The Triggering Receptor Expressed on Myeloid Cells 2 Inhibits Complement Component 1q Effector Mechanisms and Exerts Detrimental Effects during Pneumococcal Pneumonia

Phagocytosis and inflammation within the lungs is crucial for host defense during bacterial pneumonia. Triggering receptor expressed on myeloid cells (TREM)-2 was proposed to negatively regulate TLR-mediated responses and enhance phagocytosis by macrophages, but the role of TREM-2 in respiratory tract infections is unknown. Here, we established the presence of TREM-2 on alveolar macrophages (AM) and explored the function of TREM-2 in the innate immune response to pneumococcal infection in vivo. Unexpectedly, we found Trem-2(-/-) AM to display augmented bacterial phagocytosis in vitro and in vivo compared to WT AM. Mechanistically, we detected that in the absence of TREM-2, pulmonary macrophages selectively produced elevated complement component 1q (C1q) levels. We found that these increased C1q levels depended on peroxisome proliferator-activated receptor-δ (PPAR-δ) activity and were responsible for the enhanced phagocytosis of bacteria. Upon infection with S. pneumoniae, Trem-2(-/-) mice exhibited an augmented bacterial clearance from lungs, decreased bacteremia and improved survival compared to their WT counterparts. This work is the first to disclose a role for TREM-2 in clinically relevant respiratory tract infections and demonstrates a previously unknown link between TREM-2 and opsonin production within the lungs

First-Breath-Induced Type 2 Pathways Shape the Lung Immune Environment

From birth onward, the lungs are exposed to the external environment and therefore harbor a complex immunological milieu to protect this organ from damage and infection. We investigated the homeostatic role of the epithelium-derived alarmin interleukin-33 (IL-33) in newborn mice and discovered the immediate upregulation of IL-33 from the first day of life, closely followed by a wave of IL-13producing type 2 innate lymphoid cells (ILC2s), which coincided with the appearance of alveolar macrophages (AMs) and their early polarization to an IL-13-dependent anti-inflammatory M2 phenotype. ILC2s contributed to lung quiescence in homeostasis by polarizing tissue resident AMs and induced an M2 phenotype in transplanted macrophage progenitors. ILC2s continued to maintain the M2 AM phenotype during adult life at the cost of a delayed response to Streptococcus pneumoniae infection in mice. These data highlight the homeostatic role of ILC2s in setting the activation threshold in the lung and underline their implications in anti-bacterial defenses.(VLID)456113

Enhanced phagocytosis by <i>Trem-2</i>^−/− AM depends on C1q.

<p>(<b>A</b>) WT AM were adhered to C1q, control BSA or uncoated plates for 3 h prior to incubation with FITC labeled <i>S. pneumoniae</i> (MOI 100) and phagocytosis was assessed using FACS 1 h later (n = 3–4 per condition). (<b>B</b>) WT AM (n = 6–7 per condition) were pre-treated for 24 h with 10 µM PPAR-δ inhibitor GSK0660 or DMSO control after which, the cells were adhered to C1q or control plates for 3 h and phagocytosis of FITC labeled <i>S. pneumoniae</i> (MOI 100) was assessed 1 h later using FACS. (<b>C</b>) WT and <i>Trem-2</i>^−/− AM (n = 4 per genotype/condition) were pre-treated for 24 h with the indicated doses of the PPAR-δ inhibitor GSK0660 or DMSO control after which phagocytosis of FITC labeled <i>S. pneumoniae</i> (MOI 100) was assessed 1 h later using FACS. (<b>D</b>) WT and <i>Trem-2</i>^−/− AM were pre-treated for 1 h with 10 µg/ml C1q blocking antibody or isotype control and phagocytosis of FITC labeled <i>S. pneumoniae</i> (MOI 100) was assessed 1 h later using FACS (n = 4 per genotype/condition). (<b>E</b>) WT and <i>Trem-2</i>^−/− AM (n = 4–7 per condition) were adhered for 3 h to plates coated with the indicated conditions prior to incubation with FITC labeled <i>S. pneumoniae</i> (MOI 100) and phagocytosis was assessed using FACS. (<b>F</b>) WT and <i>Trem-2</i>^−/− AM (n = 4 per genotype) were incubated with FITC labeled <i>S. pneumoniae</i> (MOI 100) that were pre-opsonised with 10% WT or C1qa^−/− serum for 30 min after which phagocytosis was assessed 1 h later using FACS (<b>G</b>) WT and <i>Trem-2</i>^−/− mice (n = 17 per condition) were intranasally treated with 125 µg C1q blocking antibody or isotype control prior to infection with 6×10⁴ CFU <i>S. pneumoniae</i>. 48 h post infection lung bacterial CFUs were enumerated. Data represent mean ± SEM; ** p<0.005, *** p<0.001, **** p<0.0001. Data in (<b>A, C–F</b>) are representative of two independent experiments, (<b>B and G</b>) are pooled data from two independent experiments.</p

Cell type specific effects of TREM-2 on <i>S. pneumoniae</i> and TLR2 mediated cytokine production.

<p>(<b>A–B</b>) WT and <i>Trem-2</i>^−/− BMDM (n = 4 per genotype) were treated with 2×10⁷ CFU/ml <i>S. pneumoniae</i> or 100 ng/ml LPS for 6 h and TNF-α (<b>A</b>) or KC (<b>B</b>) levels were measured in the supernatant. (<b>C–D</b>) WT and <i>Trem-2</i>^−/− AM (n = 4 per genotype/time point) were treated with 2×10⁷ CFU/ml <i>S. pneumoniae</i> (<b>C</b>) or 10 µg/ml <i>S. pneumoniae</i> LTA (<b>D</b>) for the indicated times and KC and TNF-α levels were measured in the supernatant. (<b>E–F</b>) WT and <i>Trem-2</i>^−/− AM (n = 4 per genotype/MOI) were treated with the indicated doses of <i>S. pneumoniae</i> and TNF-α (<b>E</b>) and KC (<b>F</b>) levels were determined in the supernatant. Statistical comparisons are done versus WT cells (<b>A–B</b>) at a given timepoint (<b>C–D</b>), or at a particular MOI of <i>S. pneumoniae</i> (<b>E–F</b>), and indicated as: * p<0.05, ** p<0.005, *** p<0.001, **** p = <0.0001. All data represent mean ± SEM and are representative of two independent experiments.</p

TREM-2 deficiency improves outcome during pneumococcal pneumonia.

<p>(<b>A</b>) WT and <i>Trem-2</i>^−/− mice were intranasally infected with 10⁵ CFU <i>S. pneumoniae</i> and survival was monitored for 10 days (n = 13/genotype). (<b>B–G</b>) WT and <i>Trem-2</i>^−/− mice (n = 9 mice per genotype) were intranasally infected with 1×10⁵ CFU <i>S. pneumoniae</i> and (<b>B</b>) lung bacterial CFUs were enumerated 48 h post infection. (<b>C</b>) Blood cultures were monitored for <i>S. pneumoniae</i> (<b>D</b>), IL-6 levels were evaluated in the plasma using ELISA. (<b>E</b>) Representative H/E staining of lungs 48 h post infection. (<b>F</b>) Lung inflammation score, as described in the Methods section. (<b>G</b>) Levels of lung cytokines were evaluated using ELISA. (<b>H–J</b>) Representative Ly6G (<b>H</b>), active caspase 3 (<b>I</b>) and TUNEL (<b>J</b>) staining of lungs 48 h post infection. Magnification depicted for TUNEL stains is 20× and arrows indicate caspase 3 positive cells. (<b>K–L</b>) Thymocytes (n = 4) were treated with 1 µM dexamethasone and apoptosis was evaluated using DNA laddering (<b>K</b>) or Annexin-V/7-AAD positivity (<b>L</b>). (<b>M</b>) WT and <i>Trem-2</i>^−/− AM (n = 8 per genotype and condition), were fed CFSE labeled apoptotic cells and efferocytosis was assessed 1 h later using FACS. Data in <b>B</b>, <b>D</b>, <b>F</b>, <b>G</b> and <b>M</b> are presented as mean ± SEM, WT versus TREM-2^−/−. Data in <b>A–G</b> are representative of two independent experiments. Data in <b>M</b> is pooled data from 2 independent experiments; * p = <0.05, ** p = 0.005.</p

Elevated phagocytosis of bacteria by TREM-2 deficient AM.

<p>(<b>A</b>) WT and <i>Trem-2</i>^−/− BMDM (n = 4–5 per genotype) were incubated with FITC labeled <i>S. pneumoniae</i> (MOI 100) and after 1 h phagocytosis was assessed using FACS. (<b>B–C</b>) WT and <i>Trem-2</i>^−/− AM (n = 4 per genotype) were incubated with FITC labeled <i>S. pneumoniae</i> (<b>B</b>) or <i>E. coli</i> (<b>C</b>) (MOI of 100) and phagocytosis was assessed using FACS 1 h later. (<b>D</b>) Elevated phagocytosis of <i>S. pneumoniae</i> by <i>Trem-2</i>^−/− AM as determined using confocal microscopy as described in the M&M section. The percentage of cells that contain bacteria is depicted (n = 4–5 per genotype). (<b>E–F</b>) WT and <i>Trem-2</i>^−/− AM (n = 4–5 per genotype) were incubated with FITC labeled <i>S. pneumoniae</i> (MOI 100) under either serum free conditions (SFM) or the bacteria were pre-opsonised with 10% anti-pneumococcal serotype III capsular antibody (ST3-Ab) (<b>E</b>) or 10% pooled WT mouse serum (<b>F</b>) for 30 min before addition to the cells. Phagocytosis was assessed 1 h later. (<b>G</b>) WT and <i>Trem-2</i>^−/− AM (n = 4 per genotype) were incubated with 1 µg/ml FITC labeled BSA or FITC labeled <i>S. pneumoniae</i> (MOI 100) and phagocytosis was assessed 1 h later by FACS. (<b>H</b>) WT and <i>Trem-2</i>^−/− AM (n = 4 per genotype) were incubated with FITC labeled <i>S. pneumoniae</i> strain 19A (MOI 100) and phagocytosis was assessed 1 h later by FACS. (<b>I–L</b>) WT and <i>Trem-2</i>^−/− mice (n = 7 mice per genotype) were intranasally infected with 10⁶ CFU FITC labeled <i>S. pneumoniae</i> for 4 h and in vivo phagocytosis by AM (<b>I–J</b>) and neutrophils (<b>K–L</b>) was determined. <b>J</b> and <b>L</b> show representative FACS plots of data in <b>I</b> and <b>K</b>. (<b>M</b>) WT and <i>Trem-2</i>^−/− mice (n = 6 mice per genotype) were intranasally infected with 10⁵ CFU <i>S. pneumoniae</i> and bacterial CFUs were enumerated 24 h post infection in the lung and BALF. All data represent mean ± SEM versus WT unless otherwise indicated. Data in (<b>A–C, F and H</b>) are representative of three independent experiments and all other data are representative of two independent experiments. * p<0.05, ** p<0.005, **** p<0.0001.</p

Elevated C1q production via PPAR-δ in TREM-2 deficient AM.

<p>(<b>A</b>) Heat map from microarray data depicting baseline expression of selected genes in WT and <i>Trem-2</i>^−/− AMs. (<b>B</b>) Verification of enhanced basal expression of the opsonins <i>C1qb</i> and <i>Thbs1</i> in AM using RT-PCR (n = 4 per genotype). (<b>C</b>) Basal expression of <i>C1qb</i> in WT versus <i>Trem-2</i>^−/− AMs as determined by intracellular FACS. Green line depicts WT macrophages, pink <i>Trem-2</i>^−/− macrophages and black represents isotype control antibody. (<b>D</b>) <i>C1qb</i> and <i>Thbs1</i> basal expression was determined in WT and <i>Trem-2</i>^−/− BMDM (n = 4 per genotype) using RT-PCR. (<b>E</b>) <i>C1qb</i> expression was quantified in RAW264.7 cells over-expressing TREM-2 or GFP control (n = 4 per condition). (<b>F</b>) WT and <i>Trem-2</i>^−/− AM (n = 4 per genotype/condition) were pre-treated for 24 h with the indicated doses of the PPAR-δ inhibitor GSK0660 or DMSO control after which RT-PCR of <i>C1qb</i> was performed. (<b>G</b>) HEK cells were transfected with a PPRE reporter plasmid together with TREM-2 and DAP-12 or a vector control, stimulated with 1 µM of the PPAR-δ activator GW0742 or DMSO 24 h post transfection, and luciferase activity was assayed 48 h post transfection (n = 4 per condition). (<b>H</b>) RAW264.7 cells over-expressing TREM-2 or GFP control (n = 4 per condition), were treated with 1 µM of the PPAR-δ activator GW0742 or DMSO control for 24 h, nuclear extracts were prepared and PPAR-δ activity levels were monitored as described in the methods. (<b>I</b>) WT and <i>Trem-2</i>^−/− AM were treated with 1 µM of the PPAR-δ activator GW0742 or DMSO control for the indicated time points, nuclear and cytoplasmic extracts were prepared and blotted for PPAR-δ. All data are representative of two independent experiments except for data in (<b>E</b>), which is representative of three independent experiments, and represent mean ± SEM versus WT (<b>B and D</b>), GFP control cells (<b>E</b>), DMSO (<b>F</b>) or vector (<b>G</b>). * p<0.05, ** p<0.005, *** p<0.001, **** p<0.0001.</p

Pulmonary TREM-2 expression and function during <i>S. pneumoniae</i> induced inflammation.

<p>(<b>A</b>) TREM-1 and TREM-2 expression was evaluated in the indicated cell types using RT-PCR. (<b>B</b>) Western blot was used to evaluate TREM-2 expression on AM. (<b>C</b>) WT mice (n = 6 per time point) were intranasally inoculated with 10⁵ CFU <i>S. pneumoniae</i> and after indicated time points TREM-2 lung transcript levels were evaluated. (<b>D</b>) WT AM were treated with 2×10⁷ CFU/ml <i>S. pneumoniae</i> for indicated time points and TREM-2 RT-PCR was conducted. (<b>E–G</b>) WT and <i>Trem-2</i>^−/− mice (n = 7 per genotype) were intranasally infected with 10⁵ CFU <i>S. pneumoniae</i> for 6 h and levels of indicated cytokines were evaluated in the lung (<b>E</b>) or BALF (<b>F</b>) and neutrophil counts were determined in the BALF (<b>G</b>). Data represent mean ± SEM and are (<b>A–G</b>) representative of two independent experiments. Differences were calculated versus time point 0 (<b>C–D</b>) or versus WT (<b>E–G</b>) and are indicated as * p<0.05, ** p<0.005, **** p<0.0001.</p