4. Discussion and Conclusions
Interstitial lung disease (ILD) accompanied with pulmonary fibrosis is a progressive disease with high morbidity and early mortality. Treatment of ILD associated PF includes immunomodulatory or antifibrotic drugs, which antifibrotic drugs are mainly utilized in IPF(Johannson, Chaudhuri, Adegunsoye & Wolters, 2021). Unfortunately, immunomodulatory therapies, including azathioprine(Idiopathic Pulmonary Fibrosis Clinical Research, Raghu, Anstrom, King, Lasky & Martinez, 2012), cyclophosphamide(Hoyles et al., 2006), methotrexate(Dawson, Quah, Earnshaw, Amoasii, Mudawi & Spencer, 2021; Johannson, Chaudhuri, Adegunsoye & Wolters, 2021), etc., exhibit efficacy only in a certain subset of patients or with controversial conclusions. Therefore, immunosuppressant therapies reach limited confidence in treating ILD associated PF. Multiple antifibrotic strategies were examined in treating IPF, including bosentan, interferon gamma, etanercept, imatanib, everolimus, ambrisentan, macitentan, and warfarin, but reach a negative outcome(Idiopathic Pulmonary Fibrosis Clinical Research, Raghu, Anstrom, King, Lasky & Martinez, 2012; Lederer & Martinez, 2018; Raghu et al., 2015). Pirfenidone and nintedanib were reported to be efficient in alleviating IPF progression, but the mortality benefit is less robust(Johannson, Chaudhuri, Adegunsoye & Wolters, 2021; Nathan et al., 2017; Nathan et al., 2019). These observations together raise that there is still an urgent need to develop potential universal strategies to treat PF with high efficacy. Several novel therapies were designed to reduce the production of TGF-β1 or the activation of TGF-β signaling(Johannson, Chaudhuri, Adegunsoye & Wolters, 2021). TGF-β signaling is both required for EMT and FMT during PF(Bartis, Mise, Mahida, Eickelberg & Thickett, 2014; Moss, Ryter & Rosas, 2022). Others and our studies all identified the importance of EMT in promotion of PF progression, which the continuous epithelial damage due to multiple types of insults results in EMT in AT II cells and thus PF progression. Therefore, EMT is a shared mechanism that could be an ideal target for developing strategies to treat PF. Here, we utilized PQ poisoning-raised EMT and PF model to identify the importance of intracellular calcium homeostasis in maintaining AT II cell fates. Specifically, either intracellular calcium burden or deficiency would result in PF. And, by combination of PQ-poisoned mice model, cynomolgus model, and clinical information from PQ poisoned patients, we identified that lysine, a utilized clinical drug, as the antidote to treat PQ poisoning by normalizing PQ-raised STIM1 association with TRPC1 for excessive intracellular calcium burden in AT II cells, leading to a remarkable reduction of PQ-induced EMT and PF, further raising that strategies maintaining intracellular calcium homeostasis would be beneficial for treating PF, which requires further efforts to elucidate.
AT II cells are well characterized to differentiate into AT I cells during pulmonary injury for maintaining the function of pulmonary gas exchange(Desai, Brownfield & Krasnow, 2014). A failure of AT II cells differentiation into AT I cells have been reported to develop fibrosis in mice(Wu et al., 2020). Several signaling have been revealed to be required for AT II cell proliferation(Aspal & Zemans, 2020), however, it is remained elusive for AT II-to-AT I differentiation. WNT, Notch, BMP, and TGFβ all have been reported to participate in AT II-to-AT I differentiation, while the signaling networks of these signals inside cells are not well elucidated(Aspal & Zemans, 2020). Calcium ions, important second messengers and exhibit almost omnipotent functions, have been well recognized to participate in the above signaling cascades(Berridge, Bootman & Roderick, 2003; Gooch, Gorin, Zhang & Abboud, 2004; Kuhl, Sheldahl, Park, Miller & Moon, 2000; Song et al., 2020). Our results further revealed the importance of intracellular calcium homeostasis in maintaining AT II cell fates, which either calcium overload or deficiency would lead to EMT and fibrosis, whether dynamic calcium signaling is required to maintain the stemness of AT II to differentiate into AT I remains to be further explored. Note, intracellular calcium signaling would be dynamically modulated by fluxes from both extracellular calcium pools and calcium stores inside cells(Berridge, Bootman & Roderick, 2003). Further efforts should be made to clarify the major routes for intracellular calcium signaling and relevant mechanisms in modulation of the stemness of AT II cells.
PQ is an efficient herbicide widely utilized worldwide. However, PQ is also extremely toxic to mammals by promotion of ROS production, inducing EMT in AT II cells, leading to pulmonary fibrosis and high mortality(Subbiah & Tiwari, 2021). Till now, no efficient antidote has been developed to treat PQ toxicity, making the utilization of PQ in agriculture production be restricted in multiple countries(Dinis-Oliveira, Duarte, Sanchez-Navarro, Remiao, Bastos & Carvalho, 2008). Strategies including charcoal perfusion, reduction of oxidative stress, and anti-inflammation therapies have been well established to treat PQ poisoning in clinic, however, the efficacy is limited and mortality of PQ-poisoned patients is hard to alleviated(Okonek, Hofmann & Henningsen, 1976). Recently, Qian et al., reported a target strategy to neutralize PQ poisoning, however, with no safety evaluation be examined (Qian et al., 2021). Our previous study identified STIM1 as an important molecular target of PQ, here we further developed lysine, a safe approach already be utilized in clinic, as a potential antidote to treat PQ poisoning by restriction of STIM1 association with TRPC for excessive extracellular calcium influx. Further studies are required to examine the efficacy of lysine in treating PQ-poisoned patients either solely or with the combination of well-established strategies utilized in clinic, especially the charcoal perfusion.
In conclusion, our previous study found that PQ targets the STIM1-TRPC1 axis for extracellular calcium entry following with intracellular Ca2+ overload in pulmonary epithelial cells and thus results in pulmonary fibrosis. Here we identified lysine as an antidote for PQ poisoning-induced EMT and PF progression, from which we further emphasized the importance of calcium signals for modulation of AT II cells differentiation for fibrosis, providing potential molecular targets and safe strategies for treating PF progression.