IL-4 receptor alpha blockade dampens allergic inflammation and upregulates IL-17A expression to promote Saureus clearance in antigen sensitized mouse skin.

BACKGROUND: Skin colonization with Staphylococcus aureus aggravates atopic dermatitis and exaggerates allergic skin inflammation in mice. IL-4 receptor α (IL-4Rα) blockade is beneficial in atopic dermatitis and reduces Saureus skin colonization through unknown mechanisms. The cytokine IL-17A restrains Saureus growth. OBJECTIVES: This study sought to examine the effect of IL-4Rα blockade on Saureus colonization at sites of allergic skin inflammation in mice and determine the mechanism involved. METHODS: BALB/c mice were epicutaneously sensitized with ovalbumin (OVA). Immediately after, PSVue 794-labeled S aureus strain SF8300 or saline was applied and a single dose of anti-IL-4Rα blocking antibody, a mixture of anti-IL-4Rα and anti-IL-17A blocking antibodies, or IgG isotype controls were administered intradermally. Saureus load was assessed 2 days later by in vivo imaging and enumeration of colony forming units. Skin cellular infiltration was examined by flow cytometry and gene expression by quantitative PCR and transcriptome analysis. RESULTS: IL-4Rα blockade decreased allergic skin inflammation in OVA-sensitized skin, as well as in OVA-sensitized and Saureus-exposed skin, evidenced by significantly decreased epidermal thickening and reduced dermal infiltration by eosinophils and mast cells. This was accompanied by increased cutaneous expression of Il17a and IL-17A-driven antimicrobial genes with no change in Il4 and Il13 expression. IL-4Rα blockade significantly decreased Saureus load in OVA-sensitized and S aureus-exposed skin. IL-17A blockade reversed the beneficial effect of IL-4Rα blockade on Saureus clearance and reduced the cutaneous expression of IL-17A-driven antimicrobial genes. CONCLUSIONS: IL-4Rα blockade promotes Saureus clearance from sites of allergic skin inflammation in part by enhancing IL-17A expression.

Honing-in antigen-specific cells during antibody discovery: a user-friendly process to mine a deeper repertoire.

Immunization based antibody discovery is plagued by the paucity of antigen-specific B cells. Identifying these cells is akin to finding needle in a haystack. Current and emerging technologies while effective, are limited in terms of capturing the antigen-specific repertoire. We report on the bulk purification of antigen-specific B-cells and the benefits it offers to various antibody discovery platforms. Using five different antigens, we show hit rates of 51-88%, compared to about 5% with conventional methods. We also show that this purification is highly efficient with loss of only about 2% antigen specific cells. Furthermore, we compared clones in which cognate chains are preserved with those from display libraries in which chains either from total B cells (TBC) or antigen-specific B cells (AgSC) underwent combinatorial pairing. We found that cognate chain paired clones and combinatorial clones from AgSC library had higher frequency of functional clones and showed greater diversity in sequence and paratope compared to clones from the TBC library. This antigen-specific B-cell selection technique exemplifies a process improvement with reduced cycle time and cost, by removing undesired clones prior to screening and increasing the chance of capturing desirable and rare functional clones in the repertoire.

IL-4-BATF signaling directly modulates IL-9 producing mucosal mast cell (MMC9) function in experimental food allergy.

BACKGROUND: This study group has previously identified IL-9-producing mucosal mast cell (MMC9) as the primary source of IL-9 to drive intestinal mastocytosis and experimental IgE-mediated food allergy. However, the molecular mechanisms that regulate the expansion of MMC9s remain unknown. OBJECTIVES: This study hypothesized that IL-4 regulates MMC9 development and MMC9-dependent experimental IgE-mediated food allergy. METHODS: An epicutaneous sensitization model was used and bone marrow reconstitution experiments were performed to test the requirement of IL-4 receptor α (IL-4Rα) signaling on MMC9s in experimental IgE-mediated food allergy. Flow cytometric, bulk, and single-cell RNA-sequencing analyses on small intestine (SI) MMC9s were performed to illuminate MMC9 transcriptional signature and the effect of IL-4Rα signaling on MMC9 function. A bone marrow-derived MMC9 culture system was used to define IL-4-BATF signaling in MMC9 development. RESULTS: Epicutaneous sensitization- and bone marrow reconstitution-based models of IgE-mediated food allergy revealed an IL-4 signaling-dependent cell-intrinsic effect on SI MMC9 accumulation and food allergy severity. RNA-sequencing analysis of SI-MMC9s identified 410 gene transcripts reciprocally regulated by IL-4 signaling, including Il9 and Batf. Insilico analyses identified a 3491-gene MMC9 transcriptional signature and identified 2 transcriptionally distinct SI MMC9 populations enriched for metabolic or inflammatory programs. Employing an in vitro MMC9-culture model system showed that generation of MMC9-like cells was induced by IL-4 and this was in part dependent on BATF. CONCLUSIONS: IL-4Rα signaling directly modulates MMC9 function and exacerbation of experimental IgE-mediated food allergic reactions. IL-4Rα regulation of MMC9s is in part BATF-dependent and occurs via modulation of metabolic transcriptional programs.

Deletion of ΔdblGata motif leads to increased predisposition and severity of IgE-mediated food-induced anaphylaxis response.

BACKGROUND: Previous studies have revealed an important role for the transcription factor GATA-1 in mast cell maturation and degranulation. However, there have been conflicting reports with respect to the requirement of GATA-1 function in mast cell dependent inflammatory processes. Herein, we examine the requirement of GATA-1 signaling in mast cell effector function and IgE-mast cell-dependent anaphylaxis. OBJECTIVE: To study the requirement of GATA-1 dependent signaling in the development and severity of IgE-mast cell-dependent anaphylaxis in mice. METHODS: Wild type (Balb/c) and mutant ΔdblGata (Balb/c) mice were employed to study the role of GATA-1 signaling in in vitro IgE-mediated activation of bone marrow derived mast cells (BMMCs). Murine models of passive IgE-mediated and oral antigen-induced IgE-mediated anaphylaxis were employed in mice. Frequency of steady state mast cells in various tissues (duodenum, ear, and tongue), peritoneal cavity, and clinical symptoms (diarrhea, shock, and mast cell activation) and intestinal Type 2 immune cell analysis including CD4+ Th2 cells, type 2 innate lymphoid cells (ILC2), and IL-9 secreting mucosal mast cells (MMC9) were assessed. RESULTS: In vitro analysis revealed that ΔdblGata BMMCs exhibit a reduced maturation rate, decreased expression of FcεRIα, and degranulation capacity when compared to their wildtype (WT) counterparts. These in vitro differences did not impact tissue resident mast cell numbers, total IgE, and susceptibility to or severity of IgE-mediated passive anaphylaxis. Surprisingly, ΔdblGata mice were more susceptible to IgE-mast cell-mediated oral antigen induced anaphylaxis. The increased allergic response was associated with increased Type 2 immunity (antigen-specific IgE, and CD4+ TH2 cells), MMC9 cells and small intestine (SI) mast cell load. CONCLUSION: Diminished GATA-1 activity results in reduced in vitro mast cell FcεRIα expression, proliferation, and degranulation activity. However, in vivo, diminished GATA-1 activity results in normal homeostatic tissue mast cell levels and increased antigen-induced CD4+ Th2 and iMMC9 cell levels and heightened IgE-mast cell mediated reactions.

IL-13-induced intestinal secretory epithelial cell antigen passages are required for IgE-mediated food-induced anaphylaxis.

BACKGROUND: Food-induced anaphylaxis (FIA) is an IgE-dependent immune response that can affect multiple organs and lead to life-threatening complications. The processes by which food allergens cross the mucosal surface and are delivered to the subepithelial immune compartment to promote the clinical manifestations associated with food-triggered anaphylaxis are largely unexplored. OBJECTIVE: We sought to define the processes involved in the translocation of food allergens across the mucosal epithelial surface to the subepithelial immune compartment in FIA. METHODS: Two-photon confocal and immunofluorescence microscopy was used to visualize and trace food allergen passage in a murine model of FIA. A human colon cancer cell line, RNA silencing, and pharmacologic approaches were used to identify the molecular regulation of intestinal epithelial allergen uptake and translocation. Human intestinal organoid transplants were used to demonstrate the conservation of these molecular processes in human tissues. RESULTS: Food allergens are sampled by using small intestine (SI) epithelial secretory cells (termed secretory antigen passages [SAPs]) that are localized to the SI villous and crypt region. SAPs channel food allergens to lamina propria mucosal mast cells through an IL-13-CD38-cyclic adenosine diphosphate ribose (cADPR)-dependent process. Blockade of IL-13-induced CD38/cADPR-dependent SAP antigen passaging in mice inhibited induction of clinical manifestations of FIA. IL-13-CD38-cADPR-dependent SAP sampling of food allergens was conserved in human intestinal organoids. CONCLUSION: We identify that SAPs are a mechanism by which food allergens are channeled across the SI epithelium mediated by the IL-13/CD38/cADPR pathway, regulate the onset of FIA reactions, and are conserved in human intestine.

C5a receptor 1-/- mice are protected from the development of IgE-mediated experimental food allergy.

BACKGROUND: Food-induced anaphylaxis is a serious allergic reaction caused by Fcε-receptor activation on mast cells (MCs). The exact mechanisms breaking oral tolerance and the effector pathways driving food allergy remain elusive. As complement is activated in food-induced anaphylaxis, we aimed to assess the role of C5a in disease pathogenesis. METHODS: Oral antigen-induced food-induced anaphylaxis was induced in BALB/c wild-type (wt) and C5ar1-/- mice. Readouts included diarrhea development, changes in rectal temperature, hematocrit, antigen-specific serum IgE, MCPT-1, and intestinal MC numbers, as well as FcεR1-mediated MC functions including C5a receptor 1 (C5aR1) regulation. Further, histamine-mediated hypothermia and regulation of endothelial tight junctions were determined. RESULTS: Repeated oral OVA challenge resulted in diarrhea, hypothermia, increased hematocrit, high OVA-specific serum IgE, and MCPT-1 levels in wt mice. Male C5ar1-/- mice were completely whereas female C5ar1-/- mice were partially protected from anaphylaxis development. Serum MCPT-1 levels were reduced gender-independent, whereas IgE levels were reduced in male but not in female C5ar1-/- mice. Mechanistically, IgE-mediated degranulation and IL-6 production from C5ar1-/- BMMCs of both sexes were significantly reduced. Importantly, FcεR1 cross-linking strongly upregulated C5aR1 MC expression in vitro and in vivo. Finally, C5ar1-/- male mice were largely protected from histamine-induced hypovolemic shock, which was associated with protection from histamine-induced barrier dysfunction in vitro following C5aR targeting. CONCLUSIONS: Our findings identify C5aR1 activation as an important driver of IgE-mediated food allergy through regulation of allergen-specific IgE production, FcεR1-mediated MC degranulation, and histamine-driven effector functions preferentially in male mice.

Prevention of food allergy development and suppression of established food allergy by neutralization of thymic stromal lymphopoietin, IL-25, and IL-33.

BACKGROUND: Food allergy (FA) is an increasing problem that has no approved treatment. The pro-TH2 cytokines IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) are associated with FA, and mAbs to these cytokines are reported to suppress murine FA development. OBJECTIVE: We sought to determine whether anti-pro-TH2 cytokine mAbs can block both FA maintenance and induction. METHODS: IgE-mediated FA was induced in BALB/c mice by oral gavage with medium-chain triglycerides (MCTs) plus egg white (EW) and was characterized by increased numbers of lamina propria TH2 cells, mast cells, and eosinophils, shock (hypothermia), mast cell degranulation (increased serum mouse mast cell protease 1), increased serum IgG1 anti-EW and IgE levels, and increased IL-4 and IL-13 secretion after MCT/EW challenge. Mice were injected with anti-IL-25, IL-33 receptor, and/or TSLP mAbs before initial oral gavage with MCT/EW to suppress FA development; treatment with the same mAbs was initiated after FA development to suppress established FA. RESULTS: Injection of an mAb to IL-25, IL-33 receptor, or TSLP strongly inhibited FA development. No single mAb to a pro-TH2 cytokine could suppress established FA, and optimal FA suppression required treatment with a cocktail of all 3 anti-pro-TH2 mAbs. Treatment with the 3-mAb cocktail during initial MCT/EW immunization induced EW tolerance. CONCLUSION: All of the pro-TH2 cytokines are required to induce our model of FA, whereas any pro-TH2 cytokine can maintain established FA. Pro-TH2 cytokines prevent oral tolerance. Combined treatment with antagonists to all 3 pro-TH2 cytokines or with an inhibitor of pro-TH2 cytokine production might be able to suppress established human FA.

ILC2s activated by IL-25 promote antigen-specific Th2 and Th9 functions that contribute to the control of Trichinella spiralis infection.

IL-25, an IL-17 family cytokine, derived from epithelial cells was shown to regulate Th2- and Th9-type immune responses. We previously reported that IL-25 was important in promoting efficient protective immunity against T. spiralis infection; however, the cellular targets of IL-25 to elicit type-2 immunity during infection have not yet been addressed. Here, we investigated IL-25-responding cells and their involvement in mediating type-2 immune response during T. spiralis infection. ILC2 and CD4+ Th2 cells residing in the gastrointestinal tract of T. spiralis infected mice were found to express high levels of surface interleukin-17 receptor B (IL-17RB), a component of the IL-25 receptor. Following T. spiralis infection, activated ILC2s upregulated surface MHCII expression and enhanced capacity of effector T helper cell in producing antigen-specific Th2 and Th9 cytokines through MHCII-dependent interactions. Reciprocally, lack of CD4+ T helper cells impaired ILC2 function to produce type 2-associated cytokines in responding to IL-25 during T. spiralis infection. Furthermore, mice deficient in IL-17RB showed markedly reduced ILC2 numbers and antigen-specific Th2 and Th9 cytokine production during T. spiralis infection. The Il17rb-/- mice failed to mount effective antigen specific Th2 and Th9 functions resulting in diminished goblet cell and mast cell responses, leading to delayed worm expulsion in the intestines and muscles. Thus, our data indicated that ILC2s and CD4+ Th2 cells are the predominant cellular targets of IL-25 following T. spiralis infection and their collaborative interactions may play a key role in mounting effective antigen-specific Th2 and Th9 cytokine responses against T. spiralis infection.

IL-9-producing cells in the development of IgE-mediated food allergy.

Food allergy is a harmful immune reaction driven by uncontrolled type 2 immune responses. Considerable evidence demonstrates the key roles of mast cells, IgE, and TH2 cytokines in mediating food allergy. However, this evidence provides limited insight into why only some, rather than all, food allergic individuals are prone to develop life-threatening anaphylaxis. Clinical observations suggest that patients sensitized to food through the skin early in life may later develop severe food allergies. Aberrant epidermal thymic stromal lymphopoietin and interleukin (IL) 33 production and genetic predisposition can initiate an allergic immune response mediated by dendritic cells and CD4+TH2 cells in inflamed skin. After allergic sensitization, intestinal IL-25 and food ingestion enhance concerted interactions between type 2 innate lymphoid cells (ILC2s) and CD4+TH2 cells, which perpetuate allergic reactions from the skin to the gut. IL-4 and cross-linking of antigen/IgE/FcεR complexes induce emigrated mast cell progenitors to develop into the multi-functional IL-9-producing mucosal mast cells, which produce prodigious amounts of IL-9 and mast cell mediators to drive intestinal mastocytosis in an autocrine loop. ILC2s and TH9 cells may also serve as alternative cellular sources of IL-9 to augment the amplification of intestinal mastocytosis, which is the key cellular checkpoint in developing systemic anaphylaxis. These findings provide a plausible view of how food allergy develops and progresses in a stepwise manner and that atopic signals, dietary allergen ingestion, and inflammatory cues are fundamental in promoting life-threatening anaphylaxis. This information will aid in improving diagnosis and developing more effective therapies for food allergy-triggered anaphylaxis.

The dendritic cell-T helper 17-macrophage axis controls cholangiocyte injury and disease progression in murine and human biliary atresia.

Biliary atresia (BA) is a fibroinflammatory obstruction of the extrahepatic biliary tree in neonates. While intrahepatic bile duct proliferation is universal at diagnosis, bile duct paucity develops later. We hypothesized that polarized T helper lymphocyte responses orchestrate progression of intrahepatic biliary injury in this disease. Interleukin 17A (IL-17A)-green fluorescent protein, cluster of differentiation 11c (CD11c)/diphtheria toxin receptor, and IL-17 receptor A-/- mice were used to examine T-lymphocyte polarization, inflammatory leukocyte recruitment, and biliary injury in rhesus rotavirus-induced BA. Multiparameter flow cytometry and automated image analysis of immunostaining were applied to liver tissue samples from infants with BA. In the mouse model, activated CD4+ lymphocytes started to emerge in the liver on day 8 after viral challenge, while innate immune responses were waning. Plasma IL-17A levels rose concomitantly with hepatic accumulation of T helper 17 lymphocytes and myeloid dendritic cells. Targeted depletion of CD11c+ dendritic cells diminished hepatic IL-17A production and ameliorated intrahepatic bile duct injury. Recombinant IL-17A induced expression of chemokine (C-C motif) ligand 2 in neonatal cholangiocytes in vitro, and blockade of the corresponding chemokine (C-C motif) receptor 2 reduced recruitment of inflammatory macrophages to the liver in vivo. Genetic disruption of IL-17A signaling was associated with down-regulation of hepatic Ccl2/Ccr2 messenger RNA expression, reduced infiltration of the liver with inflammatory Ly6Chi macrophages, and improved survival. In the liver of infants with BA, cholangiocytes were found to express IL-17 receptor A, and the prevalence of IL-17A+ cells was positively correlated with the degree of CD68+ macrophage infiltration at diagnosis. Hepatic CD4+ lymphocytes were chief producers of IL-17A in patients with progressive disease undergoing liver transplantation. CONCLUSION: These findings identify the dendritic cell-T helper 17-macrophage axis as a target for the development of strategies to block progression of intrahepatic bile duct injury in patients with BA. (Hepatology 2017;65:174-188).

Developing food allergy: a potential immunologic pathway linking skin barrier to gut.

Immunoglobulin E (IgE)-mediated food allergy is an adverse reaction to foods and is driven by uncontrolled type-2 immune responses. Current knowledge cannot explain why only some individuals among those with food allergy are prone to develop life-threatening anaphylaxis. It is increasingly evident that the immunologic mechanisms involved in developing IgE-mediated food allergy are far more complex than allergic sensitization. Clinical observations suggest that patients who develop severe allergic reactions to food are often sensitized through the skin in early infancy. Environmental insults trigger epidermal thymic stromal lymphopoietin and interleukin-33 (IL-33) production, which endows dendritic cells with the ability to induce CD4 +TH2 cell-mediated allergic inflammation. Intestinal IL-25 propagates the allergic immune response by enhancing collaborative interactions between resident type-2 innate lymphoid cells and CD4 +TH2 cells expanded by ingested antigens in the gastrointestinal tract. IL-4 signaling provided by CD4 +TH2 cells induces emigrated mast cell progenitors to become multi-functional IL-9-producing mucosal mast cells, which then expand greatly after repeated food ingestions. Inflammatory cytokine IL-33 promotes the function and maturation of IL-9-producing mucosal mast cells, which amplify intestinal mastocytosis, resulting in increased clinical reactivity to ingested food allergens. These findings provide the plausible view that the combinatorial signals from atopic status, dietary allergen ingestions, and inflammatory cues may govern the perpetuation of allergic reactions from the skin to the gut and promote susceptibility to life-threatening anaphylaxis. Future in-depth studies of the molecular and cellular factors composing these stepwise pathways may facilitate the discovery of biomarkers and therapeutic targets for diagnosing, preventing, and treating food allergy.

Intratracheal myriocin enhances allergen-induced Th2 inflammation and airway hyper-responsiveness.

INTRODUCTION: Ceramide is the central substrate of sphingolipid metabolism and plays a key role in cellular signal transduction pathways, regulating apoptosis, differentiation, and chemotaxis. Alterations in airway ceramide levels are observed in multiple pulmonary diseases and recent human genetic association studies have linked dysregulation of sphingolipid regulatory genes with asthma pathogenesis. METHODS: Utilizing myriocin, a potent inhibitor of sphingolipid synthesis, we evaluated the immune regulatory role of de novo ceramide generation in vitro and in vivo. Intratracheal myriocin was administered alone or during house dust mite sensitization (HDM) of BALB/C mice and airway hyper-responsiveness (AHR) was evaluated by invasive plethysmography followed by bronchial lavage (BAL) cytology and cytokine quantification. RESULTS: Myriocin inhibits and HDM exposure activates de novo ceramide synthesis in bone marrow-derived dendritic cells. Mice receiving intratracheal myriocin developed a mild airway neutrophilic infiltrate without inducing a significant increase in AHR. CXCL1 was elevated in the BAL fluid of myriocin-treated mice while the neutrophilic chemotactic factors anaphylatoxin C5a, leukotriene B4, and IL-17 were unaffected. HDM treatment combined with myriocin led to a dramatic enhancement of AHR (63% increase over HDM alone, p < 0.001) and increased granulocyte pulmonary infiltrates versus HDM or myriocin alone. Elevated Th2 T cell counts and Th2 cytokines/chemokines (IL5, IL13, CCL17) were observed in mice treated with combined HDM/myriocin compared to HDM alone. Myriocin-treated pulmonary CD11c+ cells stimulated with HDM secreted significantly more CXCL1 than cells stimulated with HDM alone while HDM stimulated airway epithelial cells showed no change in CXCL1 secretion following myriocin treatment. CONCLUSIONS: Intratracheal myriocin, likely acting via ceramide synthesis inhibition, enhances allergen-induced airway inflammation, granulocyte and Th2 lymphocyte recruitment, and allergen-induced AHR. Sphingolipid pathways may represent novel targets for possible future anti-inflammatory asthma medications.

IL-25 and CD4(+) TH2 cells enhance type 2 innate lymphoid cell-derived IL-13 production, which promotes IgE-mediated experimental food allergy.

BACKGROUND: Food-mediated allergic reactions have emerged as a major health problem. The underlying mechanisms that promote uncontrolled type 2 immune responses to dietary allergens in the gastrointestinal tract remain elusive. OBJECTIVE: We investigated whether altering IL-25 signaling enhances or attenuates allergic responses to food allergens. METHODS: Mice of an IL-25 transgenic mouse line (iIL-25Tg mice), which constitutively overexpress intestinal IL-25, and Il17rb(-/-) mice, in which Il17rb gene expression is disrupted, were sensitized and gavage fed with ovalbumin (OVA). We assessed symptomatic characteristics of experimental food allergy, including incidence of diarrhea, incidence of hypothermia, intestinal TH2 immune response, and serum OVA-specific IgE and mast cell protease 1 production. RESULTS: Rapid induction of Il25 expression in the intestinal epithelium preceded onset of the anaphylactic response to ingested OVA antigen. iIL-25Tg mice were more prone and Il17rb(-/-) mice were more resistant to experimental food allergy. Resident intestinal type 2 innate lymphoid cells (ILC2s) were identified as the major producers of IL-5 and IL-13 in response to IL-25. Reconstituting irradiated wild-type mice with Rora(-/-) or Il17rb(-/-) bone marrow resulted in a deficiency or dysfunction of the ILC2 compartment, respectively, and resistance to experimental food allergy. Repeated intragastric antigen challenge induced a significant increase in numbers of CD4(+) TH2 cells, which enhance IL-25-stimulated IL-13 production by ILC2s ex vivo and in vivo. Finally, reconstituted IL-13-deficient ILC2s had reduced capability to promote allergic inflammation, resulting in increased resistance to experimental food allergy. CONCLUSION: IL-25 and CD4(+) TH2 cells induced by ingested antigens enhance ILC2-derived IL-13 production, thereby promoting IgE-mediated experimental food allergy.

Induction of Interleukin-9-Producing Mucosal Mast Cells Promotes Susceptibility to IgE-Mediated Experimental Food Allergy.

Experimental IgE-mediated food allergy depends on intestinal anaphylaxis driven by interleukin-9 (IL-9). However, the primary cellular source of IL-9 and the mechanisms underlying the susceptibility to food-induced intestinal anaphylaxis remain unclear. Herein, we have reported the identification of multifunctional IL-9-producing mucosal mast cells (MMC9s) that can secrete prodigious amounts of IL-9 and IL-13 in response to IL-33, and mast cell protease-1 (MCPt-1) in response to antigen and IgE complex crosslinking, respectively. Repeated intragastric antigen challenge induced MMC9 development that required T cells, IL-4, and STAT6 transcription factor, but not IL-9 signals. Mice ablated of MMC9 induction failed to develop intestinal mastocytosis, which resulted in decreased food allergy symptoms that could be restored by adoptively transferred MMC9s. Finally, atopic patients that developed food allergy displayed increased intestinal expression of Il9- and MC-specific transcripts. Thus, the induction of MMC9s is a pivotal step to acquire the susceptibility to IgE-mediated food allergy.

Collaborative interactions between type 2 innate lymphoid cells and antigen-specific CD4+ Th2 cells exacerbate murine allergic airway diseases with prominent eosinophilia.

Type-2 innate lymphoid cells (ILC2s) and the acquired CD4(+) Th2 and Th17 cells contribute to the pathogenesis of experimental asthma; however, their roles in Ag-driven exacerbation of chronic murine allergic airway diseases remain elusive. In this study, we report that repeated intranasal rechallenges with only OVA Ag were sufficient to trigger airway hyperresponsiveness, prominent eosinophilic inflammation, and significantly increased serum OVA-specific IgG1 and IgE in rested mice that previously developed murine allergic airway diseases. The recall response to repeated OVA inoculation preferentially triggered a further increase of lung OVA-specific CD4(+) Th2 cells, whereas CD4(+) Th17 and ILC2 cell numbers remained constant. Furthermore, the acquired CD4(+) Th17 cells in Stat6(-/-)/IL-17-GFP mice, or innate ILC2s in CD4(+) T cell-ablated mice, failed to mount an allergic recall response to OVA Ag. After repeated OVA rechallenge or CD4(+) T cell ablation, the increase or loss of CD4(+) Th2 cells resulted in an enhanced or reduced IL-13 production by lung ILC2s in response to IL-25 and IL-33 stimulation, respectively. In return, ILC2s enhanced Ag-mediated proliferation of cocultured CD4(+) Th2 cells and their cytokine production, and promoted eosinophilic airway inflammation and goblet cell hyperplasia driven by adoptively transferred Ag-specific CD4(+) Th2 cells. Thus, these results suggest that an allergic recall response to recurring Ag exposures preferentially triggers an increase of Ag-specific CD4(+) Th2 cells, which facilitates the collaborative interactions between acquired CD4(+) Th2 cells and innate ILC2s to drive the exacerbation of a murine allergic airway diseases with an eosinophilic phenotype.

Loss of IL-4Rα-mediated PI3K signaling accelerates the progression of IgE/mast cell-mediated reactions.

Clinical and experimental evidence indicate that polymorphisms within the interleukin 4 (IL-4) receptor (IL-4R) chain are sufficient for altered strength of IL-4/IL-13 signaling, leading to an exaggerated allergic inflammatory response and increase susceptibility to allergic phenotypes. In the present study, we show that ablation of IL-4Rα-induced phosphatidylinositol 3-kinase (PI3K) activating signal by germline point mutation within the IL-4Rα motif (Y500F) did not alter susceptibility to IgE-mediated, food-induced experimental anaphylaxis. Moreover, diarrhea occurrence, antigen-specific IgE and intestinal mastocytosis were comparable between WT and IL-4Rα(Y500F) mice. However, mice unable to stimulate IL-4Rα-mediated PI3K signaling had accelerated disease progression. Notably, the accelerated anaphylactic response was associated with more rapid histamine-induced hypovolemia. Mechanistic in vitro and in vivo analyses revealed that endothelial IL-4Rα PI3K signaling negatively regulates the histamine-induced endothelial leak response. These results define an unanticipated role for IL-4Rα-mediated PI3K signaling in negative regulation of IgE-mediated anaphylactic reactions.

Cryptococcus gattii infection dampens Th1 and Th17 responses by attenuating dendritic cell function and pulmonary chemokine expression in the immunocompetent hosts.

Cryptococcal infections are primarily caused by two related fungal species: Cryptococcus neoformans and Cryptococcus gattii. It is well known that C. neoformans generally affects immunocompromised hosts; however, C. gattii infection can cause diseases in not only immunocompromised hosts but also immunocompetent individuals. While recent studies suggest that C. gattii infection could dampen pulmonary neutrophil recruitment and inflammatory cytokine production in immunocompetent hosts, the impact of C. gattii infection on the development of their adaptive T helper cell immune response has not been addressed. Here, we report that C. neoformans infection with highly virulent and less virulent strains preferentially induced pulmonary Th1 and Th17 immune responses in the host, respectively. However, fewer pulmonary Th1 and Th17 cells could be detected in mice infected with C. gattii strains. Notably, dendritic cells (DC) in mice infected with C. gattii expressed much lower levels of surface MHC-II and Il12 or Il23 transcripts and failed to induce effective Th1 and Th17 differentiation in vitro. Furthermore, the expression levels of Ip10 and Cxcl9 transcripts, encoding Th1-attracting chemokines, were significantly reduced in the lungs of mice infected with the highly virulent C. gattii strain. Thus, our data suggest that C. gattii infection dampens the DC-mediated effective Th1/Th17 immune responses and downregulates the pulmonary chemokine expression, thus resulting in the inability to mount protective immunity in immunocompetent hosts.

Biliary repair and carcinogenesis are mediated by IL-33-dependent cholangiocyte proliferation.

Injury to the biliary epithelium triggers inflammation and fibrosis, which can result in severe liver diseases and may progress to malignancy. Development of a type 1 immune response has been linked to biliary injury pathogenesis; however, a subset of patients with biliary atresia, the most common childhood cholangiopathy, exhibit increased levels of Th2-promoting cytokines. The relationship among different inflammatory drivers, epithelial repair, and carcinogenesis remains unclear. Here, we determined that the Th2-activating cytokine IL-33 is elevated in biliary atresia patient serum and in the livers and bile ducts of mice with experimental biliary atresia. Administration of IL-33 to WT mice markedly increased cholangiocyte proliferation and promoted sustained cell growth, resulting in dramatic and rapid enlargement of extrahepatic bile ducts. The IL-33-dependent proliferative response was mediated by an increase in the number of type 2 innate lymphoid cells (ILC2s), which released high levels of IL-13 that in turn promoted cholangiocyte hyperplasia. Induction of the IL-33/ILC2/IL-13 circuit in a murine biliary injury model promoted epithelial repair; however, induction of this circuit in mice with constitutive activation of AKT and YAP in bile ducts induced cholangiocarcinoma with liver metastases. These findings reveal that IL-33 mediates epithelial proliferation and suggest that activation of IL-33/ILC2/IL-13 may improve biliary repair and disruption of the circuit may block progression of carcinogenesis.