Decreased alveolar macrophage apoptosis is associated with increased pulmonary inflammation in a murine model of pneumococcal pneumonia

Abstract

Regulation of the inflammatory infiltrate is critical to the successful outcome of pneumonia. Alveolar macrophage apoptosis is a feature of pneumococcal infection and aids disease resolution. The host benefits of macrophage apoptosis during the innate response to bacterial infection are incompletely defined. Because NO is required for optimal macrophage apoptosis during pneumococcal infection, we have explored the role of macrophage apoptosis in regulating inflammatory responses during pneumococcal pneumonia, using inducible NO synthase (iNOS)-deficient mice. iNOS(-/-) mice demonstrated decreased numbers of apoptotic macrophages as compared with wild-type C57BL/6 mice following pneumococcal challenge, greater recruitment of neutrophils to the lung and enhanced expression of TNF-alpha. Pharmacologic inhibition of iNOS produced similar results. Greater pulmonary inflammation was associated with greater levels of early bacteremia, IL-6 production, lung inflammation, and mortality within the first 48 h in iNOS(-/-) mice. Labeled apoptotic alveolar macrophages were phagocytosed by resident macrophages in the lung and intratracheal instillation of exogenous apoptotic macrophages decreased neutrophil recruitment in iNOS(-/-) mice and decreased TNF-alpha mRNA in lungs and protein in bronchial alveolar lavage, as well as chemokines and cytokines including IL-6. These changes were associated with a lower probability of mice becoming bacteremic. This demonstrates the potential of apoptotic macrophages to down-regulate the inflammatory response and for the first time in vivo demonstrates that clearance of apoptotic macrophages decreases neutrophil recruitment and invasive bacterial disease during pneumonia.

Figure 1. iNOS deficiency is associated with a reduction in AM apoptosis and bacterial killing cells after pneumococcal challenge ex vivo.

A) Percentage apoptotic cells in cultures of alveolar macrophages (AM) isolated from wild type (C57BL/6) and iNOS deficient mice (iNOS^-/-) 20 h after in vitro challenge with opsonized type 2 pneumococci (Spn), n=6. B) Concentration of bacteria in culture supernatants of C57BL/6 and iNOS^-/- mice 4 and 20 h after in vitro challenge with opsonized type 2 pneumococci (Spn), n=3. Data is derived from triplicate points and is representative of two independent experiments. Mean+SEM, *p<0.05, *** p<0.001, t-test, C57BL/6, iNOS^-/-.

Figure 2. iNOS deficiency is associated with reduction in apoptotic cells in bronchial lavage fluid after pneumococcal infection.

A) The percentage of apoptotic events (apoptotic cells and bodies) in cytospins of bronchial alveolar lavage (BAL) from wild type controls (C57BL/6) and iNOS deficient mice (iNOS^-/-) at the indicated time points after instillation of 10⁷ CFU type 2 pneumococci (Spn) or PBS Percentage B) macrophage (AM) or C) neutrophil (PMN) apoptosis (Annexin V-PE⁺/ToPro 3^-, flow cytometry) in BAL from the same experiments as A). 12h; n=6-8, 24 h; n=3-9, 48 h; n=5-16. Mean+SEM, * p<0.05, ** p<0.01, *** p<0.001, t- test, C57BL/6. PBS, iNOS^-/- PBS, C57BL/6 Spn, iNOS^-/- Spn.

Figure 3. Increased numbers of neutrophils in the bronchial alveolar lavage from lungs of iNOS^-/- mice after pneumococcal infection.

A) Numbers of neutrophils (#PMN) in bronchial alveolar lavage (BAL) from wild type controls (C57BL/6) and iNOS deficient mice (iNOS^-/-) at the indicated time points after intratracheal instillation of 10⁷ CFU type 2 pneumococci. B) Representative RNase protection assay of RNA from whole lung from wild type controls (C57BL/6) and iNOS deficient mice (iNOS^-/-) 12 h post instillation of 10⁷ CFU type 2 pneumococci. C) Levels of TNF-α, KC and monocyte chemotactic protein-3 (MCP3) mRNA as quantified by densitometry and normalised to GAPDH, n=4. D) TNF-α concentration in BAL in the same experiments as A) 12h; n=11-13, 24 h; n= 8-14, 48 h; n=13-16. Mean+SEM, * p<0.05, **p<0.01, ***p<0.001, t-test, C57BL/6, iNOS^-/-.

Figure 4. iNOS inhibition reduces apoptosis and increases pulmonary inflammation after pneumococcal infection.

A) The percentage of apoptotic events (apoptotic cells and bodies) in cytospins of bronchial alveolar lavage (BAL) from C57BL/6 mice 24 h after instillation of 10⁷ CFU type 2 pneumococci (Spn), in the absence (H₂O) or presence of aminoguanidine (AG) treatment. B) Representative dot plots to show the percentage macrophage apoptosis (Annexin V-PE⁺/ToPro 3^-cells as determined by flow cytometry), C) Percentage apoptotic macrophages, D) number of recruited neutrophils (#PMN) and E) TNF-α concentration in BAL in the same experiments as A), n=8. Mean+SEM, * p<0.05, ***p<0.001, t-test, H₂O, AG.

Figure 5. Increased lung inflammation in iNOS^-/- mice after pneumococcal infection.

A) Survival of wild type iNOS sufficient mice (C57BL/6) and iNOS deficient (iNOS^-/-) mice after intratracheal instillation of 10⁷ CFU type 2 pneumococci, n=23-24 per group. B) Concentration of bacteria in blood of C57BL/6 and iNOS^-/- mice 12 and 48 h after intratracheal instillation of 10⁷ CFU type 2 pneumococci. The percentage of C57BL/6 vs. iNOS^-/- mice with bacteremia at 12 h was 53 vs. 82% and at 48 h was 61 vs. 81%. C-D) Concentration of C) IL-6 and D) protein in bronchial alveolar lavage (BAL) 48 h after intratracheal instillation of 10⁷ CFU type 2 pneumococci n=8-16. Mean+SEM, * p<0.05, **p<0.01, ***p<0.001, t-test, C57BL/6, iNOS^-/-.

Figure 6. Histologic appearance of increased lung inflammation in iNOS^-/- mice after pneumococcal infection.

Representative appearances of lung sections stained with haematoxylin and eosin from C57BL/6 and iNOS deficient mice (iNOS^-/-) 48 h after intratracheal instillation of 10⁷ CFU type 2 pneumococci as viewed by 10x and 40x objective. Images were obtained from one of four mice in each group reviewed by two independent pathologists.

Figure 7. Macrophages phagocytose apoptotic cells during pneumococcal infection.

A) The percentage of extracellular apoptotic cells (AC) and B) intracellular AC in cytospins of bronchial alveolar lavage (BAL) from wild type controls (C57BL/6) and iNOS deficient mice (iNOS^-/-) at the indicated time points after instillation of 10⁷ CFU type 2 pneumococci (Spn) or PBS, n=4-16. Mean+SEM, **p<0.01, ***p<0.001, t-test, C57BL/6 PBS, iNOS^-/- PBS, C57BL/6 Spn, iNOS^-/- Spn.

Figure 8. Instillation of apoptotic macrophages into lungs reduces neutrophil numbers in bronchial alveolar lavage after pneumococcal infection.

A) Resident alveolar macrophages (AM) of C57BL/6 mice were labelled in vivo with PKH2 green fluorescent phagocytic cell linker compound. AM from donor mice were labelled in vitro with CellTracker Red and apoptosis induced by UV irradiation. Apoptotic AM were delivered by intra-tracheal instillation. Lungs were lavaged 30 min post instillation and cytospin preparations made from lung cells. AM nuclei were counterstained with DAPI. Arrows indicate red apoptotic donor cells located in green resident AM. B) Numbers of neutrophils (#PMN) in BAL from wild type controls (C57BL/6) iNOS deficient mice (iNOS^-/-) at the indicated time points after instillation of 10⁷ CFU type 2 pneumococci in the absence or presence of apoptotic AM (AC), n=3-7. Mean+SEM, * p<0.05, ** p<0.01, t-test, C57BL/6, C57BL/6 AC, iNOS^-/-, iNOS^-/- AC.

Figure 9. Instillation of apoptotic macrophages into lungs of iNOS^-/- mice reduces TNF-α levels in lung and bronchial alveolar lavage after pneumococcal infection.

A) Representative RNase protection assay of RNA from whole lung from wild type controls (C57BL/6) and iNOS deficient mice (iNOS^-/-) 12 h post instillation of 10⁷ CFU type 2 pneumococci in the absence or presence of apoptotic alveolar macrophages (AC). B) Levels of TNF-α mRNA as quantified by densitometry and normalised to GAPDH, n=3-4. C) Levels of TNF-α mRNA as quantified by RT-PCR and normalised to β-actin, in the same mice as B). D) Concentration of TNF-α in bronchial alveolar lavage (BAL) 12 h after instillation of 10⁷ CFU type 2 pneumococci with and without AC, n=3-4. Mean+SEM, * p<0.05, t-test, C57BL/6, C57BL/6 AC, iNOS^-/-, iNOS^-/- AC.

Figure 10. Instillation of apoptotic macrophages into lungs of iNOS^-/- mice reduces proinflammatory cytokine levels in bronchial alveolar lavage after pneumococcal infection.

Concentration of A) MIP-2, B) KC in bronchial alveolar lavage (BAL) 12 h after or C) IL-6 24 h after instillation of 10⁷ CFU type 2 pneumococci with and without AC. n=3-4. Mean+SEM, * p<0.05, t-test, C57BL/6, C57BL/6 AC, iNOS^-/-, iNOS^-/- AC.

Figure 11. Instillation of apoptotic macrophages into lungs of iNOS^-/- mice reduces bacteremia after pneumococcal infection.

Percentage of wild type controls (C57BL/6) and iNOS deficient mice (iNOS^-/-) with detectable bacteremia 12 h post instillation of 10⁷ CFU type 2 pneumococci in the absence or presence of apoptotic alveolar macrophages (AC), n=4-5. C57BL/6, C57BL/6 AC, iNOS^-/-, iNOS^-/- AC.