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Non-healing wounds pose a major burden to patients and health care systems alike. These wounds are chronically stuck in the inflammatory phase of the healing process without transitioning to the proliferative phase. They are also characterized by the excessive presence of leukocytes which are assumed to provoke the persistent inflammation observed in pathological wound healing. Recent studies suggested a beneficial role of cold physical plasma in the treatment of chronic wounds. Hence, it was the central question, whether exposure to cold physical plasma would affect the viability and/or function of human leukocytes. Cold plasma displays various properties of which the generation of reactive molecules, such as reactive oxygen and nitrogen species (ROS/RNS), where found to be central in mediating redox changes in leukocytes. Oxidative stress was present especially in lymphocytes that readily underwent apoptosis after exposure to plasma. This was largely a direct consequence of plasma-generated hydrogen peroxide but not superoxide or RNS. Amount of apoptosis was comparable among several lymphocyte subpopulations, with the wound healing-relevant γδ T cells being least affected. Lymphocyte apoptosis was accompanied by mitochondrial membrane depolarization, caspase 3 activation, DNA fragmentation, and phosphatidylserine exposure. These results are in line with previous characterizations of the intrinsic apoptotic pathway in redox biology, and suggest that plasma-induced apoptosis was not mediated by alternative molecular mechanisms. An important immune response mechanism, the proliferation of lymphocytes, was not interrupted in plasma-treated but non-apoptotic cells. In wounds, a central role of leukocytes is to orchestrate the healing response via the release of small communication molecules called cytokines. Non-healing wounds are associated with elevated amounts of pro-inflammatory IL-1β, IL-6, and TNFα, and plasma-treatment of leukocytes strongly decreased their concentrations. At the same time, the expression of anti inflammatory cytokines (IL-10, TGFβ) was markedly increased. The pro inflammatory chemokine IL-8 was the only molecule to be significantly increased in supernatants of plasma-treated cells. IL-8 is the major chemo-attractant for neutrophil granulocytes. Neutrophils are frequently associated with non-healing wounds. These professional phagocytes are the first to migrate to the site of injury where they inactivate invading pathogens by various mechanisms. Importantly, highly relevant effector functions remained mostly unaffected by plasma treatment: the phagocytosis of bacteria, the oxidative burst, and the intracellular killing of microbes. Of note, plasma induced a strong induction of neutrophil extracellular traps (NETs). Decorated with antimicrobial proteins, NETs are web-like chromatin extrusions that entrap pathogens. These results have several implications for wound healing. Plasma-treated neutrophils were still capable of eradicating bacteria, which are frequently associated with non-healing wounds. In addition, plasma-induced NETs could aid in wound healing by providing an antibacterial scaffold to safeguard against further dissemination of microorganisms. Chronic wounds display a state of sustained inflammation and plasma induced apoptosis but not necrosis in lymphocytes. This was an important finding as necrosis, the involuntary cell death, is associated with the release of intracellular content, enhancing inflammation. By contrast, apoptosis dampens it as dead cells are cleared by macrophages inducing anti inflammatory responses. Further, the cytokine signature of plasma-treated leukocytes was largely non inflammatory, which could further decrease inflammation in wounds. Altogether, this work provided first insight with regard to effects and mechanisms of cold physical plasma treatment of wound-relevant leukocytes. Generally, these cells were affected by a plasma mediated modulation of their redox state. Future studies should include the possibility of redox modulation into their experimental approach to further elucidate the role of ROS/RNS in inflammation and possibly to improve existing wound healing therapies.
The order of bats (Chiroptera) account for ~20% of all mammalian species and attracted immense global attention due to their identification as important viral reservoir. Bats can harbour a plethora of high-impact zoonotic viruses, such as filoviruses, lyssaviruses, and coronaviruses without displaying clinical signs of disease themselves. Given this striking diversity of the bat virome, their ability of self-powered flight, and global distribution, understanding chiropteran immunity is essential to facilitate assessment of future spillover events and risks.
However, scarcity of bat-specific or cross-reactive tools and standardized model systems impede progress until today. Furthermore, the richness of species led to generation of isolated datasets, hampering data interpretation and identification of general immune mechanisms, applicable for various chiropteran suborders/families. The key to unlocking bat immunity are coordinated research approaches that comprehensively define immunity in several species. In this work, an in-depth study of innate and adaptive immune mechanisms in the fructivorous Egyptian Rousette bat (Rousettus aegyptiacus, ERB) is presented.
Detailed stability analyses identified EEF1A1 as superior reference gene to ACTB, and GAPDH, which rendered unstable upon temperature increase or presence of type-I-IFN. Since the body core temperatures of pteropid bats reach from 35°C to 41°C and it has been postulated that bats display constitutive expression of IFNs, a suitable reference gene has to be stable under these physiologically relevant conditions. To study cellular innate immunity in detail, cell lines from the nasal epithelium, the olfactory compartment and the cerebrum were generated. To include immune responses of epithelia cells, essential for immunity at sites of primary viral infection, primary epithelia cells from the nasal epithelium, trachea, lung and small intestine were generated. Cellular identities were determined by comprehensive analyses of transcripts and proteins expressed by each cell line. The capacity of each cell line to produce type-I- and III-IFNs was assessed at 37°C and 40°C upon stimulation with viral mimetics. This revealed cell type-dependent differences is the capability to express IFNs upon stimulation. Furthermore, the constitutive expression of type-I- and III-IFNs was significantly elevated in higher temperatures and quantified at mRNA copy levels. To characterize ERB innate immunity upon infection with high-impact zoonotic viruses, cells from the nasal epithelium, the olfactory system, and the brain were infected with several lyssaviruses. This revealed striking differences in susceptibility: cells from the nasal epithelium rendered least whereas cells from the olfactory epithelium rendered most susceptible to viral infection and replication. Additionally, due to a lack of IFN expression in infected cells, it could be shown that LBV possibly possesses advanced strategies to ensure successful replication in ERB cells. Since the current SARS-CoV-2 pandemic put bats even further in the focus of zoonotic research, primary epithelial cells and animals were infected with this virus to monitor ERB-specific immune transcripts in cells and tissues. These studies revealed a notably early IFNG expression in the respiratory tract of infected individuals.
To understand immunomaturation in bats, the immune cell landscape in periphery and various tissue in adult and juvenile ERB was analyzed by flow cytometry and scRNA-seq, revealing intriguing, age-dependent variations in the abundance of granulocytes and lymphocytes. Flow cytometry revealed a significantly higher number of granulocytes in adults, as well as higher numbers of B cells in juveniles. scRNA-seq allowed detailed identification of different leukocyte subsets, uncovering the presence of highly-abundant NKT-like cells and a unique PLAC8 expressing B cell population. A functional characterization of phagocytic cells and lymphocytes derived from adult and juvenile ERB revealed no significant differences in cellular functionality.
In conclusion, the presented work demonstrated suitability of all established ERB cell lines to study bat immunity in vitro, which led to striking findings regarding IFN expression at steady state, or upon stimulation or viral infection. In addition, established qRT-PCR protocols allowed definition of constitutive and temperature-dependent elevation of IFN expression magnitudes, as well as insights into expression of immune-related transcripts in SARS-CoV-2 infected ERB. Finally, based on optimized scRNA-seq technologies and flow cytometry, frequencies and absolute cell counts could be determined in ERB of different ages, revealing e.g. age-dependent variations in leukocyte profile compositions.