Absence of the Lectin Activation Pathway of Complement Ameliorates Proteinuria-Induced Renal Injury.

Proteinuria is an adverse prognostic feature in renal diseases. In proteinuric nephropathies, filtered proteins exert an injurious effect on the renal tubulointerstitium, resulting in inflammation and fibrosis. In the present study, we assessed to what extent complement activation via the lectin pathway may contribute to renal injury in response to proteinuria-related stress in proximal tubular cells. We used the well-established mouse model of protein overload proteinuria (POP) to assess the effect of lectin pathway inhibition on renal injury and fibrotic changes characteristic of proteinuric nephropathy. To this end, we compared experimental outcomes in wild type mice with MASP-2-deficient mice or wild type mice treated with MASP-2 inhibitor to block lectin pathway functional activity. Multiple markers of renal injury were assessed including renal function, proteinuria, macrophage infiltration, and cytokine release profiles. Both MASP-2-deficient and MASP-2 inhibitor-treated wild type mice exhibited renoprotection from proteinuria with significantly less tubulointerstitial injury when compared to isotype control antibody treated mice. This indicates that therapeutic targeting of MASP-2 in proteinuric nephropathies may offer a useful strategy in the clinical management of proteinuria associated pathologies in a variety of different underlying renal diseases

Additional file 1: of Mannan binding lectin-associated serine protease-2 (MASP-2) critically contributes to post-ischemic brain injury independent of MASP-1

Orsini_Chrysanthou. (PDF 608 kb

MASP-2 is essential for C3 deposition on <i>S pneumoniae</i>.

<p>Serial dilutions of sera were incubated in microtiter plates coated with <i>S. pneumoniae</i> D39 or N-acetylated BSA (as a control), and C3 deposition determined by ELISA. <b>A.</b> Shows the raw data from one experiment with WT and MASP-2 deficient murine serum (means ±SEM). In <b>B</b>, the experiment was extended to include murine sera deficient in other complement components. Results are duplicates (±SD) and are normalised to the C3 deposition observed in the WT control. <b>C.</b> Time course of C3 activation on <i>S. pneumoniae</i>. 1∶40 diluted murine sera were incubated in microtiter plates coated with <i>S. pneumoniae</i> for the times indicated then C3 deposition assayed. Results are means of duplicates and are representative of three independent experiments. <b>D.</b> FACS analysis of C3 deposition on <i>S. pneumoniae</i> opsonised with MASP-2 −/− serum (black line), WT serum (red) and C4 −/− serum (green). The blue trace shows non-opsonised bacteria. <b>E.</b> Results from 3 independent FACS analyses of C3 deposition (mean fluorescent intensity ±SEM). <b>F.</b> Inhibition of MASP-2 activity with mAb AbD04211 abolished C3b deposition on <i>S. pneumoniae</i> opsonised with C4 deficient serum. (means of triplicates ±SEM; p value from Student's t-test). <b>G.</b> In human serum, MBL deficiency had no effect on C3 deposition, while C4 deficiency had no significant effect on the EC₅₀, but reduces absolute C3b deposition by about 50% (means ±SEM; n = 3 for NHS and MBL −/− serum, 1 for C4 deficient serum). <b>H.</b> Correlation between L-ficolin serum concentration and C3b deposition on <i>S. pneumoniae</i> immobilised on microtiter plates for 47 samples of NHS (solid line shows Fisher transformation of Pearson's correlation coefficient; dashed lines, 95% CI thereof).</p

Deposition of C4 breakdown products on <i>S. pneumoniae</i>.

<p>ELSIA plates were coated with <i>S. pneumoniae</i> D39, <i>E. coli</i> or mannan (as positive controls) and incubated with NHS for 1 h at 37°C. Bound C4 was detected using an antibody against C4c (which also detects C4b) (<b>A</b>) or an antibody against C4dg, the last breakdown product of C4 that remains covalently attached to the activating surface (<b>B</b>). <b>C</b> and <b>D</b>: Detection of C4c (<b>C</b>) and C4dg (<b>D</b>) on live <i>S. pneumoniae</i> D39 opsonised with NHS. Green trace shows anti-C4 antibodies; the purple shading, an isotype control Ab.</p

MASP-2 deficiency impairs phagocytosis of <i>S. pneumoniae</i> by polymorphonuclear leukocytes.

<p><i>S. pneumoniae</i> were pre-incubated with murine serum (20% v/v), mixed with freshly isolated human PMN and incubated for 2 hr before being immobilised on microscope slides and stained with eosin Y and azur II (REASTAIN), as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002793#s4" target="_blank">materials and methods</a>. Bacteria opsonised with WT serum (<b>A</b>) are internalized by PMNs, whereas bacteria opsonised with MASP-2 −/− serum (<b>B</b>) are excluded (arrows). <b>C.</b> Electron micrograph showing pneumococci opsonised with WT serum inside a PMN. <b>D.</b> Samples were removed from the <i>S. pneumoniae</i>/PMN mix at the times indicated and viable bacteria determined. WT serum (crosses) facilitated killing by PMN, whereas MASP-2 −/− serum (open squares) is severely compromised in its ability to opsonise <i>S. pneumoniae</i>. Controls were run in parallel containing non-opsonised bacteria (triangles) and bacteria opsonised with WT serum, but without PMN (circles). Results are means (±SEM) of triplicates.</p

Inhibition of MASP-2 increases the severity of <i>S. pneumoniae</i> infection.

<p>WT C57/BL6 mice were dosed <i>i.p.</i> with 1.0 mg/kg body weight of anti-MASP-2 mAb AbD04211 (open triangles) or an isotype control mAb (crosses) 12 h before being infected with 1×10⁶ cfu <i>S. pneumoniae</i> D39. MASP-2 inhibition significantly worsened survival (<b>A</b>) and there was a corresponding increase in viable <i>S. pneumoniae</i> counted in peripheral blood after infection (<b>B</b>). Treatment with 20 mg/kg body weight ceftriaxone 12 hr before infection and every 12 hr thereafter afforded complete protection from <i>S. pneumoniae</i> in animals injected with AbD04211 (filled triangles in A). Results in B are means (±SEM) of twelve animals from which blood was taken at each time point. *p<0.05 (ANOVA).</p

Binding of lectin pathway recognition molecules to <i>S. pneumoniae</i>.

<p>Microtiter plates coated with formalin-fixed <i>S. pneumoniae</i> or control substrates were used to capture lectin pathway recognition complexes from WT mouse serum or NHS. Murine MBL-A and MBL-C (<b>A</b>), murine ficolin A (<b>B</b>), murine CL-11 (<b>C</b>) human MBL (<b>D</b>), FCN1 & 3 (<b>E</b>) and FCN2 and CL-K1 (<b>F</b>) were assayed by ELISA, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002793#s4" target="_blank">materials and methods</a>. Only ficolin A, CL-11 and FCN2 (L-ficolin) bound to the bacteria. Results are means of duplicates and are representative of three independent experiments.</p

Lectin pathway deficiency significantly increases the severity of <i>S. pneumoniae</i> infection.

<p>Lectin pathway deficient mice (open squares) and WT littermates (crosses) were infected intranasally with 1×10⁶ cfu <i>S. pneumoniae</i> D39. C57/BL6 <i>^Masp2</i>^−/− (<b>A</b>) and C57/BL6 <i>^Fcna</i>^−/− (<b>B</b>) mice are impaired in their survival of <i>S. pneumoniae</i> infection (Mantel-cox log-rank test). <b>C–F</b>. Viable <i>S. pneumoniae</i> counted in lung homogenates and peripheral blood at the indicated time points after infection. Results in C–E are means (±SEM) of five animals sacrificed at each time point. *p<0.05; **p<0.01 (ANOVA).</p