lung fibroblasts and Th1 chemokine expression in dermal fibroblasts (DFs) stimulated with IFN-γ. In this study, we investigated the potential contribution of LIGHT to SSc development using clinical samples and animal models. In SSc-involved skin, LIGHT was upregulated in inflammatory cells, whereas herpesvirus entry mediator (HVEM), a receptor of LIGHT, was downregulated in DFs. Similar expression profiles of LIGHT and HVEM were reproduced in bleomycin-treated mice. Transcription factor FLI1 bound to the HVEM promoter, and FLI1 small interfering RNA suppressed HVEM expression in normal DFs. In SSc DFs, LIGHT significantly increased IL-6 production, whereas IFN-γ/LIGHT-dependent Th1 chemokine induction was decreased compared with that in normal DFs. Importantly, LIGHT small interfering RNA significantly attenuated bleomycin-induced skin fibrosis, and serum LIGHT levels were elevated in patients with diffuse cutaneous SSc and positively correlated with clinical parameters reflecting skin and pulmonary fibrosis. Taken together, these results suggest that altered response of DFs to LIGHT, namely increased IL-6 production and decreased Th1 chemokine expression, contributes to the development of skin fibrosis in SSc.

TNFSF14-Derived Molecules as a Novel Treatment for Obesity and Type 2 Diabetes.

Obesity is one of the most prevalent metabolic diseases in the Western world and correlates directly with glucose intolerance and insulin resistance, often culminating in Type 2 Diabetes (T2D). Importantly, our team has recently shown that the TNF superfamily (TNFSF) member protein, TNFSF14, has been reported to protect against high fat diet induced obesity and pre-diabetes. We hypothesized that mimics of TNFSF14 may therefore be valuable as anti-diabetic agents. In this study, we use in silico approaches to identify key regions of TNFSF14 responsible for binding to the Herpes virus entry mediator and Lymphotoxin ß receptor. In vitro evaluation of a selection of optimised peptides identified six potentially therapeutic TNFSF14 peptides. We report that these peptides increased insulin and fatty acid oxidation signalling in skeletal muscle cells. We then selected one of these promising peptides to determine the efficacy to promote metabolic benefits in vivo. Importantly, the TNFSF14 peptide 7 reduced high fat diet-induced glucose intolerance, insulin resistance and hyperinsulinemia in a mouse model of obesity. In addition, we highlight that the TNFSF14 peptide 7 resulted in a marked reduction in liver steatosis and a concomitant increase in phospho-AMPK signalling. We conclude that TNFSF14-derived molecules positively regulate glucose homeostasis and lipid metabolism and may therefore open a completely novel therapeutic pathway for treating obesity and T2D.

HVEM structures and mutants reveal distinct functions of binding to LIGHT and BTLA/CD160.

HVEM is a TNF (tumor necrosis factor) receptor contributing to a broad range of immune functions involving diverse cell types. It interacts with a TNF ligand, LIGHT, and immunoglobulin (Ig) superfamily members BTLA and CD160. Assessing the functional impact of HVEM binding to specific ligands in different settings has been complicated by the multiple interactions of HVEM and HVEM binding partners. To dissect the molecular basis for multiple functions, we determined crystal structures that reveal the distinct HVEM surfaces that engage LIGHT or BTLA/CD160, including the human HVEM-LIGHT-CD160 ternary complex, with HVEM interacting simultaneously with both binding partners. Based on these structures, we generated mouse HVEM mutants that selectively recognized either the TNF or Ig ligands in vitro. Knockin mice expressing these muteins maintain expression of all the proteins in the HVEM network, yet they demonstrate selective functions for LIGHT in the clearance of bacteria in the intestine and for the Ig ligands in the amelioration of liver inflammation.

Thymic Egress Is Regulated by T Cell-Derived LTßR Signal and via Distinct Thymic Portal Endothelial Cells.

Thymic blood vessels at the perivascular space (PVS) are the critical site for both homing of hematopoietic progenitor cells (HPCs) and egress of mature thymocytes. It has been intriguing how different opposite migrations can happen in the same place. A subset of specialized thymic portal endothelial cells (TPECs) associated with PVS has been identified to function as the entry site for HPCs. However, the cellular basis and mechanism underlying egress of mature thymocytes has not been well defined. In this study, using various conventional and conditional gene-deficient mouse models, we first confirmed the role of endothelial lymphotoxin beta receptor (LTßR) for thymic egress and ruled out the role of LTßR from epithelial cells or dendritic cells. In addition, we found that T cell-derived ligands lymphotoxin (LT) and LIGHT are required for thymic egress, suggesting a crosstalk between T cells and endothelial cells (ECs) for thymic egress control. Furthermore, immunofluorescence staining analysis interestingly showed that TPECs are also the exit site for mature thymocytes. Single-cell transcriptomic analysis of thymic endothelial cells suggested that TPECs are heterogeneous and can be further divided into two subsets depending on BST-1 expression level. Importantly, BST-1hi population is associated with thymic egressing thymocytes while BST-1lo/- population is associated with HPC settling. Thus, we have defined a LT/LIGHT-LTßR signaling-mediated cellular crosstalk regulating thymic egress and uncovered distinct subsets of TPECs controlling thymic homing and egress, respectively.

The tumor necrosis factor family molecules LIGHT and lymphotoxins in sinus mucosa of patients with chronic rhinosinusitis with or without nasal polyps.

BACKGROUND: Little is known about the role of lymphotoxins (LTs) family in the sinonasal mucosa of patients with chronic rhinosinusitis (CRS). This study aims at investigating the expression of LIGHT, LTα, LTß, and their receptors, LTßR and HVEM in normal and inflammatory sinus mucosa, and the effect of LIGHT and LTalpha1beta2 on chemokine secretion in epithelial cells, epithelial permeability, and leukocyte migration. MATERIAL AND METHODS: The expression of LTs family in sinonasal mucosa was evaluated with real-time PCR, immunohistochemistry, and western blot. In LTßR, HVEM siRNA, or control siRNA-transfected epithelial cells treated with LIGHT or LTalpha1beta2, the expression of chemokines, the epithelial permeability, and the expression of junctional complex proteins were evaluated using real-time PCR, ELISA, western blot, confocal microscopy, and FITC-dextran. In cultured endothelial cells treated with LIGHT or LTalpha1beta2, the expression of ICAM-1 and VCAM-1, and leukocyte migration were elucidated. RESULTS: LTs family was expressed in normal mucosa and their levels were increased in inflammatory mucosa of CRS patients. Recombinant LIGHT and LTalpha1beta2 induced chemokine secretion, increased epithelial permeability, and promoted leukocyte migration. However, the activity of LIGHT and LTalpha1beta2 was attenuated in cells transfected with LTßR and HVEM siRNA. CONCLUSIONS: LIGHT and LTs may participate in the ongoing process of chronic inflammation, inducing chemokine secretion, leukocyte migration, and dysregulated epithelial barrier through LTßR and HVEM in sinonasal mucosa.

Model Lipid Membranes Assembled from Natural Plant Thylakoids into 2D Microarray Patterns as a Platform to Assess the Organization and Photophysics of Light-Harvesting Proteins.

Natural photosynthetic "thylakoid" membranes found in green plants contain a large network of light-harvesting (LH) protein complexes. Rearrangement of this photosynthetic machinery, laterally within stacked membranes called "grana", alters protein-protein interactions leading to changes in the energy balance within the system. Preparation of an experimentally accessible model system that allows the detailed investigation of these complex interactions can be achieved by interfacing thylakoid membranes and synthetic lipids into a template comprised of polymerized lipids in a 2D microarray pattern on glass surfaces. This paper uses this system to interrogate the behavior of LH proteins at the micro- and nanoscale and assesses the efficacy of this model. A combination of fluorescence lifetime imaging and atomic force microscopy reveals the differences in photophysical state and lateral organization between native thylakoid and hybrid membranes, the mechanism of LH protein incorporation into the developing hybrid membranes, and the nanoscale structure of the system. The resulting model system within each corral is a high-quality supported lipid bilayer that incorporates laterally mobile LH proteins. Photosynthetic activity is assessed in the hybrid membranes versus proteoliposomes, revealing that commonly used photochemical assays to test the electron transfer activity of photosystem II may actually produce false-positive results.