The role of b cells in the pathogenesis of atherosclerosis 2

3.3.2 Plasmablasts in extrafollicular responses were IgM+ Our data, thus far, indicated that extrafollicular responses in apoE-/mice may be responsible for the generation of total IgM+ and also oxLDLspecific IgM+ plasma cells Thus, we performed immunofluorescence staining to evaluate whether IgM+ plasmablasts were generated from extrafollicular responses Our result showed that IgM+ plasmablasts were indeed colocalizing with CD11chi DCs at the bridging channel of the follicles (Figure 21A) Extending our findings, we observed these IgM+ plasmablasts that colocalized with CD11chi DCs were proliferating as they incorporated thymidine analog, EdU in a 12hr pulsed chase experiment (Figure 21B) Because of the lack of tools to evaluate if these IgM+ plasmablasts were oxLDL-specific, we immunized young WT mice with oxLDL via intravenous route to evaluate if immunization with oxLDL could elicit splenic extrafollicular responses Activated   B   cells   migration   to   the   extrafollicular   sites   could   be   detected   from   day   3.5   and   maximally   at   day   4.5   after   immunization  as  described  (Gatto  et  al.,  2009)   Therefore,   these mice were sacrificed on Day after immunization to evaluate extrafollicular responses However, the use of common adjuvant such as CFA alone could induce the increased titer of MDA-LDL specific antibodies (Khallou-Laschet et al., 2006) Therefore, the use of adjuvants was excluded in our immunization studies Our results showed that although the CD138+ plasmablasts colocalized with CD11chi DCs were not as numerous as in apoE-/- mice (data not shown), we were still able to observe an increased extent of extrafollicular responses in oxLDL-immunized mice compared to PBS-immunized controls (Figure 21C) We also evaluated if the immunization with oxLDL could elicit GC reactions   104   in these mice on Day 14 by flow cytometry However, we did not detect an increase in percentage and number of GC B cells in these oxLDL-immunized mice compared to PBS-immunized controls (Figure 21D & 21E)   105     106   Figure 21 IgM+ plasmablasts were generated in extrafollicular responses in the spleen of apoE-/- mice (A) Representative image of IgM+ plasmablasts colocalizing with CD11c+ DCs at bridging channel of follicle in spleen of apoE-/- Outer laying region (dotted lines) denotes B cell follicle regions (n=6) Data were representative of two independent experiments (B) Representative image of proliferating IgM+ plasmablasts in EdU pulse chase (n=3) White arrows denote proliferating IgM+EdU+ plasmablasts colocalizing with CD11c+ DCs at bridging channel of follicle (C) Quantification of extrafollicular responses sites per area (mm2) analyzed in spleen of i.v oxLDL immunized WT mice at Day (n=4) Data were pooled from two independent experiments (D-E) Flow cytometry analysis of GC B cells in terms of (D) percentage and (E) number in the spleen of i.v oxLDL immunized WT mice (n=5) at Day 14 ***, P < 0.001   107   3.3.3 Summary Collectively, we provide direct evidence that IgM+ plasmablasts were generated through the extrafollicular response pathway in the spleen of apoE-/mice The increased humoral IgM responses in spleen of apoE-/- mice were not due to defective antibodies class-switching from the GC reactions Also, we showed that extrafollicular responses, but not GC reactions, were elicited in WT mice when we immunized WT mice with oxLDL   108   3.4 Evaluation of molecular cues to direct extrafollicular responses in the spleen of apoE-/- mice Activated B cells that migrate to the bridging channel in extrafollicular responses requires the expression of chemokine receptor, EBI2 (Gatto et al., 2009) Until recently, the natural ligand for EBI2 was identified to be 7α, 25OHC (Hannedouche et al., 2011; Liu et al., 2011) The formation of 7α, 25OHC is by the stepwise actions of two enzymes, CH25H and CYP7B1 (Figure 2) This oxysterol could be further metabolized into 4-cholesten-7α, 25-ol-3-one by HSD3B7 (Figure 2) The deficiency of any of the three enzymes is associated with decreased antigen-specific plasma cell numbers (Hannedouche et al., 2011; Yi et al., 2012) Therefore, we investigated if the robust splenic extrafollicular responses seen in apoE-/- mice could be attributed to increased EBI2 expression and/or increased bioavailability of 7α, 25-OHC 3.4.1 Increased ch25h mRNA expression in the spleen of apoE-/- mice We examined the mRNA expression of ebi2, ch25h, cyp7b1 and hsd3b7 relative to the house keeping gene, hprt1 in spleen from WT and apoE/- mice Our analysis revealed no change in ebi2 mRNA expression in spleen of apoE-/- mice compared to WT mice (Figure 22A) However, when we examined the mRNA expression level of the enzymes critical for 7α, 25-OHC synthesis, we found statistical significant increased ch25h mRNA expression but not cyp7b1 and hsd3b7 mRNA expression (Figure 22B, 22C & 22D) Thus, our data suggests that mRNA expression of ebi2 may not account for the robust extrafollicular responses in the spleen of apoE-/- mice Furthermore,   109   the increased ch25h mRNA expression observed may translate into higher bioavailability of 7α, 25-OHC in spleen of apoE-/- mice since mRNA expression level of hsd3b7 was not elevated to indicate higher efficiency to metabolize 7α, 25-OHC into 4-cholesten-7α, 25-ol-3-one 3.4.2 Increased oxysterol in the spleen of apoE-/- mice To confirm our hypothesis, we collaborated with Dr Andreas Sailor from Novartis (Basel, Switzerland) to measure the amount of oxysterol in the spleen of apoE-/- mice compared to WT mice using high performance liquid chromatography mass spectrometry (HPLC-MS) Indeed, our data analysis showed higher amount of 25-OHC and 7α, 25-OHC oxysterol in the spleen of apoE-/- mice compared to WT mice (Figure 22E & 22F) Therefore, our data indicates the possibility that the robust splenic extrafollicular responses in apoE-/- mice may be supported, at least in part, by increased bioavailability of 7α, 25-OHC   110   Figure 22 Elevated 7α, 25-OHC oxysterol in the spleen of apoE-/- mice (A-D) Quantitative mRNA transcript expression of (A) EBI2, (B) CH25H, (C) CYP7B1 and (D) HSD3B7, relative to HPRT1 (mean ± SEM, n = 8) Data were pooled from two independent experiments (E-F) Quantitative oxysterol measurement of (E) 25-OHC and (F) 7α, 25-OHC (n=5) by LC-MS using D67α, 25-OHC as reference *, P < 0.05   111   3.4.3 Summary Our data suggests the robust extrafollicular responses in the spleen of apoE-/- mice may be due to the increased amount of EBI2 ligand, 7α, 25-OHC This was facilitated by the increased ch25h, but not hsd3b7, mRNA expression observed in the spleen of apoE-/- mice Our result also suggests changes in EBI2 expression unlikely contribute to the robust extrafollicular responses although it remains possible that B cell subpopulations may display increased ebi2 mRNA expression   112   3.5 Antibody production of B1a cells in apoE-/- mice The peritoneal cavity (PEC) is highly enriched for B1 cells and also exists in the spleen but constitute a minor B cell population (Baumgarth, 2011) B1 cell population could be divided into two sub-populations; CD19+CD5+ B1a and CD19+CD5- B1b cells Since the amount of oxLDL-specific IgM autoantibodies in circulation were elevated in apoE-/- mice and B1a cells are implicated in the production of oxidation epitope-specific antibodies in atherosclerosis (Chou et al., 2009), we examined if B1a cell population increased in apoE-/- mice These B1a cells had also been described to retain CD5+ marker before losing expression days after LPS stimulation (Yang et al., 2007) Therefore, it allows a window of opportunity to investigate B1a cells differentiating into IgM+CD138+ ASCs (Yang et al., 2007) 3.5.1 B1a cells were not expanded in PEC of apoE-/- mice Our flow cytometry analysis demonstrated that there were no differences in relative percentage and number in B1a cell population in PEC of apoE-/- mice compared to WT mice (Figure 23A & 23B) Furthermore, when we examined extracellular IgM+CD138+ B1a cell population, we also could not detect any difference in relative percentage and number when compared to WT mice (Figure 23C & 23D) Therefore, our data not support the hypothesis that an increased in B1a cell population accompanied by differentiation into IgM+ ASCs in PEC of apoE-/- mice may account for increased titer of oxLDL-specific IgM autoantibodies   113   3.7.2 Summary Collectively, our data established an accumulation of plasma cells in the bone marrow of apoE-/- mice Because we maintained the mice on BrdU in drinking water for one month, the result is also indicative that these plasma cells accumulated in the bone marrow of apoE-/- mice were long-lived Subsequent analysis revealed that the accumulated plasma cells in the bone marrow of apoE-/- mice were of IgM isotype, accompanied by observation of increased oxLDL-specific IgM ASCs   127   3.8 Evaluation of IgM+ plasmablasts migration from the spleen to bone marrow of apoE-/- mice The increased population of IgM+ plasma cells and oxLDL-specific ASCs in the bone marrow of apoE-/- mice suggests migration of these populations from the spleen To address the possibility of migration of ASCs from the spleen into the bone marrow compartment for long-term maintenance, we used two different approaches 1) performing splenectomy and 2) disrupting humoral responses through CD11c depletion 3.8.1 Bone marrow was generating humoral responses in the absence of spleen in apoE-/- mice Since our findings established the spleen as a major site in generating humoral responses via extrafollicular response pathway in apoE-/- mice, we performed an intervention experiment where apoE-/- mice before 10 weeks of age were splenectomized (Sx) and thereafter, evaluated for the humoral responses in these mice when they were at least 24 weeks old Our evaluation of antibodies responses in the plasma showed that the total IgM in circulation in Sx apoE-/- mice were similar to that of shamoperated apoE-/- mice (Figure 27A) In addition, the titer of oxLDL-specific IgM autoantibodies was also similar, if not non-statistical significantly higher than that of sham-operated apoE-/- mice (Figure 27B) in agreement with previous report that increased MDA-LDL IgM autoantibodies in Sx apoE-/mice was associated with increased atherosclerotic lesion size in the aorta compared to sham-operated mice (Caligiuri et al., 2002) This implied that the humoral responses in Sx apoE-/- mice were not affected by the lack of spleen   128   When we examined the bone marrow, we found increased frequency of total IgM and oxLDL-specific IgM ASCs in Sx apoE-/- mice compared to sham-operated apoE-/- mice in our ELISpot analysis (Figure 27C & 27D) Therefore, our data suggests that in the Sx apoE-/- mice, the bone marrow compartment was a major site of humoral responses in compensatory for the lack of spleen To confirm that the total IgM and oxLDL IgM ASCs were not generated from other sites besides the bone marrow, we re-examined the LN compartment in Sx apoE-/- mice Indeed, our ELISpot analysis did not show an increased frequency of total IgM and oxLDL-specific IgM ASCs in the Sx apoE-/- when compared to sham operated apoE-/- mice (Figure 27D & 27E) Therefore, the splenectomy experiment could not address our question whether the accumulation of IgM+ plasma cells and oxLDL-specific IgM ASCs was due to migration from the spleen because the bone marrow compartment was serving as a compensatory compartment for generating humoral responses in the absence of the spleen   129   Figure 27 The bone marrow compartment of splenectomized apoE-/- mice in generating humoral responses in the absence of spleen (A) Quantification of total IgM in the plasma of Sx apoE-/-, sham-operated apoE-/- and WT mice by ELISA (mean ± SEM; n = 5-7) Data were pooled from two independent experiments (B) Titre of anti-oxLDL IgM in plasma of Sx apoE-/-, sham-operated apoE-/- and WT mice (mean ± SEM; n = 5-7) by ELISA (C-D) Comparative ELISpot quantification on frequency of (C) total IgM and (D) anti-oxLDL IgM ASCs in the bone marrow (mean ± SEM; n = 45) (E-F) Comparative ELISpot quantification on frequency of (E) total IgM and (F) anti-oxLDL IgM ASCs in axillary and brachial LNs (mean ± SEM; n = 4-7) * P < 0.05; ** P < 0.01; *** P < 0.001   130   3.8.2 Evaluation of bone marrow compartment after disruption of humoral responses in the spleen The findings that colocalization of plasmablasts with CD11chi DCs is critical for the survival and differentiation into plasma cells (Garcia De Vinuesa et al., 1999), prompted us to deplete DCs to disrupt extrafollicular responses in the spleen by means of CD11c-DTR Thus, depletion of CD11cexpressing DCs could be achieved through DT administration However, the construction of bone marrow chimera in apoE-/- mice would reverse their hypercholesterolemia because of APOE expression from donor cells (Linton et al., 1995) Therefore, we used another atherosclerotic mouse model, ldlr-/mice for the purpose of bone marrow chimera experiment where the cholesterol level in these mice will not be affected (Linton et al., 1995) 3.8.2.1 Suitability of ldlr-/- mice Because another mouse model will be used in our investigation, we first confirmed if elevated IgM responses in circulation and splenic extrafollicular responses could also be observed in ldlr-/- mice Indeed, we did also observed elevated amount of IgM, but not IgG, antibodies in the plasma of 24 weeks old ldlr-/- mice (Figure 28A) However, unlike our observations in apoE-/- mice, we could not detect elevated titers of LDL- and oxLDLspecific IgM autoantibodies in these mice (Figure 28B & 28C, left) Because the disease progression in ldlr-/- mice was at a much slower pace than agematched apoE-/- mice, we examined the ldlr-/- mice when they were older, at 45 weeks old However, we still could not detect higher titers of oxLDLspecific IgM autoantibodies (Figure 28D, left) In addition, we did not   131   observed any differences in titers of LDL- and oxLDL-specific IgG autoantibodies at 24- and 45 weeks old ldlr-/- mice (Figure 28B, 28C & 28D, right) Despite differences in the specificity of antibodies responses, an increased extent of extrafollicular responses in spleen of ldlr-/- mice was also detected, similar to apoE-/- mice (Figure 29) Therefore, ldlr-/- mice were suitable to a certain extent for our analysis of humoral responses   132   Figure 28 Increased total IgM, but not anti-oxLDL IgM, was observed in circulation of ldlr-/- mice (A) Quantification of total IgM and IgG in 24 weeks old ldlr-/- mice (mean ± SEM, n=8) Data were pooled from two independent experiments (B-C) Titre IgM and IgG against (B) native LDL and (C) oxLDL in 24 weeks old ldlr-/mice (mean ± SEM, n=11) (D) Titre IgM and IgG against oxLDL in 45 weeks old ldlr-/- mice (mean ± SEM, n=5) ** P < 0.01   133   Figure 29 Increased extent of splenic extrafollicular responses in ldlr-/mice Quantification of extrafollicular responses sites per area (mm2) analyzed in spleen of 24-30 weeks old ldlr-/- and WT (n=2) Data were pooled from two independent experiments * P < 0.05   134   3.8.2.2 Detection of EdU+ new migrant IgM+ plasma cells in bone marrow Another important consideration is that plasmablasts incorporates thymidine analog as they proliferate in extrafollicular responses Therefore, we reasoned that we could track these cells by means of BrdU/EdU administration and their successful migration into the bone marrow Indeed, we observed a statistical significant increased population of IgM+CD138+EdU+B220- in terms of relative percentage and number in the bone marrow of apoE-/-, which we identified them as new migrant plasma cells (Figure 30A & 30B) Thus, it is anticipated that when disruption of humoral responses by means of CD11chi DCs depletion is achieved, the number of BrdU+/EdU+ plasma cells in bone marrow may decrease   135   Figure 30 Increased accumulation of newly formed plasma cells in the bone marrow of apoE-/- mice (A-B) Comparative flow cytometry analysis of (A) percentage and (B) number of EdU+B220-CD138+IgM+ plasma cells in the bone marrow (mean ± SEM; n=11-13) Data were pooled from three independent experiments * P < 0.05; ** P < 0.01   136   3.8.2.3 Evaluation of bone marrow compartment after DT administration in chimeric mice We reconstituted irradiated young ldlr-/- recipient mice with donor CD11c-GFP-DTR bone marrow cells and thereafter, placed them on high fat diet to allow atherosclerotic development (Figure 31A) To track the cell migration from the spleen to the bone marrow compartment, we administered and maintained the mice with BrdU in drinking water, and disrupted splenic humoral responses with DT administration for a week before analysis (Figure 31A) To confirm that splenic DCs were depleted, we examined the population (CD11c+GFP+) by flow cytometry analysis and did not detect them after DT administration (Figure 31B & 31C) Next, we examined the spleen by immunofluorescence staining for quantification of extrafollicular responses but we could no longer detect the responses in DT-treated mice compared to non-treated mice (Figure 31D) Therefore, our data suggests, with the CD11chi DCs depleted in the spleen, extrafollicular responses were disrupted in their generation of plasma cells Subsequently, we proceeded with examination of bone marrow compartment for the evaluation of ASCs accumulation Our ELISpot analysis revealed a statistical significant decreased frequency of total IgM+ ASCs in the bone marrow of DT-treated mice (Figure 31E) This was, at least in part, due to a decreased relative percentage of BrdU+B220-IgM+CD138+ new migrant plasma cells (Figure 31F)   137   With extrafollicular responses in the spleen disrupted and concomitant decreased frequency of total IgM+ ASCs in the bone marrow, we proceeded to investigate if the amount of total IgM antibodies in circulation was adversely affected The findings that IgM antibodies had a half-life of days (Vieira and Rajewsky, 1988) and that the duration of our DT administration regimen was for a week, allowed detection for changes readily Indeed, we detected a decreased amount of total IgM antibodies in the plasma of DT-treated mice (Figure 31G)   138     139   Figure 31 Decreased IgM+ plasma cells in bone marrow after depletion of CD11c-expressing cells (A) Schematic diagram to illustrate workflow to generate ldlr-/- chimeric mice reconstituted with CD11c-GFP-DTR BM and depletion of CD11c-expressing cells through DT administration while maintaining the mice on BrdU (B) Flow cytometry gating strategy to identify splenic CD11c+ DCs after DT treatment (C) Comparative flow cytometry analysis on splenic CD11c+ DCs after DT treatment (n=3-9) Data were pooled from two independent experiments (D) Quantification of extrafollicular responses sites per area (mm2) analyzed in spleen of non-treated and treated ldlr-/- chimeric mice (n=39) (E) Comparative ELISpot quantification on frequency of total IgM ASCs per million cells in BM of non-treated and treated ldlr-/- chimeric mice (n=3-8) Data were pooled from two independent experiments (F) Comparative flow cytometry analysis on relative percentage of B220-BrdU+CD138+IgM+ plasma cells in BM of non-treated and treated ldlr-/- chimeric mice (n=3-8) Data pooled from two independent experiments (G) Quantification of total IgM in plasma of non-treated and treated ldlr-/- chimeric mice (mean ± SEM, n=3-9) Data were pooled from two independent experiments * P < 0.05; *** P < 0.001   140   3.8.3 Summary Collectively, our data suggests that the decreased frequency of new migrant IgM+ plasma cells in the bone marrow may be the consequence of the disruption of extrafollicular responses in the spleen through DT-mediated depletion of DCs As a result, a significant decreased amount of IgM antibodies circulating in the plasma However, in the absence of the spleen, the bone marrow acts as a compensatory tissue organ in mounting humoral responses to generate total IgM and oxLDL-specific IgM+ ASCs   141   ... plasma cells in the bone marrow of apoE-/- mice Because we maintained the mice on BrdU in drinking water for one month, the result is also indicative that these plasma cells accumulated in the bone... and/or increased bioavailability of 7α, 25 -OHC 3.4.1 Increased ch25h mRNA expression in the spleen of apoE-/- mice We examined the mRNA expression of ebi2, ch25h, cyp 7b1 and hsd 3b7 relative to the. .. plasmablasts B1 cells also exists in the spleen but constituted only 1 -2% of CD19+ B cell population (Baumgarth, 20 11) Therefore, it remained possible that B1 a cells increased in the spleen of apoE-/-