Characterization of the Anti-Inflammatory Capacity of IL-10-Producing Neutrophils in Response to Streptococcus pneumoniae Infection

Abstract

Neutrophils are immune cells classically defined as pro-inflammatory effector cells. However, current accumulated evidence indicates that neutrophils have more versatile immune-modulating properties. During acute lung infection with Streptococcus pneumoniae in mice, interleukin-10 (IL-10) production is required to temper an excessive lung injury and to improve survival, yet the cellular source of IL-10 and the immunomodulatory role of neutrophils during S. pneumoniae infection remain unknown. Here we show that neutrophils are the main myeloid cells that produce IL-10 in the lungs during the first 48 h of infection. Importantly, in vitro assays with bone-marrow derived neutrophils confirmed that IL-10 can be induced by these cells by the direct recognition of pneumococcal antigens. In vivo, we identified the recruitment of two neutrophil subpopulations in the lungs following infection, which exhibited clear morphological differences and a distinctive profile of IL-10 production at 48 h post-infection. Furthermore, adoptive transfer of neutrophils from WT mice into IL-10 knockout mice (Il10^-/- ) fully restored IL-10 production in the lungs and reduced lung histopathology. These results suggest that IL-10 production by neutrophils induced by S. pneumoniae limits lung injury and is important to mediate an effective immune response required for host survival.

Keywords: IL-10-producing neutrophils; Streptococcus pneumoniae; adoptive neutrophil transfer; interleukin-10; pneumonia.

Figure 1

Neutrophils are the main source of IL-10 during the early phase of S. pneumoniae infection. Representative histograms and graphs showing the number of cells expressing IL-10 in lungs in uninfected mice (n = 5), and mice infected after 24 (n = 6) and 48 hpi (n = 6), and their corresponding MFI measurements. (A) Neutrophils. (B) Ly6C^neg monocytes. (C) Eosinophils. (D) Interstitial macrophages. WT uninfected mice were used to establish the baseline of GFP expression ( Figure S3 ). Quantification of each cell population was performed using counting beads. Statistics: Kruskal-Wallis with Dunn’s multiple comparison post-test. *P < 0.05; **P < 0.01. Comparisons of the mean of infected and uninfected mice, 24 and 48 hpi. hpi, h post-infection; MFI, Median Fluorescence Intensity; I.M., Interstitial macrophages.

Figure 2

Neutrophil subsets isolated from infected mice exhibit structural and functional differences. (A) Representative flow cytometry plots of lung cells show neutrophils gated as CD11b⁺Ly6G⁺ cells. Neutrophil subsets were gated according to FSC parameters as FSC^low and FSC^high. (B) Number of FSC^low (blue bar) and FSC^high (red bar) neutrophils expressing IL-10 in uninfected mice together with the observed neutrophils at 24 and 48hpi. (C) IL-10 expression of neutrophils (from IL-10::eGFP mice subsets in lungs at 24 and 48hpi. (D) Representative TEM images of FSC^low and FSC^high subsets obtained from 24h infected lungs. The white arrows point to the heterochromatin and the black arrows point to the euchromatin fraction. (E) Phenotypic parameters measured in FSC^low and FSC^high populations with ImageJ software. Statistics: Tukey’s test following two-way ANOVA and a two-tailed T-test. *P < 0.05; **P < 0.01; ***P < 0.001. TEM, Transmission Electron Microscopy; hpi, hours post-infection; Uninf, Uninfected.

Figure 3

S. pneumoniae induces IL-10 production by neutrophils in vitro. Bone marrow-derived neutrophils were incubated with different MOIs of heat-killed S. pneumoniae (D39 and R6 strains) to determine IL-10 production in response to stimulus. (A) Histograms showing the percentage of IL-10 neutrophils by treatment, as well as the eGFP/MFI expression evaluated by flow cytometry at 12hpi. (B) Supernatants were recovered and evaluated for IL-10 detection by ELISA at 6, 12 and 24hpi. Kruskal-Wallis with Dunn’s multiple comparison post-tests were performed for statistical analyses. *P < 0.05; **P < 0.01; ***P < 0.001 in each treatment comparison with the untreated cells. Three independent experiments.

Figure 4

Adoptive transfer of neutrophil improves Il10 ^-/- mice survival and clinical score independently of IL-10 production. Neutrophils obtained from WT and Il10 ^-/- mice were intranasally transferred to Il10 ^-/- mice and 24 h later infected with 3x10⁷ cfu of S. pneumoniae (n = 7 to 9 for each group). This experimental design was performed to evaluate the survival and clinical parameters of mice in the different groups during 10 days. (A) Survival curve (B) Disease parameters were evaluated according to a clinical score guideline including activity, weight loss, body posture and general appearance ( Table S1 ). Statistics: Log-rank (Mantel-Cox) test. (a) ****P < 0.0001 between WT and Il10 ^-/-, (b) **P < 0.01 between Il10 ^/- and Il10 ^-/-+ WT, (c) *P < 0.05 between Il10 ^-/- and Il10 ^-/-+ Il10 ^-/-. Two-way ANOVA with Tukey’s multiple comparisons post-test were performed. (a) ****P < 0.0001 between WT and Il10^-/-, (b) **P < 0.01 between Il10^/- and Il10^-/-+ WT, (c) *P < 0.05 between Il10^-/- and Il10^-/-+ Il10^-/-.

Figure 5

IL-10 produced by neutrophils does not have impact in reduction of pro-inflammatory cytokines or cells infiltration in Il10 ^-/- mice. (A) Viable monocyte and neutrophil infiltration in mouse lungs of each group were measured at 48hpi by flow cytometry. Pro-inflammatory (B–D) and anti-inflammatory (E) cytokines obtained from BALFs at 48hpi were determined by ELISA in each control and transferred group. Statistics: One-way ANOVA with Sidak’s multiple comparison post-test. *P < 0.05. Comparisons of the mean of each group with the mean of every other groups. n = 9 to 12 per group. BALFs, Bronchoalveolar Lavages; hpi, hours post infection.

Figure 6

Transfer of WT neutrophils impairs bacterial clearance. Bacterial loads were measured at 48hpi in (A) lungs, (B) blood, (C) spleen and (D) brain. Data is shown as CFU per organ. Statistics: T-test with Mann-Whitney post-test. *P < 0.05; **P < 0.01. Comparisons of the mean of each group with the mean of all the other groups. n = 9 to 12 for each group. hpi, hours post-infection; CFUs, Colony Forming Units.

Figure 7

Adoptive transfer of neutrophils from WT to Il10 ^-/- mice induces a decrease in the histopathological score at 48 hpi. (A) Representative lung tissue sections stained with H&E at 1X and 10X magnification. These images were used to evaluate histopathological changes after neutrophils adoptive transfer. (B) Histopathological score for lung damage. This score was used to quantify lung damage after neutrophils adoptive transfer. To capture photographs and analyze them, the Aperio ImageScope Software and GraphPad Prism Software were used. Statistics: Two-way ANOVA with Tukey’s multiple comparisons post-test were performed. ***P < 0.001; ****P < 0.0001.