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. 2014 Mar;141(3):457-66.
doi: 10.1111/imm.12208.

Protection against collagen-induced arthritis in mice afforded by the parasitic worm product, ES-62, is associated with restoration of the levels of interleukin-10-producing B cells and reduced plasma cell infiltration of the joints

David T Rodgers  1 , Miguel A Pineda, Mairi A McGrath, Lamyaa Al-Riyami, William Harnett, Margaret M Harnett
Affiliations

Protection against collagen-induced arthritis in mice afforded by the parasitic worm product, ES-62, is associated with restoration of the levels of interleukin-10-producing B cells and reduced plasma cell infiltration of the joints

David T Rodgers et al. Immunology. 2014 Mar.

Abstract

We have previously reported that ES-62, a molecule secreted by the parasitic filarial nematode Acanthocheilonema viteae, protects mice from developing collagen-induced arthritis (CIA). Together with increasing evidence that worm infection may protect against autoimmune conditions, this raises the possibility that ES-62 may have therapeutic potential in rheumatoid arthritis and hence, it is important to fully understand its mechanism of action. To this end, we have established to date that ES-62 protection in CIA is associated with suppressed T helper type 1 (Th1)/Th17 responses, reduced collagen-specific IgG2a antibodies and increased interleukin-10 (IL-10) production by splenocytes. IL-10-producing regulatory B cells have been proposed to suppress pathogenic Th1/Th17 responses in CIA: interestingly therefore, although the levels of IL-10-producing B cells were decreased in the spleens of mice with CIA, ES-62 was found to restore these to the levels found in naive mice. In addition, exposure to ES-62 decreased effector B-cell, particularly plasma cell, infiltration of the joints, and such infiltrating B cells showed dramatically reduced levels of Toll-like receptor 4 and the activation markers, CD80 and CD86. Collectively, this induction of hyporesponsiveness of effector B-cell responses, in the context of the resetting of the levels of IL-10-producing B cells, is suggestive of a modulation of the balance between effector and regulatory B-cell responses that may contribute to ES-62-mediated suppression of CIA-associated inflammation and inhibition of production of pathogenic collagen-specific IgG2a antibodies.

Keywords: ES-62; interleukin-10-producing B cells; parasitic helminths; rheumatoid arthritis.

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Figures

Figure 1
Figure 1
Gating strategy for analysis of B-cell subsets and phenotyping of populations. This is a modification of that based on the peripheral B cell phenotypic markers defined by Allman and Pillai. T1: CD19+ CD93+ CD21int CD23− IgDlow/− IgMhigh; T2: CD19+ CD93+ CD21int CD23+ IgDhigh IgMhigh; T3: CD19+ CD93+ CD21int CD23+ IgDhigh IgMlow; marginal zone precursor (MZP): CD19+ CD93− CD21high CD23+ CD1dhigh IgDhigh IgMhigh; marginal zone (MZ): CD19+ CD93− CD21high CD23− CD1dhigh IgDlow/− IgMhigh; Fo1: CD19+ CD93− CD21low CD23+ IgDhigh IgMlow; Fo2: CD19+ CD93− CD21low CD23+ IgDhigh IgMhigh; GC: CD19+ CD43− CD24+ GL7+. Cell populations were initially selected on the basis of ‘Lymphocyte’ size (forward scatter; FSC) and granularity (side scatter; SSC) parameters and excluding ‘doublets’ (by comparing FSC-Height and FSC-Area) and dead cells (by the Live/Dead® fixable aqua dead cell dye; Invitrogen) (data not shown). We initially gated on CD19+ CD23− and CD19+ CD23+ cells (a) to resolve MZP (CD21high CD1dhigh) from follicular (Fo) (CD21low CD1dlow) B cells (b) and MZ (CD21+ IgM+) and T1 (CD21− IgM+) cells (c), respectively. The Fo population identified (b; CD21low CD1dlow) is a heterogeneous population that contains the functionally distinct follicular type 1 (Fo1: IgDhigh IgMlow AA4·1−) and follicular type 2 (Fo2: IgDhigh IgMhigh AA4·1−) as well as the transitional 2 (T2: IgDhigh IgMhigh AA4·1+) and transitional 3 (T3: IgDhigh IgMlow AA4·1+) populations. These populations are first separated on the basis of their expression of IgM and IgD (d) and then, AA4·1 (e,f). For the identification of germinal centre (GC) B cells we first identify CD19+ CD43− cells (g) and then exclude contaminating non-B cells by gating on the GC cell-specific marker GL7 along with the pan-B-cell marker CD24 (h) before confirming expression of FAS (i) by essentially all (> 90%) CD19+ CD43− CD24+ GL7+ GC B cells; we have therefore not included this redundant marker in our analysis.
Figure 2
Figure 2
ES-62 reduces the levels of germinal centre B cells in the spleens of mice with collagen-induced arthritis (CIA). Mean articular scores (±SEM) of CIA mice treated with PBS (n = 34) or ES-62 (n = 18) at time of cull at day 28 (a). The percentage of CD19+ B cells (b); representative plots (c) and proportions (d; mean values ± SEM of individual mice where naive, n = 16; PBS, n = 31; ES-62, n = 12) of marginal zone precursor (MZP), marginal zone (MZ) and follicular (Fo) B cells as defined by their expression of CD21 and CD23; Follicular type 1 B cells (Fo1, e: CD19+ CD23+ CD21low CD1dlow IgMlow IgDhigh AA4·1−) and germinal centre cells (GC; f: CD19+ CD43− CD24+ GL7+), as derived by the gating strategy presented in Fig. 1, in spleens from mice undergoing CIA are shown.
Figure 3
Figure 3
ES-62 modulates the recruitment of B cells to the joints of mice with collagen-induced arthritis (CIA). Cells extracted from the joints of mice with CIA were analysed for the proportion (a,b) and number (c) of infiltrating CD19+ B cells (data in c are presented as the means ± SEM of four biological replicates pooled from two independent experiments) and consequently for the relative proportions of CD19+ CD23+ (d,e) and plasma cells on the basis of CD19 CD138 expression (f,g). Exclusion of myeloid and T-cell-expressing CD138 cells by use of Dump channel (CD4+ CD8+ GR1+ F4/80+ CD11b+ CD11c+; h) allowed analysis of Dump− CD19− B220− CD138+ and Dump− CD19+ B220low/− CD138+ plasma cells (i,j).
Figure 4
Figure 4
ES-62 modulates expression of Toll-like receptor 4 (TLR4) and co-stimulatory molecules on Follicular B cells. CD80, CD86, TLR4 and BAFF-R expression by CD19+ CD23high CD21low follicular B cells in the joints of PBS and ES-62 treated mice are presented as expression levels relative to isotype control (grey area), for CIA mice treated with PBS (broken line) or ES-62 (black line). Cells from at least five mice/group were pooled.
Figure 5
Figure 5
ES-62 modulates the levels of B1-like cells in the spleen and LNs of mice with collagen-induced arthritis (CIA). There is no unambiguous phenotype for B1 cells in the spleen but they have been described as CD19high CD23− CD43+ IgMhigh IgDlow/− CD5± cells where CD5+ B1a and CD5− B1b comprise ˜2% and 1% of cells in the spleen, respectively. However, following gating on CD19+ IgM+ (a), analysis of CD43+ CD5± cells has been widely used to describe B1 cells, whereas CD19+ CD5+ gating has been used to describe B1a cells. Moreover, although CD43 can be up-regulated on B2 cells, this is usually expressed at a lower level than on B1 cells and we have therefore chosen to gate only on CD43high cells (b) so as to exclude any potential CD43+ B2 cells. We have therefore phenotyped CD19high CD43+ IgMhigh B cells as CD5+ B1a-like cells and CD5− B1b-like cells and the data show their relative proportions in the spleen (c; naive, n = 10; PBS, n = 13 and ES-62, n = 11) and draining lymph node (d; naive, n = 4; PBS, n = 8 and ES-62, n = 8) of the indicated groups of mice.
Figure 6
Figure 6
ES-62 induces interleukin-10 (IL-10)-producing B cells in the spleen of mice with collagen-induced arthritis (CIA). IL-10-producing CD19+ B cell (a,c) and IL-10-producing CD19− non-B-cell subsets (a,d) in the spleen were analysed with the proportions of these cells, in spleens of individual naive mice and PBS- and ES-62-treated mice with CIA, shown respectively. Representative plots of the phenotypes of CD19+ IL-10+ B cells based on their expression of CD21 and CD23 (b) are shown for spleens of mice with CIA.
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