Low pinocytic brain endothelial cells primarily utilize membrane fusion to internalize extracellular vesicles.

Extracellular vesicles (EVs) are an emerging class of drug carriers and are primarily reported to be internalized into recipient cells via a combination of endocytic routes such as clathrin-mediated, caveolae-mediated and macropinocytosis pathways. In this work, (1) we investigated potential effects of homotypic vs. heterotypic interactions by studying the cellular uptake of homologous EVs (EV donor cells and recipient cells of the same type) vs. heterologous EVs (EV donor cells and recipient cells of different types) and (2) determined the route of EV internalization into low pinocytic/hard-to-deliver cell models such as brain endothelial cells (BECs) and phagocytic cell model as macrophages. Homotypic interactions led to a greater extent of uptake into the recipient BECs compared to heterotypic interactions. However, we did not see a complete reduction in EV uptake into recipient BECs when endocytic pathways were blocked using pharmacological inhibitors and our findings from a R18-based fusion assay suggest that EVs primarily use membrane fusion to enter low-pinocytic recipient BECs instead of relying on endocytosis. Lipophilic PKH67 dye-labeled EVs but not intravesicular esterase-activated calcein ester-labeled EVs severely reduced particle uptake into BECs while phagocytic macrophages internalized both types of EV-labeled particles to comparable extents. Our results also highlight the importance of carefully choosing labeling dye chemistry to study EV uptake, especially in the case of low pinocytic cells such as BECs.

Cell-intrinsic regulation of phagocyte function by interferon lambda during pulmonary viral, bacterial super-infection.

Influenza infections result in a significant number of severe illnesses annually, many of which are complicated by secondary bacterial super-infection. Primary influenza infection has been shown to increase susceptibility to secondary methicillin-resistant Staphylococcus aureus (MRSA) infection by altering the host immune response, leading to significant immunopathology. Type III interferons (IFNs), or IFNλs, have gained traction as potential antiviral therapeutics due to their restriction of viral replication without damaging inflammation. The role of IFNλ in regulating epithelial biology in super-infection has recently been established; however, the impact of IFNλ on immune cells is less defined. In this study, we infected wild-type and IFNLR1-/- mice with influenza A/PR/8/34 followed by S. aureus USA300. We demonstrated that global IFNLR1-/- mice have enhanced bacterial clearance through increased uptake by phagocytes, which was shown to be cell-intrinsic specifically in myeloid cells in mixed bone marrow chimeras. We also showed that depletion of IFNLR1 on CX3CR1 expressing myeloid immune cells, but not neutrophils, was sufficient to significantly reduce bacterial burden compared to mice with intact IFNLR1. These findings provide insight into how IFNλ in an influenza-infected lung impedes bacterial clearance during super-infection and show a direct cell intrinsic role for IFNλ signaling on myeloid cells.

Localized PD-1 Blockade in a Mouse Model of Renal Cell Carcinoma.

Herein we report the impact of localized delivery of an anti-mouse PD-1-specific monoclonal antibody (aPD1) on Renca tumors in the resulting T cell responses and changes in broader immune gene expression profiles. Renca is a BALB/c mice syngeneic tumor that has been used to model human renal cell carcinoma In this study, T cell subsets were examined in tumors and draining lymph nodes of mice treated with localized PD-1 with and without the addition of adenosine deaminase (ADA), an enzyme that catabolizes adenosine (ADO), identified as an immune checkpoint in several types of human cancers. The biologics, aPD1, or aPD1 with adenosine deaminase (aPD1/ADA), were formulated with the self-assembling peptides Z15_EAK to enhance retention near the tumor inoculation site. We found that both aPD1 and aPD1/ADA skewed the local immune milieu towards an immune stimulatory phenotype by reducing Tregs, increasing CD8 T cell infiltration, and upregulating IFNÉ£. Analysis of tumor specimens using bulk RNA-Seq confirmed the impact of the localized aPD1 treatment and revealed differential gene expressions elicited by the loco-regional treatment. The effects of ADA and Z15_EAK were limited to tumor growth delay and lymph node enlargement. These results support the notion of expanding the use of locoregional PD-1 blockade in solid tumors.

α-synucleinopathy exerts sex-dimorphic effects on the multipurpose DNA repair/redox protein APE1 in mice and humans.

Lewy body disorders are characterized by oxidative damage to DNA and inclusions rich in aggregated forms of α-synuclein. Among other roles, apurinic/apyrimidinic endonuclease 1 (APE1) repairs oxidative DNA damage, and APE1 polymorphisms have been linked to cases of Lewy body disorders. However, the link between APE1 and α-synuclein is unexplored. We report that knockdown or inhibition of APE1 amplified inclusion formation in primary hippocampal cultures challenged with preformed α-synuclein fibrils. Fibril infusions into the mouse olfactory bulb/anterior olfactory nucleus (OB/AON) elicited a modest decrease in APE1 expression in the brains of male mice but an increase in females. Similarly, men with Lewy body disorders displayed lower APE1 expression in the OB and amygdala compared to women. Preformed fibril infusions of the mouse OB/AON induced more robust base excision repair of DNA lesions in females than males. No fibril-mediated loss of APE1 expression was observed in male mice when the antioxidant N-acetylcysteine was added to their diet. These findings reveal a potential sex-biased link between α-synucleinopathy and APE1 in mice and humans. Further studies are warranted to determine how this multifunctional protein modifies α-synuclein inclusions and, conversely, how α-synucleinopathy and biological sex interact to modify APE1.

Heat Shock Protein 70 as a Sex-Skewed Regulator of α-Synucleinopathy.

The role of molecular chaperones, such as heat shock protein 70 (Hsp70), is not typically studied as a function of biological sex, but by addressing this gap we might improve our understanding of proteinopathic disorders that predominate in one sex. Therefore, we exposed male or female primary hippocampal cultures to preformed α-synuclein fibrils in a model of early-stage Lewy pathology. We first discovered that two mechanistically distinct inhibitors of Hsp70 function increased phospho-α-synuclein+ inclusions more robustly in male-derived neurons. Because Hsp70 is released into extracellular compartments and may restrict cell-to-cell transmission/amplification of α-synucleinopathy, we then tested the effects of low-endotoxin, exogenous Hsp70 (eHsp70) in primary hippocampal cultures. eHsp70 was taken up by and reduced α-synuclein+ inclusions in cells of both sexes, but pharmacological suppression of Hsp70 function attenuated the inhibitory effect of eHsp70 on perinuclear inclusions only in male neurons. In 20-month-old male mice infused with α-synuclein fibrils in the olfactory bulb, daily intranasal eHsp70 delivery also reduced inclusion numbers and the time to locate buried food. eHsp70 penetrated the limbic system and spinal cord of male mice within 3 h but was cleared within 72 h. Unexpectedly, no evidence of eHsp70 uptake from nose into brain was observed in females. A trend towards higher expression of inducible Hsp70-but not constitutive Hsp70 or Hsp40-was observed in amygdala tissues from male subjects with Lewy body disorders compared to unaffected male controls, supporting the importance of this chaperone in human disease. Women expressed higher amygdalar Hsp70 levels compared to men, regardless of disease status. Together, these data provide a new link between biological sex and a key chaperone that orchestrates proteostasis.

Chemically-Induced Cross-Linking of Peptidic Fibrils for Scaffolding Polymeric Particles and Macrophages.

EAK16-II (EAK) is a self-assembling peptide (SAP) that forms ß-sheets and ß-fibrils through ionic-complementary interactions at physiological ionic strengths. The soft materials can be injected in vivo, creating depots of drugs and cells for rendering pharmacological and biological actions. The scope of the applications of EAK is sought to extend to tissues through which the flow of extracellular fluid tends to be limited. In such anatomical locales the rate and extent of the fibrilization are limited insofar as drug delivery and cellular scaffolding would be impeded. A method is generated utilizing a carbodiimide cross-linker by which EAK fibrils are pre-assembled yet remain injectable soft materials. It is hypothesized that the resulting de novo covalent linkages enhance the stacking of the ß-sheet bilayers, thereby increasing the lengths of the fibrils and the extent of their cross-linking, as evidenced in Diffuse Reflectance Infrared Fourier Transform (DRIFT) spectroscopy, scanning electron microscopy, and atomic force microscopy analyses. The cross-linked EAK (clEAK) retains polymeric microspheres with an average diameter of 1 µm. Macrophages admixed with clEAK remain viable and do not produce the inflammatory mediator interleukin-1ß. These results indicate that clEAK should be investigated further as a platform for delivering particles and cells in vivo.