IgE and IL‐33−mediated triggering of human basophils inhibits TLR4−induced monocyte activation

Basophils are circulating granulocytes, best known as effector cells in allergic reactions. Recent studies in mice suggest that they might also participate in the suppression of chronic inflammation. The aim of this study was to assess the ability of purified human basophils to modulate monocyte responses upon IL‐33 and IgE triggering. Activation of human basophils with IL‐33 induced the production of IL‐4 and the release of histamine, and enhanced their IgE‐mediated activation. In addition, basophils triggered with IL‐33 and anti‐IgE significantly suppressed the LPS‐induced production of the proinflammatory cytokine TNF‐α and the upregulation of the costimulatory molecule CD80 by monocytes. These effects were mainly explained by the release of histamine, as they could be inhibited by the histamine receptor 2 antagonist ranitidine, with a smaller contribution of IL‐4. In contrast, basophil‐derived IL‐4 and histamine had opposing effects on the expression of the inhibitory Fc γ receptor IIb and the production of IL‐10 by monocytes. Our data show that basophils can influence monocyte activation and suggest a previously unrecognized role for human basophils in the modulation of monocyte‐mediated immune responses, through the balanced secretion of histamine and IL‐4.     

C‐type Lectin Receptor Expression on Human Basophils and Effects of Allergen‐Specific Immunotherapy

Basophils are emerging as immunoregulatory cells capable of interacting with their environment not only via their characteristic IgE‐mediated activation, but also in an IgE‐independent manner. Basophils are known to express and respond to stimulation via TLR2, TLR4, DC‐SIGN and DCIR, but whether basophils also express other C‐type lectin receptors (CLRs) is largely unknown. In this study, we investigate the CLR expression profile of human basophils using multicolour flow cytometry. As FcRs as well as some CLRs are associated with allergen recognition and shown to be involved in subsequent immune responses, the expression of CLRs and FcRs on peripheral blood basophils, as well as their frequency, was monitored for 1 year in subjects undergoing subcutaneous allergen‐specific immunotherapy (AIT). Here, we show that human basophils express CLECSF14, DEC205, Dectin‐1, Dectin‐2 and MRC2. Furthermore, we demonstrate that the frequencies of basophils expressing the allergy‐associated CLRs Dectin‐1 and Dectin‐2 were significantly reduced after 1 year and 8 weeks of AIT, respectively. In contrast, the frequency of basophils positive for FcγRII, as well as the fraction of total basophils, significantly increased after 1 year of AIT. The herein demonstrated expression of various CLRs on basophils, and their altered CLR and FcR expression profile upon AIT, suggest yet unexplored ways by which basophils can interact with antigens and may point to novel immunoregulatory functions targeted through AIT.     

Basophils are potent antigen‐presenting cells that selectively induce Th2 cells

Basophils and mast cells are important effector cells in helminth‐infected host and IgE‐mediated allergic inflammation. Although they have the same progenitors, basophils and mast cells complete their terminal differentiation in the bone marrow and peripheral tissues, respectively, and only basophils circulate in the blood. Although it is recognized that basophils are important for Th2 responses, and it is also well established that IL‐4 is required for Th2 differentiation from naïve CD4⁺ T cells, the nature of the cells that produce “early” IL‐4, remained elusive until recently. Three groups independently demonstrated that basophils are the predominant APC in inducing Th2 response against helminth parasites and allergens. Basophils express MHC class II and CD80/86, have the potential to take‐up and process protein Ag (particularly Ag–IgE complex) and to present peptide in the context of MHC class II, and to produce IL‐4. These Ag‐pulsed basophils induce the development of Th2 cells both in vitro and in vivo. Thus, basophils contribute to Th2/IgE response by the production of IL‐4 and presentation of MHC class II/peptide complex to naïve CD4⁺ T cells, in contrast to the Th1‐inducing action of DC. In this review, we summarize what is known regarding basophil function in allergy and parasite infection, examine the novel Ag‐presenting function of basophils and discuss potential clinical implications of this finding.     

Involvement of hypoxia‐inducible factor‐1 HiF(1α) in IgE‐mediated primary human basophil responses

Basophils play a pivotal role in regulating chronic allergic inflammation as well as angiogenesis. Here, we show for the first time that IgE‐mediated activation of primary human basophils results in protein accumulation of the α‐subunit of hypoxia‐inducible factor 1α (HIF‐1α), which is differentially regulated compared with signals controlling histamine release. HIF‐1 facilitates cellular adaptation to hypoxic conditions such as inflammation and tumour growth by controlling glycolysis, angiogenesis and cell adhesion. ERK and p38 MAPK, but not reactive oxygen species (ROS), ASK1 or PI 3‐kinase, were critical for IgE‐mediated accumulation of HIF‐1α, although the latter crucially affected degranulation. Abrogating HIF‐1α expression in basophils using siRNA demonstrated that this protein is essential for vascular endothelial growth factor (VEGF) mRNA expression and, consequently, release of VEGF protein. In addition, HIF‐1α protein alters IgE‐induced ATP depletion in basophils, thus also supporting the production of the pro‐allergic cytokine IL‐4.     

Toll‐like receptor 4 agonists adsorbed to aluminium hydroxide adjuvant attenuate ovalbumin‐specific allergic airway disease: role of MyD88 adaptor molecule and interleukin‐12/interferon‐γ axis

Background Epidemiological and experimental data suggest that bacterial lipopolysaccharides (LPS) can either protect from or exacerbate allergic asthma. Lipopolysaccharides trigger immune responses through toll‐like receptor 4 (TLR4) that in turn activates two major signalling pathways via either MyD88 or TRIF adaptor proteins. The LPS is a pro‐Type 1 T helper cells (Th1) adjuvant while aluminium hydroxide (alum) is a strong Type 2 T helper cells (Th2) adjuvant, but the effect of the mixing of both adjuvants on the development of lung allergy has not been investigated. Objective We determined whether natural (LPS) or synthetic (ER‐803022) TLR4 agonists adsorbed onto alum adjuvant affect allergen sensitization and development of airway allergic disease. To dissect LPS‐induced molecular pathways, we used TLR4‐, MyD88‐, TRIF‐, or IL‐12/IFN‐γ‐deficient mice. Methods Mice were sensitized with subcutaneous injections of ovalbumin (OVA) with or without TLR4 agonists co‐adsorbed onto alum and challenged with intranasally with OVA. The development of allergic lung disease was evaluated 24 h after last OVA challenge. Results Sensitization with OVA plus LPS co‐adsorbed onto alum impaired in dose‐dependent manner OVA‐induced Th2‐mediated allergic responses such as airway eosinophilia, type‐2 cytokines secretion, airway hyper‐reactivity, mucus hyper production and serum levels of IgE or IgG1 anaphylactic antibodies. Although the levels of IgG2a, Th1‐affiliated isotype increased, investigation into the lung‐specific effects revealed that LPS did not induce a Th1 pattern of inflammation. Lipopolysaccharides impaired the development of Th2 immunity, signaling via TLR4 and MyD88 molecules and via the IL‐12/IFN‐γ axis, but not through TRIF pathway. Moreover, the synthetic TLR4 agonists that proved to have a less systemic inflammatory response than LPS also protected against allergic asthma development. Conclusion Toll‐like receptor 4 agonists co‐adsorbed with allergen onto alum down‐modulate allergic lung disease and prevent the development of polarized T cell‐mediated airway inflammation.     

Interleukin‐4‐ and NACHT,LRR and PYD domains‐containing protein 3‐independent mechanisms of alum enhanced T helper type 2 responses on basophils

Aluminium hydroxide (alum), the most widely used adjuvant in human and animal vaccines, has long been known to promote T helper type 2 (Th2) responses and Th2‐associated humoral responses, but the mechanisms have remained poorly understood. In this study, we explored whether alum is able to directly modulate antigen‐presenting cells to enhance their potency for Th2 polarization. We found that alum treatment of dendritic cells failed to show any Th2‐promoting activities. In contrast, alum was able to enhance the capacity of basophils to induce Th2 cells. When basophils from interleukin‐4 (IL‐4) knockout mice were examined, the intrinsic Th2‐promoting activities by basophils were largely abrogated, but the alum‐enhanced Th2‐promoting activities on basophils were still detectable. More importantly, Th2‐promoting adjuvant activities by alum found in IL‐4 knockout mice were also largely reduced when basophils were depleted by antibody administration. Therefore, basophils can mediate Th2‐promoting activities by alum both in vitro and in vivo through IL‐4‐independent mechanisms. Further studies revealed that secreted soluble molecules from alum‐treated basophils were able to confer the Th2‐promoting activities, and neutralization of thymic stromal lymphopoietin or IL‐25 attenuated the IL‐4‐independent development of Th2 cells elicited by alum‐treated basophils. Finally, alum was able to activate NACHT, LRR and PYD domains‐containing protein 3 (NLRP3) inflammasome in murine basophils in the same way as alum in professional antigen‐presenting cells, but NLRP3 was not required for Th2‐promoting activities on basophils by alum in vitro. These results demonstrated that alum can enhance the capacities of basophils to polarize Th2 cells via IL‐4‐ and NLRP3‐independent pathways.     

Toll‐like receptor 2 agonist Pam3CSK4 enhances the induction of antigen‐specific tolerance via the sublingual route

Background Sublingual immunotherapy (SLIT) has been established in humans as a safe and efficacious treatment for type I respiratory allergies. Objective In this study, we compared three Toll‐like receptor (TLR) 2 ligands (Pam3CSK4, Porphyromonas gingivalis lipopolysaccharide and lipoteichoic acid) as potential adjuvants for sublingual allergy vaccines. Methods These molecules were tested in co‐cultures of adjuvant‐pre‐treated dendritic cells (DCs) with murine naïve CD4⁺ T lymphocytes. Patterns of cytokine production, phenotype, proliferation and gene expression were analysed by ELISA, cytofluorometry and quantitative PCR, respectively. TLR2 ligands were subsequently tested in a model of SLIT in BALB/c mice sensitized with ovalbumin (OVA). Results Among the three TLR2 ligands tested, the synthetic lipopeptide Pam3CSK4 is the most potent inducer of IL‐12p35 and IL‐10 gene expression in murine bone marrow‐derived DCs, as well as in purified oral myeloid DCs. Only Pam3CSK4‐treated DCs induce IFN‐γ and IL‐10 secretion by naïve CD4⁺ T cells. Sublingual administration of Pam3CSK4 together with the antigen in BALB/c mice sensitized to OVA decreases airway hyperresponsiveness as well as OVA‐specific T‐helper type 2 (Th2) responses in cervical lymph nodes dramatically. Conclusion Pam3CSK4 induces Th1/regulatory T cell responses, and as such, is a valid candidate adjuvant for sublingual allergy vaccines.     

Disordered Toll‐like receptor 2 responses in the pathogenesis of pulmonary sarcoidosis

In this study, we hypothesized that the granulomatous disorder sarcoidosis is not caused by a single pathogen, but rather results from abnormal responses of Toll‐like receptors (TLRs) to conserved bacterial elements. Unsorted bronchoalveolar lavage (BAL) cells from patients with suspected pulmonary sarcoidosis and healthy non‐smoking control subjects were stimulated with representative ligands of TLR‐2 (in both TLR‐2/1 and TLR‐2/6 heterodimers) and TLR‐4. Responses were determined by assessing resulting production of tumour necrosis factor (TNF)‐α and interleukin (IL)‐6. BAL cells from patients in whom sarcoidosis was confirmed displayed increased cytokine responses to the TLR‐2/1 ligand 19‐kDa lipoprotein of Mycobacterium tuberculosis (LpqH) and decreased responses to the TLR‐2/6 agonist fibroblast stimulating ligand‐1 (FSL)‐1. Subsequently, we evaluated the impact of TLR‐2 gene deletion in a recently described murine model of T helper type 1 (Th1)‐associated lung disease induced by heat‐killed Propionibacterium acnes. As quantified by blinded scoring of lung pathology, P. acnes‐induced granulomatous pulmonary inflammation was markedly attenuated in TLR‐2^–/– mice compared to wild‐type C57BL/6 animals. The findings support a potential role for disordered TLR‐2 responses in the pathogenesis of pulmonary sarcoidosis.     

Different interleukin‐17‐secreting Toll‐like receptor+ T‐cell subsets are associated with disease activity in multiple sclerosis

Signalling through Toll‐like receptors (TLRs) may play a role in the pathogenesis of autoimmune diseases, such as multiple sclerosis (MS). In the present study, the expression of TLR‐2, ‐4 and ‐9 was significantly higher on CD4⁺ and CD8⁺ T‐cells from MS patients compared to healthy individuals. Following in‐vitro activation, the proportion of interleukin (IL)‐17⁺ and IL‐6⁺ CD4⁺ and CD8⁺ T‐cells was higher in the patients. In addition, the proportion of IFN‐γ‐secreting TLR⁺ CD8⁺ T‐cells was increased in MS patients. Among different IL‐17⁺ T‐cell phenotypes, the proportion of IL‐17⁺ TLR⁺ CD4⁺ and CD8⁺ T‐cells producing IFN‐γ or IL‐6 were positively associated with the number of active brain lesions and neurological disabilities. Interestingly, activation of purified CD4⁺ and CD8⁺ T‐cells with ligands for TLR‐2 (Pam3Csk4), TLR‐4 [lipopolysaccharide (LPS)] and TLR‐9 [oligodeoxynucleotide (ODN)] directly induced cytokine production in MS patients. Among the pathogen‐associated molecular patterns (PAMPs), Pam3Csk4 was more potent than other TLR ligands in inducing the production of all proinflammatory cytokines. Furthermore, IL‐6, IFN‐γ, IL‐17 and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) levels produced by Pam3Csk4‐activated CD4⁺ cells were directly associated with disease activity. A similar correlation was observed with regard to IL‐17 levels released by Pam3Csk4‐stimulated CD8⁺ T‐cells and clinical parameters. In conclusion, our data suggest that the expansion of different T helper type 17 (Th17) phenotypes expressing TLR‐2, ‐4 and ‐9 is associated with MS disease activity, and reveals a preferential ability of TLR‐2 ligand in directly inducing the production of cytokines related to brains lesions and neurological disabilities.     

Emerging functions of basophils in protective and allergic immune responses

Basophils that were long thought to have a redundant role in mast cells in the effector response to allergens and parasites are now being recognized to have important roles in the regulation of adaptive immune responses. Recent data have revealed their role in the initiation of the T helper cell 2 (Th2)-mediated immune response. Not only do basophils guide the Th1–Th2 balance by providing an early source of crucial Th2-skewing cytokines, interleukin (IL)-4 and thymic stromal lymphopoietin, but recent findings have also illustrated their capacity to function as antigen-presenting cells. Thus, basophils activate and instruct naive CD4 T cells, and guide their development into Th2 cells. Not only do basophils directly interact with T cells, but new insights have illustrated that they may also directly guide antibody responses in both the primary and memory responses. These and other studies have illustrated the emerging role of basophils in the regulation of type 2 immunity.     

Basophils as a primary inducer of the T helper type 2 immunity in ovalbumin‐induced allergic airway inflammation

Antigen‐induced allergic airway inflammation is mediated by T helper type 2 (Th2) cells and their cytokines, but the mechanism that initiates the Th2 immunity is not fully understood. Recent studies show that basophils play important roles in initiating Th2 immunity in some inflammatory models. Here we explored the role of basophils in ovalbumin (OVA) ‐induced airway allergic inflammation in BALB/c mice. We found that OVA sensitization and challenge resulted in a significant increase in the amount of basophils in blood and lung, along with the up‐regulation of activation marker of CD200R. However, depletion of basophils with MAR‐1 or Ba103 antibody attenuated airway inflammation, represented by the significantly decreased amount of the Th2 subset in spleen and draining lymph nodes, interlukin‐4 level in lung and OVA‐special immunoglobulin E (sIgE) levels in serum. On the other hand, adoptive transfer of basophils from OVA‐challenged lung tissue to naive BALB/c mice provoked the Th2 immune response. In addition, pulmonary basophils from OVA‐challenged mice were able to uptake DQ‐OVA and express MHC class II molecules and CD40 in vivo, as well as to release interleukin‐4 following stimulation by IgE–antigen complexes and promote Th2 polarization in vitro. These findings demonstrate that basophils may participate in Th2 immune responses in antigen‐induced allergic airway inflammation and that they do so through facilitating antigen presentation and providing interleukin‐4.     

Type I IFN‐mediated synergistic activation of mouse and human DC subsets by TLR agonists

Novel approaches of dendritic cell (DC) based cancer immunotherapy aim at harnessing the unique attributes of different DC subsets. Classical monocyte‐derived DC vaccines are currently being replaced by either applying primary DCs or specifically targeting antigens and adjuvants to these subsets in vivo. Appropriate DC activation in both strategies is essential for optimal effect. For this purpose TLR agonists are favorable adjuvant choices, with TLR7 triggering being essential for inducing strong Th1 responses. However, mouse CD8α⁺ DCs, considered to be the major cross‐presenting subset, lack TLR7 expression. Interestingly, this DC subset can respond to TLR7 ligand upon concurrent TLR3 triggering. Nevertheless, the mechanism underlying this synergy remains obscure. We now show that TLR3 ligation results in the production of IFN‐α, which rapidly induces the expression of TLR7, resulting in synergistic activation. Moreover, we demonstrate that this mechanism conversely holds for plasmacytoid DCs that respond to TLR3 ligation when TLR7 pathway is mobilized. We further demonstrate that this mechanism of sharpening DC senses is also conserved in human BDCA1⁺ DCs and plasmacytoid DCs. These findings have important implications for future clinical trials as it suggests that combinations of TLR ligands should be applied irrespective of initial TLR expression profiles on natural DC subsets for optimal stimulation.     

Adhesion molecule cross‐linking and cytokine exposure modulate IgE‐ and non‐IgE‐dependent basophil activation

Basophils are known for their role in allergic inflammation, which makes them suitable targets in allergy diagnostics such as the basophil activation test (BAT) and the microfluidic immunoaffinity basophil activation test (miBAT). Beside their role in allergy, basophils have an immune modulatory role in both innate immunity and adaptive immunity. To accomplish this mission, basophils depend on the capability to migrate from blood to extravascular tissues, which includes interactions with endothelial cells, extracellular matrix and soluble mediators. Their receptor repertoire is well known, but less is known how these receptor–ligand interactions impact the degranulation process and the responsiveness to subsequent activation. As the consequences of these interactions are crucial to fully appreciate the role of basophils in immune modulation and to enable optimization of the miBAT, we explored how basophil activation status is regulated by cytokines and cross‐linking of adhesion molecules. The expression of adhesion molecules and activation markers on basophils from healthy blood donors was analysed by flow cytometry. Cross‐linking of CD203c, CD62L, CD11b and CD49d induced a significant upregulation of CD63 and CD203c. To mimic in vivo conditions, valid also for miBAT, CD62L and CD49d were cross‐linked followed by IgE‐dependent activation (anti‐IgE), which caused a reduced CD63 expression compared with anti‐IgE activation only. IL‐3 and IL‐33 priming caused increased CD63 expression after IgE‐independent activation (fMLP). Together, our data suggest that mechanisms operational both in the microfluidic chip and in vivo during basophil adhesion may impact basophil anaphylactic and piecemeal degranulation procedures and hence their immune regulatory function.     

Human TSLP and TLR3 ligands promote differentiation of Th17 cells with a central memory phenotype under Th2-polarizing conditions

Background Human thymic stromal lymphopoietin (TSLP) is expressed in the human asthmatic lung and activates dendritic cells (DCs) to strongly induce proallergic T‐helper type 2 (Th2) cell responses, suggesting that TSLP plays a critical role in the pathophysiology of human asthma. Th2 cells are predominantly involved in mild asthma, whereas a mixture of Th1 and Th2 cells with neutrophilic inflammation, probably induced by Th17, affects more severe asthmatic disease. Exacerbation of asthmatic inflammation is often triggered by airway‐targeting RNA viral infection; virus‐derived double‐stranded RNA, Toll‐like receptor (TLR)3 ligand, activates bronchial epithelial cells to produce pro‐inflammatory mediators, including TSLP. Objective Because TSLPR‐expressing DCs express TLR3, we examined how the relationship between TSLP and TLR3 ligand stimulation influences DC activation. Methods CD11c⁺DCs purified from adult peripheral blood were cultured in TLR ligands containing media with or without TSLP and then co‐cultured with allogeneic naïve CD4⁺T cells. Results CD11c⁺ DCs responded to a combination of TSLP and TLR3 ligand, poly(I : C), to up‐regulate expression of the functional TSLP receptor and TLR3. Although TSLP alone did not induce IL‐23 production by DCs, poly(I : C) alone primed DCs for the production of IL‐23, and a combination of TSLP and poly(I : C) primed DCs for further production of IL‐23. The addition of poly(I : C) did not inhibit TSLP‐activated DCs to prime naïve CD4⁺ T cells to differentiate into inflammatory Th2 cells. Furthermore, DCs activated by a combination of TSLP and poly(I : C) primed more naïve CD4⁺ T cells to differentiate into Th17‐cytokine–producing cells with a central memory T cell phenotype compared with DCs activated by poly(I : C) alone. Conclusions These results suggest that through DC activation, human TSLP and TLR3 ligands promote differentiation of Th17 cells with the central memory T cell phenotype under Th2‐polarizing conditions.     

IL‐33 broadens its repertoire to affect DC

IL‐33 is a novel multi‐functional IL‐1 family member that, in contrast to other family members, is associated with Th2 responses. IL‐33 signals via a heterodimer composed of its receptor, IL‐1 receptor‐like‐1 (IL‐1RL1), more commonly known as ST2L, and the IL‐1R accessory protein. ST2L is expressed by endothelial cells, mast cells, basophils, Th2 cells, and DC. IL‐33 has been associated with several immune‐mediated disorders, including asthma, arthritis, and inflammatory bowel disease. In contrast, there is evidence that IL‐33 can inhibit atherosclerosis development. A report in this issue of the European Journal of Immunology reveals a novel function of IL‐33: the ability to promote myeloid DC generation in murine BM cell cultures, by triggering GM‐CSF production by other BM cells, likely basophils. DC generated in the presence of IL‐33 are maturation resistant, with only minimal T‐cell stimulatory ability, associated with comparatively high levels of programmed death receptor ligand expression. This commentary discusses several questions raised by these findings, and provides a basis for further evaluation of IL‐33 and ST2L in regulation of APC generation and function in both innate and adaptive immunity.     

Mycobacterium tuberculosis RpfE promotes simultaneous Th1‐ and Th17‐type T‐cell immunity via TLR4‐dependent maturation of dendritic cells

Reciprocal induction of the Th1 and Th17 immune responses is essential for optimal protection against Mycobacterium tuberculosis (Mtb); however, only a few Mtb antigens are known to fulfill this task. A functional role for resuscitation‐promoting factor (Rpf) E, a latency‐associated member of the Rpf family, in promoting naïve CD4⁺ T‐cell differentiation toward both Th1 and Th17 cell fates through interaction with dendritic cells (DCs) was identified in this study. RpfE induces DC maturation by increasing expression of surface molecules and the production of IL‐6, IL‐1β, IL‐23p19, IL‐12p70, and TNF‐α but not IL‐10. This induction is mediated through TLR4 binding and subsequent activation of ERK, p38 MAPKs, and NF‐κB signaling. RpfE‐treated DCs effectively caused naïve CD4⁺ T cells to secrete IFN‐γ, IL‐2, and IL‐17A, which resulted in reciprocal expansions of the Th1 and Th17 cell response along with activation of T‐bet and RORγt but not GATA‐3. Furthermore, lung and spleen cells from Mtb‐infected WT mice but not from TLR4^?/? mice exhibited Th1 and Th17 polarization upon RpfE stimulation. Taken together, our data suggest that RpfE has the potential to be an effective Mtb vaccine because of its ability to activate DCs that simultaneously induce both Th1‐ and Th17‐polarized T‐cell expansion.