Global proteome analysis indicates STAT3 modulates lipid metabolism associated inflammatory signaling
Since redox stress management was ruled out as the main molecular event, we investigated the global plasma proteome using iTRAQ-LC-MS/MS (Table S2, Supplementary information ) to ascertain the molecular events. In all groups, 312 proteins were identified and quantified. In HD3, 32 proteins were up-regulated and 42 were down-regulated. In HD7, 48 proteins were up-regulated and 36 proteins were down-regulated. In HD14, 60 proteins (the highest) were up-regulated and 31 proteins (the lowest) were down-regulated (Fig. 4a). This is in contrast to the lung proteome where across all groups, down-regulation of protein expression was dominant. The most significant molecular and cellular processes across groups were the same along with the same proteins involved in those processes (Fig. 4b). Cellular movement, Cell death and survival and Cell-to-cell signaling and interaction were also observed to be significant in the lung proteome. Protein synthesis and cellular compromise are unique to the plasma proteome. The trends of protein expression in the global proteome across HD3, HD7 and HD14 (relative to Normoxia) was pictured using hierarchical clustergram (Fig. 4c). The most significant canonical pathways were Acute phase response signaling (APRS), LXR/RXR Activation, FXR/RXR Activation, Coagulation system and Complement system (Fig. 4d). LXR/RXR remained down-regulated throughout the hypoxia exposure. Coagulation system remained up-regulated throughout the environmental hypoxia exposure. Complement system is up-regulated sequentially in HD7 and HD14 after being neutral in HD3. FXR/RXR remains non-directional throughout the environmental hypoxia exposure. APRS, quite interestingly, is strongly up-regulated in HD3, gets down-regulated at HD7. In HD14, it becomes up-regulated again.
Since STAT3 is a regulatory protein for Acute phase response, we investigated the APRS with overlaid expression data from our plasma dataset (Fig. 5a). Furthermore, an integrated network created using the top five canonical pathways (Fig. 5b) to identify the upstream regulators. We observed an interplay of RXR with HNF4A, complement proteins, apolipoproteins and coagulation proteins. This was indicative of perturbations in lipid metabolism associated with perturbations in inflammatory signaling and coagulation cascades during extended exposures to low pO2. To further verify these perturbations (in addition to their links with acute phase response) and assess antioxidant status we performed immunoblotting of proteins like glutathione peroxidase 3 (GPx3), plasminogen, C3, C4b, transthyretin (TTR), apolipoproteins A1 and H (Fig. 5c). GPx-3 was observed to be higher in HD3, similar to Normoxia in HD7 while HD14 showed lowest levels. C3 levels sequentially increased across the groups, reaching highest levels at HD14. C4b sequentially increased in HD3 and HD7, reaching its highest levels. In HD14, C4b levels were similar to HD3. Ttr levels decreased in HD3 and remained nearly constant in HD7 and HD14. Apo A1 levels were progressively and notably lower across HD3 to HD14 as compared to control group. Apo H levels were notably low throughout the environmental hypoxia exposure. Overall, upon increasing durations of low pO2 exposures, particularly past day 7, there is increased inflammatory signaling, declining antioxidant reserves and adverse effects on lipid metabolism.
Indicators of cytoskeletal remodeling and inflammation in altitude acclimatized humans
The real-world manifestation of lowered pO2 exposure for extended durations is observed during high altitude explorations. We observed that during extended exposure to low pO2 in SD rats, lipid metabolism was favored while TCA cycle was subdued (Fig. 3c & d; RXR and MDH) indicating perturbed systemic energy homeostasis. However, results also indicate lowered HDL levels (incumbent on Apo A1 expression) as exposure of duration to lower pO2increased (Fig. 5c). We found that it was associated with the lung cytoskeletal re-arrangements (Fig. 3d). The perturbed energy homeostasis resulted in innate immune activation, mostly inflammatory signaling, which was observed via acute phase response signaling (Fig. 5). Based on these aspects, we selected the following proteins that were specific for a signaling network/molecular process: alpha-1-antitrypsin, cofilin-1 and S100A8. These may not be called translated markers owing to the different time points at altitude as well as a different altitude zone as compared to the experimental conditions of the SD rats. The small sample size may also be considered a disqualification for the same. However, the results obtained (Fig. 6) do provide qualitative hints at the major processes that may be further analyzed for finding biomarkers of altitude acclimatization/disease and therapeutic targets in human samples.
Since innate immune activation and calcium associated proteins were observed in rat plasma (Table S1; Supplementary information), we measured S100A8 in human plasma (Fig. 6a). S100A8 inhibits neutrophil activation and adhesion and also modulates inflammatory processes (Raquil, Anceriz, Rouleau, & Tessier, 2008). It is a component of calprotectin (Yui, Nakatani, & Mikami, 2003), a fecal biomarker of gastrointestinal inflammation in irritable bowel syndrome (Van Rheenen, Van de Vijver, & Fidler, 2010). There were no major changes throughout HAD7, HAD30 and HAD120 as compared to Normoxia. This is indicative that innate immune processes, particularly inflammatory processes, are well-balanced in individuals acclimatized to low pO2levels at high altitude. Next, we evaluated cofilin-1 levels in human plasma (Fig. 6b). Along with Lim kinase, cofilin signaling is essential for remodeling of cytoskeleton (Danen et al., 2005; Wang, Eddy, & Condeelis, 2007). Higher levels were observed in HAD7 suggesting increased cytoskeletal re-modeling corroborated by increased redox stress in HD3 in rats. Its near Normoxic levels in HAD30 and HAD120 suggest a balanced impetus for actin dynamics. However, till day 7 cofilin-1 levels are higher than control group. To delve further into this aspect, alpha-1-antitrypsin (Tables S1 and S2; Supplementary information) was also assayed in human plasma (Fig. 6c). It was observed in the Acute phase response signaling in SD rats. It is known to inhibit elastase and thus prevents lung tissue damage during various conditions (Brantly et al., 1988; Garratt et al., 2016; Gøtzsche & Johansen, 2016). In a previous study, we had shown that Acute phase response signaling is a homologous pathway between rat and human exposed to hypoxia (Paul, Bhargava, & Ahmad, 2017). Alpha-1-antitrypsin levels were investigated in human plasma based on our findings that cytoskeletal re-arrangement was taking place in the rat lung. We observed that plasma alpha-1-antitrypsin levels are lowered throughout the high altitude exposure in humans till HAD30 suggesting increased chances of lung tissue damage/re-modeling. However, it’s increased levels at HAD120 suggest better adjusted lung cytoskeletal dynamics in agreement with recovering cofilin-1 levels.