The lung is a vital organ responsible for the gaseous exchange between the body and external environment. Exposure to airborne debris, pathogens, and chemicals may lead to acute or chronic injury of the lung tissues [1
]. Influenza virus infection is one of the most common global infectious diseases, afflicting millions of people worldwide during influenza epidemics [2
]. During seasonal influenza infection, the epithelial and immune cells in the upper respiratory tract are targeted [3
], resulting in inflammation and death of these cells. In healthy individuals, the infection is typically self-limiting, and they usually recover within 7 days [5
]. However, pandemic strains of influenza virus (such as the 1918-H1N1 Spanish Flu and the 1957-H2N2 Asian Flu), and occasionally seasonal strains, can infect the lower respiratory tract, resulting in the inflammation of alveolar air sacs, giving rise to influenza pneumonitis. This condition may cause diffuse alveolar damage, necrosis and hemorrhage of the alveolar epithelium [6
], leading to acute respiratory distress syndrome (ARDS) and even death. While the damage to the lungs may be severe, patients who survive are usually able to achieve partial or full recovery within two years [8
], demonstrating the ability of the lungs to regenerate following pneumonia.
Stem cells in the lower respiratory tract have been described to participate in lung repair and regeneration. These include the basal cells in the trachea and bronchus with dual expression of Trp63 (P63) and cytokeratin 5 (KRT5) [11
], and the bronchio-alveolar stem cells (BASCs) in the bronchioalveolar duct junction (BADJ) that express uteroglobin (CC10) and surfactant protein C (SPC) [12
]. In the alveoli, two populations of putative stem cells have been identified. The first population of alveolar stem cells is thought to be proliferating alveolar type II (AT2) cells that express SPC [13
]. Recently, it was suggested that these proliferating AT2 cells are the main stem cells in the lungs for replacing the alveolar epithelium during influenza pneumonia [16
]. However, another distinct group of stem cells has been documented in the regenerating lung following sub-lethal infection of a mouse-adapted H1N1 influenza virus strain (PR8). Similar to the basal cells in the trachea, these cells known as distal airway stem cells (DASCs) [17
] or lineage-negative epithelial progenitors (LNEPs) [18
], also express P63 and KRT5. DASCs are believed to be of a different lineage from the tracheal basal cells, and are shown to differentiate into alveolar epithelial cells in a mouse lineage-tracing model [17
]. However, other studies suggested that these DASCs did not conclusively give rise to the regeneration of the alveolar epithelial cells destroyed following ARDS [18
Studies that concurrently compare both populations of putative stem cells in the lungs during early recovery from influenza pneumonia are lacking, and we sought to analyze the relative contribution of these two cell types to early alveolar regeneration of the distal airways. Thus, to shed light on such early events, we employed a wild-type BALB/c mouse model of pneumonia induced by PR8 infection to simultaneously delineate and quantify the short-term spatial and temporal changes of DASCs and proliferating AT2 cells in the lungs from the time of infection till early recovery from influenza pneumonia. We found that by 25 days following infection, when mice regained their physical healthy state, their lungs still exhibited signs of damage but the DASCs had not differentiated into alveolar epithelial cells. On the other hand, we observed that AT2 cells proliferated to replace the AT2 cells that were destroyed during infection, suggesting that proliferating AT2 cells contribute positively to regeneration of alveolar epithelium [16
]. In addition, proteomic analyses of global lung proteins revealed prominent innate immune responses at 7 dpi, which shifted towards adaptive immune responses by 15 dpi. In summary, our findings provide a better understanding of the early events of the cellular and molecular regenerative processes in the lungs in response to injury from influenza viral infection.
The lung is a very complex tissue, beginning from the trachea, branching into bronchi that divide into smaller bronchioles, and finally ending as alveoli. In view of its intricate structure, it is likely that there are different populations of stem cells that are separated spatially and temporally, and that respond to specific types of injury to mediate the lung regeneration process [45
]. For example, in pneumonia, where the distal alveolar epithelial cells are damaged by infection and/or the concomitant inflammation, it is unlikely for basal cells in the trachea to repopulate the alveolar epithelial cells due to spatial constraints. Indeed, studies identified potential stem cells in the distal area of the lungs that are able to differentiate and give rise to alveolar epithelial cells following various injuries. One population of stem cells constitutes DASCs that express P63 and KRT5 [17
]. A KRT5 lineage-tracing model was utilized to isolate DASCs which were transplanted into syngeneic mice infected with influenza. The transplanted DASCs could differentiate into alveolar type I and II cells in the recipient mice. Proliferating AT2 cells are considered to be another stem cell population [13
]. An SPC lineage-tracing model revealed that AT2 cells proliferated following injury, and eventually differentiated to generate type I cells. This was further substantiated in an influenza pneumonia model [16
In our sub-lethal influenza pneumonia model, we observed the formation of DASCs (expressing P63 and KRT5) that started migrating out from the bronchioles from 9 dpi, giving rise to obvious DASC pods at 11 dpi, which was also previously reported [19
]. However, these DASCs did not co-express alveolar epithelial cell markers at 25 dpi, when the mice had appeared to recover from pneumonia. In fact, the earliest time-point that DASCs were found to have differentiated into AT2 cells was 90 dpi [17
]. Therefore, it was possible that the DASCs had yet to differentiate into AT2 cells by 25 dpi in our model. However, another study demonstrated that these DASCs did not conclusively differentiate into AT2 cells even up to 200 dpi, but instead evolved into cysts lined with KRT5-positive cells [20
]. Taken together with our findings, this suggests that DASCs may not serve as a major source of alveolar epithelial cell renewal in the lungs following influenza-induced pneumonia.
We next addressed whether there are other stem cell populations that can replace the AT2 cells lost during the initial phase of infection, since they were not replenished by DASCs. Interestingly, the population of proliferating AT2 cells increased, even as the animals were recovering from their weight loss. These cells are typically present in low numbers in the alveoli under normal conditions [13
], and the sub-lethal infection would be unlikely to damage all AT2 cells. Hence, existing proliferating AT2 cells that are already near or within the damaged area in the lungs may likely be the “first responder” in the lung regeneration process. Indeed, our data suggested that these proliferating AT2 cells that survived the damage induced by infection and inflammation would then begin to divide to generate new AT2 cells. These proliferating AT2 cells eventually stopped dividing once sufficient numbers of AT2 cells were regenerated, thus explaining the reduction in the overall number of cells co-expressing SPC and PCNA proliferation marker, but an increase in cells expressing SPC only.
An interesting question to address is the relationship between proliferating AT2 cells and DASCs. Our data and lineage-tracing evidence [16
] suggested that these two cell populations were independent of each other. Although pneumonia may result in severe acute diffuse alveolar damage in the lungs, we found that infected mice were able to recover relatively quickly. Alveolar epithelial cells are crucial in mediating gaseous exchange, with AT2 cells secreting surfactant to reduce surface tension and increase lung compliance, while type I cells facilitate gaseous diffusion. To achieve recovery from pneumonia would require the timely replenishment of both alveolar epithelial cell types in the lungs. Although both DASCs [17
] and AT2 cells [13
] have been shown to give rise to both type I and II cells, our data suggested that the proliferating AT2 cells were the major drivers of alveolar epithelial cell regeneration during the early recovery phase of influenza pneumonia. Based on the spatial distribution of DASCs, it is possible that the role of DASCs was to expand, to line the alveolar spaces to fill the gaps in alveoli arising from the destruction of type I and II cells, in order to mitigate the infiltration of inflammatory cells and fluid infiltrating into the alveolar air spaces. The fact that the DASCs only appeared during severe but not in milder lung injury [20
] lends credence to the possibility that the DASCs act as an “emergency repair” mechanism during severe lung injury to counter the inflammation-induced damage. This may also account for the independence of the two cell populations. While it is possible that DASCs may differentiate into type I and II cells during the later phase until complete recovery, we did not observe this phenomenon at least in the immediate repair phase following pneumonia.
It is also noteworthy that these cells involved in lung repair only emerged when the inflammatory process began to shift towards adaptive immune responses. Inflammation is a vital process triggered by infection, and can be divided into three different phases: (a) pro-inflammatory response; (b) switch towards tissue repair; and (c) tissue homeostasis [46
]. At 7 dpi when innate immune responses dominated, the DASC pods were not yet formed, and the AT2 cells had not started to proliferate. This is unsurprising in view of the hostile microenvironment expected at the site of inflammation and innate immunity (such as the release of reactive oxygen species and hypoxia), rendering it unfavorable for the cells to begin the repair process. Towards the later stages of pneumonia at around 15 dpi when there was a shift away from the innate immune response, we observed peak AT2 cell proliferation and distinct DASC pods. Inflammation is a key driver of lung tissue regeneration [47
] through the production of growth factors by lung macrophages such as hepatocyte growth factor or HGF [50
]. Such growth factors promote proliferation and differentiation of the relevant cells [55
], and may explain the appearance of DASCs and proliferating AT2 cells, during the transition of inflammation towards the tissue repair phase. From this, we can appreciate the importance of balancing of the immune response before and during the recovery phase, and the relationship between inflammatory and regenerative processes in the lungs. Hitherto, it is still unclear how inflammation drives the emergence of these DASCs and proliferating AT2 cells to initiate the tissue repair process. Moreover, the appearance of DASCs is documented in various lung injury models such as bleomycin (to represent acute inflammation) [18
] and idiopathic pulmonary fibrosis (chronic inflammation) [57
], but not during the normal alveolar turnover process. While inflammation may considerably influence the appearance of DASCs, the degree of lung injury may also determine whether DASCs are required for the repair process [20
Spatio-temporal quantification of lung cells can enhance our understanding of the dynamic cellular processes of recovery from influenza pneumonia [58
]. While we have described the spatial and temporal characteristics of DASCs, and how the proliferating AT2 cell population reacted to alveolar damage induced by influenza pneumonia, there remain unanswered questions concerning alveolar regeneration.
Firstly: Is there a link between the resultant inflammation and the appearance of stem cells in the lungs? One limitation of our study was that the proteomic analysis of the lungs during recovery was conducted on whole lung tissue. Any molecular processes related to regeneration may be masked by the extensive inflammatory processes occurring at the same time. Notwithstanding this, our data indicated that inflammation is a critical process in pulmonary regeneration, consistent with reports describing the vital role of macrophages in alveolar regeneration following infection and pneumonectomy [59
]. There is increasing evidence that lung resident macrophages contribute significantly to the development of the adult stem cells in the lungs in response to influenza pneumonia.
Secondly: Would the proliferation of AT2 cells be sufficient for lung repair for varying degrees of severity of influenza injury? In our model, we employed the PR8 strain that is specifically mouse-adapted and known to cause severe damage to murine lungs [61
]. However, DASCs were not observed using another influenza virus (H3N2 strain X31) which resulted in less severe damage to the lungs [20
]. Given that different influenza virus subtypes result in different disease severity [62
], the severity of damage may drive differential epithelial regeneration and repair processes, an area worthy of future exploration.
Thirdly: What is the relationship between the proliferating AT2 cells, DASCs and the newly regenerated alveolar epithelial cells during the entire regenerative process? We only described the reported stem cells in the lungs up to 25 dpi, which could be considered to be an early phase of recovery due to the simultaneous presence of lung damage and regeneration. An extended study up to 60 dpi or even longer to study the mid- to even late stage of recovery and tissue repair would elucidate how the different stem cells interact and contribute to alveolar regeneration following influenza pneumonia. Finally, the influence of crucial host factors such as aging and DNA repair processes on lung regeneration also merits consideration [63