Research ArticleIMMUNOLOGY

Inhaled GM-CSF in neonatal mice provides durable protection against bacterial pneumonia

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Science Advances  14 Aug 2019:
Vol. 5, no. 8, eaax3387
DOI: 10.1126/sciadv.aax3387
  • Fig. 1 Administration of rGM-CSF increases AMs in LPL−/− mice.

    (A) rGM-CSF (20 ng) in 6 μl of PBS was administered via intranasal (i.n.) instillation to neonatal pups on DOB, PND1, and PND2. Mice were evaluated at indicated times after rGM-CSF administration. (B) Representative flow cytometry of whole-lung homogenates from PND3 WT and LPL−/− pups treated intranasally with PBS or rGM-CSF. (C) Quantification of the distribution of monocytes, pre-AMs, AMs, and total CD11c+ (maturing) cells in PND3 WT and LPL−/− pups. (D) Representative flow cytometry from BAL fluid obtained from adult WT and LPL−/− mice that had received neonatal rGM-CSF therapy. (E) Percentage and number of AMs recovered from BAL fluid from adult WT and LPL−/− mice that had received neonatal rGM-CSF treatment. (C and E) n of each group is provided below x axes. Data were obtained from four independent cohorts of animals. P values are determined using the Mann-Whitney U test. Kruskal-Wallis test comparing four groups revealed P = 0.0014 for AM % (top) and P = 0.015 for AM numbers (bottom). The “n” of AM numbers (bottom) in some groups is lower than AM % (top) because cell numbers in one experiment were counted manually rather than by cytometer acquisition. Only cell counts obtained by the same method (cytometer acquisition) are included in data shown here.

  • Fig. 2 Adult LPL−/− mice that received neonatal rGM-CSF therapy are protected from pneumococcal infection.

    (A) Experimental design: Neonatal WT and LPL−/− pups were intranasally given 20 ng of rGM-CSF or PBS (control) on DOB, PND1, and PND2. After reaching adulthood, mice were challenged intratracheally (i.t.) with pneumococcus. (B) Adult WT (gray) or LPL−/− (black) mice that had received neonatal rGM-CSF therapy (open symbols) or PBS (control; closed symbols) were infected intratracheally with pneumococcus and monitored for survival. Survival curves show data combined from three independent experiments (WT + PBS, n = 19; WT + rGM-CSF, n = 7; LPL−/− + PBS, n = 11; LPL−/− + rGM-CSF, n = 16) and were compared using Mantel-Cox log-rank test. (C) Quantitative blood culture obtained from adult mice 24 hours after intratracheal instillation of pneumococci. Comparison of all groups by one-way analysis of variance (ANOVA) (Kruskal-Wallis test), P = 0.0007. (D) Distribution of neutrophils [polymorphonuclear leukocytes (PMNs)], monocytes, and B cells in the peripheral blood of WT and LPL−/− mice challenged with pneumococcus. Mice had received neonatal rGM-CSF therapy (GM) or PBS (P; control). (E) Number of AMs recovered from BAL fluid of WT or LPL−/− adult mice that survived 2 weeks after intratracheal pneumococcal infection. Grossly bloody BAL fluids were excluded from analysis (clotted). (F) Distribution of DCs, eosinophils, neutrophils (PMNs), and monocytes in whole-lung homogenates from uninfected adult WT and LPL−/− mice that had received neonatal rGM-CSF therapy or PBS (control). Percentages of total CD45+ cells are given. (C to F) Data are combined from three (C to E) or two (F) independent experiments. n of each group is given below x axes. P values of comparisons between two groups are determined using the Mann-Whitney U test.

  • Fig. 3 rGM-CSF administration increased pre-AM proliferation in LPL−/− pups.

    (A) Representative flow cytometry of BrdU incorporation into AMs and precursors (monocytes and pre-AMs, defined as shown) in whole-lung homogenates from PND3 WT and LPL−/− pups receiving rGM-CSF therapy or PBS (control). Percentage of cells in each gate incorporating BrdU listed in the top right-hand corner. (B) Quantification of the percentage of monocytes, pre-AMs, or AMs incorporating BrdU in PND3 WT or LPL−/− pups receiving neonatal rGM-CSF therapy (gray symbols) or PBS (control; open symbols). Each symbol represents one animal. Data are combined from three independent experiments. Line shows median value. P values are determined using the Mann-Whitney U test. (C) TGF-β concentration in whole-lung homogenates from PND3 WT and LPL−/− pups receiving neonatal rGM-CSF therapy (gray bars) or PBS (open bars). Line shows the median value. n of each group is listed below the x axis. Data are from two independent cohorts of animals.

  • Fig. 4 Neonatal rGM-CSF corrects increased SP-D in LPL−/− mice.

    (A) SP-D or (B) SP-A concentrations in whole-lung homogenates obtained from PND3 pups or adult WT or LPL−/− mice that had received neonatal rGM-CSF therapy (gray bars) or PBS (control; open bars). Line shows the median value. P values are determined using the Mann-Whitney U test. n of each group is listed below x axes. Data are combined from two independent cohorts of animals.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/8/eaax3387/DC1

    Fig. S1. No increase in mature AMs following the subcutaneous administration of rGM-CSF (20 ng) to LPL−/− neonatal pups on DOB, PND1, and PND2.

    Fig. S2. No disruption of alveolarization observed after intranasal neonatal rGM-CSF therapy.

    Fig. S3. Increased SP-D in LPL−/− PND3 neonatal pups.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. No increase in mature AMs following the subcutaneous administration of rGM-CSF (20 ng) to LPL−/− neonatal pups on DOB, PND1, and PND2.
    • Fig. S2. No disruption of alveolarization observed after intranasal neonatal rGM-CSF therapy.
    • Fig. S3. Increased SP-D in LPL−/− PND3 neonatal pups.

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