Abstract
The qualities of antibody (Ab) responses provided by B lymphocytes and their plasma cell (PC) descendants are crucial facets of responses to vaccines and microbes. Metabolic processes and products regulate aspects of B cell proliferation and differentiation into germinal center (GC) and PC states as well as Ab diversification. However, there is little information about lymphoid cell-intrinsic functions of enzymes that mediate ether lipid biosynthesis, including a major class of membrane phospholipids. Imaging mass spectrometry (IMS) results had indicated that concentrations of a number of these phospholipids were substantially enhanced in GC compared to the background average in spleens. However, it was not clear if biosynthesis in B cells was a basis for this finding, or whether such cell-intrinsic biosynthesis contributes to B cell physiology or Ab responses. Ether lipid biosynthesis can involve the enzyme PexRAP, the product of the Dhrs7b gene. Using combinations of IMS and immunization experiments in mouse models with inducible Dhrs7b loss-of-function, we now show that B lineage-intrinsic expression of PexRAP promotes the magnitude and affinity maturation of a serological response. Moreover, the data revealed a Dhrs7b-dependent increase in ether phospholipids in primary follicles with a more prominent increase in GC. Mechanistically, PexRAP impacted B cell proliferation via enhanced survival associated with controlling levels of ROS and membrane peroxidation. These findings reveal a vital role of this peroxisomal enzyme in B cell homeostasis and the physiology of humoral immunity.
Introduction
The qualities and concentrations of antigen (Ag)-specific antibodies (Ab) are key elements of immunity and the pathophysiology of diverse conditions involving inflammation (1–3). Ab are secreted by cells in the B lymphocyte lineage, but the exact sources are diverse (4–7). Many derive from a subset termed B1 B cells, which are thought to having characteristics of innate responses (4, 5), but progeny of follicular and marginal zone B cells (FoB and MZB, respectively) can differentiate into Ab-secreting plasma cells (PC) after activation and proliferation (6–8). In addition to Ab concentrations and affinity for Ag, the efficacy of pathogen clearance can depend on the isotype to which B lymphocytes may transfer the Ag-combining variable region from an IgM heavy chain constant region to a new immunoglobulin (Ig) heavy chain (9–12). Importantly, the affinities of serum Ab for target Ag can increase over time after an immune exposure, especially after recurrent encounter(s) (8, 9, 13, 14).
Many sources and forms of Ab can be protective, though in several autoimmune diseases these molecules and the somatic mutations that diversify an initial BCR repertoire drive pathology (15, 16). A micro-anatomic structure termed the secondary follicle or germinal center (GC) provides one mechanism to increase spectrum of Ab affinities over time and repeated Ag exposure (17–19). Although diversification can, like PC production, occur independent from GC, results with different forms of vaccination support the practical importance of T cell help (20) and of this micro-anatomic process (21). Current evidence holds that most Ig class-switching is executed before GC entry of proliferating B cells (22). GC reactions are initiated after B cells in a primary lymphoid follicle encounter Ag, migrate to interact with activated helper T cells after extensive proliferation (23). GC formation, size, and function depend on factors that include both the efficiency of population growth for B cells in a phase before they enter and take on characteristics of GC B cells, and on homeostatic and differentiative processes while in the secondary follicle [reviewed in (24)]. Proliferation and selection end in development as Ab-secreting plasma cells or memory B cells [reviewed in (14, 18, 19)]. In line with a selection process, substantial rates of B cell death have been measured in GC (25), so that determinants of survival efficiency affect their outputs. As such, the qualities and quantities of Ab elicited by immunization (or vaccination) depend on B cell proliferation and survival both prior to GC entry as well as during GC reactions.
Accumulating evidence indicates that metabolic reprogramming and intermediary metabolism play pivotal roles in survival, proliferation, differentiation and function in the pre-immune populations that provide immune protection (26, 27), including B cells (28, 29). Among core metabolic processes, several converge on oxidative metabolism fed by glycolytic generation of pyruvate, anaplerotic use of amino acids such as glutamine, and fatty acid oxidation [reviewed in (29, 30)]. Compared to resting populations such as naive B cells, rates of such metabolism in B cells appear to be increased after activation, including in GC B cells [31-36; reviewed in (29)] although in one case at a rate that may be less heightened in this specialized subset than after T-independent B cell activation (33). Such increases support the generation of substrates for anabolic processes in the dividing cell population and perhaps the energy demands thereof.
Mitochondria have been the focus of investigations exploring molecular mechanisms for oxidative metabolism to generate energy and substrates in lymphocytes during immune responses [reviewed in (37, 38)]. Oxidative phosphorylation in and dynamics of this organelle appear to be important in determining rates of somatic hypermutation that characterize GC B cell biology (33–35), albeit by a molecular mechanism that has not yet been identified. Use of the Krebs TCA cycle to feed an electron transport chain (ETC) and efficient conversion of ADP to ATP as a high-energy phosphate donor is among specialized functions of this highly dynamic subcellular organelle. Along with other sources, mitochondria generate reactive oxygen species (ROS) via the ETC at a rate determined by structural and biochemical features (39, 40). Rates of ROS production, release, and resolution require regulation at multiple levels because these labile species and their reactions with cellular molecules are important for both signaling and cell proliferation but undermine cell function or survival when excessive.
B cells in most GC exhibit inadequacy of oxygen delivery relative to demands (31, 41–43). Of note, genome-wide screening for genes that help cells adapt to hypoxia revealed a requirement for peroxisomes and an ER enzyme involved in synthesizing plasmalogen ether lipids (EL) (44). Using an unbiased lipidomic analysis that combined imaging mass spectrometry (IMS) and techniques for identifying GC in sections of spleen from immunized mice, we had found that after immunization, local concentrations of a subset of EL were heightened in GC relative to other portions of the spleen (45). Ether phospholipids (EPL) have a fatty acid linked to the glycerol backbone with an alkyl or a vinyl ether bond at the sn-1 position (46, 47). Synthesis of ether phospholipids depends on the generation of precursors in peroxisomes and final processing in the ER (48, 49). However, EL biosynthesis is prominent in the liver, and in theory these species or their precursors might distribute to tissues from a primary site of biosynthesis or from the diet (49–53). Thus, the finding that secondary follicles had greater densities of some - but not all - ether phospholipids left several key questions unanswered – (1) Does the capacity to synthesize and increase ether lipids in B cell follicles – primary or secondary (i.e., GC) - affect antibody responses? (2) Are any of the EL dependent on B lineage-intrinsic metabolism to generate precursors or final products? Moreover, there is debate whether EL or their plasmalogen subset promote cell death or resistance, and whether inactivation of biosynthesis causes defects in establishment or maintenance of hematopoietic cells (46, 53–55).
Peroxisomal Reductase Activating PPARγ (PexRAP, encoded by the Dhrs7b gene) catalyzes the reduction of alkyl-dihydroxyacetonephosphate (DHAP) to 1-alkyl-G3P (46, 48, 56), which then is transferred into endoplasmic reticulum (ER) and further metabolized to generate EL. Recent work notes that this enzyme may substantially function in the ER in addition to the peroxisome (56). Widespread inactivation of Dhrs7b in mature mice decreased hepatic production of ether lipid species and reduced the population of erythrocytes and neutrophils (46). Although questioned based on inborn errors of metabolism affecting other aspects of ether lipid biosynthesis (54), this body of work indicated that PexRAP can be essential in some aspects of ether lipid metabolism and may function in hematopoietic cells. Diverse functions of ether lipids in general, and the plasmalogen subset in particular, have been proposed or supported by prior work [reviewed in (57–61)]. The ether linkage may provide a sink for oxygen radicals - thereby acting directly to resolve the reactivity and contribute to ROS homeostasis, although there is debate as to the physiological impact of this chemical property (62). Whether or not this lipid subset influences adaptive immunity or conventional lymphocyte lineages, however, is unclear.
Herein, we tested the hypotheses that (i) the ether lipid profiles in activated B cells depend on their expression of the biosynthetic enzyme PexRAP [alternatively referred to as acyl/alkyl-DHAP reductase (ADHAPR) (56)], and (ii) this enzyme promotes B cell physiology. To do so, we combined conventional lipidomics, Imaging Mass Spectrometry (IMS), and immunization experiments with genetic models in which loss-of-function for Dhrs7b is induced in adult mice. We provide evidence that PexRAP impacts B cell proliferation via enhanced survival associated with controlling levels of ROS and membrane peroxidation. The results also indicate that beyond an impact on B cell homeostasis, GC are reduced in its absence, and the magnitude and affinity maturation of a serological response depend on B cell expression of PexRAP. Taken together, these finding support a function of B cell-intrinsic biosynthesis of ether lipids in B lymphocyte homeostasis, the production of Ab, and in promoting GC reactions.
Results
PexRAP promotes homeostatic maintenance and proliferation of B cells
By use of IMS, we had reported that at least a dozen ether phospholipids - including plasmalogens - were enriched in splenic germinal centers of immunized mice when compared to the rest of the tissue (45).
While previous work provided evidence that these lipid molecules were important for the homeostasis of short-lived neutrophils (46), this effect was questioned based on inborn errors of metabolism (54). Moreover, the impact of these molecules on adaptive immune cells or functions is not known. Accordingly, we sought to test for effects of an intervention that interferes with a key biosynthetic pathway used for endogenous synthesis of these molecules. To do so, we started with analyses using induced loss-of-function for Dhrs7bf/f and the widely expressed Rosa26-CreERT2 transgene (63, 64) used in (46), which reduced hepatic generation of a subset of ether lipids and acts in all hematopoietic cells as well.
We modeled initial experiments on the published work with imaging mass spectrometry (45). Mice were treated with tamoxifen using conditions less intense than a regimen shown to have no effect on pre-immune splenic populations or B lineage (65, 66), immunized with SRBC [akin to the analyses in (31, 36, 41, 45)], and analyzed 7 d after immunization (Fig. 1A). Functional inactivation of Dhrs7b in T and B lymphocytes was confirmed by Western blot analysis using anti-PexRAP Ab (Fig. 1B). The frequencies and numbers of CD19+ B220+ B cells in Dhrs7bf/f; Rosa26-CreERT2 - only a small minority of which would have been activated by the immunization - were approximately 0.6 the values for controls (Dhrs7b+/+ Rosa26-CreERT2, i.e., wild-type locus) (Fig. 1C, D). In contrast to B cell populations, TCRβ+ CD4+ T cells and TCRβ+ CD4- CD8 T cells were intact in tamoxifen-treated and SRBC-immunized Dhrs7bf/f; Rosa26-CreERT2 mice (Supplemental Fig. 1A-C). These results suggest that Dhrs7b is non-redundantly required for fully functional B cell maintenance but indicate that the enzyme is dispensable for the main populations of conventional T cells. In light of the earlier finding that a subset of ether lipids was more prevalent in secondary follicles, we analyzed GC in these samples. Microscopy and flow cytometry analyses after immunofluorescent staining found that less GC B cells were present after immunization of Dhrs7bf/f; Rosa26-CreERT2 mice, i.e., less IgD- CD95+ GL7+ (GC-phenotype) B cells (Fig. 1E-I; Supplemental Fig. 1D). Quantitation of the immune fluorescence micrography with spleens from immunized mice revealed that the numbers of GC per spleen were halved in Dhrs7bΔ/Δ mice compared with controls (Fig. 1I; Supplemental Fig. 1E). The sizes of those GC that did form were substantially reduced as well (Fig. 1I; Supplemental Fig. 1F). The lower number of B cells found in immunized mice - while a less profound effect than the reduction in GC - tempers the result, and it was possible that the function of helper CD4 T cells was impaired even their though numbers were not affected. While not addressing whether or not B cells are part of the requirement for PexRAP in promoting GC, these results indicate that Dhrs7b is necessary for a normal-sized geminal center response.
To determine if there are B lineage-specific functions of PexRAP, we used a conditionally active Cre transgene that is expressed specifically in mature B cells (67). Dhrs7b f/f, huCD20-CreERT2 mice and huCD20-CreERT2 were treated with tamoxifen to induce Cre-mediated recombination of the floxed alleles after initial establishment of normal B cell populations (Fig. 2A). Western blot analyses showed an almost complete absence of PexRAP protein from B cells purified from tamoxifen-treated Dhrs7bf/f; huCD20-CreERT2(hereinafter, Dhrs7bΔ/Δ-B) mice compared to similarly treated huCD20-CreERT2 controls (Fig. 2B). The enzyme encoded by this gene catalyzes the synthesis of a lipid precursor to many - but not all - plasmalogens and other ether phospholipids (46–48), and Dhrs7b deficiency impacted neutrophil survival (46). When we tested if Dhrs7b inactivation affected the steady-state B cell population, the frequencies and total numbers of CD19+ B220+ B cells in viable lymphocyte gates were about 20% lower in tamoxifen-treated Dhrs7bΔ/Δ-B mice compared to tamoxifen-injected, huCD20-CreERT2, Dhrs7b+/+ controls (Fig. 2C). With loss-of-function after production of mature B cells, i.e., inactivation of Dhrs7b at ages over 6 wk, we observed balanced decreases in the numbers of multiple subsets within the B lineage without evidence of selectivity among main sub-classes of B cell (Supplemental Fig 1G-I). Thus, the frequencies of MZB and FOB amidst the smaller population of splenic B cells in tamoxifen-treated Dhrs7bf/f; huCD20-CreERT2 mice were similar (Supplemental Fig 1H), and frequencies of B1 B cells in the peritoneal cavity also were unaffected (Supplemental Fig 1I). Prior work documents normal frequencies and numbers of developing and mature B cells under harsher conditions of tamoxifen-induced deletion with CreERT2 (65). De novo B cell production rates are too low to yield the ∼20% decrease observed one week after gene disruption, and few splenic B cells are in cell cycle. Accordingly, we infer that after maturation of a B cell, Dhrs7b promotes its survival, albeit to a modest degree.
B cell population growth can be impacted by increased cell death. To test if PexRAP affects proliferation in vivo, Dhrs7bΔ/Δ B cells were stained with CellTrace Violet (CTV) and adoptively transferred into B cell deficient µMT recipient mice. Strikingly fewer PexRAP-depleted B cells were recovered (Fig. 2D), and the frequencies of Dhrs7bΔ/Δ B cells that were IgD- (i.e., had been activated) were half those of controls (Fig. 2E; supplemental Fig. S1J). As compared to control B cells, CTV partitioning analyses suggested that division rates in vivo were modestly lower for the Dhrs7bΔ/Δ B cells (Fig. 2F, G). Mitogen-stimulated Dhrs7bΔ/Δ B cells that proliferated in culture exhibited substantially less robust division: frequencies of viable cells that had undergone >3 divisions were halved in Dhrs7bΔ/Δ B cells) and yielded ∼1/3 as large a progeny population (Fig. 2G, H). Collectively, these data indicate that the Dhrs7b gene product supports homeostatic maintenance of a quiescent (pre-immune) B cell population in vivo and effective proliferation of B cells.
B cell expression of PexRAP is required for achieving normally distributed concentrations of ether phospholipids
As noted, lipidomic analyses that used 2-dimensional image mass-spectrometry (2D-IMS) discovered that local concentrations of a subset of ether lipids are enriched in GC compared with the area outside of GC after immunization (45). To measure the extent to which PexRAP expression within mature B cells can affect their lipid content and composition, including their ether- and plasmalogen phospholipids, LC-MS-MS analyses were performed with B cells directly isolated from spleens as well as those cultured after mitogenic activation. These analyses showed substantial reductions in many ether phospholipids - as well as lysophosphatidylethanolamines (LPE) generated by phospholipase cleavage of the R2 fatty acid sidechain of an ether phospholipid - in PexRAP-deficient B cells (Fig. 3A, B; Supplemental Fig. 2A, B). While beyond the scope of this work, this evidence suggests that generation of potential signaling molecules via phospholipase(s) such as PLA2 may be reduced. Of note, whereas differences were modest or absent in the naive population, the greatest impact of PexRAP on quantitative amounts of phospholipids was found in the activated B cells, for which tamoxifen treatment and CreERT2 made minimal differences (Supplemental Fig. 2C, D). Several species, skewed towards those with the most detected ions, were at less than 1/5th the level of non-deleted control B cells (Fig. 3C, D). There is no in vitro surrogate that faithfully represents GC or the GC B cell, but collectively these data provide strong evidence that the capacity to rapidly produce increased levels of many ether lipid molecules after lymphocyte activation depends on a functional Dhrs7b gene in B cells. Although additional uptake or biosynthesis pathways may add to the overall pool, as previously noted (46, 47) - and especially in resting B cells that are out of cycle - the data indicate that Dhrs7b in B cells enhances cell-autonomous biosynthesis. The straightforward inference is that PexRAP catalyzes the generation of ether lipid precursors in amounts crucial for regulating the overall ether phospholipid pools, especially after B cell activation.
Our earlier work (45) reported only ion mode features in negative mode and did not analyze the primary follicle (the vast majority of which are resting B cells) relative to the extrafollicular white pulp, red pulp, and GC. To investigate these issues and ultimately the requirement for specific expression of PexRAP, we started with AID-GFP mice to enhance spatial localization. These measurements confirmed that a substantial number of both positive and negatively charged ions whose exact masses identified them as plasmalogens were substantially concentrated in GC as compared to the rest of the B cell follicle (Table 1; Supplemental Fig. 3A, B). Accordingly, we tested if the differential enrichment of any ether lipid species in primary or secondary follicles depends on biosynthesis within a specific lymphoid lineage. Mice with conditional mutations were tamoxifen-treated, immunized with SRBC, harvested seven days thereafter, and analyzed by IMS (Fig. 4A, B). With deletion driven by the widely expressed Rosa26-CreERT2 transgene, as in earlier work (46) and Fig. 1, substantial reductions of the GC enrichment pattern were observed (Fig. 4C). For instance, two representative ions identified in negative ion mode (i.e. m/z 752.5545 and m/z 776.5556) were again enriched in GC regions of spleens from immunized WT mice. These features barely increased in splenic samples of immunized Dhrs7bΔ/Δ (tamoxifen-injected Dhrs7bf/f; Rosa26-CreERT2) mice (Fig. 4C).
To determine if the levels of particular ether phospholipids in GC regions were affected by PexRAP expression in B cells, Dhrs7bf/f; huCD20-CreERT2 mice and huCD20-CreERT2 controls were analyzed after tamoxifen injections followed by immunization, and compared to samples from unimmunized mice. IMS using both positive and negative ion modes identified at least eight ions - including m/z 752.5545, m/z 776.5556, and m/z 872.5749 - much more substantially (∼2-3-fold) concentrated in GC of immunized control mice (Fig. 4D, E; Table 2) than in the primary follicle of unimmunized mice. Notably, these increased signals in secondary follicles (GC) were reduced or almost completely eliminated in GCs of mice immunized after B cell-specific PexRAP depletion (Dhrs7bΔ/Δ-B) (Fig. 4D, E; Table 2; Supplemental Fig. 3C).
Collectively, these results indicate that Dhrs7b gene expression in activated B cells regulates the spectrum of ether lipids in the micro-anatomic locale of a secondary follicle. Interestingly, immunization also led to modest increases in the levels of some ether and conventional phospholipids in the B cell-rich primary follicle regions (Table 2). This finding extends the previous report (45), which focused on the relationship between the AID-GFPhi region (i.e., GC) and lipid features. Moreover, the absence of PexRAP blunted most of the immunization-induced increases observed in the primary follicles. These findings and those of Fig. 3 suggest that there are secondary consequences of Dhrs7b inactivation (e.g., some di-acyl phospholipids are affected), but provide direct evidence that the enhancement of many ether phospholipid signals in the GC depends on PexRAP in B cells.
B cell-intrinsic role of Dhrs7b in Ab affinity and quantity
As key components of adaptive immunity, progeny of an activated B cell can differentiate into Ab-secreting plasma cells (PC) or into GC B cells - whose physiology diversifies the affinity for and breadth of antigen recognized by the original clone and improves properties of memory [(14, 17–19, 68). PC that develop after a second encounter with antigen yield affinity maturation, and the higher quality and proper quantity of Ag-specific Abs from plasma cells are integral to humoral immune responses (68). An increasing body of evidence indicates that metabolic reprogramming and intermediary metabolites modulate immune cell differentiation and function (26–36). The observed B cell-intrinsic function of Dhrs7b in the accumulation of many ether lipid species in both primary and secondary follicles prompted us to test the effect of B cell type-restricted PexRAP depletion on GC responses and Ag-specific Ab production. Tamoxifen-injected Dhrs7bf/f; huCD20-CreERT2 and control mice were immunized with NP-ovalbumin (NP-OVA), then boosted with NP-OVA to elicit affinity-matured Ab, and harvested 1 week thereafter (Fig. 5A). Although PexRAP-deficient B cell numbers were reduced less than 20% a week after starting gene inactivation prior to immunization (i.e., were over 0.8-fold those of controls) (Fig 2A-C), frequencies and numbers of IgDneg B cells were halved in Dhrs7bΔ/Δ-B mice at harvest (4 wk after completion of the induced deletion) (Fig. 5B, C). Moreover, the GL7+ CD95+ fraction of the B cell population in the dump / IgDneg gate was substantially reduced (Fig. 5D), such that numbers of GL7+ CD95+ GC B cells were dramatically decreased in Dhrs7bΔ/Δ-B mice (Fig. 5E). Consistent with these findings, GC in Dhrs7bΔ/Δ-B mice were reduced after SRBC immunization, with a modest change in the DZ/LZ ratio (Supplemental Fig. 4A, B).
Ab class-switch recombination (CSR) and affinity maturation can occur through extrafollicular response, but the GC reaction significantly increases Ab diversification and high-affinity Ab production, in part later in a primary response but also upon secondary exposure to antigens (6, 8, 9, 17–19). ELISA on sera at 3 wk post-immunization (Fig. 6A-D), just before the boost, showed that B cell-specific depletion of PexRAP prior to immunization reduced NP-specific IgM and the switched isotype IgG1 (Fig. 6A, C). Of note, the capacity to generate high-affinity Ab, detected with low valency NP2, was even more severely undermined than the overall response - especially for IgG1 (Fig. 6B, D). A week after a second immunization, Ab-secreting cells (ASCs) in the spleen (Fig. 6E) and circulating anti-NP IgM concentrations were diminished in Dhrs7bΔ/Δ-B mice compared with controls that were huCD20-CreERT2, Dhrs7b+/+ treated with tamoxifen in parallel to the mice with induced loss of PexRAP (Fig. 6F). The ratio of high-affinity (NP2-binding) to all-affinity (NP20) IgM Ab also was substantially lower in Dhrs7bΔ/Δ-B mice (Fig. 6G), as were the serum levels of high-affinity IgG1 and IgG2c, class-switched isotypes (Supplemental Fig. S4C, D). These data reinforce and extend the conclusion that the expression of PexRAP in mature B cells is important for their physiology and function.
Consistent with in vivo results finding reduced Ag-specific ASCs in Dhrs7bΔ/Δ-B mice compared with controls (Fig. 6E), the attenuated population of PexRAP-deficient B lineage cells recovered after transfer into recipient mice (Fig. 2D) yielded lower frequencies of CD138+ progeny (Fig. 6H, I) along with evidence of reduced activation [i.e., lower frequencies of IgD- progeny (Supplemental Fig. 1I). Mitogen-stimulated Dhrs7bΔ/Δ B cells also yielded lower frequencies of CD138+ cells in vitro, but the division-specific frequencies of CD138+ cells showed only modest decreases in Dhrs7bΔ/Δ B cells (Supplemental Fig. 4E). Thus, the reduction of CD138+ cell differentiation appears to be due mostly to its dependence on survival to a sufficient division count.
Dhrs7b inactivation was initiated prior to immunization in the preceding experiments. Therefore, the impact of PexRAP deficiency on GC and the Ab response in such a setting might be due exclusively to impairment of B cells, e.g., their reduced population expansion, prior to their entry into GC. Alternatively, the effects could also in part involve a requirement for PexRAP-dependent metabolites within GC B cells. To test if Dhrs7b functions within GC, we used conditional deletion of Dhrs7b driven by the S1pr2-CreERT2 transgene whose expression at high levels marks GC B cells (69). Of note, experiments with a fate-marking reporter allele showed that activated B cells that lack the GC B phenotype were not marked by this conditional Cre after immunization with the NP-carrier approach (69). Dhrs7bf/f; S1pr2-CreERT2and control S1pr2-CreERT2 (control) mice were immunized with SRBC, and tamoxifen was injected at a time point after the initiation of GC to assure more fully that the inactivation of Dhrs7b would be in GC B cells rather than pre-GC blasts (Figure 7A). Frequencies of GC B cells were substantially lower in tamoxifen-treated Dhrs7bf/f; S1pr2-CreERT2 mice (Figure 7B, C), whereas the overall populations of CD138+ cells, and CD19+ B220+ plasmablasts (PBs) among them - almost all of which would have developed prior to the induced loss-of-function - were normal (Figure 7D, E). These data provide evidence that Dhrs7b expression within GC B cells influences them during an Ab response. Collectively, these data indicate that an optimal GC response requires PexRAP function in B cells, and the B cell-intrinsic functions of Dhrs7b promote the quantity and affinity maturation of Ab responses, in part via net proliferation of B cells.
Dhrs7b contributes to modulation of ROS and their impact on B cell population growth
Increased susceptibility to cell death after activation would be a mechanism that could cause the reduced population expansion, GC, and Ab production. Consistent with earlier analyses of LPS-stimulated B cells (70), we had noted that ROS levels were higher in GC B cells, memory B cells (MBCs) and ASCs than in the naive B2 B cell pool (36). ROS can be produced in activated B cells via diverse sources that include mitochondrial electron transport chain function, NADPH oxidase activated by BCR engagement, and fatty acid oxidation in peroxisomes (71–73). ROS can positively mediate signal transduction, but their steady-state concentrations need to be calibrated because overproduction can have deleterious effect on cells, for instance via lipid peroxidation at excessive rates (74–76). Endogenous antioxidant properties have been imputed to plasmalogens because the vinyl ether bond is susceptible to reaction with reactive oxygen, thereby scavenging ROS (48, 57–59). When we tested if Dhrs7b function is critical for redox control in B cells, mitogen-stimulated Dhrs7bΔ/Δ B cells exhibited ∼3 fold higher signal and about a doubling when stained with fluorescent sensors of cellular and mitochondrial ROS, respectively, compared to WT B cells in vitro (Fig. 8A-D). Iron- and copper-dependent lipid peroxidation is considered to be a biological mechanism for ROS-mediated cell death (75, 76). Lipid peroxidation measured by a fluorescent indicator (C11-Bodipy) in vitro was enhanced in mitogen-activated Dhrs7bΔ/Δ B cells compared with WT controls (Fig. 8E). One independent means of driving increased ROS and lipid peroxidation in B cells (supplemental Fig. S5A) rapidly led to reduced cell viability and B cell numbers (Fig. 8F). Furthermore, in vitro analyses indicated that B cells lacking PexRAP also exhibited increases in the activated executioner caspase, cleaved caspase-3 (CC3) along with early apoptotic cells that are annexin V+ but exclude 7-aminoactinomycin D (Fig. 8G, H). These data indicate that Dhrs7b function supports not only protection against cell death, but also control of cellular and mitochondrial ROS levels and their impact on lipid modification. Of note, while H2O2 did not increase this early apoptotic population (Supplemental Fig. SC), increasing ROS with menadione drove B cell death preceded by annexin V+ state without an increase in C11-Bodipy signal (Supplemental Fig. S5D-G). We infer that rather than a single mode of death, increased ROS resulting from PexRAP depletion stimulates at least two distinct modes of B cell death.
Consistent with the increased steady-state ROS and death, activated Dhrs7bΔ/Δ B cells exhibited a defect in B cell population growth in vivo and in vitro (Fig. 2; Fig. 8A-H). The well-established ROS scavenger, N-acetyl-L-cysteine (NAC), exerted a concentration-dependent effect that improved the survival and population growth of PexRAP-deficient B cells (Fig. 8I).
We exploited the sub-maximal rescue by a lower concentration of NAC (1 mM) to explore if peroxisomal oxidative metabolism might contribute to the toxicity observed when B cells are PexRAP-depleted. Although thioridazine on its own provided no increase in the B cell population growth, its combination with 1 mM NAC treatment improved growth of the Dhrs7bΔ/Δ B cell population compared to either agent on its own (Fig. 8J). These data indicate that sufficient generation of PexRAP-dependent products is crucial for B cell survival. Moreover, this enzyme contributes - directly or indirectly - to a resolution or detoxification of ROS that is critical for population growth of activated B cells.
Dhrs7b in B cells affects their oxidative metabolism and ER mass
Our finding that mitochondrial ROS were increased prompted us to investigate how PexRAP affects physiological functions by measuring respiration in activated B cells. Mitochondrial stress tests with in vitro activated B lymphoblasts measured small but definite decreases in both basal and maximal respiration (oxygen consumption rates, or OCR) (Fig. 9A-C). The altered performance of mitochondria was associated with reductions in calculated ATP generation and proton leak as well as a small decrease in spare respiratory capacity (SRC) (Fig. 9D-F, respectively). In contrast, the rates of glucose-stimulated extracellular acidification - cytosolic reactions and a surrogate that can approximate glycolytic activity - were unaffected (Fig. 9G).
Thus, the altered respiration did not reflect a decrease in all cell metabolism. Inasmuch as peroxisomes and mitochondria form contacts and functionally interact with the ER, we measured relative ER mass and found that the signal of the fluorophore ERTracker was consistently reduced in PexRAP-depleted B cells (Fig. 9H, I). We infer that both mitochondrial function and ER mass in B cells are promoted by their expression of PexRAP.
Discussion
We have shown herein that B lymphocyte expression of an enzyme essential for biosynthesis of a subset of ether phospholipids is crucial for part of the increases in their levels localized to GC. Moreover, the genetic approach used to deplete B cells of PexRAP allowed us to show that the B cell type-restricted gene, Dhrs7b, promotes B cell survival, proliferation, and GC size along with affinity maturation and serum concentrations of Ag-specific Ab after immunization. Taken together, this work establishes the predictive value of the discovery approach that identified an unexpected feature of cellular biochemistry in GC and indicates that B cell-autonomous generation of ether lipid species is crucial for B cell survival and the qualities of the Ab elicited by immunization.
Using imaging mass spectrometry for discovery-based hypothesis generation, we had reported that the concentrations of at least a dozen ether phospholipids are increased in splenic GC relative to the remainder of the tissue (45). The analyses presented here confirm and extend the observations, and provide evidence that after immunization, these concentrations increase even in the primary B cell follicle, albeit to a modest degree when compared to the magnitude of increases in GC, and in B cells activated ex vivo. The cell type-specific gene inactivation after establishment of a pre-immune population establishes that an enzyme crucial for generation of a number of plasmalogens has substantial effects on B lymphocyte physiology and function.
Plasmalogens and other ether lipids and phospholipids have long been shown to be major constituents of lipid bilayers and cell membranes (48, 49, 57). However, remarkably little is known about whether or not cell-intrinsic synthesis of ether lipids or the balance among their specific molecular species matters for hematopoietic cells or in immunity. Several inborn errors of metabolism in humans are attributable to loss-of-function mutations of genes encoding peroxisomal proteins that impact ether lipid (and hence plasmalogen) synthesis (58) but affect other critical processes as well [reviewed in (48, 58–62)]. Several of these - such as Zellweger Syndrome and rhizomelic chondrodysplasia punctata (RCDP) - and the mouse models generated to study mechanisms in these human disease, lead to severe neurological defects and early post-partum death (57, 58). Whether or not lymphocyte development, homeostasis or function were affected in these studies is not clear. Deficient generation of ether lipids is among many abnormalities caused by elimination of fatty acid synthase (FAS) (46, 47). Acute inactivation of Fasn, the gene encoding this enzyme, in young mature mice caused a decrease in spleen size disproportionate to the reduction in neutrophils, which was the most profound hematological consequence (46). In addition, circulating lymphocytes were reduced despite normal steady-state representation in the marrow. In-trans cell-extrinsic functions of ether lipids have been noted previously - specifically, as ligands for the invariant natural killer T cell receptor (77) or for a G-protein coupled receptor on natural killer cells (78). As such, the widespread loss of FAS function and the potential for indirect and pleiotropic effects of this enzyme deficiency left unresolved what cells actually depend on their own synthesis of ether lipids.
PexRAP, the enzyme encoded by the Dhrs7b gene, generates intermediates vital for the synthesis of some - but not all - ether lipids in cells such as neutrophils (46). Analyses of neutrophil numbers and survival provided evidence that this enzyme can be crucial for a normal membrane composition. Moreover, PexRAP promoted viability of this very short-lived cell type whose membranes have a high plasmalogen content (46). The bloodborne population of lymphocytes was reduced in the Rosa26-CreERT2, Dhrs7bf/f mice (46), but the specific types of lymphoid cell were not reported and the circulating cell number does not necessarily correlate with that in spleen or other organs. Of note, although subject to points articulated in (53), the interpretation that the neutrophil phenotype is due to insufficiency of ether lipids or plasmalogens has been questioned based on alternative gene disruption results (54). Several models of unconditional gene inactivation or mutation that drastically reduced the phospholipid end-products were noted to have normal neutrophils [summarized in (54), albeit at older ages (4 - 7 mo) than those used in (46). As noted, however, there may be age-related changes in the processes analyzed herein using young (age ∼ 2 mo) mice (53). Moreover, cellular and organismal selection for adaptation variants makes the results of acute loss-of-function inherently different from unconditional loss and heterozygote parentage (53). The potential for toxicity induced by 4OHT activation of CreERT2 was among concerns mooted about the earlier study of neutrophils (54). This concern may have been extrapolated from papers that noted impairment in earlier hematopoiesis when Rosa26-CreERT2 was combined with intensive regimens of tamoxifen administration to infant mice (65, 79). Of note, such studies used individual doses over 4-fold higher that those used herein, and delivered by gavage five times on a daily basis, yet the IgM+ B cell population sizes in spleen and marrow were normal even in the face of reduced erythropoiesis and thymopoiesis (65). Moreover, the analyses herein compared B lineage-specific deletion to CreERT2+/-, Dhrs7b+/+ mice identically dosed in parallel with the test subjects. Thus, the work herein indicates that PexRAP is essential for normal physiology and function of mature B lymphocytes, in particular after their activation.
The findings are most consistent with a capacity for PexRAP function to contribute towards B lymphocyte survival and effective proliferation by restraining both executioner caspase activation and lipid peroxidation due to excessive ROS levels. These findings are consistent with those obtained with neutrophils (46). On the surface such results might appear to present a conundrum relative to work indicating that both fatty acyl-CoA reductase 1 and the enzyme that later generates an alkenyl bond at a terminal step in plasmalogen biosynthesis are crucial for the susceptibility of HT-1080 and 786-O tumor cell lines to ferroptosis (55). However, our data are consistent with published evidence that PexRAP is needed for a narrower subset of ether lipids than the broad requirement for FAS or fatty acyl-CoA reductases (46–49, 80, 81). Moreover, molecular dissection of mechanisms in various non-hematopoietic cell types (including the cancer line 786-O) showed that ether lipid biosynthesis can either promote susceptibility or resistance to ferroptosis (82). Collectively, our findings suggest that for B cells the inactivation of PexRAP tilts the balance toward death and reduced Ab - perhaps involving lower fractions of poly-unsaturated fatty acids (PUFA) (82), or a lower ratio of plasmalogens (alkenyl linkages) relative to alkyl-linked ether lipids.
PexRAP localizes to peroxisomes (35–37), and prior work has suggested both that peroxisomes are increased in GC B cells and likely contribute to FAO by these cells (26). In line with the possibility that this organelle may affect PexRAP-dependent survival, our data indicated that specific inhibition of peroxisomal FAO could collaborate with sub-optimal ROS scavenging to mitigate the reduced growth of activated B cells. However, recent findings show that PexRAP also is expressed in the ER and is capable of contributing to ether lipid biosynthesis via catalysis in this organelle in addition to or instead of the peroxisome (56). Thus, although ROS generated in peroxisomes likely contribute to the stress experienced by PexRAP-deficient B cells, the phenotypes observed herein may not reflect the need for the peroxisomal fraction of the enzyme.
Our findings with an intermediary in ether lipid biosynthesis add to an increasing body of evidence that metabolism in B lymphocytes affects their differentiation or function (28–36). Many sources and forms of Ab can be protective, while in several autoimmune diseases these molecules can drive pathology (1, 2). The capacity to generate increased affinities and circulating concentrations of Ab is an important factor both in protective immunity and auto-immune disease. Antigen-independent, TLR-driven PC differentiation requires increased oxidative phosphorylation (70), while oxidative metabolism in GC B cells promotes affinity maturation by unknown mechanisms (33–35). While not the exclusive determinant of such properties, GC are major sources of higher affinity Ab with a broader spectrum of specificities (9, 13, 14). GC formation, size, and function depend on the efficiency of population growth for B cells in a phase before they enter and take on characteristics of GC B cells, and then homeostatic and differentiative processes while in the secondary follicle. Thus, while the findings with the post-immunization inactivation of Dhrs7b using S1pr2-CreERT2 provide evidence that the expression of PexRAP is important within GC B cells, the decreases of GC and affinity-matured Ab are likely also to involve a pre-GC phase of clonal expansion. In any case, the rapidity of the effects suggests that pharmacological inhibition of PexRAP may be a target in inflammatory disease in which B cells participate, particularly in light of the potential for impacts on pro-inflammatory neutrophils.
Materials & methods
Reagents
Monoclonal antibodies (mAb) against mouse CD40, CD90.2, B220 (CD45R), and other mAbs (purified, biotinylated, or fluorophore-conjugated) were from BD Biosciences or Tonbo Biosciences (San Diego CA) unless otherwise indicated. BAFF was from AdipoGen (San Diego, CA). Recombinant mouse IL-4 and recombinant mouse IL-5 were from Peprotech (Rocky Hill NJ). Glucose, 2-deoxyglucose, oligomycin, rotenone, antimycin A, carbonyl cyanide 4-phenylhydrazone (FCCP), N-acetyl-cysteine (NAC), thioridazine hydrochloride, H2O2, and 4-hydroxytamoxifen (4-OHT) were from Sigma-Aldrich Chemicals (St. Louis MO), and menadione from Cayman Chemicals (Ann Arbor, MI). Tamoxifen (Tmx) was from APExBio Technology (Houston TX). NP-BSA and NP-PSA (for capture in ELISA) as well as NP-OVA (for immunization) were obtained from Biosearch Technology (Novato CA). SRBCs (sheep red blood cells) were from Thermo Fisher Scientific (Waltham MA). CellTraceTM Violet cell proliferation kit, CM-H2DCFDA, MitoSOXTM Red, and BODIPYTM 581/591 C11 were obtained from Invitrogen (Waltham, MA).
Mice, immunizations, ELISA and ELISpot
All animal protocols were reviewed and approved by the Vanderbilt University Institutional Animal Care and Use Committee. Mice were housed in ventilated micro-isolators under Specified Pathogen-Free conditions in a Vanderbilt University Medical Center mouse facility and used both male and female mice at 6–8 weeks of age. For cell type specific inactivation of Dhrs7b genes, Dhrs7bf/f mice (46) were crossed with Rosa26- CreERT2 (46), huCD20-CerERT2 (30), or S1pr2-CreERT2 strains (51, 52), all on C57/BL6 genetic background. Tamoxifen was administered as previously reported (25, 30). To control for potential Cre toxicity, CreERT2 mice were similarly injected to use as wild-type (WT) controls.
Mice (ages ∼ 8wk) were immunized with SRBCs (2×108 cells per mouse) and analyzed 1 week after immunization as described (25, 45). Alternatively, mice were immunized and boosted by i.p. injections [each of 100 µg NP16-OVA (Biosearch Technologies, Novato, CA) emulsified in 100 µL of ImjectTM Alum (Thermo Fisher Scientific, Pittsburgh, PA)] as described previously (25, 30), and harvested for analyses 1 wk after the 2nd injection. Isotype-specific relative levels of Ag-specific Ab were quantitated by capture ELISA using Ag (NP20-BSA or NP2-PSA for all- or high-affinity hapten-specific Abs, respectively) followed by SBA Clonotyping System (Southern Biotech, Birmingham AL), as described previously (25, 30). As previously described (30), frequencies of Ab-secreting cells (ASCs) were analyzed by ELISpot and quantitated using an ImmunoSpot Analyzer (Cellular Technology, Shaker Heights OH).
Cell cultures, proliferation assay, and reversion of population growth
Splenic B cells were purified (90-95%) using negative selection as previously described (30) or by positive selection with anti-mouse B220 microbeads (Miltenyi Biotech, Auburn, CA). B cells (5 × 105 cells in 1 ml) were activated with anti-CD40 (1 μg/mL) and cultured with BAFF (10 ng/mL), IL-4 (10 ng/mL), IL-5 (10 ng/mL) and 4-OHT (50 nM) in Iscoves Modified Dulbecco’s Medium (IMDM) supplemented with 10% Fetal Bovine Serum (FBS), 100 U/mL penicillin 100 µg/mL streptomycin (Invitrogen), 3 mM L-glutamine, nonessential amino acids (Invitrogen), 0.1 mM 2-ME (Sigma-Aldrich). To analyze the effect of ROS scavenger and inhibition of peroxisomal lipid oxidation on population growth, bead-purified B cells from tamoxifen-treated huCD20-CerERT2 or Dhrs7bf/f; huCD20-CerERT2 mice were activated with anti-CD40, cultured with BAFF, IL-4 and IL-5 in the presence or absence of NAC (1 mM and 5 mM) and/or thioridazine·HCl (100 µM) for 5 days. For enhancement of ROS in WT B cells, purified B cells were analyzed 3 d after activation as for ROS scavenging, but vehicle, H2O2 (200 μM) or menadione (8 μM) was added at three different time points (overnight, 3 hours, and 1 hour prior to processing for flow cytometry to counting and measurements of DCFDA, annexin V, and C11 Bodipy signals.
For the analysis of proliferation in vitro, B cells were purified by depleting CD90.2+ T cells and CD138+ cells using biotinylated anti-CD90.2 Ab and biotinylated anti-CD138 Ab followed by streptavidin-conjugated microbeads (iMagTM; BD Bioscience), labeled with 5 µM CellTraceTM Violet (CTV, Invitrogen), activated and cultured as above. To analyze proliferation in vivo, B cells were purified, labeled with CTV, and injected intravenously into B cell deficient µMT recipient mice. Mice were harvested at 4 days after adoptive transfer.
Flow cytometry - general and measurements of ROS, lipids peroxidation, and ER
GC B cells were identified as GL7+ CD95+ events in the viable B220+ dump- gate (dump channel consisting of one fluorophore for CD11b, CD11c, F4/80, Gr1, TCRβ, and 7AAD), while plasmablasts were defined as B220+ CD138+ TACI+ dump- cells. For flow analyses of total intracellular ROS and mitochondrial superoxide, B cells (1 × 106 cells) were washed with PBS and stained with 1.25 µM CM-H2DCFDA or 5 µM MitoSOXTM Red in PBS (20 min at 37°C), respectively, then washed with 1% BSA containing PBS, and further stained with anti-B220, anti-CD19, anti-CD138, and Ghost-BV510. To measure the lipid peroxidation, cultured B cells (1 × 106) were washed with PBS and stained with 1.25 µM C11-BODIPYTM in PBS (20 min at 37°C), and washed with 1% BSA containing PBS followed by surface staining as above. To analyze the level of endoplasmic reticulum (ER), B cells (1 × 106) were stained with 0.5 µM ER-TrackerTM Green (Molecular Probes, Eugene OR) in Hank’s Balanced Salt Solution with calcium and magnesium (HBSS/Ca/Mg; Gibco) for 30 min at 37°C.
For measurements of cell death (78), cultured B cells (1 x 106) were washed twice with PBS, once with Annexin V binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2), and then incubated (15 min at 20 C in the dark) with Annexin V-rPE, 7AAD, APC-conjugated CD138, APC-Cy7-conjugated B220, e450-CD19. Cells were then washed with Annexin V binding buffer and analyzed on the flow cytometer. To measure levels of cleaved caspase-3 (25), an activated apoptosis executor, the cultured cells were rinsed with PBS after harvest, stained (15 min at 4 C) with 7AAD, APC-conjugated CD138, APC-Cy7-conjugated B220, e450-CD19, washed (FBS, 1% v/v in PBS), then fixed with paraformaldehyde (4% w/v, 10 min at 20 C), permeabilized with permeabilization buffer (saponin, 0.2% w/v with FBS 1% in PBS), stained with FITC-conjugated Ab specific for cleaved caspase-3 (BD Biosciences), rinsed, and analyzed by flow cytometry.
Immunohistochemistry, MALDI-IMS, and IMS data analysis
Mice were immunized with SRBC and spleens were harvested at 1 week after immunization. Spleens were snap frozen on dry ice, and mounted to a cryostat chuck with a minimal amount of OCT (Thermo Fisher Scientific, San Jose, CA). Frozen spleens were cut at 12 µm thickness using a Leica CM3050S (Leica, IL, USA) at -20 °C, fixed with 1% paraformaldehyde for 10 min, washed twice with PBS, and blocked with M.O.MTM (Vector Lab) followed by incubation with GL7-FITC, anti-IgD-PE, and anti-CD35-biotin Ab followed by streptavidin-conjugated Alex647at 4 °C. After the tile scanning of spleen sections, GC size and numbers were quantified with FIJI Image J software.
For Matrix-Assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS), frozen spleens were cut as above, and mounting onto indium tin oxide (ITO) coated microscope slides (Delta Technologies ETC). Slides were then vacuum desiccated for at least 30 minutes before matrix application. Pre-IMS autofluorescence (AF) images were acquired prior to matrix application on a Zeiss Axio Scan Z1 Slide Scanner using the brightfield channel and the DAPI (ex. 340-380 nm; em. 435-485 nm, blue), EGFP (ex. 450-490 nm; em. 500-550 nm, green), DsRed (ex. 538-562 nm; em. 570-640 nm, red) filter cubes. After taking AF images, a custom in-house developed sublimation device was used to apply the matrices 2,5-dihydroxyacetophenone (DHA) and 1,5-diaminonaphthalene (DAN) (Sigma Aldrich, St. Louis, MO, USA) to tissue sections for positive and negative ion mode analyses, respectively (78). For immunized AID-GFP mice, the fluorescence emissions identified GC localization within the section used next for IMS. Alternatively, two serial sections of immunized mice spleens were used, one for 2D-IMS and the contiguous section for immunohistochemistry (IHC). Fluorescence data were registered with the 2D-IMS visualizations of phospholipids to align images (45), allowing quantitation of the ion intensities in specific m/z peaks within defined micro-anatomic portions of the spleen (Fig. 4B). MALDI IMS data were acquired with a 10 µm pixel size (laser spot size 8 µm) in full scan mode using a Bruker trapped ion mobility time-of-flight (timsTOF) Flex mass spectrometer (Bruker Daltonics Billerica, MA, USA). Data were acquired with 250 shots per pixel and a mass range of m/z 200-2000. SCiLS (software Bruker Daltonics) was used to process the data, normalize ion intensity, visualize ion images, and merge the images.
LC-MS for lipidomics
To prepare samples, a one-phase method was used to extract lipids (85). Briefly, 0.5 mL of MeOH/MTBE/CHCl3 mix (1.3:1:1) was added to a frozen pellet of B-cells (1x106 cells total), spiked with 10 uL EquiSPLASH-lipidomics internal standard mix (Avanti Research), briefly vortexed and shaken gently for 20 min, followed by centrifugation at 20,000 x g for 15 min at 10°C. The supernatant was transferred to a clean Eppendorf tube, evaporated under a gentle stream of N2 gas, and resuspended in 100 uL methanol/CHCl3 (9:1) and 2uL were used for LC-HRMS (high-resolution MS) analysis. Each sample was injected two times - one injection in positive ESI mode followed by one in negative mode. Pooled QCs were injected to assess the performance of the LC and MS instruments at the beginning, in the middle and at the end of each sequence. Discovery lipidomics data were acquired using a Vanquish UHPLC (ultrahigh performance liquid chromatography) system interfaced to a Q Exactive HF quadrupole/orbitrap mass spectrometer (Thermo Fisher Scientific).
Chromatographic separation was performed with a reverse-phase Acquity BEH C18 column (1.7 mm, 2.1x150mm, Waters, Milford, MA) at a flow rate of 250 ul/min. Mobile phases were made up of 10 mM ammonium formate and 0.1% formic acid in (A) H2O/CH3CN (40:60) and in (B) CH3CN/ iPrOH (10:90). Gradient conditions were as follows: 0–1 min, B = 20 %; 1–8 min, B = 20– 100 %; 8–10 min, B = 100 %; 10–10.5 min, B = 100–20 %; 10.5–15 min, B = 20%. The total chromatographic run time was 15 min. Mass spectra were acquired over a precursor ion scan range of m/z 200 to 1,600 at a resolving power of 60,000 using the following HESI-II source parameters: spray voltage 4 kV (3 kV in negative mode); capillary temperature 250°C; S-lens RF level 60 V; N2 sheath gas 40; N2 auxiliary gas 10; auxiliary gas temperature 350°C. MS/MS spectra were acquired for the top-seven most abundant precursor ions with an MS/MS AGC target of 1e5, a maximum MS/MS injection time of 100 ms, and a normalized collision energy of 15, 30, 40. High resolution mass spectrometry data were processed with MS-DIAL version 4.70 in lipidomics mode (86). MS1, and MS2 tolerances were set to 0.01 and 0.025 Da respectively. Minimum peak height was set to 30,000 to decrease the number of false positive hits. Peaks were aligned on a quality control (QC) reference file with RT tolerance of 0.1 min and mass tolerance of 0.015 Da. Default lipid library was used (Msp20210527163602_converted.lbm2), solvent type was set to HCOONH4 to match the solvent used for separation, and the identification score cut off was set to 80%. All lipid classes were made available for the search. After lipid identification was completed, MS-DIAL results were exported into Excel and cleaned using minimum RSD for QC samples set to 20% and minimum ratio of QC to Blank set to 10. For species identified in both PexRAP-depleted (Dhrs7b Δ/Δ) B cells and controls, mean levels (areas under peak curves) and their variance were analyzed in Excel.
Metabolic flus analyses
Oxygen Consumption Rate (OCR) were measured using Seahorse XF96 extracellular flux analyzer (Agilent Technology, Santa Clara, CA) as described previously (30). Briefly, in vitro activated B cells were washed twice, resuspended in XF Base Media (Agilent Technologies) supplemented with 2 mM L-glutamine, and equal numbers of B cells (2 × 105) were plated on extracellular flux assay plates (Agilent Technologies) coated with 2.5 µg/mL CellTak (Corning) according to the manufacturer’s protocol. Before extracellular flux analysis, B cells were rested (25 minutes at 37°C, atmospheric CO2) in XF Base Media. OCR and ECAR were measured before and after the sequential addition of 1.5 µM oligomycin, 0.5 µM FCCP and 0.5 µM rotenone/antimycin A.
Supplemental information
Supplemental Figures
Supplemental Table 1. Immunization-induced increases of selected ether lipids in GC. Shown are (a) a sample of m/z features characteristic of the indicated phospholipid species identified by the LIPIDMAPS database, and (b) the mean ±SEM ion intensity / counts in IMS data generated from spleens of AID-GFP mice, immunized or not (UI), after mapping to the indicated regions of interest (primary follicles or GC, i.e. secondary follicles) using fluorescent images. Shown are ions more accumulated in GC regions compared to primary follicles (8 ions from negative ion mode, and 5 ions from positive ion mode, all p<0.05 for comparison of 10 follicle to UI (c), or GC to 10 follicle (d). P values were calculated by Mann-Whitney U test.
Supplemental Table 2. Impact of PexRAP on relative quantities of selected lipid species in primary and secondary follicles (GC). As in Table 1 except that B cells of immunized mice were either WT or lacked PexRAP (cKO, i.e., Dhrs7bΔ/Δ-B). Shown are (a) a sample of m/z features characteristic of the indicated phospholipid species. (b) the mean ±SEM ion intensity counts in IMS data after mapping to the indicated regions of interest as in Table 1. (c-f) indicate that p<0.05 for the null hypothesis in considering a difference between WT mouse spleens after immunization versus UI controls (c), 10 B cell follicles in spleens of immunized WT vs Dhrs7bΔ/Δ-B mice (d), GC vs in 10 B cell follicles in spleens of immunized WT mice (e), and GC of immunized WT vs Dhrs7bΔ/Δ-B mice (f) in the ions counts for designated m/z features. P values were calculated by Mann-Whitney U test.
Acknowledgements
The experimental work was funded by NIH Grants to VUMC [R01 AI113292 (M.R.B), R01 HL106812 (M.R.B.), followed by R21 AI164760 (M.R.B.) and departmental funds]. Mass spectrometry imaging was supported in part by NIH grant P41 GM103391 to Vanderbilt University (R.M.C.). Additional support for M. A. J. was provided by the National Science Foundation, NSF DGE-1445197. NIH Shared Instrumentation Grant 1S10OD018015 as well as scholarships via the Cancer Center Support Grant (CA068485) and Diabetes Research Center (DK0205930) helped defray costs of Vanderbilt Cores. We thank J. Cyster for generously expediting shipment of S1pr2-CreERT2 breeding stock, and Vanderbilt institutional cores (High-Throughput Screening; Flow Cytometry Shared Resource; Mass Spectrometry Shared Resource; Cell & Developmental Biology) for equipment, expertise, and assistance.
References
- 1.Physiological and pathological inflammation induced by antibodies and pentraxinsCells 10
- 2.Immunoregulation by antibody secreting cells in inflammation, infection, and cancerImmunol Rev 303:103–118
- 3.Beyond binding: antibody effector functions in infectious diseasesNat Rev Immunol 18:46–61
- 4.Peripheral B cell subsetsCurr Opin Immunol 20:149–157
- 5.B-cell subsets and the mature preimmune repertoire. Marginal zone and B1 B cells as part of a “natural immune memoryImmunol Rev 175:70–79
- 6.Extrafollicular antibody responsesImmunol Rev 194:8–18
- 7.B cell clones that sustain long-term plasmablast growth in T-independent extrafollicular antibody responsesProc Natl Acad Sci USA 103:5905–5510
- 8.Clonal selection and learning in the antibody systemNature 81:751–758
- 9.Molecular regulation of peripheral B cells and their progeny in immunityGenes Dev 33:26–48
- 10.T-box transcription factor T-bet, a key player in a unique type of B-cell activation essential for effective viral clearanceProc Natl Acad Sci USA 110:E3216–24
- 11.Human neutrophil Fc gamma receptors: different buttons for different responsesJ Leukoc Biol 114:571–584
- 12.Immunoglobulin class-switch recombination: induction, targeting, and beyondNat Rev Immunol 12:517–531
- 13.Germinal Center and extrafollicular B cell responses in vaccination, immunity, and autoimmunityImmunity 53:1136–1150
- 14.Plasticity and heterogeneity in the generation of memory B cells and long-lived plasma cells: the influence of germinal center interactions and dynamicsJ Immunol 185:3117–3125
- 15.Anti-DNA antibodies from autoimmune mice arise by clonal expansion and somatic mutationJ Exp Med 171:265–292
- 16.Somatically mutated B cell pool provides precursors for insulin antibodiesJ Immunol 157:763–771
- 17.Dynamics of B cells in germinal centresNat Rev Immunol 15:137–148
- 18.B cell responses: Cell interaction dynamics and decisionsCell 177:524–540
- 19.Germinal CentersAnnu Rev Immunol 40:413–442
- 20.Cognate stimulatory B-cell-T-cell interactions are critical for T-cell help recruited by glyco-conjugate vaccinesInfect Immun 67:6375–6384
- 21.Germinal centre-driven maturation of B cell response to mRNA vaccinationNature 604:141–145
- 22.Class-switch recombination occurs infrequently in Germinal CentersImmunity 51:337–350
- 23.Clonal selection in the germinal centre by regulated proliferation and hypermutationNature 509:637–40
- 24.Follicular helper T cellsAnnu Rev Immunol 34:335–368
- 25.The micro-anatomic segregation of selection by apoptosis in the germinal centerScience 358
- 26.Metabolic adaptation of lymphocytes in immunity and diseaseImmunity 55:14–30
- 27.Nutrient inputs and social metabolic control of T cell fateCell Metab 36:10–20
- 28.Metabolic control of B cell immune responsesCurr Opin Immunol 63:21–28
- 29.Supplying the trip to antibody production-nutrients, signaling, and the programming of cellular metabolism in the mature B lineageCell Mol Immunol 19:352–369
- 30.A guide to immunometabolism for immunologistsNat Rev Immunol 16:553–565
- 31.Germinal centre hypoxia and regulation of antibody qualities by a hypoxia response systemNature 537:234–237
- 32.Germinal center B cells selectively oxidize fatty acids for energy while conducting minimal glycolysisNat Immunol 21:331–342
- 33.Coupled analysis of transcriptome and BCR mutations reveals role of OXPHOS in affinity maturationNat Immunol 22:904–913
- 34.Mitochondrial respiration in B lymphocytes is essential for humoral immunity by controlling the flux of the TCA cycleCell Rep 39
- 35.Dynamic mitochondrial transcription and translation in B cells control germinal center entry and lymphomagenesisNat Immunol 24:991–1006
- 36.Plasma cell differentiation, antibody quality, and initial germinal center B cell population depend on glucose influx rateJ Immunol 212:43–56
- 37.Biochemical underpinnings of immune cell metabolic phenotypesImmunity 46:703–713
- 38.Regulation of redox balance in cancer and T cellsJ Biol Chem 293:7499–7507
- 39.Mitochondria and reactive oxygen speciesHypertension 53:885–892
- 40.Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS releasePhysiol Rev 94:909–950
- 41.Gsk3 is a metabolic checkpoint regulator in B cellsNat Immunol 18:303–312
- 42.Regulation of humoral immune response by HIF-1α-dependent metabolic reprogramming of the germinal center reactionCell Immunol 367
- 43.Epi-microRNA mediated metabolic reprogramming ensures affinity maturationbioRxiv
- 44.Genetic screen for cell fitness in high or low oxygen highlights mitochondrial and lipid metabolismCell 181:716–727
- 45.Discovering new lipidomic features using cell type-specific fluorophore expression to provide spatial and biological specificity in a multimodal workflow with MALDI Imaging Mass SpectrometryAnal Chem 92:7079–7086
- 46.Peroxisomal lipid synthesis regulates inflammation by sustaining neutrophil membrane phospholipid composition and viabilityCell Metab 21:51–64
- 47.Inhibiting adipose tissue lipogenesis reprograms thermogenesis and PPARγ activation to decrease diet-induced obesityCell Metab 16:189–201
- 48.Structural and functional roles of ether lipidsProtein Cell 9:196–206
- 49.Regulation of plasmalogen biosynthesis in mammalian cells and tissuesBrain Res Bull 194:118–123
- 50.Normal plasmalogen levels are maintained in tissues from mice with hepatocyte-specific deletion in peroxin 5Brain Res Bull 193:158–165
- 51.Regulation of plasmalogen metabolism and traffic in mammals: The fog begins to liftFront Cell Dev Biol 10
- 52.Exogenous ether lipids predominantly target mitochondriaPLoS One 7
- 53.Acute ether lipid deficiency affects neutrophil biology in miceCell Metab 21:652–653
- 54.Ether lipid deficiency does not cause neutropenia or leukopenia in mice and menCell Metab 21:650–651
- 55.Peroxisome-driven ether-linked phospholipids biosynthesis is essential for ferroptosisCell Death Differ 28:2536–2551
- 56.Distinct functions of acyl/alkyl dihydroxyacetonephosphate reductase in peroxisomes and endoplasmic reticulumFront Cell Dev Biol 8
- 57.Functions of plasmalogen lipids in health and diseaseBiochim Biophys Acta 1822:1442–1452
- 58.The ether lipid-deficient mouse: tracking down plasmalogen functionsBiochim Biophys Acta 1763:1511–1526
- 59.On the road to unraveling the molecular functions of ether lipidsFEBS Lett 93:2378–2389
- 60.Bioactive ether lipids: Primordial modulators of cellular signalingMetabolites 11
- 61.Plasmalogens and chronic inflammatory diseasesFront Physiol 12
- 62.Plasmalogens the neglected regulatory and scavenging lipid speciesChem Phys Lipids 164:573–589
- 63.Promoter traps in embryonic stem cells: A genetic screen to identify and mutate developmental genes in miceGenes Devel 5:1513–1523
- 64.Blimp-1 is required for maintenance of long-lived plasma cells in the bone marrowJ Exp Med 202:1471–1476
- 65.Direct hematological toxicity and illegitimate chromosomal recombination caused by the systemic activation of CreERT2J Immunol 182:5633–5640
- 66.Comment on “Direct hematological toxicity and illegitimate recombination caused by the systemic activation of CreERT2“J Immunol 183
- 67.B cell receptor signal transduction in the GC is short-circuited by high phosphatase activityScience 336:1178–1181
- 68.Memory B cellsNat Rev Immunol 24:5–17
- 69.Regulated selection of germinal-center cells into the memory B cell compartmentNat Immunol 17:861–869
- 70.Progressive upregulation of oxidative metabolism facilitates plasmablast differentiation to a T-independent antigenCell Rep 23:3152–3159
- 71.HVCN1 modulates BCR signal strength via regulation of BCR-dependent generation of reactive oxygen speciesNat Immunol 11:265–272
- 72.Prolonged production of reactive oxygen species in response to B cell receptor stimulation promotes B cell activation and proliferationJ Immunol 189:4405–4416
- 73.Involvement of reactive oxygen species (ROS) in BCR signaling as a second messengerAdv Exp Med Biol 1254:37–46
- 74.Ferroptosis: mechanisms, biology and role in diseaseNat Rev Mol Cell Biol 22:266–282
- 75.Peroxisomal metabolism and oxidative stressBiochimie 98:56–62
- 76.Ferroptosis at the intersection of lipid metabolism and cellular signalingMol Cell 82:2215–2227
- 77.Peroxisome-derived lipids are self antigens that stimulate invariant natural killer T cells in the thymusNat Immunol 3:474–480
- 78.Plasmalogen-mediated activation of GPCR21 regulates cytolytic activity of NK cells against the target cellsJ Immunol 209:310–325
- 79.Warning regarding direct hematological toxicity of tamoxifen-activated CreERT2 in young Rosa26-CreERT2 miceSci Rep 13
- 80.Peroxisomes: a nexus for lipid metabolism and cellular signalingCell Metab 19:380–392
- 81.Peroxisomes as cellular adaptors to metabolic and environmental stressTrends Cell Biol 31:656–670
- 82.Plasticity of ether lipids promotes ferroptosis susceptibility and evasionNature 585:603–608
- 83.Microscopy-Directed Imaging Mass Spectrometry for Rapid High Spatial Resolution Molecular Imaging of GlomeruliJ Am Soc Mass Spectrom 34:1305–1314
- 84.Requirement for Rictor in homeostasis and function of mature B lymphoid cellsBlood 122:2369–2379
- 85.LC/MS lipid profiling from human serum: a new method for global lipid extractionAnal Bioanal Chem 406:7937–7948
- 86.A lipidome atlas in MS-DIAL 4Nat Biotechnol 38:1159–1163
- 87.Reactive oxygen species oxidize STING and suppress interferon productionElife 9https://doi.org/10.7554/eLife.57837
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