6 research outputs found
Enhanced phagocytosis by <i>Trem-2</i><sup>β/β</sup> 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 (nβ=β3β4 per condition). (<b>B</b>) WT AM (nβ=β6β7 per condition) were pre-treated for 24 h with 10 Β΅M PPAR-Ξ΄ 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><sup>β/β</sup> AM (nβ=β4 per genotype/condition) were pre-treated for 24 h with the indicated doses of the PPAR-Ξ΄ 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><sup>β/β</sup> AM were pre-treated for 1 h with 10 Β΅g/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 (nβ=β4 per genotype/condition). (<b>E</b>) WT and <i>Trem-2</i><sup>β/β</sup> AM (nβ=β4β7 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><sup>β/β</sup> AM (nβ=β4 per genotype) were incubated with FITC labeled <i>S. pneumoniae</i> (MOI 100) that were pre-opsonised with 10% WT or C1qa<sup>β/β</sup> serum for 30 min after which phagocytosis was assessed 1 h later using FACS (<b>G</b>) WT and <i>Trem-2</i><sup>β/β</sup> mice (nβ=β17 per condition) were intranasally treated with 125 Β΅g C1q blocking antibody or isotype control prior to infection with 6Γ10<sup>4</sup> CFU <i>S. pneumoniae</i>. 48 h post infection lung bacterial CFUs were enumerated. Data represent mean Β± SEM; ** p<0.005, *** p<0.001, **** p<0.0001. Data in (<b>A, CβF</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>AβB</b>) WT and <i>Trem-2</i><sup>β/β</sup> BMDM (nβ=β4 per genotype) were treated with 2Γ10<sup>7</sup> CFU/ml <i>S. pneumoniae</i> or 100 ng/ml LPS for 6 h and TNF-Ξ± (<b>A</b>) or KC (<b>B</b>) levels were measured in the supernatant. (<b>CβD</b>) WT and <i>Trem-2</i><sup>β/β</sup> AM (nβ=β4 per genotype/time point) were treated with 2Γ10<sup>7</sup> CFU/ml <i>S. pneumoniae</i> (<b>C</b>) or 10 Β΅g/ml <i>S. pneumoniae</i> LTA (<b>D</b>) for the indicated times and KC and TNF-Ξ± levels were measured in the supernatant. (<b>EβF</b>) WT and <i>Trem-2</i><sup>β/β</sup> AM (nβ=β4 per genotype/MOI) were treated with the indicated doses of <i>S. pneumoniae</i> and TNF-Ξ± (<b>E</b>) and KC (<b>F</b>) levels were determined in the supernatant. Statistical comparisons are done versus WT cells (<b>AβB</b>) at a given timepoint (<b>CβD</b>), or at a particular MOI of <i>S. pneumoniae</i> (<b>EβF</b>), and indicated as: * p<0.05, ** p<0.005, *** p<0.001, **** pβ=β<0.0001. All data represent mean Β± 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><sup>β/β</sup> mice were intranasally infected with 10<sup>5</sup> CFU <i>S. pneumoniae</i> and survival was monitored for 10 days (nβ=β13/genotype). (<b>BβG</b>) WT and <i>Trem-2</i><sup>β/β</sup> mice (nβ=β9 mice per genotype) were intranasally infected with 1Γ10<sup>5</sup> 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>HβJ</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 20Γ and arrows indicate caspase 3 positive cells. (<b>KβL</b>) Thymocytes (nβ=β4) were treated with 1 Β΅M 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><sup>β/β</sup> AM (nβ=β8 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 Β± SEM, WT versus TREM-2<sup>β/β</sup>. Data in <b>AβG</b> are representative of two independent experiments. Data in <b>M</b> is pooled data from 2 independent experiments; * pβ=β<0.05, ** pβ=β0.005.</p
Elevated phagocytosis of bacteria by TREM-2 deficient AM.
<p>(<b>A</b>) WT and <i>Trem-2</i><sup>β/β</sup> BMDM (nβ=β4β5 per genotype) were incubated with FITC labeled <i>S. pneumoniae</i> (MOI 100) and after 1 h phagocytosis was assessed using FACS. (<b>BβC</b>) WT and <i>Trem-2</i><sup>β/β</sup> AM (nβ=β4 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><sup>β/β</sup> AM as determined using confocal microscopy as described in the M&M section. The percentage of cells that contain bacteria is depicted (nβ=β4β5 per genotype). (<b>EβF</b>) WT and <i>Trem-2</i><sup>β/β</sup> AM (nβ=β4β5 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><sup>β/β</sup> AM (nβ=β4 per genotype) were incubated with 1 Β΅g/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><sup>β/β</sup> AM (nβ=β4 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>IβL</b>) WT and <i>Trem-2</i><sup>β/β</sup> mice (nβ=β7 mice per genotype) were intranasally infected with 10<sup>6</sup> CFU FITC labeled <i>S. pneumoniae</i> for 4 h and in vivo phagocytosis by AM (<b>IβJ</b>) and neutrophils (<b>KβL</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><sup>β/β</sup> mice (nβ=β6 mice per genotype) were intranasally infected with 10<sup>5</sup> CFU <i>S. pneumoniae</i> and bacterial CFUs were enumerated 24 h post infection in the lung and BALF. All data represent mean Β± SEM versus WT unless otherwise indicated. Data in (<b>AβC, 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
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 (nβ=β6 per time point) were intranasally inoculated with 10<sup>5</sup> 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 2Γ10<sup>7</sup> CFU/ml <i>S. pneumoniae</i> for indicated time points and TREM-2 RT-PCR was conducted. (<b>EβG</b>) WT and <i>Trem-2</i><sup>β/β</sup> mice (nβ=β7 per genotype) were intranasally infected with 10<sup>5</sup> 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 Β± SEM and are (<b>AβG</b>) representative of two independent experiments. Differences were calculated versus time point 0 (<b>CβD</b>) or versus WT (<b>EβG</b>) and are indicated as * p<0.05, ** p<0.005, **** p<0.0001.</p
Elevated C1q production via PPAR-Ξ΄ 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><sup>β/β</sup> AMs. (<b>B</b>) Verification of enhanced basal expression of the opsonins <i>C1qb</i> and <i>Thbs1</i> in AM using RT-PCR (nβ=β4 per genotype). (<b>C</b>) Basal expression of <i>C1qb</i> in WT versus <i>Trem-2</i><sup>β/β</sup> AMs as determined by intracellular FACS. Green line depicts WT macrophages, pink <i>Trem-2</i><sup>β/β</sup> 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><sup>β/β</sup> BMDM (nβ=β4 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 (nβ=β4 per condition). (<b>F</b>) WT and <i>Trem-2</i><sup>β/β</sup> AM (nβ=β4 per genotype/condition) were pre-treated for 24 h with the indicated doses of the PPAR-Ξ΄ 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 Β΅M of the PPAR-Ξ΄ activator GW0742 or DMSO 24 h post transfection, and luciferase activity was assayed 48 h post transfection (nβ=β4 per condition). (<b>H</b>) RAW264.7 cells over-expressing TREM-2 or GFP control (nβ=β4 per condition), were treated with 1 Β΅M of the PPAR-Ξ΄ activator GW0742 or DMSO control for 24 h, nuclear extracts were prepared and PPAR-Ξ΄ activity levels were monitored as described in the methods. (<b>I</b>) WT and <i>Trem-2</i><sup>β/β</sup> AM were treated with 1 Β΅M of the PPAR-Ξ΄ activator GW0742 or DMSO control for the indicated time points, nuclear and cytoplasmic extracts were prepared and blotted for PPAR-Ξ΄. 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 Β± 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