doi:10.1111/cei.12695
MOLECULAR AND CELLULAR ASPECTS OF B CELL BIOLOGY REVIEW SERIES
Clinical and Experimental Immunology
R EVI EW A RT I C L E
Eosinophils: important players in humoral immunity
C. Berek
Summary
B cell Immunology, Deutsches Rheuma Forschungszentrum, Berlin, Germany
Accepted for publication 19 August 2015 Correspondence: C. Berek, Deutsches Rheuma Forschungszentrum, Chariteplatz 1, 10117 Berlin, Germany. E-mail: [email protected]
Eosinophils perform numerous tasks. They are involved in inflammatory reactions associated with innate immune defence against parasitic infections and are also involved in pathological processes in response to allergens. Recently, however, it has become clear that eosinophils also play crucial non-inflammatory roles in the generation and maintenance of adaptive immune responses. Eosinophils, being a major source of the plasma cell survival factor APRIL (activation and proliferation-induced ligand), are essential not only for the long-term survival of plasma cells in the bone marrow, but also for the maintenance of these cells in the lamina propria which underlies the gut epithelium. At steady state under non-inflammatory conditions eosinophils are resident cells of the gastrointestinal tract, although only few are present in the major organized lymphoid tissue of the gut – the Peyer’s patches (PP). Surprisingly, however, lack of eosinophils abolishes efficient class-switching of B cells to immunoglobulin (Ig)A in the germinal centres of PP. Thus, eosinophils are required to generate and to maintain mucosal IgA plasma cells, and as a consequence their absence leads to a marked reduction of IgA both in serum and in the gut-associated lymphoid tissues (GALT). Eosinophils thus have an essential part in longterm humoral immune protection, as they are crucial for the longevity of antibody-producing plasma cells in the bone marrow and, in addition, for gut immune homeostasis. Keywords: eosinophil, IgA, mucosal immunity, plasma cell
Introduction Eosinophils are short-lived cells, which are generated continuously from haematopoietic stem cells in the bone marrow. They are generally thought of as being cells of the innate immune system, and they accumulate typically in the tissues in response to parasitic infection or other inflammatory conditions. On the downside, eosinophils have a critical role in allergen-induced inflammatory responses, such as asthmatic lung disease or atopic dermatitis [1–3]. Eosinophils are regarded as end-stage effector cells which, when activated, degranulate and release highly cytotoxic substances, such as the major basic protein or the eosinophil cationic protein. In the case of an infection these granule proteins may act directly against parasites; however, in an allergic situation they contribute to tissue destruction and in patients with atopic asthma the damage to the lung epithelium is correlated with the number of influxing eosinophils [4,5]. Furthermore, fully activated
eosinophils may respond by ejecting extracellular traps consisting of mitochondrial DNA and tissue destructive granular proteins [6]. In this way eosinophils, like neutrophils, are able to trap bacteria and kill them. However, this cytotoxic reaction will occur only under inflammatory conditions, when eosinophils are highly stimulated by cytokines such as interferon (IFN)-g and, in addition, high levels of interleukin (IL)-5 are required to induce DNA net formation [6]. In addition to these inflammatory functions, eosinophils also play a beneficial role in regulating and modulating immune responses. They do this, at least in part, by synthesizing and secreting a surprisingly broad spectrum of different cytokines and immune mediators [2]. In immune responses, activated eosinophils show an enhanced expression of cytokines [7–9]. Although activation can be induced by adjuvants alone, a stable activation is achieved only in antigen-induced immune responses. It is possible
C 2015 British Society for Immunology, Clinical and Experimental Immunology, 183: 57–64 V
57
C. Berek
MOLECULAR AND CELLULAR ASPECTS OF B CELL BIOLOGY REVIEW SERIES
that immunoglobulin (Ig) secreted by plasma cells contributes to the stable and enhanced cytokine expression of eosinophils [7]. As is characteristic for cells of the innate immune system the responses they mount to activation are immediate, as many of the mediators are already preformed in the cell and stored in granules or in granule-derived small transport vesicles [10]. Receptor-mediated specific recruitment of cytokines allows release of mediators in a specific and controlled fashion. Under physiological conditions this selective process of piecemeal degranulation does not result in cell death [10]. Recent data suggest that eosinophils are required for tissue integrity and are necessary for tissue remodelling [11]. Under normal non-inflammatory conditions eosinophils are recruited in an eotaxin-dependent fashion into various tissues. Eosinophils are found in the pubertal uterus and may have a role in the onset of the oestrous cycle and in preparing the uterus for pregnancy. Similarly, in prepubertal mammary glands, eosinophils together with macrophages regulate normal breast ductal development [11,12]. Furthermore, eosinophils accumulate in the thymus of the newborn and it has been suggested that they contribute to class I restricted thymocyte selection [11,13,14]. At steady state, the large majority of eosinophils in the body are located in the intestinal tissues. In particular, they are resident in the lamina propria of the small intestine, caecum, colon and stomach, where they localize to the submucosa [15–17]. The presence of these eosinophils in the intestinal tissues is independent of overt infection or inflammation. Indeed, eosinophils already accumulate in the gut-associated lymphoid tissue (GALT) during embryonic development, long before bacteria colonize the gut, so that at birth a full complement of eosinophils is already present in the lamina propria [18]. This is quite different from the situation with lymphocytes, where a full complement is reached in the GALT only at approximately 2 years of age [18], and the immigration of T and B cells to establish the mucosal immune tissues is dependent upon the presence of a microbiota in the gut lumen. The constitutive expression of eotaxin in the gastrointestinal tract is required for the population of the gut mucosal tissue with eosinophils and their maintenance there is supported by type 2 innate lymphoid cells (ILC-2), which constitutively secrete IL-5 and IL-13 [15]. Signalling through the common g chain of the IL-5 and IL-13 receptors prolongs the survival of eosinophils in the gastrointestinal tissues where, in contrast to blood or other tissues, they survive for more than 2 weeks [16]. Altogether, the lamina propria seems to provide a microenvironment supportive for eosinophil maintenance. Both plasma cells and gut epithelial cells express CD47, a ligand for the inhibitory receptor signal regulatory protein a (SIRPa) (CD172a) [19,20], which is expressed on eosinophils where its engagement with CD47 has been shown to inhibit degranulation and 58
apoptosis [20,21]. Furthermore, human eosinophils bind secretory IgA via the FcaR receptor; this prevents apoptosis and hence prolongs eosinophil survival [22]. In recent years it has become increasingly apparent that the various granulocyte populations have a broader role than merely that of aggressive end-stage effector cells [23,24]. Neutrophils, basophils and eosinophils present antigen to T cells, suggesting an important impact upon T cell activation. In addition, neutrophils function as B helper cells by inducing Ig class-switch somatic hypermutation and differentiation of marginal zone B cells into plasma cells [23]. What, then, is the function of eosinophils at steady state under non-inflammatory physiological conditions? This review will discuss recent data showing that eosinophils have a major role in protective humoral immunity, as they are a main source of activation and proliferation-induced ligand (APRIL) and of IL-6, which are the key cytokines essential for the survival of antibodyproducing plasma cells, both in the bone marrow and in the lamina propria [25]. Furthermore, eosinophils have a crucial role in the regulation of mucosal immune processes and in the control of immune homeostasis at mucosal sites. The use of eosinophil-deficient mice has demonstrated the important function of these cells in the generation of IgAexpressing B cells, and thus in the development of the IgA plasma cells which reside in the lamina propria [26,27]. Although the mechanisms by which eosinophils perform these functions are not yet understood, it is now clear that these cells have essential roles in adaptive immunity, as they are required for the long-term survival of plasma cells in the bone marrow and for the generation and maintenance of IgA-secreting plasma cells at mucosal sites.
In T cell-dependent immune responses eosinophils enhance early B cell activation Efficient immunization or vaccination requires the use of adjuvants [28]. In particular, the precipitation of antigen with aluminum hydroxide (alum) has an enhancing effect on the immune response, and alum administration has been shown to stimulate eosinophil development in the bone marrow and to promote their accumulation in the spleen [9]. In T cell-dependent immune responses, B cell activation can result in an immediate extrafollicular response to antigenic challenge, whereby antigen-activated B cells differentiate rapidly into IgM-secreting plasma cells. This early immediate B cell response to antigenic challenge requires the presence of eosinophils [8,9], whereas the induction of splenic germinal centres (GC) and the generation of high-affinity IgG-secreting plasma cells seem to be independent of them [25]. Recently it was suggested that danger signals, induced by cell stress and/or cell death, might initiate the recruitment of eosinophils [11]. One site at which many cells are driven into apoptosis is the GC where the T-dependent immune
C 2015 British Society for Immunology, Clinical and Experimental Immunology, 183: 57–64 V
MOLECULAR AND CELLULAR ASPECTS OF B CELL BIOLOGY REVIEW SERIES Eosinophils and humoral immunity
response develops. Development in the GC involves the rapid expansion of an antigen-specific B cell clone, hypermutation of the B cell receptor (BCR) and the selection of mutants with increased affinity for the antigen. In the socalled dark zone of the GC B cells divide every 6–8 h, and within a few days a single B cell will have generated a large clone of several 1000 cells [29]. Most of these cells will die by apoptosis, and only the few high-affinity mutants are selected to differentiate into B memory and plasma cells. Eosinophils seem not to be involved in these processes, as eosinophil-deficient DdblGATA-1 mice have normal GC reactions, antibody-affinity maturation is not affected and the differentiation of B cells into IgG1 secreting plasma cells is comparable to that of wild-type animals [25].
Eosinophils and megakaryocytes are required for efficient homing of plasma cells to bone marrow Plasma cells generated during a GC reaction leave the follicular structures and may home to the bone marrow, where they can survive and secrete antibody for years or even decades [30–32]. After primary immunization with a T cell-dependent antigen, there is only a slight increase in the number of antigen-specific plasma cells in the bone marrow, but in secondary responses this influx of plasma cells is considerable [25,33,34]. A histological analysis of the bone marrow of mice after secondary immunization showed that most of the plasma cells are in close contact with stromal cells, and this network of vascular cell adhesion protein 1 (V-CAM)1 reticular stromal cells seems to be crucial for the organization of the plasma cell survival niche in the bone marrow. Macrophages, megakaryocytes, B and T cells and also neutrophils are found in the vicinity of plasma cells; however, a statistical evaluation of the data showed that only eosinophils have a specific co-localization with the plasma cells [35–37]. The question arises as to which cells are required for the homing of plasma cells to the bone marrow and which are required for long-term survival of plasma cells. Analysis of c-mpl-deficient mice, which have reduced numbers of megakaryocytes, showed that the megakaryocytes play a role in the retention of plasma cells in the bone marrow [37]. In these mice the number of plasma cells in the bone marrow was reduced significantly during the first days after antigenic challenge; however, by 4 weeks the numbers had normalized. This suggests that the homing of the newly generated plasma cells to the bone marrow is aided by the presence of megakaryocytes. However, while these immediate interactions affect the initial rate of entry of plasma cells into the bone marrow, they are not essential for the establishment of a long-term plasma cell survival niche. Immunization of eosinophil-deficient DdblGATA-1 mice showed that, in comparison with wild-type BALB/c mice, many fewer plasma cells home to the bone marrow and 8
weeks after secondary challenge almost no antigen-specific plasma cells were detected [25]. Reconstitution of DdblGATA-1 mice with eosinophils prior to secondary challenge with antigen resulted in enhanced homing of plasma cells to the bone marrow. As eosinophils are shortlived cells the effect was transient [25], but these data imply that eosinophils play crucial roles both in the homing of plasma cells to the bone marrow and their retention.
The establishment of the plasma cell survival niche The establishment of the plasma cell survival niche in the bone marrow is a complex and time-consuming process. In the first weeks after secondary immunization, the newly generated plasma blasts (immature plasma cells) are found mainly in the vicinity of the sinuses in the bone marrow, and many of these cells are in close contact with eosinophils [36]. At this time-point single eosinophils are found in contact with single plasma blasts, and these direct interactions seem to be necessary for the development of plasma blasts into mature long-lived plasma cells. Only a few plasma cells are found in the bone marrow in eosinophil-deficient mice, and these seem not to be fully developed, as they secrete less antibody than do the plasma cells in the bone marrow of wild-type control animals. Four weeks after antigenic challenge plasma cells are now found deep in the parenchyma embedded in nests of eosinophils (Fig. 1) [36]. This suggests that these eosinophil nests are the survival niches, which support longevity of plasma cells. Eosinophils in the bone marrow are short-lived and therefore have a high turnover [16,35], while plasma cells, in contrast, are long-lived [30–32]. Although plasma cells express high levels of CD47, which inhibits eosinophil degranulation and protects them from apoptosis [19,20], in-vivo pulse-chase labelling with the thymidine analogue 5-ethynyl-20 -deoxyuridine (EdU) has shown that the eosinophil survival time is very much shorter than that of plasma cells [35]. This implies that the plasma cell survival niche in the bone marrow is a dynamic niche, where dying eosinophils are replaced constantly with newly generated ones [36].
Eosinophils are the main source of plasma cell survival factors Stromal cells in the bone marrow secrete the chemokine CXCL12, which attracts both CXCR4-expressing plasma cells and eosinophils. In-vitro cultures have shown that while the chemokine CXCL12 helps to support the maintenance of plasma cells [38], the crucial survival factor for the long-term maintenance of plasma cells is APRIL [39]. In the lymph node and the spleen, plasma cell survival is supported by macrophages expressing APRIL [36,40]. However, in the bone marrow, where the vast majority of plasma cells reside, macrophages alone are not sufficient, as
C 2015 British Society for Immunology, Clinical and Experimental Immunology, 183: 57–64 V
59
C. Berek
MOLECULAR AND CELLULAR ASPECTS OF B CELL BIOLOGY REVIEW SERIES
Fig. 1. The plasma cell survival niche in the bone marrow. Mice were immunized with a T cell-dependent antigen and 2 months after secondary challenge with antigen, bone marrow was taken and frozen tissue sections (6 lm) prepared. To visualize eosinophils and plasma cells, sections were stained with major basic protein-specific antibodies (red) and with anti immunoglobulin (Ig)G (green), respectively. Sections were counterstained with 40 ,6-diamidino-2phenylindole (DAPI).
depletion of eosinophils by injection of Siglec F-specific antibody induces a rapid loss both of eosinophils and of plasma cells [25]. Furthermore, at steady state only a few plasma cells are found in the bone marrow of eosinophildeficient mice, and when these animals are immunized with a T cell-dependent antigen almost no plasma cells home to the bone marrow and practically no long-lived plasma cells are detected [25]. Thus, eosinophils are essential for the maintenance of long-lived plasma cells in the bone marrow.
Eosinophils are required for the maintenance of plasma cells in the lamina propria At steady state large numbers of eosinophils are found in the lamina propria, in particular in the small intestine and the caecum. As in the bone marrow, intestinal eosinophils express high levels of the plasma cell survival factors APRIL and IL-6 [26]. Co-staining of tissue sections with antibodies specific for APRIL showed that epithelial cells are the main producers within the lamina propria, followed by eosinophils, while dendritic cells (DC) express much less. Neutrophils, which were shown to express APRIL and B cell-activating factor (BAFF) and thus support the differentiation of marginal zone B cells [23], do not seem to have 60
an important function in mucosal B cell activation at steady state. A significant influx of neutrophils is induced only under inflammatory conditions. When animals are injected with Siglec F-specific antibodies, which induce apoptosis in eosinophils, a rapid loss of plasma cells follows. As soon as the eosinophils are replenished, plasma cells, which are being generated continuously in the intestinal immune tissue, also reappear [26]. Thus, both in the bone marrow and in the lamina propria, plasma cell survival is dependent upon the presence of eosinophils. In the lamina propria of eosinophil-deficient mice the overall level of the plasma cell survival factors APRIL and IL-6 is not reduced significantly [27], most probably because epithelial cells express these cytokines abundantly. Nevertheless, abundant though this epithelial cell expression of APRIL and IL-6 is, it is not sufficient for the maintenance of plasma cells. It is well possible that the effective promotion of plasma cell maintenance requires close contact with the source of survival factors. Alternatively, the eosinophils in the intestinal mucosa may provide additional signals through direct cell contact with plasma cells, as has also been proposed for the survival niche in the bone marrow [36]. Normally, IgA is the most abundant antibody class in the mucosa and has a crucial function in the maintenance of immune homeostasis of the gut-associated tissues. Deficiencies in IgA production also have a strong effect in shaping the microbial community in the gut lumen [41,42]. Because, in eosinophil-deficient mice, the amount of IgAsecreting plasma is strongly reduced, there is a dramatic effect on the levels of IgA both in the circulation and at mucosal sites. It is therefore not surprising that in the absence of eosinophils there is a disruption of mucosal homeostasis, which includes both considerable changes to the mucus layer of the gut and to changes in the microorganism populations in the gut lumen [26,27].
The generation of mucosal IgA1 B and plasma cells The majority of IgA1 B and plasma cells in the lamina propria are generated in the organized lymphoid structures of the Peyer’s patches (PP), where Ig class-switch recombination requires help from T cells [43,44]. To what extent B cells belonging to the B1 subset contribute to the generation of intestinal IgA plasma cells is currently a matter of controversy [45–47]. The activation of these B1 cells and the induction of class-switch recombination to IgA have been shown to be T cell-independent [45]. Similarly, B cell activation in isolated lymphoid follicles (ILF) is independent of T cell help. The absence of eosinophils affects not only the number of IgA plasma cells, but also results in a strong reduction in the number of IgA1 B cells in the lamina propria and in ILF. In addition, there is a strong reduction in the number
C 2015 British Society for Immunology, Clinical and Experimental Immunology, 183: 57–64 V
MOLECULAR AND CELLULAR ASPECTS OF B CELL BIOLOGY REVIEW SERIES Eosinophils and humoral immunity
of IgA1 B cells in the germinal centres of PP and, in contrast to wild-type BALB/c mice, the majority of IgA1 B cells in the lamina propria belong to the B1 subset. As the overall number of IgM1 B cells in the lamina propria of eosinophil-deficient mice was normal, this suggests that eosinophils in the GALT have an important role in the differentiation of IgM1 B cells to IgA1 B cells.
Eosinophils may directly support IgA class-switch of B cells The question arises as to how lack of eosinophils impairs the generation of IgA1 B cells in the lamina propria, in PP and also in the ILF of eosinophil-deficient mice (Fig. 2a). Eosinophils in the lamina propria express transforming growth factor b (TGF-b) as well as APRIL and BAFF which, together, support synergistically the T cellindependent switch to IgA [48]. TGF-b is produced in an inactive form, which requires processing by metalloproteases for its activation, and eosinophils are a major source of the matrix metalloproteinases (MMP)-2 and MMP-9, as shown by a significant reduction of these enzymes in the lamina propria of eosinophil-deficient mice [26]. Eosinophils isolated from the lamina propria of myeloid differentiation primary response gene 88/TIR-domain-containing adapter-inducing interferon-b (MyD88/TRIF) double-deficient mice show decreased expression of the cytokines TGF-b, APRIL, BAFF and the metalloproteases MMP-2 and MMP-9, suggesting that in wild-type animals eosinophils in the gut mucosa are activated by bacterial antigens in the lamina propria via Toll-like receptors (TLRs). In-vitro co-cultures of splenic IgD1 B cells and eosinophils with bacterial antigens showed that only eosinophils from wild-type animals but not those from MyD88/ TRIF double-deficient mice were able to promote switching
to IgA [26]. Nevertheless, further work will be required to demonstrate whether eosinophils support B cell Ig classswitching in vivo.
Eosinophils are required for T cell-independent generation of IgA The relationship between the microbiota, B cell activation in the lamina propria and T cell-independent IgA class switch is not well understood [43–45,49]. Recent work suggested a role for ILC-expressing membrane-bound lymphotoxin alpha (LTa) in the T cell-independent generation of IgA [50,51]. It was shown that the interaction of membrane-bound LTa with its receptor LTbR on lamina propria stromal cells is required to establish a microenvironment that supports T-independent IgA class-switch. In the absence of LTa or LTbR the efficiency of B1 cells homing to the lamina propria is reduced, leading to a considerable reduction in the number of mucosal B cells. The level of mucosal IgA is consequently lower than in normal mice [51]. In addition, DC from LTa-deficient mice were less potent in supporting IgA class-switching than were those from wild-type controls [50]. Eosinophils may play a role here, because they have been shown to express high levels of IL-1b, a cytokine that promotes proliferation of ILC, and in addition enhances the expression of LTa [27]. Loss of eosinophil-derived IL-1b may thus play an indirect role in T cell-independent IgA switching in the lamina propria [27]. However, the analysis of B cell subsets in the lamina propria of eosinophil-deficient mice showed that this can only be part of the story, as the numbers of IgA1 B1 cells were reduced only marginally, whereas IgA1 B2 cells were reduced strongly [26]. Thus, it is primarily the T celldependent IgA class-switch in the GC of PP that is affected by the lack of eosinophils [17,26].
(a)
(b)
(c)
Fig. 2. Eosinophils promote immune homeostasis in the gastrointestinal tract. C 2015 British Society for Immunology, Clinical and Experimental Immunology, 183: 57–64 V
61
C. Berek
MOLECULAR AND CELLULAR ASPECTS OF B CELL BIOLOGY REVIEW SERIES
Eosinophils are required for T cell-dependent generation of IgA An analysis of B cell development in the PP of eosinophildeficient DdblGATA-1 mice showed normal development of the GC, but only a few IgA B cells were detected. A surprising finding was that the B cells in these PP switch; however, in contrast to wild-type animals, they switch preferentially to IgG1 rather than to IgA. This finding is even more surprising as under steady state conditions eosinophils are practically absent from PP and hence cannot exert direct effects on the generation of IgA1 B cells. In PP, the few eosinophils which are present do not colocalize with B cells, but instead tend to be located in the outer T cell zone together with DC [52]. In PP T follicular helper cells (TFH) play a crucial role by providing cognate help to GC B cells and promote Ig class-switch recombination and B cell differentiation into memory B cells and plasma cells. TFH in the GC of PP apparently develop from forkhead box protein 3 (FoxP3)1 T cells or T helper type 17 (Th17) cells, and thus provide a specific cytokine milieu which favours B cell Ig class-switch recombination to IgA [53–55]. In particular, synergy between the cytokines TGF-b1 and IL-21, both expressed strongly by the TFH of PP, is required for the preferential switch to IgA [56]. The combination of these two cytokines promotes the down-regulation of the chemokine receptor CXCR5 and up-regulation of CCR10, which allows the newly generated IgA plasma cells to leave the PP and home to the lamina propria [56]. In the PP of eosinophil-deficient mice, TFH have aberrantly enhanced expression of the transcription factor GATA3, which is indicative of Th2 helper cells, and accordingly express increased levels of Th2 type cytokines. This is consistent with their support of a preferential switch to IgG1, rather than to IgA [26]. The development of both FoxP31 T cells and Th17 cells requires TGF-b. Lack of eosinophils in DdblGATA-1 mice leading to a deficiency of active TGF-b may prevent the normal development of the TFH cells required to support class-switch recombination to IgA in the PP. In line with this interpretation is the finding that TGF-b-deficient mice have a similar phenotype to the eosinophil-deficient mice, in that there is a shift in TFH cell cytokine expression and that B cells switch preferentially to IgG [57]. Importantly, and in striking contrast to the situation in TGF-b-deficient mice, these effects in eosinophil-deficient mice are restricted to the PP, whereas in the spleen cytokine expression of TFH was not affected [26,57]. It is a characteristic of mucosal tissues that they contain a subset of T cells, expressing CD103, the a chain of a TGF-b inducible integrin. Similarly, a subset of DC expressing CD103 are found in mucosal tissues. In eosinophil-deficient mice the frequency of the CD1031 populations of CD41 T cells was reduced specifically and 62
the frequency of CD1031 DC was also diminished. These effects on the T cell and DC compartments were seen in the lamina propria and PP, but not in mesenteric lymph nodes [26], supporting the notion that the absence of eosinophils leads to a specific reduction in the levels of active TGF-b in the GALT.
Eosinophils may have a broader function than simply the maintenance of plasma cells The mechanisms responsible for impaired generation of IgA1 B cells and, as a consequence, IgA deficiency observed in the absence of eosinophils is not currently understood (Fig. 2). The explanation cannot be due to some minor detail affecting one particular mouse strain, because reduced levels of IgA are seen in eosinophil-lineage ablated DdblGATA-1 and in eosinophil-deficient PHIL animals as well as in mice with impaired homing of eosinophils to the gastrointestinal tract due to a deficiency in eotaxins 1 and 2 or in the eotaxin receptor CCR3 [26,27]. Similarly, in CD47-deficient mice, in which the numbers of eosinophils are reduced, the level of IgA production is also impaired [21]. Multiple changes in the GALT of eosinophil-deficient mice are seen, and it would be interesting to know whether these effects are simply a consequence of the reduced IgA levels or whether the absence of eosinophils has a more direct effect on the development of the gastrointestinal immune compartments (Fig. 2b) [26,27]. The GC reaction in PP is triggered by bacterial antigens, which are constantly transported through highly specialized epithelial cells (M cells) and DCs into the immune tissues. Furthermore, small soluble food antigens and bacterial products were shown to be transported by CX3CR11 macrophages and also by goblet cells from the gut lumen to the mucosal tissues, where CD1031 CD11c1 DC capture them and present them to T cells [49,58]. As IgA-deficiency affects the microbiota, this may feed back to the development of T cell and DC subsets in lamina propria and PP [59] (Fig. 2a). Alternatively, eosinophils in co-operation with ILC may have a regulatory function in the establishment and organization of the intestinal immune compartments (Fig. 2c). In favour of this interpretation is that eosinophils already home to the intestinal tissues before birth and before colonization of the gut with the commensal flora. In contrast, T and B cells will home to the intestinal tissues only when the microbiota is present, and only then will the organized lymphoid structures develop in the anlagen of the Peyer’s patches (Fig. 2c). As eosinophils are present from the start of immune tissue development in the gastrointestinal tract, this provides an explanation for the broad changes seen in the immune compartments of the lamina propria and the PP in eosinophil-deficient mice.
C 2015 British Society for Immunology, Clinical and Experimental Immunology, 183: 57–64 V
MOLECULAR AND CELLULAR ASPECTS OF B CELL BIOLOGY REVIEW SERIES Eosinophils and humoral immunity
Concluding remarks From recent work it has become apparent that eosinophils are more than simply end-stage effector cells of the innate immune system. They play crucial, although as yet incompletely understood roles, in adaptive immune responses. In the bone marrow eosinophils support long-term plasma cell viability, and thus they have an essential impact on humoral immunity and long-term protection of the organism. In the intestinal tissues eosinophils are involved in the development of IgA1 B cells and in the maintenance of plasma cells. It will be an important goal in future to determine whether they effect IgA class-switch recombination directly, or whether they promote indirectly the generation of IgA1 B cells through providing essential cytokines to other cells.
Acknowledgements I would like to thank V. T. Chu for his outstanding work and R. S. Jack for critical discussion and help in the preparation of the manuscript. The work was supported by DFG grant (BE 1171/2). The DRFZ, an Institute of the Leibniz Gemeinschaft, is supported by the Berlin Senate of Research and Education.
Disclosure There are no competing interests.
References 1 Weller PF. Eosinophils: structure and functions. Curr Opin Immunol 1994; 6: 85–90. 2 Rothenberg ME, Hogan SP. The eosinophil. Annu Rev Immunol 2006; 24: 147–74. 3 Gleich GJ. Mechanisms of eosinophil-associated inflammation. J Allergy Clin Immunol 2000; 105: 651–63. 4 Bystrom J, Amin K, Bishop-Bailey D. Analysing the eosinophil cationic protein – a clue to the function of the eosinophil granulocyte. Respir Res 2011; 12: 10. 5 Amin K, Ludviksdottir D, Janson C et al. Inflammation and structural changes in the airways of patients with atopic and nonatopic asthma. BHR Group. Am J Respir Crit Care Med 2000; 162: 2295–301. 6 Yousefi S, Gold JA, Andina N et al. Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense. Nat Med 2008; 14: 949–53. 7 Chu VT, Berek C. Immunization induces activation of bone marrow eosinophils required for plasma cell survival. Eur J Immunol 2012; 42: 130–7. 8 Jordan MB, Mills DM, Kappler J, Marrack P, Cambier JC. Promotion of B cell immune responses via an alum-induced myeloid cell population. Science 2004; 304: 1808–10. 9 Wang HB, Weller PF. Pivotal advance: eosinophils mediate early alum adjuvant-elicited B cell priming and IgM production. J Leukoc Biol 2008; 83: 817–21.
10 Melo RC, Spencer LA, Dvorak AM, Weller PF. Mechanisms of eosinophil secretion: large vesiculotubular carriers mediate transport and release of granule-derived cytokines and other proteins. J Leukoc Biol 2008; 83: 229–36. 11 Lee JJ, Jacobsen EA, McGarry MP, Schleimer RP, Lee NA. Eosinophils in health and disease: the LIAR hypothesis. Clin Exp Allergy 2010; 40: 563–75. 12 Gouon-Evans V, Pollard JW. Eotaxin is required for eosinophil homing into the stroma of the pubertal and cycling uterus. Endocrinology 2001; 142: 4515–21. 13 Throsby M, Herbelin A, Pleau JM, Dardenne M. CD11c1 eosinophils in the murine thymus: developmental regulation and recruitment upon MHC class I-restricted thymocyte deletion. J Immunol 2000; 165: 1965–75. 14 Tulic MK, Sly PD, Andrews D et al. Thymic indoleamine 2,3dioxygenase-positive eosinophils in young children: potential role in maturation of the naive immune system. Am J Pathol 2009; 175: 2043–52. 15 Jung Y, Rothenberg ME. Roles and regulation of gastrointestinal eosinophils in immunity and disease. J Immunol 2014; 193: 999–1005. 16 Carlens J, Wahl B, Ballmaier M, Bulfone-Paus S, Forster R, Pabst O. Common gamma-chain-dependent signals confer selective survival of eosinophils in the murine small intestine. J Immunol 2009; 183: 5600–7. 17 Rothenberg ME, Mishra A, Brandt EB, Hogan SP. Gastrointestinal eosinophils. Immunol Rev 2001; 179: 139–55. 18 Mishra A, Hogan SP, Lee JJ, Foster PS, Rothenberg ME. Fundamental signals that regulate eosinophil homing to the gastrointestinal tract. J Clin Invest 1999; 103: 1719–27. 19 Delogu A, Schebesta A, Sun Q, Aschenbrenner K, Perlot T, Busslinger M. Gene repression by Pax5 in B cells is essential for blood cell homeostasis and is reversed in plasma cells. Immunity 2006; 24: 269–81. 20 Verjan Garcia N, Umemoto E, Saito Y et al. SIRPalpha/CD172a regulates eosinophil homeostasis. J Immunol 2011; 187: 2268–77. 21 Westlund J, Livingston M, Fahlen-Yrlid L, Oldenborg PA, Yrlid U. CD47-deficient mice have decreased production of intestinal IgA following oral immunization but a maintained capacity to induce oral tolerance. Immunology 2012; 135: 236–44. 22 Bartemes KR, Cooper KM, Drain KL, Kita H. Secretory IgA induces antigen-independent eosinophil survival and cytokine production without inducing effector functions. J Allergy Clin Immunol 2005; 116: 827–35. 23 Puga I, Cols M, Barra CM et al. B cell-helper neutrophils stimulate the diversification and production of immunoglobulin in the marginal zone of the spleen. Nat Immunol 2012; 13: 170–80. 24 Sokol CL, Medzhitov R. Role of basophils in the initiation of Th2 responses. Curr Opin Immunol 2010; 22: 73–7. 25 Chu VT, Frohlich A, Steinhauser G et al. Eosinophils are required for the maintenance of plasma cells in the bone marrow. Nat Immunol 2011; 12: 151–9. 26 Chu VT, Beller A, Rausch S et al. Eosinophils promote generation and maintenance of immunoglobulin-A-expressing plasma cells and contribute to gut immune homeostasis. Immunity 2014; 40: 582–93. 27 Jung Y, Wen T, Mingler MK et al. IL-1beta in eosinophilmediated small intestinal homeostasis and IgA production. Mucosal Immunol 2015; 8: 930–42.
C 2015 British Society for Immunology, Clinical and Experimental Immunology, 183: 57–64 V
63
C. Berek
MOLECULAR AND CELLULAR ASPECTS OF B CELL BIOLOGY REVIEW SERIES
28 Marrack P, McKee AS, Munks MW. Towards an understanding of the adjuvant action of aluminium. Nat Rev Immunol 2009; 9: 287–93. 29 Allen CD, Okada T, Cyster JG. Germinal-center organization and cellular dynamics. Immunity 2007; 27: 190–202. 30 Tangye SG. Staying alive: regulation of plasma cell survival. Trends Immunol 2011; 32: 595–602. 31 Slifka MK, Ahmed R. Long-lived plasma cells: a mechanism for maintaining persistent antibody production. Curr Opin Immunol 1998; 10: 252–8. 32 Manz RA, Thiel A, Radbruch A. Lifetime of plasma cells in the bone marrow. Nature 1997; 388: 133–4. 33 Benner R, Hijmans W, Haaijman JJ. The bone marrow: the major source of serum immunoglobulins, but still a neglected site of antibody formation. Clin Exp Immunol 1981; 46: 1–8. 34 Smith KG, Hewitson TD, Nossal GJ, Tarlinton DM. The phenotype and fate of the antibody-forming cells of the splenic foci. Eur J Immunol 1996; 26: 444–8. 35 Zehentmeier S, Roth K, Cseresnyes Z et al. Static and dynamic components synergize to form a stable survival niche for bone marrow plasma cells. Eur J Immunol 2014; 44: 2306–17. 36 Chu VT, Berek C. The establishment of the plasma cell survival niche in the bone marrow. Immunol Rev 2013; 251: 177–88. 37 Winter O, Moser K, Mohr E et al. Megakaryocytes constitute a functional component of a plasma cell niche in the bone marrow. Blood 2010; 116: 1867–75. 38 Cassese G, Arce S, Hauser AE et al. Plasma cell survival is mediated by synergistic effects of cytokines and adhesion-dependent signals. J Immunol 2003; 171: 1684–90. 39 Belnoue E, Pihlgren M, McGaha TL et al. APRIL is critical for plasmablast survival in the bone marrow and poorly expressed by early-life bone marrow stromal cells. Blood 2008; 111: 2755–64. 40 Mohr E, Serre K, Manz RA et al. Dendritic cells and monocyte/ macrophages that create the IL-6/APRIL-rich lymph node microenvironments where plasmablasts mature. J Immunol 2009; 182: 2113–23. 41 Suzuki K, Maruya M, Kawamoto S et al. The sensing of environmental stimuli by follicular dendritic cells promotes immunoglobulin A generation in the gut. Immunity 2010; 33: 71–83. 42 Hooper LV, Littman DR, Macpherson AJ. Interactions between the microbiota and the immune system. Science 2012; 336: 1268–73. 43 Cerutti A. Location, location, location: B-cell differentiation in the gut lamina propria. Mucosal Immunol 2008; 1: 8–10. 44 Fagarasan S, Kawamoto S, Kanagawa O, Suzuki K. Adaptive immune regulation in the gut: T cell-dependent and T cellindependent IgA synthesis. Annu Rev Immunol 2010; 28: 243–73. 45 Macpherson AJ, Gatto D, Sainsbury E, Harriman GR, Hengartner H, Zinkernagel RM. A primitive T cell-independent
64
46
47
48
49
50
51
52
53
54
55
56
57
58
59
mechanism of intestinal mucosal IgA responses to commensal bacteria. Science 2000; 288: 2222–6. Thurnheer MC, Zuercher AW, Cebra JJ, Bos NA. B1 cells contribute to serum IgM, but not to intestinal IgA, production in gnotobiotic Ig allotype chimeric mice. J Immunol 2003; 170: 4564–71. Suzuki K, Maruya M, Kawamoto S, Fagarasan S. Roles of B-1 and B-2 cells in innate and acquired IgA-mediated immunity. Immunol Rev 2010; 237: 180–90. Litinskiy MB, Nardelli B, Hilbert DM et al. DCs induce CD40independent immunoglobulin class switching through BLyS and APRIL. Nat Immunol 2002; 3: 822–9. Miller MJ, Knoop KA, Newberry RD. Mind the GAPs: insights into intestinal epithelial barrier maintenance and luminal antigen delivery. Mucosal Immunol 2014; 7: 452–4. Kruglov AA, Grivennikov SI, Kuprash DV et al. Nonredundant function of soluble LTalpha3 produced by innate lymphoid cells in intestinal homeostasis. Science 2013; 342: 1243–6. Kang HS, Chin RK, Wang Y et al. Signaling via LTbetaR on the lamina propria stromal cells of the gut is required for IgA production. Nat Immunol 2002; 3: 576–82. Mishra A, Hogan SP, Brandt EB, Rothenberg ME. Peyer’s patch eosinophils: identification, characterization, and regulation by mucosal allergen exposure, interleukin-5, and eotaxin. Blood 2000; 96: 1538–44. Hirota K, Turner JE, Villa M et al. Plasticity of TH17 cells in Peyer’s patches is responsible for the induction of T celldependent IgA responses. Nat Immunol 2013; 14: 372–9. Kato LM, Kawamoto S, Maruya M, Fagarasan S. Gut TFH and IgA: key players for regulation of bacterial communities and immune homeostasis. Immunol Cell Biol 2014; 92: 49–56. Tsuji M, Komatsu N, Kawamoto S et al. Preferential generation of follicular B helper T cells from Foxp31 T cells in gut Peyer’s patches. Science 2009; 323: 1488–92. Dullaers M, Li D, Xue Y et al. A T cell-dependent mechanism for the induction of human mucosal homing immunoglobulin A-secreting plasmablasts. Immunity 2009; 30: 120–9. van Ginkel FW, Wahl SM, Kearney JF et al. Partial IgAdeficiency with increased Th2-type cytokines in TGF-beta 1 knockout mice. J Immunol 1999; 163: 1951–7. Mazzini E, Massimiliano L, Penna G, Rescigno M. Oral tolerance can be established via gap junction transfer of fed antigens from CX3CR1(1) macrophages to CD103(1) dendritic cells. Immunity 2014; 40: 248–61. Geem D, Medina-Contreras O, McBride M, Newberry RD, Koni PA, Denning TL. Specific microbiota-induced intestinal Th17 differentiation requires MHC class II but not GALT and mesenteric lymph nodes. J Immunol 2014; 193: 431–8.
C 2015 British Society for Immunology, Clinical and Experimental Immunology, 183: 57–64 V
MOLECULAR AND CELLULAR ASPECTS OF B CELL BIOLOGY REVIEW SERIES
Clinical and Experimental Immunology
R EVI EW A RT I C L E
Eosinophils: important players in humoral immunity
C. Berek
Summary
B cell Immunology, Deutsches Rheuma Forschungszentrum, Berlin, Germany
Accepted for publication 19 August 2015 Correspondence: C. Berek, Deutsches Rheuma Forschungszentrum, Chariteplatz 1, 10117 Berlin, Germany. E-mail: [email protected]
Eosinophils perform numerous tasks. They are involved in inflammatory reactions associated with innate immune defence against parasitic infections and are also involved in pathological processes in response to allergens. Recently, however, it has become clear that eosinophils also play crucial non-inflammatory roles in the generation and maintenance of adaptive immune responses. Eosinophils, being a major source of the plasma cell survival factor APRIL (activation and proliferation-induced ligand), are essential not only for the long-term survival of plasma cells in the bone marrow, but also for the maintenance of these cells in the lamina propria which underlies the gut epithelium. At steady state under non-inflammatory conditions eosinophils are resident cells of the gastrointestinal tract, although only few are present in the major organized lymphoid tissue of the gut – the Peyer’s patches (PP). Surprisingly, however, lack of eosinophils abolishes efficient class-switching of B cells to immunoglobulin (Ig)A in the germinal centres of PP. Thus, eosinophils are required to generate and to maintain mucosal IgA plasma cells, and as a consequence their absence leads to a marked reduction of IgA both in serum and in the gut-associated lymphoid tissues (GALT). Eosinophils thus have an essential part in longterm humoral immune protection, as they are crucial for the longevity of antibody-producing plasma cells in the bone marrow and, in addition, for gut immune homeostasis. Keywords: eosinophil, IgA, mucosal immunity, plasma cell
Introduction Eosinophils are short-lived cells, which are generated continuously from haematopoietic stem cells in the bone marrow. They are generally thought of as being cells of the innate immune system, and they accumulate typically in the tissues in response to parasitic infection or other inflammatory conditions. On the downside, eosinophils have a critical role in allergen-induced inflammatory responses, such as asthmatic lung disease or atopic dermatitis [1–3]. Eosinophils are regarded as end-stage effector cells which, when activated, degranulate and release highly cytotoxic substances, such as the major basic protein or the eosinophil cationic protein. In the case of an infection these granule proteins may act directly against parasites; however, in an allergic situation they contribute to tissue destruction and in patients with atopic asthma the damage to the lung epithelium is correlated with the number of influxing eosinophils [4,5]. Furthermore, fully activated
eosinophils may respond by ejecting extracellular traps consisting of mitochondrial DNA and tissue destructive granular proteins [6]. In this way eosinophils, like neutrophils, are able to trap bacteria and kill them. However, this cytotoxic reaction will occur only under inflammatory conditions, when eosinophils are highly stimulated by cytokines such as interferon (IFN)-g and, in addition, high levels of interleukin (IL)-5 are required to induce DNA net formation [6]. In addition to these inflammatory functions, eosinophils also play a beneficial role in regulating and modulating immune responses. They do this, at least in part, by synthesizing and secreting a surprisingly broad spectrum of different cytokines and immune mediators [2]. In immune responses, activated eosinophils show an enhanced expression of cytokines [7–9]. Although activation can be induced by adjuvants alone, a stable activation is achieved only in antigen-induced immune responses. It is possible
C 2015 British Society for Immunology, Clinical and Experimental Immunology, 183: 57–64 V
57
C. Berek
MOLECULAR AND CELLULAR ASPECTS OF B CELL BIOLOGY REVIEW SERIES
that immunoglobulin (Ig) secreted by plasma cells contributes to the stable and enhanced cytokine expression of eosinophils [7]. As is characteristic for cells of the innate immune system the responses they mount to activation are immediate, as many of the mediators are already preformed in the cell and stored in granules or in granule-derived small transport vesicles [10]. Receptor-mediated specific recruitment of cytokines allows release of mediators in a specific and controlled fashion. Under physiological conditions this selective process of piecemeal degranulation does not result in cell death [10]. Recent data suggest that eosinophils are required for tissue integrity and are necessary for tissue remodelling [11]. Under normal non-inflammatory conditions eosinophils are recruited in an eotaxin-dependent fashion into various tissues. Eosinophils are found in the pubertal uterus and may have a role in the onset of the oestrous cycle and in preparing the uterus for pregnancy. Similarly, in prepubertal mammary glands, eosinophils together with macrophages regulate normal breast ductal development [11,12]. Furthermore, eosinophils accumulate in the thymus of the newborn and it has been suggested that they contribute to class I restricted thymocyte selection [11,13,14]. At steady state, the large majority of eosinophils in the body are located in the intestinal tissues. In particular, they are resident in the lamina propria of the small intestine, caecum, colon and stomach, where they localize to the submucosa [15–17]. The presence of these eosinophils in the intestinal tissues is independent of overt infection or inflammation. Indeed, eosinophils already accumulate in the gut-associated lymphoid tissue (GALT) during embryonic development, long before bacteria colonize the gut, so that at birth a full complement of eosinophils is already present in the lamina propria [18]. This is quite different from the situation with lymphocytes, where a full complement is reached in the GALT only at approximately 2 years of age [18], and the immigration of T and B cells to establish the mucosal immune tissues is dependent upon the presence of a microbiota in the gut lumen. The constitutive expression of eotaxin in the gastrointestinal tract is required for the population of the gut mucosal tissue with eosinophils and their maintenance there is supported by type 2 innate lymphoid cells (ILC-2), which constitutively secrete IL-5 and IL-13 [15]. Signalling through the common g chain of the IL-5 and IL-13 receptors prolongs the survival of eosinophils in the gastrointestinal tissues where, in contrast to blood or other tissues, they survive for more than 2 weeks [16]. Altogether, the lamina propria seems to provide a microenvironment supportive for eosinophil maintenance. Both plasma cells and gut epithelial cells express CD47, a ligand for the inhibitory receptor signal regulatory protein a (SIRPa) (CD172a) [19,20], which is expressed on eosinophils where its engagement with CD47 has been shown to inhibit degranulation and 58
apoptosis [20,21]. Furthermore, human eosinophils bind secretory IgA via the FcaR receptor; this prevents apoptosis and hence prolongs eosinophil survival [22]. In recent years it has become increasingly apparent that the various granulocyte populations have a broader role than merely that of aggressive end-stage effector cells [23,24]. Neutrophils, basophils and eosinophils present antigen to T cells, suggesting an important impact upon T cell activation. In addition, neutrophils function as B helper cells by inducing Ig class-switch somatic hypermutation and differentiation of marginal zone B cells into plasma cells [23]. What, then, is the function of eosinophils at steady state under non-inflammatory physiological conditions? This review will discuss recent data showing that eosinophils have a major role in protective humoral immunity, as they are a main source of activation and proliferation-induced ligand (APRIL) and of IL-6, which are the key cytokines essential for the survival of antibodyproducing plasma cells, both in the bone marrow and in the lamina propria [25]. Furthermore, eosinophils have a crucial role in the regulation of mucosal immune processes and in the control of immune homeostasis at mucosal sites. The use of eosinophil-deficient mice has demonstrated the important function of these cells in the generation of IgAexpressing B cells, and thus in the development of the IgA plasma cells which reside in the lamina propria [26,27]. Although the mechanisms by which eosinophils perform these functions are not yet understood, it is now clear that these cells have essential roles in adaptive immunity, as they are required for the long-term survival of plasma cells in the bone marrow and for the generation and maintenance of IgA-secreting plasma cells at mucosal sites.
In T cell-dependent immune responses eosinophils enhance early B cell activation Efficient immunization or vaccination requires the use of adjuvants [28]. In particular, the precipitation of antigen with aluminum hydroxide (alum) has an enhancing effect on the immune response, and alum administration has been shown to stimulate eosinophil development in the bone marrow and to promote their accumulation in the spleen [9]. In T cell-dependent immune responses, B cell activation can result in an immediate extrafollicular response to antigenic challenge, whereby antigen-activated B cells differentiate rapidly into IgM-secreting plasma cells. This early immediate B cell response to antigenic challenge requires the presence of eosinophils [8,9], whereas the induction of splenic germinal centres (GC) and the generation of high-affinity IgG-secreting plasma cells seem to be independent of them [25]. Recently it was suggested that danger signals, induced by cell stress and/or cell death, might initiate the recruitment of eosinophils [11]. One site at which many cells are driven into apoptosis is the GC where the T-dependent immune
C 2015 British Society for Immunology, Clinical and Experimental Immunology, 183: 57–64 V
MOLECULAR AND CELLULAR ASPECTS OF B CELL BIOLOGY REVIEW SERIES Eosinophils and humoral immunity
response develops. Development in the GC involves the rapid expansion of an antigen-specific B cell clone, hypermutation of the B cell receptor (BCR) and the selection of mutants with increased affinity for the antigen. In the socalled dark zone of the GC B cells divide every 6–8 h, and within a few days a single B cell will have generated a large clone of several 1000 cells [29]. Most of these cells will die by apoptosis, and only the few high-affinity mutants are selected to differentiate into B memory and plasma cells. Eosinophils seem not to be involved in these processes, as eosinophil-deficient DdblGATA-1 mice have normal GC reactions, antibody-affinity maturation is not affected and the differentiation of B cells into IgG1 secreting plasma cells is comparable to that of wild-type animals [25].
Eosinophils and megakaryocytes are required for efficient homing of plasma cells to bone marrow Plasma cells generated during a GC reaction leave the follicular structures and may home to the bone marrow, where they can survive and secrete antibody for years or even decades [30–32]. After primary immunization with a T cell-dependent antigen, there is only a slight increase in the number of antigen-specific plasma cells in the bone marrow, but in secondary responses this influx of plasma cells is considerable [25,33,34]. A histological analysis of the bone marrow of mice after secondary immunization showed that most of the plasma cells are in close contact with stromal cells, and this network of vascular cell adhesion protein 1 (V-CAM)1 reticular stromal cells seems to be crucial for the organization of the plasma cell survival niche in the bone marrow. Macrophages, megakaryocytes, B and T cells and also neutrophils are found in the vicinity of plasma cells; however, a statistical evaluation of the data showed that only eosinophils have a specific co-localization with the plasma cells [35–37]. The question arises as to which cells are required for the homing of plasma cells to the bone marrow and which are required for long-term survival of plasma cells. Analysis of c-mpl-deficient mice, which have reduced numbers of megakaryocytes, showed that the megakaryocytes play a role in the retention of plasma cells in the bone marrow [37]. In these mice the number of plasma cells in the bone marrow was reduced significantly during the first days after antigenic challenge; however, by 4 weeks the numbers had normalized. This suggests that the homing of the newly generated plasma cells to the bone marrow is aided by the presence of megakaryocytes. However, while these immediate interactions affect the initial rate of entry of plasma cells into the bone marrow, they are not essential for the establishment of a long-term plasma cell survival niche. Immunization of eosinophil-deficient DdblGATA-1 mice showed that, in comparison with wild-type BALB/c mice, many fewer plasma cells home to the bone marrow and 8
weeks after secondary challenge almost no antigen-specific plasma cells were detected [25]. Reconstitution of DdblGATA-1 mice with eosinophils prior to secondary challenge with antigen resulted in enhanced homing of plasma cells to the bone marrow. As eosinophils are shortlived cells the effect was transient [25], but these data imply that eosinophils play crucial roles both in the homing of plasma cells to the bone marrow and their retention.
The establishment of the plasma cell survival niche The establishment of the plasma cell survival niche in the bone marrow is a complex and time-consuming process. In the first weeks after secondary immunization, the newly generated plasma blasts (immature plasma cells) are found mainly in the vicinity of the sinuses in the bone marrow, and many of these cells are in close contact with eosinophils [36]. At this time-point single eosinophils are found in contact with single plasma blasts, and these direct interactions seem to be necessary for the development of plasma blasts into mature long-lived plasma cells. Only a few plasma cells are found in the bone marrow in eosinophil-deficient mice, and these seem not to be fully developed, as they secrete less antibody than do the plasma cells in the bone marrow of wild-type control animals. Four weeks after antigenic challenge plasma cells are now found deep in the parenchyma embedded in nests of eosinophils (Fig. 1) [36]. This suggests that these eosinophil nests are the survival niches, which support longevity of plasma cells. Eosinophils in the bone marrow are short-lived and therefore have a high turnover [16,35], while plasma cells, in contrast, are long-lived [30–32]. Although plasma cells express high levels of CD47, which inhibits eosinophil degranulation and protects them from apoptosis [19,20], in-vivo pulse-chase labelling with the thymidine analogue 5-ethynyl-20 -deoxyuridine (EdU) has shown that the eosinophil survival time is very much shorter than that of plasma cells [35]. This implies that the plasma cell survival niche in the bone marrow is a dynamic niche, where dying eosinophils are replaced constantly with newly generated ones [36].
Eosinophils are the main source of plasma cell survival factors Stromal cells in the bone marrow secrete the chemokine CXCL12, which attracts both CXCR4-expressing plasma cells and eosinophils. In-vitro cultures have shown that while the chemokine CXCL12 helps to support the maintenance of plasma cells [38], the crucial survival factor for the long-term maintenance of plasma cells is APRIL [39]. In the lymph node and the spleen, plasma cell survival is supported by macrophages expressing APRIL [36,40]. However, in the bone marrow, where the vast majority of plasma cells reside, macrophages alone are not sufficient, as
C 2015 British Society for Immunology, Clinical and Experimental Immunology, 183: 57–64 V
59
C. Berek
MOLECULAR AND CELLULAR ASPECTS OF B CELL BIOLOGY REVIEW SERIES
Fig. 1. The plasma cell survival niche in the bone marrow. Mice were immunized with a T cell-dependent antigen and 2 months after secondary challenge with antigen, bone marrow was taken and frozen tissue sections (6 lm) prepared. To visualize eosinophils and plasma cells, sections were stained with major basic protein-specific antibodies (red) and with anti immunoglobulin (Ig)G (green), respectively. Sections were counterstained with 40 ,6-diamidino-2phenylindole (DAPI).
depletion of eosinophils by injection of Siglec F-specific antibody induces a rapid loss both of eosinophils and of plasma cells [25]. Furthermore, at steady state only a few plasma cells are found in the bone marrow of eosinophildeficient mice, and when these animals are immunized with a T cell-dependent antigen almost no plasma cells home to the bone marrow and practically no long-lived plasma cells are detected [25]. Thus, eosinophils are essential for the maintenance of long-lived plasma cells in the bone marrow.
Eosinophils are required for the maintenance of plasma cells in the lamina propria At steady state large numbers of eosinophils are found in the lamina propria, in particular in the small intestine and the caecum. As in the bone marrow, intestinal eosinophils express high levels of the plasma cell survival factors APRIL and IL-6 [26]. Co-staining of tissue sections with antibodies specific for APRIL showed that epithelial cells are the main producers within the lamina propria, followed by eosinophils, while dendritic cells (DC) express much less. Neutrophils, which were shown to express APRIL and B cell-activating factor (BAFF) and thus support the differentiation of marginal zone B cells [23], do not seem to have 60
an important function in mucosal B cell activation at steady state. A significant influx of neutrophils is induced only under inflammatory conditions. When animals are injected with Siglec F-specific antibodies, which induce apoptosis in eosinophils, a rapid loss of plasma cells follows. As soon as the eosinophils are replenished, plasma cells, which are being generated continuously in the intestinal immune tissue, also reappear [26]. Thus, both in the bone marrow and in the lamina propria, plasma cell survival is dependent upon the presence of eosinophils. In the lamina propria of eosinophil-deficient mice the overall level of the plasma cell survival factors APRIL and IL-6 is not reduced significantly [27], most probably because epithelial cells express these cytokines abundantly. Nevertheless, abundant though this epithelial cell expression of APRIL and IL-6 is, it is not sufficient for the maintenance of plasma cells. It is well possible that the effective promotion of plasma cell maintenance requires close contact with the source of survival factors. Alternatively, the eosinophils in the intestinal mucosa may provide additional signals through direct cell contact with plasma cells, as has also been proposed for the survival niche in the bone marrow [36]. Normally, IgA is the most abundant antibody class in the mucosa and has a crucial function in the maintenance of immune homeostasis of the gut-associated tissues. Deficiencies in IgA production also have a strong effect in shaping the microbial community in the gut lumen [41,42]. Because, in eosinophil-deficient mice, the amount of IgAsecreting plasma is strongly reduced, there is a dramatic effect on the levels of IgA both in the circulation and at mucosal sites. It is therefore not surprising that in the absence of eosinophils there is a disruption of mucosal homeostasis, which includes both considerable changes to the mucus layer of the gut and to changes in the microorganism populations in the gut lumen [26,27].
The generation of mucosal IgA1 B and plasma cells The majority of IgA1 B and plasma cells in the lamina propria are generated in the organized lymphoid structures of the Peyer’s patches (PP), where Ig class-switch recombination requires help from T cells [43,44]. To what extent B cells belonging to the B1 subset contribute to the generation of intestinal IgA plasma cells is currently a matter of controversy [45–47]. The activation of these B1 cells and the induction of class-switch recombination to IgA have been shown to be T cell-independent [45]. Similarly, B cell activation in isolated lymphoid follicles (ILF) is independent of T cell help. The absence of eosinophils affects not only the number of IgA plasma cells, but also results in a strong reduction in the number of IgA1 B cells in the lamina propria and in ILF. In addition, there is a strong reduction in the number
C 2015 British Society for Immunology, Clinical and Experimental Immunology, 183: 57–64 V
MOLECULAR AND CELLULAR ASPECTS OF B CELL BIOLOGY REVIEW SERIES Eosinophils and humoral immunity
of IgA1 B cells in the germinal centres of PP and, in contrast to wild-type BALB/c mice, the majority of IgA1 B cells in the lamina propria belong to the B1 subset. As the overall number of IgM1 B cells in the lamina propria of eosinophil-deficient mice was normal, this suggests that eosinophils in the GALT have an important role in the differentiation of IgM1 B cells to IgA1 B cells.
Eosinophils may directly support IgA class-switch of B cells The question arises as to how lack of eosinophils impairs the generation of IgA1 B cells in the lamina propria, in PP and also in the ILF of eosinophil-deficient mice (Fig. 2a). Eosinophils in the lamina propria express transforming growth factor b (TGF-b) as well as APRIL and BAFF which, together, support synergistically the T cellindependent switch to IgA [48]. TGF-b is produced in an inactive form, which requires processing by metalloproteases for its activation, and eosinophils are a major source of the matrix metalloproteinases (MMP)-2 and MMP-9, as shown by a significant reduction of these enzymes in the lamina propria of eosinophil-deficient mice [26]. Eosinophils isolated from the lamina propria of myeloid differentiation primary response gene 88/TIR-domain-containing adapter-inducing interferon-b (MyD88/TRIF) double-deficient mice show decreased expression of the cytokines TGF-b, APRIL, BAFF and the metalloproteases MMP-2 and MMP-9, suggesting that in wild-type animals eosinophils in the gut mucosa are activated by bacterial antigens in the lamina propria via Toll-like receptors (TLRs). In-vitro co-cultures of splenic IgD1 B cells and eosinophils with bacterial antigens showed that only eosinophils from wild-type animals but not those from MyD88/ TRIF double-deficient mice were able to promote switching
to IgA [26]. Nevertheless, further work will be required to demonstrate whether eosinophils support B cell Ig classswitching in vivo.
Eosinophils are required for T cell-independent generation of IgA The relationship between the microbiota, B cell activation in the lamina propria and T cell-independent IgA class switch is not well understood [43–45,49]. Recent work suggested a role for ILC-expressing membrane-bound lymphotoxin alpha (LTa) in the T cell-independent generation of IgA [50,51]. It was shown that the interaction of membrane-bound LTa with its receptor LTbR on lamina propria stromal cells is required to establish a microenvironment that supports T-independent IgA class-switch. In the absence of LTa or LTbR the efficiency of B1 cells homing to the lamina propria is reduced, leading to a considerable reduction in the number of mucosal B cells. The level of mucosal IgA is consequently lower than in normal mice [51]. In addition, DC from LTa-deficient mice were less potent in supporting IgA class-switching than were those from wild-type controls [50]. Eosinophils may play a role here, because they have been shown to express high levels of IL-1b, a cytokine that promotes proliferation of ILC, and in addition enhances the expression of LTa [27]. Loss of eosinophil-derived IL-1b may thus play an indirect role in T cell-independent IgA switching in the lamina propria [27]. However, the analysis of B cell subsets in the lamina propria of eosinophil-deficient mice showed that this can only be part of the story, as the numbers of IgA1 B1 cells were reduced only marginally, whereas IgA1 B2 cells were reduced strongly [26]. Thus, it is primarily the T celldependent IgA class-switch in the GC of PP that is affected by the lack of eosinophils [17,26].
(a)
(b)
(c)
Fig. 2. Eosinophils promote immune homeostasis in the gastrointestinal tract. C 2015 British Society for Immunology, Clinical and Experimental Immunology, 183: 57–64 V
61
C. Berek
MOLECULAR AND CELLULAR ASPECTS OF B CELL BIOLOGY REVIEW SERIES
Eosinophils are required for T cell-dependent generation of IgA An analysis of B cell development in the PP of eosinophildeficient DdblGATA-1 mice showed normal development of the GC, but only a few IgA B cells were detected. A surprising finding was that the B cells in these PP switch; however, in contrast to wild-type animals, they switch preferentially to IgG1 rather than to IgA. This finding is even more surprising as under steady state conditions eosinophils are practically absent from PP and hence cannot exert direct effects on the generation of IgA1 B cells. In PP, the few eosinophils which are present do not colocalize with B cells, but instead tend to be located in the outer T cell zone together with DC [52]. In PP T follicular helper cells (TFH) play a crucial role by providing cognate help to GC B cells and promote Ig class-switch recombination and B cell differentiation into memory B cells and plasma cells. TFH in the GC of PP apparently develop from forkhead box protein 3 (FoxP3)1 T cells or T helper type 17 (Th17) cells, and thus provide a specific cytokine milieu which favours B cell Ig class-switch recombination to IgA [53–55]. In particular, synergy between the cytokines TGF-b1 and IL-21, both expressed strongly by the TFH of PP, is required for the preferential switch to IgA [56]. The combination of these two cytokines promotes the down-regulation of the chemokine receptor CXCR5 and up-regulation of CCR10, which allows the newly generated IgA plasma cells to leave the PP and home to the lamina propria [56]. In the PP of eosinophil-deficient mice, TFH have aberrantly enhanced expression of the transcription factor GATA3, which is indicative of Th2 helper cells, and accordingly express increased levels of Th2 type cytokines. This is consistent with their support of a preferential switch to IgG1, rather than to IgA [26]. The development of both FoxP31 T cells and Th17 cells requires TGF-b. Lack of eosinophils in DdblGATA-1 mice leading to a deficiency of active TGF-b may prevent the normal development of the TFH cells required to support class-switch recombination to IgA in the PP. In line with this interpretation is the finding that TGF-b-deficient mice have a similar phenotype to the eosinophil-deficient mice, in that there is a shift in TFH cell cytokine expression and that B cells switch preferentially to IgG [57]. Importantly, and in striking contrast to the situation in TGF-b-deficient mice, these effects in eosinophil-deficient mice are restricted to the PP, whereas in the spleen cytokine expression of TFH was not affected [26,57]. It is a characteristic of mucosal tissues that they contain a subset of T cells, expressing CD103, the a chain of a TGF-b inducible integrin. Similarly, a subset of DC expressing CD103 are found in mucosal tissues. In eosinophil-deficient mice the frequency of the CD1031 populations of CD41 T cells was reduced specifically and 62
the frequency of CD1031 DC was also diminished. These effects on the T cell and DC compartments were seen in the lamina propria and PP, but not in mesenteric lymph nodes [26], supporting the notion that the absence of eosinophils leads to a specific reduction in the levels of active TGF-b in the GALT.
Eosinophils may have a broader function than simply the maintenance of plasma cells The mechanisms responsible for impaired generation of IgA1 B cells and, as a consequence, IgA deficiency observed in the absence of eosinophils is not currently understood (Fig. 2). The explanation cannot be due to some minor detail affecting one particular mouse strain, because reduced levels of IgA are seen in eosinophil-lineage ablated DdblGATA-1 and in eosinophil-deficient PHIL animals as well as in mice with impaired homing of eosinophils to the gastrointestinal tract due to a deficiency in eotaxins 1 and 2 or in the eotaxin receptor CCR3 [26,27]. Similarly, in CD47-deficient mice, in which the numbers of eosinophils are reduced, the level of IgA production is also impaired [21]. Multiple changes in the GALT of eosinophil-deficient mice are seen, and it would be interesting to know whether these effects are simply a consequence of the reduced IgA levels or whether the absence of eosinophils has a more direct effect on the development of the gastrointestinal immune compartments (Fig. 2b) [26,27]. The GC reaction in PP is triggered by bacterial antigens, which are constantly transported through highly specialized epithelial cells (M cells) and DCs into the immune tissues. Furthermore, small soluble food antigens and bacterial products were shown to be transported by CX3CR11 macrophages and also by goblet cells from the gut lumen to the mucosal tissues, where CD1031 CD11c1 DC capture them and present them to T cells [49,58]. As IgA-deficiency affects the microbiota, this may feed back to the development of T cell and DC subsets in lamina propria and PP [59] (Fig. 2a). Alternatively, eosinophils in co-operation with ILC may have a regulatory function in the establishment and organization of the intestinal immune compartments (Fig. 2c). In favour of this interpretation is that eosinophils already home to the intestinal tissues before birth and before colonization of the gut with the commensal flora. In contrast, T and B cells will home to the intestinal tissues only when the microbiota is present, and only then will the organized lymphoid structures develop in the anlagen of the Peyer’s patches (Fig. 2c). As eosinophils are present from the start of immune tissue development in the gastrointestinal tract, this provides an explanation for the broad changes seen in the immune compartments of the lamina propria and the PP in eosinophil-deficient mice.
C 2015 British Society for Immunology, Clinical and Experimental Immunology, 183: 57–64 V
MOLECULAR AND CELLULAR ASPECTS OF B CELL BIOLOGY REVIEW SERIES Eosinophils and humoral immunity
Concluding remarks From recent work it has become apparent that eosinophils are more than simply end-stage effector cells of the innate immune system. They play crucial, although as yet incompletely understood roles, in adaptive immune responses. In the bone marrow eosinophils support long-term plasma cell viability, and thus they have an essential impact on humoral immunity and long-term protection of the organism. In the intestinal tissues eosinophils are involved in the development of IgA1 B cells and in the maintenance of plasma cells. It will be an important goal in future to determine whether they effect IgA class-switch recombination directly, or whether they promote indirectly the generation of IgA1 B cells through providing essential cytokines to other cells.
Acknowledgements I would like to thank V. T. Chu for his outstanding work and R. S. Jack for critical discussion and help in the preparation of the manuscript. The work was supported by DFG grant (BE 1171/2). The DRFZ, an Institute of the Leibniz Gemeinschaft, is supported by the Berlin Senate of Research and Education.
Disclosure There are no competing interests.
References 1 Weller PF. Eosinophils: structure and functions. Curr Opin Immunol 1994; 6: 85–90. 2 Rothenberg ME, Hogan SP. The eosinophil. Annu Rev Immunol 2006; 24: 147–74. 3 Gleich GJ. Mechanisms of eosinophil-associated inflammation. J Allergy Clin Immunol 2000; 105: 651–63. 4 Bystrom J, Amin K, Bishop-Bailey D. Analysing the eosinophil cationic protein – a clue to the function of the eosinophil granulocyte. Respir Res 2011; 12: 10. 5 Amin K, Ludviksdottir D, Janson C et al. Inflammation and structural changes in the airways of patients with atopic and nonatopic asthma. BHR Group. Am J Respir Crit Care Med 2000; 162: 2295–301. 6 Yousefi S, Gold JA, Andina N et al. Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense. Nat Med 2008; 14: 949–53. 7 Chu VT, Berek C. Immunization induces activation of bone marrow eosinophils required for plasma cell survival. Eur J Immunol 2012; 42: 130–7. 8 Jordan MB, Mills DM, Kappler J, Marrack P, Cambier JC. Promotion of B cell immune responses via an alum-induced myeloid cell population. Science 2004; 304: 1808–10. 9 Wang HB, Weller PF. Pivotal advance: eosinophils mediate early alum adjuvant-elicited B cell priming and IgM production. J Leukoc Biol 2008; 83: 817–21.
10 Melo RC, Spencer LA, Dvorak AM, Weller PF. Mechanisms of eosinophil secretion: large vesiculotubular carriers mediate transport and release of granule-derived cytokines and other proteins. J Leukoc Biol 2008; 83: 229–36. 11 Lee JJ, Jacobsen EA, McGarry MP, Schleimer RP, Lee NA. Eosinophils in health and disease: the LIAR hypothesis. Clin Exp Allergy 2010; 40: 563–75. 12 Gouon-Evans V, Pollard JW. Eotaxin is required for eosinophil homing into the stroma of the pubertal and cycling uterus. Endocrinology 2001; 142: 4515–21. 13 Throsby M, Herbelin A, Pleau JM, Dardenne M. CD11c1 eosinophils in the murine thymus: developmental regulation and recruitment upon MHC class I-restricted thymocyte deletion. J Immunol 2000; 165: 1965–75. 14 Tulic MK, Sly PD, Andrews D et al. Thymic indoleamine 2,3dioxygenase-positive eosinophils in young children: potential role in maturation of the naive immune system. Am J Pathol 2009; 175: 2043–52. 15 Jung Y, Rothenberg ME. Roles and regulation of gastrointestinal eosinophils in immunity and disease. J Immunol 2014; 193: 999–1005. 16 Carlens J, Wahl B, Ballmaier M, Bulfone-Paus S, Forster R, Pabst O. Common gamma-chain-dependent signals confer selective survival of eosinophils in the murine small intestine. J Immunol 2009; 183: 5600–7. 17 Rothenberg ME, Mishra A, Brandt EB, Hogan SP. Gastrointestinal eosinophils. Immunol Rev 2001; 179: 139–55. 18 Mishra A, Hogan SP, Lee JJ, Foster PS, Rothenberg ME. Fundamental signals that regulate eosinophil homing to the gastrointestinal tract. J Clin Invest 1999; 103: 1719–27. 19 Delogu A, Schebesta A, Sun Q, Aschenbrenner K, Perlot T, Busslinger M. Gene repression by Pax5 in B cells is essential for blood cell homeostasis and is reversed in plasma cells. Immunity 2006; 24: 269–81. 20 Verjan Garcia N, Umemoto E, Saito Y et al. SIRPalpha/CD172a regulates eosinophil homeostasis. J Immunol 2011; 187: 2268–77. 21 Westlund J, Livingston M, Fahlen-Yrlid L, Oldenborg PA, Yrlid U. CD47-deficient mice have decreased production of intestinal IgA following oral immunization but a maintained capacity to induce oral tolerance. Immunology 2012; 135: 236–44. 22 Bartemes KR, Cooper KM, Drain KL, Kita H. Secretory IgA induces antigen-independent eosinophil survival and cytokine production without inducing effector functions. J Allergy Clin Immunol 2005; 116: 827–35. 23 Puga I, Cols M, Barra CM et al. B cell-helper neutrophils stimulate the diversification and production of immunoglobulin in the marginal zone of the spleen. Nat Immunol 2012; 13: 170–80. 24 Sokol CL, Medzhitov R. Role of basophils in the initiation of Th2 responses. Curr Opin Immunol 2010; 22: 73–7. 25 Chu VT, Frohlich A, Steinhauser G et al. Eosinophils are required for the maintenance of plasma cells in the bone marrow. Nat Immunol 2011; 12: 151–9. 26 Chu VT, Beller A, Rausch S et al. Eosinophils promote generation and maintenance of immunoglobulin-A-expressing plasma cells and contribute to gut immune homeostasis. Immunity 2014; 40: 582–93. 27 Jung Y, Wen T, Mingler MK et al. IL-1beta in eosinophilmediated small intestinal homeostasis and IgA production. Mucosal Immunol 2015; 8: 930–42.
C 2015 British Society for Immunology, Clinical and Experimental Immunology, 183: 57–64 V
63
C. Berek
MOLECULAR AND CELLULAR ASPECTS OF B CELL BIOLOGY REVIEW SERIES
28 Marrack P, McKee AS, Munks MW. Towards an understanding of the adjuvant action of aluminium. Nat Rev Immunol 2009; 9: 287–93. 29 Allen CD, Okada T, Cyster JG. Germinal-center organization and cellular dynamics. Immunity 2007; 27: 190–202. 30 Tangye SG. Staying alive: regulation of plasma cell survival. Trends Immunol 2011; 32: 595–602. 31 Slifka MK, Ahmed R. Long-lived plasma cells: a mechanism for maintaining persistent antibody production. Curr Opin Immunol 1998; 10: 252–8. 32 Manz RA, Thiel A, Radbruch A. Lifetime of plasma cells in the bone marrow. Nature 1997; 388: 133–4. 33 Benner R, Hijmans W, Haaijman JJ. The bone marrow: the major source of serum immunoglobulins, but still a neglected site of antibody formation. Clin Exp Immunol 1981; 46: 1–8. 34 Smith KG, Hewitson TD, Nossal GJ, Tarlinton DM. The phenotype and fate of the antibody-forming cells of the splenic foci. Eur J Immunol 1996; 26: 444–8. 35 Zehentmeier S, Roth K, Cseresnyes Z et al. Static and dynamic components synergize to form a stable survival niche for bone marrow plasma cells. Eur J Immunol 2014; 44: 2306–17. 36 Chu VT, Berek C. The establishment of the plasma cell survival niche in the bone marrow. Immunol Rev 2013; 251: 177–88. 37 Winter O, Moser K, Mohr E et al. Megakaryocytes constitute a functional component of a plasma cell niche in the bone marrow. Blood 2010; 116: 1867–75. 38 Cassese G, Arce S, Hauser AE et al. Plasma cell survival is mediated by synergistic effects of cytokines and adhesion-dependent signals. J Immunol 2003; 171: 1684–90. 39 Belnoue E, Pihlgren M, McGaha TL et al. APRIL is critical for plasmablast survival in the bone marrow and poorly expressed by early-life bone marrow stromal cells. Blood 2008; 111: 2755–64. 40 Mohr E, Serre K, Manz RA et al. Dendritic cells and monocyte/ macrophages that create the IL-6/APRIL-rich lymph node microenvironments where plasmablasts mature. J Immunol 2009; 182: 2113–23. 41 Suzuki K, Maruya M, Kawamoto S et al. The sensing of environmental stimuli by follicular dendritic cells promotes immunoglobulin A generation in the gut. Immunity 2010; 33: 71–83. 42 Hooper LV, Littman DR, Macpherson AJ. Interactions between the microbiota and the immune system. Science 2012; 336: 1268–73. 43 Cerutti A. Location, location, location: B-cell differentiation in the gut lamina propria. Mucosal Immunol 2008; 1: 8–10. 44 Fagarasan S, Kawamoto S, Kanagawa O, Suzuki K. Adaptive immune regulation in the gut: T cell-dependent and T cellindependent IgA synthesis. Annu Rev Immunol 2010; 28: 243–73. 45 Macpherson AJ, Gatto D, Sainsbury E, Harriman GR, Hengartner H, Zinkernagel RM. A primitive T cell-independent
64
46
47
48
49
50
51
52
53
54
55
56
57
58
59
mechanism of intestinal mucosal IgA responses to commensal bacteria. Science 2000; 288: 2222–6. Thurnheer MC, Zuercher AW, Cebra JJ, Bos NA. B1 cells contribute to serum IgM, but not to intestinal IgA, production in gnotobiotic Ig allotype chimeric mice. J Immunol 2003; 170: 4564–71. Suzuki K, Maruya M, Kawamoto S, Fagarasan S. Roles of B-1 and B-2 cells in innate and acquired IgA-mediated immunity. Immunol Rev 2010; 237: 180–90. Litinskiy MB, Nardelli B, Hilbert DM et al. DCs induce CD40independent immunoglobulin class switching through BLyS and APRIL. Nat Immunol 2002; 3: 822–9. Miller MJ, Knoop KA, Newberry RD. Mind the GAPs: insights into intestinal epithelial barrier maintenance and luminal antigen delivery. Mucosal Immunol 2014; 7: 452–4. Kruglov AA, Grivennikov SI, Kuprash DV et al. Nonredundant function of soluble LTalpha3 produced by innate lymphoid cells in intestinal homeostasis. Science 2013; 342: 1243–6. Kang HS, Chin RK, Wang Y et al. Signaling via LTbetaR on the lamina propria stromal cells of the gut is required for IgA production. Nat Immunol 2002; 3: 576–82. Mishra A, Hogan SP, Brandt EB, Rothenberg ME. Peyer’s patch eosinophils: identification, characterization, and regulation by mucosal allergen exposure, interleukin-5, and eotaxin. Blood 2000; 96: 1538–44. Hirota K, Turner JE, Villa M et al. Plasticity of TH17 cells in Peyer’s patches is responsible for the induction of T celldependent IgA responses. Nat Immunol 2013; 14: 372–9. Kato LM, Kawamoto S, Maruya M, Fagarasan S. Gut TFH and IgA: key players for regulation of bacterial communities and immune homeostasis. Immunol Cell Biol 2014; 92: 49–56. Tsuji M, Komatsu N, Kawamoto S et al. Preferential generation of follicular B helper T cells from Foxp31 T cells in gut Peyer’s patches. Science 2009; 323: 1488–92. Dullaers M, Li D, Xue Y et al. A T cell-dependent mechanism for the induction of human mucosal homing immunoglobulin A-secreting plasmablasts. Immunity 2009; 30: 120–9. van Ginkel FW, Wahl SM, Kearney JF et al. Partial IgAdeficiency with increased Th2-type cytokines in TGF-beta 1 knockout mice. J Immunol 1999; 163: 1951–7. Mazzini E, Massimiliano L, Penna G, Rescigno M. Oral tolerance can be established via gap junction transfer of fed antigens from CX3CR1(1) macrophages to CD103(1) dendritic cells. Immunity 2014; 40: 248–61. Geem D, Medina-Contreras O, McBride M, Newberry RD, Koni PA, Denning TL. Specific microbiota-induced intestinal Th17 differentiation requires MHC class II but not GALT and mesenteric lymph nodes. J Immunol 2014; 193: 431–8.
C 2015 British Society for Immunology, Clinical and Experimental Immunology, 183: 57–64 V
