Pituitary Adenylate Cyclase-Activating Polypeptide Ameliorates Experimental Acute Ileitis and Extra-Intestinal Sequelae

Background The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) plays pivotal roles in immunity and inflammation. So far, potential immune-modulatory properties of PACAP have not been investigated in experimental ileitis. Methodology/Principal Findings Mice were perorally infected with Toxoplasma (T.) gondii to induce acute ileitis (day 0) and treated daily with synthetic PACAP38 from day 1 to 6 post infection (p.i.; prophylaxis) or from day 4 to 6 p.i. (therapy). Whereas placebo-treated control mice suffered from acute ileitis at day 7 p.i. and succumbed to infection, intestinal immunopathology was ameliorated following PACAP prophylaxis. PACAP-treated mice exhibited increased abundance of small intestinal FOXP3+ cells, but lower numbers of ileal T lymphocytes, neutrophils, monocytes and macrophages, which was accompanied by less ileal expression of pro-inflammatory cytokines such as IL-23p19, IL-22, IFN-γ, and MCP-1. Furthermore, PACAP-treated mice displayed higher anti-inflammatory IL-4 concentrations in mesenteric lymph nodes and liver and higher systemic anti- inflammatory IL-10 levels in spleen and serum as compared to control animals at day 7 p.i. Remarkably, PACAP-mediated anti-inflammatory effects could also be observed in extra-intestinal compartments as indicated by reduced pro- inflammatory mediator levels in spleen (TNF-α, nitric oxide) and liver (TNF-α, IFN-γ, MCP-1, IL-6) and less severe histopathological sequelae in lungs and kidneys following prophylactic PACAP treatment. Strikingly, PACAP prolonged survival of T. gondii infected mice in a time-of-treatment dependent manner. Conclusion/Significance Synthetic PACAP ameliorates acute small intestinal inflammation and extra-intestinal sequelae by down-regulating Th1-type immunopathology, reducing oxidative stress and up-regulating anti-inflammatory cytokine responses. These findings provide novel potential treatment options of inflammatory bowel diseases

Immunomodulatory Effects of the Neuropeptide Pituitary Adenylate Cyclase-Activating Polypeptide in Acute Toxoplasmosis

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is an endogenous neuropeptide with distinct functions including the regulation of inflammatory processes. PACAP is able to modify the immune response by directly regulating macrophages and monocytes inhibiting the production of inflammatory cytokines, chemokines and free radicals. Here, we analyzed the effect of exogenous PACAP on peripheral immune cell subsets upon acute infection with the parasite Toxoplasma gondii (T. gondii). PACAP administration was followed by diminished innate immune cell recruitment to the peritoneal cavity of T. gondii-infected mice. PACAP did not directly interfere with parasite replication, instead, indirectly reduced parasite burden in mononuclear cell populations by enhancing their phagocytic capacity. Although proinflammatory cytokine levels were attenuated in the periphery upon PACAP treatment, interleukin (IL)-10 and Transforming growth factor beta (TGF-β) remained stable. While PACAP modulated VPAC1 and VPAC2 receptors in immune cells upon binding, it also increased their expression of brain-derived neurotrophic factor (BDNF). In addition, the expression of p75 neurotrophin receptor (p75NTR) on Ly6Chi inflammatory monocytes was diminished upon PACAP administration. Our findings highlight the immunomodulatory effect of PACAP on peripheral immune cell subsets during acute Toxoplasmosis, providing new insights about host-pathogen interaction and the effects of neuropeptides during inflammation

Intestinal immune cell responses following ileitis induction in PACAP-treated mice.

<p>Ileitis was induced by peroral infection of mice with <i>T. gondii</i> at day 0. The average number of cells positive for (A) CD3 (T lymphocytes), (B) FOXP3 (regulatory T cells, Tregs), (C) MPO7 (myeloperoxidase-7, neutrophils and monocytes), and (D) F4/80 (macrophages) from at least six high power fields (HPF, 400× magnification) per animal were determined microscopically in immunohistochemically stained ileum sections derived from mice following PACAP prophylactic treatment (Proph, white bars), PACAP therapy (Therap, gray bars), or placebo application (black bar) at day 7 post infection as compared to naïve controls (lined bars). Numbers of analyzed animals are given in parentheses. Mean values, standard errors of the mean, and significance levels as determined by the Mann-Whitney-U test are indicated. Data are pooled from three independent experiments.</p

Amelioration of acute ileitis in PACAP-treated mice.

<p>Ileitis was induced by peroral infection of mice with <i>T. gondii</i> at day 0. (A) Relative body weight loss of mice (in %), (B) relative small intestinal shortening (in %), and (C) histopathological scores of the terminal ileum following PACAP prophylactic treatment (Proph, white bars), PACAP therapy (Therap, gray bars) or placebo application (black bar) at day 7 post infection. Numbers of analyzed animals are given in parentheses. Mean values, standard errors of the mean, and significance levels as determined by the Mann-Whitney-U test are indicated. Data are pooled from three independent experiments. (D) Representative photomicrographs (100× magnification; scale bar 100 µm) of histopathological changes in ileal paraffin sections from three independent experiments are shown. Naïve, uninfected mice served as controls.</p

Survival rates of PACAP treated mice.

<p>Ileitis was induced by peroral infection of mice with <i>T. gondii</i> at day 0 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108389#s2" target="_blank">methods</a>. Survival of animals following prophylactic (Proph, black line; n = 10) or therapeutic (Therap, gray line; n = 10) PACAP treatment as compared to placebo controls (PLC, dotted line; n = 16) was monitored twice daily until day 28 post infection (p.i.). Significance levels (as compared to respective groups) were determined by Kaplan-Meier analysis. Data are pooled from three independent experiments.</p

Less severe extra-intestinal histopathology following PACAP prophylaxis.

<p>Paraffin section of (A) lung and (B) kidney samples were obtained from naïve animals (right panel) and from mice following PACAP prophylactic treatment (PACAP, middle panel) and compared to placebo controls (PLC, left panel) at day 7 post infection. Representative photomicrographs of H&E stained paraffin sections (upper panel: 100× magnification; scale bar 100 µm; lower panel: 400× magnification; scale bar 20 µm) from three independent experiments are shown. White arrow head points towards hemosiderin deposit (in A), black arrows indicate an inflammatory peri-bronchial cuff (in A) and a pyknotic, degenerated, apoptotic glomerulum (in B).</p

Pro-inflammatory cytokine responses in ilea of PACAP-treated mice.

<p>Ileitis was induced by peroral infection of mice with <i>T. gondii</i> at day 0. (A) IL-23p19 and (B) IL-22 mRNA expression levels (by RT-PCR) as well as (C) IFN-γ and (D) MCP-1 protein concentrations (by CBA) were determined in <i>ex vivo</i> ileum biopsies obtained from mice following PACAP prophylactic treatment (Proph, white bars), PACAP therapy (Therap, gray bars), or placebo application (black bars) at day 7 post infection as compared to naïve animals (lined bars). mRNA expression levels are expressed as fold changes relative to HPRT mRNA expression (Arbitrary Units). Numbers of analyzed animals are given in parentheses. Mean values, standard errors of the mean, and significance levels as determined by the Mann-Whitney-U test are indicated. Data are pooled from three independent experiments.</p

Pro-inflammatory cytokine responses in mesenteric lymph nodes of PACAP-treated mice.

<p>Ileitis was induced by peroral infection of mice with <i>T. gondii</i> at day 0. (A) IFN-γ and (B) nitric oxide secretion were determined in overnight cultures of <i>ex vivo</i> biopsies derived from mesenteric lymphnodes (MLNs) of mice following PACAP PACAP prophylactic treatment (Proph, white bars), PACAP therapy (Therap, gray bars), or placebo application (black bars) at day 7 post infection as compared to naïve animals (lined bars). Numbers of analyzed animals are given in parentheses. Mean values, standard errors of the mean, and significance levels as determined by the Mann-Whitney-U test are indicated. Data are pooled from three independent experiments.</p

Pro-inflammatory cytokine responses in extra-intestinal compartments of PACAP-treated mice.

<p>Ileitis was induced by peroral infection of mice with <i>T. gondii</i> at day 0. Hepatic (A) IFN-γ, (B) TNF-α, (C) MCP-1, (D) IL-6, and (E) splenic TNF-α secretion were determined in overnight cultures of <i>ex vivo</i> biopsies derived from respective organs of mice following PACAP PACAP prophylactic treatment (Proph, white bars), PACAP therapy (Therap, gray bars), or placebo application (black bars) at day 7 post infection as compared to naïve animals (lined bars). Numbers of analyzed animals are given in parentheses. Mean values, standard errors of the mean, and significance levels as determined by the Mann-Whitney-U test are indicated. Data are pooled from three independent experiments.</p