S100A9 is indispensable for survival of pneumococcal pneumonia in mice

S100A8/A9 has important immunomodulatory roles in antibacterial defense, but its relevance in focal pneumonia caused by Streptococcus pneumoniae (S. pneumoniae) is understudied. We show that S100A9 was significantly increased in BAL fluids of patients with bacterial but not viral pneumonia and correlated with procalcitonin and sequential organ failure assessment scores. Mice deficient in S100A9 exhibited drastically elevated Zn$^{2+}$ levels in lungs, which led to bacterial outgrowth and significantly reduced survival. In addition, reduced survival of S100A9 KO mice was characterized by excessive release of neutrophil elastase, which resulted in degradation of opsonophagocytically important collectins surfactant proteins A and D. All of these features were attenuated in S. pneumoniae-challenged chimeric WT→S100A9 KO mice. Similarly, therapy of S. pneumoniae-infected S100A9 KO mice with a mutant S100A8/A9 protein showing increased half-life significantly decreased lung bacterial loads and lung injury. Collectively, S100A9 controls central antibacterial immune mechanisms of the lung with essential relevance to survival of pneumococcal pneumonia. Moreover, S100A9 appears to be a promising biomarker to distinguish patients with bacterial from those with viral pneumonia. Trial registration: Clinical Trials register (DRKS00000620)

Effect of S100A9 deletion or reconstitution on lung antibacterial immunity in <i>S</i>. <i>pneumoniae</i>-infected bone-marrow chimeric mice.

(A) Endogenous S100A9 levels in BALF of chimeric mice on day 1 after pneumococcal challenge (n = 7–10 mice per group). (B,C) Bacterial loads in BAL fluids (B) and lung tissue (C) of S. pneumoniae-challenged chimeric mice on day 1 post-infection (n = 8–9 mice per group). (D) Zinc levels in lungs of S. pneumoniae-infected chimeric mice on day 1 post-infection, relative to controls (n = 8 mice per treatment group). Data are shown as means ± SD and are representative of two independently performed experiments. (E,F) Survival of chimeric mice infected with S. pneumoniae during an observation period of 10 days (n = 9–10 mice per group). The data represent two independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001, ****p ≤ 0.0001 compared to chimeric controls, +p ≤ 0.05; ++p ≤ 0.01 compared to untreated controls (Kruskal-Wallis test, log-rank test).</p

Endogenous S100A9 levels and its correlation with clinical parameters in BALF of pneumonia patients, as well as S100A9 levels in plasma and BALF of <i>S</i>. <i>pneumoniae</i>-infected WT mice.

(A) S100A9 levels were determined in BALF of patients with bacterial or viral pneumonia and healthy controls. Data are shown as mean ± SD of n = 7–19 patients. Significant correlation between BAL fluid levels of S100A9 and procalcitonin (B) or SOFA scores at BAL with best GCS (C) in patients with bacterial pneumonia. GCS, Glasgow coma score (n = 17 patients). WT mice were left untreated (CL) or were infected with S. pneumoniae. Endogenous S100A9 levels were determined in plasma (D) and BAL fluids (E) of mice at days 1, 2, 3 and 4 post-infection by ELISA. Data are shown as mean ± SD of n = 5–8 mice per time point and treatment group and are representative of two independently performed experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001, ****p ≤ 0.0001 compared to controls or patients with viral pneumonia (Mann-Whitney U test).</p

Neutrophil elastase-dependent degradation of SP-A and SP-D in lungs of bone-marrow chimeric mice.

(A) Numbers of neutrophils in lungs of S. pneumoniae-challenged bone-marrow chimeric mice on day 1 post-infection. Data are shown as mean ± SD of 8–9 mice per treatment group. (B-D) Neutrophil elastase (B), SP-A (C) and SP-D (D) proteins in BAL fluids of bone-marrow chimeric WT→WT, KO→KO, WT→KO and KO→WT mice on day 1 after pneumococcal challenge as indicated. The data are representative of two independently performed experiments.</p

Schematic concept of how lung antibacterial immunity is affected by S100A8/A9 deficiency.

(A) S100A8/A9 is released by neutrophils upon activation. By complexing divalent metal ions S100A8/A9 inhibits bacterial outgrowth. As a consequence, less neutrophil recruitment and neutrophil-dependent NE release is observed. This protects alveolar collectins SP-A and SP-D from NE-dependent degradation. (B) S100A8/A9 deficient neutrophils are recruited to the site of infection. In the absence of S100A8/A9, divalent metal ions foster bacterial outgrowth, resulting in excessive neutrophil recruitment and release of NE. As a consequence, the increased proteolytic NE burden in lungs promotes degradation of opsonophagocytically important collectins SP-A and SP-D, thereby impairing lung antibacterial immunity against S. pneumoniae. S. pneumoniae-driven neutrophil activation and NE release are diminished in infection experiments involving S. pneumoniae ΔPly.</p

Phagocytosis and burst induction in <i>S</i>. <i>pneumoniae</i>-infected alveolar macrophages and neutrophils of WT and S100A9 KO mice.

Cells were purified as described in Materials and Methods followed by infection with S. pneumoniae at a multiplicity of infection (MOI) of 25. (A,B) Phagocytosis capacity of resident AM (A) and bone marrow-derived neutrophils (BM-PMN) (B) at 30 or 60 minutes after infection of cells with S. pneumoniae. (C) Burst induction in purified BM-PMN of WT and S100A9 KO mice by S. pneumoniae (MOI 5). Data are representative of two independently performed experiments. (TIF)</p

Growth of <i>S</i>. <i>pneumoniae</i> under defined growth conditions <i>in vitro</i>.

To achieve defined growth conditions, THB medium was treated with Chelex 100 resin. (A-G) Pneumococci were not able to grow in depleted THB medium + 1% FCS (A-G, green line). (A-C) Depleted THB medium was supplemented with increasing concentrations of certain divalent cations. (A) Addition of MgCl2 to depleted THB medium + 1% FCS restored growth of S. pneumoniae in a dose dependent manner. Treatment of depleted THB + 1% FCS with different concentrations of either MnSO4 (B) or ZnCl2 (C) alone was not able to retrieve pneumococcal growth in vitro. (D) Pneumococcal growth in medium supplemented with 200 μM Mg2+ + 50 μM Mn2+ or 200 μM Mg2+ + 50 μM Zn2+ was nearly identical to pneumococcal growth in normal THB + 1% FCS. Additionally, depleted THB + 1% FCS medium supplemented with either Mg2+ alone (E), Mg2+ + Mn2+ (F) or Mg2+ + Zn2+ (G) was further supplemented with recombinant S100A8/A9 (50 μg/ml). S. pneumoniae was then added and bacterial growth was monitored hourly over a time period of six hours. Untreated THB medium supplemented with 1% FCS served as positive control in all growth experiments (A-G). The Data are representative of two independently performed experiments.</p

Patient characteristics and pathogenic agents in the bronchoalveolar lavage fluid of patients with severe bacterial or viral ARDS.

Patient characteristics and pathogenic agents in the bronchoalveolar lavage fluid of patients with severe bacterial or viral ARDS.</p

NE dependent degradation of alveolar collectins SP-A and SP-D in the lungs of <i>S</i>. <i>pneumoniae</i>-challenged S100A9 KO mice.

(A) Neutrophil elastase in BAL fluids of untreated or S. pneumoniae-infected WT versus S100A9 KO mice at day 1 post-infection as indicated. Equal amounts of BAL protein (15 μg) were used for western blot analysis. (B) Quantification of NE protein levels in BAL fluids of mice of the respective treatment groups by ELISA. (C) Caseinolytic activity in BALFs of untreated and S. pneumoniae-infected WT and S100A9 KO mice on day 1 post-infection. (D,E) SP-A (D) and SP-D (E) protein levels in BALFs of untreated and S. pneumoniae-challenged WT versus S100A9 KO mice on day 1 and day 2 post-infection, as indicated. Recombinant SP-A or SP-D protein serving as positive control (lane 11 in D,E). (F) Quantification of SP-D protein in BAL fluids of the respective treatment groups. Data are shown as mean ± SD of 5–8 mice per time point and group and are representative of two independently performed experiments. (G,H) Incubation of exogenous NE with BALF of S. pneumoniae-infected WT or S100A9 KO mice led to degradation of SP-D (G, H, lanes 1–5), while pre-incubation of NE with specific inhibitor Sivelestat prevented SP-D degradation in vitro (G,H, lanes 6–9). (I,J) Just 0.5 μg NE are sufficient to degrade rSP-A (I) and rSP-D (J) in vitro. *p ≤ 0.05; **p ≤ 0.01 compared to WT mice, ++p ≤ 0.01 compared to day 2 (Mann-Whitney U test).</p

Leukocyte recruitment and cytokine expression in lungs of <i>S</i>. <i>pneumoniae</i>-challenged WT and S100A9 KO mice.

(A) Representative immunofluorescence analysis of recruited neutrophils and corresponding CD11b expression in frozen lung tissue sections of untreated and S. pneumoniae-infected WT and S100A9 KO mice on day 1 post-infection. Lung tissue sections were stained with AF647-conjugated anti-Ly6G antibody (red) and BV421-conjugated anti-CD11b antibody (blue). Green autofluorescence was recorded for topographical purposes. Merge profiles identify Ly6G and CD11b-positive neutrophils in lung tissue (purple fluorescence staining) (n = 4 mice per group, scale bar: 50 μm). (B-D) Differential leukocyte counts in BAL fluid of WT and S100A9 KO mice challenged with S. pneumoniae on day 1 and 2 post-infection (n = 5–6 mice per time point and treatment group). (E-G) Cytokine expression in BALF of untreated and S. pneumoniae-infected WT and S100A9 KO mice (n = 5–9 mice per time point and treatment group). Values are shown as means ± SD and are representative of two independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 compared to WT mice (Mann-Whitney U test).</p