A novel type I IFN-producing cell subset in murine lupus

Abstract

Excess type I IFNs (IFN-I) have been linked to the pathogenesis of systemic lupus erythematosus (SLE). Therapeutic use of IFN-I can trigger the onset of SLE and most lupus patients display up-regulation of a group of IFN-stimulated genes (ISGs). Although this "IFN signature" has been linked with disease activity, kidney involvement, and autoantibody production, the source of IFN-I production in SLE remains unclear. 2,6,10,14-Tetramethylpentadecane-induced lupus is at present the only model of SLE associated with excess IFN-I production and ISG expression. In this study, we demonstrate that tetramethylpentadecane treatment induces an accumulation of immature Ly6C(high) monocytes, which are a major source of IFN-I in this lupus model. Importantly, they were distinct from IFN-producing dendritic cells (DCs). The expression of IFN-I and ISGs was rapidly abolished by monocyte depletion whereas systemic ablation of DCs had little effect. In addition, there was a striking correlation between the numbers of Ly6C(high) monocytes and the production of lupus autoantibodies. Therefore, immature monocytes rather than DCs appear to be the primary source of IFN-I in this model of IFN-I-dependent lupus.

Figure 1. TMPD induces elevated expression of IFN-I and accumulation of Ly6C^hi monocytes

Comparisons of A) IFN-I expression (conventional PCR) and B) ISG expression (RT-PCR) in peritoneal cells after TMPD or MO treatment. C) Flow cytometry of peritoneal cells in wild-type 129Sv mice treated with PBS, TMPD (2 weeks), mineral oil (2 weeks), thioglycollate (3 days), and cecal-puncture ligation (3 days). Boxes indicate Ly6C^hi immature monocytes (R1), CD11b⁺ Ly6C^mid granulocytes (R2), and Ly6C⁻ mature monocytes/macrophages (R3). D) Quantification and E) morphologic analysis of peritoneal cell populations. F) Flow cytometry of surface makers on Ly6C^hi immature monocytes. G) Flow cytometry of peritoneal cells from wild-type 129Sv mice treated with TMPD or mineral oil for 1 day. Shaded region (panel F) represents isotype control staining and boxes indicate Ly6C^hi immature monocytes. Each bar (panels B and D) represents the mean of 4 animals and error bars indicate standard deviation (s.d.). * p < 0.05 (Student’s t-test).

Figure 2. TMPD induces the direct recruitment of circulating Ly6C^hi monocytes

A) Propidium idodide cell cycle analysis of peritoneal cells two weeks after TMPD treatment. B) Flow cytometry of Ly6C expression on peripheral blood monocytes (gated on CD11b⁺Ly6G⁻ cells) and quantification of peripheral blood Ly6C^hi and Ly6C⁻ monocytes following TMPD treatment (n = 4 per group). C) Flow cytometry of peripheral blood and peritoneal monocytes (gated on CD11b⁺Ly6G⁻ cells) in TMPD-treated mice 24 hr after administration of DID-liposomes. Data are representative of 3 independent experiments.

Figure 3. Ly6C^hi monocytes are major producers of IFN-I

Analysis of A) IFN-I expression (conventional PCR) and B) ISG expression (RT-PCR) in magnetic bead-sorted peritoneal Ly6C^hi monocytes, granulocytes and the negative fraction (lymphocytes and DCs) from wild-type 129sv mice treated with TMPD (2 weeks). C) Flow cytometry of TMPD-elicited peritoneal cells 2 days following treatment with clodronate-containing liposomes (clo-lip). Box indicates Ly6C^hi monocytes. D) IFN-I expression (conventional PCR) and E) ISG expression (RT-PCR) in peritoneal cells after clodronate-liposome or PBS treatment. Each bar represents the mean of 5 animals and error bars indicate s.d. * p < 0.05 (Student’s t-test).

Figure 4. Dendritic cells are not required for IFN-I production induced by TMPD

A) Flow cytometry of peritoneal DCs after TMPD treatment. Box indicate CD11c⁺ I-A⁺ DCs. B) Depletion in CD11c-DTR mice 2 days after diphtheria toxin (DT) injection. Box indicates CD11c⁺ I-A⁺ DCs. C) Quantification of peritoneal dendritic cells and Ly6C^hi monocytes. D) Quantification of splenic DC depletion. MDCs were defined as CD11c^hi I-A⁺ CD11b⁺ cells and PDCs were defined as CD11c⁺ B220⁺ PDCA-1⁺. E) IFN-I expression (conventional PCR) and F) ISG expression (RT-PCR) in peritoneal exudates cells. Each bar represents the mean of 6 animals and error bars indicate s.d. * p < 0.05 (Student’s t-test).

Figure 5. Accumulation of Ly6C^hi monocytes is correlated with the frequency of anti-Sm/RNP autoantibodies and upregulation of ISG expression

A) Flow cytometry of peritoneal cells in wild-type 129Sv mice treated with n-hexadecane (2 weeks) or squalene (2 weeks). Boxes indicate Ly6C^hi immature monocytes. Correlation of the levels of Ly6C^hi monocyte accumulation with B) the frequency of anti-Sm/RNP autoantibodies and C) ISG expression induced by TMPD, n-hexadecane (HEX), squalene (SQ), and mineral oil (MO) and PBS. Autoantibody frequencies represent the mean of >10 mice per group from previous studies. Percentage of Ly6C^hi monocytes and ISG expression index represent the mean of 4 mice. Error bars indicate s.d. ISG expression index was calculated as follows: mean relative expression of (Mx-1 + MCP + IP-10)/3.

Figure 6. Ly6C expression on immature monocytes is not dependent on IFN-I signaling

Flow cytometry analysis of monocyte subsets in the peripheral blood (upper) and bone marrow (lower) of TMPD-treated wild-type 129sv and IFN-I receptor-deficient mice. Boxes and ovals indicate peripheral blood Ly6C^hi monocytes and bone marrow Ly6C^hi monocyte precursors, respectively.