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 Ig

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 specific IgM+ plasma cells Thus, we performed immunofluorescence staining

oxLDL-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 A

sacrificed on Day 4 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

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)

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 4 (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

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

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, 25-OHC (Hannedouche et al., 2011; Liu et al., 2011) The formation of 7α, 25-OHC 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,

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

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 D6-

7α, 25-OHC as reference *, P < 0.05

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

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 5 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 do 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

3.5.2 Increased splenic B1a cells differentiation into IgM + plasmablasts

B1 cells also exists in the spleen but constituted only 1-2% of CD19+ B cell population (Baumgarth, 2011) Therefore, it remained possible that B1a

cells increased in the spleen of apoE -/- mice and differentiated into IgM+

plasma cells in apoE -/- mice

Our preliminary analysis of CD19+CD5+ B1a cell population showed

that apoE -/- mice had higher relative percentage of B1a in the spleen (Figure 24A) However, we did not detect an increase in relative cell number of B1a

population in the spleen of apoE -/- mice (Figure 24B) Therefore, these sets of

observations suggest increased frequency of splenic B1a cell population was due to changes in frequency of other lymphocyte sub-populations instead of

indications that there was B1a cell population expansion in apoE -/- mice

Next, we examined if there were more B1a cells differentiating into IgM+ ASCs in apoE -/- mice Preliminarily, we observed a non-statistical significant increase in relative percentage but a statistical significant increase

in relative number of extracellular IgM+CD138+ B1a cells in apoE -/- mice

(Figure 24C & 24D) On the contrary, when we examined for intracellular

IgM+CD138+ B1a cells, we found significant decrease in relative percentage

and number in the spleen of apoE -/- mice (Figure 24E & 24F) Two

possibilities could explain the decreased IgM+CD138+ B1a plasma cells; 1) the

plasma cells died in situ or, 2) the plasmablasts migrated to the bone marrow

for long-term maintenance With more CD11chi DCs colocalizing with IgM+plasmablasts in extrafollicular responses to aid in the survival and successful

differentiation of plasmablasts in apoE -/- mice, it is unlikely to explain that

these IgM+CD138+CD19+CD5+ died rapidly to account for the decreased population We favoured the latter possibility that these IgM+ plasmablasts migrate to the bone marrow for long-term maintenance that we will explore in the subsequent sections

Figure 23 No difference in B1a cell population in peritoneal cavity of

apoE -/- mice

(A-B) Comparative flow cytometry analysis of relative (A) percentage and (B)

number of CD19+CD5+ B1a cells (mean ± SEM, n = 9) Data were pooled

from three independent experiments (C-D) Comparative flow cytometry

analysis of relative percentage and number of extracellular IgM+CD138+CD19+CD5+ B1a cells (mean ± SEM, n = 6) Data were pooled from two independent experiments

Figure 24 Increased population of B1a cells differentiating into IgM +

plasmablasts in the spleen of apoE -/- mice

(A-B) Comparative flow cytometry analysis of relative (A) percentage and (B)

number of CD19+CD5+ B1a cells in the spleen (mean ± SEM, n=9) Data were

pooled from three independent experiments (C-D) Comparative flow cytometry analysis of relative (C) percentage and (D) number of extracellular

IgM+CD138+CD19+CD5+ B1a cells in the spleen (mean ± SEM, n=6) Data

were pooled from two independent experiments (E-F) Comparative flow cytometry analysis of relative (E) percentage and (F) number of intracellular

IgM+CD138+CD19+CD5+ B1a cells in the spleen (mean ± SEM, n=9) Data were pooled from three independent experiments * P < 0.05; ** P < 0.01

3.5.3 Summary

Collectively, our data suggests that PEC B1a cells were not affected in

the apoE -/- mice In addition, our preliminary data suggests that although B1a

cells did not expand in the spleen of apoE -/- mice, we did detect increased

differentiation of B1a cells into IgM+CD138+ plasmablasts and their

subsequent disappearance/ decrease in relative percentage and number as

intracellular IgM+CD138+ B1a plasma cells in the spleen

3.6 Evaluation of the impact of splenic CD138 + ASCs on atherosclerosis

Elegant studies by Caliguri et al, demonstrated that the adoptive transfer of splenic B cells, but not T cells, from old apoE -/- mice donor into

young apoE -/- mice recipient led to decreased lesion size in the aorta (Caligiuri

et al., 2002) They also showed that this protective effect was not observed

when adoptive transfer of splenic B cells were from young apoE -/- mice (Caligiuri et al., 2002) This prompted us to investigate if the protective effect

of B cells could be due to ASCs existing in the old apoE -/- mice

3.6.1 Adoptive transfer of splenic CD138 + ASCs into apoE -/- mice

To investigate this, we carried out a pilot experiment in which sorted CD138+ splenic ASCs from apoE -/- or WT mice were adoptively transferred

via i.v route into 10 weeks old apoE -/- recipient mice Due to cell number limitation ( < 1.0 x 106 cells) after sorting from six spleens from donor mice, only one recipient mouse per group was used Recipient mice were sacrificed

at 28 weeks old and aorta analysis for lesion was carried out We performed Oil Red-O staining to visualize lipid distribution throughout the aortic tree of the mice As reported, aortic arches of the aorta are lesion prone sites and most

affected in disease severity (Figure 25A) Our observation of the whole mount

Oil Red-O stained aortas suggested that less amount of lipids were

accumulated in the aortic arch region of apoE -/- ASCs recipient mouse (Figure 25A) To confirm, we sectioned the aortic arch and performed

immunofluorescence staining with α-actin and DAPI to visualize smooth muscle cells and necrotic core respectively, to aid visualization of lesion area

within the lumen of aortic arch (Figure 25B) At the same time, we performed

quantitative analysis of the lesion size but we did not detect any differences

between recipient mouse receiving apoE -/- ASCs and recipient mouse

receiving WT ASCs or apoE -/- control mice (Figure 25C)

Figure 25 No differences in lesion size of apoE -/- mice after adoptive transfer of ASCs

(A) Representative whole mount images of aorta from apoE -/- mice (n=2), WT

mice (n=3), apoE -/- mice recipient for apoE -/- ASCs (n=1) and apoE -/- mice

recipient for WT ASCs (n=1) (B) Representative immunofluorescence image

of aorta staining to reveal lesion size Scale bar represents 200µm (C)

Quantification of lesion size in aorta of apoE -/- mice (n=2), WT mice (n=3),

apoE -/- mice recipient for apoE -/- ASCs (n=1) and apoE -/- mice recipient for

WT ASCs (n=1) (mean ± SEM)