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. 2016 Sep 19;213(10):2167-85.
doi: 10.1084/jem.20150282. Epub 2016 Sep 12.

The soluble pattern recognition receptor PTX3 links humoral innate and adaptive immune responses by helping marginal zone B cells

Alejo Chorny  1 , Sandra Casas-Recasens  1 , Jordi Sintes  2 , Meimei Shan  1 , Nadia Polentarutti  3 , Ramón García-Escudero  4 , A Cooper Walland  1 , John R Yeiser  1 , Linda Cassis  2 , Jorge Carrillo  5 , Irene Puga  2 , Cristina Cunha  6 , Hélder Bastos  7 , Fernando Rodrigues  6 , João F Lacerda  8 , António Morais  9 , Rebeca Dieguez-Gonzalez  1 , Peter S Heeger  10 , Giovanni Salvatori  11 , Agostinho Carvalho  6 , Adolfo Garcia-Sastre  12 , J Magarian Blander  13 , Alberto Mantovani  14 , Cecilia Garlanda  14 , Andrea Cerutti  15
Affiliations

The soluble pattern recognition receptor PTX3 links humoral innate and adaptive immune responses by helping marginal zone B cells

Alejo Chorny et al. J Exp Med. .

Erratum in

Abstract

Pentraxin 3 (PTX3) is a fluid-phase pattern recognition receptor of the humoral innate immune system with ancestral antibody-like properties but unknown antibody-inducing function. In this study, we found binding of PTX3 to splenic marginal zone (MZ) B cells, an innate-like subset of antibody-producing lymphocytes strategically positioned at the interface between the circulation and the adaptive immune system. PTX3 was released by a subset of neutrophils that surrounded the splenic MZ and expressed an immune activation-related gene signature distinct from that of circulating neutrophils. Binding of PTX3 promoted homeostatic production of IgM and class-switched IgG antibodies to microbial capsular polysaccharides, which decreased in PTX3-deficient mice and humans. In addition, PTX3 increased IgM and IgG production after infection with blood-borne encapsulated bacteria or immunization with bacterial carbohydrates. This immunogenic effect stemmed from the activation of MZ B cells through a neutrophil-regulated pathway that elicited class switching and plasmablast expansion via a combination of T cell-independent and T cell-dependent signals. Thus, PTX3 may bridge the humoral arms of the innate and adaptive immune systems by serving as an endogenous adjuvant for MZ B cells. This property could be harnessed to develop more effective vaccines against encapsulated pathogens.

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Figures

Figure 1.
Figure 1.
PTX3 is released by NBh cells expressing an immune activation–related gene signature. (A) Microarray analysis of human NC and NBh cells. The Venn diagram summarizes comparably and differentially expressed genes, whereas the heat map provides a global view of differentially expressed genes. Colors correspond to significant fold change (FC) expression. Red, high expression; blue, low expression. (B) Gene ontology analysis showing the top 10 biological processes up-regulated (red) and down-regulated (blue) in human NBh cells compared with NC cells. Numbers indicate genes. (C) Scatter plot illustrating gene expression signal intensity in human NC and NBh cells. Circles indicate some of the top differentially expressed genes. (D) GSEA comparing genes expressed by NBh cells and GM-CSF–activated NC cells. Normalized enrichment score (NES) indicates correlation between individual gene sets. Positive correlation, NES >0 (red bars); negative correlation, NES <0 (blue bars). The top 10 genes similarly up-regulated in NBh cells and GM-CSF–treated NC cells are listed. FDR, false discovery rate. (E) qRT-PCR of mRNAs for EGR-1, IκBζ (NFKBIZ), TNFAIP3, and EDN-1 in human NC and NBh cells. Results are normalized to mRNA for β-actin and presented as relative expression (RE) compared with a reference set of NC cells. (F) qRT-PCR of PTX3 mRNA (top) and ELISA of PTX3 protein (bottom) from human NC cells cultured with or without GM-CSF and/or LPS for 1 d. PTX3 mRNA is normalized to mRNA for β-actin and presented as relative expression compared with NC cells cultured with medium alone (Ctrl). (G and H) IFA of human spleens stained for elastase (ELA; green), PTX3 (red), and IgD or LPS (blue). FO, follicle; PFZ, perifollicular zone. Bars: (G) 200 µm; (H) 40 µm; (H, right inset) 6 µm. (I and J) qRT-PCR of PTX3 mRNA (I) and ELISA of PTX3 protein (J) from human NC and NBh cells. PTX3 mRNA is normalized to β-actin mRNA and shown as relative expressions compared with NC cells. ELISA was performed after 2 d of culture in medium alone. Data show one experiment with six biological replicates for each cell type (A–D), one representative experiment of at least four with similar results (E–H), or two experiments with at least three samples in each experimental group (I and J). Error bars, SEM. *, P < 0.05; **, P < 0.01 (two-tailed Student’s t test).
Figure 2.
Figure 2.
PTX3 from NBh cells binds to B cells and enhances CSR from IgM to IgG. (A) IFA of human splenic tissue stained for IgD (green), PTX3 (red), and DNA (blue). Boxes and arrowheads highlight IgD+ B cells proximal to NBh cells, including areas of IgD-PTX3 colocalization. Bars: (top) 25 µm; (bottom insets) 5 µm. (B) Flow cytometric analysis of PTX3 and control BSA bound to human circulating CD19+ B cells. (C and D) Flow cytometric analysis of fluorochrome-labeled PTX3 bound to human circulating naive, MZ, and memory B cells before (C) and after (D) preincubation with unlabeled PTX3 or control (Ctrl) IgG. MFI, mean fluorescence intensity. (E) Flow cytometric analysis of PTX3 bound to human MZ B cells after exposure to NC cells incubated with medium alone (control), LPS, GM-CSF, or both LPS and GM-CSF for 4 h. The dashed line indicates mean fluorescence intensity of PTX3 binding to MZ B cells cultured with medium alone. (F) Flow cytometric analysis of PTX3 bound to human splenic MZ B cells stimulated with medium alone (control) or CpG DNA for 1 d. (G) Flow cytometric analysis of PTX3 binding to human circulating IgD+ B cells in the presence or absence of C1q. Binding of BSA is shown as the control. (H) IFA of human splenic tissue stained for C1q (green), PTX3 (red), and IgD (blue). CA, central arteriole; FO, follicle; PFZ, perifollicular zone. Bar, 100 µm. (I) Southern blotting of Iμ-Cμ, Iγ1-Cγ1, Iγ2-Cγ2, and Iα1-Cα1 germline transcripts (GTs) and Iγ-Cμ and switch Iα-Cμ circle transcripts (CTs) amplified by standard reverse transcription PCR from human circulating IgD+ B cells cultured with or without PTX3 for 2 d (germline transcripts) or 4 d (circle transcripts). Iμ-Cμ was the loading control. (J) qRT-PCRs of mRNAs for AID (AICDA) and BLIMP-1 from human circulating IgD+ B cells exposed to PTX3 for 2 d. Results are normalized to mRNA for β-actin and presented as relative expression (RE) compared with B cells incubated with medium alone. (K) ELISA of total IgG from human circulating IgD+ B cells cultured for 4 d with medium alone (control), BAFF, or IL-10 in the presence or absence of PTX3. Data show one representative experiment of at least four with similar results (A, B, D, H, and I) or one to two experiments with at least three samples in each experimental group (C, E–G, and I–K). Error bars, SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-tailed Student’s t test).
Figure 3.
Figure 3.
PTX3 binds to MZ B cells independently of TLR4 and FcγRs. (A) IFA of mouse spleen stained for MMP9 (green), Ly6G (red), and IgM or DNA (blue). FO, follicle; PALS, periarteriolar lymphoid sheath. Bars: (top) 100 µm; (inset) 10 µm. (B) Confocal microscopy of mouse tissue stained for MMP9 (green), B220 (red), and DNA (blue). Bar, 3 µm. (C) qRT-PCR of RNAs for BAFF (Tnfsf13b), APRIL (Tnfsf13), CD40L (Cd40lg), IL-10, and IL-6 in mouse neutrophils from bone marrow and spleen. Results are normalized to mRNA for HPRT and presented as relative expression compared with bone marrow neutrophils. The color code indicates high (red), mean (black), and low (green) expression. (D) qRT-PCR of mRNA for PTX3 from mouse NBh cells stimulated with LPS for 4 h. Results are normalized to mRNA for HPRT and presented as relative expression (RE) compared with NBh cells incubated with medium alone. (E) ELISA of serum PTX3 from WT and MyD88−/−Trif−/− mice. (F) ELISA of serum PTX3 from MyD88−/−Trif−/− mice before (control [Ctrl]) and after adoptive transfer of NBh cells from WT mice. (G) Flow cytometric analysis of PTX3 or BSA binding to MZ and FO B cells from WT mice. MFI, mean fluorescence intensity. (H and I) Flow cytometric analysis of PTX3-binding to splenic B220+AA4-1+ transitional B cells (T B cell), including B220+AA4-1+CD23−IgMhi T1 B cells, B220+AA4-1+CD23+IgMhi T2 B cells, and B220+AA4-1+CD23+IgMlo T3 B cells (H) and of PTX3 or BSA binding to MZ B cells from WT, FcγR−/−, or Tlr4−/− mice (I). Data show one representative experiment of at least four with similar results (A, B, H, and I) or one to two experiments with at least three samples in each experimental group (C–G). Error bars, SEM. *, P < 0.05; ***, P < 0.001 (two-tailed Student’s t test).
Figure 4.
Figure 4.
PTX3 enhances homeostatic IgM and IgG responses to CPS. (A) Flow cytometric analysis of frequency and absolute number of total B220+ B cells, B220+CD21hiCD23− MZ B cells, B220+CD21+CD23+ FO B cells, B220+CD21−CD23−CD43+ B-1 cells, and B220−CD138+ PBs/PCs from spleens of WT and Ptx3−/− mice. (B) ELISA of total IgM and specific IgM to phosphorylcholine (PCh-IgM), CPS serotype 9, CPS serotype 14, and lipoteichoic acid (LTA) from serum of WT and Ptx3−/− mice. OD was at 450 nm. Data show serum total and antigen-specific Igs from 7–10 mice in each experimental group. (C) qRT-PCR of mRNA for BLIMP-1 (Prdm1) in MZ B cells from WT and Ptx3−/− mice. Results are normalized to mRNA for HPRT and presented as relative expression (RE) compared with MZ B cells from WT mice. (D) IFA of spleens from WT and Ptx3−/− mice stained for MMP9 (green), Ly6G (red), and MOMA-1 (blue). Bars, 100 µm. (E) Flow cytometric quantification of NBh cells from spleens of WT and Ptx3−/− mice. (F) ELISA of total and CPS-reactive IgG in BAL fluids from PTX3-sufficient h1/h1 and h1/h2 patients and PTX3-deficient h2/h2 patients. Data show one representative experiment of at least two with similar results (A and D), summarize one to two experiments with at least eight mice in each experimental group (A and C–E), or show one experiment with samples from at least eight donors in each experimental group (F). Error bars, SEM. *, P < 0.05; **, P < 0.01 (two-tailed Student’s t test).
Figure 5.
Figure 5.
PTX3 enhances MZ B cell proliferation and CSR from IgM to IgG in response to encapsulated bacteria. (A) IFA of spleens from WT mice stained for MMP9 (green), IgM (red), and MOMA-1 (blue) before (control [Ctrl]) and after (d3) i.v. injection of live SP. Bars, 100 µm. (B) Confocal microscopy of mouse splenic tissue stained for MMP9 (green), B220 (red), and DNA (blue) after i.v. injection (d3) of live SP. The right image corresponds to the magnified boxed tissue area from the left image, which depicts B cell–interacting NBh cells. Arrowheads point to B220 captured by an MMP9+ NBh cell. Bars: (left) 10 µm; (right) 3 µm. (C) ELISA of serum PTX3 from WT mice after i.v. injection of live SP. (D) Flow cytometric quantification of NBh cells from WT and Ptx3−/− mice after i.v. injection of live SP. (E and F) Flow cytometric quantification of frequency of proliferating Ki-67+ MZ B cells, B-1a cells, and B-1b cells (E) and absolute number of total MZ B cells, B-1a cells, and B-1b cells (F) from spleens of WT and Ptx3−/− mice after i.v. injection (d5) of live SP. Dashed lines show the mean frequency or absolute number in nonimmunized WT mice. Similar values were obtained in nonimmunized Ptx3−/− mice. FSC, forward side scatter. (G) qRT-PCR of mRNAs for AID (Aicda), Iγ2b-Cγ2b, and Iγ2c-Cγ2c transcripts in MZ B cells from WT and Ptx3−/− mice after i.v. injection (d5) of live SP. Results are normalized to mRNA for HPRT and presented as relative expression (RE) compared with MZ B cells from WT mice. (H) Absolute numbers of class-switched B cells expressing IgG2b, IgG2c, or IgG3 from spleens of WT and Ptx3−/− mice after i.v. injection (d5) of live SP. Dashed lines show the mean frequency or absolute number in nonimmunized WT mice. Similar values were obtained in nonimmunized Ptx3−/− mice. Data show one representative experiment of at least three with similar results (A and B) or summarize two experiments with at least three mice in each experimental group (C–H). Error bars, SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-tailed Student’s t test).
Figure 6.
Figure 6.
PTX3 enhances PB and PC expansion as well as IgG production in response to encapsulated bacteria. (A and B) ELISA of serum SP-reactive IgG2b, IgG2c, IgG3, and IgM (A) and ELISPOT of splenic SP-reactive antibody-secreting cells (ASCs; B) from WT and Ptx3−/− mice after i.v. injection (d7) of live SP. RU, relative units. (C) Flow cytometric quantification of frequency (left cytograms) and absolute number (right graphs) of B220−CD138+ PBs/PCs expressing IgM or class-switched IgG2b, IgG2c, and IgG3 from WT and Ptx3−/− mice after i.v. injection (d7) of live SP. Dashed lines show the mean frequency or absolute number in nonimmunized WT mice. Similar values were obtained in nonimmunized Ptx3−/− mice. (D) qRT-PCR of mRNA for AID (Aicda) in splenic B220−CD138+ PBs/PCs from WT and Ptx3−/− mice after i.v. injection (d7) of live SP. Results are normalized to mRNA for HPRT and are presented as relative expression (RE) compared with PBs/PCs from WT mice. (E) Flow cytometric quantification of frequency of splenic B220− PBs expressing extracellular (extra) but not intracellular (intra) IgG2b, IgG2c, or IgG3 (left) as well as splenic B220− PCs expressing both extracellular and intracellular IgG2b, IgG2c, or IgG3 (right) from WT and Ptx3−/− mice after i.v. injection (d7) of live SP. Dashed lines show the mean frequency or absolute number in nonimmunized WT mice. Similar values were obtained in nonimmunized Ptx3−/− mice. (F) IFA of spleens stained for MMP9 (green), IgG2c (red), and MOMA-1 (blue) from WT and Ptx3−/− mice after i.v. injection (d7) of live SP. Bars, 50 µm. (G) IFA of splenic tissue from SP-infected WT mice stained for MMP9 (green), IgG2c (red), and DNA (blue) from WT mice after i.v. injection (d7) of live SP. Bar, 3 µm. Data summarize at least two experiments with at least three mice in each experimental group (A–C and E) or one representative experiment of at least three with similar results (D, F, and G). Error bars, SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-tailed Student’s t test).
Figure 7.
Figure 7.
PTX3 can enhance IgG responses to microbial carbohydrates by activating a TI pathway. (A) ELISA of serum SP-reactive IgG2b, IgG2c, and IgG3 from Tcrb−/− and Tcrb−/−Ptx3−/− mice after i.v. injection (d7) of live SP. RU, relative units. (B) Flow cytometric analysis of frequency (left) and absolute number (right) of splenic class-switched PBs expressing IgG2b or IgG2c and splenic NBh cells from Tcrb−/− and Tcrb−/−Ptx3−/− mice after i.v. injection (d7) of live SP. (C) ELISA of serum SP-reactive IgG2b, IgG2c, and IgG3 from Tcrb−/− mice after i.v. injection (d7) of live SP with or without exogenous PTX3. (D) ELISA of serum SP-reactive IgG2b and IgG3 from Tcrb−/−Ptx3−/− mice after i.v. injection (d7) of SP in the presence of exogenous PTX3. (E) ELISA of serum Pneumovax-specific IgM from WT, Ptx3−/−, and PTX3-treated Ptx3−/− mice at day 7 after i.v. immunization with Pneumovax. (F) ELISA of serum TNP-reactive IgG2b and IgG2c from WT mice after i.v. injection (d7) of TNP-LPS with or without exogenous PTX3. Data summarize at least two experiments with at least three mice in each experimental group. Error bars, SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-tailed Student’s t test).
Figure 8.
Figure 8.
PTX3 deficiency mitigates IgG responses to particulate but not soluble TD antigens. (A) ELISA of serum NP-specific IgG2b and IgG2c from WT and Ptx3−/− mice at day 14 after i.p. immunization with NP-OVA and alum. RU, relative units. (B) IFA of spleens stained for MMP9 (green) and B220 (red) from WT mice i.n. infected with PR8 influenza virus. Ctrl, control. Bars, 50 µm. (C and D) Flow cytometric analysis of absolute numbers of NBh cells (C) and MZ B cells (D) from WT and Ptx3−/− mice i.n. infected with PR8 influenza virus. (E) Serum PR8-specific IgG2c in WT and Ptx3−/− mice i.n. infected with PR8 influenza virus. Data summarize one to two experiments with at least three mice in each experimental group (A and C–E) or show one representative experiment of three with similar results (A). Error bars, SEM. *, P < 0.05 (two-tailed Student’s t test).
Figure 9.
Figure 9.
PTX3 from NBh cells delivers NF-κB–inducing signals and elicits IgG CSR and production in MZ B cells. (A) qRT-PCR of mRNAs for AID (Aicda) and Iγ2b-Cγ2b and ELISA of IgG2b in MZ B cells from WT mice co-cultured with or without NBh cells from WT or Ptx3−/− mice in the presence or absence of LPS for 2 d (mRNAs) or 5 d (IgG). Results are normalized to mRNA for the B cell–specific molecule CD79a and presented as relative expression (RE) as compared with MZ B cells incubated with medium alone. (B) qRT-PCR array of NF-κB–dependent gene products in MZ B cells isolated from 2-d co-cultures with NBh cells from WT or Ptx3−/− mice. In these co-cultures, NBh cells from Ptx3−/− mice were supplemented or not supplemented with exogenous PTX3. Results are normalized to mRNA for β-actin and presented relatively to mean expression. The color code indicates high (red), mean (black), and low (green) expression. (C–E) ELISA of serum SP-specific IgG2b, IgG2c, and IgG3 (C) and flow cytometric analysis of frequencies of MZ, transitional (T)/B-1 and FO B cells (D and E) after i.v. injection of live SP in WT mice, Ptx3−/− mice adoptively transferred with PTX3-sufficient (from WT mice) NBh cells, or Ptx3−/− mice adoptively transferred with PTX3-deficient (from Ptx3−/− mice) NBh cells. RU, relative units. Data summarize at least two experiments with at least three mice in each experimental group (A and C–E) or show one representative experiment of two with similar results (B). Error bars, SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-tailed Student’s t test).
Figure 10.
Figure 10.
NBh cell depletion impairs the expansion of splenic IgG class-switched PBs and PCs after polysaccharide immunization. (A) IFA of spleens stained for MMP9 (green), TNP (red), and MOMA-1 or B220 (blue) from WT mice before (d0) and after (d3) i.v. immunization with TNP-Ficoll. Bars, 100 µm. (B) Flow cytometric analysis of the frequency of splenic Ly6G+Ly6C+ NBh cells and Ly6GloLy6C+ monocytes/macrophages before and after treatment of WT mice with control (Ctrl) or 1A8 mAbs for 3 d. FSC, forward side scatter; SSC, side scatter. (C and D) Flow cytometric analysis of frequencies and absolute numbers of total B220−CD138+ PBs/PCs and class-switched B220−CD138+ PBs/PCs expressing IgG2b, IgG2c, or IgG3 from spleens of WT mice i.v. immunized with TNP-Ficoll (d7) after continuous i.p. treatment with nonneutrophil-depleting control mAb or neutrophil-depleting 1A8 mAb from day −3 before immunization to day 7 after immunization. (E) ELISA of serum TNP-specific IgG2b, IgG2c, IgG3, and IgM from WT mice treated and immunized as in A. Late 1A8 mAb indicates treatment with 1A8 mAb starting at day 3 instead of day −3. RU, relative units. Data show one representative experiment of at least three with similar results (A and B) or summarize at least two experiments with at least four mice in each experimental group (C–E). Error bars, SEM. *, P < 0.05; **, P < 0.01 (two-tailed Student’s t test).
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