UBXN3B is crucial for B lymphopoiesis

Abstract

Background: The ubiquitin regulatory X (UBX) domain-containing proteins (UBXNs) are putative adaptors for ubiquitin ligases and valosin-containing protein; however, their in vivo physiological functions remain poorly characterised. We recently showed that UBXN3B is essential for activating innate immunity to DNA viruses and controlling DNA/RNA virus infection. Herein, we investigate its role in adaptive immunity.

Methods: We evaluated the antibody responses to multiple viruses and pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza in tamoxifen-inducible global and constitutive B cell-specific Ubxn3b knockout mice; quantified various immune populations, B lineage progenitors/precursors, B cell receptor (BCR) signalling and apoptosis by flow cytometry, immunoblotting and immunofluorescence microscopy. We also performed bone marrow transfer, single-cell and bulk RNA sequencing.

Findings: Both global and B cell-specific Ubxn3b knockout mice present a marked reduction in small precursor B-II (>60%), immature (>70%) and mature B (>95%) cell numbers. Transfer of wildtype bone marrow to irradiated global Ubxn3b knockouts restores normal B lymphopoiesis, while reverse transplantation does not. The mature B population shrinks rapidly with apoptosis and higher pro and activated caspase-3 protein levels were observed following induction of Ubxn3b knockout. Mechanistically, Ubxn3b deficiency leads to impaired pre-BCR signalling and cell cycle arrest. Ubxn3b knockout mice are highly vulnerable to respiratory viruses, with increased viral loads and prolonged immunopathology in the lung, and reduced production of virus-specific IgM/IgG.

Interpretation: UBXN3B is essential for B lymphopoiesis by maintaining constitutive pre-BCR signalling and cell survival in a cell-intrinsic manner.

Funding: United States National Institutes of Health grants, R01AI132526 and R21AI155820.

Keywords: B cell; COVID-19; Haematopoiesis; Lymphopoiesis; UBXN.

Fig. 1

UBXN3B controls B cell development in a B cell-intrinsic manner. a, b) Irradiated wild type (WT, CD45.1) recipient mice were transplanted with ERT2-Cre⁺Ubxn3b^flox/flox bone marrow (CD45.2). The mice were then treated with tamoxifen (TMX) to delete Ubxn3b in haematopoietic cells (designated Ubxn3b^−/− BM–WT) or corn oil (designated Ubxn3b^+/+ BM–WT). c, d) Irradiated Cre⁺Ubxn3b^flox/flox and Ubxn3b^flox/flox (CD45.2) mice were transplanted with wild type (WT, CD45.1) bone marrow. The mice were then treated with TMX to delete Ubxn3b (designated WT BM–Ubxn3b^−/−) or not (designated WT BM–Ubxn3b^+/+) in non-haematopoietic cells. Data shown in a-d) are the cellularity of major blood immune populations at 45 days after TMX. e) The cellularity of blood immune cells in Mb1-Cre^−/−Ubxn3B^WT/WT (Mb1^WTUbxn3b^WT), Mb1-Cre^−/+Ubxn3B^WT/WT (Mb1^HetUbxn3b^WT), Mb1-Cre^−/+Ubxn3B^flox/WT (Mb1^HetUbxn3b^Het), Mb1-Cre^+/+Ubxn3B^WT/WT (Mb1^KOUbxn3b^WT), and Mb1-Cre^−/+Ubxn3B^flox/flox (Mb1^HetUbxn3b^KO) mice. RBC: red blood cells, WBC: white blood cells. The horizontal line indicates the median of the result. Each symbol = one mouse. In a, b) N = 5, c, d) N = 4, e) N = 3 mice/genotype. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 [multiple unpaired Mann–Whitney tests with Holm-Šídák correction for a; multiple unpaired Welch t-tests with Holm-Šídák correction for b-d; two-way ANOVA with two-stage step-up method of Benjamini, Krieger and Yekutieli multiple comparisons test after log-transformation for e].

Fig. 2

UBXN3B is essential for the proliferation of large pre-BII. a) The cellularity of terminally differentiated immune cells, b) the flow cytometry gating strategy, c) cellularity and percentage of B lineage fractions in the bone marrow of inducible global Ubxn3b knockout and wildtype littermates derived from ERT2-Cre⁺Ubxn3b^flox/flox. d) The cellularity of B lineage fractions in the bone marrow of B cell specific knockout mice derived from Mb1-Cre^−/+/Ubxn3b^flox/+. Neu: neutrophil, Mono: monocyte, pre-pro-B: pre-progenitor B, Fraction B: progenitor B (pro-B), C: precursor BI (pre-BI), C’: large (L) pre-BII, D: small (S) pre-BII, E: immature B (Imm-B), F: mature B. The horizontal line indicates the median of the result. Each symbol = one mouse. N = 3 mice/genotype. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 [multiple unpaired Welch t-tests with Holm-Šídák correction for a, c; two-way ANOVA with two-stage step-up method of Benjamini, Krieger and Yekutieli multiple comparisons test after log-transformation for d].

Fig. 3

Pre-BCR signalling is impaired in large pre-BII. a-b) ERT2-Cre⁺Ubxn3b^flox/flox mice were treated with tamoxifen (TMX) (Ubxn3b^−/−) or corn oil (Ubxn3^+/+) every two days for 7 days. At 14 days after the first dose of TMX, bone marrow B cells were analysed by flow cytometry. a) Representative histograms of the indicated phosphorylated proteins in the large (L) pre-BII fraction. X-axis: fluorescence intensity, Y-axis: normalised to mode. b) The mean fluorescence intensity (MFI) of indicated phosphorylated proteins and percentage of positive L-pre-BII cells (%). Each symbol = one animal. N = 3 mice/genotype. The horizontal line indicates the mean of the results. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 [multiple unpaired Welch t-tests with Holm-Šídák correction]. c) The immunoblots of phosphorylated (p) and total proteins in human NALM6 pre-B cells stimulated with an anti-human IgM. d) The ratio of ΔF (the difference of calcium load between any a given time after anti-IgM treatment and time point zero F0) to F0 in NALM6. Each dot represents the ratio of mean ΔF/F0 of all the cells recorded at a given time (every second). The results are representative of two independent experiments or mice (c, d). e) The top pathways enriched from significantly downregulated genes in the Ubxn3b^−/− B fraction with high expression of pre-BCR surrogate light chains (SCL^hi). The whole transcriptomes (pooled from 3 mice/genotype) are analysed by single cell (sc) RNA-seq. The statistical method for p-values is Binomial Test, and for FDR (false discovery rate) is Benjamini-Hochberg.

Fig. 4

UBXN3B is crucial for mature B cell survival. ERT2-Cre⁺Ubxn3b^flox/flox mice were treated with tamoxifen (TMX) (Ubxn3b^−/−) or corn oil (Ubxn3^+/+) every two days for 7 days. a) At 14 days after the first treatment, the whole transcriptomes in the bone marrow mature B fraction (N = 2 Ubxn3^+/+, 3 Ubxn3b^−/−) are analysed by bulk RNA-seq. Shown are the top pathways enriched from significantly downregulated genes in Ubxn3b^−/−. The statistical method for p-values is Binomial Test, and for FDR (false discovery rate) is Benjamini-Hochberg. b) The blood B cell ratios of TMX to corn oil-treated ERT2-Cre⁺Ubxn3b^flox/flox mice at various timepoints. Data point: mean ± SD, N = 4 mice/genotype, ∗∗p < 0.01, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 [repeated measures two-way ANOVA Bonferroni's multiple comparison test with Greenhouse-Geisser correction]. c) The flow cytometry gating strategy and d) percentage of apoptosis (Annexin V positivity) in the splenic B, T, or myeloid populations at 14 days after the first TMX/oil treatment. N = 3 mice/genotype, ∗p < 0.05 [multiple unpaired Welch t-tests with Holm-Šídák correction]. e) Immunofluorescence staining for apoptotic (TUNEL) and B cells (CD19) in the spleens of mice at 14 days after the first TMX/oil treatment. Objective: 20×. Scale bar: 50 μM. DNA is stained with DAPI. f) The numbers of TUNEL-positive cells per microscopic field. Each symbol = one field. N = 11 fields from 3 Ubxn3^+/+, N = 17 fields from 3 Ubxn3b^−/− spleens. ∗∗∗∗p < 0.0001 [unpaired Welch t-test]. g, h) The immunoblots of indicated proteins in g) splenic B and non-B immune cells, h) splenic B cells stimulated with an anti-mouse IgM antibody or isotype control. Spleens are from the mice at 14 days after the first TMX/oil treatment.

Fig. 5

UBXN3B limits influenza pathogenesis. Sex- and age-matched B cell specific Ubxn3b^KO (Mb1^HetUbxn3b^KO) and matched Ubxn3b^WT (Mb1^HetUbxn3b^WT) were infected with 350 CCID50 (cell culture infectious dose 50% assay) influenza A PR/8/34H1N1 intranasally. a) The body weight changes over day 0. N = 5 Ubxn3b^WT, 3 Ubxn3b^KO. b) The viral RNA loads in lungs at 9 days after infection. N = 3 mice per genotype. c) Representative microscopic images of iron-staining (blue) of lung sections at 24 days p.i. (n = 3 mice per genotype). The arrow heads indicate blue cells with haemosiderosis. d) The cell counts of various immune cell populations in the blood at 7 days p.i. N = 5 Ubxn3b^WT, 4 Ubxn3b^KO. e) The concentrations of IAV-specific serum IgM and IgG presented as optical density (OD) at λ_450nm. N = 4 Ubxn3b^WT, 3 Ubxn3b^KO. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 [repeated measures two-way ANOVA Bonferroni's multiple comparisons test a; unpaired Welch t-test for b; multiple unpaired Welch t-tests with Holm-Šídák correction for d; repeated measures two-way ANOVA with Bonferroni's multiple comparisons test for e].