The Axonal Guidance Receptor Neogenin Promotes Acute Inflammation

Neuronal guidance proteins (NGP) were originally described in the context of axonal growth and migration. Yet recent work has demonstrated that NGPs also serve as guidance cues for immune competent cells. A crucial target receptor for NGPs during embryonic development is the neogenin receptor, however its role during acute inflammation is unknown. We report here that neogenin is abundantly expressed outside the nervous system and that animals with endogenous repression of neogenin (Neo1−/−) demonstrate attenuated changes of acute inflammation. Studies using functional inhibition of neogenin resulted in a significant attenuation of inflammatory peritonitis. In studies employing bone marrow chimeric animals we found the hematopoietic presence of Neo1−/− to be responsible for the attenuated inflammatory response. Taken together our studies suggest that the guidance receptor neogenin holds crucial importance for the propagation of an acute inflammatory response and further define mechanisms shared between the nervous and the immune system

Neogenin is expressed outside the CNS.

<p><b>A</b>) Neogenin (<i>Neo 1</i>) mRNA expression in the brain (Br), lung (Lu), liver (Li), spleen (Sp) and intestine (In) of WT animals <b>B</b>) Pooled Western blot analysis of four independent animals demonstrating <i>Neo1</i> expression in (Br), lung (Lu), liver (Li), spleen (Sp) and intestine (In) <b>C</b>) Flow-cytometry of murine blood stained with anti-Neo1, anti-CD45 or isotype-matched control antibody (Data are Mean ± SEM, n = 4 per condition).</p

Functional inhibition of neogenin dampens acute inflammatory peritonitis.

<p>WT animals were injected i.p. with NaCl or 1 mg of zymosan A (ZyA) and subsequently i.v. with either IgG control or anti-Neogenin (Anti-Neo1) antibody (1 µg) and peritoneal lavage obtained after 8 hours <b>A</b>) Cell count in peritoneal lavage of <i>Neo1^−/−</i> and WT animals <b>B</b>) Myeloperoxidase (MPO) activity in peritoneal lavage <b>C</b>) Protein content in peritoneal lavage of <i>Neo1^−/−</i> and WT animals <b>D</b>) Representative histological analysis of the peritoneum, the mesenterial fat and cytospin samples of the peritoneal lavage in <i>Neo1^−/−</i> and WT animals 8 hours following intraperitoneal NaCl or ZyA injection. Sections prepared with hematoxylin-eosin staining (Magnification ×400, insert ×1000; Data are Mean ± SEM, n = 6 per group, *P<0.05, **P<0.01, ***P<0.001 as indicated).</p

Loss of haematopoietic neogenin decreases acute peritoneal inflammation.

<p>Chimeric animals and controls were injected i.p. with zymosan A (ZyA) and peritoneal lavage obtained after 8 hours <b>A</b>) Cell count within the peritoneal fluid in chimeric animals and controls 8 h after exposure to ZyA <b>B</b>) Myeloperoxidase (MPO) activity in peritoneal lavage <b>C</b>) Protein content in peritoneal lavage of chimeric animals and controls <b>D</b>) Representative histological analysis of the peritoneum, the mesenterial fat and cytospin samples of the peritoneal lavage of chimeric animals and controls 8 hours following intraperitoneal ZyA injection. Sections prepared with hematoxylin-eosin staining (Magnification ×400, insert ×1000). (Data are Mean ± SEM, n = 6 per group, *P<0.05, **P<0.01, ***P<0.001 as indicated).</p

Loss of haematopoietic neogenin dampens cytokine release during acute inflammatory peritonitis.

<p>Chimeric animals and controls were injected i.p. with 1 mg of zymosan A (ZyA) and samples taken after 8 hours <b>A</b>) TNF-α concentration in peritoneal lavage <b>B</b>) IL-1β concentration in peritoneal lavage <b>C</b>) IL-6 concentration in peritoneal lavage <b>D</b>) KC concentration in peritoneal lavage of chimeric animals and controls injected with zymosan A (Data are Mean ± SEM, n = 6 per group, *P<0.05, **P<0.01, ***P<0.001 as indicated).</p

Neogenin enhances acute inflammatory peritonitis.

<p><i>Neo1^−/−</i> and WT animals were injected i.p. with NaCl or 1 mg of zymosan A (ZyA) and peritoneal lavage obtained after 8 hours <b>A</b>) Cell count in peritoneal lavage of <i>Neo1^−/−</i> and WT animals <b>B</b>) Myeloperoxidase (MPO) activity in peritoneal lavage <b>C</b>) Protein content in peritoneal lavage of <i>Neo1^−/−</i> and WT animals <b>D</b>) Representative histological analysis of the peritoneum, the mesenterial fat and cytospin samples of the peritoneal lavage in <i>Neo 1^−/−</i> and WT animals 8 hours following intraperitoneal NaCl 0.9% or ZyA injection. Sections prepared with hematoxylin-eosin staining (Magnification ×400, insert ×1000; Data are Mean ± SEM, n = 6 per group, *P<0.05, **P<0.01, ***P<0.001 as indicated).</p

Functional inhibition of neogenin dampens release of inflammatory cytokines <i>in-vivo</i>.

<p>WT animals were injected i.p. with NaCl or 1 mg of zymosan A (ZyA) and subsequently i.v. with either IgG control or anti-Neogenin (Anti-Neo1) antibody (1 µg) and peritoneal lavage obtained after 8 hours <b>A</b>) TNF-α concentration in peritoneal lavage <b>B</b>) IL-1β concentration in peritoneal lavage <b>C</b>) IL-6 concentration in peritoneal lavage <b>D</b>) KC concentration in peritoneal lavage of WT animals injected with either vehicle or Anti-Neo1 (Data are Mean ± SEM, n = 6 per group, *P<0.05, **P<0.01, ***P<0.001 as indicated).</p

Neogenin increases inflammatory cytokine release <i>in-vivo</i>.

<p><i>Neo1^−/−</i> and <i>WT</i> animals were injected i.p. with NaCl or 1 mg of zymosan A (ZyA) and peritoneal lavage obtained after 8 hours <b>A</b>) TNF-α concentration in peritoneal lavage of <i>Neo1^−/−</i> and WT animals <b>B</b>) IL-1β concentration in peritoneal lavage of <i>Neo1^−/−</i> and WT animals <b>C</b>) IL-6 concentration in peritoneal lavage of <i>Neo1^−/−</i> and WT animals <b>D</b>) KC concentration in peritoneal lavage of <i>Neo1^−/−</i> and WT animals 8 hours following intraperitoneal NaCl or ZyA injection (Data are Mean ± SEM, n = 6 per group, *P<0.05, **P<0.01, ***P<0.001 as indicated).</p