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.