doi: 10.1038/mi.2015.128. Epub 2015 Dec 2.
Lung epithelium and myeloid cells cooperate to clear acute pneumococcal infection
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- PMID:26627460
- PMCID: PMC4990776
- DOI: 10.1038/mi.2015.128
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Lung epithelium and myeloid cells cooperate to clear acute pneumococcal infection
M Dudek et al. Mucosal Immunol.2016 Sep.
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Abstract
The Gram-positive bacterium Streptococcus pneumoniae causes life-threatening infections, especially among immunocompromised patients. The host's immune system senses S. pneumoniae via different families of pattern recognition receptors, in particular the Toll-like receptor (TLR) family that promotes immune cell activation. Yet, while single TLRs are dispensable for initiating inflammatory responses against S. pneumoniae, the central TLR adapter protein myeloid differentiation factor 88 (MyD88) is of vital importance, as MyD88-deficient mice succumb rapidly to infection. Since MyD88 is ubiquitously expressed in hematopoietic and non-hematopoietic cells, the extent to which MyD88 signaling is required in different cell types to control S. pneumoniae is unknown. Therefore, we used novel conditional knockin mice to investigate the necessity of MyD88 signaling in distinct lung-resident myeloid and epithelial cells for the initiation of a protective immune response against S. pneumoniae. Here, we show that MyD88 signaling in lysozyme M (LysM)- and CD11c-expressing myeloid cells, as well as in pulmonary epithelial cells, is critical to restore inflammatory cytokine and antimicrobial peptide production, leading to efficient neutrophil recruitment and enhanced bacterial clearance. Overall, we show a novel synergistic requirement of compartment-specific MyD88 signaling in S. pneumoniae immunity.
Figures
Myeloid differentiation factor 88 (MyD88) signaling in hematopoietic and non-hematopoietic cells is necessary for survival and chemokine-dependent neutrophil recruitment. Lethally irradiated recipient Ly5.1 (wild type mice expressing the congenic marker Ly5.1) or MyD88−/− mice were reconstituted with bone marrow (BM) cells from donor wild type (WT), Ly5.1 or MyD88−/− mice. After 8 weeks of reconstitution, mice were intranasally inoculated with either a low dose (a) or a high dose (b–e) ofStreptococcus pneumoniae. (a) Survival curve ofS. pneumoniae–infected BM chimeric mice. Data are pooled from four individual experiments with three to eight mice per group. Statistics were calculated using log-rank (Mantel-Cox) test; **P<0.01 and ***P<0.001. (b)S. pneumoniae burden in total lung homogenates 18 h post infection (p.i.) (c) Tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and (d) macrophage inflammatory protein 2 (MIP-2) and keratinocyte chemoattractant (KC) levels in total lung homogenates 18 h p.i. measured by enzyme-linked immunosorbent assay (ELISA). (e) Fluorescence-activated cell sorting (FACS) analysis of CD11c− MHCII− CD11bhigh Ly6Cint neutrophils (PMNs; excluding CD11c− MHCII− CD11bint Ly6Chi monocytes). Shown are total cell numbers in lungs from mice 18 h p.i. (b–e) Data points depict individual mice. Bars indicate mean values. Data represent two pooled experiments with two to four mice per group. Statistics were calculated using Mann-Whitney test; *P<0.05, **P<0.01, ***P<0.001, and ns, not significant.
Myeloid differentiation factor 88 (MyD88) signaling in club cells contributes to proinflammatory cytokine production and control of bacterial burden. Wild type (WT), club cell secretory protein (CCSP)–MyD88ON and MyD88OFF mice were intranasally inoculated with either a low dose (a) or a high dose (b–g) ofStreptococcus pneumoniae. (a) Survival curve ofS. pneumoniae–infected mice. Data show two pooled individual experiments with five to nine mice per group. Statistics were calculated using log-rank (Mantel-Cox) test; ***P<0.001 and ns, not significant. (b)S. pneumoniae burden in total lung homogenates 18 h post infection (p.i.). (c) Tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and (d) macrophage inflammatory protein 2 (MIP-2) and keratinocyte chemoattractant (KC) levels in total lung homogenates 18 h p.i. measured by enzyme-linked immunosorbent assay (ELISA). (e) Fluorescence-activated cell sorting (FACS) analysis of CD11c− MHCII− CD11bhigh Ly6Cint neutrophils (PMNs; excluding CD11c− MHCII− CD11bint Ly6Chi monocytes). Shown are total cell numbers in lungs from WT, CCSP–MyD88ON, and MyD88OFF mice 18 h p.i. Evaluation of (f) hematoxylin and eosin (H&E) and (g) myeloperoxidase (MPO) staining of lung tissue from uninfected andS. pneumoniae–infected mice 18 h p.i. Tissue sections were evaluated in a blinded manner. Dot plots depict (f) histopathological scoring and (g) MPO+ PMNs counted within 10 high power fields (h.p.f.). (b–e) Data represent three pooled individual experiments with three to five mice per group. (f,g) Data are pooled from two individual experiments with two to three mice per group. (b–g) Data points depict individual mice. Bars indicate mean value. Statistics were calculated using Mann-Whitney test; *P<0.05, **P<0.01, and ***P<0.001.
Club cells produce proinflammatory cytokines and surfactant protein D (SP-D) dependent on myeloid differentiation factor 88 (MyD88) signaling. (a) Cryosections from lungs of naive wild type (WT) and club cell secretory protein–red fluorescent protein (CCSP–RFP) mice were stained with anti-RFP antibody. Representative immunofluorescence pictures from one mouse of each group are shown. Bar = 50 μm. (b) CCSP–RFP mice were either intranasally inoculated with a high dose ofStreptococcus pneumoniae (Spn T4) or treated with phosphate-buffered saline (PBS, uninfected). CCSP–RFP+ cells were fluorescence-activated cell sorting-(FACS) sorted and analyzed forTnfa,Il6, andMip2 gene expression levels by quantitative PCR. Gene expression is shown relative toβ-actin expression. (c) WT, CCSP–MyD88ON, and MyD88OFF mice were intranasally inoculated with a high dose ofS. pneumoniae and bronchoalveolar lavage (BAL) was taken from uninfected and infected mice 18 h post infection (p.i.) BAL proteins were analyzed by western blot for SP-D content. SP-D band densities were quantified with ImageJ gel analyzer and depicted relative to the mean value of SP-D levels of uninfected mice. (a) Data show one mouse out of two mice from each group from one experiment. (b) Data are pooled from three individual experiments with two to four mice per group. (c) Data represent three pooled individual experiments with one to four mice per group. (b,c) Data points depict individual mice. Bars indicate mean values. Statistics were calculated using Mann-Whitney test; **P<0.01, ***P<0.001, and ns, not significant.
Myeloid differentiation factor 88 (MyD88) reactivation in CD11c or lysozyme M (LysM)–positive cells initiates early innate immune responses leading to improved survival and reduced bacterial burden. Wild type (WT), CD11c–MyD88ON, LysM–MyD88ON, and MyD88OFF mice were intranasally inoculated with either a low dose (a) or a high dose (b–h) ofStreptococcus pneumoniae. (a) Survival curve ofS. pneumoniae–infected mice. Data show two pooled individual experiments with 6–10 mice per group. Statistics were calculated using log-rank (Mantel-Cox) test; **P<0.01 and ***P<0.001. (b)S. pneumoniae burden in total lung homogenates 18 h post infection (p.i.). (c) Tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and (d) macrophage inflammatory protein 2 (MIP-2) and keratinocyte chemoattractant (KC) levels in total lung homogenates 18 h p.i. measured by enzyme-linked immunosorbent assay (ELISA). (e) Fluorescence-activated cell sorting (FACS) analysis of CD11c− MHCII− CD11bhigh Ly6Cint neutrophils (PMNs; excluding CD11c− MHCII− CD11bint Ly6Chi monocytes). Shown are total cell numbers in lungs from mice 18 h p.i. Uninfected group represents pooled data from three to four phosphate-buffered saline (PBS)–treated mice from each mouse line: WT, CD11c–MyD88ON, LysM–MyD88ON, and MyD88OFF. Evaluation of (f) hematoxylin and eosin (H&E) and (g) myeloperoxidase (MPO) staining of lung tissue from uninfected andS. pneumoniae–infected mice 18 h p.i. Tissue sections were evaluated in a blinded manner. Dot plots depict (f) histopathological scoring and (g) MPO+ PMNs counted within 10 high power fields (h.p.f.). (h) Bronchoalveolar lavage (BAL) was taken from uninfected and infected mice 18 h p.i. BAL proteins were analyzed by western blot for surfactant protein D (SP-D) content. SP-D band densities were quantified with ImageJ gel analyzer and depicted relative to the mean value of SP-D levels of uninfected mice. (b–e) Data represent two pooled individual experiments with three to four mice per group. (f,g) Data are pooled from two individual experiments with two to four mice per group. (h) Data show two pooled individual experiments with one to three mice per group. (b–h) Data points depict individual mice. Bars indicate mean values. Statistics were calculated using Mann-Whitney test; *P<0.05, **P<0.01, ***P<0.001, and ns, not significant.
Lung epithelium, alveolar macrophages (AMs), and neutrophils (PMNs) act synergistically to clear pneumococcal infection in a myeloid differentiation factor 88 (MyD88)–dependent manner. Lethally irradiated recipient (a–e) wild type (WT) or (f) club cell secretory protein (CCSP)–MyD88ON mice were reconstituted with bone marrow (BM) from donor CD11c–MyD88ON, LysM–MyD88ON, or MyD88OFF mice. After 8 weeks of reconstitution, mice were intranasally inoculated with either a high dose (a–d) or low dose (e,f) ofStreptococcus pneumoniae. (a) Tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and (b) macrophage inflammatory protein 2 (MIP-2) and keratinocyte chemoattractant (KC) levels in total lung homogenates 18 h post infection (p.i.) measured by enzyme-linked immunosorbent assay (ELISA). (c) Fluorescence-activated cell sorting (FACS) analysis of CD11c− MHCII− CD11bhigh Ly6Cint neutrophils (PMNs; excluding CD11c− MHCII− CD11bint Ly6Chi monocytes) and autofluorescence+ MHCIIint CD11c+ SiglecF+ AMs. Shown are total cell numbers in lungs from mice 18 h p.i. (d)S. pneumoniae burden in total lung homogenates 18 h p.i. (e,f) Survival curve ofS. pneumoniae–infected BM chimeric mice. (a–d) Data represent two pooled individual experiments with two to six mice per group. Data points depict individual mice. Bars indicate mean values. Statistics were calculated using Mann-Whitney test; *P<0.05, **P<0.01, and ***P<0.001. (e,f) Data are pooled from two individual experiments with three to six mice per group. Statistics were calculated using log-rank (Mantel-Cox) test; *P<0.05, **P<0.01, ***P<0.001, and ns, not significant.
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