Acellular Human Amniotic Fluid-Derived Extracellular Vesicles as Novel Anti-Inflammatory Therapeutics against SARS-CoV-2 Infection.

The ongoing COVID-19 pandemic caused by SARS-CoV-2 is associated with acute respiratory distress syndrome (ARDS) and fatal pneumonia. Excessive inflammation caused by SARS-CoV-2 is the key driver of ARDS and lethal disease. Several FDA-approved drugs that suppress virus replication are in clinical use. However, despite strong evidence for the role of virus-induced inflammation in severe COVID-19, no effective anti-inflammatory drug is available to control fatal inflammation as well as efficiently clear the virus. Therefore, there is an urgent need to identify biologically derived immunomodulators that suppress inflammation and promote antiviral immunity. In this study, we evaluated acellular human amniotic fluid (acAF) containing extracellular vesicles (hAF-EVs) as a potential non-toxic and safe biologic for immunomodulation during COVID-19. Our in vitro results showed that acAF significantly reduced inflammatory cytokine production in TLR2/4/7 and SARS-CoV-2 structural protein-stimulated mouse macrophages. Importantly, an intraperitoneal administration of acAF reduced morbidity and mortality in SARS-CoV-2-infected mice. A detailed examination of SARS-CoV-2-infected lungs revealed that the increased protection in acAF-treated mice was associated with reduced viral titers and levels of inflammatory myeloid cell infiltration. Collectively, our results identify a novel biologic that has potential to suppress excessive inflammation and enhance survival following SARS-CoV-2 infection, highlighting the translational potential of acAF against COVID-19.

Human amniotic fluid derived extracellular vesicles attenuate T cell immune response.

Introduction: Extracellular vesicles isolated from human amniotic fluid (AF-EVs) have previously been found to modulate inflammation and macrophage infiltration in a mouse model. However, the effects of acellular amniotic fluid (acAF) or AF-EVs on the T-Cell immune response have not been explored. Methods: In this study, we investigated the effects of acAF and AF-EVs on the T cell immune response in an in vitro cell culture model. Peripheral Blood Mononuclear Cells (PBMCs) were stimulated with Phytohemagglutinin (PHA) to induce the immune response and were subsequently treated with either serum-free media (vehicle), acAF, or concentrated AF-EVs. Results: Both acAF and AF-EV treatment suppressed PHA-induced T cell proliferation and PHA-induced T cell activation; however, treatment with concentrated AF-EVs had a greater effect. Additionally, both acAF and AF-EVs reduced PBMC pro-inflammatory cytokine release. AF-EVs were found to be taken up by both CD4+ and CD8+ effector T cell subsets. Conclusion: Overall, this data demonstrates that AF-EVs have a robust immunomodulatory effect on T cells and suggests AF-EVs could be used as an immunotherapeutic tool.

TNFR2 promotes Treg-mediated recovery from neuropathic pain across sexes.

Tumor necrosis factor receptor 2 (TNFR2) is a transmembrane receptor that is linked to immune modulation and tissue regeneration. Here, we show that TNFR2 essentially promotes long-term pain resolution independently of sex. Genetic deletion of TNFR2 resulted in impaired neuronal regeneration and chronic nonresolving pain after chronic constriction injury (CCI). Further, pharmacological activation of TNFR2 using the TNFR2 agonist EHD2-sc-mTNFR2 in mice with chronic neuropathic pain promoted long-lasting pain recovery. TNFR2 agonist treatment reduced neuronal injury, alleviated peripheral and central inflammation, and promoted repolarization of central nervous system (CNS)-infiltrating myeloid cells into an antiinflammatory/reparative phenotype. Depletion of regulatory T cells (Tregs) delayed spontaneous pain recovery and abolished the therapeutic effect of EHD2-sc-mTNFR2 This study therefore reveals a function of TNFR2 in neuropathic pain recovery and demonstrates that both TNFR2 signaling and Tregs are essential for pain recovery after CCI. Therefore, therapeutic strategies based on the concept of enhancing TNFR2 signaling could be developed into a nonopioid therapy for the treatment of chronic neuropathic pain.

Tumor necrosis factor receptor 1 inhibition is therapeutic for neuropathic pain in males but not in females.

Tumor necrosis factor (TNF) is a proinflammatory cytokine, which is involved in physiological and pathological processes and has been found to be crucial for pain development. In the current study, we were interested in the effects of blocking Tumor necrosis factor receptor 1 (TNFR1) signaling on neuropathic pain after peripheral nerve injury with the use of transgenic mice and pharmacological inhibition. We have previously shown that TNFR1 mice failed to develop neuropathic pain and depressive symptoms after chronic constriction injury (CCI). To investigate the therapeutic effects of inhibiting TNFR1 signaling after injury, we delivered a drug that inactivates soluble TNF (XPro1595). Inhibition of solTNF signaling resulted in an accelerated recovery from neuropathic pain in males, but not in females. To begin exploring a mechanism, we investigated changes in N-methyl-D-aspartate (NMDA) receptors because neuropathic pain has been shown to invoke an increase in glutamatergic signaling. In male mice, XPro1595 treatment reduces elevated NMDA receptor levels in the brain after injury, whereas in female mice, NMDA receptor levels decrease after CCI. We further show that estrogen inhibits the therapeutic response of XPro1595 in females. Our results suggest that TNFR1 signaling plays an essential role in pain induction after CCI in males but not in females.

The effects of spinal cord injury on bone loss and dysregulation of the calcium/parathyroid hormone loop in mice.

OBJECTIVE: To map the progression of osteoporosis following spinal cord injury in mice in specific areas and analyze changes in parathyroid hormone (PTH) and ion levels which could be responsible for overall bone loss. SUMMARY OF BACKGROUND DATA: Spinal cord injury rapidly induces severe bone loss compared to other conditions, yet the cause of this bone loss has not been identified. Studies suggest the bone loss after injury is not solely due to disuse. METHODS: To quantify bone loss we weighed individual bones and measured bone mineral density using dual energy X-ray absorptiometry at acute (1 week) and chronic (4 week) time points following a T9 contusion. An ELISA was used to measure blood PTH levels at 1 and 4 weeks after injury. Calcium and phosphate levels were also analyzed at 4 weeks following injury at the University of Miami pathology core. RESULTS: We observed a significant decrease in bone mineral density in hind limbs after an acute injury, and found this bone loss to progress over time. Furthermore, following chronic injury a decrease in bone mineral density is also observed in bones above the level of injury and in the total bone mineral density. We observed a significant decrease in parathyroid hormone levels in injured mice at the chronic time point, but not at the acute time point which suggests this could be involved in the global bone loss following injury. We also observed a significant increase in serum calcium levels following injury which could account for the imbalance of PTH levels.

A muscle-specific p38 MAPK/Mef2/MnSOD pathway regulates stress, motor function, and life span in Drosophila.

Molecular mechanisms that concordantly regulate stress, life span, and aging remain incompletely understood. Here, we demonstrate that in Drosophila, a p38 MAP kinase (p38K)/Mef2/MnSOD pathway is a coregulator of stress and life span. Hence, overexpression of p38K extends life span in a MnSOD-dependent manner, whereas inhibition of p38K causes early lethality and precipitates age-related motor dysfunction and stress sensitivity, that is rescued through muscle-restricted (but not neuronal) add-back of p38K. Additionally, mutations in p38K are associated with increased protein carbonylation and Nrf2-dependent transcription, while adversely affecting metabolic response to hypoxia. Mechanistically, p38K modulates expression of the mitochondrial MnSOD enzyme through the transcription factor Mef2, and predictably, perturbations in MnSOD modify p38K-dependent phenotypes. Thus, our results uncover a muscle-restricted p38K-Mef2-MnSOD signaling module that influences life span and stress, distinct from the insulin/JNK/FOXO pathway. We propose that potentiating p38K might be instrumental in restoring the mitochondrial detoxification machinery and combating stress-induced aging.