ARTICLE
Received 24 Sep 2011 | Accepted 8 Mar 2013 | Published 23 Apr 2013
Jun Kunisawa1,2,3,4,5, Masashi Gohda1,2, Eri Hashimoto1, Izumi Ishikawa1, Morio Higuchi1,6, Yuji Suzuki1,
Yoshiyuki Goto1,5,7, Casandra Panea7, Ivaylo I. Ivanov7, Risa Sumiya1, Lamichhane Aayam1,2, Taichi Wake1,6, So Tajiri1,2, Yosuke Kurashima1,5,6, Shiori Shikata1, Shizuo Akira8, Kiyoshi Takeda5,9 & Hiroshi Kiyono1,2,3,5,6
Intestinal plasma cells predominantly produce immunoglobulin (Ig) A, however, their functional diversity remains poorly characterized. Here we show that murine intestinal IgA plasma cells can be newly classied into two populations on the basis of CD11b expression, which cannot be discriminated by currently known criteria such as general plasma cell markers, B cell origin and T cell dependence. CD11b IgA plasma cells require the lymphoid structure of
Peyers patches, produce more IgA than CD11b IgA plasma cells, proliferate vigorously, and require microbial stimulation and IL-10 for their development and maintenance. These features allow CD11b IgA plasma cells to mediate early-phase antigen-specic intestinal
IgA responses induced by oral immunization with protein antigen. These ndings reveal the functional diversity of IgA plasma cells in the murine intestine.
1 Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan. 2 Department of Medical Genome Science, Graduate School of Frontier Science, The University of Tokyo, Chiba 277-8562, Japan. 3 International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan. 4 Laboratory of Vaccine Materials, National Institute of Biomedical Innovation, Osaka 567-0085, Japan. 5 Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Tokyo 102-0075, Japan. 6 Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan. 7 Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York 10032, USA. 8 Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan. 9 Laboratory of Immune Regulation, Department of Microbiology and Immunology, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan. Correspondence and requests for materials should be addressed to J.K.(email: mailto:[email protected]
Web End [email protected] ).
NATURE COMMUNICATIONS | 4:1772 | DOI: 10.1038/ncomms2718 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications 1
& 2013 Macmillan Publishers Limited. All rights reserved.
DOI: 10.1038/ncomms2718 OPEN
Microbe-dependent CD11b IgA plasma cells mediate robust early-phase intestinal IgA responses in mice
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2718
Immunoglobulin (Ig) A is an antibody found predominantly in the intestinal lumen, where it protects the host against pathogenic infections1,2. It also has an important role in the
creation and maintenance of immunological homoeostasis by shaping homeostatic communities of commensal bacteria35. Indeed, some patients with IgA deciency show marked susceptibility to infections with pathogens such as Giardia lamblia, Campylobacter, Clostridium, Salmonella and rotavirus; they also have increased incidences of intestinal immune diseases such as coeliac disease and inammatory bowel diseases6.
Peyers patches (PPs) are the major sites for the initiation of antigen-specic intestinal IgA production, mainly in a T cell-dependent manner7. Intestinal IgA also originates from B1 cells. B1 cells differ from B2 cells in terms of origin, surface markers (for examples, B220, IgM, IgD, CD5, CD11b and CD23), growth properties and VH repertoire810. B1 cells are predominantly present in the peritoneal cavity (PerC) and trafc into the intestinal compartment for the production of IgA against T cell-independent antigens such as DNA and phosphatidylcholine11. T cell independent antigen-specic IgA responses are also initiated in the isolated lymphoid follicles (ILFs), which are small clusters of B2 cells in the intestine12.
Upon Ig class switching from m to a, IgA B cells acquire the expression of type 1 sphingosine-1-phosphate receptor, CCR9 and a4b7 integrin, allowing them to migrate out from the PPs or
PerC and trafc to the intestinal lamina propria (iLP)11,13,14. In the iLP, they further differentiate into IgA-secreting plasma cells (PCs) under the inuence of terminal differentiation factors (for example, IL-6)15. As these locally produced IgA antibodies are continuously transported and secreted by epithelial cells as a form of secretory IgA into the intestinal lumen, stably high levels of IgA production are required for the maintenance of sufcient amounts of IgA; this production is determined by the generation, survival and function of IgA PCs.
Several lines of evidence have demonstrated that the function and survival of PCs in the systemic compartments (for example, spleen and bone marrow (BM)) are not only determined by intrinsic factors but are regulated by the presence of environmental niches16. As with systemic PCs, differentiation of IgA PCs in the iLP is regulated by exogenous factors such as IgA-enhancing cytokines (for example, interleukin (IL)-5, IL-6,
IL-10, IL-15, a proliferation-inducing ligand (APRIL) and B cell activating factor (BAFF))7,15. In addition, microbial stimulation is required for the full effects of intestinal IgA. Indeed, germ-free (GF) mice have decreased intestinal IgA responses with immature structures of PPs and ILFs17,18. Previous studies in mono-associated GF mice have indicated that only a small proportion of the total amount of intestinal IgA is reactive to monoassociated bacteria; microbe-dependent IgA production is therefore mediated by polyclonal stimulation through innate immune receptors such as toll-like receptors, rather than through B cell receptors specic for microbial antigens19,20. Accumulating evidence has revealed the molecular and cellular pathways of IgA production mediated by innate immunity, including the involvement of myeloid differentiation primary response gene 88 (MyD88) in the regulation of tumour necrosis factor/inducible nitric oxide synthase-producing DCs in the iLP21 and follicular DCs in the PPs22. However, the effects of microbial stimulation on the regulation of differentiated IgA PCs remain to be investigated. Here, we identied unique microbe-dependent subsets of IgA PCs, which add a new level of complexity to the intestinal IgA system of mice.
ResultsMicrobe dependency of intestinal IgA cells. To examine the immunological elements of intestinal IgA production associated with commensal bacteria, we initially compared the IgA cells of specic pathogen-free (SPF) and GF mice. Flow cytometric analysis showed that CD11b IgA cells accounted for about 30% of IgA cells, and we found a lack of CD11b IgA cells in the iLP of GF mice (Fig. 1a). Similarly, the numbers of intestinal CD11b IgA cells were reduced in both antibiotic-treated SPF mice and MyD88 KO mice (Fig. 1bd). Immunohistological analysis indicated that CD11b IgA cells were dispersed throughout the iLP of wild-type (WT) mice (Fig. 1d), although their frequency appeared lower than expected from the ow cytometric data, probably because of difference in methodological sensitivity. These ndings collectively suggest that CD11b IgA cells are unique subset that requires MyD88-dependent microbial stimulation for its development and maintenance.
SPF CD11b+ IgA+ CD11b IgA+ CD11b+ IgA+
0 + +
GF
Mock
Antibiotics
CD11b IgA+
104
104
104
6.9 0.8
20
104
10 P=0.008 P=0.042
6.6
2.7
10
P<0.001 P=0.62
20
103
103
8
Percentage
Percentage
15
103
103
8
15
102
102
6
10
102
102
Percentage
6
10
4
4
101
101
2
5
101
101
2
5
CD11b
100100
100
0
100
100
101
102
104
100 101 102 104 100 101 102 100 101
0 SPF GF SPF GF
CD11b
104 104
102
14.4 11.4
103 103
14.0 13.3
Antibiotics
0
Antibiotics
IgA
IgA
WT
MyD88 KO
CD11b+ IgA+ CD11b IgA+
WT
MyD88 KO
104
104
10
20
WT KO WT KO
P=0.81
103
8
P<0.001
6.0 1.8
103
15
IgA CD11b DAPI
6
102
102
10
4
101
101
2
5
0
0
CD11b
100 100
100
101
102
103 103
13.8 14.2
104 100 101 104
102
IgA
Figure 1 | Intestinal CD11b IgA cells require microbial stimulation. (ac) Mononuclear cells were isolated from the small intestines of SPF or GF mice (a), mock- or antibiotic-treated SPF mice (b), or MyD88 WTor knockout (KO) mice (c) for analysis of IgA and CD11b expression by ow cytometry. Graphs show data from individual mice, and bars indicate median. Statistical analyses were performed with MannWhitneys U-test. (d) Specimens of small intestinal tissues of WT and MyD88 KO mice were stained for IgA and CD11b, and counterstained with 40,6-diamidino-2-phenylindole. Data are representative of three independent experiments. Scale bars, 50 mm.
2 NATURE COMMUNICATIONS | 4:1772 | DOI: 10.1038/ncomms2718 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications
& 2013 Macmillan Publishers Limited. All rights reserved.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2718 ARTICLE
Intestinal CD11b IgA cells are PCs. We next aimed to characterize the CD11b and CD11b IgA cells in the iLP. In addition to a gating strategy to exclude the possibility that the
CD11b IgA cells detected by ow cytometry were doublets (Supplementary Fig. S1), we further performed a cytospin analysis and conrmed that both CD11b and CD11b IgA cells had homogeneous morphology that was the same as that of PCs (for example, large irregular nuclei with prominent nucleoli), whereas CD11bhi IgA cells were composed of different kinds of cells, including eosinophils and macrophages (Fig. 2a). We also conrmed that both CD11b and CD11b IgA cells did not express markers for macrophages (F4/80), DCs (CD11c) or eosinophils (CCR3) (Fig. 2b). Thus, CD11b IgA cells are neither doublets nor myeloid cells decorated by bound IgA on their surfaces.
CD11b and CD11b IgA cells were identical in cell size and density, as determined by forward scatter (FSC) and side scatter (SSC), respectively, and by their surface expression patterns (CD19int, B220, CD138 , CD38hi and CD40int) (Fig. 2c).
Although PCs in the systemic compartments (for example, the spleen) generally express little or no surface immunogloblin23, we previously conrmed that CD38 CD138 cells in the iLP express IgA both on the cell surface and in the intracellular compartment (Supplementary Fig. S2)13. These ndings indicated that both CD11b and CD11b IgA cells could be classically
categorized as PCs. This view was further supported by our nding that both populations expressed equal levels of Blimp1, a master transcription factor for PCs (Fig. 2c)23.
The phenotypes of IgA cells in the iLP differed from those of IgA cells in the spleen. Splenic CD11b IgA cells exclusively had a memory phenotype (that is, B220 , CD138, CD38int and
CD40hi), whereas splenic CD11b IgA cells contained almost equal amounts of B220 CD138 CD38int CD40hi memory cells and B220 CD138 CD38hi CD40low PCs (Supplementary
Fig. S3). These results indicated that CD11b IgA cells in the iLP were unique PCs that had an immunologically different status from splenic CD11b IgA cells.
Intestinal CD11b IgA PCs require PP lymphoid structure. CD11b IgA PCs expressed CD18 (Supplementary Fig. S4), which associates with CD11b and acts as a ligand for intercellular adhesion molecule-1 (ICAM-1)24. As ICAM-1 is an endothelial adhesion molecule that regulates cell trafcking24,25, we considered that CD11b IgA PCs were recent emigrants from IgA-inductive tissues (for example, PPs and PerC) and had migrated into the iLP. To test this possibility, we employed FTY720 to inhibit the trafcking of IgA-committed B cells from PPs and PerC into the iLP. As we previously reported11,13, FTY720 treatment reduced the numbers of intestinal IgA PCs,
CD11b+ IgA+
CD11b+ IgA
104
10 100
Cell number
100
94.5
95.1
50
103
102
104
0 100 101 102 103 104
100 101 102 103
104
CD11b IgA+
F4/80 CD11c
101
CD11b
0
Control
CD11b IgA+ CD11b+ IgA+
CD11b+ IgA
101
102
103
104
Cell number
100
50
IgA
CCR3
Cell number
100
50
0 0 1,023 0 1,023
0 100 101 102 103
100 101 102 103
104
100 101 102 103
104
Control
CD11b IgA+
CD11b+ IgA+
Cell number
100
50
FSC SSC CD19 B220
CD138 CD38 CD40 Blimp1
104
0 100 101 102 103 104
100 101 102 103 104
100 101 102 103 104
100 101 102 103
Figure 2 | Both CD11b and CD11b IgA cells in the intestine are categorized as plasma cells. (a) Cells were puried by cell sorting from the iLP, and their morphology was examined by haematoxylin and eosin staining after cytospin. Data are representative of three independent experiments.
(b) Cells were isolated from the iLP for the analysis of F4/80, CD11c and CCR3 expression on CD11b IgA , CD11b IgA and CD11b IgA cells. Grey indicates isotype control. Similar results were obtained from three separate experiments. (c) Cells were isolated from the iLP for comparisons between CD11b and CD11b IgA cells in terms of cell size (FSC) and density (SSC), and expression of CD19, B220, CD138, CD38, CD40 and Blimp1.
Grey indicates isotype control. Similar results were obtained from ve separate experiments.
NATURE COMMUNICATIONS | 4:1772 | DOI: 10.1038/ncomms2718 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications 3
& 2013 Macmillan Publishers Limited. All rights reserved.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2718
but the effect was not specic to CD11b IgA PCs (Fig. 3a). These data suggested that CD11b IgA PCs were not recent emigrants from IgA inductive tissues (for example, PPs and
PerC).
The second possibility was that CD11b IgA PCs originated from B1 cells, because CD11b is a marker of peritoneal B1 cells26.
To test this possibility, peritoneal CD11b B1 cells were puried and adoptively transferred into severe combined immunodeciency mice. As we reported previously11, adoptively transferred CD11b B1 cells migrated into the intestine, where they differentiated into IgA PCs. Although we transferred
B cells expressing CD11b, they lost their CD11b expression in the iLP (Supplementary Fig. S5). Although only a few cells were detected in the iLP under these experimental conditions, CD11b expression was likely to be reversible on B cells and was thus not to be a marker of PCs originating from peritoneal CD11b
B1 cells.
As a third possibility for discriminating between CD11b and CD11b IgA PCs, we examined the T cell dependency of their differentiation and IgA production. For this, we employed TCRbd mice. Although TCRb d mice had decreased levels of intestinal IgA cells, the ratio between CD11b and CD11b IgA PCs did not differ between the WT mice and the TCRb d mice (Fig. 3b).
We also examined the production of IgA against T cell dependent and T cell independent antigens by CD11b and
CD11b IgA PCs. For the analysis of T cell dependent antigen, mice were orally immunized with ovalbumin (OVA) plus cholera toxin (CT). Following three oral immunizations, substantial amounts of OVA-specic IgA antibody-forming cells (AFCs) were detected in the iLP by enzyme-linked immunosorbent spot (ELISPOT) assay; this production was reduced by almost 50% when either the CD11b IgA or the CD11b IgA cells were removed before the ELISPOT assay (Fig. 3c). Similar results were
obtained when we enumerated IgA AFCs against phosphorylcholine, a typical TI antigen, induced by commensal bacteria (Fig. 3c)27. These results collectively suggested that both CD11b IgA and CD11b IgA cells almost equally included
IgA AFCs producing IgA antibodies specic for T cell dependent and T cell independent antigens.
Next, to examine the involvement of PPs, we established PP-null mice by in utero treatment with anti-IL-7Ra antibody28 and found that PP-null mice had reduced numbers of CD11b IgA
PCs in the iLP (Fig. 3d). In addition, CD11b was not expressed on IgA B cells in the PPs (Fig. 3e). We treated mice with anti-IL-7Ra antibody only once in utero and conrmed that it did not affect the ILFs28. Although it is still possible that CD11b IgA
PCs specically require IL-7, the most plausible conclusion based on our current ndings is that CD11b IgA B cells require the lymphoid structure of PPs, and CD11b IgA B cells acquire
CD11b expression in the iLP.
As in antibiotic-treated and MyD88 KO mice (Fig. 1), the numbers of CD11b IgA PCs changed little in PP-null mice (Fig. 3d), suggesting that it is unlikely that CD11b IgA PCs differentiate back into CD11b IgA cells in the iLP. This view is further supported by the results of in vitro analysis. When puried CD11b and CD11b IgA PCs were separately cultured with different kinds of stimulants (for example, phorbol 12-myristate 13-acetate plus ionomycin, or lipopolysaccharide) little change was noted in CD11b expression (Supplementary Fig. S6). Although the origin of these cells remains to be rmly established, it is plausible that CD11b IgA PCs act as a separate lineage once they differentiate in the iLP.
High proliferation activity of CD11b IgA PCs. We next performed a gene microarray analysis to assess the uniqueness of CD11b IgA PCs in the iLP. Gene ontology enrich-
P=0.036
P=0.009
OVA-specific IgA AFCs
7.21.4 / 12.82.6
5.41.8 / 9.32.3
Phosphorylcholine-
specific IgA AFCs
500 P<0.001
30 P=0.015
Mock
FTY720
0 5
WT
3.60.2 / 14.42.5
1.50.4 / 6.22.3
400
TCR KO
15
10 0 5 30
25
20
15
10
20
25
100
IgA+ cells (%) IgA+ cells (%)
CD11b(+)
CD11b()
CD11b(+) CD11b()
0 None
CD11b()
CD11b(+)
0 None
CD11b()
CD11b(+)
Depletion
Depletion
Control Ab Anti-IL-7R Ab CD11b+ IgA+ CD11b IgA+
104
103
4.3
104
103
0.8
20
P=0.39
104
103
0.1
8
10 P=0.002
15
102
102
Percentage
6
10
102
4
101
101
5
101
CD11b
2
10 100
0
100
0
CD11b
100
101
102 103
104 100 101 102 104 100 101 102 104
103
14.7
0 Cont Cont
Peyers patch
3.0
IL-7R IL-7R
103
14.4
IgA
IgA
Figure 3 | CD11b IgA cells require the lymphoid structure of Peyers patches. (a) Mice were treated with FTY720 every day for 5 days. The day after the nal treatment, the proportions of CD11b and CD11b IgA cells were measured by ow cytometry. Data are presented as meanss.d. from four mice. Similar results were obtained from three separate experiments. (b) Proportions of CD11band CD11b IgA cells in the iLP of WT and TCRbd
KO mice were measured by ow cytometry. Data are presented as meanss.d. from four mice. Similar results were obtained from three separate experiments. (c) After three oral immunizations with OVA plus cholera toxin, cells were isolated from the iLP and used in an ELISPOT assay to enumerate OVA-specic IgA AFCs. In some groups of mice, CD11b or CD11b IgA cells were depleted by cell sorting before application of ELISPOT assay.
Phosphorylcholine-specic IgA AFCs were measured. Graphs show data from individual mice, and bars indicate median. Statistical analyses were performed with MannWhitneys U-test. (d) Mononuclear cells were isolated from the iLP of Peyers patch (PP)-normal (control Ab) and -null (anti-IL-7Ra
Ab) mice for analysis of IgA and CD11b expression by ow cytometry. Graphs show data from individual mice. Statistical analyses were performed with MannWhitneys U-test. (e) Mononuclear cells were isolated from PPs for analysis of CD11b and CD11b IgA cells by ow cytometry. Similar results were obtained from three separate experiments.
4 NATURE COMMUNICATIONS | 4:1772 | DOI: 10.1038/ncomms2718 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications
& 2013 Macmillan Publishers Limited. All rights reserved.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2718 ARTICLE
ment score computation analysis showed that the activity of cell-cycle-associated pathways was higher in CD11b IgA PCs than in CD11b IgA PCs (Supplementary Table S1). Consistent with this nding, higher expression of cell-cycle-associated genes was noted in CD11b IgA PCs than in CD11b IgA
PCs; these genes included members of the cell division cycle family (Fig. 4a and Supplementary Table S2). In line with this, these cells expressed higher levels of the proliferation marker Ki67 than did CD11b IgA PCs (Fig. 4a and Supplementary
Table S2). Additionally, CD11b IgA PCs showed greater uptake of bromodeoxyuridine (BrdU) than did CD11b IgA
PCs (Fig. 4b). CD11b IgA PCs were preferentially removed by treatment with cyclophosphamide (CPM), which selectively targets proliferating cells (Fig. 4c). These data collectively suggested that CD11b IgA PCs possessed greater proliferating activity than did CD11b IgA PCs in the iLP.
Microarray analysis further identied CD150 (also known as signalling lymphocytic activation molecule family member 1, SLAMF1)29, b1 integrin and CD168 (also known as hyaluronan-mediated motility receptor)30 as possible candidates uniquely expressed on CD11b IgA PCs (Supplementary Table S3).
Flow cytometric analysis conrmed that CD11b IgA PCs expressed higher levels of CD150 than did CD11b IgA PCs, whereas CD11b IgA and CD11b IgA PCs identically expressed b1 integrin and no CD168 (Supplementary Fig. S7).
IL-10 is essential for intestinal CD11b IgA cells. We next aimed to identify key molecules for inducing and maintaining
CD11b IgA PCs in the iLP. As CD11b IgA PC numbers were reduced in MyD88 mice (Fig. 1c), and MyD88 is expressed in not only hematopoietic cells, including B cells, but also nonhematopoietic cells, including epithelial cells31, we performed BM chimeric experiments to determine whether MyD88 in nonhematopoietic cells, hematopoietic cells, or both, was required for the generation of CD11b IgA cells. Similar levels of CD11b
IgA cells were observed in irradiated WT mice receiving WT or MyD88 BM cells and in irradiated MyD88 mice receiving WT
BM cells (Supplementary Fig. S8), suggesting that MyD88-dependent molecules commonly expressed in both non-
hematopoietic and hematopoietic cells are involved in the microbe-dependent induction of CD11b IgA PCs.
We then examined the involvement of cytokines known to enhance IgA responses. Among several IgA-enhancing cytokines (for example, IL-5, IL-6, IL-10 and APRIL/BAFF)7,15, we found that neutralization of IL-10 resulted in preferential reduction in CD11b IgA PCs, whereas blocking of other cytokines induced a reduction in IgA cell numbers regardless of CD11b expression (Fig. 5a). Additionally, CD11b IgA cell numbers were preferentially reduced in IL-10 KO mice (Fig. 5b). As normal differentiation into IgA B cells was observed in the PPs and PerC of IL-10 KO mice (Supplementary Fig. S9), it is plausible that IL-10 targets the maintenance of CD11b IgA cells in the iLP, but not the induction of IgA cells in inductive tissues such as PPs and PerC.
Early-phase robust IgA responses by proliferating IgA PCs. To examine the immunological importance of proliferating IgA
PCs present mainly in CD11b IgA PCs, mice were orally immunized with OVA plus CT. In this assay, one group received
CPM treatment during immunization and the second group received CPM treatment 4 days after the nal immunization (Fig. 6a). Because of the high cell-proliferation activity, CPM treatment during oral immunization resulted in efcient killing of peanut agglutinin (PNAhi) B220 GC B cells and thus a reduction in the numbers of IgA IgM plasmablasts in the PPs (Supplementary Fig. S10). Thus, treatment with CPM during oral immunization led to an B90% reduction in the numbers of
OVA-specic IgA AFCs (Fig. 6b); this was associated with almost complete disappearance of faecal IgA produced against OVA (Fig. 6c). On the other hand, when mice were treated with CPM 4 days after the nal immunization to remove proliferating cells mainly present in CD11b IgA cells in the iLP, the reduction in numbers of OVA-specic IgA AFCs in the iLP was only about 50% (Fig. 6b). This nding was consistent with our current nding that CD11b IgA PCs accounted for half the number of OVA-specic IgA AFCs (Fig. 3c). Thus, CPM treatment after the last immunization preferentially depleted CD11b IgA cells, with little inuence on CD11b
CD11b+ IgA+ CD11b IgA+
Mock CPM
Control
CD11b IgA+
CD11b+ IgA+
104
7.2 1.6
104
10 P<0.001 P=0.11
20
Ccnb1
Cks1b
Cdc6 Ccna2
Sgol1 CD11b
Aurkb Mki67 Incenp
Cdc2a Cdca5
Aurkb Cenpn
Cdca8
Cenpl Cenpp
Cdca8
Crkrs Cdc25c
CD11b +
103
103
8
15
Cell number
100
6
50
102
102
Percentage
10
4
101
101
2
5
CD11b
0 100 101 102 103 104 100 101 102 103 104
100100
100
0 + +
0
101
102
103
104
12.9 9.9
CPM
CPM
BrdU
IgA
3.5 6.9 10.4
Figure 4 | CD11b IgA cells are proliferating cells. (a) mRNA was puried from small intestinal CD11b and CD11b IgA cells and used for microarray analysis. Data related to the cell cycle and proliferation are shown. Data are representative of two independent experiments. (b) Mice were treated with BrdU, and uptake of BrdU by CD11b and CD11b IgA cells was determined by ow cytometry. Data are representative of four independent experiments. (c) Cells were isolated from the intestinal lamina propria of mice receiving CPM to analyse CD11b IgA cells. Similar results were obtained from four separate experiments. Graphs show data from individual mice. Statistical analyses were performed with MannWhitneys U-test.
NATURE COMMUNICATIONS | 4:1772 | DOI: 10.1038/ncomms2718 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications 5
& 2013 Macmillan Publishers Limited. All rights reserved.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2718
136.518.4 / 44.7213.3
63.215.6 / 19.210.6
81.514.5 / 34.63.6
105.310.6 / 16.92.95
104.711.9 / 47.47.3
41.54.6 / 19.13.2
Cell number (104)
WT
IL-10 KO
13.5
Mock
104
104
103
103
IL-5 Ab
IL-6 Ab
IL-10 Ab
TACI-Ig
102
102
101
101
CD11b
100 100
100
101
102
103
104 100 101 102 103 104
0 50 100 150 200
6.4 1.7
13.2
IgA
CD11b+ IgA+ CD11b IgA+
Cont
10
Percentage
P=0.032 P=0.42
0 WT KO WT KO
20
8
15
6
10
4
0 150
10
5
200
2
5
0
CD11b(+)
CD11b()
Figure 5 | Role of IL-10 in the maintenance of CD11b IgA cells in the iLP. (a) Mice were treated with antibodies to block IL-5, IL-6, IL-10 or antagonistic TACI-immunoglobulin (TACI-Ig) fusion protein. Mononuclear cells were isolated from the iLP and used for analysis of CD11b and CD11b
IgA cells by ow cytometry. Data are presented as meanss.d. (n 4). (b) Mononuclear cells were isolated from the iLP of WT or IL-10 KO mice
for analysis of IgA and CD11b expression by ow cytometry. Graphs show data from individual mice. Statistical analyses were performed with MannWhitneys U-test.
Oral immunization with OVA-CT
P=0.019
P<0.001
CPM treatment
OVA-specific IgA AFCs
2.0
500 P=0.002
400
NoneAfter imm. During imm.
1.5
1.0
0.5
CPM (during) CPM (after)
0 7 14 21
18 20
ELISA/ELISPOT
19 25
CT challenge
Absorbance (OD 450)
100
**
*
0 0 1 2
3
None
During
imm.
After
imm.
4 6 8 9
7
5
10
Dilution (log2)
P=0.037
P<0.001
P=0.76 P=0.23
P=0.51
1,000
600
400
200
2.0
P=0.008
Absorbance (OD 450)
Mock
P=0.004
2.0
1.5
14
10
1.0
CTB-specific IgA AFCs
Water volume (ml)
Spot size (Log2)
15
13
11
12
800
P=0.006
P=0.008
P=0.032
1.5
***
1.0
0.5
0.5
CPM
0 7 8 9 10 11 12
0 None
CPM
Dilution (log2)
CD11b(+)
CD11b()
0 Naive
Mock
CPM
Immunized
9 None
CD11b(+)
CD11b()
Depletion
Depletion
Figure 6 | Proliferating IgA cells mediate early-phase IgA responses to oral antigen. (a) Experimental schedule for oral immunization and CPM treatment. Mice were orally immunized with OVA plus CT on days 0, 7 and 14. One group received CPM during oral immunization (days 0, 7 and 14) and another received CPM after the last immunization (days 18, 19 and 20). (b,c) One week after the nal immunization (day 21), mononuclear cells were isolated from the iLP to quantify OVA-specic IgA-forming cells by ELISPOT (b). Simultaneously, faeces (c,d) were collected and were used for the detection of the (c) OVA-or (d) B subunit of CT (CTB)-specic IgA by enzyme-linked immunosorbent assay. Data are from individual mice and bars indicate median (b) and represent meanss.d. (n 10) from two separate experiments (c,d). *Po0.001, **Po0.01, ***Po0.05 (two tailed unpaired
t-test). (e) Mononuclear cells were isolated from the iLP of mock- or CPM-treated mice 1 week after the nal immunization to quantify CTB-specic IgA-forming cells by ELISPOT. In some groups of mock-treated mice, CD11b or CD11b IgA cells were depleted by cell sorting before application of
ELISPOTassay. Graphs show data from individual mice, and bars indicate median. (f) On day 21, mice were orally challenged with 100 mg CT. After 15 h, the volume of intestinal uid was measured. Graphs show data from individual mice, and bars indicate median. Similar results were obtained from two separate experiments. (g) Spot sizes of CTB-specic IgA AFCs were measured by Zeiss KS ELISPOT software. Graphs show data from individual mice, and bars indicate median. Statistical analyses were performed with MannWhitneys U-test (eg).
IgA cells. Of note, these mice showed B90% reduction in OVA-specic IgA content in the faeces compared with mice not treated with CPM (Fig. 6c). We also conrmed that CPM treatment 4 days after nal immunization induced a reduction in the production of IgA specic to the B subunit of CT (that is, CTB), which was associated with the halving of the abundance of
CTB-specic IgA AFCs in the intestine (Fig. 6d,e). Like OVA-specic IgA responses (Figs. 3c and 6b), similar levels of reduction of CTB-specic IgA AFCs were noted when CD11b IgA cells were depleted before ELISPOT assay (Fig. 6e). These mice showed reduced resistance to oral challenge with CT and developed watery diarrhoea (Fig. 6f and Supplementary Fig. S11).
6 NATURE COMMUNICATIONS | 4:1772 | DOI: 10.1038/ncomms2718 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications
& 2013 Macmillan Publishers Limited. All rights reserved.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2718 ARTICLE
These ndings led us to hypothesize that CD11b IgA PCs are capable of producing more IgA than are CD11b IgA PCs.
To test this hypothesis, we measured the size of each spot in CTB-specic IgA AFCs in an ELISPOT assay. The cells in the CD11b
IgA cell-enriched fraction (depletion of CD11b IgA cells) were bigger than those in the CD11b IgA cell-enriched fraction (depletion of CD11b IgA cells) (Fig. 6g). Furthermore, an adoptive transfer experiment demonstrated higher intestinal IgA production in severe combined immunodeciency mice receiving CD11b IgA PCs than in those receiving
CD11b IgA PCs (Supplementary Fig. S12), presumably because of both high IgA production and proliferating activity of
CD11b IgA PCs. Although some possibilities (for example, proliferation and CD11b expression of IgA cells might be changed during immunization) cannot be excluded, it is plausible that the actual production of IgA secreted into the intestinal lumen was derived mainly from CD11b IgA PCs in the early phase of the IgA response against orally immunized antigen.
DiscussionPCs could secrete antibodies to provide antigen-specic humoral immune responses in both systemic and mucosal tissues. Here, we demonstrated that intestinal IgA PCs in mice could be categorized into two populations on the basis of CD11b expression. CD11b is an integrin aM that non-covalently associates with CD18 to form aMb2 integrin (Mac-1) and binds to ICAM-1 (ref. 24). We therefore expected that CD11b IgA
PCs were newly migrating cells whose migration was mediated by endothelial cells expressing ICAM-1, but in fact they were not. We also found no uptake of opsonized bacteria in either CD11b or CD11b IgA cells (Supplementary Table S4 and Supplementary Fig. S13a), although CD11b is a receptor for complement (iC3b)24. In addition, unlike in human CD11b B cells, which stimulate T cells strongly32, major histocompatibility complex (MHC) class II (I-Ad) and costimulatory molecules (for example, CD80) were identically expressed on both CD11b and CD11b
IgA cells (Supplementary Table S4 and Supplementary Fig. S13b).
A similar subset of CD11b IgA cells was observed in the systemic murine compartments (for example, spleen), but the immunological characteristics of these cells differed from those of the cells in the intestine. Indeed, intestinal CD11b IgA cells consisted exclusively of PCs, but not memory B cells, whereas splenic CD11b IgA cells included both PCs and memory
B cells. We further found that CD11b could not be used as a marker of B1 cells in the intestine. Our current ndings show for the rst time that CD11b could be a specic marker for discriminating IgA PCs that require microbial stimulation and IL-10, and presumably contribute to the early phase of the intestinal IgA response in mice.
We have identied unique CD11b IgA PCs in mice; the next question is whether or not the same population of IgA PCs exists in humans. Our preliminary experiments have shown that no human intestinal IgA cells express CD11b, but that some
IgA cells express Ki67, a marker of proliferating cells (unpublished data). One possible explanation for this difference between human and mice is difference in the composition of commensal bacteria. In this regard, we examined the involvement of segmented lamentous bacteria (SFB), which are a known major IgA stimulus in mice, but has not yet been conrmed as part of the human microbiota19. As expected, SFB stimulated IgA production following colonization of SFB-decient C57BL/6 mice from the Jackson laboratory (JAX mice) with bacterial suspensions from SFB-monoassociated mice (JAX SFB mice)33;
however, we found that CD11b is expressed on IgA cells
independently of SFB colonization (Supplementary Fig. S14). It is possible that other commensal bacteria such as Lactobacillus (abundant in mice) and Bidobacterium (abundant in human) are responsible for the species-specic expression of CD11b on IgA cells. It is important to recognize the differences between the mouse and human immune systems, but it is obvious that proliferating IgA cells are present in the iLP of both mouse and human. The immunological function of human proliferating IgA cells in the intestine will therefore be the subject of our next study.
In the initial step of the antibody response to T cell dependent antigens, B cells are activated by antigens and form GCs in the lymph nodes7. As depleting antigen-specic GC B cells by CPM treatment during oral immunization resulted in complete loss of the IgA response to orally immunized antigen, it is likely that both CD11b and CD11b IgA PCs against T cell dependent antigen are derived from GC B cells. We also found that depletion of proliferating CD11b IgA PCs by CPM treatment after nal immunization led to a decrease in the early-phase IgA response, although it is possible that proliferation activity and/or CD11b expression on IgA cells might be wobble during immunization.
Our in vivo ndings indicated that the reduction in CD11b IgA PC numbers in MyD88 KO, IL-10 KO and PP-null mice did not affect the numbers of CD11b IgA PCs (Figs 1c, 3d and 5b). These ndings, together with our in vitro data (Supplementary
Fig. S6), indicate that it is likely that CD11b IgA PCs act as a separate lineage once they differentiate in the iLP.
Proliferating CD11b IgA PCs required microbial stimulation in the intestine. As proliferation is one of the characteristics of plasmablasts, it was possible that CD11b IgA cells have been recently committed to the PC fate. Notably, intestinal IgA cells expressed MHC class II molecules; this expression is one of the unique characteristics of plasmablasts. Therefore, it is likely that intestinal IgA PCs partly retain their plasmablast features.
However, our ndings indicated that CD11b and CD11b IgA cells expressed identical levels of Blimp-1 and MHC class II. In addition, similar reduction was noted in CD11b and
CD11b IgA cells when cell trafcking from IgA inductive tissues (for example, PPs and the PerC) into the iLP was inhibited by treatment with FTY720. Thus, our ndings suggest that CD11b IgA cells uniquely exhibit high proliferating and IgA-producing activity, although their other immunological features as PCs are similar to those of CD11b IgA PCs.
Proliferating CD138 PCs have been detected in the spleens of NZB/W mice with signs of systemic lupus erythematosus, but not in naive mice34. In contrast, the number of non-proliferating CD138 PCs is unchanged in the intestines of GF mice, as it is in the spleens of NZB/W mice34. These ndings suggest that
MyD88-dependent homeostatic stimulation of commensal bacteria determines the fate of proliferating CD11b IgA
CD138 PCs in the intestine. Several lines of evidence have revealed the cellular and molecular mechanisms of microbe-dependent initiation of IgA responses. B cells express several toll-like receptors, and B cell-intrinsic MyD88-mediated signalling has been implicated in enhanced antibody production in some studies35,36. However, our current ndings indicated that MyD88-mediated signalling in hematopoietic cells, including B cells, was not essential for intestinal CD11b IgA PC production. Additionally, we found IL-10 as a key molecule inducing CD11b IgA PC production. Previous studies have demonstrated that IL-10 promotes the proliferation of activated
B cells and subsequent IgA production in vitro37,38, which are consistent with our current ndings of high-level proliferation of, and IgA production by, CD11b IgA PCs. Thus, our current ndings proved that IL-10 functions in IgA production in vivo and that CD11b IgA PCs are the main targets in this
NATURE COMMUNICATIONS | 4:1772 | DOI: 10.1038/ncomms2718 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications 7
& 2013 Macmillan Publishers Limited. All rights reserved.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2718
pathway. Despite these ndings, our preliminary study demonstrated that treatment of CD11b or CD11b IgA PCs with
IL-10 alone did not induce their reciprocal differentiation into each other, and IL-10 KO mice with colitis possessed CD11b
IgA PCs (J.K., unpublished data). Thus, IL-10 is redundant in some cases and additional factors are required for the maintenance of CD11b IgA PCs. Our current ndings identied CD150 as a surface molecule that is highly expressed on
CD11b IgA PCs. CD150 is a 70-kDa glycoprotein expressed on some B and T cells, thymocytes and macrophages29.
Homophilic interaction of CD150 induces proliferation of, and antibody synthesis by, B cells39, and notably IL-10 synergistically enhances CD150-mediated B cell proliferation39. Thus, it is likely that, at least partly, IL-10 and CD150 determine the unique features (for example, proliferation and high IgA production) of CD11b IgA PCs in the iLP. In addition, accumulating evidence has revealed an important immunological function of stromal cells as survival niches for PCs in the BM40 and intestine41,42. It is possible that complex immunological communications among commensal ora, epithelial and stromal cells, and the cells involved in innate and acquired immunity determine the differentiation and maintenance of IgA PCs in the intestine.
Taken together, our results provide new insights into the nature of IgA PCs in the murine intestine, and especially into the regulation of the early-phase IgA responses to intestinal antigens and requirement of microbe-dependent stimulation, IL-10, and the PP lymphoid structure. These ndings add a new level of complexity to the intestinal IgA system of mice.
Methods
Mice. SPF and GF Balb/c mice were obtained from Japan CLEA (Tokyo, Japan). MyD88 KO mice, IL-10 mice (Balb/c background) and TCRbd mice (C57/BL6 background) were maintained under SPF conditions at the Experimental Animal Facility, The Institute of Medical Science, The University of Tokyo, and WT littermates were used as controls. To deplete gut commensal bacteria, mice received broad-spectrum antibiotics, namely ampicillin (1 g l 1; Sigma-Aldrich, St Louis,
MO), vancomycin (500 mg l 1; Shionogi, Osaka, Japan), neomycin sulphate(1 g l 1; Sigma-Aldrich) and metronidazole (1 g l 1; Sigma-Aldrich), in their drinking water for 4 weeks43. To establish BM chimeric mice, we injected g-irradiated (960 rad, Gammacell 40, Atomic Energy of Canada Limited, Ontario,
Canada) recipient mice with 5 106 BM cells through the tail vein and used them
in experiments 8 weeks after injection. Under our experimental conditions, the reconstitution efcacy was about 9095%. To obtain PP-null mice, pregnant BALB/ c mice were injected intravenously and subcutaneously with 1 mg anti-IL-7Ra antibody (A7R34, BioLegend, San Diego, CA) at 14.5 days post coitus, as described previously28. We conrmed the disruption of organized PPs and the existence of ILFs in the offspring, as described previously28. To neutralize cytokines, mice were treated intraperitoneally with 250 mg of monoclonal antibodies specic for IL-5 (TRFK5), IL-6 receptor (D7715A7) or IL-10 (JES5.16E3) (BioLegend, San Diego, CA); control antibody (Rat IgG2b); or 100 mg of soluble TACI-Fc fusion protein (R&D Systems, Minneapolis, MN) every second day for 2 weeks44,45. For assessing the role of SFB, mice purchased from the Jackson laboratory were orally inoculated with bacterial suspensions obtained by homogenizing faecal pellets from SFB-monoassociated mice in water. SFB colonization was conrmed by quantitative PCR33 and CD11b IgA cells were analysed in the small intestine 2 weeks post gavage by ow cytometry. All experiments followed the guidelines of the Animal
Care and Use Committee, The University of Tokyo and Columbia University.
Oral immunization. Mice were given sodium bicarbonate solution to neutralize stomach acid11,13. Thirty minutes later, the mice were orally immunized with 1 mg OVA (Sigma-Aldrich) and 10 mg CT (List Biological Laboratories, Campbell, CA).
This procedure was conducted on days 0, 7 and 14. In some groups, mice were intraperitoneally given CPM (35 mg kg 1 each time, Sigma-Aldrich). One week after the nal immunization, faecal samples and mononuclear cells from the iLP were collected for enumeration of OVA-specic antibody responses by enzyme-linked immunosorbent assay and ELISPOT, respectively13. In vivo CT challenge was performed by oral challenge of naive or immunized mice with 100 mg of CT as previously described46.
Cell isolation. To isolate mononuclear cells from PPs, we stirred the tissues in RPMI-1640 medium containing 2% fetal calf serum plus 0.5 mg ml 1 collagenase
(Wako, Osaka, Japan)11,13. To isolate mononuclear cells from the iLP, PPs were carefully removed and the remaining intestines including ILFs were opened longitudinally, washed with RPMI-1640, cut into 2-cm pieces and stirred for20 min at 37 C into RPMI-1640 containing 0.5 mM EDTA and 2% fetal calf serum to remove epithelial cells and intraepithelial lymphocytes11,13. The tissues were then stirred three times in 0.5 mg ml 1 collagenase for 20 min before undergoing discontinuous Percoll gradient centrifugation (40 and 75%). Peritoneal cells were obtained by peritoneal ushing with 8 ml ice-cold phosphate-buffered saline (PBS)11,13.
Flow cytometry and cell sorting. Mononuclear cells were preincubated with10 mg ml 1 anti-CD16/32 antibody (BD Biosciences, San Diego, CA). They were then reacted with the following antibodies: Pacic blue-rat anti-mouse CD45R (B220) (RA3-6B2, 0.8 mg ml 1), phycoerythrin (PE)-rat anti-mouse CD11b (M1/70, 0.1 mg ml 1), PE-Cy7-hamster anti-mouse CD11c (HL3, 0.4 mg ml 1),
PE-rat anti-mouse CD18 (C71/16, 0.8 mg ml 1), PE-rat anti-mouse CD19 (1D3,0.8 mg ml 1), PE-rat anti-mouse CD38 (90, 0.13 mg ml 1), FITC-rat anti-mouse IgA (C10-3, 2 mg ml 1), PE-Cy7-rat anti-mouse IgM (R6-60.2, 1 mg ml 1), PE-anti-mouse I-Ad (AMS-32.1, 0.4 mg ml 1), APC-Cy7-rat anti-mouse CD11b (M1/
70, 1 mg ml 1), APC-Cy7-anti-mouse b1-integrin (HMb1-1, 4 mg ml 1), APC-anti-mouse CD40 (3/23, 2 mg ml 1), Pacic blue-anti-mouse CD11b (M1/70,1 mg ml 1), PE-Cy7-anti-mouse F4/80 (BM8, 0.4 mg ml 1) and biotin mouse anti-
CD138 (281-2, 10 mg ml 1) (all antibodies from BD Biosciences) followed by incubation with streptavidin-APC (1 mg ml 1, BD Biosciences), PE-anti-mouse
CD150 (TC15-12F12.2, 0.1 mg ml 1), Alexa Fluor 647-anti-mouse CD80 (16-10A1, 1 mg ml 1) (BioLegend, San Diego, CA), anti-mouse CD267 (TACI)
(8F10-3, 4 mg ml 1) (eBioscience, San Diego, CA), PE-mouse CCR3 (83101,0.5 mg ml 1) (R&D Systems) or biotinylated anti-peanut agglutinin lectin(1 mg ml 1, Vector Laboratories, Burlingame, CA), followed by staining with streptavidin PE (1 mg ml 1, BD Biosciences). For staining for Blimp-1, cells were xed and permeabilized with a Cytox/Cytoperm kit (BD Biosciences) and stained with PE-conjugated anti-Blimp1 goat polyclonal IgG (0.4 mg ml 1, Santa Cruz
Biotechnology, Santa Cruz, CA). FSC-H and FSC-A discrimination was used to exclude doublet cells, and ViaProbe cell-viability solution (BD Biosciences) was used to discriminate between dead and living cells. To detect proliferating cells, mice received 1 mg BrdU intraperitoneally 24 h before analysis; the BrdU signal was detected with the manufacturers protocol (BD Biosciences). Concentration-matched isotype antibodies were used as negative controls. Flow-cytometric analysis and cell sorting were performed with FACSCanto II and FACSAria (BD Biosciences), respectively. We conrmed that cell purity was about 95% (Fig. 2a).
Immunohistological analysis. Intestines were xed in 4% paraformaldehyde for 15 h at 4 C, washed with PBS and treated sequentially in 10 and 20% sucrose for 12 h at 4 C13. The tissues were embedded in OCT compound (Sakura Fine-technical Co., Tokyo, Japan). Cryostat sections (7 mm) were pre-blocked with anti-
CD16 and CD32 antibody for 15 min at room temperature and stained for 15 h at 4 C with FITC-rat anti-mouse IgA (C10-3, 2 mg ml 1) and biotin anti-mouse
CD11b antibody (M1/70, 1 mg ml 1). This was followed by incubation with horseradish peroxidase (HRP)-conjugated streptavidin (Pierce, Rockford, IL) for 30 min at 4 C and amplication of the uorescent signal with Cy3-tyramide (TSA-Direct kit; PerkinElmer, Waltham, MA)13. We conrmed that no signal was detected when the specimens were stained with the concentration-matched isotype antibodies. They were then counterstained with 40,6-diamidino-2-phenylindole (Sigma-Aldrich). Deconvoluted uorescence images of specimens were obtained by uorescence microscopy (BZ9000, Keyence, Osaka, Japan).
Detection of antibody responses by enzyme-linked immunosorbent assay and ELISPOT. To measure OVA- or CTB-specic IgA levels in faecal extracts, faeces were homogenized in PBS by vigorous vortexing11,13. After centrifugation of the extracts (9,000g for 15 min) the supernatants were used as faecal extracts. Plates were coated with 1 mg ml 1 OVA or 2 mg ml 1 CTB in PBS; this was followed by blocking for 1 h at room temperature with 200 ml PBS containing 1% (w/v) bovine serum albumin. After extensive washing of the plates with PBS containing 0.05% Tween 20, serial sample dilutions were added for incubation overnight at 4 C. Samples were then incubated for 1 h at room temperature with optimally diluted HRP-conjugated goat anti-mouse IgA (SouthernBiotech, Birmingham, AL). After sample washing, the colour reaction was developed at room temperature with 3,30,5,50-tetramethylbenzidine (Moss, Pasadena, MD) and terminated by adding0.5 M HCl. The colour reaction was measured as the optical density (wavelength 450 nm).
ELISPOT assay was used to enumerate IgA-producing AFCs in the iLP11,13. Briey, various concentrations of mononuclear cells were cultured at 37 C for4 h in 96-well nitrocellulose membrane plates (Millititer HA; Millipore, Bedford, MA) coated with 1 mg ml 1 OVA and 5 mg ml 1 bovine serum albumin-conjugated phosphorylcholine (Biosearch Technologies, Novato, CA). After vigorous washing of the plates with PBS and PBS containing 0.05% Tween 20, HRP-conjugated goat anti-mouse IgA was added; the plates were then incubated overnight at 4 C. Spots of AFCs were developed with 2-amino-9-ethylcarbazole
8 NATURE COMMUNICATIONS | 4:1772 | DOI: 10.1038/ncomms2718 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications
& 2013 Macmillan Publishers Limited. All rights reserved.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2718 ARTICLE
(Polysciences, Warrington, PA). The size of each spot was measured with Zeiss KS ELISPOT software (Oberkochen, Germany).
In vitro culture. CD11b IgA or CD11b IgA PCs (104 cells per well) were puried from the iLP and cultured with 100 ng ml 1 phorbol 12-myristate 13-acetate plus 300 ng ml 1 ionomycin, or 10 mg ml 1 lipopolysaccharide (all from Sigma-Aldrich), for 24 h.
For the bacteria uptake assay, uorescent Staphylococcus aureus was opsonized in accordance with the manufacturers protocol (Molecular Probes). Mononuclear cells isolated from the iLP (2 105 cells) were incubated with 1 105 opsonized
bacteria for 90 min. After being washed, the cells were stained with antibodies for PE-IgA (mA-6E1, 0.5 mg ml 1, eBioscience) and Pacic Blue CD11b, and the bacterial uptake by each population was examined by ow cytometry.
Microarray analysis. Microarray analysis was performed as we previously reported47. Briey, CD11b IgA and CD11b IgA cells were isolated from the iLP, and total RNA was extracted from them with an RNeasy kit (Qiagen,
Dusseldorf, Germany). cRNA was hybridized with DNA probes on a GeneChip Mouse Genome 430 2.0 array (Affymetrix), washed and uorescence-labelled in accordance with the standard amplication protocol developed by Affymetrix. The uorescence intensity of each probe was taken to represent the raw expression level and was quantied with GeneChip Operating software (Affymetrix). Data obtained from two independent experiments were analysed with GeneSpring 7.3.1 software (Silicon Genetics). All microarray data have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/
Web End =www.ncbi.nlm.nih.gov/geo/) under the accession no. GSE37225.
Statistics. Results were compared by a non-parametric MannWhitneys U-test and unpaired t-test (two tailed) (GraphPad Software, San Diego, CA).
References
1. Macpherson, A. J., McCoy, K. D., Johansen, F. E. & Brandtzaeg, P. The immune geography of IgA induction and function. Mucosal Immunol. 1, 1122 (2008).
2. Brandtzaeg, P. Function of mucosa-associated lymphoid tissue in antibody formation. Immunol. Invest. 39, 303355 (2010).
3. Fagarasan, S. Intestinal IgA synthesis: a primitive form of adaptive immunity that regulates microbial communities in the gut. Curr. Top Microbiol. Immunol. 308, 137153 (2006).
4. Macpherson, A. J. & Slack, E. The functional interactions of commensal bacteria with intestinal secretory IgA. Curr. Opin. Gastroenterol. 23, 673678 (2007).
5. Slack, E. et al. Innate and adaptive immunity cooperate exibly to maintain host-microbiota mutualism. Science 325, 617620 (2009).
6. Woof, J. M. & Kerr, M. A. The function of immunoglobulin A in immunity.J. Pathol. 208, 270282 (2006).7. Fagarasan, S., Kawamoto, S., Kanagawa, O. & Suzuki, K. Adaptive immune regulation in the gut: T cell-dependent and T cell-independent IgA synthesis. Annu. Rev. Immunol. 28, 243273 (2010).
8. Hayakawa, K., Hardy, R. R. & Herzenberg, L. A. Progenitors for Ly-1 B cells are distinct from progenitors for other B cells. J. Exp. Med. 161, 15541568 (1985).
9. Kantor, A. B. & Herzenberg, L. A. Origin of murine B cell lineages. Annu. Rev. Immunol. 11, 501538 (1993).
10. Montecino-Rodriguez, E., Leathers, H. & Dorshkind, K. Identication of a B-1 B cell-specied progenitor. Nat. Immunol. 7, 293301 (2006).
11. Kunisawa, J. et al. Sphingosine 1-phosphate regulates peritoneal B-cell trafcking for subsequent intestinal IgA production. Blood 109, 37493756 (2007).
12. Tsuji, M. et al. Requirement for lymphoid tissue-inducer cells in isolated follicle formation and T cell-independent immunoglobulin A generation in the gut. Immunity 29, 261271 (2008).
13. Gohda, M. et al. Sphingosine 1-phosphate regulates the egress of IgA plasmablasts from Peyers patches for intestinal IgA responses. J. Immunol. 180, 53355343 (2008).
14. Mora, J. R. et al. Generation of gut-homing IgA-secreting B cells by intestinal dendritic cells. Science 314, 11571160 (2006).
15. Cerutti, A., Chen, K. & Chorny, A. Immunoglobulin responses at the mucosal interface. Annu. Rev. Immunol. 29, 273293 (2011).
16. Radbruch, A. et al. Competence and competition: the challenge of becoming a long-lived plasma cell. Nat. Rev. Immunol. 6, 741750 (2006).
17. Weinstein, P. D. & Cebra, J. J. The preference for switching to IgA expression by Peyers patch germinal center B cells is likely due to the intrinsic inuence of their microenvironment. J. Immunol. 147, 41264135 (1991).
18. Hamada, H. et al. Identication of multiple isolated lymphoid follicles on the antimesenteric wall of the mouse small intestine. J. Immunol. 168, 5764 (2002).
19. Talham, G. L., Jiang, H. Q., Bos, N. A. & Cebra, J. J. Segmented lamentous bacteria are potent stimuli of a physiologically normal stateof the murine gut mucosal immune system. Infect. Immun. 67, 19922000 (1999).
20. Cebra, J. J., Jiang, H. Q., Boiko, N. V. & Tlaskalva-Hogenova, H. The role of mucosal microbiota in the development, maintenance, and pathologies of the mucosal immune system. in Mucosal Immunology 3rd edn (Mestecky, J. et al. eds) 335368 (Academic Press, 2005).
21. Tezuka, H. et al. Regulation of IgA production by naturally occurring TNF/ iNOS-producing dendritic cells. Nature 448, 929933 (2007).
22. Suzuki, K. et al. The sensing of environmental stimuli by follicular dendritic cells promotes immunoglobulin A generation in the gut. Immunity 33, 7183 (2010).
23. Shapiro-Shelef, M. & Calame, K. Regulation of plasma-cell development. Nat. Rev. Immunol. 5, 230242 (2005).
24. Patarroyo, M. et al. Leukocyte-cell adhesion: a molecular process fundamental in leukocyte physiology. Immunol. Rev. 114, 67108 (1990).
25. Pabst, O. et al. Cutting edge: egress of newly generated plasma cells from peripheral lymph nodes depends on beta 2 integrin. J. Immunol. 174, 74927495 (2005).
26. Kunisawa, J. & Kiyono, H. A marvel of mucosal T cells and secretory antibodies for the creation of rst lines of defense. Cell Mol. Life Sci. 62, 13081321 (2005).
27. Shroff, K. E., Meslin, K. & Cebra, J. J. Commensal enteric bacteria engender a self-limiting humoral mucosal immune response while permanently colonizing the gut. Infect. Immun. 63, 39043913 (1995).
28. Kunisawa, J. et al. Lack of antigen-specic immune responses in anti-IL-7 receptor alpha chain antibody-treated Peyers patch-null mice following intestinal immunization with microencapsulated antigen. Eur. J. Immunol. 32, 23472355 (2002).
29. Schwartzberg, P. L., Mueller, K. L., Qi, H. & Cannons, J. L. SLAM receptors and SAP inuence lymphocyte interactions, development and function. Nat. Rev. Immunol. 9, 3946 (2009).
30. Maxwell, C. A., McCarthy, J. & Turley, E. Cell-surface and mitotic-spindle RHAMM: moonlighting or dual oncogenic functions? J. Cell Sci. 121, 925932 (2008).
31. Cario, E. et al. Lipopolysaccharide activates distinct signaling pathways in intestinal epithelial cell lines expressing Toll-like receptors. J. Immunol. 164, 966972 (2000).
32. Grifn, D. O. & Rothstein, T. L. A small CD11b( ) human B1 cell
subpopulation stimulates T cells and is expanded in lupus. J. Exp. Med. 208, 25912598 (2011).33. Ivanov, I. I. et al. Induction of intestinal Th17 cells by segmented lamentous bacteria. Cell 139, 485498 (2009).
34. Hoyer, B. F. et al. Short-lived plasmablasts and long-lived plasma cells contribute to chronic humoral autoimmunity in NZB/W mice. J. Exp. Med. 199, 15771584 (2004).
35. Pasare, C. & Medzhitov, R. Control of B-cell responses by Toll-like receptors. Nature 438, 364368 (2005).
36. Hou, B. et al. Selective utilization of Toll-like receptor and MyD88 signaling in B cells for enhancement of the antiviral germinal center response. Immunity 34, 375384 (2011).
37. Rousset, F. et al. Interleukin 10 is a potent growth and differentiation factor for activated human B lymphocytes. Proc. Natl Acad. Sci. USA 89, 18901893 (1992).
38. Defrance, T. et al. Interleukin 10 and transforming growth factor beta cooperate to induce anti-CD40-activated naive human B cells to secrete immunoglobulinA. J. Exp. Med. 175, 671682 (1992).39. Punnonen, J. et al. Soluble and membrane-bound forms of signaling lymphocytic activation molecule (SLAM) induce proliferation and Ig synthesis by activated human B lymphocytes. J. Exp. Med. 185, 9931004 (1997).
40. Tokoyoda, K., Hauser, A. E., Nakayama, T. & Radbruch, A. Organization of immunological memory by bone marrow stroma. Nat. Rev. Immunol. 10, 193200 (2010).
41. Fagarasan, S., Kinoshita, K., Muramatsu, M., Ikuta, K. & Honjo, T. In situ class switching and differentiation to IgA-producing cells in the gut lamina propria. Nature 413, 639643 (2001).
42. Kang, H. S. et al. Signaling via LTbetaR on the lamina propria stromal cells of the gut is required for IgA production. Nat. Immunol. 3, 576582 (2002).
43. Rakoff-Nahoum, S., Paglino, J., Eslami-Varzaneh, F., Edberg, S. & Medzhitov,R. Recognition of commensal microora by toll-like receptors is required for intestinal homeostasis. Cell 118, 229241 (2004).44. Hiroi, T., Yanagita, M., Ohta, N., Sakaue, G. & Kiyono, H. IL-15 and IL-15 receptor selectively regulate differentiation of common mucosal immune system-independent B-1 cells for IgA responses. J. Immunol. 165, 43294337 (2000).
NATURE COMMUNICATIONS | 4:1772 | DOI: 10.1038/ncomms2718 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications 9
& 2013 Macmillan Publishers Limited. All rights reserved.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2718
45. Yan, M. et al. Identication of a receptor for BLyS demonstrates a crucial role in humoral immunity. Nat. Immunol. 1, 3741 (2000).
46. Nochi, T. et al. Rice-based mucosal vaccine as a global strategy for cold-chain- and needle-free vaccination. Proc. Natl Acad. Sci. USA 104, 1098610991 (2007).
47. Terahara, K. et al. Comprehensive gene expression proling of Peyers patch M cells, villous M-like cells, and intestinal epithelial cells. J. Immunol. 180, 78407846 (2008).
Author contributions
J.K. planned the research and experiments, analysed data, wrote the paper and directed the research; M.G., E.H., I.I., M.H., Y.S., Y.G., C.P., I.I.I., R.S., L.A., T.W., S.S., Y.K. and S.S. conducted the immunological experiments; K.T. and S.A. provided key materials; and H.K. wrote the paper.
Additional information
Accession codes: Microarray data have been deposited in the National Center for Bio-technology Information Gene Expression Omnibus database under series accession code GSE37225.
Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications
Web End =http://www.nature.com/ http://www.nature.com/naturecommunications
Web End =naturecommunications
Competing nancial interests: The authors declare no competing nancial interests.
Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/
Web End =http://npg.nature.com/ http://npg.nature.com/reprintsandpermissions/
Web End =reprintsandpermissions/
How to cite this article: Kunisawa, J. et al. Microbe-dependent CD11b IgA plasma cells mediate robust early-phase intestinal IgA responses in mice. Nat. Commun. 4:1772 doi: 10.1038/ncomms2718 (2013).
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/
Web End =http://creativecommons.org/licenses/by-nc-sa/3.0/
Acknowledgements
This work was supported by grants from the Program for Promotion of Basic and Applied Research for Innovations in Bio-oriented Industry (BRAIN to J.K.), the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grants-in-Aid for Young Scientists A (22689015 to J.K.), for Scientic Research on Innovative Areas (23116506 to J.K.), for Scientic Research S (23229004 to H.K.), for Scientic Research on Priority Area (19059003 to H.K.), for Challenging Exploratory Research (24659217 to J.K.) and for the Leading-edge Research Infrastructure Program (to J.K. and H.K.)), and the Young Researcher Overseas Visits Program for Vitalizing Brain Circulation (Japan Society for the Promotion of Science) (to J.K., H.K., Y.K. and Y.G.); grants for JSPS Fellows (021-07124 to Y.K.); and grants from the Ministry of Health and Welfare of Japan (J.K. and H.K.), the New Energy and Industrial Technology Development Organization (to H.K.), the Global Center of Excellence Program of the Center of Education and Research for Advanced Genome-based Medicine (to H.K.), the Yakult Bio-Science Foundation (to J.K.) and DK085329 from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK, to I.I.).
10 NATURE COMMUNICATIONS | 4:1772 | DOI: 10.1038/ncomms2718 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications
& 2013 Macmillan Publishers Limited. All rights reserved.
You have requested "on-the-fly" machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Show full disclaimer
Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer
Copyright Nature Publishing Group Apr 2013
Abstract
Intestinal plasma cells predominantly produce immunoglobulin (Ig) A, however, their functional diversity remains poorly characterized. Here we show that murine intestinal IgA plasma cells can be newly classified into two populations on the basis of CD11b expression, which cannot be discriminated by currently known criteria such as general plasma cell markers, B cell origin and T cell dependence. CD11b+ IgA+ plasma cells require the lymphoid structure of Peyer's patches, produce more IgA than CD11b- IgA+ plasma cells, proliferate vigorously, and require microbial stimulation and IL-10 for their development and maintenance. These features allow CD11b+ IgA+ plasma cells to mediate early-phase antigen-specific intestinal IgA responses induced by oral immunization with protein antigen. These findings reveal the functional diversity of IgA+ plasma cells in the murine intestine.
You have requested "on-the-fly" machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Show full disclaimer
Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer