IL-12 p80-dependent macrophage recruitment primes the host for increased survival following a lethal respiratory viral infection

Abstract

A protective immune response to a respiratory viral infection requires a series of coordinated cellular and molecular responses. We have previously demonstrated that increased expression of airway epithelial cell interleukin (IL)-12 p80, a macrophage chemoattractant, is associated with human respiratory viral infection and mediates post-viral mortality in the mouse. To better understand the role of IL-12 p80-dependent macrophage chemotaxis in mediating viral immunity, we generated a transgenic mouse strain utilizing a promoter to drive IL-12 p40 gene expression in the airway epithelium. This transgenic strain secreted biologically active IL-12 p80 in a lung-specific manner, and demonstrated a selective increase in the number of resident, unactivated airway macrophages at baseline. Following infection with a sublethal dose of mouse parainfluenza virus type 1 (Sendai virus), the transgenic mice demonstrated an earlier peak and decline in the number of airway inflammatory cells. The transgenic mice were resistant to a lethal dose of virus and this viral resistance was dependent on the increased number of airway macrophages at baseline as partial depletion prior to infection abrogated this phenotype. The survival advantage in the transgenic mice was independent of viral load but was associated with a more rapid decline in the number of airway inflammatory cells and concentrations of multiple chemokines including the CC chemokine ligand 2 (CCL2)/JE, CCL3/macrophage inflammatory protein (MIP)-1alpha, CCL4/MIP-1beta, and CCL5/RANTES. Collectively, these results suggest that IL-12 p80-driven increases in the number of resident airway macrophages prime the host for a protective immune response that can confer increased survival following a lethal respiratory viral infection.

Figure 1

p80/p40 transgenic mice demonstrate lung-specific expression of biologically active interleukin (IL)-12 p80. (a) Bronchoalveolar lavage (BAL) or serum from 7-week-old wild-type (WT) or IL-12 p80/p40 transgenic (p80/p40 Tg) littermates was analysed for IL-12, IL-12 p80/40 and IL-23 by enzyme-linked immunosorbent assay (ELISA). Values represent mean ± standard deviation (SD) of duplicate samples (n = 5–6). (b) Recombinant IL-23, IL-12 p40 monomer (p40), IL-12 and IL-12 p80 (p80) standards, and concentrated BAL fluid from WT and p80/p40 Tg (Tg⁺) mice were subjected to western analysis against anti-IL-12 p40 antibody (Ab) under non-reducing conditions (top) or anti-mouse immunoglobulin G (IgG) Ab under reducing conditions (bottom). (c) Wild-type C57BL/6J or C57BL/6J IL-12 receptor β-1-deficient (IL-12Rβ1 −/−) peritoneal macrophages were placed in the upper compartment of a modified Boyden chamber apparatus. Media, concentrated BAL from WT or p80/p40 Tg mice, or media with the chemoattractant JE (10⁻⁸ m) were placed in the lower compartment and the apparatus was incubated for 2 hr at 37°. Migrating cells were counted and values represent mean ± SD for five high-power fields (HDF) (magnification × 400) (n = 9–12). For panels (b) and (d) a significant difference from WT is indicated (*P < 0·05).

Figure 2

Increased numbers of resident airway macrophages in the p80/p40 transgenic mice. (a) Lung tissue from wild-type (WT) or p80/p40 transgenic (p80/p40 Tg) littermates was immunolabelled with anti-Mac3 antibody (Ab) (top row) or control immunoglobulin G (IgG) (bottom row) and detected with 3,3′-diaminobenzidine (brown colour). Arrows indicate alveolar macrophages shown at higher magnification in the insert. Representative photomicrographs are shown (n = 5). Bar, 20 μm. (b) Bronchoalveolar lavage (BAL) fluid from WT or p80/p40 Tg littermates was analysed for total and differential cell numbers. Values represent mean ± standard deviation (SD) (n = 4–6). (c) Blood from WT or p80/p40 Tg littermates underwent total and differential cell counts using an automated veterinary haematological analyser. Values represent mean ± SD (n = 10). (d) Total lung cells from WT or p80/p40 Tg littermate lungs were counted, immunolabelled, and examined by flow cytometry for conventional dendritic cells (DCs; based on scatter characteristics, high CD11c expression and B220⁻) and plasmacytoid dendritic cells (pDCs; scatter characteristics, low CD11c⁺ and high B220 expression). Values represent mean ± SD of cells per lung (n = 4). Macs, macrophages; Lymphs, lymphocytes; Mono, monocytes; PMNs, polymorphonuclear leucocytes; Eos, eosinophils.

Figure 3

Enhanced resolution of viral-dependent airway inflammation in the p80/p40 transgenic mice. (a) Wild-type (WT) or p80/p40 transgenic (p80/p40 Tg) littermates were inoculated with Sendai virus 5000 egg infectious dose 50% (Sendai 5 K) and day 3, 5 and 8 post-inoculation lung sections were stained with haematoxylin and eosin. Representative photomicrographs are shown (n = 5). Bar, 100 μm. (b–e) BAL from day 3, 5, 8 and 21 post viral inoculation was analysed for total and differential cell numbers. Values represent mean ± standard deviation (n = 4–6), and a significant difference from WT is indicated (*P < 0·05). Macs, macrophages; Lymphs, lymphocytes; PMNs, polymorphonuclear leucocytes; Eos, eosinophils.

Figure 4

Resistance to a lethal Sendai virus infection in the p80/p40 transgenic mice is abrogated by macrophage depletion. (a) Wild-type (WT; n = 25) or p80/p40 Tg (n = 21) littermates were inoculated with Sendai virus 50 000 egg infectious dose 50% (Sendai 50 K) and monitored for survival by Kaplan–Meier analysis. A significant increase in survival of p80/p40 Tg mice (by Wilcoxon rank-sum test) is indicated (*P < 0·05). (b) p80/p40 Tg mice were inoculated with empty liposomes (lipid; n = 28) or clodronate-containing liposomes (lipid + clodronate; n = 26) 5 days prior to inoculation with Sendai virus 50 000 EID₅₀ (Sendai 50 K). Mice were monitored for survival by Kaplan–Meier analysis and a significant decrease in survival of mice treated with clodronate (by Wilcoxon rank-sum test) is indicated (*P < 0·05).

Figure 5

Resistance to a lethal Sendai virus infection in the p80/p40 transgenic mice is associated with enhanced resolution of viral-dependent airway inflammation. (a) Wild-type (WT) or p80/p40 transgenic (p80/p40 Tg) littermates were inoculated with Sendai virus 50 000 egg infectious dose 50% (Sendai 50 K) and day 3, 5 and 8 post-inoculation lung sections were stained with haematoxylin and eosin. Representative photomicrographs are shown (n = 8). Bar, 100 μm. (b–e) BAL from day 3, 5, 8 and 21 post viral inoculation was analysed for total and differential cell numbers. Values represent mean ± standard deviation (n = 6–8), and a significant difference from WT is indicated (*P < 0·05). Macs, macrophages; Lymphs, lymphocytes; PMNs, polymorphonuclear leucocytes; Eos, eosinophils.

Figure 6

Resistance to a lethal Sendai virus infection in the p80/p40 transgenic mice is independent of viral load. (a) Wild-type (WT; top) or p80/p40 Tg (bottom) littermates were inoculated with Sendai virus 50 000 egg infectious dose 50% (50 K) and day 5 post-inoculation lung sections were immunolabelled with anti-Sendai antibody (Ab) [detected with fluorescein isothiocyanate (FITC); green] and anti-CD68 Ab (detected with Alexa Fluor 555; red). Top and bottom rows are identical views photographed with a filter for the green channel (column 1), a filter for the red channel (column 2), and a merged image (column 3). Control immunoglobulin G (IgG) Ab gave no signal above background (not shown). Arrows indicate dual-labelled cells. Representative photomicrographs are shown (n = 4). Bar, 20 μm. (b) Wild-type (WT) and p80/p40 Tg littermates were inoculated without Sendai virus or with Sendai virus 50 K and day 3, 5, 8 and 12 whole-lung homogenates were analysed for Sendai plaque-forming units (PFU)/g of lung tissue. Values represent mean ± standard deviation for duplicate samples (n = 6–8). (c) Wild-type and p80/p40 Tg littermates were inoculated without Sendai virus or with Sendai virus 50 K or 5 K and whole-lung RNA from day 3, 5 and 8 post-inoculation was analysed for Sendai virus-specific and GAPDH RNA by one-step fluorogenic reverse transcriptase–polymerase chain reaction (RT-PCR). The mean of duplicate measurements of Sendai virus-specific RNA was normalized to GAPDH and reported as the Sendai to GAPDH ratio. A significant difference from WT is indicated (*P < 0·05).

Figure 7

Enhanced resolution of viral-dependent airway inflammation in the p80/p40 transgenic mice is associated with altered expression of airway chemokines. (a–d) Wild-type (WT) or p80/p40 transgenic (p80/p40 Tg) littermates were inoculated without or with Sendai virus 50 000 egg infectious dose 50% (EID₅₀) and at days 0, 3, 5 and 8 cell-free bronchoalveolar lavage (BAL) supernatants were analysed for CC chemokine ligand 2 (CCL2)/JE [murine homologue of human monocyte chemoattractant protein (MCP)-1] (a), CCL3/macrophage inflammatory protein (MIP)-1α (b), CCL4/MIP-1β (c) and CCL5/RANTES (d). Values represent mean ± standard deviation for duplicate samples (n = 6–8) and a significant difference from WT is indicated (*P < 0·05).