TLR-2 and TLR-4 but not TREM-1, regulate activation of macrophages <i>in vitro</i> by kidney DAMPs.

<p><b>(A–B)</b><b> </b> Q-PCR showing <i>Il-1β</i> and <i>Trem1</i> expression in BMDMφ stimulated for 8, 16 or 24h with soluble factors prepared from normal kidney (control) or disease kidney (kidney DAMPs). <b>(C–D)</b> Graphs showing <i>Il-1β</i> and <i>Trem1</i> expression by Q–PCR, 16h after BMDMφ were stimulated with kidney DAMPs and (<b>C</b>) activating anti-TREM-1 antibodies, or (<b>D</b>) TREM1-Fc, which blocks TREM-1 receptor by competing for ligands. (<b>E–F</b>) Q-PCR showing (<b>E</b>) <i>Il-1β</i> and (<b>F</b>) <i>Trem1</i> expression in BMDMφ isolated from WT, <i>Tlr2^−/−, Tlr4^−/−, Tlr2–4^−/−,</i> and <i>Myd88^−/−</i> mice stimulated with kidney DAMPs for 16h. Q-PCR results were normalized to their respective control group. (*P<0.05, n = 5–7/group, 3 independent experiments; ns, p is not significant).</p

Treatment with soluble TREM1-Fc does not prevent macrophage activation, injury and fibrosis in sterile kidney injury.

<p>(<b>A</b>) Schema showing experimental design. Mice were subjected to unilateral ischemia and reperfusion injury (U-IRI) and treated daily with 40 µg/mouse of TREM1-Fc or hIgG, as control. (<b>B</b>) Western blot showing presence of TREM1-Fc (approximately 56kD) in 2 µl of plasma collected at day 2 from mice treated daily with 40 µg of TREM1-Fc. Anti-mouse IgG was used as endogenous control. (<b>C</b>) Q-PCR for different inflammatory transcripts (left) or pro-fibrotic transcripts, Collagen1a1 <i>(Col1a1)</i> and alpha smooth muscle actin (<i>Acta2</i>), from whole kidney day 5 after U-IRI. (<b>D</b>) Representative images (left) and quantitative graphs (right) showing+F4/80 cells (green),+αSMA (red) or collagen deposition (Sirius Red staining) day 5 after U-IRI. (*P<0.05, n = 5–7/group, 3 independent experiments; Bar marker = 50 µm; Q-PCR results were normalized to sham +hIgG).</p

The TLR-2/TLR-4/MyD88 pathways play a role in fibrosis in the U-IRI model of sterile kidney injury.

<p><b>(A–C)</b><i>Tlr2–4^−/−</i><b>(D–F)</b><i>Myd88^−/−</i> mice or respective controls were subjected to U-IRI and kidney harvested for tissue analysis 5 days later. Q-PCR <b>(A,D)</b> for different inflammatory transcripts, <b>(B,E)</b> pro-fibrotic transcripts, collagen1a1 (<i>col1a1</i>) and alpha smooth muscle actin (<i>Acta2</i>), and the tubule injury marker, kidney injury molecule-1 (<i>Kim-1</i>) from whole kidney day 5 after U-IRI. <b>(C,F)</b> Representative fluorescent images (left) and quantitative graphs (right) showing+αSMA (red) cells and+F4/80 cells (green). (*P<0.05, n = 5–7/group, 3 independent experiments; ns, p is not significant; Bar = 50 µm; Q-PCR results were normalized to wild type control).</p

The TLR-2/TLR-4/MyD88 pathway is dispensable in macrophage activation in kidney fibrosis, but important in mesenchyme cell activation.

<p>(<b>A–C</b>) <i>Csf1R-icre; Myd88^fl/fl</i> mice or respective controls were subjected to U-IRI and kidney harvested for tissue analysis 5 days later. Q-PCR (A) for different inflammatory transcripts, (<b>B</b>) pro-fibrotic transcripts, collagen1a1 (<i>col1a1</i>) and alpha smooth muscle actin (<i>Acta2</i>), and the tubule injury marker, kidney injury molecule-1 (<i>Kim-1</i>) from whole kidney day 5 after U-IRI. (<b>C</b>) Representative fluorescent images (left) and quantitative graphs (right) showing+αSMA (red) cells and+F4/80 cells (green). (<b>D–E</b>) Primary pericytes were isolated from <i>Myd88^−/−</i> and <i>Tlr2–4^−/−</i> mice and stimulated <i>in vitro</i> for 8h with kidney DAMPs. (<b>D</b>) Graph showing <i>Il-6</i> and <i>Col1a1</i> transcript expression by Q-PCR. (<b>E</b>) Graph showing IL-6 and MCP-1 concentration in supernatant by ELISA. (*P<0.05, n = 5–7/group, 3 independent experiments; ns, p is not significant; Bar = 50 µm; Q-PCR results were normalized to wild type control).</p

TREMs are highly expressed in macrophages during kidney injury.

<p>(<b>A</b>) Q-PCR for <i>Trem1</i> expression in whole kidney 0, 5 and 10 days after UUO and U-IRI. (<b>B</b>) Q-PCR for TREM family transcript expression in blood monocytes, and different sub-populations of kidney macrophages purified at day 5 after UUO. (<b>C</b>) Fluorescence images showing CD11b (green), Ly6C (green) and TREM-1 (red) expression in tissue sections from control kidney (sham), day 3 and 10 after UUO (a, arteriole; Bar = 25 µm; arrowhead shows interstitial CD11b+ and TREM-1+ cells; arrow shows autofluorescent arteriole internal elastic lamina). (<b>D</b>) Western blot of whole kidney lysates detecting TREM-1 (23kD) and β-Actin (43kD), 0, 3, 7, 10 and 14 days after UUO. (<b>E</b>) TREM-1 protein densitometry normalized to endogenous control β-Actin. (n = 3–5/group, 3 independent experiments; *P<0.05).</p

Oligonucleotides sequence.

<p>Oligonucleotides sequence.</p

Pro and anti-inflammatory molecule expression in B1KO and wild type animals.

<p>All molecule expressions were measured by real-time PCR at 24 hours of reperfusion. B1KO group had lower pro-inflammatory molecule expression (T-bet and IL-1β) (A and B) and higher anti-inflammatory response (GATA-3, IL-4 and IL-10) (C, D and E). Statistical analyses were performed using the t-test.* B1KO <i>versus</i> B1B2WT, p<0.05.</p

Renal IRI and bradykinin receptors expression.

<p>Bradykinin receptors were analyzed by real-time PCR. In B1B2WT, receptors expressions were cross-modulated (A). In B2KO, B1R expression was increased at 4 and 24 hours (B). Statistical analyses were performed by ANOVA. *B1R <i>versus</i> B2R, p<0.05. # B2KO <i>versus</i> B1B2WT, p<0.05.</p

Pro and anti-inflammatory molecules expression after B1R antagonist and agonist treatment.

<p>All molecule expressions were estimated by real-time PCR at 24 hours of reperfusion. B1R antagonism (R-954) resulted in lower pro-inflammatory molecule expression (T-bet, IL-1β and MCP-1) (A, B and C) and higher anti-inflammatory response (GATA-3, IL-4, IL-10 and HO-1) (D, E, F and G). Molecule expression after B1R agonism (DABK) were similar to non-treated mice. Statistical analyses were performed using ANOVA.*IR+HOE-140 <i>versus</i> IR, p<0.05.</p

Cell death modulation under B1R-knockout.

<p>Apoptosis was estimated by Bcl-2 and Bad expression and caspase-3 activity, at 24 hours of reperfusion. Bcl-2 and Bad expression were measured by real-time PCR, and caspase-3 activity by fluorimetric assay. B1KO animals presented higher Bcl-2 expression (A) along with lower Bad expression (B) and caspase-3 activity (C), indicating a lower degree of apoptosis. Statistical analyses were performed using ANOVA.* B1KO <i>versus</i> B1B2WT and B2KO, p<0.05.</p