Granulocyte-macrophage colony stimulating factor-mediated innate responses in tuberculosis

Abstract

The mechanisms by which GM-CSF mediates bacterial clearance and inflammation during mycobacterial infection are poorly understood. The objective of this work was to determine how GM-CSF alters pulmonary mycobacterial infection in vivo. Differences in GM-CSF levels in the lungs of normal mice (GM(+/+)), transgenic GM-CSF-deficient (GM-CSF(-/-)), and transgenic mice with high GM-CSF expression only in lung epithelial cells (SP-C-GM-CSF(+/+)/GM(-/-)) did not affect pulmonary infection rates caused by either the attenuated Mycobacterium bovis BCG or the virulent Mycobacterium tuberculosis H37Rv. However, in contrast to findings with BCG, all GM-CSF(-/-) and SP-C-GM-CSF(+/+)/GM(-/-) mice succumbed prematurely to virulent H37Rv. Granuloma formation was impaired in both GM-CSF(-/-) and SP-C-GM-CSF(+/+)/GM(-/-) mice regardless of mycobacterial virulence. However, H37Rv-infected GM-CSF(-/-) mice suffered broncho-alveolar destruction, edema, and necrosis while only short-lived granulomas were observed in SP-C-GM-CSF(+/+)/GM(-/-) mice. Bone marrow-derived macrophages, but not dendritic cells of SP-C-GM-CSF(+/+)/GM(-/-) mice, were hypo-responsive to mycobacterial infection. Surfactant protein levels were differentially influenced by BCG and H37Rv. We conclude that GM-CSF has an essential protective role first in preserving alveolar structure and second in regulating macrophages and dendritic cells to facilitate containment of virulent mycobacteria in pulmonary granulomas. However, precise regulation of lung GM-CSF is vital to effective control of M. tuberculosis.

Figure 1

Expression profiles of macrophage differentiation markers in naïve mice. (A) Flow cytometric analysis shows expression of ER-MP20 in GMKO but not in WT or GMOE alveolar macrophages. Data shown are representative of three separate experiments. Open histograms: isotype control IgG2b and shaded gray histograms: anti-ER-MP20. (B) Northern blot hybridization indicates different expression levels of 1.9 kb MARCO mRNA in WTand GMOE lungs. MARCO is not expressed in GMKO lungs. The expression level of MARCO mRNA in spleen is inversely correlated to the level of GM-CSF in the lung. Each lane was loaded with 20µg of total RNA and the results are representative of two independent experiments. Equivalent loading of RNA is indicated by the similar intensity of 28S rRNA on corresponding ethidium bromide-stained gels prior to Northern transfer.

Figure 2

Infection and survival profiles from pulmonary mycobacterial infections. (A) Similar biphasic reduction of intranasal BCG infection from WT, GMOE, and GMKO lungs. Viable BCG cfu were determined after serial dilution and culture of lung homogenates. Data are means ±S.E.M. Data shown are the combined results from two independent experiments and n = 14 WT mice, 8 GMKO mice, and 8 GMOE mice per time point until day 29. For day 70 n = 3. (B) Similar pulmonary infection and proliferation of aerosolized H37Rv in WT, GMOE, and GMKO mice until day 22. The GMKO lungs had significantly higher burden of H37Rv on day 29. Viable H37Rv cfu were quantitated after serial dilution and culture of lung homogenates. Data are means ±S.E., n = 4 per time point. ****p < 0.001, GMKO versus WT and GMOE on day 29. (C) All WT and GMOE and 80% of GMKO survived the intranasal BCG infection over 150 days of observation. Two GMKO mice were found dead each on days 17 and 19 post-infection without obvious sign of disease. N = 10 for each mouse group. (D) All GMKO and GMOE mice succumbed to H37Rv between 30–35 and 100–150 after infection, respectively. All WT mice survived. WT, n = 21; GMKO, n = 18; and GMOE, n = 22.

Figure 3

Granuloma formation by intranasal BCG infection. Lung inflammation resulting from intranasal infection of 1.5 × 10⁷ BCG is shown for days 9 (A, E), 14 (B, F), 22 (C, G, I, and J) and 29 (D, H, and K) for WT (A–D), GMKO (E– I and J), and GMOE mice (K). Progressive granulomatous inflammation was observed in WT mice (A–D, arrows). Granulomatous inflammation was attenuated in GMKO mice (E-H, arrows). Two of eight GM^−/− mice had encapsulated lesions (I) with a necrotic core (I, oval arrow) surrounded by cellular boundaries (I, open arrow) and fibroblastic granulation tissue (I, closed arrow). Adjacent areas in these lungs contained dense eosinophilic material (I, star), abscesses (I, diamond arrow), and intravascular infiltrates (I, box) rich in plasma cells containing Russell bodies (J, open arrows). The DIP pathology of SP-C-GM^+/+ lungs on day 29 is shown on panel (K). Representative images from n = 8 per mouse genotype per time point are shown. Magnification × 2 for images (A–I), × 50 for image (J), and × 10 for image (K).

Figure 4

Granuloma formation by aerosol infection with H37Rv. Lung inflammation of H37Rv-infected WT (A-E), GMKO (F-J), and GMOE (K-O) mice was evaluated on weekly intervals. Progressive granuloma formation in WT mice (B-E, closed arrows) was typified by the appearance of small interstitial infiltrates on day 7 that became enlarged thereafter, and in close proximity with bronchial and vascular structures. Granuloma formation was completely abrogated in GMKO mice as indicated by the lack of focused inflammation in the parenchyma. Over time, the inflammation in GMKO mice was distinct with scattered alveolar infiltrates (G, closed arrow), peribronchial and perivascular inflammation (H and I, diamond arrows), epithelial necrosis (H, oval arrow), and collapse of broncho-alveolar structures into amorphous necrotic nodules (J, oval arrow). In contrast, GMOE mice developed intense but unstable granulomas in the parenchyma (L-O, closed arrows) and diffuse interstitial inflammation (O, oval arrow). The images shown represent the findings from four mice per group at each time point. Magnification × 10.

Figure 5

Secretion of IL-12p70 in lung and spleen. (A) Concentration of IL-12p70 in lung homogenates at indicated times after intranasal BCG infection in WT, GMKO, and GMOE mice. Data are means ±S.E.M. from n = 4–6 mice per time point. (B) Concentration of IL-12p70 in splenocytes from naїve or infected mice 70 days after intranasal BCG infection. Splenocytes were cultured in media (open bars) or in the presence BCG (moi 5:1) (strippled and black bars) for 48h before assessment of IL-12 p40. Spontaneous secretions of IL-12 p40 in media (open bars) are combined data from splenocytes of naïve and infected mice. Data shown are means ±S.E.M. from 4 to 5 mice. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 6

Effect of BCG and H37Rv on lung protein and GM-CSF levels. (A) Protein concentration in filtered homogenates of H37Rv infected mice was assessed using the BCA assay data are means ±S.D. from 3 to 4 mice. *p < 0.05 and **p < 0.01. The concentration of GM-CSF was measured by ELISA in lung homogenates from BCG (B) and H37Rv-infected (C) GMOE mice. Data shown are means ±S.E.M. from 3 to 4 mice per time point *p < 0.05 and **p < 0.001.

Figure 7

Expression of SP-A and SP-D after BCG infection. Western blot analysis of lung homogenates from WT (A), GMKO (B), and GMOE (C) following intranasal BCG infection. Each lane was loaded with 20 µg of protein. Proteins were separated on 10% SDS–PAGE gels and blotted nitrocellulose. Blots were probed with anti-SP-A or anti-SP-D antibodies overnight and then with HRP-conjugated goat anti-rabbit antibodies. SP-A and SP-D were visualized by enhanced chemiluminescence for 5–10s on Kodak X-ray film.

Figure 8

Secretion of TNFα and IL-12p70 in bone marrow and spleen cells. BMMφ from WT, GMKO, and GMOE mice (A, C, and D) were differentiated in either M-CSF (A) or GM-CSF (C and D). Bone marrow-derived dendritic cells (BMDC) cells were differentiated in the presence of GM-CSF and IL-4 and immuno-magnetically separated using anti-CD11c antibodies (C and D). Splenocytes (B) were isolated from either naїve or infected mice 70 days after intranasal immunization with 1 × 10⁵ BCG. Cells were challenged with an moi of 1:1 BCG to cell ratio (A-C), or 100ng/mL LPS (D). The concentrations of TNFα (A and B) or IL-12 p40 (B and D) were measured in supernatants 48h after addition of stimulants. Data shown are means ±S.E.M. from 4 to 5 independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.