Lipoxin B4 Enhances Human Memory B Cell Antibody Production via Upregulating Cyclooxygenase-2 Expression

Abstract

Vaccination has been the most effective way to prevent or reduce infectious diseases; examples include the eradication of smallpox and attenuation of tetanus and measles. However, there is a large segment of the population that responds poorly to vaccines, in part because they are immunocompromised because of disease, age, or pharmacologic therapy and are unable to generate long-term protection. Specialized proresolving mediators are endogenously produced lipids that have potent proresolving and anti-inflammatory activities. Lipoxin B₄ (LXB₄) is a member of the lipoxin family, with its proresolving effects shown in allergic airway inflammation. However, its effects on the adaptive immune system, especially on human B cells, are not known. In this study, we investigated the effects of LXB₄ on human B cells using cells from healthy donors and donors vaccinated against influenza virus in vitro. LXB₄ promoted IgG Ab production in memory B cells and also increased the number of IgG-secreting B cells. LXB₄ enhanced expression of two key transcription factors involved in plasma cell differentiation, BLIMP1 and XBP1. Interestingly, LXB₄ increased expression of cyclooxygenase-2 (COX2), an enzyme that is required for efficient B cell Ab production. The effects of LXB₄ are at least partially COX2-dependent as COX2 inhibitors attenuated LXB₄-stimulated BLIMP1 and Xpb-1 expression as well as IgG production. Thus, our study reveals for the first time, to our knowledge, that LXB₄ boosts memory B cell activation through COX2 and suggests that LXB₄ can serve as a new vaccine adjuvant.

Figure 1.. LXB₄ enhances memory B cell IgG production.

(A) Purified CD19+ B cells were pretreated with 10 or 100 nM LXB₄, followed by stimulation with different polyclonal activators as shown (MBCC, memory B cell-inducing cocktail). IgG was measured in the supernatant by ELISA after 7–8 days. (B) Purified B cells were treated with LXB₄ alone (50nM) or CpG + anti-IgM as a positive control, for 7 days, and IgG was measured in the supernatant. (C–F) PBMCs or purified CD19+ B cells were stimulated with the memory B cell-inducing cocktail and treated with the indicated concentration of LXB₄. IgG, IgM or IgE levels were measured in the culture supernatants after 7–8 days by ELISA. For (A) and (B), results shown are mean±S.D. for triplicate wells from one representative donor of 3 donors tested. For (C-F), each symbol represents an individual donor and represents the mean of triplicate or quadruplicate cultures. Data analyzed by repeated measures one-way ANOVA with Tukey’s posttest, *p≤0.05, **p≤0.01, ***p≤0.001.

Figure 2.. LXB₄ promotes reactivation of memory B cells from donors recently vaccinated against influenza virus.

PBMCs (A), or CD19+ B cells (B) were isolated from donors 7 days after influenza vaccination. PBMCs were pretreated with LXB₄, followed by stimulation with the memory B cell-inducing cocktail for 7–8 days (A). B cells pretreated with LXB₄ without stimulation were cultured for 24 hours (B). The last day, PBMCs or B cells were transferred to anti-IgG- or HA (from A/California/7/2009)-coated ELISpot plates and further cultured overnight. The number of spots representing the number of ASC was calculated using an ImmunoSpot Series 5 Analyzer. The absolute number of HA-specific or total IgG secreting cells or the fold change induced by LXB₄ are shown. Representative image from ELISpot assay is also shown. Each symbol represents an individual donor and represents means of at least triplicate cultures. Data analyzed by RM one-way ANOVA with Tukey’s posttest, *p≤0.05.

Figure 3.. LXB₄ enhances the expression of two transcription factors important for plasma cell differentiation.

CD19+ B cells from healthy individuals were pretreated with LXB₄, and then stimulated with the memory B cell-inducing cocktail (MBCC )for the number of days indicated. (A) [³H] Thymidine incorporation assay in B cells pretreated with or without LXB₄ (100nM), stimulated with memory B cell-inducing activators is done at different time points. Results shown are mean±S.D. for triplicate wells from one representative donor of 3 donors tested. (B) B cell viability with LXB₄ treatment was measured at different time points (day 1–6) in B cells from 3 different donors using 7-AAD staining and flow cytometry, mean±S.D. is shown. The effects of LXB₄ on BLIMP1 and XBP1 mRNA levels (C-D) or total protein levels (E-F) were measured using RT-qPCR or western blot, respectively. (C-D) Normalized expression of BLIMP1 and XBP1 mRNA with or without LXB₄ (100 nM) were measured and shown for one representative donor. HPRT was used as a control. Each qPCR reaction was run in triplicate and the results shown are mean±SEM for 3 different donors. (E-F) Changes in total protein levels of BLIMP1 and XBP1 at day 6 post-stimulation were measured using western blot. β-tubulin was used as a control. (E) One representative western blot of 3 donors tested. (F) Densitometry for 3 donors (mean±SEM), samples normalized to vehicle control (no treatment). Data analyzed by repeated measures one-way ANOVA with Tukey’s posttest, *p≤0.05.

Figure 4.. LXB₄ enhances cyclooxygenase-2 (COX2) expression in B cells.

CD19+ B cells from healthy individuals were pretreated with LXB₄, and then stimulated with the memory B cell-inducing cocktail (MBCC) for the indicated length of time. (A) The kinetics of COX2 protein expression in B cells stimulated with the memory B cell-inducing cocktail without any other treatments was assessed using western blot. One representative western blot is shown of 3 donors tested. (B) Changes in total COX2 protein levels with LXB₄ were measured at day 6 post-stimulation in 4 different donors. One representative western blot is shown, the densitometry is mean ± SEM for 4 donors. Data analyzed by RM one-way ANOVA with Tukey’s posttest, *p≤0.05. (C) B cells were subjected to intracellular staining for COX2 followed by flow cytometric analysis. The gating strategy is shown. The percentage of B cells expressing COX2 and mean fluorescence intensity for COX2 staining was determined. In the diagram, black dots represent B cells stimulated without any treatment, and gray dots represent B cells pretreated with LXB₄ and stimulated. Data shown from one representative donor of 4 donors tested.

Figure 5.. The stimulatory effect of LXB₄ on memory B cells is mediated by COX2 activity.

(A) CD19+ B cells from healthy donors were stimulated with the memory B cell-inducing cocktail and treated with 10μM celecoxib or NS-398, prior to LXB₄ treatment. Cell culture supernatants were collected at day 7–8, and IgG levels were measured by ELISA. Results shown are mean ± S.D. for triplicate wells from one representative donor of 2 donors tested. (B) B cells were stimulated with the memory B cell-inducing cocktail and treated with celecoxib at the indicated concentrations, and IgG levels were measured. Each panel is a single donor and the results shown are mean±S.D. of triplicate wells. Data analyzed by two-way ANOVA with Tukey’s posttest, *p≤0.05, **p≤0.01, ***p≤0.001 compared to vehicle alone. #p≤0.05, ##p≤0.01, ###p≤0.01 compared to vehicle plus 100 nM LXB₄.