IL‐4Rα‐expressing CD11c+ cells contribute to driving optimal cellular responses during Schistosoma mansoni infection in mice

Abstract Development of IL‐4 receptor alpha (IL‐4Rα)‐dependent cellular immunity regulates host protection against acute schistosomiasis. In this study, we investigated the importance of IL‐4Rα‐expressing CD11c+ cells in driving the development of optimal cellular responses to Schistosoma mansoni infection by using CD11ccreIL‐4Rα−/lox BALB/c mice, which lacked IL‐4Rα expression on dendritic cells and alveolar macrophages. Abrogation of IL‐4Rα expression on CD11c+ cells affected activation of CD4+ T cells, resulting in reduced numbers of effector CD4+ T cells and impaired production of Th1 and Th2 cytokines by CD4+ T cells ex vivo. However, secretion of both type 1 and type 2 Ab isotypes was unchanged in infected CD11c‐specific IL‐4Rα‐deficient mice compared to littermate controls. Together, these data demonstrate that IL‐4Rα‐expressing CD11c+ cells play an important role in maintaining cellular immunity during schistosomiasis in mice.


INTRODUCTION
Dendritic cells (DCs) are the most specialized APCs, capable of recognizing and processing foreign Ag, migrating to the lymph nodes, and efficiently initiating T cell activation. [1][2][3] The ability of DCs to orchestrate Th1/Th17 immunity in response to bacterial and viral pathogens Abbreviations: AST, aspartate transaminase; BMDCs, bone marrow-derived dendritic cells; DC, dendritic cell; IL-4R , IL-4 receptor alpha; MLN, mesenteric lymph node; pLN, popliteal lymph node; SEA, schistosome egg antigen Ag (BMDCs) have been shown to respond to IL-4 by up-regulating alternative activation markers such as Ym1/2 and RELM , and abrogation of IL-4 receptor alpha (IL-4R ) expression on DCs resulted in impaired IFN-production in both Th1 and Th2 settings. 7 Interestingly, IL-4 has been shown to instruct BMDCs to produce IL-12p70 in the presence of bacterial LPS or CpG in vitro. [8][9][10][11] The instruction theory was shown to be relevant in vivo because the treatment of susceptible BALB/c mice with IL-4 conferred host protection to Leishmania major infection. 8 Recently, it was shown that DCs from mice carrying a specific deletion of IL-4R on CD11c + cells had reduced IL-12p40 expression and increased IL-10 expression during L. major infection, coinciding with impaired Th1-type responses and severe disease characterized by footpad necrosis. 12 Furthermore, IL-4R expression on CD11c + cells was shown to promote allergen-induced Th2 effector responses in allergic airway disease in mice. 13 DCs IL-4R expression therefore appears to play an important role in both Th1-and Th2-associated diseases.
Schistosomiasis is an important parasitic disease that infects more than 200 million people, mostly in developing countries, and causes an estimated 280,000 deaths per annum in sub-Saharan Africa alone. 14,15 During infection in both humans and mice, S. mansoni eggs trapped in the host tissue (particularly in the liver) induce a strong granulomatous inflammatory response that is accompanied by augmented production of Th2 cytokines, eosinophilia, goblet cell hyperplasia, and increased IgG1 and IgE production. 5,16,17 Infection of IL-4 −/− and IL-4/IL-13 −/− mice with S. mansoni revealed a crucial protective role for Th2 cytokine and granuloma formation. [18][19][20] IL-4 and IL-13 are key Th2 cytokines that signal though the common receptor chain, the IL-4R . 21,22 IL-4 exclusively signals through the type 1 receptor composed of IL-4R and the common gamma chain, while both IL-4 and IL-13 signal through the type II receptor comprising of IL-4R and IL-13R 1 chains. 21,22 Mice deficient in IL-4R signaling quickly succumb to S. mansoni infection due to impaired granuloma formation, impaired Th2 polarization, increased liver inflammation, and destruction of the gut integrity that results in endotoxemia and septic shock. 18,23,24 Therefore, IL-4 and IL-13 responsiveness plays a key role in down-regulating organ injury induced by S. mansoni eggs.
Generation of cell-specific IL-4R deficient mouse strains has proven to be an invaluable tool for dissecting the mechanisms conferring host protection or susceptibility to S. mansoni infection.
Previous studies from our laboratory have demonstrated a pivotal role for IL-4R expressing macrophages in host protective responses during acute schistosomiasis. 24 Mice deficient in IL-4R expression on macrophages (LysM cre IL-4R −/lox ) were extremely susceptible to S. mansoni infection due to increased hepatocellular damage and intestinal inflammation that led to endotoxemia and septic shock. 25 Furthermore, using mice that lacked IL-4R expression on pan-T cells (iLck cre IL-4R −/lox ), we showed that IL-4/IL-13 responsive non-CD4 + T cells contribute to resistance against acute schistosomiasis by controlling excessive liver inflammation. 23 In contrast, mice deficient in IL-4R expression specifically on CD4 + T cells (Lck cre IL-4R −/lox ) survived S. mansoni infection despite augmented Th1 granulomatous pathology. 26 Other possible IL-4/IL-13 responsive cellular populations contributing to the mechanism of host resistance to S. mansoni infection are yet to be identified. Therefore, we investigated whether IL-4R signaling on CD11c + cells plays a role in orchestrating cellular responses and host protection to S. mansoni infection, by utilizing a transgenic mouse strain (CD11c cre IL-4R −/lox ) that lacked IL-4R expression specifically on CD11c + cells. 12 We found that effector CD4 + T cells and B cells numbers were drastically reduced in the draining mesenteric lymph nodes (MLNs) of CD11c cre IL-4R −/lox mice compared to littermate controls. Together, these data demonstrate that IL-4R signaling on CD11c + cells is important for driving cellular immunity during acute schistosomiasis.

Impaired Th1 and Th2 cytokine responses in CD11 cre IL-4R −/lox mice challenged with S. mansoni eggs in the footpad
To determine the effect of CD11c + cell IL-4R expression on Th1/Th2 type responses to S. mansoni, we challenged CD11c cre IL-4R −/lox and IL-4R −/lox littermate control mice with 2,500 S. mansoni eggs in the left hind footpad and collected popliteal lymph nodes (pLNs) 7 d postchallenge. We stimulated total pLN cells with either Schistosome egg Ag (SEA) or -CD3 and analyzed cytokine secretion by sandwich ELISA. Secretion of IL-4 and IL-13 was reduced in total pLN cells from CD11c cre IL-4R −/lox mice compared to control mice after either Ag-specific or mitogenic restimulation ( Fig. 1A and B), despite unaltered frequencies of CD19 + B cells or CD4 + T cells (Supplementary Fig. S1A and B). Moreover, production of the Th1 cytokine IFNwas reduced in -CD3-stimulated cells from CD11c cre IL-4R −/lox mice compared to control mice (Fig. 1B). Little or no cytokine expression was detected in naïve mutant or control mice after antigenic or mitogenic restimulation (Fig. S2). We also found that the frequency of CD19 + B cells secreting IL-4, IL-13, and IFN-after stimulation with PMA/Ionomycin ex vivo was reduced in CD11c cre IL-4R −/lox mice compared to littermate control mice (Fig. 1C, Supplementary Fig.   S3A). Together, these data suggest that specific deletion of IL-4R on CD11c + cells impairs secretion of both Th1 and Th2 cytokines during synchronous eggs challenge in the footpad. Together, these data suggest that IL-4R -expressing CD11c + cells play a role in initiating and maintaining cytokine production by CD4 +

T cells in vivo.
We also analyzed the numbers of lymphocytes in the MLN after infection using flow cytometry. We found that the total number of MLN cells was significantly reduced (60%) in CD11c cre IL-4R −/lox mice compared to IL-4R −/lox littermate control mice at 7 wk postinfection (Fig. 2E). This corresponded with a striking decrease in the total number of CD4 + T cells (Fig. 2F) and effector CD4 + T cells ( Fig. 2G and H) in CD11c cre IL-4R −/lox mice compared to littermate control mice.
Therefore, these data demonstrate that IL-4R -expressing CD11c + cells are required for expansion of CD4 + T cells in the MLN during S. mansoni infection.

Production of specific IgG and total IgE during S. mansoni infection is unaffected by deletion of IL-4R on CD11c + cells
Depletion of CD11c + DCs has been shown to affect B cell development during S. mansoni infection. 6 To investigate the impact of impairing IL-4R signaling specifically on CD11c + cells, Ag-specific Ab isotypes (IgG1, IgG2a, and IgG2b) and total IgE were determined by ELISA. Abrogation of IL-4R expression on CD11c + cells did not impair isotype class switching, as shown by the similar levels of

CD11c cre IL-4R −/lox mice show reduced perioval liver fibrogranulomatous inflammation after S. mansoni infection
A major site for egg entrapment during schistosomiasis is the liver, where a considerable part of the fibrogranulomatous inflammation takes place. 24 Therefore, we investigated the contribution of (D-F) Liver was homogenized, protein content determined using Pierce BCA assay kit, cytokines detected by ELISA, and normalized to tissue weight. (G) Formalin-fixed liver sections (100×) stained with H&E for morphological analysis or CAB for collagen content. Data are representative of 2 independent experiments; n = 4-6 mice. *P < 0.05, **P < 0.01, and ***P < 0.001 using two-tailed Mann-Whitney nonparametric Student's t-test IL-4R -expressing CD11c + cells in modulating granulomatous liver inflammation in mice. Interestingly, abrogation of IL-4R signaling on CD11c + cells resulted in smaller granulomas (Fig. 4A), reduced liver fibrosis, indicated by reduced hydroxyproline concentrations (Fig. 4B), and normal serum aspartate transaminase (AST) levels compared to infected IL-4R −/lox littermate control mice (Fig. 4C). We found comparable egg burdens in the liver and small intestines in both strains of infected mice (Supplementary Fig. S4), suggesting that the progression of infection was similar. Moreover, CD11c cre IL-4R −/lox mice had reduced levels of tissue IL-10 and IFN-and unaltered levels of tissue IL-33 compared to littermate control mice (Fig. 4D-F). Histological examination of liver sections confirmed that CD11c cre IL-4R −/lox mice developed smaller granulomas around parasite eggs than the littermate control mice (Fig. 4F). We found comparable levels of IL-10, IFN-, and IL-33 in the small intestines between mutant mice (Supplementary Fig. S5A-C). Finally, we observed comparable gut pathology in both the infected mutant mice during acute schistosomiasis ( Supplementary Fig. S5D). Therefore, these data suggest that IL-4R -expressing CD11c + cells partake in driving fibrogranulomatous perioval liver inflammation during schistosomiasis.  Fig. S6C and D). We also investigated whether impairing IL-4R expression on DCs altered their ability to produce IL-12p70 and IL-10 production. Intracellular flow cytometric analysis revealed that CD11c + MHCII + cells from CD11c cre IL-4R −/lox mice exhibited a trend toward reduced production of IL-12p70 and IL-10 compared to littermate control mice, although the differences were not significant ( Supplementary Fig. S6E). Despite the increased frequency of eosinophils (CD11c − Siglec-F + ; Supplementary   Fig. S6F), the proportions of neutrophils ( Supplementary Fig. S6H) as well as the number of both eosinophils (Supplementary Fig. S6G) and neutrophils (Fig. S6I) were comparable between both infected strains in the liver. Together, these data suggest that deleting IL-4R expression on CD11c + cells may affect their activation status during S. mansoni infection.

DISCUSSION
CD11c-expressing cells include DCs, which are important Ag presenters required to educate and activate naïve T cells during infection. 1,3 Prior studies aimed at elucidate the contribution of CD11c + DCs on the development of sufficient Th2 immune responses during S. mansoni infection, relied on depleting CD11c + cells by administering diphtheria toxin in CD11c-diphtheria toxin receptor mice. 6 This study demonstrated that depleting CD11c + cells tilted the immune balance toward Th1 immunity by up-regulating the production of IFN-, while simultaneously impairing the production of Th2 cytokines after synchronous S. mansoni eggs challenge in the footpad or during active infection. 6 A subsequent study from the same group showed that CD11c + DCs respond to IL-4 by up-regulating multiple markers that were associated with alternative activation of macrophage and that DCs expressing IL-4R driving optimal production of IFN-in both Th1 and Th2 settings, via a feedback loop in which IL-4 further instructs DC to produce IL-12. 7 We have previously shown that IL-4R -expressing DCs are crucial for rendering host protection to L. major infection 12 and house dust mite induced allergic asthma. 29 In this study, we report on the direct contribution of IL-4R -expressing CD11c + cells in orchestrating optimal cellular immunity to S. mansoni infection and modulating tissue granulomatous pathology in vivo.
Previously, we found that T cells of CD11c cre IL-4R −/lox mice show the same ability to proliferate and differentiate as T cells of littermate control mice ex vivo. 29  disease setting. 6,7,29 Therefore, these data suggest that CD11c + cells are crucial for driving optimal cellular immunity during schistosomiasis in mice. We observed a more pronounced effect on cytokine production in CD11c cre IL-4R −/lox mice after synchronous eggs challenge in the footpads compared to an active S. mansoni infection. Together, these data suggest that CD11c + cells play a key role early during infection to educate T cells to produce both Th1 and Th2 cytokines during  6 suggesting that IL-4R expression on CD11c + cells is particularly important in driving these responses. The expression of MHCII on B cells was shown to be essential for the development of sufficient cell-mediated immunity to Heligomosomoides polygyrus infection using mixed bone marrow chimera mice that lacked specific expression of MHCII on B cells. 30 Moreover, the protective effect offered by B cells expressing MHCII was shown to be independent of Ab production. 30 In our study, too, Ab responses were unaffected in mice lacking CD11c cells expressing IL-4R , yet our data suggest that IL-4R -expressing CD11c + cells are important for optimal B cell responses in the gut-draining lymphatic tissue.
As expected, infected CD11c cre IL-4R −/lox mice had reduced tissue granulomatous pathology indicated by reduced granuloma size and fibrosis compared to littermate control mice, in line with the observed impaired Th2 immunity. It is an established fact in the literature that Th2 cytokines, particularly IL-4 and IL-13, are critical for liver granuloma formation. 18,23,24,[31][32][33][34] Moreover, IL-13 has been shown to be a master cytokine mediating fibrogenesis in tandem with other type 2 cytokines during schistosomiasis. 35,36 In support of this, we consistently found significantly reduced production of IL-13 during both synchronous S. mansoni eggs footpad challenge and active infection in Dectin-1, 7 markers previously associated with alternatively activated macrophages. 27,28 Expression of Relm-by DCs is also required to prime expression of IL-10 and IL-13 by CD4 + T cells in a Th2 setting. 7 In contrast, lymph node DCs from CD11c cre IL-4R −/lox mice suffering from cutaneous leishmanisis caused by L. major infection showed a shift toward alternative activation with reduced expression of iNOS but increased expression of Arginase-1 compared to infected littermate control mice. 12 CD11c cre IL-4R −/lox BALB/c mice were found to be hypersusceptible to L. major infection due to up-regulated Th2 cytokine responses, increased type 2 Ab production, and impaired classical activation of macrophages. 12 This was due to reduced IL-12 and increased IL-10 production by IL-4R -unresponsive DCs from L. majorinfected mice. 12 These results confirmed and extended IL-4 instruction of DCs to produce IL-12p70 in the presence of LPS or CpG in vitro or in vivo during L. major infection in BALB/c mice. 8 In this study, we observed a trend toward reduced IL-10 and IL-12 expression by CD11c + MHCII + cells suggesting that the instruction theory may be irrelevant in the context of helminth infections.
In summary, we found that deleting IL-4R expression on CD11c + cells down-modulates fibrogranulomatous perioval liver inflammation.
This coincided with reduced production of Th2 cytokines particularly IL-13, reduced number of CD4 + T cells, and effector CD4 + T cells draining into MLN in infected CD11c cre IL-4R −/lox mice compared to control mice. Moreover, B cell responses were significantly abrogated in mice lacking IL-4R expression on CD11c + cells compared to control mice, suggesting that IL-4R -expressing CD11c + cells play an important role in the development of adaptive immune responses during schistosomiasis. As CD11c can be expressed not only on DCs but also on various other myeloid cell subsets during inflammatory diseases, identifying which CD11c + myeloid cells are involved in driving optimal adaptive immune responses and supporting liver fibrogranulomatous inflammation during schistosomiasis would be useful for developing novel control strategies.

Ethics statement
This study was performed in strict accordance with the recommenda-

Footpad model
Mice were challenged by injection of 2,500 S. mansoni eggs into the right hind footpad and killed 7 d postchallenge. S. mansoni eggs were purchased from the Theodor Bilharz Research Institute (Schistosome Biological Supply Center, Egypt) and stored at -80 • C until use.
The integrity and viability of the eggs were evaluated using a light microscope prior to use.
Staining specificity was verified by appropriate isotype matched Ab controls. Cells were acquired on a FACSCalibur machine (BD Immunocytometry system, San Jose, CA) and data were analyzed using Flowjo software (Treestar, Ashland, OR).

Cell preparation and ex vivo restimulation
Single cell suspensions were prepared by pressing the draining lymph nodes through 70 M cell strainers. Cells were resuspended in complete IMDM (Gibco Walthan, MA, USA) supplemented with 10% FCS (Gibco) and penicillin and streptomycin (100 U/ml and 100 g/ml, Gibco). The cells were cultured at 2 × 10 6 cells/ml in 48-well plates coated with -CD3 (20 g/ml) or soluble egg Ag (SEA, 20 g/ml) and incubated at 37 • C in a humidified atmosphere containing 5% CO 2 .
Supernatants were collected after 72 h and cytokines were measured by ELISA. Quantities of IL-4, IL-5, IL-10, IL-13, and IFN-were measured by sandwich ELISA as previously described. 37

Enzyme-linked immunosorbent assays
Cytokines in supernatants were measured by sandwich ELISA as previously described. 37 For detection of Ab isotypes, blood was collected in serum separator tubes (BD Bioscience, San Diego, CA) and serum was separated by centrifugation at 8,000 × g for 10 min at 4 • C. Titers of SEA-specific IgG1, IgG2a, IgG2b, and total IgE were determined as previously described. 37

Hydroxyproline assay
Hydroxyproline content as a measure of collagen production was determined using a modified protocol. 38 Briefly, weighed liver sam-ples were hydrolyzed and added to a 40 mg Dowex/Norit mixture. The supernatants were neutralized with 1% phenolphthalein and titrated against 10 M NaOH. An aliquot was mixed with isopropanol and added to chloramine-T/citrate buffer solution (pH 6.5). Erlich's reagent was added and absorbance was read at 570 nm. Hydroxyproline levels were calculated using 4-hydroxy-L-proline (Calbiochem, San Diego, CA, USA) as a standard, and results were expressed as moles hydroxyproline per weight of tissue that contained 10 4 eggs.

AST measurement
Serum was sent to the Department of Surgery at the University of Cape Town (Histology facility) to measure AST levels.

Histology
Liver and gut samples were fixed in 4% (v/v) formaldehyde in PBS, embedded in wax, and processed. Sections (5-7 m) were stained with hematoxylin and eosin (H&E) and analine blue solution (CAB) and counterstained with Wegert's hematoxylin for collagen staining. Micrographs of liver granuloma were captured using a Nikon 5.0 mega pixel color digital camera (DCT DS-SMc). The diameter of each granuloma containing a single egg was measured with the ImageJ 1.34 software.
An average of 25 granulomas per mouse were included in the analyses.

Statistics
Statistical analysis was conducted using GraphPad Prism 4 software.