The Aging Lacrimal Gland of Female C57BL/6J Mice Exhibits Multinucleate Macrophage Infiltration Associated With Lipid Dysregulation.

Purpose: Loss of function of the lacrimal gland (LG), which produces the aqueous tear film, is implicated in age-related dry eye. To better understand this deterioration, we evaluated changes in lipid metabolism and inflammation in LGs from an aging model. Methods: LG sections from female C57BL/6J mice of different ages (young, 2-3 months; intermediate, 10-14 months; old,  ≥24 months) were stained with Oil Red-O or Toluidine blue to detect lipids. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis and western blotting of LG lysates determined differences in the expression of genes and proteins related to lipid metabolism. A photobleaching protocol to quench age-related autofluorescence was used in LG sections to evaluate changes in immunofluorescence associated with NPC1, NPC2, CTSL, and macrophages (F4/80, CD11b) with age using confocal fluorescence microscopy. Results: Old LGs showed increased lipids prominent in basal aggregates in acinar cells and in extra-acinar sites. LG gene expression of Npc1, Npc2, Lipa, and Mcoln2, encoding proteins involved in lipid metabolism, was increased with age. NPC1 was also significantly increased in old LGs by western blotting. In photobleached LG sections, confocal fluorescence microscopy imaging of NPC1, NPC2, and CTSL immunofluorescence showed age-associated enrichment in macrophages labeled to detect F4/80. Although mononuclear macrophages were detectable in LG at all ages, this novel multinucleate macrophage population containing NPC1, NPC2, and CTSL and enriched in F4/80 and some CD11b was increased with age at extra-acinar sites. Conclusions: Lipid-metabolizing proteins enriched in F4/80-positive multinucleated macrophages are increased in old LGs adjacent to sites of lipid deposition in acini.

Supra-lacrimal protein-based carriers for cyclosporine A reduce Th17-mediated autoimmunity in murine model of Sjögren's syndrome.

Sjögren's syndrome (SS) is a multifactorial autoimmune disease with principal symptoms including inflammation and loss of function of lacrimal glands (LG) and salivary glands. While glandular infiltrates includes both B- and T-cells, CD4+ T cells are strongly implicated. Utilizing the male non-obese diabetic (NOD) mouse model of SS, this work: 1) identifies clinically-relevant elevations in cytokines (IL-17A, IL-2) in LG-derived CD4+ T cells; and 2) explores tissue-specific immunosuppression of SS using a novel protein-based drug carrier to concentrate cyclosporine A (CsA) directly in the LG. As a potent immunosuppressant, topical ophthalmic CsA is approved for dry eye disorders; however, it cannot effectively resolve inflammation due to limited accumulation in the LG. Systemic CsA has dose-limiting side effects that also limit its ability to block LG inflammation. Using elastin-like polypeptides (ELPs) fused genetically to cyclophilin, the intracellular cognate receptor of CsA, this manuscript reports a sustained-release formulation of CsA that maintains therapeutic drug concentrations in the LG and extends intervals between doses. This formulation blocked both in vitro Th17 cell differentiation and IL-17A secretion. In vivo treatment significantly decreased the abundance of Th17.1 cells, a helper cell population sharing phenotypes of both Th17 and Th1, in the LG of diseased NOD mice. Treatment with even a single dose of the sustained-release formulation was effective enough to improve basal levels of tear production. Thus, this sustained-release formulation suppressed local LG inflammation driven through IL-17 dependent pathways, while improving ocular surface function.

Live long and active: Polypeptide-mediated assembly of antibody variable fragments.

Antibodies possess multiple biologically relevant features that have been engineered into new therapeutic formats. Two examples include the adaptable specificity of their variable (Fv) region and the extension of plasma circulation times through their crystallizable (Fc) region. Since the invention of the single chain variable fragment (scFv) in 1988, antibody variable regions have been re-engineered into a wide variety of multifunctional nanostructures. Among these strategies, peptide-mediated self-assembly of variable regions through heterologous expression has become a powerful method to produce homogenous, functional biomaterials. This manuscript reviews recent reports of antibody fragments assembled through fusion with peptides and proteins, including elastin-like polypeptides (ELPs), collagen-like polypeptides (CLPs), albumin, transmembrane proteins, leucine zippers, silk protein, and viruses. This review further discusses the current clinical status of engineered antibody fragments and challenges to overcome.

Cathepsin S activation contributes to elevated CX3CL1 (fractalkine) levels in tears of a Sjögren's syndrome murine model.

Autoimmune dacryoadenitis and altered lacrimal gland (LG) secretion are features of Sjögren's syndrome (SS). Activity of cathepsin S (CTSS), a cysteine protease, is significantly and specifically increased in SS patient tears. The soluble chemokine, CX3CL1 (fractalkine), is cleaved from membrane-bound CX3CL1 by proteases including CTSS. We show that CX3CL1 is significantly elevated by 2.5-fold in tears (p = 0.0116) and 1.4-fold in LG acinar cells (LGAC)(p = 0.0026) from male NOD mice, a model of autoimmune dacryoadenitis in SS, relative to BALB/c controls. Primary mouse LGAC and human corneal epithelial cells (HCE-T cells) exposed to interferon-gamma, a cytokine elevated in SS, showed up to 9.6-fold (p ≤ 0.0001) and 25-fold (p ≤ 0.0001) increases in CX3CL1 gene expression, and 1.9-fold (p = 0.0005) and 196-fold (p ≤ 0.0001) increases in CX3CL1 protein expression, respectively. Moreover, exposure of HCE-T cells to recombinant human CTSS at activity equivalent to that in SS patient tears increased cellular CX3CL1 gene and protein expression by 2.8-fold (p = 0.0021) and 5.1-fold (p ≤ 0.0001), while increasing CX3CL1 in culture medium by 5.8-fold (p ≤ 0.0001). Flow cytometry demonstrated a 4.5-fold increase in CX3CR1-expressing immune cells (p ≤ 0.0001), including increased T-cells and macrophages, in LG from NOD mice relative to BALB/c. CTSS-mediated induction/cleavage of CX3CL1 may contribute to ocular surface and LG inflammation in SS.