Influenza viruses target epithelial cell subsets of the distal murine lung to different extent after intratracheal infection

It is well established that IV infect different subsets of the airways and alveoli, particularly ciliated and goblet cells, club cells and AECII [5–7]. However, recent advances in the field resulted in the definition of more specialized subsets of lung epithelial cells, some of which display stem/progenitor cell characteristics and contribute to repair of the injured organ [17, 19, 27]. To address which of these epithelial cell compartments were infected by IV, we fractionated distal lung epithelial cells into different subsets, after dissection of large airways and vessels and depletion of leukocytes and endothelial cells, according to surface expression levels of EpCam and integrin α6 [19], and the lineage markers CD24 (differentiated airway epithelial cells) [19], CC10 (club cells), pro-SPC (AEC II) and T1α (AEC I), by flow cytometry. We identified a high-frequent EpCam^lowα6^low fraction (91.3 ± 1.8%) and a low frequent EpCam^highα6^high fraction, the latter of which consisted of a CD24^low and CD24^high population (1.7 ± 0.3% and 6.3 ± 1.8%, respectively, Fig 1A). The majority of the most abundant EpCam^lowα6^low cells showed a granular cytoplasm typically observed in AEC II, with approximately 95% of the cells expressing the AEC II signature pro-SPC^highT1α^neg and around 5% expressing an AEC I signature (SPC^negT1α⁺) (Fig 1B). EpCam^highα6^highCD24^high cells contained pro-SPC^negCC10^neg differentiated small airway epithelial cells (SAEC, 70%), composed of both β-tubulin⁺ ciliated and mucin5AC⁺ goblet cells, and pro-SPC^negCC10⁺ club cells (30%) (Fig 1C). EpCam^highα6^highCD24^low cells were cells of homogeneous morphology and stained positive for the stem cell antigen Sca-1⁺ (Fig 1D).

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Fig 1. Characterization of distal lung epithelial cell subpopulations and analysis of their infection rates in vivo.

(A) Gating strategy of three epithelial cell subsets in CD31 and CD45 depleted lung homogenates of wt mice according to the expression of EpCam, α6 integrin and CD24. (B) Pappenheim stained cytospins of flow-sorted EpCam^lowα6^low epithelial cells and flow cytometric subgating of this fraction with proSPC and T1α. (C) Characterization of the EpCam^highα6^highCD24^high subpopulation by flow cytometry reveals a CC10⁺ and a CC10^neg fraction. Pappenheim stained cytospins of flow-sorted EpCam^highα6^highCD24^highCC10^neg epithelial cells (arrows indicate ciliated cells) and immunofluorescence stainings of this cell subset with mucin5ac and β-tubulin after 4d of culture. (D) Flow-sorted and Pappenheim stained cytospins of the EpCam^highα6^highCD24^low epithelial cell population (left). Further flow cytometric phenotype characterization of the EpCam^highα6^highCD24^low population revealed that it is Sca-1⁺ (middle) and localizes to the β4 integrin⁺ and CD200⁺ fraction of EpCam⁺ cells (right, EpCam^highα6^highCD24^low population depicted in red). (E-F) Wt mice were infected with 500pfu of the indicated influenza virus strains and the fractions of influenza virus infected (nucleoprotein⁺, NP⁺) cells of the different EpCam⁺ subpopulations were determined by FACS at d4 pi. (G) Cytospins of the flow-sorted EpCam^highα6^highCD24^low population or of tracheal digests (positive control) from uninfected mice were stained for krt5 and p63 (left). Quantification of p63 and krt5 mRNA levels of flow-sorted EpCam^highα6^highCD24^low at d14 pi from PR/8 infected mice (right). Bar graphs represent fold induction compared to mock-infected controls. Bar graphs represent means ± SD of n = 4 independent experiments; * p<0.05; **p<0.01; ***p<0.001.

https://doi.org/10.1371/journal.ppat.1005544.g001

To analyse which of these epithelial cell subset were targeted by IV, we infected C57BL/6 mice using 500pfu of IV strains of increasing pathogenicity, i.e. low-pathogenic H3N2 (x-31), pandemic pH1N1 strain (A/Hamburg/04/09), causing mild to moderate lung injury at this dose in mice, and the highly pathogenic mouse-adapted PR/8 strain [28, 29]. Quantification of the infection rates by staining for IV nucleoprotein (NP) revealed that EpCam^lowα6^highCD24^high and EpCam^lowα6^low cells were infected with a frequency of ∼11% and ∼6%, respectively, by d4 pi after PR/8 infection, a time point where PR/8 replication in the lung reaches a peak [28]. Subfractionation into differentiated alveolar and airway epithelial cells revealed that rates of PR/8 infection in AEC I and AEC II ranged at ∼8% and ∼4%, whereas club cells and ciliated/goblet cells displayed similar PR/8 infection rates of ∼10%. Of note, EpCam^highα6^highCD24^low cells were infected by IV to high amounts (around 15% of all EpCam^highα6^highCD24^low after PR/8 infection), and the proportion of infected epithelial cell subsets at d4 pi correlated with the level of pathogenicity of the IV strain used (Fig 1E and 1F). To further address whether PR/8 revealed increased tropism to EpCam^highα6^highCD24^low cells, AEC, SAEC and EpCam^highα6^highCD24^low cells were flow-sorted, seeded into culture plates at equal numbers and infected ex vivo with x-31, PR/8 and pH1N1 at an MOI of 2, respectively. After one replication cycle (6h), excluding de novo infection by progeny virions, the infection rate was determined, reflecting the capacity of each virus strain to infect the respective cell population. Similar to our in vivo results, we observed that PR/8 reveals an enhanced tropism to EpCam^highα6^highCD24^low cells (S1 Fig).

Highly infectable EpCam^highα6^highCD24^lowSca-1^pos cells reveal epithelial stem cell characteristics and generate lung-like organoids in an Fgf10-dependent manner

Given that the EpCam^highα6^highCD24^lowSca-1^pos cells which revealed the highest rates of infection were previously described as epithelial stem/progenitor population giving rise to airway and alveolar epithelium [19], we aimed to further characterize their phenotype and stemness properties. Further analyses using established stem cell markers [17] revealed that they were integrin β4⁺CD200⁺, a signature which has been confined to a distal lung stem cell phenotype known to engage the krt5/p63 regeneration program [17] (Fig 1D). In accordance, these cells were negative for krt5 and p63 in healthy lungs, but highly upregulated krt5 and p63 gene expression after IV-induced injury (Fig 1G), suggesting that they contain epithelial stem/progenitor cells (EpiSPC).

To verify these stem/progenitor cell characteristics ex vivo, EpCam⁺ cell fractions were flow-sorted and seeded in organotypic 3D cultures [30]. As opposed to AEC I/II, and SAEC/club cells, EpiSPC developed typical large organoid spheres with cystic or saccular outgrowth in the presence of the growth factors Fgf10 and Hgf (hepatocyte growth factor), a characteristic feature of lung stem/progenitor cells [19] (S2A Fig). A robust clonogenic potential of EpiSPC was demonstrated by repetitive cycles of serial passaging of digested organospheres by single-cell sorting and detection of de novo sphere formation after one week, respectively (S2B Fig), and by use of clonality assays where tdtomato⁺ EpiSPC were mixed with wildtype (wt) EpiSPC at a defined ratio and cultured in matrix, resulting in pure tdtomato⁺ and tdtomato^neg colonies indicative of clonal expansion (S2C Fig).

Given that Fgf10-Fgfr2b signaling is indispensable for epithelial stem cell outgrowth ex vivo [18, 23, 30], we aimed to further define whether EpiSPC expansion and differentiation were Fgf10-dependent. To understand cellular cross-talk mechanisms involved in activation of the regenerative Fgfr2b axis after IV-induced lung injury we sought to define the predominant cellular source of Fgfr2b ligands, Fgf7 and 10 under homeostatic conditions and in the acute phase of IV-induced lung injury, at the peak of EpiSPC proliferation (d7 pi). Lung digests of mock- or PR/8-infected mice (d7 pi) were therefore fractionated by FACS sorting into four main lineages, including endothelial cells (CD31⁺CD45^negEpCam^neg, R1), leukocytes (CD31^negCD45⁺EpCam^neg, R2), epithelial cells (CD31^negCD45^negEpCam⁺, R3) and CD31^negCD45^negEpCam^negSca-1^high cells (S3A Fig, left, R4). mRNA expression of Fgfr2b ligands in these four populations revealed that both Fgf7 and Fgf10 expression was significantly increased in the R4 fraction (CD31^negCD45^negEpCam^negSca-1^high) compared to the other three major lineages of the lung (endothelial cells, epithelial cells, leukocytes), independent of IV infection (S3A Fig, right). Previous data suggested that Sca-1^high expression in EpCam^neg cells was associated with the fibroblast lineage [20]. To address whether Fgf10-expressing resident mesenchymal cells (rMCs) would support organosphere generation, flow-sorted EpiSPC and rMC were co-cultured for several days in absence of growth factor supplementation. As shown in S3B Fig, presence of rMC was sufficient to drive early organosphere formation at d5 of culture. Furthermore, rMCs mediated saccular outgrowth of EpiSPC spheres at d10 and formation of lung-like structures at d16 of culture, as compared to EpiSPC mono-cultures in supplemented medium, and this response was abrogated early in the cystic phase when Fgf10 was blocked by neutralizing antibodies (S3D Fig). Of note, co-culture of EpiSPC with either flow-sorted CD45⁺ (R2) or CD31⁺ (R1) cells did not result in organosphere formation (S3C Fig). Finally, lung-like structures derived from EpiSPC-rMA co-cultures significantly upregulated markers of terminal airway and alveolar cell differentiation, such as T1α and β-tubulin (S3E Fig). Together, these data indicate that EpiSPC both self-renew and differentiate to distal lung epithelial cell subsets in an Fgf10-dependent manner during organotypic culture.

High renewal capacity of EpiSPC after IV-induced lung injury is mediated by the Fgf10-Fgfr2b axis

Analyses of the proliferative response of various distal lung epithelial cell populations after PR/8 infection revealed that the EpiSPC population showed the highest proliferation capacity compared to the AEC and SAEC subsets between d7 to d14 after injury (Figs 2A and S4). Of note, comparison of EpiSPC and AEC proportions under homeostatic conditions and at d7 pi revealed an increase of the EpiSPC pool from 1.7 ± 0.3% to 5.7 ± 1.8% after IV- infection, whereas the high frequent AEC pool is reduced from 91.3 ± 1.8% to 84.7 ± 4.4%. Quantification of phosphatidylserine externalization by Annexin V expression of non-infected and PR/8 infected wt mice at d7 pi, a time point where apoptotic epithelial injury is most prominent in the lungs [8, 28], revealed that EpiSPC were resistant to IV-induced apoptosis, whereas the other EpCam⁺ populations showed high levels of apoptosis in response to infection (Fig 2B). These findings suggest that a damage-resistant cell population with high proliferative capacity is contained in the EpiSPC fraction, which might contribute to renewal of both bronchiolar and alveolar epithelial tissue [19].

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Fig 2. EpiSPC are resistant to apoptosis and show a high proliferative response after PR/8 infection which is mediated by Fgf10/Fgfr2b signaling.

(A) Proliferation rates of the given epithelial cell subsets was analysed in PR/8 infected wt mice by FACS quantification of Ki67⁺ cells at the indicated time points pi. (B) Apoptosis of each EpCam⁺ subset was quantified by FACS (Annexin V⁺ proportions) at d7 post PR/8 infection and of non-infected wt mice. (C) Expression of Fgfr2b on EpiSPC at the given time points post PR/8 or mock infection was quantified by FACS and is given as MFI (median fluorescence intensity) of Fgfr2b ab minus MFI of matched isotype control. The proliferative response of the EpCam⁺ cell subsets was quantified by FACS at d7 pi in Rosa26^rtTA/+;tet(O)sFgfr2b/+ (D) Rosa26^rtTA/+;tet(O)Fgf10/+ mice (E) and Fgf7^-/- mice (F) compared to non-dox-induced or wt littermates. Bar graphs represent means ± SD of n = 4–6 independent experiments; * p<0.05; **p<0.01; +dox, doxycycline food; -dox, normal diet.

https://doi.org/10.1371/journal.ppat.1005544.g002

Given that Fgf10 is an indispensable growth factor for EpiSPC outgrowth ex vivo and that genetic deletion of Fgf10 or of the Fgf10 receptor Fgfr2b results in failure of embryonic lung development [25], we speculated that this pathway might be reactivated to drive EpiSPC proliferation for epithelial regeneration after infectious injury in adult mice. In fact, Fgfr2b surface expression was significantly upregulated on EpiSPC in the course of severe PR/8 infection compared to mock-infected mice, most prominent at d7 post infection (pi) when the proliferative response was highly increased (Fig 2C). To decipher the functional role of Fgfr2b and its ligands Fgf10 and Fgf7 in this EpiSPC renewal response, transgenic mice with inducible overexpression of either soluble dominant negative Fgfr2b or overexpression of the ligand Fgf10, were PR/8-infected and proliferation of all EpCam⁺ subsets was quantified at d7 pi. Attenuation of Fgfr2b signaling by doxycycline induction of a dominant-negative Fgfr2b (scavenging soluble Fgf10) resulted in significant impairment of EpiSPC proliferation capacity compared to non-induced littermates, whereas AEC and SAEC revealed no or little, Fgfr2b-independent proliferation (Fig 2D). Similarly, the proliferating proportion of EpiSPC was significantly increased in mice with induced overexpression of Fgf10 at d7 pi, compared to non-induced litters (Fig 2E). Of note, Fgf7^-/- mice exhibited only slightly but not significantly reduced EpiSPC proliferation in comparison to Fgf7^+/+ mice (Fig 2F). To verify that the Fgf10-Fgfr2b axis is indeed a key pathway in the epithelial regenerative response of the distal lung following IV-induced injury, we determined re-establishment of barrier function and outcome in doxycycline (dox)-induced versus non-induced Rosa26^rtTA/+;tet(O)sFgfr2b/+ mice. Blockade of Fgfr2b signaling resulted in significantly increased lung permeability (as determined by alveolar albumin leakage) during the repair phase at d14 pi (S5A Fig), indicating that this pathway is crucial for re-establishment of gas exchange function. Concomitantly, the surviving sFgfr2b overexpressing mice showed decreased body weight compared to controls during the regeneration phase at d11 to d20 pi, and did not fully regain weight until d21 (S5B Fig). Together, these findings demonstrate that IV infection induces activation of an Fgfr2b-dependent signaling pathway, which largely mediates the EpiSPC proliferative response and barrier repair after IV-induced injury.

Highly pathogenic IV inhibit Fgfr2b-dependent renewal in EpiSPC by blockade of β-catenin mediated transcription

Given that the EpiSPC cell fraction harbored stem/progenitor cells crucial for Fgfr2b-drived lung repair and at the same time represented primary targets of high pathogenic PR/8 in the distal lung, we sought to address whether infection of these cells would result in an impaired renewal response. We therefore infected C57BL/6 mice using 500pfu of IV strains of increasing pathogenicity, i.e. x-31, pH1N1, and the highly pathogenic PR/8 [28, 29]. After 21 days, when mice had apparently recovered from IV infection, x-31 and pH1N1 infected mice showed a restored distal lung epithelial architecture, whereas mice infected with the highly pathogenic PR/8 still presented with thickened alveolar walls and incomplete re-epithelialization (Fig 3A), suggesting that IV of high pathogenicity impaired the regenerative response of the lung. Quantification of Ki67⁺ fractions in infected (NP⁺) versus non-infected (NP^neg) EpiSPC within the same PR/8 infected lungs revealed that the proliferative response was lost in infected EpiSPC (Fig 3B), associated with loss of Fgfr2b upregulation in NP⁺ EpiSPC (Fig 3C). Finally, infection of flow-sorted EpiSPC ex vivo with increasing doses of PR/8 followed by organotypic culture resulted in significantly reduced formation of organospheres depending on the multiplicity of infection (MOI) applied (Figs 3D and S6). This was not due to infection-induced death of EpiSPC (as analysed by live/dead staining). Importantly and in line with these data, intratracheal transplantation of non-infected (viral hemagglutinin-neg; HA^neg) EpiSPC, flow-sorted from the lungs of IV-infected tdtomato⁺ mice, into IV-infected wildtype mice, resulted in integration into and in de novo generation of distal lung tissue (including AEC I) between d7-d14 post transplantation, whereas infected (HA⁺) EpiSPC or SAEC showed incorporation into distal lung tissue, but only limited expansion and did not give rise to distal lung tissue (Figs 3E, S7A and S7B). These data indicate that EpiSPC indeed contain precursors of alveolar tissue in vivo, and IV infection of the EpiSPC niche results in defective tissue repair after IV-induced injury (Figs 3E and S7B). Of note, transplanted flow-sorted EpiSPC of non-infected tdtomato⁺ mice can be visualized at d14 post transplantation in the lung tissue of IV-infected wt mice, but do not expand to generate tissue de novo (S7B and S7C Fig), suggesting that factors expressed within the stem cell niche during IV-induced injury play a crucial role in early activation of quiescent EpiSPC for tissue repair (e.g. via IV-induced upregulation of the p63/krt5 regeneration program, Fig 1G).

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Fig 3. Influenza virus infected EpiSPC are impaired in their regenerative response due to restricted Fgfr2b expression.

(A) Wt mice were infected with 500pfu of the indicated influenza virus strains, or mock-infected, and lung sections were stained with hematoxylin-eosin at d21 pi (arrows indicate areas of non-epithelialized tissue). (B) Infected and non-infected epithelial cell subsets of PR/8 infected wt mice were quantified by flow cytometry for their proliferative response. (C) Quantification of Fgfr2b expression in infected (NP⁺) and non-infected (NP^-) EpiSPC by flow cytometry at d4 pi. (D) Flow-sorted EpiSPC were ex vivo infected with the indicated MOI of PR/8 and seeded in matrix for 3D cultures. At d6 of culture, the number of formed organoids was quantified. (E) Infected (hemagglutinin⁺; HA⁺) or non-infected (hemagglutinin^neg; HA^-) EpiSPC or control HA^- SAEC were flow-sorted from the lungs of PR/8-infected tdtomato⁺ mice at d4 pi for intrapulmonary transplantation into 7d PR/8-infected wt mice. Lung sections were obtained at d7 and d14 after transplantation. Representative micrographs show overlays of brightfield and red staining of tdtomato⁺ transplanted cells. Overlay of tdtomato⁺ transplanted cells (red) and the type I AEC cell marker T1α (green) is shown in the right panels (arrows indicate co-expression of T1α and tdtomato); bars = 100μm. Bar graphs represent means ± SD of n = 3–4 independent experiments; * p<0.05; **p<0.01; ***p<0.001; HA, hemagglutinin; Tx, transplantaion.

https://doi.org/10.1371/journal.ppat.1005544.g003

We next addressed the putative mechanism of inhibition of Fgfr2b upregulation in infected EpiSPC. Previous data revealed that Fgfr2b expression is dependent on Wnt/β-catenin signaling in the developing lung [31]. Indeed, conditional knockout of β-catenin by tamoxifen treatment in adult distal lung epithelial cells from Rosa26^{ERTCre/ERTCre};Ctnnb1^flox/flox mice grown ex vivo (Fig 4A, left) resulted in impaired upregulation of fgfr2b mRNA expression after PR/8 infection (Fig 4A, right). Recent data suggest that β-catenin is involved in expression of interferon-dependent genes and that IV block β-catenin transcriptional activity in vitro as part of an antiviral escape strategy [32]. In fact, activation of the Wnt/β-catenin pathway by LiCl resulted in widely reduced IV replication in ex vivo cultured distal lung epithelial cells, whereas inhibition increased replication, as demonstrated by immunofluorescence and quantification of the viral m segment expression (Fig 4B). Concomitantly, expression of the β-catenin target genes Axin and Ccnd1, and of Fgfr2b, was reduced by ∼50 to 100-fold in infected (HA⁺) compared to non-infected (HA^neg) EpiSPC flow-sorted from PR/8-challenged mice at d7 pi (Fig 4C). These data indicate that EpiSPC tropism of IV represents a key factor of pathogenicity. IV interfere with β-catenin-dependent gene transcription in infected EpiSPC, likely to escape β-catenin anti-viral properties, which results in impaired Fgfr2b expression and reduced renewal capacity in infected EpiSPC.

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Fig 4. β-catenin dependent transcription mediates upregulation of Fgfr2b expression, which is inhibited in PR/8-infected, but not in non-infected lung epithelial cells.

(A) EpCam⁺ lung epithelial cells derived from Rosa26^{ERTCre/ERTCre};Ctnnb1^flox/flox mice were grown to confluency and treated with tamoxifen or DMSO control prior to infection with PR/8 (MOI = 0.1; 24h). mRNA expression of β-catenin (Ctnnb1) (left) or of Fgfr2b (right) was quantified and normalized to values of DMSO-treated control. (B) Wt distal lung epithelial cells in confluent culture were PR/8-infected (MOI 0.1) and treated with an activator (LiCl) or inhibitor (XAV939) of β-catenin signaling. Expression of the viral M segment was quantified at 16 h pi and normalized to LiCl-treated cultures (left). The right plot shows representative photomicrographs of these cultures stained for IV nucleoprotein (NP) after 6 h of PR/8 infection. (C) Wt mice were infected with PR/8 for 7d and infected (IV hemagglutinin⁺, HA⁺) vs. non-infected (HA-) EpCam⁺ cells were flow-sorted. Expression of the β-catenin-dependent transcripts Axin2, Fgfr2b, and Ccnd1 was quantified in HA- cells and normalized to values from HA⁺ cells. All bar graphs represent means ± SD of n = 3–4 independent experiments; * p<0.05; **p<0.01; Tam, tamoxifen.

https://doi.org/10.1371/journal.ppat.1005544.g004

Exogenous application of excess Fgf10 compensates impaired regeneration of the lung epithelial barrier and improves outcome after severe IV infection

To evaluate whether alveolar deposition of excess Fgf10 would counteract the impaired Fgfr2b-mediated renewal response in IV infected mice by increased recruitment of the non-infected Fgfr2b^high EpiSPC, we applied recombinant Fgf10 or PBS to IV-infected C57BL/6 mice at d6 pi. Indeed, Fgf10 treatment resulted in significantly increased proliferation of EpiSPC compared to PBS-treated mice at d7 pi (Fig 5A). IV-infected mice showed a severely disturbed lung architecture with distinct cellular infiltrates, areas of extensive atelectasis and loss of epithelial cells at d10 pi, which was partially reverted by Fgf10 treatment (Fig 5B, left). By d21, Fgf10-treated mice revealed an almost normal lung structure with re-epithelialized bronchioli and alveoli (Fig 5B, right, arrowheads), whereas PBS-treated controls still presented with areas of atelectasis, cellular infiltrates and epithelial injury, indicating failure of epithelial renewal and persisting injury-associated inflammatory responses (Fig 5B, right, arrows). To verify that Fgf10 indeed impacted re-establishment of bronchiolar and alveolar epithelial structures, E-cadherin stainings of lung sections were performed and revealed that Fgf10 treatment resulted in complete re-establishment of the epithelium at d21 pi, associated with increased numbers of Ki67⁺ proliferating cells, whereas PBS-treated mice showed only partial epithelial renewal (Fig 5C). These findings were confirmed by quantification of the total numbers of EpCam⁺ cells in these treatment groups (Fig 5D). Of note, Fgf10-treated murine lungs showed increased expression of krt5, a marker of stem cell-induced repair, at d21 pi (Fig 5E) [16, 17]. Finally, Fgf10-mediated epithelial repair resulted in improved barrier function at d14 pi, and improved survival until d21 compared to controls (Fig 5F and 5G), highlighting the therapeutic potential of Fgf10 to improve EpiSPC-dependent epithelial regeneration.

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Fig 5. Therapeutic treatment with recombinant Fgf10 improves influenza virus-induced lung injury and improves re-epithelialization and barrier repair.

Wt mice were infected with PR/8 and treated with a single dose of either 5μg recombinant Fgf10 (rFgf10) or diluent (PBS^-/-) at d6 pi. (A) The proliferative response of EpCam⁺ epithelial cell subsets was determined by flow cytometry at d7 pi. (B) Lung sections were stained with hematoxylin-eosin at d10 and d21 pi. Arrows depict non-epithelialized alveolar tissue; arrowheads depict areas of ongoing re-epithelialization. (C) Immunofluorescence staining of lung sections for E-cadherin (green), Ki67 (red), and Dapi (blue) at d21 pi. The top row shows lung tissue from mock-infected, untreated mice at d21. (D) Quantification of total lung epithelial cells (EpCam⁺) in lung homogenates at d14 pi. (E) Lung sections were stained for krt5 (green) and Dapi (blue) at d21 pi. (F) Lung barrier function was analysed by quantification of alveolar leakage of FITC-labeled albumin at d14 pi. Values are given in arbitrary units (AU) and represent ratios of FITC fluorescence in BALF and serum. (G) Survival of n = 8 mice per treatment group was analysed until d21 pi. Bar graphs represent means ± SD of n = 5–6 independent experiments; * p<0.05; **p<0.01. Photomicrographs are representative for n = 3–4 independent experiments; bars = 200 μm. (H) Summary: IV with high pathogenicity infect a substantial fraction of EpiSPC, resulting in inhibition of β-catenin-dependent Fgfr2b upregulation and impaired epithelial repair mediated by rMC-expressed Fgf10. Therapeutic application of excess Fgf10 antagonizes IV-induced regeneration failure by engagement of non-infected, Fgfr2b^high EpiSPC.

https://doi.org/10.1371/journal.ppat.1005544.g005

Mice

Wildtype C57BL/6 mice were purchased from Charles River Laboratories. CMV-Cre mice [47] were crossed with rtTA^flox mice [48] to generate mice expressing rtTA under the ubiquitous Rosa26 promoter. This constitutive Rosa26^rtTA/rtTA mouse line was then crossed with tet(O)sFgfr2b/+ or tet(O)Fgf10/+ responder lines to generate Rosa26^rtTA/+;tet(O)sFgfr2b/+ and Rosa26^rtTA/+;tet(O)Fgf10/+ double heterozygous animals on a mixed genetic background, allowing ubiquitous expression of dominant-negative soluble Fgfr2b [49, 50] or of Fgf10 [18, 46] by administration of doxycycline-containing normal rodent diet with 0.0625% doxycycline (Harlan Teklad). Mice were genotyped as described previously [46, 50–52]. Fgf7^-/- mice were obtained from Jackson Laboratory and backcrossed for several generations on a C57BL/6 background (strain #4161). B6.129(Cg)-Gt(ROSA)26Sor^{tm4(ACTB-tdTomato,-EGFP)Luo}/J (mTmG) mice in C57BL/6 genetic background, a tamoxifen-responsive driver mouse line, and Ctnnb1^flox/flox mice were obtained from Jackson Laboratory (strains #7676, #3309 and #4152) and the latter bred to generate homozygous Rosa26^{ERTCre/ERTCre};Ctnnb1^flox/flox mice on a C57BL/6 background allowing induction of a β-catenin knockout by application of tamoxifen. Mice were housed under pathogen-free conditions and experiments were performed according to the regional institutions´ guidelines.

Reagents

The following antibodies were used for flow cytometric analyses, cell sorting or immunofluorescence: CD49f PE or Pacific Blue (clone: GoH3), CD326 (EpCam) APC-Cy7 or FITC (clone G8.8), CD24 PE-Cy7 (clone: M1/69), Ly-6A/E (Sca-1) PerCP/Cy5.5 or Pacific Blue (clone: D7), CD31 Alexa fluor 488 or PE (clone: MEC13.3), CD45 FITC or APC-Cy7 (clone: 30-F11), CD200 PE (clone: OX-90), T1α/podoplanin APC (clone: 8.1.1.) and corresponding isotype controls syrian hamster IgG (clone SHG-1); all Biolegend. Influenza A virus nucleoprotein (NP) FITC (clone: 431, Abcam), Fgfr2b (clone: 133730) and corresponding isotype control IgG2a (clone: 54447, both R&D Systems), p63 (Life Span Biotechnology), CC10 (clones T-18 and S-20) and isotype-matched normal goat IgG (Santa Cruz Biotechnology), CD104 Alexa fluor 647 (clone: 346-11A, AbD SeroTec), Ki67 FITC or PE (clone: B56) and corresponding isotype control IgG1κ FITC or PE (clone: MOPC-21, both BD Bioscience), Annexin V Alexa fluor 647 (Invitrogen), E-cadherin (clone: DECMA-1, Abcam), p63 Alexa fluor 555 (clone: P51A, Bioss), Podoplanin (clone: RTD4E10, Abcam) or corresponding isotype control syrian hamster IgG (clone: SHG-1, Abcam), beta IV tubulin (clone: ONS.1A6) and corresponding isotype control IgG1 (clone: CT6, both Abcam), cytokeratin 5 FITC (Bioss), Uteroglobin (clone: EPR12008, abcam) and isotype-matched monoclonal rabbit IgG (abcam), purified Ki67 (Thermo Scientific), purified pro-surfactant protein C (Millipore), biotinylated mucin 5AC (clone: 45M1, Abcam), anti-influenza NP (clone: 1331, Meridian Life Science). Secondary antibodies used were anti-Streptavidin APC (Becton Dickinson), anti-rabbit Alexa fluor 488/555, anti-goat Alexa fluor 647, anti-mouse Alexa fluor 555/647, anti-rat Alexa fluor 488/647, anti-hamster Alexa fluor 488 (all Molecular Probes). Magnetic separation was performed using biotinylated rat anti-mouse CD45, CD16/32 and CD31 mAb (BD Bioscience, Pharmingen). For ex vivo neutralization assays anti-fgf10 (clone: C-17) or normal goat IgG (Santa Cruz Biotechnology) were used at a concentration of 5μg/ml. For flow cytometric analyses, cells were routinely stained with 7-AAD (Biolegend) or fixable live/dead stain reagents (Molecular Probes) for dead cell exclusion.

In vivo treatment protocols

Mice were anaesthesized and intratracheally inoculated with 500pfu (plaque forming units) of A/PR/8/34 (H1N1, mouse-adapted), A/x-31 (H3N2), or A/Hamburg/5/09 (pandemic H1N1), grown and quantified in Madin Darby Canine Kidney (MDCK) cells (obtained from American Type Culture Collection) and diluted in a total volume of 70 μl in sterile PBS^-/-. In the treatment approach, 5 μg recombinant Fgf10 (R&D Systems) dissolved in sterile PBS^-/- or PBS^-/- alone were intratracheally applied to IV infected mice. Venous blood and BALF were collected as described previously [53]. Lung permeability was determined by i.v. injection of 100 μl FITC-labeled albumin (Sigma-Aldrich) and quantification of FITC fluorescence ratios in BALF and serum with a fluorescence reader (FLX800, Bio-Tek instruments) as described elsewhere [28]. In selected experiments, 20,000 flow-sorted HA⁺ or HA^neg EpiSPC from IV infected mTmG mice were intratracheally applied into IV infected wt mice at d7 pi. Virus titers from BALF were determined by immunohistochemistry-based plaque assay on confluent MDCK cells in 6-well plates in duplicates as previously described [54]. Infected mice were monitored 1–3 times per day and a morbidity score was calculated from weight loss, general appearance, breathing frequency/dyspnea, and body temperature. Mice with a score ≥ 20 were moribund, sacrificed and classified as dead in mortality studies.

Isolation of murine distal lung cells

Lung homogenates of distal lung cell suspensions were obtained by instillation of dispase (BD Biosciences) and 0.5 ml low-melting agarose (Sigma) through the trachea into the HBSS (Gibco) perfused lung, followed by incubation in dispase for 40 min as previously described [55]. After gelling of the agarose and removal of the agarose-filled trachea and proximal bronchial tree, the lung was homogenized (GentleMACS, MACS Miltenyi Biotech) in DMEM/2.5% HEPES with 0.01% DNase (Serva) and filtered through 100μm and 40μm nylon filters. Cell suspensions were incubated with biotinylated rat anti-mouse CD45, CD16/32 and CD31 mAb for 30 min at 37°C followed by incubation with biotin-binding magnetic beads and magnetic separation to deplete leukocytes and endothelial cells prior to flow cytometric analysis and cell sorting or to further culture.

Flow cytometry and cell sorting

Multicolor flow cytometry or high speed cell sorting was performed with an LSR Fortessa or an Aria III cell sorter using DIVA software (BD Bioscience). For analytical measurements 1–5 x 10⁵ cells were freshly stained with fluorochrome-labeled antibodies for 20 min at 4°C in BD FACS buffer. For intracellular stainings (NP, proSPC), permeabilization of cells was achieved by previous incubation with 0.2% saponin in PBS^-/- for 15 min at 4°C, followed by incubation with anti-NP FITC, anti-proSPC, or respective isotype control mAbs for 20 min at 4°C. When non-labeled primary mAb were used, a fluorescent labeled secondary Ab was added and incubated for 20 min at 4°C in FACS buffer. Doublets and dead cells were routinely excluded from the analyses (the latter by using 7AAD). Annexin V staining was performed on fresh, non-permeabilized cells. Prior to antibody incubation, cells were washed and resuspended in Annexin V buffer (BD Bioscience) and incubated with Annexin V 647 and further mAbs in Annexin V buffer for 20 min at 4°C. The stained cells were washed and resuspended in Annexin V buffer. Cell sorting was performed with an 85 or 100μm nozzle. Single cell sorting was performed using the automated cell deposition unit (ACDU) with a 24-well plate and 12mm cell culture inserts (0.4μm pore size, Millipore). Purity of flow-sorted cells was always > 95%.

Culture of flow-sorted lung cells in matrix and clonogenic assays

Mono- or co-culture of EpiSPC and rMC was performed as described previously [30]. In brief, flow sorted cells were counted, resuspended and mixed with growth factor reduced matrix (BD Biosciences) diluted with EpiSPC medium (α-MEM, 10% FCS, 1x pen/strep, 1x insulin/transferrin/selenium, 2mM L-glutamine, 0.0002% heparin) at a 1:1 ratio. Cell suspensions were seeded in 12mm cell culture inserts (0.4μm pore size, Millipore) in a 24-well plate and incubated for 5 min at 37°C, 5% CO₂ to allow gelling. EpiSPC medium was then added to the bottom wells of the plate. In selected experiments, 50ng/ml recombinant Fgf10 and 30ng/ml recombinant Hgf (both R&D systems) or anti-Fgf10 or control Ab were added to the medium at day 2 of culture. For matrix digestion a preheated dispase/collagenase I (Boehringer, Gibco) mixture (3 mg/ml) was added and a single cell suspension was obtained for re-seeding or single-cell sorting. Images were taken with a DM IL LED microscope and a corresponding camera MC170 HD (Leica).

Immunofluorescence and immunohistochemistry

To obtain lung cryosections, lungs were perfused with HBSS and filled with 1.5ml TissueTek (Sakura) mixed with PBS^-/- at a 1:1 ratio as described [56]. Lungs were removed, snap-frozen and 4–10 μm sections were prepared using a Leica cryotome. In selected experiments, lungs were filled with a TissueTek/PBS^-/- mixture containing 1% paraformaldehyde and were incubated in 1% paraformaldehyde after removal. Lung cryosections were stained with Hematoxylin-Eosin or fixed with 4% paraformaldehyde for 20 min, blocked with 0.05% Tween 20, 5% BSA, 5% horse serum in PBS^-/- for 30 min and stained with fluorochrome-labeled mAb diluted in PBS^-/-, 0.1% BSA, 0.2% Triton X-100 for 2 h. After washing, secondary mAbs were added for 2 h, followed by mounting with Dapi containing mounting medium (Vectashield, Vector Labs). Epithelial or mesenchymal cells cultured in chamber slides (Nunc) were fixed in a 1:1 ratio of cold acetone/methanol for 5 min and blocked with 3% BSA in PBS for 30 min prior to staining. Cytospins were additionally stained with Pappenheim stain. Analysis was performed with a Leica DM 2000 or with the Evos Fl Auto (Invitrogen) microscope.

Ex vivo infection of primary cells

Primary murine distal lung cells contained >90% epithelial cells as determined by FACS. Cells were grown in 24-well plates (Greiner) or in chamber slides (Nunc) in DMEM enriched with HEPES, L-Glutamine, FCS, and pen/strep, until confluency and infected with PR/8 at the indicated MOI, as described previously [55]. PR/8 was diluted in PBS^-/- containing BSA and pen/strep and added to the cells for 1 h, until the inoculum was removed and changed to infection medium (DMEM supplemented with BSA, pen/strep, L-Glutamine and trypsin) for further incubation. For inhibition of β-catenin, isolated distal lung epithelial cells of Rosa26^{ERTCre/ERTCre};Ctnnb1^flox/flox mice were treated with 1 μM tamoxifen (Sigma-Aldrich) for 24 hours prior to PR/8 infection. Wildtype cells were treated with either 50 mM LiCl (Abcam) or 10 μM XAV939 (Abcam) directly after PR/8 infection, followed by RNA extraction or immunofluorescence staining. For infection of FACS-sorted lung cells (AEC, EpiSPC, SAEC), the cells were counted, seeded in wells or infected in tubes for 1 hour with the given virus strain and MOI, and were either seeded in matrix, or further incubated at 37°C, 5% CO₂ with EpiSPC medium and processed for FACS analysis at the given time point pi.

Quantitative real-time PCR

RNA from sorted or cultivated cells was isolated using RNeasy Kit (Qiagen) according to manufacturer's manual. cDNA synthesis was performed, as described previously [56] or with RiboSPIA kit (NuGen) according to manual. Actin or ribosomal protein subunit S-18 (RPS-18) expression served as normalization controls for the qRT-PCR, and the reactions were performed with SYBR green I (Invitrogen) in the AB Step one plus Detection System (Applied Bioscience). The following intron spanning primers were used: Actin (FP, 5′-ACCCTAAGGCCAACCGTGA-3′; RP, 5′-CAGAGGCATACAGGGACAGCA-3′), Rps-18 (FP, 5’- CCGCCATGTCTCTAGTGATCC-3′; RP, 5’- TTGGTGAGGTCGATGTCTGC-3′), p63 (FP, 5’-CAAAGAACGGCGATGGTACG-3′; RP 5’-CCTCTCACTGGTAGGTACAGC-3′), Krt5 (FP, 5’-CCTTCGAAACACCAAGCACG-3′; RP 5’-AGGTTGGCACACTGCTTCTT-3′), β-tubb (FP, 5’-CCACCACCATGCGGGAAA-3′; RP, 5’-CTGATGACCTCCCAGAACTTG-3′), Fgf10 (FP, 5’-CCATGAACAAGAAGGGGAAA-3′; RP 5’-CCATTGTGCTGCCAGTTAAA-3′), Fgf7 (FP, 5’- TCGCACCCAGTGGTACCTG-3′; RP, 5’- ACTGCCACGGTCCTGATTTC-3’), Axin2 (FP, 5’-AAGCCCCATAGTGCCCAAAG-3′; RP, 5’-GGGTCCTGGGTAAATGGGTG-3′), Fgfr2b (FP, 5’- AAGAGGACCAGGGATTGGCA-3′; RP, 5’- GTACGGTGCTCCTTCTGGTTC-3′), Ctnnb1 (FP, 5’- ACTTGCCACACGTGCAATTC-3′; RP, 5’-ATGGTGCGTACAATGGCAGA-3′), Ccnd1 (FP, 5’- GCGTACCCTGACACCAAT-3′; RP, 5’- GGTCTCCTCCGTCTTGAG-3′), Pdpn (FP, 5’-CCCCAATAGAGATAATGCAGGGG-3′; RP, 5’-GCCAATGGCTAACAAGACGC-3′), Influenza Virus M segment (5’-GGACTGCAGCGTAGACGC-3′; 5’ CATCCTGTTGTATATGAG-3′; 5’-CATTCTGTTGTATATGAG-3′). The relative gene abundance compared to the reference gene Actin or Rps-18) was calculated as dCt value (Ct_reference−Ct_target). Data are presented as dCT, ddCt (dCt_reference—dCt_target) or fold change of gene expression (2^ddCt).

Ethics statement

Animal experiments performed at the UGMLC were approved by the regional authorities of the State of Hesse (Regierungspräsidium Giessen; reference numbers 100–2012, 48–2013, 26–2013, 09–2009) and conducted according to the legal regulations of the German federal animal protection law (Tierschutzgesetz).

Statistics

All data are given as mean ± SD. Statistical significance between 2 groups was estimated using the unpaired Student’s t test or ANOVA and post-hoc Tukey for comparison of 3 groups and calculated with GraphPadPrism. A p value less than 0.05 was considered significant.