Lipoxin A₄ modulates adaptive immunity by decreasing memory B-cell responses via an ALX/FPR2-dependent mechanism

Abstract

Specialized proresolving mediators are endogenous bioactive lipid molecules that play a fundamental role in the regulation of inflammation and its resolution. Lipoxins and other specialized proresolving mediators have been identified in important immunological tissues including bone marrow, spleen, and blood. Lipoxins regulate functions of the innate immune system including the promotion of monocyte recruitment and increase macrophage phagocytosis of apoptotic neutrophils. A major knowledge gap is whether lipoxins influence adaptive immune cells. Here, we analyzed the actions of lipoxin A₄ (LXA₄) and its receptor ALX/FPR2 on human and mouse B cells. LXA₄ decreased IgM and IgG production on activated human B cells through ALX/FPR2-dependent signaling, which downregulated NF-κB p65 nuclear translocation. LXA₄ also inhibited human memory B-cell antibody production and proliferation, but not naïve B-cell function. Lastly, LXA₄ decreased antigen-specific antibody production in an OVA immunization mouse model. To our knowledge, this is the first description of the actions of lipoxins on human B cells, demonstrating a link between resolution signals and adaptive immunity. Regulating antibody production is crucial to prevent unwanted inflammation. Harnessing the ability of lipoxins to decrease memory B-cell antibody production can be beneficial to threat inflammatory and autoimmune disorders.

Figure 1. Human B cells express ALX/FPR2

A) Human CD19⁺ B cells were isolated by magnetic bead purification and sorted for CD19⁺ cells. B cell ALX/FPR2 mRNA expression was amplified by nested semi-quantitative PCR. Differentiated HL-60 and HEK293 cell lines were used as positive and negative controls, respectively. B) To exclude the presence of contaminating ALX/FPR2 mRNA derived from other peripheral blood mononuclear cells (PBMCs), expression levels of CD3, CD11b and CD14 was measured by real-time PCR. CD3, CD11b and CD14 expressions were normalized to 7S. C) PBMCs were isolated from healthy subjects and ALX/FPR expression was analyzed by flow cytometry. Cells were gated on the live lymphocyte gate and B cells (CD19⁺) and monocytes (CD14⁺) were analyzed. D) Quantification of ALX/FPR2 expression on primary B cells and monocytes showed as mean fluorescence intensity (MFI) (gray dotted line represents background signal). E) JeKo-1 cells were transfected with ALX/FPR2 shRNA and mRNA knockdown was confirmed by nested PCR analysis. F) ALX/FPR2 protein knockdown was confirmed in JeKo-1 cells by flow cytometry analysis. G) Quantification of ALX/FPR2 expression on JeKo-1 cells. H) Primary B cells were either left non-stimulated or activated with CpG ODN 2395 plus anti-IgM. Here is shown a representative histogram of ALX/FPR2 expression analyzed by flow cytometry. Cells were gated on the live lymphocyte gate CD19⁺ population. I) Quantification of ALX/FPR2 expression. Gray dotted line represents background signal.

Figure 2. LXA₄ reduces IgM and IgG production on human B cells via ALX/FPR2 signaling

Purified B cells from healthy donors were pretreated with LXA₄, followed by stimulation with CpG ODN 2395 plus anti-IgM. Cells were cultured for 6 days at which time supernatants were collected. A) IgM and B) IgG antibody levels were measured by ELISA (n=8). C–D) Isolated B cells were pretreated with the ALX/FPR2 antagonist Boc-2 (1 µM). Cells were then treated with LXA₄, followed by activation with CpG ODN 2395 plus anti-IgM. Supernatants were collected at day 6 of culture, C) IgM and D) IgG production were measured by ELISA (n=3). Statistical analysis done using one-way ANOVA with a Tukey’s post test (*p≤0.05, **p≤0.01, n.s. not significant).

Figure 3. LXA₄ signaling decreases NF-κB translocation to the nucleus

CD19⁺ B cells were treated with LXA₄ or vehicle control and stimulated with CpG ODN 2395 plus anti-IgM, cells were then harvested and analyzed for NF-κB p65 translocation to the nucleus (n=3). A) Nuclear protein extracts were purified and analyzed by Western blot, β-tubulin and total actin were used as a nuclear purity control and loading control respectively. B) B cells were fixed, permeabilized and stained with the nuclear dye DRAQ5 (blue), anti-p65 (orange) and analyzed by ImageStream. Left panel shows representative dot plots of p65/DRAQ5 nuclear colocalization. Right panel shows representative images from single cells from either p65/DRAQ5 colocalized or non-colocalized populations. C) Representative histogram of p65/DRAQ5 colocalization (R1 represents colocalization positive gate). D) Quantification of p65 nuclear translocation normalized to freshly isolated B cells. Data were analyzed by two-way ANOVA with a Bonferroni post test (*p≤0.05).

Figure 4. ALX/FPR2 is differentially expressed in naïve and memory B cells

Purified B cells were surface stained and either FACS sorted or analyzed by flow cytometry (n=3). A) Representative histograms of ALX/FPR2 expression on naïve (CD19⁺ CD27 IgD) and memory (CD19⁺ CD27⁺ IgD) B cells, all cells were gated on live lymphocyte gate. B) B cells were stained and FACS sorted into naïve B cells (CD19⁺ CD27) and memory B cells (CD19⁺ CD27⁺) fractions. Naïve and memory B cells were stimulated with CpG ODN 2395 plus anti-Ig, pansorbin, or left un-stimulated for up 72 hours. At each time point cells were fixed and stained for ALX/FPR and analyzed by flow cytometry. Representative histograms of one donor are shown.

Figure 5. LXA₄ decreases antibody production by memory B cells but not naïve B cells

CD19⁺ B cells were stained and sorted into naïve B cells (CD19⁺ CD27) and memory B cells (CD19⁺ CD27⁺). Naïve and memory cells were pretreated with LXA₄ and stimulated with CpG ODN 2395 plus anti-Ig (n=4). A–D) After 6 days of culture, supernatants were collected. Naïve B cell (A) IgM and (B) IgG as well as memory B cell (C) IgM and (D) IgG levels measured by ELISA. E–I) Antibody-secreting cells were counted by ELISpot. E) Representative images of IgM secreting cells in naïve and memory B cell cultures. F–I) Quantification of IgM and IgG antibody-secreting cells in (F–G) naïve and (H–I) memory B cell cultures. Results expressed as mean ± SEM and analyzed using a one-way ANOVA with a Tukey’s post test (*p≤0.05, **p≤0.01, n.s. not significant).

Figure 6. LXA₄ decreases proliferation of memory B cells but not naïve B cells

Naïve (CD19⁺ CD27) and memory (CD19⁺ CD27⁺) B cells were sorted, pretreated with LXA₄ and activated with CpG ODN 2395 plus anti-Ig (n=4). A–B) Naïve and memory B cell proliferation was measured by [³H] thymidine incorporation assay, presented as counts per minute (cpm). C–D) Naïve and memory B cell viability was measured at day 6 using 7-AAD exclusion dye and analyzed by flow cytometry. Proliferation data were analyzed by two-way ANOVA with a Bonferroni posttest. Cell death results were analyzed by one-way ANOVA with a Tukey’s post test (*p≤0.05, n.s. not significant). Results expressed as mean ± SEM.

Figure 7. LXA₄ decreases mouse B cell antibody production and proliferation in vitro

Mouse splenocytes were stained for flow cytometry analysis (n=3). A) Representative histogram of ALX/FPRL1 expression on B cells (CD19+ B220+) and T cells (CD3⁺ B220). B) Quantification of ALX/FPRL-1 expression shown as MFI (gray dotted line represent background signal). C–D) Splenocytes were treated with LXA₄ or vehicle control, followed by LPS stimulation. After 6 days of culture C) IgM and D) IgG antibody production was measured in the supernatants (n=7). E) Proliferation was measured by [³H] thymidine incorporation assay over 5 days and presented as counts per minute (cpm) (n=5). F) Quantification of live cells measured by 7-AAD exclusion at day 5 of treatment (n=5). Results expressed as mean ± SEM. ALX/FPRL1 MFI results were analyzed using a paired Student t-test. Antibody production and cell viability results were analyzed using one-way ANOVA with a Tukey’s post test. Proliferation results were analyzed by two-way ANOVA with a Bonferroni post test (*p≤0.05, **p≤0.01, ***p≤0.001).

Figure 8. LXA₄ decreases antigen-specific antibody production in vivo

C57BL/6J mice were immunized with both OVA and vehicle control or LXA₄ (n=6). Two weeks after injections mice were bled and sera were isolate and use for OVA-specific A) IgM and B) IgG ELISA. Ten weeks after initial immunization mice were rechallenged with OVA, bled 2 weeks after secondary immunization and sera were use for OVA-specific C) IgM and D) IgG ELISA. Results expressed as mean ± SEM. Data were analyzed using an unpaired t-test (*p≤0.05, n.s. not significant).