MicroRNA-146a deficiency enhances host protection against murine cytomegalovirus

Abstract

Natural killer (NK) cells are innate lymphoid cells that protect a host from viral infections and malignancies. MicroRNA-146a (miR-146a) is an important regulator of immune function that is highly expressed in NK cells and is further upregulated during murine cytomegalovirus (MCMV) infection. Here we utilized mice with a global targeted deletion of miR-146a to understand its impact on the innate immune responses to MCMV infection. MiR-146a^-/- mice were protected from lethal MCMV infection, which was intrinsic to the hematopoietic compartment based on bone marrow chimera experiments. NK cell depletion abrogated this protection, implicating NK cells as critical for the miR-146a^-/- protection from MCMV. Surprisingly, NK cells from miR-146a-deficient mice were largely similar to control NK cells with respect to development, maturation, trafficking, and effector functions. However, miR-146a^-/- mice had increased NK cell numbers and frequency of the most mature Stage IV (CD27^-CD11b⁺) NK cells in the liver at baseline, enhanced STAT1 phosphorylation, and increased selective expansion of Ly49H⁺ NK cells and T cells during MCMV infection. This study demonstrates a critical role for miR-146a in the host response to MCMV, arising from mechanisms that include increased NK cell numbers and early T-cell expansion.

Keywords: Innate immunity; NK cells.

Figure 1.. MiR-146a deficiency provides protection against MCMV infection

(A-B) Mice were infected with 2.35 × 10⁵ pfu MCMV, i.p.. (A) Survival curves of miR-146a−/− mice compared to wild-type controls after lethal MCMV infection. N=18–22 mice per group total from 3 independent experiments. (B) Bone-Marrow chimera mice were generated by transferring bone marrow from miR-146a−/− or Control mice into lethally irradiated recipient mice. 3 Months later, mice were infected with lethal MCMV. N=7–8 mice per group total from 2 independent experiment. (C) Survival curves of miR-146a−/− mice after lethal MCMV infection in the presence of NK cells (PBS control) or following NK cell depletion (α-NK1.1 injection). Mice received two doses of either PBS or 100µg of anti-NK1.1 antibody (α-NK1.1, PK136) i.p. in the week prior to MCMV infection. N=8–10 mice per group from 2 independent experiments. Statistical significance was determined by Mantel-Cox test; *p < 0.05; ***p < 0.001.

Figure 2.. Loss of miR-146a increases NK cell numbers in the liver but not in other tissues.

(A) The percentage of NK cells (CD45⁺, NK1.1⁺, CD3⁻ lymphocytes; gating strategy shown in Supplemental Figure 2) was determined by flow cytometry. (B) Absolute NK cell counts were calculated using the NK cell percentage and the total cells per organ. (C) NK cell maturation stages were determined by the following maturation stage identifications: stage I, CD27⁻CD11b⁻; stage II, CD27⁺CD11b⁻; stage III, CD27⁺CD11b⁺; stage IV, CD27⁻CD11b⁺. (D) Absolute NK cell numbers. Total NK cell numbers for blood are represented as percentage of CD45 positive cells, and bone marrow cell counts represent the total cells per femur. N=6–15 mice per group from 3–4 independent experiments. Summary data are shown as mean ± SEM. Statistical significance was determined by t-test; *p < 0.05; ***p < 0.001; ****p < 0.0001.

Figure 3.. Loss of miR-146a does not significantly impact NK cell IFN-γ production 36 hours post-MCMV infection.

(A) Experimental Schema. miR-146a^−/− and control mice were challenged with sublethal MCMV infection (5 × 10⁴ pfu MCMV, i.p.). Spleens were harvested and analyzed 36 hours post-infection. (B) Total cell counts from spleen, (C) percentage of NK cells in spleen, and (D) total number of splenic NK cells. (E) Summary data of percentage of IFN-γ positive NK cells. N=8–9 mice per group total from 2 independent experiments. Summary data are shown as mean ± SEM; no statistically significant differences were found by t-test.

Figure 4.. In vitro NK cell functional responses were not significantly impacted but STAT1 signaling is enhanced by miR-146a deficiency.

Splenocytes were isolated from miR-146a−/− and wild-type control mice. (A-B) Summary data of IFN-γ and (C) CD107a expression in NK cells under the indicated stimulation conditions. N=5–8 mice per group total from 2 independent experiments. (D) Granzyme B expression of NK cells was determined by flow cytometry after culturing for two days in media containing IL-15 or IL-15 + IL-18. N=3–6 mice per group total from 2 independent experiments. (E-G) Splenocytes were stimulated with IFN-α for 60 minutes and phophorlyated(p)-STAT1 in NK cells was assessed by flow cytometry. (E) Representative flow plot. (F-G) Summary data of p-STAT1 Median Florescent intensity (MFI) in (F) bulk NK and (G) NK cells in indicated maturation stage. N=7 mice per group from 3 independent experiments. Summary data are shown as mean + SEM; Statistical significance was determined by t-test in (A-D) and by 2-way ANOVA in (F-G) ; *p < 0.05; ***p < 0.001; ****p < 0.0001.

Figure 5.. Loss of miR-146a promotes MCMV-specific NK cell expansion during innate immunity-mediated phase of MCMV infection and early T-cell expansion.

(A) Percent of Ly49H+ NK cells in the spleen and liver of control and miR-146a−/− mice at baseline. N=6–9 mice per group from 2–3 independent experiments. (B) Experiment Schema for (C-E). Mice were infected with 5 × 10⁴ pfu, a sublethal dose of MCMV. Spleens and livers were assessed at 7 dpi and 28 dpi by flow cytometry. (C) Absolute number of Ly49H+ NK cells in the spleen and liver on 7 days post-infection (dpi) (D) Absolute numbers of T cells in infected mice at 7 dpi and (E) 28 dpi. N=9–10 mice per group total, 2 independent experiments. Summary data are shown as mean ± SEM. Statistical significance was determined by t-test. *p < 0.05; **p < 0.01; ****p < 0.0001.