Saraiva, E. | Search bioRxiv

bioRxiv2025

Neutrophil Extracellular Traps Mediate In Vitro and In Vivo Degradation of α-Synuclein Amyloid Fibrils

Neutrophil extracellular traps (NETs) are chromatin-derived structures decorated with neutrophil enzymes such as elastase and myeloperoxidase. Our group has previously demonstrated that amyloid fibrils (AFs), regardless of their protein composition, induce NET release in vitro in human neutrophils through a process dependent on reactive oxygen species (ROS) generation by NADPH oxidase 2 (NOX-2). Moreover, the proteases embedded in NETs were shown to degrade AFs into smaller, potentially toxic species. The present study aimed to determine whether amyloid fibrils composed of -synuclein (SF) can induce NET formation in vivo and to investigate the role of NETs in modulating amyloid-associated pathology. To this end, we employed gp91phox knockout (KO) mice, which lack NOX-2 activity and are therefore unable to release NETs. SF was instilled into the lungs of both WT and KO mice (males and females), leveraging the lungs robust immune cell recruitment - particularly of neutrophils-as a model system. Eight hours after SF instillation, both WT and KO animals exhibited marked neutrophil infiltration in the lungs causing inflammation. However, NET formation-evidenced by the presence of citrullinated histones and myeloperoxidase - was detected only in WT mice. Interestingly, while Congo red-positive amyloid-like structures persisted in the lungs of KO mice, they were absent in the lungs of WT animals, suggesting that NET-associated proteases facilitate the clearance of AFs from lung tissue. Lung function was assessed by measuring elastance and resistance. Our data showed that, while AFs were still present in the lungs of both WT and KO mice, elastance was impaired. As AFs were cleared from the lungs of WT mice, lung function recovered. In contrast, KO animals, in which AFs persisted, continued to exhibit compromised elastance. Together, our findings demonstrate that AFs impair lung function, and that NETs, induced in response to these fibrils, promote their degradation and thereby protect lung tissue from further damage.

biochemistry↗

bioRxiv2019

Protection induced by anti-PD-1 and anti-PD-L1 treatment in Leishmania amazonensis-infected BALB/c mice

Leishmaniasis is a neglected disease, for which current treatment presents numerous issues. Leishmania amazonensis is the etiological agent of cutaneous and diffuse cutaneous leishmaniasis. The roles of the programmed death-1 (PD-1) receptor on lymphocytes and its ligand (PD-L1) on antigen-presenting cells have been well studied in tumor and other infection models; but little is known about their roles in non-healing cutaneous leishmaniasis. Our previous report of L. amazonensis-induced PD-L1 expression on dendritic cells, in combination with decreased IFN-{gamma} production by CD4+ T cells in C57BL/6 mice, led to a hypothesis that the formation of the PD-1/PD-L1 complex contributes to down-modulation of immune responses, especially T cell suppression, enabling parasite survival and persistence. In this study, we tested the therapeutic potential of anti-PD-1 and anti-PD-L1 monoclonal antibodies (MoAbs) against a non-healing L. amazonensis infection in BALB/c mice. We observed that L. amazonensis induced PD-1 expression on both CD4+ and CD8+ T cells, and that anti-PD-1 and anti-PD-L1 treatment significantly increased IFN-{gamma}-producing CD4+ and CD8+ T cells, respectively. Compared with infection controls, mice that received treatment with anti-PD-1 and anti-PD-L1, but not anti-PD-L2, displayed bigger lesions with significantly lower parasite loads. Treatment did not affect anti-Leishmania antibody or IL-10 production, but anti-PD-1 treatment reduced both IL-4 and TGF-{beta} production. Together, our results highlight the therapeutic potential of an anti-PD-1-based treatment in promoting the reinvigoration of T cells for the control of parasite burden.

immunology↗