Alveolar epithelial cells orchestrate DC function in murine viral pneumonia

Abstract

Influenza viruses (IVs) cause pneumonia in humans with progression to lung failure. Pulmonary DCs are key players in the antiviral immune response, which is crucial to restore alveolar barrier function. The mechanisms of expansion and activation of pulmonary DC populations in lung infection remain widely elusive. Using mouse BM chimeric and cell-specific depletion approaches, we demonstrated that alveolar epithelial cell (AEC) GM-CSF mediates recovery from IV-induced injury by affecting lung DC function. Epithelial GM-CSF induced the recruitment of CD11b+ and monocyte-derived DCs. GM-CSF was also required for the presence of CD103+ DCs in the lung parenchyma at baseline and for their sufficient activation and migration to the draining mediastinal lymph nodes (MLNs) during IV infection. These activated CD103+ DCs were indispensable for sufficient clearance of IVs by CD8+ T cells and for recovery from IV-induced lung injury. Moreover, GM-CSF applied intratracheally activated CD103+ DCs, inducing increased migration to MLNs, enhanced viral clearance, and attenuated lung injury. Together, our data reveal that GM-CSF-dependent cross-talk between IV-infected AECs and CD103+ DCs is crucial for effective viral clearance and recovery from injury, which has potential implications for GM-CSF treatment in severe IV pneumonia.

Figures

Figure 1. AEC GM-CSF attenuates acute lung injury and increases survival in PR8 infection.

(A) Gm-csf mRNA expression in primary murine (m-) or human (h-) AECs infected with PR8 in vitro. (B) Gm-csf mRNA induction in purified AECs from PR8-infected WT mice at 3 dpi. Expression of the type II AEC marker Sftpc was additionally determined. (C) GM-CSF release in LHs of PR8-infected WT, Gm-csf–/–, SPC-GM, Gm-csf–/–→WT, and WT→Gm-csf–/– mice. (D) AEC apoptosis, determined by quantification of Annexin V binding to CD45–CD31–EpCam+ LH cells. (E) Alveolar albumin leakage analyzed after PR8 infection. The ratio of BALF and serum FITC fluorescence is expressed in AU. (F) Arterial oxygen saturation (SO2). (G) Survival of WT and Gm-csf–/– mice infected with 500 PFU PR8 (n = 8 per group), WT and SPC-GM mice infected with 2,000 PFU PR8 (n = 8 per group), and Gm-csf–/–→WT and WT→Gm-csf–/– mice infected with 500 PFU PR8 (n = 9 per group). Data are mean ± SD. *P < 0.05, ***P < 0.005 vs. mock-infected control (ctl) or as indicated by brackets.

Figure 2. Lung-protective effects of AEC GM-CSF are mediated by pulmonary DCs in SPC-GM mice after PR8 infection.

(A) FACS quantification of macrophages (Mac; CD45+CD11c+SiglecF+MHCIIint; solid line) and DCs (CD45+CD11c+SiglecF–MHCIIhi; dashed line) in the lungs of uninfected or PR8-infected (7 dpi) WT, Gm-csf–/–, and SPC-GM mice. (B) Mice were i.t. treated with clodronate liposomes (CL) to deplete alveolar macrophages or with empty liposomes (EL) 48 hours prior to PR8 infection, and AEC apoptosis and alveolar albumin leakage were determined at 7 dpi. (C) Treatment protocol. WT and SPC-GM mice were lethally irradiated and transplanted 1 × 106 CD11c+/DTR BM cells to generate CD11c+/DTR→WT or CD11c+/DTR→SPC-GM chimeric mice. 26 days later, when >90% of lung DCs were of CD11c+/DTR donor phenotype (whereas alveolar macrophages were mainly of recipient CD11c+/+ phenotype), chimeras were treated with clodronate or empty liposomes i.t. and with DTX or PBS i.p. to deplete lung macrophages and DCs, respectively, then infected with PR8 48 hours later. (D) Depletion efficacy in CD11c+/DTR→WT chimeras at 28 dpi, determined by FACS. Fractions of alveolar (CD45+CD11c+MHCIIintSiglecF+; solid line) and lung tissue (CD45+CD11c+SiglecF+MHCIIint) macrophages and of alveolar DCs (CD45+CD11c+MHCIIhiSiglecF–; dashed line) were determined from BALF and LH, respectively, and fractions of CD45+CD11c+MHCIIhiCD103+ (red gates) and CD45+CD11c+MHCIIhiCD11b+ (blue gates) DCs were determined from LH and MLNs. (E) Survival of clodronate liposome/DTX–treated CD11c+/DTR→WT mice and empty liposome/PBS–, clodronate liposome/PBS–, or clodronate liposome/DTX–treated CD11c+/DTR→SPC-GM mice after PR8 infection (n = 7–8). Data are mean ± SD. *P < 0.05, ***P < 0.005.

Figure 3. AEC GM-CSF is required for accumulation of DC populations in the lung under steady-state conditions and after PR8 infection.

SPC-GM, Gm-csf–/–, and WT mice were PR8 infected, and the number of total CD45+CD11c+MHCIIlo/hiSiglecF– DCs (A), CD11b+ DCs (CD45+CD11c+MHCIIhiB220–CD103–CD11b+; B), mo-DCs (CD45+CD11c+MHCIIloB220–CD103–CD11b+; C), and CD103+ DCs (CD45+CD11c+MHCIIhiB220–CD103+; D) were quantified by counting LH cells and flow cytometric quantification of the respective proportions (see Supplemental Figure 3 for gating strategy). (E) Representative dot plots for lung DC subpopulations from uninfected (CD11b+, blue gates; CD103+, red gates, gated from CD45+CD11c+MHCIIhiB220– LH cells) and PR8-infected (CD11b+, blue gates; mo-DC, green gates, gated from CD45+CD11c+MHCIIhi/loB220– LH cells) mice. Data are mean ± SD. *P < 0.05, ***P < 0.005.

Figure 4. AEC GM-CSF is required for CD103+ DC activation and migration to draining MLNs under steady-state conditions and upon PR8 infection.

(A and B) WT, Gm-csf–/–, SPC-GM, WT→Gm-csf–/–, and Gm-csf–/–→WT mice were PR8 infected, and the fractions of CD11b+ DCs (A) and CD103+ DCs (B) in MLNs were quantified by flow cytometry using the gating strategy in C (red gates, CD103+ DCs; blue gates, CD11b+ DCs). (D) Migratory CD103+ DCs from LH of WT mice were additionally stained for IV NP or control IgG at 5 dpi. Representative FACS plot depicts the NP+ fraction of CD45+CD11c+MHCIIhiCD103+ DCs. (E) Comparative flow cytometric quantification of CD80 and CD86 expression on lung and MLN CD103+ DCs after PR8 infection. Values are given as mean intensities ×1,000 of PE (CD80) and PE-Cy7 (CD86) fluorescence. Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.005.

Figure 5. AEC GM-CSF is required for alveolar antiviral CD8+ T cell responses and PR8 clearance from mouse lungs.

(A) Absolute numbers of CD4+ and CD8+ T lymphocytes in BALF were determined by counting BALF cells and by flow cytometric quantification of the CD45+SSCloCD3+CD4+ and CD45+SSCloCD3+CD8+ fractions, respectively, after PR8 infection in WT, Gm-csf–/–, and SPC-GM mice. (B) Antigen-specific CD4+ and CD8+ T cell numbers in BALF or spleen, determined by analysis of the NPpeptide/H2-Db dextramer+ or the IFN-γ+ fraction of CD45+SSCloCD3+CD4+ and CD45+SSCloCD3+CD8+ cells, respectively, after PR8 infection (7 dpi). (C) Representative FACS plots of the IFN-γ analysis; percent values of the respective IFN-γ+ proportions are shown in each plot. (D) PR8 titers in WT, Gm-csf–/–, SPC-GM, WT→Gm-csf–/–, and Gm-csf–/–→WT mice, quantified from BALF at the indicated dpi by standard plaque assay and given as PFU/ml × log10 (detection limit, 101 PFU/ml × log10). Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.005. SSC, side scatter.

Figure 6. Lung-protective effects of AEC GM-CSF are mediated by CD103+ lung DCs.

(A) Treatment protocol. WT or SPC-GM mice were transplanted 1 × 106 Langerin+/DTR BM cells to generate Langerin+/DTR→WT or Langerin+/DTR →SPC-GM chimeric mice. 26 days later, when >90% of lung DCs were of Langerin+/DTR donor phenotype, mice were treated with either DTX or PBS to deplete CD103+ DCs, then infected with PR8 48 hours later for further analyses at 35 dpi. (B) At 28 dpi, efficacy of CD103+ DC depletion after PBS or DTX treatment was analyzed by FACS. The CD103+ fraction of CD45+CD11c+MHCIIhi LH cells was determined after gating on the CD45+CD11c+MHCIIhi population; percentage values are given. At 7 dpi, the fractions of CD103+ and CD11b+ DCs in MLNs were quantified (C), using the gating strategy in Figure 3C; the proportions of IFN-γ+ of CD4+ and CD8+ T cells in BALF were quantified (D), as outlined in Figure 5C; and the fractions of NP+ EpCam+CD45– epithelial cells in LHs were determined by FACS (E), depicted as absolute numbers in Langerin+/DTR→WT and Langerin+/DTR→SPC-GM chimeric mice after PBS or DTX application. (F) AEC apoptosis in chimeric mice after PBS or DTX application was analyzed by FACS quantification of annexin V binding to CD45–CD31–EpCam+ LH cells at 7 dpi, and (G) total protein was determined in BALF. (H) Survival of PR8-infected chimeric mice after PBS or DTX treatment (n = 8–13). Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.005.

Figure 7. GM-CSF application i.t. increases CD103+ DC migration, alveolar T lymphocyte recruitment, and viral clearance, which is associated with attenuated lung injury after PR8 infection.

WT mice were PR8 infected (or mock infected in selected experiments), followed by i.t. application of 5 μg GM-CSF or PBS plus 0.1% BSA. (A) At 7 dpi, the fraction of CD103+ DCs in MLNs was quantified by flow cytometry. (B) Absolute numbers of CD4+ and CD8+ T lymphocytes in BALF were determined by counting BALF cells and flow cytometric quantification of the CD45+SSCloCD3+CD4+ and CD45+SSCloCD3+CD8+ fractions, respectively, and (C) PR8 titers were determined from BALF by standard plaque assay. (D–F) At 7 dpi, AEC apoptosis (D), alveolar albumin leakage (given as ratio of BALF and serum FITC fluorescence in arbitrary units; E), and arterial oxygen saturation and partial CO2 pressure (pCO2; F) were determined. Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.005.

Figure 8. AEC GM-CSF mediates recovery from IV-induced lung injury by improving antiviral host defense functions of pulmonary CD103+ DCs.

IV infection induces GM-CSF in AECs, which is particularly required for activation and migration of CD103+ DCs to the draining MLNs. By its effects on CD103+ DCs, epithelial GM-CSF increases the recruitment of IFN-γ+CD4+ and IFN-γ+CD8+ T cells to the air spaces, accelerates IV clearance, and mediates recovery from epithelial injury.