Role of Interferon-γ-Producing Th1 Cells in a Murine Model of Type I Interferon-Independent Autoinflammation Resulting From DNase II Deficiency

Abstract

Objective: Patients with hypomorphic mutations in DNase II develop a severe and debilitating autoinflammatory disease. This study was undertaken to compare the disease parameters in these patients to those in a murine model of DNase II deficiency, and to evaluate the role of specific nucleic acid sensors and identify the cell types responsible for driving the autoinflammatory response.

Methods: To avoid embryonic death, Dnase2^-/- mice were intercrossed with mice that lacked the type I interferon (IFN) receptor (Ifnar^-/- ). The hematologic changes and immune status of these mice were evaluated using complete blood cell counts, flow cytometry, serum cytokine enzyme-linked immunosorbent assays, and liver histology. Effector cell activity was determined by transferring T cells from Dnase2^-/- × Ifnar^-/- double-knockout (DKO) mice into Rag1^-/- mice, and 4 weeks after cell transfer, induced changes were assessed in the recipient mice.

Results: In Dnase2^-/- × Ifnar^-/- DKO mice, many of the disease features found in DNase II-deficient patients were recapitulated, including cytopenia, extramedullary hematopoiesis, and liver fibrosis. Dnase2^+/+ × Rag1^-/- mice (n > 22) developed a hematologic disorder that was attributed to the transfer of an unusual IFNγ-producing T cell subset from the spleens of donor Dnase2^-/- × Ifnar^-/- DKO mice. Autoinflammation in this murine model did not depend on the stimulator of IFN genes (STING) pathway but was highly dependent on the chaperone protein Unc93B1.

Conclusion: Dnase2^-/- × Ifnar^-/- DKO mice may be a valid model for exploring the innate and adaptive immune mechanisms responsible for the autoinflammation similar to that seen in DNASE2-hypomorphic patients. In this murine model, IFNγ is required for T cell activation and the development of clinical manifestations. The role of IFNγ in DNASE2-deficient patient populations remains to be determined, but the ability of Dnase2^-/- mouse T cells to transfer disease to Rag1^-/- mice suggests that T cells may be a relevant therapeutic target in patients with IFN-related systemic autoinflammatory diseases.

Figure 1.. Unc93B1-dependent type I IFN-independent hematological defects in Dnase2^−/− mice.

(A) Total WBC, lymphocytes, platelets and hematocrit of Het, DKO and Unc93B1 TKO mice in blood collected from 5–7 wk old mice (n=6). EPO serum levels of 10-week-old mice (n=4–6); spleen weight of 4-week-old mice (n=12–19). (B) FACS analysis of total spleen from 10-week-old mice to identify: Erythroid progenitors (ProE, CD71⁺ Ter119^lo, n=12–14); Erythroid lineage cells (Ter119^hi, n=13–16); and EryA erythroblasts (Ter119⁺ CD71⁺ FSC-A^hi, n=12–14). (C) Number of cells recovered from 4 leg bones/mouse and EryB erythroblasts (Ter119⁺ CD71⁺ FSC-A^lo) in total BM of 10 wk old mice (n=7–16 mice/group).

Figure 2.. Lymphocyte and myeloid cell abnormalities in DKO mice.

(A) FACS analysis of Ter119^neg spleen cells stained for B cell marker CD19 (top) and FACS analysis of total BM cells stained for B220 and AA4.1 to identify immature (AA4.1^hi) and mature (AA4.1^lo) B cells (bottom). (B) Analysis of Ter119^neg spleen cells stained for CD3, CD4, CD8, CD69 (activated T cell), and effector memory subsets (CD44⁺, CD62L⁻). (C) Analysis of Ter119^neg spleen cells to identify myeloid cells (CD11b⁺) and neutrophils (CD11b⁺ Ly6G⁺) in spleen (top) and BM (bottom). Bar graphs for A-C summarize 5 independent experiments using 10 wk old mice (n=5–22 mice/group).

Figure 3.. Proinflammatory cytokines and liver fibrosis in DKO mice.

(A) Cytokine levels in sera from 8-week-old Het (white bars), DKO (black bars) and Unc93B1 TKO (grey bars) mice (n=5). (B) Trichrome stain of liver sections from 6 mon old mice (representative of n=3). Magnification is 40x and enlarged insert is 100x.

Figure 4.. Induction of autoinflammation in Rag1^−/− mice.

(A) Rag1^−/− mice were transplanted under the kidney capsule with spleen fragments from Het or DKO mice and tissues were collected 5 mons post-transplant. The weight of the host spleen was determined upon euthanasia. The % erythroblasts and % myeloid cells in the spleen and BM of unmanipulated (grey bars) and transplanted (white and black bars) Rag1^−/− mice was determined by flow cytometry (n=5–9 mice/group). (B) Rag1^−/− mice were injected i.v. with 10⁷ RBC-depleted spleen cells from Het, DKO, Unc93B1 TKO or STING TKO mice and tissues collected 4 wks post-injection. Bar graphs summarize 4–5 experiments (n=3–13 mice/group). (C) Rag1^−/− mice were injected i.v. with 10⁷ total RBC-depleted DKO spleen cells or B220-depleted, CD4/CD8-depleted DKO spleen cells, or 3 × 10⁶ bead purified spleen cells and tissues collected at 4 wks post injection. Spleen weight, % erythroblasts, % myeloid cells and % pro-B cells in the spleen and BM were compared to uninjected Rag1^−/− (Ctrl) mice. Bar graphs summarize 4–5 experiments (n=18 for depleted cells and n=3–14 for purified cells).

Figure 5.. Induction of Autoinflammation by DKO CD4⁺ T cells transfer into Rag1^−/− mice.

(A) Rag1−/− mice were injected with 10⁷ magnetic bead-purified CD4+ T cells isolated from either Het or DKO mice and the spleen and bones were obtained from euthanized mice 4 weeks later and compared to tissues from uninjected Rag1^−/− mice. (B, C) Bone marrow and spleen cell suspensions isolated from the same mice were analyzed by flow cytometry. Plots are representative of 3–5 mice/group and results are summarized in the bottom row.

Figure 6.. Role of TLRs and IFNγ in autoinflammation.

(A) Immunofluorescent stain of HEp-2 cells by sera from 4–5-month-old mice of the indicated strains (left). Spleen weights of 4–10-week-old mice of the indicated strains (n=8–31 mice/group) (right). (B) RNA was extracted from Het, DKO and Unc93B1 TKO CD4-bead purified T cells and expression levels were quantified using the Nanostring Immune Code Set. Morpheus software used to generate heat map of Th1-related genes and additional genes upregulated 10–100 fold in the DKO vs Het CD4+ cells (n=3). (C) Comparison of 5 wk old mice from the indicated strains (n=3–10 mice/group). (D) Comparison of uninjected Rag1^−/− mice, either Rag1^−/− or Rag1^−/− IFNγR^−/− mice injected with DKO spleen cells (n=3–19). (E) Comparison of uninjected Rag1^−/− mice, Rag1^−/− mice injected with DKO T cells or Rag1^−/− IFNγR^−/− mice injected with in vitro generated Th1 cells (n=3–17).