Lung airway MIWI2 expressing multiciliated cells and influenza A pathogenesis
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Abstract
Influenza is one of the most common respiratory infections globally, causing significant morbidity and mortality each year. Despite the availability of seasonal vaccines and antiviral therapies, their inconsistent efficacy, particularly among high-risk populations, underscores the need for a deeper understanding of host responses to influenza and the identification of novel therapeutic targets. Influenza A virus (IAV) primarily infects multiciliated cells that line the lung airway. Our lab identified a distinct subset of these multiciliated cells that uniquely express the MIWI2 protein, the mouse ortholog of human p-element induced wimpy testes-like 4 (PIWIL4), referred to as MIWI2 multiciliated (M2MC) cells. While MIWI2 is recognized for its role in PIWI-interacting RNA (piRNA) binding and retrotransposon suppression during male fetal gonadogenesis, and the PIWI proteins in insects are known to interact with viral RNAs, its function(s) in somatic mammalian cells remains largely unexplored. The overarching goal of this dissertation is to investigate MIWI2 in M2MC cells during IAV infection and its broader influence on airway epithelial responses and host defense.To achieve this, we generated a MIWI2 targeted knockout model using MIWI2 haplosufficient (Miwi2+/tom) and deficient (Miwi2tom/tom) mice, which allows for the isolation of M2MC cells via tdTomato expression. Following intratracheal infection with mouse-adapted influenza A/Puerto Rico/8/1934 (PR8), Miwi2tom/tom mice exhibited reduced viral titers and lower viral RNA levels by 7 days post-infection (dpi) and faster weight recovery compared to wild-type (Miwi2+/+) and Miwi2+/tom mice. These findings indicated MIWI2 expression is exacerbating viral pathogenesis. Our central hypothesis is during initial exposure, MIWI2 is regulating key pathways that is driving IAV pathogenesis.
We next explored the possibility that M2MC cells were preferentially infected at early stages and promoted viral propagation. Using an mNeon-hemagglutinin (mNeon-HA) reporter IAV, we infected Miwi2+/tom mice at 3 dpi. M2MC cells were not more likely to be infected than nonMIWI2 multiciliated (nonM2MC) cells. Similarly, MIWI2 expression did not alter the proportions of infected alveolar type I, type II, club and multiciliated cells at later stages of infection. Preliminary proteomic analysis of bronchoalveolar lavage fluid (BALF) revealed increased PIEZO1 levels in PR8-infected Miwi2tom/tom mice compared to Miwi2+/+.
Considering MIWI2’s known roles in host RNA regulation and piRNA interaction, we profiled PR8 and saline treated M2MC and nonM2MC cells from digested lungs of Miwi2+/tom and Miwi2tom/tom mice at 3 dpi. Although retrotransposon and viral RNA expression were independent of MIWI2, MIWI2 deficiency was associated with a selected decrease in mitochondrial and ribosomal gene expression in M2MC cells during PR8 infection. Following up, evaluation of mitochondrial reactive oxygen species (ROS) generation revealed a significant increase in intracellular ROS in the entire multiciliated cell population of PR8 infected Miwi2tom/tom mice. Lastly, a decrease in a species of small non-coding RNAs derived from nuclear mitochondrial (NuMT) DNA was observed in saline-treated MIWI2 expressing cells.
This study provides the first evidence that MIWI2 regulates a repertoire of small non-coding RNAs and mitochondrial oxidant generation within a somatic cell population, highlighting a previously unrecognized role for MIWI2’s function outside of the germline. These findings underscore MIWI2’s involvement in viral pathogenesis and MIWI2/PIWIL4 as a potential host susceptibility factor for severe respiratory infections. This work offers new insights into influenza biology and host-pathogen interactions, establishing a foundation for the development of novel therapeutic strategies.
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2025