The targeted delivery of therapies to diseased tissues offers a safe opportunity to achieve optimal efficacy while limiting systemic exposure. These considerations apply to many disease indications but are especially relevant for rheumatoid arthritis (RA), as RA is a systemic autoimmune disease which affects multiple joints. We have identified an antibody that is specific to damaged arthritic cartilage (anti-ROS-CII) that can be used to deliver treatments specifically to arthritic joints, yielding augmented efficacy in experimental arthritis. In the current study, we demonstrate that scaffolds enriched with bioactive payloads can be delivered precisely to an inflamed joint and achieve superior efficacy outcomes consistent with the pharmacological properties of these payloads. As a scaffold, we have used extracellular vesicles (EVs) prepared from human neutrophils (PMNs), which possess intrinsic anti-inflammatory properties and the ability to penetrate inflamed arthritic cartilage. EV fortified with anti-ROS-CII (EV/anti-ROS-CII) retained anti-ROS-CII specificity and bound exclusively to the damaged cartilage. Following systemic administration, EV/anti-ROS-CII (a) exhibited the ability to localize specifically in the arthritic joint in vivo and (b) was able to specifically target single (viral IL-10 or anti-TNF) or combined (viral IL-10 and anti-TNF) anti-inflammatory treatments to the arthritic joint, which accelerated attenuation of clinical and synovial inflammation. Overall, this study demonstrates the attainability of targeting a pro-resolving biological scaffold to the arthritic joint. The potential of targeting scaffolds such as EV, nanoparticles, or a combination thereof alongside combined therapeutics is paramount for designing systemically administered broad-spectrum of anti-inflammatory treatments.

Background: CD4+ T cells that express the chemokine receptor, CCR5, are the most important target of HIV-1 infection, but their functions, phenotypes and anatomical locations are poorly understood. We aimed to use multiparameter flow cytometry to better define the full breadth of these cells. Methods: High-parameter fluorescence flow and mass cytometry were optimized to analyse subsets of CCR5+ memory CD4+ T cells, including CD25highCD127dim Tregs, CXCR3+CCR6− Th1-like, CCR6+CD161+CXCR3− Th17-like, integrins α4+ß7+ gut-homing, CCR4+ skin-homing, CD62L+ lymph node-homing, CD38+HLA-DR+ activated cells, and CD27−CD28− cytotoxic T lymphocytes, in a total of 22 samples of peripheral blood, ultrasound-guided fine needle biopsies of lymph nodes and excised tonsils. CCR5+ antigen-specific CD4+ T cells were studied using the OX40 flow-based assay. Results: 10–20% of CCR5+ memory CD4+ T cells were Tregs, 10–30% were gut-homing, 10–30% were skin-homing, 20–40% were lymph node-homing, 20–50% were Th1-like and 20–40% were Th17-like cells. Up to 30% were cytotoxic T lymphocytes in CMV-seropositive donors, including cells that were either CCR5highGranzyme K+ or CCR5dimGranzyme B+. When all possible phenotypes were exhaustively analysed, more than 150 different functional and trafficking subsets of CCR5+ CD4+ T cells were seen. Moreover, a small population of resident CD69+Granzyme K+CCR5+ CD4+ T cells was found in lymphoid tissues. CMV− and Mycobacterium tuberculosis-specific CD4+ T cells were predominantly CCR5+. Conclusion: These results reveal for the first time the prodigious heterogeneity of function and trafficking of CCR5+ CD4+ T cells in blood and in lymphoid tissue, with significant implications for rational approaches to prophylaxis for HIV-1 infection and for purging of the HIV-1 reservoir in those participants already infected.

Neutrophil trafficking is a key component of the inflammatory response. Here, we have investigated the role of the immunomodulatory lectin Galectin‐9 (Gal‐9) on neutrophil recruitment. Our data indicate that Gal‐9 is upregulated in the inflamed vasculature of RA synovial biopsies and report the release of Gal‐9 into the extracellular environment following endothelial cell activation. siRNA knockdown of endothelial Gal‐9 resulted in reduced neutrophil adhesion and neutrophil recruitment was significantly reduced in Gal‐9 knockout mice in a model of zymosan‐induced peritonitis. We also provide evidence for Gal‐9 binding sites on human neutrophils; Gal‐9 binding induced neutrophil activation (increased expression of β2 integrins and reduced expression of CD62L). Intra‐vital microscopy confirmed a pro‐recruitment role for Gal‐9, with increased numbers of transmigrated neutrophils following Gal‐9 administration. We studied the role of both soluble and immobilized Gal‐9 on human neutrophil recruitment. Soluble Gal‐9 significantly strengthened the interaction between neutrophils and the endothelium and inhibited neutrophil crawling on ICAM‐1. When immobilized, Gal‐9 functioned as an adhesion molecule and captured neutrophils from the flow. Neutrophils adherent to Gal‐9 exhibited a spread/activated phenotype that was inhibited by CD18 and CD44 neutralizing antibodies, suggesting a role for these molecules in the pro‐adhesive effects of Gal‐9. Our data indicate that Gal‐9 is expressed and released by the activated endothelium and functions both in soluble form and when immobilized as a neutrophil adhesion molecule. This study paves the way for further investigation of the role of Gal‐9 in leukocyte recruitment in different inflammatory settings.