In this study, we report that SR-BI deficiency leads to impaired lymphocyte homeostasis characterized by splenomegaly and imbalanced expansion of T and B lymphocytes. Importantly, T and B lymphocytes in SR-BI null mice exhibited a heightened active status as shown by 3–4-fold increases in activated T and B lymphocytes. More importantly, in line with the accumulation of the activated T and B lymphocytes, SR-BI null mice developed systemic autoimmune disorders characterized by the presence of autoantibodies in circulation, the deposition of immune complexes in glomeruli, and the leukocyte infiltration in kidney.
To understand the mechanisms of how SR-BI deficiency contributes to the impaired lymphocyte homeostasis, we assessed the effect of SR-BI on lymphocyte proliferation and cytokine production. We found that SR-BI is moderately expressed in both T and B cells and its deficiency enhances lymphocyte proliferation in basal status and causes imbalanced IFN-g/IL-4 production in stimulated status. Furthermore, HDL from SR-BI null mice exhibited less capability of suppressing lymphocyte proliferation. An early report by Zhu et al demonstrated that SR-BI negatively regulates TLR9-dependent B cell activation induced by CpG.21
Combined with the current finding, these studies suggest that SR-BI has both intrinsic and extrinsic effects on lymphocyte activation and function.
As SR-BI has been shown to suppress inflammatory cytokine production by macrophages via regulating TLR4/NF-κB signaling pathway, 24
and the NF-κB signaling in macrophages plays an important role in lymphocyte activation, 30
we elucidated the effect of SR-BI on macrophage populations and proinflammatory cytokine production. We found a significant increase in the number of splenic monocytes/macrophages, marked increases in TNF-α, IL-6 and iNOS expression and significant increases in serum TNF-α, IL-6 and NOx levels in mice lacking SR-BI.
Taken together, these findings suggest that SR-BI regulates lymphocyte homeostasis likely through multiple ways by modulating the proliferation of lymphocytes, the cytokine production by lymphocytes and macrophages, and the function of HDL.
Glucocorticoid is an immunosuppressive hormone. As an HDL receptor, SR-BI plays an essential role in providing cholesterol for glucocorticoid synthesis in stressed conditions such as endotoxemia, sepsis and long-term fasting.23–24, 34
However, SR-BI deficiency does affect glucocorticoid production in physiological condition,23–24, 34
which suggests that the impaired lymphocyte homeostasis may not be caused by a change in glucocorticoid levels.
Recent studies revealed that excess accumulation of cellular cholesterol due to a defect in HDL-mediated cholesterol efflux disrupts lymphocyte homeostasis in mice lacking LXRβ, ABCA1/ABCG1 or ApoA-1/LDL receptor31, 35–38
. SR-BI is an HDL receptor which mediates intracellular uptake of cholesterol from HDL. This raises a possibility that SR-BI may modulate lymphocyte homeostasis via regulating cellular cholesterol levels. To address this speculation, we isolated T and B cells from the spleen and analyzed their cholesterol contents. No difference in free or esterified cholesterol levels was found between SR-BI null and wild type littermates (Supplement Fig. 5a
). We also assessed the plasma membrane cholesterol level of T and B cells by filipin staining and no difference was observed between SR-BI null and wild type control mice (Supplement Fig. 5b
). Our finding is consistent with a recent report by Ji et al demonstrating that SR-BI expression enhances both cell cholesterol efflux and cholesterol influx from HDL, but does not lead to altered cellular cholesterol mass.39
These data suggest that the impaired lymphocyte homeostasis may not be caused by a change in cellular cholesterol contents.
Regulatory T cells (Tregs) and regulatory B cells (Bregs) play important role in lymphocyte proliferation and activation. To examine whether SR-BI deficiency affects Tregs or Bregs, we quantified Tregs and Bregs using CD4+
as markers, respectively. As shown in Supplement Fig. 6
, SR-BI null mice displayed no change in the percentage of Trges and a moderate increase in the number of Tregs; the percentage of Bregs was significantly decreased but the number of Bregs remained unchanged. Further investigation is required to determine a role of SR-BI in regulatory T cells and regulatory B cells and their contribution to lymphocyte homeostasis.
Early studies showed that mice lacking SR-BI exhibit extramedullary erythropoiesis due to defects in erythrocyte maturation40–41
. To assess the contribution of the erythrocytes to the observed splenic hypercellularity, we quantified erythrocytes (Ter119+
) with FACS in the splenocyte suspension after ACK treatment and found that about 14% of the splenocytes in SR-BI null mice were Ter119+
cells while only 3% of the splenocytes in wild type littermates were Ter119+
cells (Supplement Fig. 7a
). Further analysis showed that most of the Ter119+
cells were premature erythrocytes (Supplement Fig. 7b
). Thus, the accumulation of erythrocytes accounted for about a 10% increase in splenocyte populations after ACK treatment. We also prepared single splenocyte suspension without ACK lysis to determine erythrocyte populations. As shown in supplemental Fig. 7c and d
, SR-BI null mice had a 20% increase in Ter119+ cells compared with wild type littermates (48.2% ± 9.6% in SR-BI−/− vs.
28.5% ± 3.2% in SR-BI+/+
), and a great portion of the erythrocytes were premature, as illustrated by a significant increase in the ratio of early (Ter119high
, region II)-to-late (Ter119high
, region IV) phase erythrocytes in SR-BI null mice. Thus, accumulation of erythrocytes accounted for about 20% increase in total splenic cellularity observed in SR-BI null mice. These data confirmed the presence of extramedullary erythropoiesis in the spleens of SR-BI null mice, but it only partly contributed to the observed splenic hypercellularity. It is of interest to determine whether and how extramedullary erythropoiesis contributes to the impaired lymphocyte homeostasis in SR-BI null mice. With the current available evidence, it is difficult to establish a causal relationship between impaired lymphocyte homeostasis and extramedullary erythropoiesis, which warrants further investigations.
In summary, SR-BI plays critical roles in modulating lymphocyte activation, proliferation and cytokine production and its deficiency leads to impaired lymphocyte homeostasis and autoimmune disorders. Our findings reveal a previously unrecognized role of SR-BI in adaptive immunity.