Fig 2: Ufbp1 promotes development of plasma cells by suppressing PKR-like ER protein kinase (PERK). a, b BLIMP1 and CD138 expression by splenocytes (a) and bone marrow cells (b) from indicated mice was analyzed by flow cytometry (n = 3 mice/genotype). c, d Enumeration of frequency of plasma cells (BLIMP1+CD138high) in a and b respectively (n = 3 mice/genotype). e Naive B cells from indicated mice were stimulated with lipopolysaccharide (LPS), interleukin (IL)-4, and IL-5 as in Fig. 3a. Four days later expression of BLIMP1 and CD138 was analyzed by flow cytometry. f Enumeration of frequency of BLIMP1+CD138high cells in e (n = 3 mice/genotype). g ER-tracker staining of splenic plasma cells (CD138+TACI+ cells) from indicated mice. h Quantification of ER-tracker staining of plasma cells in g (n = 3 mice/genotype). i Transmission electron microscope images of CD138+ cells sorted from LPS-stimulated B cell cultures from indicated mice. Scale bars are 1 µm. j CD138+ cells (104 cells/well) sorted from LPS-stimulated B cell cultures from indicated mice were cultured for 4 h and IgM production in culture supernatants was analyzed by enzyme-linked immunosorbent assay (ELISA). k Bone marrow plasma cells (4 × 103 cells/well) from indicated mice were cultured overnight and IgM production in culture supernatants was analyzed by ELISA. l Enumeration of antibody-secreting cells by ELISpot in CD138+ population sorted from LPS-stimulated B cell cultures from indicated mice. Error bars represent mean ± standard error. **P < 0.01, ***P < 0.001. Unpaired Student’s two-tailed t-test was used. A representative of two experiments is shown

Fig 3: SLex mediated rolling of eosinophils on P-selectin-expressing cells without affecting the migration. (A) The schedule of eosinophil induction. The mouse hematopoietic progenitor cells were isolated from the bone marrow using the MojosortTM hematopoietic progenitor cell isolation kit. FLT3-Ligand and SCF were added to the culture medium on days 0 and 2. The medium was partly exchanged with medium supplemented with IL-5 on days 4, 7, and 10. (B) The harvested eosinophils were analyzed using flow cytometry. The mature eosinophils were defined as CD45+Siglec-F+CD11b+CCR3High. The experiment was independently repeated twice. (C) The rolling assays were performed, and the videos were taken using a CMOS camera equipped on a microscope at the speed of 20 photos per second for 30 s. In total, the stuck of 600 pictures were analyzed using Trackmate in Fiji ImageJ after subtracting the background. The rolling cells are circled with purple, and their rolling tracks are drawn in yellow by the software. The experiments were repeated twice independently. (D) The speed of the rolling cells was calculated using the Trackmate in Fiji ImageJ. In the WT eosinophil group, most of the cells moved between 5 and 15 μm/s. No rolling cells were detected in the DKO eosinophil (DKO Eosinophils) and F2-treated WT eosinophil groups (WT Eosinophils with F2). (E) A migration assay was performed using transwells, and the percentage of migrated eosinophils into the lower chamber was shown. Cells from the murine bone marrow was pretreated with or without F2 mAb and used for the migration assay. The lower chamber contained mCCL11 or medium only. ** p < 0.01. A p-value of less than 0.05 was considered significant.

Fig 4: Administration of mAb F2 suppressed the allergic immune response in the lungs and serum. (A) The mRNA expression in the lung of IL-4, IL-5, IL-13, IL-33, CCL11, and MBP was examined using quantitative-PCR. Samples from 2 independent experiments were examined. (B) The concentration of the Th2 cytokine IL-4 and IL-5 in the BALF was determined by ELISA. (C) The concentration of IL-5 and the amounts of OVA-specific antibodies in the serum were determined using ELISA. The experiment was repeated twice independently. * p < 0.05, ** p < 0.01. A p-value of less than 0.05 was considered significant.

Fig 5: Possible mechanism underlying mAb F2-mediated suppression of the positive feedback loop in the asthma model. A hypothetical mechanism of the suppressive effects of F2 in the asthma model is shown in the figure. The initial exposure of the airway to environmental allergens can lead to the activation of pathogenic Th2 cells (upper left in purple). Th2 cells can then release Th2 cytokines, including IL-4, IL-5, and IL-13. IL-4 and IL-13 facilitate IgE class switching to activate local immune cells and cause damage to the bronchus. IL-5 is released into the peripheral blood and helps eosinophils to proliferate and mature in the bone marrow (bottom). The mature eosinophils translocate into the blood and infiltrate the sites of inflammation through the binding of sLex to P-selectin. The infiltrated eosinophils release tissue-damaging proteins such as MBP and cytokines, including IL-4 and IL-13. As a result, a positive feedback loop is formed, such that an increasing number of eosinophils are recruited to the lungs. However, when mAb F2 is administered (upper right), eosinophil infiltration is prevented in the lungs, which breaks the whole positive feedback loop to aggravate asthmatic symptoms, leading to a possible treatment for asthma.