T cell–independent (TI) B cell responses to nonprotein Ags involve multiple cues from the innate immune system. Neutrophils express complement receptors and activated neutrophils can release BAFF, but mechanisms effectively linking neutrophil activation to TI B cell responses are incompletely understood. Using germline and conditional knockout mice, we found that TI humoral responses involve alternative pathway complement activation and neutrophil-expressed C3a and C5a receptors (C3aR1/C5aR1) that promote BAFF-dependent B1 cell expansion and TI Ab production. Conditional absence of C3aR1/C5aR1 on neutrophils lowered serum BAFF levels, led to fewer Peyer’s patch germinal center B cells, reduced germinal center B cells IgA class-switching, and lowered fecal IgA levels. Together, the results indicate that sequential activation of complement on neutrophils crucially supports humoral TI and mucosal IgA responses through upregulating neutrophil production of BAFF.

Visual Abstract

T cell–independent (TI) humoral immune responses to polysaccharide Ags involve B1 cell activation and differentiation in the absence of germinal centers (GC). B1 cells in secondary lymphoid organs and in close proximity to mucosal surfaces of lungs, gut, and peritoneum are responsible for early IgM responses to pathogens and contribute to mucosal IgA immune responses (1, 2).

The complement system is intricately associated with the development of adaptive T cell and B cell immune responses (3). Complement activation initiates via the classical, mannan-binding lectin, and alternative pathways, which converge to form C3 convertases that cleave C3 into C3a, C3b, and, ultimately, C3dg. C3 convertases also facilitate assembly of C5 convertases that cleave C5 into C5a and C5b, the latter required for C5b-9 membrane attack complex formation. Surface-expressed and soluble complement regulators, including decay-accelerating factor (DAF; CD55), complement receptor (CR) 1, murine Crry, and membrane inhibitor of reactive lysis (CD59) (3), control complement activation.

C3 deficiency results in severe defects in TI and T cell–dependent (TD) immune responses (46). Mechanistically, C3dg-coated Ags coligate the B cell coreceptor and CR2-CD19-CD81, lowering BCR activation thresholds (6, 7). C3b-opsonized Ag facilitates trafficking to follicular dendritic cells, which use CR2/C3dg ligations to present the Ags to GC B cells during affinity maturation (8, 9). GC B cells undergo coordinate downregulation of surface-expressed CD55/DAF to permit local complement activation and autocrine C3aR1/C5aR1 signaling required for GC B cell–positive selection and Ab affinity maturation (10). The unanticipated role for complement regulators and receptors during TD responses raised the possibility that C3aR1/C5aR1 signaling may also affect TI IgM and mucosal IgA B cell responses, hypotheses that we addressed in this study.

C57BL/6J (B6), B6;129S4-C3tm1Crr/J (C3tm1Crr, C3−/−), C57BL/6-Tg(IghelMD4)4Ccg/J (MD4), B6.129P2-Lyz2tm1(cre)Ifo/J (LysM-Cre), B6.Cg-Tg(Cd4-cre)1Cwi/BfluJ (CD4-Cre), and B6.Cg-Tg(S100A8-cre,-EGFP)1Ilw/J (S100-Cre) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). We generated B6 C3aR1−/−C5aR1−/−, C3ar1fl/fl, C3ar1fl/fl, and C3ar1fl/flC3ar1fl/fl mice (10, 11). We crossed C5ar1fl/fl and C3ar1fl/fl mice to CD4-Cre+, LySM-Cre+, or S100-Cre+ transgenic mice and intercrossed their progeny to produce C3ar1fl/fl/C5ar1fl/fl × CD4-Cre+/− (C3aR1/C5aR1ΔCD4), C3ar1fl/fl/C5ar1fl/fl × LysM-Cre+/− (C3aR1/C5aR1ΔLySM), C5ar1fl/fl × S100-Cre+/− (C5aR1ΔS100), and C3ar1fl/fl/C5ar1fl/fl × S100-Cre+/− (C3aR1/C5aR1ΔS100) mice. B6 factor B−/− (fB; M. Pekna, Gothenburg, Sweden) and CR1/2−/− mice (M Carroll, Harvard Medical School) were kind gifts. We housed all animals in the Center for Comparative Medicine and Surgery at Mount Sinai under institutional animal care in accordance with guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International (approval IACUC-2018-0014). Experiments were performed with groups of age- (6–12 wk old) and sex-matched (both sexes) mice, using littermates or animals maintained in the same room and cohoused for >2 wk within the same cages from the age of 4 wk.

We immunized mice with 50 μg of 2,4,6, trinitrophenyl-LPS (TNP-LPS; Innovative Research, Novi, MI) i.p. (TI responses) or with 100 μg of TNP hapten–keyhole limpet hemocyanin in aluminum hydroxide (Immject Alum; Thermo Fisher Scientific) i.p. (TD responses). In some experiments, 4 × 107 enriched splenic IgMb+ wild-type (WT) or C3aR1−/−C5aR1−/− B cells (MagniSort; STEMCELL Technologies) were injected into IgMa+ MD4 BCR-transgenic recipients retro-orbitally and immunized with TNP-LPS as described above.

We quantified anti-TNP Abs (10), fecal and serum IgA levels (12), murine BAFF, anti-phosphatidylcholine, and anti-capsular polysaccharide 9 and 14 (13) by ELISA.

Peyer’s patches (PP) fixed in 1.6% paraformaldehyde/20% sucrose at 4°C were washed in PBS, embedded OCT compound (Tissue-Tek; Sakura Finetek USA, Torrance, CA), cut into 8-μm sections, washed with PBS, blocked with 5% BSA (Sigma-Aldrich) and Fc-block (1:200), and then sequentially stained with anti-Ly6G and anti-BAFF Abs overnight. Sections were then stained with anti-rat F(ab′)2-FITC (for Ly6G) and anti-rat IgG-PE (for BAFF) (Jackson ImmunoResearch Laboratories). We acquired images on a Zeiss LSM 780 (confocal) and processed them with Zen 2.3 software (Zeiss).

Single-cell suspensions of spleen cells, lavaged peritoneal leukocytes (5 ml PBS–1% FCS), and excised PP cells (incubated at 37°C for 10 min in 5 mM EDTA, 15 mM HEPES, and FCS 20% in HBSS) were stained (see Supplemental Table I for Ab clones) as indicated. Preparations were analyzed by flow cytometry (gating strategy in Supplemental Fig. 1) using FACSLyric or Canto II cytometers (BD Biosciences, San Jose, CA), and analysis was performed with FlowJo software (Tree Star, Ashland, OR).

We used GraphPad Prism software (version 8.4.2) for all statistical analyses (see figure legends for details). Each figure panel represents two to three or more independent experiments with multiple biological replicates (single mice or tissue samples, n > 3) and three to five technical replicates.

Upon immunization of WT and congenic B6 C3aR1−/−, C5aR1−/−, and C3aR1−/−C5aR1−/− mice i.p. with TNP-LPS (10 d; (Fig. 1A, Supplemental Fig. 1A, 1B), we observed lower frequencies of total and TNP-reactive peritoneal B1 B cells and reduced anti-TNP IgM Ab titers in mice lacking C3aR1, C5aR1, or both, without differences among the knockout strains. We also observed lower titers of natural IgM reactive to phosphatidylcholine and capsular polysaccharides in unimmunized C3aR1−/−C5aR1−/− mice versus WT controls (Supplemental Fig. 1C). Reduced frequencies of B1 cells and lower Ab titers in immunized C3−/−, CR2−/−, and, specifically, fB−/− mice (Fig. 1B) implicated involvement of the alternative complement pathway.

FIGURE 1.

TI responses require alternative complement pathway and C3aR/C5aR on neutrophils. Mice were injected with 50 μg TNP-LPS i.p., sacrificed on day 10 after immunization. Peritoneal B1 cell counts (top) and serum concentration of anti-TNP IgM Abs (bottom) were analyzed in WT, C3aR1−/−, C5aR1−/−, and C3aR1−/−C5aR1−/− (A) and WT, C3−/−, CR2−/−, and fB−/− (B) mice. (C) Kinetics of DAF expression on peritoneal B1 cells (mean fluorescence intensity [MFI] via flow cytometry); n = 4/experiment. (D) Donor peritoneal B1 cell count (IgMaCD19+B220lowCD11b+) and concentration of anti-TNP IgM in MD4+ mice on day 10 after transfer of 4 × 107 WT or C3aR1−/−C5aR1−/− B cells and TNP-LPS immunization. Peritoneal B1 cell count–naive (E) and serum anti-TNP IgM concentration–naive and TNP-LPS–immunized (F) groups of mice as indicated. Graphs show mean ± SEM from two or more independent experiments, with *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 by nonparametric ANOVA with Bonferroni posttest (A–C, E and F) or Student t test (D). In (E) and (F), responses in immunized mice were greater than in naive controls (p < 0.001 for each) unless otherwise indicated.

FIGURE 1.

TI responses require alternative complement pathway and C3aR/C5aR on neutrophils. Mice were injected with 50 μg TNP-LPS i.p., sacrificed on day 10 after immunization. Peritoneal B1 cell counts (top) and serum concentration of anti-TNP IgM Abs (bottom) were analyzed in WT, C3aR1−/−, C5aR1−/−, and C3aR1−/−C5aR1−/− (A) and WT, C3−/−, CR2−/−, and fB−/− (B) mice. (C) Kinetics of DAF expression on peritoneal B1 cells (mean fluorescence intensity [MFI] via flow cytometry); n = 4/experiment. (D) Donor peritoneal B1 cell count (IgMaCD19+B220lowCD11b+) and concentration of anti-TNP IgM in MD4+ mice on day 10 after transfer of 4 × 107 WT or C3aR1−/−C5aR1−/− B cells and TNP-LPS immunization. Peritoneal B1 cell count–naive (E) and serum anti-TNP IgM concentration–naive and TNP-LPS–immunized (F) groups of mice as indicated. Graphs show mean ± SEM from two or more independent experiments, with *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 by nonparametric ANOVA with Bonferroni posttest (A–C, E and F) or Student t test (D). In (E) and (F), responses in immunized mice were greater than in naive controls (p < 0.001 for each) unless otherwise indicated.

Close modal

To determine whether complement regulator and/or receptor levels change on B1 cells upon activation or class-switching after immunization [as observed in TD-induced GC B cells (10)], we phenotyped peritoneal B1 cells after TNP-LPS immunization. B1 cells in naive mice expressed high levels of DAF (Fig. 1C), which underwent only modest and transient reduction 6–12 h following immunization. At 24 h postimmunization, analyses showed higher expression of C3aR1, C5aR1, CR1/2, and CD59 on B1 cells (Supplemental Fig. 1D), all of which returned to baseline levels by day 7.

We tested for a functional role of B cell–expressed C3aR1/C5aR1 in TI humoral immunity by adoptively transferring splenic C3aR1−/−C5aR1−/− or WT B cells into MD4 BCR-transgenic recipients (IgMa+IgDa+ B cells, specific for hen egg lysozyme; do not respond to TNP) and subsequent immunization with TNP-LPS (Fig. 1D). Donor B1 cell count and concentration of anti-TNP IgM showed no differences between groups.

To discern which cells require C3aR1/C5aR1 signaling to support TI responses, we immunized mice lacking C3aR1/C5aR1 in myeloid cells (C3aR1/C5aR1ΔLysM) or neutrophils (C5aR1ΔS100 and C3aR1/C5aR1ΔS100). These animals all showed significant defects in B1 responses (Fig. 1E) and anti-TNP Ab titers (Fig. 1F). Controls showed no differences between mice lacking C3aR1/C5aR1 on T cells (C3aR1/C5aR1ΔCD4) and WT. Marginal zone B cells and follicular B cells in naive C3aR1/C5aR1ΔLySM and C3aR1/C5aR1ΔS100 did not differ from controls (Supplemental Fig. 1E, 1F). In contrast to TI immunization, TD immunization with TNP–keyhole limpet hemocyanin induced equivalent anti-TNP IgG titers in C3aR1/C5aR1ΔLySM and control C3aR1/C5aR1fl/fl mice (Supplemental Fig. 1G). Although frequencies of splenic B1 cells did not differ between the knockout and control strains (Supplemental Fig. 1H), we observed fewer peritoneal B1 cells in naive C3aR1/C5aR1ΔS100 versus naive S100-Cre+/− mice (Fig. 1E), suggesting a migratory defect (14). Additional controls showed that the neutrophils (gating strategy in Supplemental Fig. 2A) from C3aR1/C5aR1ΔLysM, C5aR1ΔS100, and C3aR1/C5aR1ΔS100 mice lacked C3aR1/C5aR1, whereas neutrophil-expressed DAF, CD59, CR1/2, and Crry did not differ among strains (Supplemental Fig. 2B, 2C). Splenic neutrophil counts did not differ among naive C3aR1/C5aR1ΔLysM, C3aR1/C5aR1ΔS100, and controls (Supplemental Fig. 2D).

Because BAFF can amplify TI responses and neutrophils can produce BAFF (15, 16), we tested for potential mechanistic links among neutrophils, C5aR1, and BAFF-dependent Ab production. After TNP-LPS immunization (Fig. 2A), i.p. C5a rapidly increased followed by neutrophil influx and an associated increase in serum BAFF. Twelve hours after TNP-LPS immunization, we observed lower neutrophil (Fig. 2B, 2C, top) and serum (Fig. 2C bottom) BAFF concentration in C3aR1/C5aR1ΔLySM and C5aR1ΔS100 mice. Conversely, C3a or C5a i.p. injections increased neutrophil BAFF (Fig. 2D, 2E, top) and serum BAFF (Fig. 2E, bottom) in control, but not C3aR1/C5aR1ΔLySM mice. Administration of exogenous BAFF (100 ng/d i.p. for 5 d) to TNP-LPS-immunized mice (Fig. 2F) rescued the Ab defects observed in the C3aR1/C5aR1ΔLySM and C5aR1ΔS100 mice. These findings mechanistically link C3aR1 and/or C5aR1 signaling on granulocytes/neutrophils to BAFF release, which in turn drives TI B1 cell responses. Previous studies by others (17, 18) had connected neutrophil C5aR1 signaling to NF-κB–mediated gene transcription, which is a crucial transcription factor for BAFF production.

FIGURE 2.

Neutrophil C3aR1/C5aR1 ligations drive BAFF-dependent IgM responses to TNP-LPS. (A) Kinetics of i.p. C5a (pg/ml × ml per lavage), neutrophil (CD11b+Ly6G+) influx, and serum BAFF levels following TNP-LPS immunization of WT mice (n = 3 to 4/experiment). Intracellular BAFF expression in neutrophils (mean fluorescence intensity [MFI], flow cytometry): representative histograms (B), quantification (C, top), and serum BAFF concentration (C, bottom) 12 h after TNP-LPS immunization. Neutrophil intracellular BAFF MFI: representative histogram (D), quantification (E, top), and serum BAFF levels (E, bottom) 12 h after 5 μg recombinant murine (rm)C3a and rmC5a, i.p. (F) Concentration of anti-TNP IgM on day 10 after TNP-LPS immunization of groups of mice injected with 100 ng rmBAFF daily for 5 d. Graphs show mean ± SEM from two or more independent experiments with *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 by nonparametric ANOVA with Bonferroni posttest (B–F).

FIGURE 2.

Neutrophil C3aR1/C5aR1 ligations drive BAFF-dependent IgM responses to TNP-LPS. (A) Kinetics of i.p. C5a (pg/ml × ml per lavage), neutrophil (CD11b+Ly6G+) influx, and serum BAFF levels following TNP-LPS immunization of WT mice (n = 3 to 4/experiment). Intracellular BAFF expression in neutrophils (mean fluorescence intensity [MFI], flow cytometry): representative histograms (B), quantification (C, top), and serum BAFF concentration (C, bottom) 12 h after TNP-LPS immunization. Neutrophil intracellular BAFF MFI: representative histogram (D), quantification (E, top), and serum BAFF levels (E, bottom) 12 h after 5 μg recombinant murine (rm)C3a and rmC5a, i.p. (F) Concentration of anti-TNP IgM on day 10 after TNP-LPS immunization of groups of mice injected with 100 ng rmBAFF daily for 5 d. Graphs show mean ± SEM from two or more independent experiments with *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 by nonparametric ANOVA with Bonferroni posttest (B–F).

Close modal

Mucosal IgA production in the gastrointestinal tract requires TD and TI mechanisms involving BAFF and TACI (1921). Prior evidence also indicated roles for granulocytes: eosinophils, which support the survival of IgA-secreting cells in the lamina propria (22); and PP, which typically support TD IgA production, can contain neutrophils (23). To test whether neutrophil-expressed C3aR1/C5aR1 contributed to steady-state mucosal IgA responses, we quantified frequencies of IgA+ GC B cells (gating strategy in Supplemental Fig. 3A) and total GC B cell numbers in PP and measured concentrations of fecal IgA in WT and various germline knockout mice (Fig. 3A, 3B). These analyses showed that all readouts were reduced in C3−/− and fB−/− animals and revealed similar defects in C3aR1−/−, C5aR1−/−, and C3aR1−/−C5aR1−/− mice. Antibiotic treatment to limit potential microbiome effects (Supplemental Fig. 3B) reduced PP GC B cells by ∼10-fold while maintaining IgA+ GC B cell differences between WT and C3aR1−/−C5aR1−/− mice, indicating the C3aR1/C5aR1 effects are microbiome-independent.

FIGURE 3.

Normal IgA mucosal responses require neutrophil C3aR1/C5aR1 activation and can be rescued by exogenous BAFF. Percentage of IgA+ GC B cells (top row), absolute GC B cell count in PPs (middle row), and concentration of fecal IgA (bottom row) in WT, C3−/−, and fB−/− mice (A), WT, C3aR1−/−, C5aR1−/−, and C3aR1−/−C5aR1−/− mice (B), and C3aR1/C5aR1fl/fl, C3aR1/C5aR1ΔLySM, and C5aR1ΔS100 (C) mice. Mice in (C) were injected daily for 5 d with PBS or 100 ng murine BAFF as indicated, and PP percentages of IgA+ GC B (middle) and GC B number (bottom) were quantified on day 6. (D) Intracellular BAFF expression in neutrophils from PP of C3aR1/C5aR1fl/fl and C3aR1/C5aR1ΔLySM mice quantified as mean fluorescence intensity (MFI) by flow cytometry. All graphs show mean ± SEM from three or more independent experiments, with **p < 0.01, ***p < 0.005, ****p < 0.001 by nonparametric ANOVA with Bonferroni posttest (A–C) or Student’s t test (D).

FIGURE 3.

Normal IgA mucosal responses require neutrophil C3aR1/C5aR1 activation and can be rescued by exogenous BAFF. Percentage of IgA+ GC B cells (top row), absolute GC B cell count in PPs (middle row), and concentration of fecal IgA (bottom row) in WT, C3−/−, and fB−/− mice (A), WT, C3aR1−/−, C5aR1−/−, and C3aR1−/−C5aR1−/− mice (B), and C3aR1/C5aR1fl/fl, C3aR1/C5aR1ΔLySM, and C5aR1ΔS100 (C) mice. Mice in (C) were injected daily for 5 d with PBS or 100 ng murine BAFF as indicated, and PP percentages of IgA+ GC B (middle) and GC B number (bottom) were quantified on day 6. (D) Intracellular BAFF expression in neutrophils from PP of C3aR1/C5aR1fl/fl and C3aR1/C5aR1ΔLySM mice quantified as mean fluorescence intensity (MFI) by flow cytometry. All graphs show mean ± SEM from three or more independent experiments, with **p < 0.01, ***p < 0.005, ****p < 0.001 by nonparametric ANOVA with Bonferroni posttest (A–C) or Student’s t test (D).

Close modal

Analyses of PP IgA+ GC B cells (Fig. 3C) and fecal IgA (Supplemental Fig. 3C) from groups of C3aR1/C5aR1ΔLySM and C5aR1ΔS100 mice phenocopied the findings from the germline C3aR1−/−C5aR1−/− animals, confirming a role for granulocyte-expressed C5aR1 in mucosal IgA production. Serum IgA levels and the proportion of IgM+ and IgG+ GC B cells in PP of C3aR1/C5aR1ΔLySM and C5aR1ΔS100 mice did not differ from controls (Supplemental Fig. 3D–F). BAFF administration rescued PP responses in the C3aR1/C5aR1ΔLySM and C5aR1ΔS100 mice to levels observed in control animals (Fig. 3C). Immunofluorescence microscopy of PP in WT mice (Supplemental Fig. 3G) showed BAFF colocalizing with Ly6G+ cells in the subepithelial PP dome. The immunofluorescence staining suggested reduced BAFF within the Ly6G+ neutrophils of the C3aR1/C5aR1ΔLySM animals, a result confirmed by flow cytometry (Fig. 3D, Supplemental Fig. 3H). Thus, C3aR1/C5aR1 signaling on intestinal lamina propria neutrophils is critical for optimal production of BAFF, which supports mucosal IgA production and PP responses.

Our new observations are distinct from known mechanisms linking complement to B cell immunity (10). Although previous studies suggested that C5aR1 signaling can modulate B1 cell function (14) and neutrophils express C5aR1 and can produce BAFF (24), mechanistic links were not reported previously for B1 responses or mucosal IgA production. Our findings are also distinct from prior work showing that macrophage C3aR1/C5aR1 ligations release G-CSF (25), which stimulates neutrophil BAFF production (16).

BAFF regulates B cells (26) by ligating BAFF receptor, TACI, and BCMA (27, 28) to activate canonical (via TACI) and alternative (via BAFF-R causing class-switch recombination) NF-κB–induced survival signals (20, 26, 29). Elevated plasma BAFF levels in TACI−/− mice also associate with an increase in IgA+ GC B cells and GC size in PP (19), consistent with our results. Because IgA switching is not dependent on TACI in PPs, we think that neutrophil-derived BAFF may be mediating its effect through the BAFF receptor in TD responses in PP. BAFF could also drive TI IgA outside of PP, contributing to the production of fecal IgA.

In sum, to our knowledge, our findings newly identify a neutrophil-C5aR1-BAFF axis that crucially supports humoral TI and mucosal TD responses. This fundamental mechanism has implications for designing strategies to improve polysaccharide and mucosal vaccines and/or for therapy of Ab-mediated diseases.

We thank the Mouse Genetics Core at the Icahn School of Medicine at Mount Sinai for their assistance in generating the conditional knockout animals, and Denise Peace and Tina Yao for assistance with animal husbandry.

This work was supported by the National Institute of Allergy and Infectious Diseases Grant R01 AI141434 awarded to P.S.H. and D.D.-S. E.C. received support from National Institute of Allergy and Infectious Diseases Grant T32 AI078892, E.K.G. from Crohn’s & Colitis Foundation Career Development Award 877970, and A.C. a fellowship grant from the American Society of Transplantation. S.M.-W.Y. received support from National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases Division of Diabetes, Endocrinology, and Metabolic Diseases Grant T32 DK007757 and the Agena Physician Scientist Training Program at Icahn School of Medicine at Mount Sinai.

The online version of this article contains supplemental material.

Abbreviations used in this article:

B6

C57BL/6

CR

complement receptor

DAF

decay-accelerating factor

fB

factor B−/−

PP

Peyer’s patch

TD

T cell–dependent

TI

T cell–independent

TNP

2,4,6,trinitrophenyl

WT

wild-type

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The authors have no financial conflicts of interest.

Supplementary data