Murine norovirus inhibits B cell development in the bone marrow of STAT1-deficient mice

Abstract

Noroviruses are a leading cause of gastroenteritis in humans and it was recently revealed that noroviruses can infect B cells. We demonstrate that murine norovirus (MNV) infection can significantly impair B cell development in the bone marrow in a signal transducer and activator of transcription 1 (STAT1) dependent, but interferon signaling independent manner. We also show that MNV replication is more pronounced in the absence of STAT1 in ex vivo cultured B cells. Interestingly, using bone marrow transplantation studies, we found that impaired B cell development requires Stat1^-/- hematopoietic cells and Stat1^-/- stromal cells, and that the presence of wild-type hematopoietic or stromal cells was sufficient to restore normal development of Stat1^-/- B cells. These results suggest that B cells normally restrain norovirus replication in a cell autonomous manner, and that wild-type STAT1 is required to protect B cell development during infection.

Keywords: B cells; B lymphocytes; Calicivirus; Murine norovirus; Norovirus; Stat1.

Fig. 1

MNV-4 infection decreases developing bone marrow B cells in a STAT1-dependent manner. Wild-type (WT) 129 mice (B) and Stat1^-/- mice (A and C) were infected with MNV-4 and the bone marrow B cells evaluated by flow cytometry at approximately 3 weeks post infection. Developing B cells were separated into pro-B/pre-B (Fractions A-C’), pre-B/immature B cells (Fraction D-E), and long-lived mature B cells (Fraction F) based on B220 and CD43 surface antigen staining. Representative of 1 of 2 independent experiments, n = 3–10 mice per group. Bars represent mean ± SEM, * = P < 0.05, ns = not significant.

Fig. 2

MNV-4 infection increases granulocytes and macrophages in the bone marrow ofStat1^-/-mice but not in wild-type mice. Wild-type (WT) 129 mice (A) and Stat1^-/- mice (B) were infected with MNV-4 and the bone marrow cells evaluated by flow cytometry at approximately 3 weeks post infection by GR1 and CD11b surface antigen staining. n = 3–5 mice per group. Bars represent mean ± SEM, * = P < 0.05, ns = not significant.

Fig. 3

MNV-4 infection results in decreased pro-B/pre-B and immature B cells at day 7 PI, and decreased mature B cells at day 3 PI, in the bone marrow ofStat1^-/-mice. Stat1^-/- mice were infected with MNV-4 and the bone marrow B cells evaluated by flow cytometry at 3, 5, 7 and 21 days post infection. Developing B cells were separated into pro-B/pre-B (Fractions A-D), immature B cells (Fraction E), and long-lived mature B cells (Fraction F) based on B220 and IgM surface antigen staining. Days 3, 5 and 7: n = 3–5 mice per group at each time point; day 21: n = 5–10 mice per group. Bars represent mean ± SEM, * = P < 0.05.

Fig. 4

Enteric bacteria are not required for MNV-4 to decrease developing bone marrow B cells inStat1^-/-mice.Stat1^-/- mice were depleted of enteric bacterial by antibiotic treatment, infected with MNV-4, and the bone marrow B cells evaluated by flow cytometry at 3 weeks post infection. Developing B cells were separated into pro-B/pre-B (Fractions A-C’), pre-B/immature B cells (Fraction D-E), and long-lived mature B cells (Fraction F) based on B220 and CD43 surface antigen staining. n = 5 mice per group, bars represent mean ± SEM, * = P < 0.05, ns = not significant.

Fig. 5

Infection with a novel MNV strain (MNV-UW) decreases bone marrow B cells and increases granulocytes and macrophages inStat1^-/-mice.Stat1^-/- mice were infected with a novel MNV-UW strain isolated from the mesenteric lymph node of a sentinel mouse and bone marrow cells were evaluated by flow cytometry at 3 weeks post infection. Developing B cells were separated into pro-B/pre-B (Fractions A-C’), pre-B/immature B cells (Fraction D-E), and long-lived mature B cells (Fraction F) based on B220 and CD43 surface antigen staining, while granulocytes and macrophages were characterized by GR1 and CD11b surface antigen staining. n = 3–5 mice per group, bars represent mean ± SEM, * = P < 0.05.

Fig. 6

MNV-4 replicates in cultured B cells isolated from the spleens ofStat1^-/-mice but does not induce cell death. B cells were isolated from the bone marrow (A) or spleen (B) of n = 3 uninfected Stat1^-/- mice and infected in vitro with MNV-4 at an MOI of 0.5 or mock infected with lysate control. Cells were evaluated at 2, 24 and 48 h for cell death by flow cytometry and Ghost Dye staining. (C) MNV genome copy number evaluated by quantitative RT-PCR in B cells isolated from the spleens of uninfected Stat1^-/- and wild-type 129 mice and then infected in vitro at an MOI of 0.5. Fig. C is pooled data from 2 independent experiments. Bars represent mean ± SEM, * = P < 0.05.

Fig. 7

MNV-4 infection decreases developing bone marrow B cells inStat1^-/-mice treated with IL-7. Bone marrow-derived macrophages were isolated and differentiated from uninfected wild-type and Stat1^-/- mice, infected in vitro with MNV-4 or mock infected, and then evaluated for mRNA expression of IL-7 by quantitative RT-PCR (A). Stat1^-/- mice were treated with recombinant mouse IL-7 or PBS daily and infected with MNV-4 or mock infected on the first day of treatment (B). Bone marrow B cells were evaluated by flow cytometry at 7 days post infection. Developing B cells were separated into pro-B/pre-B (Fractions A-C’), pre-B/immature B cells (Fraction D-E), and long-lived mature B cells (Fraction F) based on B220 and CD43 surface antigen staining. n = 3–5 mice per group. A one-way ANOVA with Sidak multiple comparison test was used to evaluate Control PBS vs. MNV-4 PBS, Control PBS vs. Control IL-7, Control IL-7 vs. MNV-4 IL-7, and MNV-4 PBS vs. MNV-4 IL-7. Bars represent mean ± SEM, * = P < 0.05.

Fig S1

MNV genome copies detected in total bone marrow by qRT-PCR. (A) MNV genome copies per 10⁶ total bone marrow cells at 21 days PI in mice: WT, Stat1^-/-, IfnαβγR^-/-, Stat1^-/- treated with antibiotics (Abx), and Stat1^-/- infected with MNV-UW. Each group was compared to WT mice by Student’s t-test. (B) MNV-4 genome copies per 10⁶ total bone marrow cells at day 3, 7, and 21 days PI in infected Stat1^-/- mice. Groups were compared by one-way ANOVA. n = 3–10 mice per group, bars represent mean ± SEM, * = P < 0.05.

Fig S2

MNV-4 infection does not alter developing bone marrow B cells inIfnαβγR^-/-mice.IfnαβγR^-/- mice were infected with MNV-4 and the bone marrow B cells evaluated by flow cytometry at approximately 3 weeks post infection. Developing B cells were separated into pro-B/pre-B (Fractions A-C’), pre-B/immature B cells (Fraction D-E), and long-lived mature B cells (Fraction F) based on B220 and CD43 surface antigen staining. n = 5 mice per group, bars represent mean ± SEM, ns = not significant.

Fig S3

MNV-4 infection decreases developing bone marrow B cells independent of mouse sex and background strain. Male Stat1^-/- mice on a 129 background (A) and female Stat1^-/- mice on a C57BL/6 background (B) were infected with MNV-4 and the bone marrow B cells evaluated by flow cytometry at 3 weeks post infection. Developing B cells were separated into pro-B/pre-B (Fractions A-C’), pre-B/immature B cells (Fraction D-E), and long-lived mature B cells (Fraction F) based on B220 and CD43 surface antigen staining. n = 5 mice per group. Bars represent mean ± SEM, * = P < 0.05.

Fig S4

MNV-4 infection does not increase caspase staining (apoptosis) in developing bone marrow B cells inStat1^-/-mice.Stat1^-/- mice were infected with MNV-4 and the bone marrow B cells evaluated by flow cytometry at 5 days post infection. Apoptosis was evaluated based on caspase and ghost dye staining in total B220⁺ cells (A), and in developing B cells (B) separated into pro-B/pre-B (Fractions A-D), immature B cells (Fraction E), and long-lived mature B cells (Fraction F) based on B220 and IgM surface antigen staining. n = 3–5 mice per group. Bars represent mean ± SEM, ns = not significant.

Fig S5

MNV-4 infection does not alterStat1^-/-or wild-type bone marrow B cells inRag2^-/-/Il2rg^-/-recipient bone marrow chimeric mice.Rag2^-/-/Il2rg^-/- mice were irradiated and administered Stat1^-/- bone marrow cells (A), or a 1:1 mixture of wild-type (WT, CD45.1) and Stat1^-/- (CD45.2) bone marrow cells (B) intravenously. After 10 weeks to allow for bone marrow reconstitution, mice were infected with MNV-4 and the bone marrow B cells evaluated by flow cytometry at 3 weeks post infection. Developing B cells were separated into pro-B/pre-B (Fractions A-C’), pre-B/immature B cells (Fraction D-E), and long-lived mature B cells (Fraction F) based on B220 and CD43 surface antigen staining, and also evaluated by CD45.1 (WT cells) and CD45.2 (Stat1^-/- cells) antigens. n = 5–6 mice per group. Bars represent mean ± SEM, ns = not significant.

Fig S6

MNV-4 infection does not alterStat1^-/-or wild-type bone marrow B cells inStat1^-/-recipient bone marrow chimeric mice.Stat1^-/- mice were irradiated and administered a 1:1 mixture of wild-type (WT, CD45.1) and Stat1^-/- (CD45.2) bone marrow cells intravenously. After 10 weeks to allow for bone marrow reconstitution, mice were infected with MNV-4 and the bone marrow B cells evaluated by flow cytometry at 3 weeks post infection. Developing B cells were separated into pro-B/pre-B (Fractions A-C’), pre-B/immature B cells (Fraction D-E), and long-lived mature B cells (Fraction F) based on B220 and CD43 surface antigen staining, and also evaluated by CD45.1 (WT cells) and CD45.2 (Stat1^-/- cells) antigens. n = 5 mice per group. Bars represent mean ± SEM, ns = not significant.