Cutting edge: Type I IFN drives emergency myelopoiesis and peripheral myeloid expansion during chronic TLR7 signaling

Abstract

Mice overexpressing TLR7 (TLR7.1 mice) are a model of systemic lupus erythematosus pathogenesis and exhibit peripheral myeloid expansion. We show that TLR7.1 mice have a dramatic expansion of splenic cells that derive from granulocyte/macrophage progenitors (GMP) compared with wild-type mice. In the bone marrow, TLR7.1 mice exhibited hallmarks of emergency myelopoiesis and contained a discrete population of Sca-1(+) GMP, termed emergency GMP, which are more proliferative and superior myeloid precursors than classical Sca-1(-) GMP. The emergency myelopoiesis and peripheral myeloid expansion in TLR7.1 mice was dependent on type I IFN signaling. TLR7 agonist administration to nontransgenic mice also drove type I IFN-dependent emergency myelopoiesis. TLR7.1 plasmacytoid dendritic cells were cell-intrinsically activated by TLR7 overexpression and constitutively produced type I IFN mRNA. This study shows that type I IFN can act upon myeloid progenitors to promote the development of emergency GMP, which leads to an expansion of their progeny in the periphery.

Fig. 1. TLR7.1 mice exhibit emergency myelopoiesis in the bone marrow

(A) Gating strategy and representative flow cytometry plots for BM LSK, CMP, GMP and eGMP. (B) Number of LT-HSC, ST-HSC and MPP from 3–5 experiments. (C, D) Number of CMP, GMP (C) and eGMP (D) from 5 independent experiments. Significance determined by two-tailed, unpaired student’s t-test.

Fig. 2. TLR7.1 myeloid progenitors exhibit a type I IFN signature in the bone marrow where pDC are constitutively activated and generating type I IFN mRNA

(A) ISG mRNA from BM progenitors, sorted as in Fig. 1A and assayed by qPCR. Data are from 2 independent experiments. (B) BM pDC from WT and TLR7.1 mice were gated on as CD11b⁻Siglec-H⁺BST-2⁺ and a histogram of MHCII expression is shown, representative of 5 independent experiments. (C) BM pDC from mixed BM chimeras generated with a 1:1 ratio of TLR7.1 and B6.SJL BM were gated as CD45.2⁺ and CD45.1⁺, respectively. MHCII expression on CD45.2⁺ and CD45.1⁺ BM pDC from the same mouse are shown, representative of 3 independent experiments. (D) BM cells were sorted (pDC as in Fig. 2B; inflammatory monocytes CD11b⁺Ly6C⁺Ly6G^-; neutrophils CD11b⁺Ly6C⁺Ly6G⁺) and used for qPCR. Data are representative of two experiments. n.d. not detected.

Fig. 3. TLR7.1 emergency myelopoiesis depends on type I IFN signaling

(A) Spleen weight. (B) Number of splenic myeloid cells were gated on as in Fig. S1A and B. (C–D) BM progenitor cells were gated on as in Fig. 1A. All data represent the mean of 12–15 mice assessed in at least 3 independent experiments. Significance determined by one-way ANOVA with a tukey post-test.

Fig. 4. Serial TLR7 agonist administration drives type I IFN-dependent emergency myelopoiesis

(A) Identification of eGMP in WT and IFNARKO mice treated with or without 10 μg gardiquimod i.v. every other day for 6 days. (B) Number of eGMP. Data show 6 mice per group from 2 independent experiments with each dot representing an individual mouse. Significance determined by one-way ANOVA with a tukey post-test.

Fig. 5. eGMP are superior myeloid precursors and more proliferative than GMP

(A–C) 250 WT GMP, TLR7.1 GMP or TLR7.1 eGMP were plated in methylcellulose media with SCF, IL-3 and IL-6. (A) Number of colony forming units (CFU) after 5 days. (B) Absolute cell number generated after 6 days in culture from 1 experiment. (C) Fold change in cell yield over WT GMP. (A, C) Data are representative of cells sorted from 3 WT or TLR7.1 mice in 2 independent experiments. (D) Percent BrdU⁺ GMP and eGMP in WT and TLR7.1 mice 1 h after injection of BrdU. Data are representative of 2 experiments. Each dot represents an individual mouse with bar representing the mean. (A–D) Significance determined by one-way ANOVA with a tukey post-test, two-tailed, paired or unpaired student’s t-test, where appropriate.