Abstract:
Neutrophils, being the most abundant leukocyte population in circulation and mediating the first line of innate immunity, exert different host mechanisms for pathogen clearance, such as the release of neutrophil extracellular traps (NETs). Although first described as a mechanism to trap and kill bacteria, NETs have been additionally reported to be released upon detection of damage-associated molecular patterns (DAMPs). DAMPs consist of non-microbial molecules and can be of exogenous or endogenous origin, e.g. occur after apoptotic cell death. Hence, NETs can be advantageous for the host by fighting invading microbes but also detrimental, as implied by their association with many diseases, such as psoriasis. They are known as drivers of persisting inflammation, although some of the exact underlying mechanisms remain to be explored. With the first report about NET-associated RNA (naRNA) by Herster et al. in 2020, a novel, potential driver of sustained, self-mediated NET formation was introduced. However, until now, naRNA has not been well characterized and its effects in immune modulation not been investigated into detail. This study therefore aimed to gain novel insights into the features and role of naRNA in the human immune system.
First, naRNA was observed to be a common, i.e. canonical, component of NETs, independent of the NET formation pathway (suicidal vs. vital) induced by various stimuli. Furthermore, naRNA was found to be the crucial driver of the self-amplifying properties of NETs, namely extracellular trap release in naïve PMNs. This process was dependent on the RNA receptors TLR8 (in human cells) and Tlr13 (in mouse). Of note, this effect was associated with the complexation of naRNA with the antimicrobial peptide LL37, as isolated, ‘naked’ naRNA was no sufficient NET stimulus, whereas LL37 was able to restore its pro-inflammatory properties. It was therefore investigated if naRNA-LL37 complexes can be found in NETs, since LL37 is known to be a highly abundant component of the latter. Surprisingly, in co-localization analysis and high-resolution electron microscopy imaging, naRNA-LL37 complexes were not only found in NETs, but also in unstimulated PMNs. The pre-assembly in vesicles of resting neutrophils indicated that these complexes not just ‘accidentally’ combine after release of both components during the NET formation process. Moreover, naRNA was not observed to be essential for the well-studied antibacterial properties of NETs, as characterized by a killing assay. Thus, the pro-inflammatory roles of naRNA continued to be a focus of this underlying study. It was hereby observed that NETs mediate IL-8 release in macrophages in a TLR8- and naRNA-dependent fashion. Moreover, NETs induced naRNA-dependent NK cell activation, measured by the secretion of IFN-γ. Additionally, incubation of keratinocyte cultures and of a 3D human skin equivalent with NETs led to a naRNA-dependent activation of these cells, characterized by cytokine ELISA and qPCR. Therefore, naRNA does not only modulate classical immune cells but also epidermal cells with immune functions, such as keratinocytes. Hence, naRNA was considered to play an important role in psoriasis, a disease manifesting mainly in cutaneous inflammation. In vivo intradermal injection of naRNA-containing NET content indeed induced psoriasis-like skin inflammation in mice, which was reduced in Tlr13 KO animals that lack RNA-sensing. Using the well-established murine imiquimod psoriasis model further showed that RNA-sensing was essential for disease progression, since Tlr13-deficient mice were protected from sustained inflammation.
In conclusion, this study characterizes naRNA as a novel composite DAMP, which is pre-packed in neutrophil vesicles and gets extruded during NET formation. This re-defines extracellular trap release from an antimicrobial host defense mechanism to a primary source of inflammatory agents: our data provide evidence for NETs as an abundant origin of immunostimulatory extracellular RNAs. The observation that naRNA modulates various immune responses in a TLR8-dependent manner additionally highlights this TLR and its downstream signaling as novel potential therapeutic target for treatment of NET-related diseases.
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