Induction of myeloid cell arginase activity by <i>T. brucei</i> PRF.

<p>Arginase activity was determined in myeloid cells from non-infected mice incubated with (<b>A</b>) WT parasites separated or not by an insert, or WT parasite-released factors (PRF), (<b>B</b>) PRF from WT parasites in presence of indicated antibodies and (<b>C</b>) PRF from WT or <i>TbKHC1</i> KO parasites, or material purified on anti-PRF antibody affinity column, in presence of indicated antibodies. Heat treatment was at 45°C for 30 min. Data are means ± SEM of 3–4 individual mice of one representative from 3 independent experiments (A, B), or of triplicate cultures of one representative from 2 independent experiments (C). * p<0.05 compared to non-stimulated (-) cells.</p

SIGN-R1 receptor contribution to myeloid cell activation and liver injury.

<p>(<b>A,B</b>) Effects of rTbKHC1 on myeloid cells from non-infected control (Ctrl) or <i>SIGN-R1</i> KO mice (<b>A</b>: relative expression of <i>Arg1</i>, <i>Arg2</i>, <i>Il10</i> and <i>iNOS</i> genes; <b>B</b>: arginase activity in presence of indicated antibodies or sugars). Data are means ± SEM of 3 individual mice of one representative from 3 independent experiments. * p<0.05 compared to non-stimulated (-) cells. (<b>C</b>) Effects of rTbKHC1 on liver injury: microscopic analysis (hematoxylin-eosin staining, magnification 20×) of sections from WT- and <i>TbKHC1</i> KO-infected mice at day 30 p.i. Anoxic infarcts (ai), periportal (>>) and lobular (>) mononuclear cell infiltrates were representative of 8 animals tested in 2 independent experiments. (<b>D</b>) Spontaneous NO and IL-10 secretions in spleen myeloid cell supernatants from WT- and <i>TbKHC1</i> KO-infected mice treated with D-NAME or L-NAME (day 30 p.i.). (<b>E</b>) Idem as <b>D</b> in <i>SIGN-R1</i> KO and control (Ctrl) mice. Data are means ± SEM of 3–4 individual mice of one representative from 3 independent experiments. ∇ p<0.05 comparing WT or <i>TbKHC1</i> KO- infected mice to non-infected mice; £ p<0.05 comparing L-NAME and D-NAME treatment in WT- or <i>TbKHC1</i> KO-infected mice; # p<0.05 comparing WT- and <i>TbKHC1</i> KO-infected mice.</p

Mechanism of arginase activity induction by <i>T. brucei</i> PRF and rTbKHC1.

<p>Myeloid cells from non-infected WT (<b>A-D,F, G</b>) or IL-4Rα KO (<b>E</b>) mice were incubated with PRF or rTbKHC1. (<b>A</b>) IL-10 production induced by PRF. (<b>B</b>) Arginase activity induced by PRF incubated with indicated antibodies or sugars. (<b>C</b>) Arginase activity and IL-10 secretion induced by rTbKHC1 incubated with indicated antibodies or sugars. (<b>D–G</b>) Relative expression level of M2 genes after incubation with rTbKHC1. In <b>F</b>, M2 IL-10-dependent genes are indicated, and in <b>G</b>, rTbKHC1 was incubated with the indicated antibodies. Data are means ± SEM of 3 individual mice of one representative from 3 independent experiments. * p<0.05 compared to non-stimulated (-) cells.</p

Effects of TbKHC1 on <i>T. brucei</i> parasitaemia.

<p>WT and <i>TbKHC1</i> KO parasitaemias were monitored in various mice and conditions: (<b>A</b>) WT mice treated with L-NAME or D-NAME; (<b>B</b>) <i>iNOS</i> KO and WT mice; (<b>C,D</b>) myeloid cell <i>Arg1</i> KO mice (KO1 = <i>LysM</i>^cre<i>Arg1</i>^-/lox; KO2 = <i>Tie2</i>^cre<i>Arg1</i>^-/lox) and controls (<i>Arg1</i>^lox/lox); (<b>E,F</b>) WT mice treated with L-ornithine, D-mannose or D-galactose; (<b>G</b>) <i>MMR</i> KO and WT mice; (<b>H</b>) <i>SIGN-R1</i> KO and control (Ctrl) mice. Data are means ± SEM of 4 individual mice of one representative from 3 independent experiments. * p<0.05 comparing D-NAME and L-NAME treated mice (A) or WT and <i>iNOS</i> KO mice (B) infected with WT parasites; ∇ p<0.05 comparing <i>Arg1</i>^lox/lox and <i>LysM</i>^cre<i>Arg1</i>^-/lox or <i>Tie2</i>^cre<i>Arg1</i>^-/lox mice infected with WT parasites; # p<0.05 comparing L-ornithine treated and non treated mice infected with <i>TbKHC1</i> KO parasites; ¥ p<0.05 comparing D-mannose and D-galactose-treated mice infected with WT parasites; £ p<0.05 comparing <i>SIGN-R1</i> KO and control mice infected with WT parasites.</p

Expression and localization of <i>T. brucei</i> TbKHC1.

<p>(<b>A</b>) Detection of <i>TbKHC1</i> transcripts by Northern blotting of RNA from procyclic forms (PF), bloodstream (BF) slender (SL) and stumpy (ST) forms, BF <i>TbKHC1</i> knock-out (KO), overexpressor (OV) and <i>TbKHC1</i> knock-down (RNAi) trypanosomes (d: days after doxycyclin induction; ds RNA: double-stranded RNA; histone H2B mRNA: loading control). (<b>B</b>) Detection of TbKHC1 with anti-rTbKHC1 polyclonal antibody in whole extracts from the indicated trypanosome forms (Tub: Tubulin loading control). (<b>C</b>) Parasite staining with anti-PRF monoclonal 2C12 antibody. Nucleus (N) and kinetoplast (K) are stained in blue with 4′,6-Diamidino-2-phenylindole (DAPI). (<b>D</b>) Localization of TbKHC1 on WT, KO and OV trypanosomes with anti-rTbKHC1 polyclonal antibody. (<b>E</b>) Immunoprecipitation of TbKHC1, actin, enolase and PDI-2 from ³⁵S-metabolically labelled WT parasite total extracts (1) or supernatants (2).</p

Effects of TbKHC1 on <i>T. brucei</i> growth and infection in mice.

<p>(<b>A,B</b>) <i>In vitro</i> parasite growth (<b>A</b>: monomorphic trypanosomes, either WT or induced and non-induced <i>TbKHC1</i> KD; <b>B</b>: pleomorphic trypanosomes, either WT or <i>TbKHC1</i> KO and rescued <i>TbKHC1</i> KO). Data are means ± SEM of one representative from 3 independent experiments (<b>C,D</b>) Parasitemia in C57Bl/6 mice infected with pleomorphic WT and <i>TbKHC1</i> KO parasites (<b>C</b>: intraperitoneal injection; <b>D</b>: injection through the bite of infected tsetse flies). Data are means ± SEM of 4 individual mice of one representative from 6 independent experiments. * p<0.05 comparing <i>TbKHC1</i> KO- and WT-infected mice. (<b>E–G</b>) TbKHC1-mediated myeloid cell activation. At day 6 p.i. with WT and <i>TbKHC1</i> KO parasites, mice were treated with anti-IL-10R (anti-IL-10R Ab) or control antibody (Ctrl Ab), and at day 7 spleen myeloid cells were analyzed for arginase activity (<b>E</b>), spontaneous NO secretion (<b>F</b>) and IL-10 secretion (<b>G</b>). Data are means ± SEM of 3 individual mice of one representative from 3 independent experiments. ∇ p<0.05 comparing WT- or <i>TbKHC1</i> KO-infected to non-infected mice; £ p<0.05 comparing WT- or <i>TbKHC1</i> KO-infected mice treated with anti-IL-10R antibody to WT- or <i>TbKHC1</i> KO-infected control antibody-treated mice; # p<0.05 comparing <i>TbKHC1</i> KO- and WT-infected mice treated with control antibody.</p

Survival and liver pathology in <i>T. brucei</i> WT- and <i>TbKHC1</i> KO- infected mice.

<p>Survival and liver pathology in <i>T. brucei</i> WT- and <i>TbKHC1</i> KO- infected mice.</p