Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model.

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders. Here, we provide a detailed protocol on EAU induction in the C57BL/6J mouse - the most widely used model organism with susceptibility to this disease. Clinical assessment of disease severity and progression will be demonstrated using fundoscopy, histological examination and fluorescein angiography. The induction procedure involves subcutaneous injection of an emulsion containing a peptide (IRBP1-20) from the ocular protein interphotoreceptor retinoid binding protein (also known as retinol binding protein 3), Complete Freund's Adjuvant (CFA) and supplemented with killed Mycobacterium tuberculosis. Injection of this viscous emulsion on the back of the neck is followed by a single intraperitoneal injection of Bordetella pertussis toxin. At the onset of symptoms (day 12-14) and under general anesthesia, fundoscopic images are taken to assess disease progression through clinical examination. These data can be directly compared with those at later timepoints and peak disease (day 20-22) with differences analyzed. At the same time, this protocol allows the investigator to assess potential differences in vessel permeability and damage using fluorescein angiography. EAU can be induced in other mouse strains - both wildtype or genetically modified - and combined with novel therapies offering flexibility for studying drug efficacy and/or disease mechanisms.

CCL4 induces inflammatory signalling and barrier disruption in the neurovascular endothelium.

BACKGROUND: During neuroinflammation many chemokines alter the function of the blood-brain barrier (BBB) that regulates the entry of macromolecules and immune cells into the brain. As the milieu of the brain is altered, biochemical and structural changes contribute to the pathogenesis of neuroinflammation and may impact on neurogenesis. The chemokine CCL4, previously known as MIP-1ß, is upregulated in a wide variety of central nervous system disorders, including multiple sclerosis, where it is thought to play a key role in the neuroinflammatory process. However, the effect of CCL4 on BBB endothelial cells (ECs) is unknown. MATERIALS AND METHODS: Expression and distribution of CCR5, phosphorylated p38, F-actin, zonula occludens-1 (ZO-1) and vascular endothelial cadherin (VE-cadherin) were analysed in the human BBB EC line hCMEC/D3 by Western blot and/or immunofluorescence in the presence and absence of CCL4. Barrier modulation in response to CCL4 using hCMEC/D3 monolayers was assessed by measuring molecular flux of 70 â€‹kDa RITC-dextran and transendothelial lymphocyte migration. Permeability changes in response to CCL4 in vivo were measured by an occlusion technique in pial microvessels of Wistar rats and by fluorescein angiography in mouse retinae. RESULTS: CCR5, the receptor for CCL4, was expressed in hCMEC/D3 cells. CCL4 stimulation led to phosphorylation of p38 and the formation of actin stress fibres, both indicative of intracellular chemokine signalling. The distribution of junctional proteins was also altered in response to CCL4: junctional ZO-1 was reduced by circa 60% within 60 â€‹min. In addition, surface VE-cadherin was redistributed through internalisation. Consistent with these changes, CCL4 induced hyperpermeability in vitro and in vivo and increased transmigration of lymphocytes across monolayers of hCMEC/D3 cells. CONCLUSION: These results show that CCL4 can modify BBB function and may contribute to disease pathogenesis.

LRG1 destabilizes tumor vessels and restricts immunotherapeutic potency.

BACKGROUND: A poorly functioning tumor vasculature is pro-oncogenic and may impede the delivery of therapeutics. Normalizing the vasculature, therefore, may be beneficial. We previously reported that the secreted glycoprotein leucine-rich α-2-glycoprotein 1 (LRG1) contributes to pathogenic neovascularization. Here, we investigate whether LRG1 in tumors is vasculopathic and whether its inhibition has therapeutic utility. METHODS: Tumor growth and vascular structure were analyzed in subcutaneous and genetically engineered mouse models in wild-type and Lrg1 knockout mice. The effects of LRG1 antibody blockade as monotherapy, or in combination with co-therapies, on vascular function, tumor growth, and infiltrated lymphocytes were investigated. FINDINGS: In mouse models of cancer, Lrg1 expression was induced in tumor endothelial cells, consistent with an increase in protein expression in human cancers. The expression of LRG1 affected tumor progression as Lrg1 gene deletion, or treatment with a LRG1 function-blocking antibody, inhibited tumor growth and improved survival. Inhibition of LRG1 increased endothelial cell pericyte coverage and improved vascular function, resulting in enhanced efficacy of cisplatin chemotherapy, adoptive T cell therapy, and immune checkpoint inhibition (anti-PD1) therapy. With immunotherapy, LRG1 inhibition led to a significant shift in the tumor microenvironment from being predominantly immune silent to immune active. CONCLUSIONS: LRG1 drives vascular abnormalization, and its inhibition represents a novel and effective means of improving the efficacy of cancer therapeutics. FUNDING: Wellcome Trust (206413/B/17/Z), UKRI/MRC (G1000466, MR/N006410/1, MC/PC/14118, and MR/L008742/1), BHF (PG/16/50/32182), Health and Care Research Wales (CA05), CRUK (C42412/A24416 and A17196), ERC (ColonCan 311301 and AngioMature 787181), and DFG (CRC1366).