IFN-I Promotes T Cell-Independent Immunity and RBC Autoantibodies Via Modulation of B-1 Cell Subsets in Murine SCD

Hemolysis is a key feature of sickle cell disease (SCD). We have recently demonstrated that hemolysis triggers type I interferon (IFN-I) response, resulting in elevated plasma levels of IFN-I in a mouse model of SCD and sickle patients. While IFN-Is play a critical role in both innate and adaptive immunity, their signaling also promotes autoimmunity including the production of autoantibodies. In this study, we investigated whether the increased levels of IFN-I have an impact on the immune responses and production of anti-red blood cell (RBC) autoantibodies in a mouse model of SCD.

To systematically evaluate immune functions in SCD, we immunized SCD mice with T cell-independent (TI) and T cell-dependent (TD) antigens. We found significantly enhanced TI immune responses in SCD mice, while TD immune responses were similar or slightly reduced depending on the routes of antigen administration. In addition, the direct anti-globin test performed by measuring anti-mouse antibodies bound on circulating RBCs by flow cytometry revealed significantly increased levels of anti-RBC autoantibodies in SCD mice.

To investigate the mechanisms by which they increase the levels of TI immune responses in SCD, we analyzed the B-1 and marginal zone B (MZB) cells, key cell populations responsible for the TI immune responses. The frequencies of splenic MZB cells in SCD mice were significantly lower compared to those of control mice. On contrary, the frequencies and numbers of B-1b cells, a subset of B-1 cell, were significantly increased in SCD mice, which support a potential role for B-1b cells in the markedly enhanced TI immune responses and anti-RBC autoantibody levels in SCD mice. To further investigate the contribution of B-1 cell subsets, we performed an intraperitoneal hypotonic lysis. Treatment of Milli-Q water led to a decreased levels of B-1a cells while the levels of B-1b cells were increased in both AS and SS mice compared to those of PBS-treated control mice. Consistent with the increased B-1b cells, the levels of TI-antigen-specific IgG and anti-RBC autoantibodies were both significantly increased in mice treated with Milli-Q water. These data support the idea that B-1b cells are responsible for the enhanced TI immune response and levels of anti-RBC autoantibodies in SS mice.

To evaluate whether IFN-I promotes TI immune response and production of anti-RBC autoantibody, we generated SCD mice deficient in IFNAR1 by crossing SCD mice with Ifnar1^-/- mice. The levels of TI-antigen-specific antibodies and anti-RBC autoantibodies were significantly reduced in the absence of IFNAR1 signaling in Ifnar1^-/-SCD mice. Strikingly, the high frequencies of B-1b cells shown in the peritoneal cavity of SCD mice were completely reversed in the absence of IFNAR1 signaling in Ifnar1^-/-SCD mice. These data uncovered a critical role for IFN-I in the enhanced TI-2 immune responses and the increased production of anti-RBC autoantibodies by modulating the innate B-1 cell subsets in SCD mice.

Given that the levels of IFN-I also increased in patients with SCD, we investigated whether there is an alteration of MZB cells and B-1 cells in patients with SCD. We found that the frequencies of MZB cells were significantly reduced while the frequencies of B-1 cells were significantly increased in patients with SCD compared to those of HD. In addition, we detected a statistically significant increase in the levels of anti-RBC autoantibodies in SCD patients. Significantly, the anti-RBC autoantibody levels correlated strongly with the numbers of peripheral B-1 like cells (r=0.82, p<0.0001), supporting a role for increased B-1 cells in the regulation of anti-RBC autoantibodies in SCD patients.

Overall, our study provides evidence that the modulation of B-1 cells and IFN-I can regulate TI immune responses and the levels of anti-RBC autoantibodies in SCD. Based on our study, either modulation of the levels of IFN-I or other treatment that can alter the B-1 cell subsets can be used as a potential intervention to control RBC autoantibodies in SCD patients. In addition, our data shed light on the mechanisms underlying the immune response to TI-2 antigens, such as polysaccharides of Streptococcus pneumoniae, which support for the use of potential adjuvants that increases IFN-I to boost TI-2 vaccine responses.