Figure 1. Host IRE1α is required for Brucella infection in vivo.

Innate cytokine production of IL-1β (A), IL-6 (B), and TNF-α (C) in bone marrow-derived macrophages (BMDMs) from the wild-type (WT, wt-IRE1α) control and Ern1 conditional knockout (CKO, m-IRE1α) mice. The BMDMs were stimulated with LPS (100 ng/ml) and at 6 hr poststimulation the cytokine production of the treated cells was determined. Colony-forming unit (CFU) assay for B. melitensis 16M (Bm16M) intracellular survival in spleens and livers of wt- and m-IRE1α mice at 7 (D) or 14 (E) days post infection (dpi). (F) Histopathology of representative hematoxylin and eosin (H&E) stained sections of spleen and liver from Bm16M-infected wt- and m-IRE1α mice at 14 dpi. Bar: 100 μm. Quantification of inflammation of spleens (G) or livers (H) at 14 dpi. (I) CFU assays of CD11b⁺ cells from Bm16M-infected wt- or m-IRE1α mice. (J) CFU assay for B. abortus S2308 (BaS2308) intracellular survival in spleens and livers in wt-IRE1α control or m-IRE1α mice at 7 dpi. (K) Bm16M invasion and intracellular replication in BMDMs from m-IRE1α and control mice. h.p.i.: hours post infection. CFU assays of Bm16M infection of WT BMDMs (L) or RAW264.7 macrophages (M). Host cells were pretreated with 4μ8C (50 μM) 1 hr before and during infection; CFUs of the infected cells were determined at the indicated h.p.i. (N) CFU assays for Bm16M infection of BMDMs from WT and Xbp1 knockout (ΔXbp1) mice at the indicated h.p.i. CFU assay for Bm16M intracellular survival in spleen (O) or liver (P) in WT or ΔXbp1 mice at 14 dpi. Immunoblotting assay for IRE1α expression (Q) and quantification of the expression levels (R) in BMDMs during a time course (48 hr) of Bm16M infection. Bm16M infection induces phosphorylation of host IRE1α (S) and quantification of the phosphorylated levels of IRE1α during a time course (24 hr) of infection (T). Images/blots are representative of three independent experiments. Statistical data represent the mean ± standard error of mean (SEM) from three independent experiments. *, **, and *** indicate significance at p < 0.05, 0.01, and 0.001, respectively.

Figure 1—figure supplement 1. Characterization of Ern1 conditional knockout (CKO) and control mice.

(A) Strategy for generating Ern1 CKO (m-IRE1α) mice. (B) Bone marrow-derived macrophages (BMDMs) from m-IRE1α mice fail to induce Xbp1 splicing in the presence of ER stress inducer tunicamycin (Tm) at the indicated hours post treatment. wt-IRE1α: mice harboring the wild-type (WT) Ern1. (C) Comparison of the levels of lymphocytes and myeloid cells in the wt-IRE1α control and m-IRE1α mice. Spleen weight of Brucella melitensis Bm16M-infected mice at 7 (D) or 14 (E) days post infection (dpi). Data represent the mean ± standard error of mean (SEM) from three independent experiments.

Figure 1—figure supplement 2. IRE1α is required for B. melitensis intracellular replication.

(A) Immunoblotting detection of IRE1α expression in the WT and Ern1 knockout (KO, Ern1^−/−) murine embryonic fibroblasts (MEFs). Colony-forming unit (CFU) assay for Bm16M infection of WT and Ern1^−/− MEFs (B) and IRE1α expression in these cells during a time course (48 hr) of Bm16M infection (C). (D) The expression levels of the truncated IRE1α in m-IRE1α BMDMs during Bm16M infection. (E) Disruption of the endonuclease domain of IRE1α does not impair its kinase activity in response to Bm16M infection. h.p.i.: hours post infection. Images are representative of three independent experiments. Data represent the mean ± standard error of mean (SEM) from three independent experiments. ***Significance at p < 0.001.