Paracrine regulations of IFN-γ secreting CD4 + T cells by lumican and biglycan are protective in allergic contact dermatitis.

The extracellular matrix (ECM) is known to regulate innate immune cells but its role in T cell functions is poorly understood. Here, we show a protective role for ECM proteoglycans, lumican and biglycan in hapten-induced contact dermatitis that is achieved through limiting proinflammatory CD4 + T cells. Lumican and biglycan-null mice develop significant inflammation with greater numbers of CD4 + T cells in hapten-challenged ear pinnae, while their draining lymph nodes show increased T-bet-STAT1 signaling, Th1 commitment, and IFN-γ secreting CD4 + T cell proliferation. Wild type mouse lymph node fibroblastic reticular cells secrete lumican, biglycan and decorin, a related proteoglycan, while none are expressed by naive or activated T cells. In vitro , lumican and biglycan co-localize with LFA-1 on T cell surfaces, and all three proteoglycans suppress LFA-1 mediated T cell activation. Overall, this study elucidates a novel paracrine regulation of Th1 cells by ECM proteoglycans to limit inflammation and tissue damage.

Regulatory T cells and IFN-γ-producing Th1 cells play a critical role in the pathogenesis of Sjögren's Syndrome.

Objectives: Sjögren's Disease (SjD) is an autoimmune disorder characterized by progressive dysfunction, inflammation and destruction of salivary and lacrimal glands, and by extraglandular manifestations. Its etiology and pathophysiology remain incompletely understood, though a role for autoreactive B cells has been considered key. Here, we investigated the role of effector and regulatory T cells in the pathogenesis of SjD. Methods: Histological analysis, RNA-sequencing and flow cytometry were conducted on glands, lungs, eyes and lymphoid tissues of mice with regulatory T cell-specific deletion of stromal interaction proteins (STIM) 1 and 2 ( Stim1/2 Foxp3 ), which play key roles in calcium signaling and T cell function. The pathogenicity of T cells from Stim1/2 Foxp3 mice was investigated through adoptively transfer into lymphopenic host mice. Additionally, single-cell transcriptomic analysis was performed on peripheral blood mononuclear cells (PBMCs) of patients with SjD and control subjects. Results: Stim1/2 Foxp3 mice develop a severe SjD-like disorder including salivary gland (SG) and lacrimal gland (LG) inflammation and dysfunction, autoantibodies and extraglandular symptoms. SG inflammation in Stim1/2 Foxp3 mice is characterized by T and B cell infiltration, and transcriptionally by a Th1 immune response that correlates strongly with the dysregulation observed in patients with SjD. Adoptive transfer of effector T cells from Stim1/2 Foxp3 mice demonstrates that the SjD-like disease is driven by interferon (IFN)-γ producing autoreactive CD4 + T cells independently of B cells and autoantiboodies. scRNA-seq analysis identifies increased Th1 responses and attenuated memory Treg function in PBMCs of patients with SjD. Conclusions: We report a more accurate mouse model of SjD while providing evidence for a critical role of Treg cells and IFN-γ producing Th1 cells in the pathogenesis of SjD, which may be effective targets for therapy.

Three-Dimensional Modeling of CpG DNA Binding with Matrix Lumican Shows Leucine-Rich Repeat Motif Involvement as in TLR9-CpG DNA Interactions.

Lumican is an extracellular matrix proteoglycan known to regulate toll-like receptor (TLR) signaling in innate immune cells. In experimental settings, lumican suppresses TLR9 signaling by binding to and sequestering its synthetic ligand, CpG-DNA, in non-signal permissive endosomes. However, the molecular details of lumican interactions with CpG-DNA are obscure. Here, the 3-D structure of the 22 base-long CpG-DNA (CpG ODN_2395) bound to lumican or TLR9 were modeled using homology modeling and docking methods. Some of the TLR9-CpG ODN_2395 features predicted by our model are consistent with the previously reported TLR9-CpG DNA crystal structure, substantiating our current analysis. Our modeling indicated a smaller buried surface area for lumican-CpG ODN_2395 (1803 Å2) compared to that of TLR9-CpG ODN_2395 (2094 Å2), implying a potentially lower binding strength for lumican and CpG-DNA than TLR9 and CpG-DNA. The docking analysis identified 32 amino acids in lumican LRR1-11 interacting with CpG ODN_2395, primarily through hydrogen bonding, salt-bridges, and hydrophobic interactions. Our study provides molecular insights into lumican and CpG-DNA interactions that may lead to molecular targets for modulating TLR9-mediated inflammation and autoimmunity.

Molecular cues for immune cells from small leucine-rich repeat proteoglycans in their extracellular matrix-associated and free forms.

In this review we highlight emerging immune regulatory functions of lumican, keratocan, fibromodulin, biglycan and decorin, which are members of the small leucine-rich proteoglycans (SLRP) of the extracellular matrix (ECM). These SLRPs have been studied extensively as collagen-fibril regulatory structural components of the skin, cornea, bone and cartilage in homeostasis. However, SLRPs released from a remodeling ECM, or synthesized by activated fibroblasts and immune cells contribute to an ECM-free pool in tissues and circulation, that may have a significant, but poorly understood foot print in inflammation and disease. Their molecular interactions and the signaling networks they influence also require investigations. Here we present studies on the leucine-rich repeat (LRR) motifs of SLRP core proteins, their evolutionary and functional relationships with other LRR pathogen recognition receptors, such as the toll-like receptors (TLRs) to bring some molecular clarity in the immune regulatory functions of SLRPs. We discuss molecular interactions of fragments and intact SLRPs, and how some of these interactions are likely modulated by glycosaminoglycan side chains. We integrate findings on molecular interactions of these SLRPs together with what is known about their presence in circulation and lymph nodes (LN), which are important sites of immune cell regulation. Recent bulk and single cell RNA sequencing studies have identified subsets of stromal reticular cells that express these SLRPs within LNs. An understanding of the cellular source, molecular interactions and signaling consequences will lead to a fundamental understanding of how SLRPs modulate immune responses, and to therapeutic tools based on these SLRPs in the future.

3D modeling of CpG DNA binding with matrix lumican shows leucine-rich repeat motif involvement as in TLR9-CpG DNA interactions.

Lumican is an extracellular matrix proteoglycan, known to regulate toll-like receptor (TLR) signaling in innate immune cells. In experimental settings, lumican suppresses TLR9 signaling by binding to, and sequestering its synthetic ligand, CpG-DNA, in non-signal permissive endosomes. However, the molecular details of lumican interactions with CpG-DNA are obscure. Here, the 3-D structure of the 22 base-long CpG-DNA (CpG ODN_2395) bound to lumican or TLR9 were modeled using homology modeling and docking methods. Some of the TLR9-CpG ODN_2395 features predicted by our model are consistent with the previously reported TLR9-CpG DNA crystal structure, substantiating our current analysis. Our modeling indicated a smaller buried surface area for lumican-CpG ODN_2395 (1803 Å2) compared to that of TLR9-CpG ODN_2395 (2094 Å2), implying a potentially lower binding strength for lumican and CpG-DNA than TLR9 and CpG-DNA. The docking analysis identified 32 amino acids in lumican LRR1-11 interacting with CpG ODN_2395, primarily through hydrogen bonding, salt-bridges and hydrophobic interactions. Our study provides molecular insights into lumican and CpG-DNA interactions that may lead to molecular targets for modulating TLR9 mediated inflammation and autoimmunity.

Genetic Disorders of the Extracellular Matrix: From Cell and Gene Therapy to Future Applications in Regenerative Medicine.

Metazoans have evolved to produce various types of extracellular matrix (ECM) that provide structural support, cell adhesion, cell-cell communication, and regulated exposure to external cues. Epithelial cells produce and adhere to a specialized sheet-like ECM, the basement membrane, that is critical for cellular homeostasis and tissue integrity. Mesenchymal cells, such as chondrocytes in cartilaginous tissues and keratocytes in the corneal stroma, produce a pericellular matrix that presents optimal levels of growth factors, cytokines, chemokines, and nutrients to the cell and regulates mechanosensory signals through specific cytoskeletal and cell surface receptor interactions. Here, we discuss laminins, collagen types IV and VII, and perlecan, which are major components of these two types of ECM. We examinegenetic defects in these components that cause basement membrane pathologies such as epidermolysis bullosa, Alport syndrome, rare pericellular matrix-related chondrodysplasias, and corneal keratoconus and discuss recent advances in cell and gene therapies being developed for some of these disorders.

Pathogenesis of keratoconus: NRF2-antioxidant, extracellular matrix and cellular dysfunctions.

Keratoconus (KC) is a degenerative disease associated with cell and extracellular matrix (ECM) loss that causes gradual thinning and steepening of the cornea and loss of vision. Collagen cross linking with ultraviolet light treatment can strengthen the ECM and delay weakening of the cornea, but severe cases require corneal transplantation. KC is multifactorial and multigenic, but its pathophysiology is still an enigma. Multiple approaches are being pursued to elucidate the molecular changes that underlie the corneal phenotype to identify relevant genes for tailored candidate searches and to develop potential biomarkers and targets for therapeutic interventions. Recent proteomic and transcriptomic studies suggest dysregulations in oxidative stress, NRF2-regulated antioxidant programs, WNT-signaling, TGF-ß, ECM and matrix metalloproteinases. This review aims to provide a broad update on the transcriptomic and proteomic studies of KC with a focus on findings that relate to oxidative stress, and dysregulations in cellular and extracellular matrix functions.

Single cell RNA-seq of human cornea organoids identifies cell fates of a developing immature cornea.

The cornea is a protective and refractive barrier in the eye crucial for vision. Understanding the human cornea in health, disease, and cell-based treatments can be greatly advanced with cornea organoids developed in culture from induced pluripotent stem cells. While a limited number of studies have investigated the single-cell transcriptomic composition of the human cornea, its organoids have not been examined similarly. Here, we elucidated the transcriptomic cell fate map of 4-month-old human cornea organoids and human donor corneas. The organoids harbor cell clusters that resemble cells of the corneal epithelium, stroma, and endothelium, with subpopulations that capture signatures of early developmental states. Unlike the adult cornea where the largest cell population is stromal, the organoids contain large proportions of epithelial and endothelial-like cells. These corneal organoids offer a 3D model to study corneal diseases and integrated responses of different cell types.

Matrix lumican endocytosed by immune cells controls receptor ligand trafficking to promote TLR4 and restrict TLR9 in sepsis.

Infections and inflammation are profoundly influenced by the extracellular matrix (ECM), but their molecular underpinnings are ill defined. Here, we demonstrate that lumican, an ECM protein normally associated with collagens, is elevated in sepsis patients' blood, while lumican-null mice resolve polymicrobial sepsis poorly, with reduced bacterial clearance and greater body weight loss. Secreted by activated fibroblasts, lumican promotes Toll-like receptor (TLR) 4 response to bacterial lipopolysaccharides (LPS) but restricts nucleic acid-specific TLR9 in macrophages and dendritic cells. The underlying mechanism involves lumican attachment to the common TLR coreceptor CD14 and caveolin 1 (Cav1) in lipid rafts on immune cell surfaces via two epitopes, which may be cryptic in collagen-associated lumican. The Cav1 binding epitope alone is sufficient for cell surface enrichment of Cav1, while both are required for lumican to increase cell surface TLR4, CD14, and proinflammatory cytokines in response to LPS. Endocytosed lumican colocalizes with TLR4 and LPS and promotes endosomal induction of type I interferons. Lumican-null macrophages show elevated TLR9 in signal-permissive endolysosomes and increased response, while wild types show lumican colocalization with CpG DNA but not TLR9, consistent with a ligand sequestering, restrictive role for lumican in TLR9 signaling. In vitro, lumican competes with CD14 to bind CpG DNA; biglycan, a lumican paralog, also binds CpG DNA and suppresses TLR9 response. Thus, lumican and other ECM proteins, synthesized de novo or released from collagen association during ECM remodeling, may be internalized by immune cells to regulate their transcriptional programs and effector responses that may be harnessed in future therapeutics.

Transcriptomic and Immunohistochemical Analysis of Progressive Keratoconus Reveal Altered WNT10A in Epithelium and Bowman's Layer.

Purpose: To identify global gene expression changes in the corneal epithelium of keratoconus (KC) patients compared to non-KC myopic controls. Methods: RNA-sequencing was performed on corneal epithelium samples of five progressive KC and five myopic control patients. Selected results were validated using TaqMan quantitative PCR (qPCR) on 31 additional independent samples, and protein level validation was conducted using western blot analysis on a subset. Immunohistochemistry was performed on tissue microarrays containing cores from over 100 KC and control cases. WNT10A transcript levels in corneal epithelium were correlated with tomographic indicators of KC disease severity in 15 eyes. Additionally, WNT10A was overexpressed in vitro in immortalized corneal epithelial cells. Results: WNT10A was found to be underexpressed in KC epithelium at the transcript (ratio KC/control = 0.59, P = 0.02 per RNA-sequencing study; ratio = 0.66, P = 0.03 per qPCR) and protein (ratio = 0.07, P = 0.06) levels. Immunohistochemical analysis also indicated WNT10A protein was decreased in Bowman's layer of KC patients. In contrast, WNT10A transcript level positively correlated with increased keratometry (Kmax ρ = 0.57, P = 0.02). Finally, WNT10A positively regulated COL1A1 expression in corneal epithelial cells. Conclusions: A specific Wnt ligand, WNT10A, is reduced at the mRNA and protein level in KC epithelium and Bowman's layer. This ligand positively regulates collagen type I expression in corneal epithelial cells. The results suggest that WNT10A expression in the corneal epithelium may play a role in progressive KC.

Pathogenic alleles in microtubule, secretory granule and extracellular matrix-related genes in familial keratoconus.

Keratoconus is a common corneal defect with a complex genetic basis. By whole exome sequencing of affected members from 11 multiplex families of European ancestry, we identified 23 rare, heterozygous, potentially pathogenic variants in 8 genes. These include nonsynonymous single amino acid substitutions in HSPG2, EML6 and CENPF in two families each, and in NBEAL2, LRP1B, PIK3CG and MRGPRD in three families each; ITGAX had nonsynonymous single amino acid substitutions in two families and an indel with a base substitution producing a nonsense allele in the third family. Only HSPG2, EML6 and CENPF have been associated with ocular phenotypes previously. With the exception of MRGPRD and ITGAX, we detected the transcript and encoded protein of the remaining genes in the cornea and corneal cell cultures. Cultured stromal cells showed cytoplasmic punctate staining of NBEAL2, staining of the fibrillar cytoskeletal network by EML6, while CENPF localized to the basal body of primary cilia. We inhibited the expression of HSPG2, EML6, NBEAL2 and CENPF in stromal cell cultures and assayed for the expression of COL1A1 as a readout of corneal matrix production. An upregulation in COL1A1 after siRNA inhibition indicated their functional link to stromal cell biology. For ITGAX, encoding a leukocyte integrin, we assayed its level in the sera of 3 affected families compared with 10 unrelated controls to detect an increase in all affecteds. Our study identified genes that regulate the cytoskeleton, protein trafficking and secretion, barrier tissue function and response to injury and inflammation, as being relevant to keratoconus.

RNA sequencing of corneas from two keratoconus patient groups identifies potential biomarkers and decreased NRF2-antioxidant responses.

Keratoconus is a highly prevalent (1 in 2000), genetically complex and multifactorial, degenerative disease of the cornea whose pathogenesis and underlying transcriptomic changes are poorly understood. To identify disease-specific changes and gene expression networks, we performed next generation RNA sequencing from individual corneas of two distinct patient populations - one from the Middle East, as keratoconus is particularly severe in this group, and the second from an African American population in the United States. We conducted a case: control RNA sequencing study of 7 African American, 12 Middle Eastern subjects, and 7 controls. A Principal Component Analysis of all expressed genes was used to ascertain differences between samples. Differentially expressed genes were identified using Cuffdiff and DESeq2 analyses, and identification of over-represented signaling pathways by Ingenuity Pathway Analysis. Although separated by geography and ancestry, key commonalities in the two patient transcriptomes speak of disease - intrinsic gene expression networks. We identified an overwhelming decrease in the expression of anti-oxidant genes regulated by NRF2 and those of the acute phase and tissue injury response pathways, in both patient groups. Concordantly, NRF2 immunofluorescence staining was decreased in patient corneas, while KEAP1, which helps to degrade NRF2, was increased. Diminished NRF2 signaling raises the possibility of NRF2 activators as future treatment strategies in keratoconus. The African American patient group showed increases in extracellular matrix transcripts that may be due to underlying profibrogenic changes in this group. Transcripts increased across all patient samples include Thrombospondin 2 (THBS2), encoding a matricellular protein, and cellular proteins, GAS1, CASR and OTOP2, and are promising biomarker candidates. Our approach of analyzing transcriptomic data from different populations and patient groups will help to develop signatures and biomarkers for keratoconus subtypes. Further, RNA sequence data on individual patients obtained from multiple studies may lead to a core keratoconus signature of deregulated genes and a better understanding of its pathogenesis.

A Guide to the Development of Human CorneaOrganoids from Induced Pluripotent Stem Cells in Culture.

The cornea is the outermost transparent and refractive barrier surface of the eye necessary for vision. Development of the cornea involves the coordinated production of extracellular matrix, epithelial differentiation, and endothelial cell expansion to produce a highly transparent tissue. Here we describe the production of multilayered three-dimensional organoids from human-induced pluripotent stem cells. These organoids have the potential for multiple downstream applications which are currently unattainable using traditional in vitro techniques.

Moderate Loss of the Extracellular Matrix Proteoglycan Lumican Attenuates Cardiac Fibrosis in Mice Subjected to Pressure Overload.

INTRODUCTION: The heart undergoes myocardial remodeling during progression to heart failure following pressure overload. Myocardial remodeling is associated with structural and functional changes in cardiac myocytes, fibroblasts, and the extracellular matrix (ECM) and is accompanied by inflammation. Cardiac fibrosis, the accumulation of ECM molecules including collagens and collagen cross-linking, contributes both to impaired systolic and diastolic function. Insufficient mechanistic insight into what regulates cardiac fibrosis during pathological conditions has hampered therapeutic so-lutions. Lumican (LUM) is an ECM-secreted proteoglycan known to regulate collagen fibrillogenesis. Its expression in the heart is increased in clinical and experimental heart failure. Furthermore, LUM is important for survival and cardiac remodeling following pressure overload. We have recently reported that total lack of LUM increased mortality and left ventricular dilatation, and reduced collagen expression and cross-linking in LUM knockout mice after aortic banding (AB). Here, we examined the effect of LUM on myocardial remodeling and function following pressure overload in a less extreme mouse model, where cardiac LUM level was reduced to 50% (i.e., moderate loss of LUM). METHODS AND RESULTS: mRNA and protein levels of LUM were reduced to 50% in heterozygous LUM (LUM+/-) hearts compared to wild-type (WT) controls. LUM+/- mice were subjected to AB. There was no difference in survival between LUM+/- and WT mice post-AB. Echocardiography revealed no striking differences in cardiac geometry between LUM+/- and WT mice 2, 4, and 6 weeks post-AB, although markers of diastolic dysfunction indicated better function in LUM+/- mice. LUM+/- hearts revealed reduced cardiac fibrosis assessed by histology. In accordance, the expression of collagen I and III, the main fibrillar collagens in the heart, and other ECM molecules central to fibrosis, i.e. including periostin and fibronectin, was reduced in the hearts of LUM+/- compared to WT 6 weeks post-AB. We found no differences in collagen cross-linking between LUM+/- and WT mice post-AB, as assessed by histology and qPCR. CONCLUSIONS: Moderate lack of LUM attenuated cardiac fibrosis and improved diastolic dysfunction following pressure overload in mice, adding to the growing body of evidence suggesting that LUM is a central profibrotic molecule in the heart that could serve as a potential therapeutic target.

Mapping Keratoconus Molecular Substrates by Multiplexed High-Resolution Proteomics of Unpooled Corneas.

Keratoconus (KCN) is a leading cause for cornea grafting worldwide. Keratoconus is a multifactorial disease that causes progressive thinning of the cornea and whose etiology is poorly understood. Several studies have used proteomics on patient tear fluids to identify potential biomarkers. However, proteome of the cornea itself has not been investigated fully. We report here new findings from a case-control study using multiplexed mass spectrometry (MS) on individual (unpooled) corneas to gain deeper insights into proteins and biomarkers relevant to keratoconus. We employed a high-pressure approach to extract total protein from individual corneas from five cases and five controls, followed by trypsin digestion and tandem mass tag (TMT) labeling. The MS-derived data were searched using the Human NCBI RefSeq protein database v92, with peptides and proteins filtered at 1% false discovery rate. A total of 3132 proteins were detected, of which 627 were altered significantly (p ≤ 0.05) in keratoconus corneas. The increases were overwhelmingly in the mTOR/PI3/AKT signal-mediated regulations of cell survival and proliferation, nonsense-mediated decay of transcripts, and proteasomal pathways. The decreases were in several extracellular matrix proteins and in many members of the complement system. Importantly, this multiplexed proteomic study of keratoconus corneas identified, to our knowledge, the largest number of corneal proteins. The novel findings include changes in pathways that regulate transcript stability, proteasomal degradation, and the complement system in corneas with keratoconus. These observations offer new prospects toward future discovery of novel molecular targets for diagnostic and therapeutic innovations for patients with keratoconus.

Small Molecule Modulation of the Integrated Stress Response Governs the Keratoconic Phenotype In Vitro.

Purpose: The degenerative corneal disease keratoconus is a leading indicator for corneal transplant with an unknown etiology. We recently identified the activation of the integrated stress response (ISR) in ex vivo human corneas and in vitro cell culture. Utilizing small molecules to modulate the ISR we sought to investigate the effects of stimulating the ISR in healthy cells to recapitulate aspects of the in vitro keratoconic phenotype and whether relieving the ISR signaling would recover the disease phenotype. Methods: Corneal fibroblasts were extracted from patients undergoing corneal transplant or unaffected cadaverous donor limbal rings. Cells were exposed to the DNA damage-inducible protein (GADD34) inhibitor SAL003 to stimulate the ISR, or Trans-ISRIB to relieve ISR signaling pathway. Collagen production was assessed through hydroxyproline production, Sirius Red incorporation, or quantitative (q)PCR. Western blotting, hydroxyproline, and qPCR were used to assess components of the ISR pathway and collagen production. Results: ISR stimulation through SAL003 resulted in significant decrease of hydroxyproline and COL1A1 transcription and eventual apoptosis in normal fibroblasts. Patient (KC) fibroblast production of hydroxyproline was increased in response to ISRIB, while matrix metalloproteinase (MMP)9 production was lowered. The prospective biomarker of keratoconus prolactin-inducible factor was also upregulated in KC fibroblast cultures in response to ISRIB. Inflammatory markers TNFα and IL-1ß were unaffected. Conclusions: Activation of the ISR is sufficient to recapitulate many key aspects of the KC phenotype in unaffected cells in vitro. Inhibition of the ISR also relieves many of the hallmarks of KC in affected cells. Therefore, targeting of the ISR through small molecules is a potential therapeutic path for small molecule treatment of keratoconus.

The extracellular matrix proteoglycan lumican improves survival and counteracts cardiac dilatation and failure in mice subjected to pressure overload.

Left ventricular (LV) dilatation is a key step in transition to heart failure (HF) in response to pressure overload. Cardiac extracellular matrix (ECM) contains fibrillar collagens and proteoglycans, important for maintaining tissue integrity. Alterations in collagen production and cross-linking are associated with cardiac LV dilatation and HF. Lumican (LUM) is a collagen binding proteoglycan with increased expression in hearts of patients and mice with HF, however, its role in cardiac function remains poorly understood. To examine the role of LUM in pressure overload induced cardiac remodeling, we subjected LUM knock-out (LUMKO) mice to aortic banding (AB) and treated cultured cardiac fibroblasts (CFB) with LUM. LUMKO mice exhibited increased mortality 1-14 days post-AB. Echocardiography revealed increased LV dilatation, altered hypertrophic remodeling and exacerbated contractile dysfunction in surviving LUMKO 1-10w post-AB. LUMKO hearts showed reduced collagen expression and cross-linking post-AB. Transcriptional profiling of LUMKO hearts by RNA sequencing revealed 714 differentially expressed transcripts, with enrichment of cardiotoxicity, ECM and inflammatory pathways. CFB treated with LUM showed increased mRNAs for markers of myofibroblast differentiation, proliferation and expression of ECM molecules important for fibrosis, including collagens and collagen cross-linking enzyme lysyl oxidase. In conclusion, we report the novel finding that lack of LUM attenuates collagen cross-linking in the pressure-overloaded heart, leading to increased mortality, dilatation and contractile dysfunction in mice.

Syndecan-1 Regulates Psoriasiform Dermatitis by Controlling Homeostasis of IL-17-Producing γδ T Cells.

IL-17 is a potent proinflammatory cytokine that drives pathogenesis of multiple autoimmune diseases, including psoriasis. A major source of pathogenic IL-17 is a subset of γδ T cells (Tγδ17) that acquires the ability to produce IL-17 while developing in the thymus. The mechanisms that regulate homeostasis of Tγδ17 cells and their roles in psoriasis, however, are not fully understood. In this paper, we show that the heparan sulfate proteoglycan syndecan-1 (sdc1) plays a critical role in regulating homeostasis of Tγδ17 cells and modulating psoriasis-like skin inflammation in mice. sdc1 was predominantly expressed by Tγδ17 cells (but not IL-17- Tγδ cells) in the thymus, lymph nodes, and dermis. sdc1 deficiency significantly and selectively increased the frequency and absolute numbers of Tγδ17 cells by mechanisms that included increased proliferation and decreased apoptosis. Adoptive transfer experiments ruled out a significant role of sdc1 expressed on nonhematopoietic cells in halting expansion and proliferation of sdc1-deficient Tγδ17 cells. When subjected to imiquimod-induced psoriasiform dermatitis, Tγδ17 cells in sdc1KO mice displayed heightened responses accompanied by significantly increased skin inflammation than their wild-type counterparts. Furthermore, transferred sdc1-deficient γδ T cells caused more severe psoriasiform dermatitis than their sdc1-sufficient counterparts in TCR-ßδ KO hosts. The results uncover a novel role for sdc1 in controlling homeostasis of Tγδ17 cells and moderating host responses to psoriasis-like inflammation.

Integrated Stress Response and Decreased ECM in Cultured Stromal Cells From Keratoconus Corneas.

Purpose: Keratoconus (KC) is a multifactorial disease where progressive thinning and weakening of the cornea leads to loss of visual acuity. Although the underlying etiology is poorly understood, a major endpoint is a dysfunctional stromal connective tissue matrix. Using multiple individual KC corneas, we determined that matrix production by keratocytes is severely impeded due to an altered stress response program. Methods: KC and donor (DN) stromal keratocytes were cultured in low glucose serum-free medium containing insulin, selenium and transferrin. Fibronectin, collagens and proteins related to their chaperone, processing and export, matrix metalloproteinase, and stress response related proteins were investigated by immunoblotting, immunocytochemistry, hydroxyproline quantification, and gelatin zymography. Multiplexed mass spectrometry was used for global proteomic profiling of 5 individual DN and KC cell culture. Transcription of selected proteins was assayed by qPCR. Results: DN and KC cells showed comparable survival and growth. However, immunoblotting of selected ECM proteins and global proteomics showed decreased fibronectin, collagens, PCOLCE, ADAMTS2, BMP1, HSP47, other structural and cytoskeletal proteins in KC. Phosphorylated (p) eIF2α, a translation regulator and its target, ATF4 were increased in KC cultured cells and corneal sections. Conclusions: The profound decrease in structural proteins in cultured KC cells and increase in the p-eIF2α, and ATF4, suggest a stress related blockade in structural proteins not immediately needed for cell survival. Therefore, this cell culture system reveals an intrinsic aggravated stress response with consequent decrease in ECM proteins as potential pathogenic underpinnings in KC.

Pathophysiology of Keratoconus: What Do We Know Today.

Keratoconus is a common corneal ectasia that leads to progressive visual impairment. Numerous studies have shown abnormal protein expression patterns in keratoconic corneas. However, the specific mechanisms causing this disease remain ambiguous. This review aims to provide an update on morphological studies of the keratoconic cornea, relate these early studies with current findings from proteomic, biochemical and cell culture studies and to postulate possible pathogenic pathways.