The role of IL-4Ra during acute schistosomiasis

Includes bibliographical references.During an infection with Schistosoma mansoni, immunopathology is associated with a dominant TH-2 type cytokine expression, tissue cosinophilia and high levels of serum immunoglobulin E. It is postulated that in addition to egg specific CD4+T cells, an IL-4Rα+ non-T cell effector population is required to prevent tissue pathology. The role of this dissertation was to examine the functional role of IL-Rα expression on the cells of the myeloid and lymphoid lineages during an acute Schistosoma mansoni infection and its requirement for the extent pf schistosome egg induced lung inflammation and pathology

IL-4/IL-13 independent goblet cell hyperplasia in experimental helminth infections

BACKGROUND: Intestinal mucus production by hyperplasic goblet cells is a striking pathological feature of many parasitic helminth infections and is related to intestinal protection and worm expulsion. Induction of goblet cell hyperplasia is associated with TH2 immune responses, which in helminth infections are controlled primarily by IL-13, and also IL-4. In the study presented here we examine the goblet cell hyperplasic response to three experimental parasitic helminth infections; namely Nippostrongylus brasiliensis, Syphacia obvelata and Schistosoma mansoni. RESULTS: As expected N. brasiliensis infection induced a strong goblet cell hyperplasia dependent on IL-4/IL-13/IL-4Ralpha expression. In contrast, and despite previously published transiently elevated IL-4/IL-13 levels, S. obvelata infections did not increase goblet cell hyperplasia in the colon. Furthermore, induction of goblet cell hyperplasia in response to S. mansoni eggs traversing the intestine was equivalent between BALB/c, IL-4/IL-13-/- and IL-4Ralpha-/- mice. CONCLUSION: Together these data demonstrate that intestinal goblet cell hyperplasia can be independent of TH2 immune responses associated with parasitic helminth infections

IL-4Rα-Independent Expression of Mannose Receptor and Ym1 by Macrophages Depends on their IL-10 Responsiveness

IL-4Rα-dependent responses are essential for granuloma formation and host survival during acute schistosomiasis. Previously, we demonstrated that mice deficient for macrophage-specific IL-4Rα (LysMcreIl4ra−/lox) developed increased hepatotoxicity and gut inflammation; whereas inflammation was restricted to the liver of mice lacking T cell-specific IL-4Rα expression (iLckcreIl4ra−/lox). In the study presented here we further investigated their role in liver granulomatous inflammation. Frequencies and numbers of macrophage, lymphocyte or granulocyte populations, as well as Th1/Th2 cytokine responses were similar in Schistosoma mansoni-infected LysMcreIl4ra−/lox liver granulomas, when compared to Il4ra−/lox control mice. In contrast, a shift to Th1 responses with high IFN-γ and low IL-4, IL-10 and IL-13 was observed in the severely disrupted granulomas of iLckcreIl4ra−/lox and Il4ra−/− mice. As expected, alternative macrophage activation was reduced in both LysMcreIl4ra−/lox and iLckcreIl4ra−/lox granulomas with low arginase 1 and heightened nitric oxide synthase RNA expression in granuloma macrophages of both mouse strains. Interestingly, a discrete subpopulation of SSChighCD11b+I-A/I-EhighCD204+ macrophages retained expression of mannose receptor (MMR) and Ym1 in LysMcreIl4ra−/lox but not in iLckcreIl4ra−/lox granulomas. While aaMφ were in close proximity to the parasite eggs in Il4ra−/lox control mice, MMR+Ym1+ macrophages in LysMcreIl4ra−/lox mice were restricted to the periphery of the granuloma, indicating that they might have different functions. In vivo IL-10 neutralisation resulted in the disappearance of MMR+Ym1+ macrophages in LysMcreIl4ra−/lox mice. Together, these results show that IL-4Rα-responsive T cells are essential to drive alternative macrophage activation and to control granulomatous inflammation in the liver. The data further suggest that in the absence of macrophage-specific IL-4Rα signalling, IL-10 is able to drive mannose receptor- and Ym1-positive macrophages, associated with control of hepatic granulomatous inflammation

IL-4Ralpha responsive CD4+CD25−CD103+FoxP3− cells control Schistosoma mansoni egg-induced inflammation by secreted IL-10

IL-4Ralpha signalling drives Th2-type responses that mediate resistance to parasitic helminth infections. We generated a novel mouse model lacking IL-4Ralpha expression specifically on all T cells (iLckcreIl4ra−/lox) to investigate IL-4Ralpha-dependent T cell responses during Schistosoma mansoni egg-driven inflammation. These mice showed higher mortality during acute schistosomiasis compared with Il4ra−/lox controls and previously established CD4+ T cell specific IL-4Ralpha deficient mice (LckcreIl4ra−/lox). iLckcreIl4ra−/lox mice developed a liver restricted pathology associated with drastic reductions of both Th2/type 2 responses and alternative macrophage activation within the granulomas. Additionally, iLckcreIl4ra−/lox mice had (i) increased FoxP3+ Treg cell responses in the granulomas, which was explained by IL-4 mediated inhibition of FoxP3 induction, and (ii) reduction of antigen-specific production of IL-10 by CD4+CD103+FoxP3− cells. In a footpad model of S. mansoni egg-induced inflammation with subsequent IL-10 neutralisation and adoptive cell transfer experiments we found evidence that the increased inflammation in iLckcreIl4ra−/lox mice was due to the impaired development of IL-10-secreting CD4+CD25−CD103+FoxP3− cells. Together, these data demonstrate that IL-4Ralpha responsiveness by T cells promote IL-10-secreting CD4+CD25−CD103+FoxP3− cells and alternatively activated macrophages, which act in concert to control egg-induced inflammation

Localisation of mannose receptor and Ym1-expressing granuloma macrophages in close contact with <i>S. mansoni</i> eggs depends on their IL-4Rα signalling.

<p>Livers were collected at 8 weeks p.i. from <i>Il4ra^−/lox</i>, <i>LysM^creIl4ra^−/lox</i>, <i>iLck^creIl4ra^−/lox</i> and <i>Il4ra^−/−</i> mice and immunofluorescent stainings performed on cryosections. (A) Representative micrographs of MMR (panels v–viii), Ym1 (panels xiii–xvi), CD204 (panels xxi–xxiv) and iNOS (panels xxix–xxxii) stainings of liver cryosections as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000689#s2" target="_blank">Methods</a>. Stainings with secondary antibody (MMR, iNOS), streptavidin alone (Ym1) or isotype-control (CD204) is shown for each corresponding staining (MMR = i–iv, Ym1 = ix–xii, CD204 = xvii–xx, iNOS = xxv–xxviii). Note that MMR⁺ and Ym1⁺ cells are restricted to the periphery of <i>LysM^creIl4ra^−/lox</i> granulomas. Outlined regions represent the areas magnified in B. (B) Representative micrographs of liver cryosections stained with scavenger receptor (CD204) for macrophages detection (panels i–viii, green) or iNOS for classically activated macrophages detection (panels ix–xvi, green); and co-stained for MMR (panels i–iv and ix–xii, red) or Ym1 (panels v–viii and xiii–xvi, red). Note the low frequency of CD204⁺ macrophages co-expressing MMR⁺ or Ym1⁺ cells around the parasite eggs (panels ii and vi) but the high levels of iNOS⁺ cells (panels x and xiv) in <i>LysM^creIl4ra^−/lox</i> mice, suggesting these macrophages to be classically activated. White arrows indicate the parasite eggs. Original magnification: 400×. Data represent one of three independent experiments.</p

IL-4Rα-expressing macrophages do not affect cytokine responses in granulomas.

<p><i>Il4ra^−/lox</i>, <i>LysM^creIl4ra^−/lox</i>, <i>iLck^creIl4ra^−/lox</i> and <i>Il4ra^−/−</i> mice were infected with 100 <i>S. mansoni</i> cercariae and killed 8 weeks later. Mesenteric lymph node (mLN) cells or liver granuloma-associated leukocytes (Liver) were then isolated and restimulated overnight with 20 µg/ml SEA before analyzed for <i>ex vivo</i> cytokine production capability. Staining of surface CD4 was followed by detection of IL-4, IL-10 and IFN-γ secretion in a cytokine catch assay or detection of intracellular levels of IL-13 after 4h of monensin treatment, as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000689#s2" target="_blank">Methods</a>. Live gate was placed on lymphocytes according to their forward- and side-scatter by flow cytometry. (A) Representative contour plots of IFN-γ, IL-4, IL-10 and IL-13-producing lymphocytes. Quadrant bars were set up on unstimulated cells and numbers represent percent cells in each quadrant. Data represent one out of three independent experiments with similar results. (B) Percentages of IFN-γ, IL-4, IL-10 or IL-13-producing cells were determined in gated CD4⁺ or non-CD4⁺ cell populations from mLN or liver granuloma-associated lymphocytes (Liver) (mean±SEM, <i>n</i> = 4). Data are representative of three independent experiments. *<i>p</i><0.05; **<i>p</i><0.001; ***<i>p</i><0.001 compared to control <i>Il4ra^−/lox</i> mice.</p

IL-10 signalling drives mannose receptor and Ym1 expression in macrophages independently of their IL-4Rα expression.

<p>Peritoneal cells were harvested 7 days after injection of 3000 <i>S. mansoni</i> purified eggs in the peritoneum of <i>Il4ra^−/lox</i>, <i>LysM^creIl4ra^−/lox</i>or <i>Il4ra^−/−</i> mice. (A) Representative histograms of macrophage mannose receptor (MMR) or Ym1 expression by peritoneal macrophages gated on FSC^highSSC^lowCD11b⁺F4/80⁺ cells after exclusion of peritoneal eosinophils (FSC^lowSSC^highCD11b⁺F4/80⁺) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000689#pntd.0000689-Taylor1" target="_blank">[37]</a>. Black, isotype control; red, <i>Il4ra^−/lox</i>; blue, <i>LysM^creIl4ra^−/lox</i>; green, <i>Il4ra^−/−</i>. Broken lines show threshold for positive signal. Data are representative of two independent experiments of pooled samples (<i>n</i> = 3). (B) Anti-IL-10 receptor treatment. Four mice out of eight in each group received 4µg of anti-IL-10R i.p. at day 0, 4 and 6 post-injection. Overlays of representative monoparametric histograms of macrophage mannose receptor (MMR) or Ym1 expression by peritoneal macrophages are shown as described in A. Bracketed line indicates positive signal. Dotted line, isotype control; greyscale, untreated mice; bold line, mice treated with anti-IL-10R. (C) Percent and mean fluorescent intensities of peritoneal macrophages expressing MMR or Ym1 based on flow cytometry analysis in B. Data is representative of two independent experiments with mean±SEM (<i>n</i> = 3). *<i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p

Flow cytometry analysis of liver granuloma during acute schistosomiasis.

<p>BALB/c mice were infected with 100 <i>S. mansoni</i> cercariae, killed 8 weeks later and liver granuloma-associated leukocytes isolated before 4-colour flow cytometry analysis. (A) Gating strategy for analysis of surface expression of CD11b and I-A/I-E on SSC^high and live (7-AAD⁻) liver granuloma-associated leukocytes. Outlined regions define CD11b⁺I-A/I-E⁻ and CD11b⁺I-A/I-E^high, respectively. (B) CD11b⁺I-A/I-E⁻, and CD11b⁺I-A/I-E^high gated populations in A were analyzed for F4/80, Gr-1, Siglec-F, CD204, MMR, Dectin-1, CD68, CD80, CD86 and IL-4Rα expression, respectively. Greyscale histograms show relevant isotype control. (C) 4-colour flow cytometry analysis of liver granuloma-associated leukocytes gated on CD11b⁺I-A/I-E⁻ cells as described in A. F4/80 and Gr-1 double staining contour plot is shown. Outlined regions were sorted for cytospin and morphological analysis. (D) 4-colour flow cytometry analysis of liver granuloma-associated leukocytes gated on CD11b⁺F4/80⁺ cells as described in A. MHC-II (I-A/I-E) and scavenger receptor-A (CD204) double staining contour plot is shown. Outlined region was sorted for cytospin and morphological analysis. Data are representative of three independent experiments with similar results.</p