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

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

TLR7 ligand R848 prevents mouse graft-versus-host disease and cooperates with anti-IL-27 antibody for maximal protection and Treg upregulation.

In spite of considerable therapeutic progress, acute graft-versus-host disease still limits allogeneic hematopoietic cell transplantation. We recently reported that mouse infection with nidovirus lactate dehydrogenase elevating virus impairs disease in non-conditioned B6D2F1 recipients of parental B6 spleen cells. As this virus activates TLR7, we tested pharmacologic TLR7 ligand R848 in this model and observed complete survival if donor and recipients were treated before transplantation. Mixed lymphocyte culture performed 48 h after R848-treatment of normal mice demonstrated that both T cell allo-responsiveness and antigen presentation by CD11b+ and CD8α+ dendritic cells were inhibited. These inhibitions were dependent on IFNAR-1 signaling. In the B6 to B6D2F1 transplantation model, R848 decelerated, but did not abrogate, donor T cell implantation and activation. However, it decreased IFNγ, TNFα and IL-27 while upregulating active TGF-β1 plasma levels. In addition, donor and recipient Foxp3+ regulatory T cell numbers were increased in recipient mice and their elimination compromised disease prevention. R848 also strongly improved survival of lethally irradiated BALB/c recipients of B6 hematopoietic cells and this also correlated with an upregulation of CD4 and CD8 Foxp3+ regulatory T cells that could be further increased by IL-27 inhibition. The anti-IL-27p28 monoclonal antibody and R848 combination also showed strong synergy in preventing disease in the B6 to B6D2F1 transplantation model when recipients were sublethally irradiated and this also correlated with Treg upregulation. We conclude that R848 modulates multiple aspects of graft-versus-host disease and offers potential for safe allogeneic bone marrow transplantation that can be further optimized by IL-27 inhibition

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

IL-4R{alpha}-responsive smooth muscle cells increase intestinal hypercontractility and contribute to resistance during acute Schistosomiasis.

Interleukin-(IL)-4 and IL-13 signal through heterodimeric receptors containing a common IL-4 receptor-alpha (IL-4Ralpha) subunit, which is important for protection against helminth infections, including schistosomiasis. Previous studies demonstrated important roles for IL-4Ralpha-responsive hematopoietic cells, including T cells and macrophages in schistosomiasis. In this study, we examined the role of IL-4Ralpha responsiveness by nonhematopoietic smooth muscle cells during experimental acute murine schistosomiasis. Comparative Schistosoma mansoni infection studies with smooth muscle cell-specific IL-4Ralpha-deficient (SM-MHC(cre)IL-4Ralpha(-/flox)) mice, heterozygous control (IL-4Ralpha(-/flox)) mice, and global IL-4Ralpha-deficient (IL-4Ralpha(-/-)) mice were conducted. S. mansoni-infected SM-MHC(cre)IL-4Ralpha(-/flox) mice showed increased weight loss and earlier mortalities compared with IL-4Ralpha(-/flox) mice, despite comparable T(H)2/type 2 immune responses. In contrast to highly susceptible IL-4Ralpha-deficient mice, increased susceptibility in SM-MHC(cre)IL-4Ralpha(-/flox) mice was not accompanied by intestinal tissue damage and subsequent sepsis. However, both susceptible mutant mouse strains failed to efficiently expel eggs, demonstrated by egg reduction in the feces compared with control mice. Reduced egg expulsion was accompanied by impaired IL-4/IL-13-mediated hypercontractile intestinal responses, which was present in the more resistant control mice. Together, we conclude that IL-4Ralpha responsiveness by smooth muscle cells and subsequent IL-4- and IL-13-mediated hypercontractility are required for host protection during acute schistosomiasis to efficiently expel S. mansoni eggs and to prevent premature mortality

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

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