Recent advances in understanding basophil functions in vivo

Basophils are mainly known as pro-inflammatory effector cells associated with allergy and helminth infections. Although they were identified over 130 years ago, their in vivo functions are still poorly understood. New insights into basophil development and function have been gained by the development of various transgenic mouse lines and staining techniques to detect and purify these cells from different organs. Several studies over the past few years have identified unexpected functions for basophils, including immunomodulatory properties and interactions with other immune cells. Here, I summarize and discuss the main findings.


Introduction
Basophils belong to the group of granulocytes which constitute rather short-lived effector cells of the innate immune system. They usually represent less than 1% of all leukocytes in the peripheral blood, but they have potent effector functions. In contrast to many other cell types of the immune system, it is poorly understood how basophils develop and execute their effector functions. Not too long ago, it was even questioned whether mice contain a bona fide basophil population mainly because mouse basophils contain fewer granules as compared to human basophils 1 . After establishing staining protocols and genetically engineered mouse strains basophils could be identified, isolated, and functionally characterized in various settings of immune responses. Basophils and mast cells share the expression of the high-affinity receptor for IgE (FcεRI), histamine, and a few other effector molecules, yet basophils do not represent a subset or precursor population of mast cells; rather, they constitute a distinct cell lineage with a very different gene expression profile 2 . Basophils can be efficiently depleted in vivo with the monoclonal antibody MAR-1 directed against FcεRI or Ba103 which binds to another activating receptor named CD200R3. However, since the recognized antigens of both antibodies are not exclusively expressed on basophils, this approach can cause bystander effects that interfere with clear interpretations of such depletion experiments. Transgenic mice expressing the Cre recombinase, GFP, or human diphtheria-toxin receptor under control of regulatory elements of the Mcpt8 gene have been developed by several groups over the past few years (reviewed in 3). Mcpt8 encodes the mouse mast cell protease 8 (mMCP-8), a serine protease which is highly expressed in basophils but not mast cells 4 . The genetically modified mouse strains facilitate the functional characterization of basophils in vivo. However, it is important to keep in mind that mouse and human basophils differ in many respects, and it remains to be determined to what extent findings in mouse models can be translated to the human immune system. In the following paragraphs, I summarize some major new findings regarding the in vivo functions of basophils published during the past few years.

Basophil development and critical transcription factors
Interleukin (IL)-3 is the most potent cytokine to promote basophil proliferation, but high concentrations of thymic stromal lymphopoietin (TSLP) can have similar effects 5 . IL-3 and TSLP-elicited basophils differ in terms of their gene expression profiles and may resemble different states of activation rather than representing stable subpopulations of basophils. The receptors for IL-3 and TSLP are linked to the STAT5 signaling pathway, and it was recently shown that STAT5 binds to regulatory elements of Gata2, which is another critical transcription factor for basophil development 6 . In addition, it was found that IRF8 can also promote Gata2 expression in precursor cells and thereby drive basophil differentiation 7 . Other transcription factors including Gata1, P1-Runx1, c/EBPα, and MITF also play an important role in basophil development and maintenance (reviewed in 8).

Relevance of antigen-presentation by basophils
Basophils were found to display low levels of major histocompatibility complex class II (MHC-II) on the cell surface, and antibody-mediated depletion of basophils resulted in poor Th2 polarization 9-11 . However, genetically basophil-depleted Mcpt8Cre mice showed normal expansion of Th2 cells during primary infection with the helminths Nippostrongylus brasiliensis 12 , Heligmosomoides polygyrus 13 , and Schistosoma mansoni 14 . Ovalbumin (OVA)-alum immunized and challenged Mcpt8Cre mice also showed an unimpaired Th2 response and eosinophilia in the lung 12 . Furthermore, papain+OVA-induced T-cell proliferation and Th2 polarization in draining lymph nodes was normal in Mcpt8Cre mice while genetically dendritic cell (DC)-depleted mice showed a severely impaired response 12 . With another genetically basophil-depleted mouse model (Basoph8 x Rosa-DTa mice), it was shown that footpad immunization with S. mansoni eggs results in normal Th2 priming in the absence of basophils 15 . In contrast, diphtheria toxin (DT)-mediated depletion of DCs causes impaired Th2 priming upon S. mansoni egg immunization and diminished Th2 cell accumulation in the liver of S. mansoniinfected mice 16 . It was further shown that DCs are required and sufficient for the Th2 response to house dust mite antigens 17 . This study also demonstrated that a subset of monocyte-derived DCs express FcεRI. These cells will therefore also be depleted with the anti-FcεRI antibody MAR-1 frequently used to deplete basophils in vivo.
Basophils lack the machinery for antigen uptake and processing, although they can contain MHC-II molecules on the cell surface which may be loaded with exogenous peptides and are then capable of stimulating T cells 9,18,19 . Two other studies provide evidence that basophils and DCs cooperate to promote Th2 polarization. It was shown that subcutaneous papain injection induced reactive oxygen species that indirectly activated DCs to promote basophil recruitment into lymph nodes and subsequent Th2 polarization 20 . Another study showed that TSLP-elicited dermal DCs express OX40L to induce IL-3 secretion from T cells leading to the recruitment of basophils which then promote Th2 polarization 21 . The Th2-promoting activity of basophils in both studies might be explained by basophil-derived IL-4 rather than direct antigen recognition on basophils.
Recent evidence shows that basophils can in fact acquire MHC-II from DCs by the uptake of plasma membrane patches, a process termed trogocytosis 19 . Clearly, further studies are needed to determine whether MHC-II trogocytosis by basophils has functional consequences for T-cell activation or memory formation or other processes that are regulated by antigen recognition.

Basophil functions in type 2 immune responses Lung inflammation
In a mouse model of allergic lung inflammation induced by the administration of the cysteine protease papain, it was found that basophil-derived IL-4 promotes the secretion of IL-5, IL-9, and IL-13 from type 2 innate lymphoid cells (ILC2s) in the lung and thereby induces lung eosinophilia 22 . Another type 2 immunityinducing property of basophils was observed in a commonly used model for allergic lung inflammation which is based on alum adjuvant-mediated priming of the Th2 response. It was recently reported that alum enhanced the ability of basophils to induce Th2 polarization by the release of TSLP and IL-25 but independently of IL-4 secretion 23 . However, we and others observed no impairment in lung Th2 responses with N. brasiliensis infection or OVA/alum immunization in genetically basophil-depleted mice 12,15 .
Local inflammatory responses in the skin Similar to reports from papain-induced lung inflammation, it was reported that basophil-derived IL-4 induces ILC2 accumulation and proliferation in the skin after topical application of MC903, a vitamin D analog 24 . MC903 elicits high levels of TSLP expression in the skin and causes pathology reminiscent of skin lesions of atopic dermatitis patients. Anti-TSLP antibodies can block the accumulation of basophils in the skin 25 but also inhibit peripheral TSLP-induced basophilia during Trichinella spiralis infection 26 . Experiments with mixed bone marrow chimeras revealed that basophil accumulation in the MC903-treated ear does not require direct recognition of TSLP by basophils 5 . Basophils were further found to cooperate with fibroblasts to promote the recruitment of eosinophils in a murine model of irritant contact dermatitis 27 . Related to this, it was reported that basophils regulate the entry of eosinophils into the skin by the induction of VCAM-1 expression on endothelial cells 28 .
Local activation of basophils in the ear skin via FcεRI causes an ear swelling response termed "chronic allergic inflammation" or IgE-CAI, which peaks at day 2-3 and resolves by day 6. This IgE-CAI response is strictly dependent on basophils 29 . Using this model, Egawa et al. reported that the release of IL-4 from basophils promotes the differentiation of alternatively activated or "M2" macrophages which have anti-inflammatory and tissue repair properties, arguing for a role of basophils in the resolution of inflammation 30 . Others observed that α(1,3) fucosyltransferases IV and VII are essential for the initial recruitment of basophils in the IgE-CAI model 31 . Basophils express various proteases including mMCP-8 and mMCP-11. By analyzing mMCP-11-deficient mice, Iki et al. showed that mMCP-11 promotes the ear swelling response in IgE-CAI 32 . However, basophils can also have anti-inflammatory properties, as shown in a model of skin contact hypersensitivity where UVB irradiation reduced the hapten-induced ear swelling response. In this model, it could be demonstrated that basophilrestricted expression of amphiregulin, a cytokine with tissue repair activities, was required for the suppressive effect of UVB irradiation 33 . Increased numbers of basophils were also found in skin biopsies from patients with various skin disorders, indicating that these cells also regulate inflammatory responses in human skin 34 .

Protection against parasites in the skin and intestine
The presence of basophils in the skin indicates that they could be involved in protective immunity against helminths that enter their hosts via the skin or against ectoparasites like ticks. Using basophil-depleted mice, Wada et al. showed that basophils impair tick feeding upon secondary engorgement 35 . Furthermore, basophils promote the trapping of larvae in the skin upon secondary N. brasiliensis infection 36 . In addition, basophils accumulate in the small intestine and induce rapid expansion of Th2 cells and protection against N. brasiliensis or H. polygyrus during secondary infection by IgE-induced secretion of IL-4/IL-13 13 . Basophils further promote a Th2 response in a TSLP-dependent manner during T. spiralis and Trichuris muris infections 25,26 . In contrast, basophils play only a minor role in the control of Strongyloides ratti infection 37 . Anti-CD200R3-mediated depletion of basophils resulted in smaller granulomas around S. mansoni eggs in the liver 38 , while this effect was not observed in genetically basophildepleted Mcpt8Cre mice 14 . This difference might be explained by secondary effects caused by the injected antibody, including mast cell activation and Fc receptor-mediated modulation of phagocytes.

Basophils in food allergy and eosinophilic esophagitis
An interesting role for basophils during skin sensitization followed by oral challenge to elicit a food allergic response has been described. Mice developed a severe food allergic response when chicken OVA was first applied in combination with MC903 to the ear skin and later given intragastrically by oral gavage 39 . The authors further showed that basophils and TSLP are required for this effect. In a subsequent study, it was further revealed that basophil-derived IL-4 promoted the IgE-mediated food allergic response and eosinophils were dispensable for this effect 40 . A role for basophils, TSLP, and IL-33 to elicit an anaphylactic response to oral antigens was also shown in another model where OVA was applied to skin pretreated with 4% SDS instead of MC903 41 .
The impaired skin barrier function in atopic dermatitis may facilitate allergic sensitization that can lead to eosinophilic esophagitis (EoE). Pathology reminiscent of EoE can be elicited by intranasal OVA challenge of MC903+OVA skin-sensitized mice 42 . Using this model, Noti et al. showed that IgE is dispensable for EoE while basophils and TSLP are required 42 . Furthermore, IL-33 was identified as a critical cytokine to promote the accumulation of basophils in the esophagus and the recruitment of eosinophils 43 .

Basophils in other inflammatory settings, allograft transplantation, and tumor control
Intravenous immunoglobulin (IVIG) therapy, the intravenous administration of high doses of purified IgG, is used as a therapeutic approach to treat autoantibody-mediated inflammation in various clinical settings. The anti-inflammatory mechanism of IVIG therapy is incompletely understood. Using a mouse model of serum-induced arthritis, researchers showed that IVIG elicits IL-33 secretion, which in turn promotes IL-4 release from basophils. Basophil-derived IL-4 then upregulates the inhibitory Fc receptor FcγRIIB on macrophages and thereby ameliorates pathology 44 . However, others found no role for basophils in IVIGinduced suppression of serum-induced arthritis 45 . The apparent discrepancies between these observations remain to be resolved.
Systemic lupus erythematosus (SLE) is another autoantibodymediated disease. SLE patients were found to have higher serum levels of IgE and activated basophils 46 . Another study showed that basophils from SLE patients promoted Th17 differentiation in vitro, probably by the secretion of IL-6 47 . Depletion of basophils from SLE-prone MRL-lpr/lpr mice resulted in ameliorated pathology and an extended lifespan, while adoptive transfer of basophils had the opposite effect 47 . Further evidence for an important role of basophil-derived IL-6 in Th17 differentiation is based on a cholera toxin-induced lung inflammation model. Here it was shown that Th17-associated lung inflammation was reduced in the absence of basophils and could be restored by the transfer of wild-type but not IL-6-deficient basophils 48 .
Basophils were also found to regulate the rejection of allogeneic transplants. In a mouse model of pancreatic islet allotransplantation, it was reported that the depletion of basophils results in improved graft survival 49 . Furthermore, basophil-derived IL-4 was found to act on myofibroblasts and promote fibrosis in a cardiac allotransplantation model 50 . In vitro studies with human basophils further revealed their potential to inhibit TLR4-induced monocyte activation 51 and to induce the differentiation of alternatively activated macrophages 52 .
Recently, basophils were further found to modulate immune responses against solid tumors. One study described the recruitment of basophils into tumor-draining lymph nodes in correlation with a Th2-biased immune response and poor survival of pancreatic cancer patients 53 . In contrast, basophils were also shown to promote tumor rejection by recruiting CD8+ T cells.

Future perspective
Our current understanding of basophil development and effector functions has improved considerably over the past few years. We realize now that basophils not only function as pro-inflammatory cells during allergic responses and helminth infections but also modulate the immune system in many ways. Most of our knowledge is still based on mouse models. It will be important to translate these findings to the human immune system in order to develop new therapeutic approaches for the treatment of inflammatory diseases where basophils may play an important role.

Competing interests
The author declares that he has no competing interests.

Grant information
Work on basophils in the Voehringer lab is currently supported by grant Vo944/7-1 from the Deutsche Forschungsgemeinschaft (DFG).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.