Fig 2: hZC3H12D contributes to the uptake of nex-hIL1ß-mRNA with function.a Scheme for two splicing isoforms of hZC3H12D. The long and short forms encode 58 and 36 kDa proteins, respectively. Both have a common N-terminal sequence, including a ZF domain (green), and the long form has a proline-rich domain (orange; top). IHC analysis of ZC3H12D in the long form (hZC58)- and short form (hZC36)-overexpressing 786-O cells (bottom). Experiments were repeated twice with similar results. b Representative confocal microscopy images of hIL1ß-mRNA-FITC uptake in the hZC58 or hZC36 cell nucleus (top). Quantitative signals of hIL1ß-mRNA-FITC in the hZC58 or hZC36 cell nucleus (bottom; 3D images per group: n = 38 for hZC58 and n = 50 for hZC36). c Uptake of hIL1ß-mRNA-FITC into CD56+CD3-NK cell nucleus 3 h after applying 50 ng/mL RNA with 200 U/mL IL-2 protein. Confocal microscopy imaging showing the DAPI-stained nucleus in single images (top) and 3D stacks combining 15 images from top to bottom (middle). 3D stack images show a comparison of nuclear uptake between hIL1ß-mRNA and hßactin-mRNA (bottom). d Nuclear signals of hIL1ß-mRNA-FITC in CD56+CD3-NK cells treated with siRNA. Control (con) is a nontargeting siRNA (n = 13, 10, and 11 cells for control, ZC3H12D-siRNA and ZC3H12A-siRNA, respectively. Each cell image is composed of 15-stacked 3D images). e IHC analysis of IFN-? induction 48 h after RNA application with 200 U/mL IL2 to CD56+CD3-NK cells. The positive control was incubated with 20 ng/mL IL12 protein in an IL2 culture medium (IFN-? signals from n = 16, 21, and 20 ZC3H12D+ cells for no, IL1ß-RNA, and IL2 protein, respectively). f IHC analysis of IFN-? induction 3 and 6 days after stimulation of hIL1ß-mRNA and IL2 protein for MDAMB231-TCM-pretreated CD56dimCD3-NK cells. The assay system is shown (top). Two donor NK cells were independently used. no, control basal culture medium (bottom left, n = 42, 45, 51, and 51 cells for no, IL1ß-RNA, and IL2 protein, and IL1ß-RNA plus IL2 protein, respectively. Bottom right, n = 34, 44, 37, and 60 cells for no, IL1ß-RNA, and IL2 protein, and IL1ß-RNA plus IL2 protein, respectively). Bars, 5 µm. In the graphs, the averages ± SEM, and the results of Student’s t-tests (two-sided) or one-way ANOVA with Bonferroni correction are shown. The P values are shown in the figure. Source data are provided as a Source Data file.

Fig 3: (a) Temporal upregulation of mHsp90 compared to total Hsp90 levels in T cells stimulated with CD3/CD28 antibodies over a 96-h period. The data were analyzed by 2-way ANOVA with Sidak’s multiple comparison test, *p < 0.05, **p < 0.01, ***p < 0.001. Experiments were performed with 6 biological replicates. (b) Flow cytometric analysis of CD3, CD25, and CD69 T-cell populations relative mHsp90 expression compared to baseline. (c) Upregulation of total Hsp90 in activated T cell populations. Experiments were performed with 3 biological replicates for each timepoint (b, c). (d) Mean (± SD) CD25 expression on T cells 24–96 h post CD3/28 stimulation. Temporal upregulation of mHsp90 (green line) post T cell stimulation. CD25 expression; 6–8 biological replicates per time point. mHsp90 expression 6 biological replicates for each timepoint. (e) mHsp90 and total Hsp90 expression in CD3+ T cells isolated from PBMC’s were stimulated with IL-2 (60 IU/mL) for 24 h. Data represent mean ± SD. The data were analyzed by 2way ANOVA with Sidaks multiple comparison test. Experiments were performed once with 3 biological replicates. (f) Cytokine expression 24 h post Th1 or Th2 T cell stimulation treated with or without HS-131 (1 μM). Data represent mean ± SEM. The data were analyzed by One-way ANOVA with Sidaks multiple comparison test *p < 0.05,**p < 0.01, ***p < 0.001. Experiment represents 3–4 biological replicates per treatment.

Fig 4: GM-CSF-Producing ILCs in Synovial Fluid of RA Patients(A) The presence of ILCs (defined as CD45+CD3−CD4−CD8−CD11b−CD11c−CD19−CD56−) from peripheral blood (PB) or synovial fluid (SF) of a patient with RA or OA (left). The percentages of ILCs in PB and SF from individual RA (n = 13) or OA (n = 6) patients. The lines indicate the sample pairs of the same patients (right).(B) Total numbers of ILCs in 1 mL of SF from OA and RA patients (n = 6). Vertical bars indicate SD.(C) Flow cytometry analysis of IFN-γ, IL-13, IL-17, and GM-CSF expression by ILCs (gated as in A) in PB or SF of a RA patient (top). The percentages of cytokine-producing ILCs from individual RA patients (n = 11) (bottom).(D) Gating strategies for GM-CSF+CD45+ lineage markers-negative (ILCs), GM-CSF+CD45+CD3−CD11b+ (myeloid cells), and GM-CSF+CD45+CD11b−CD3+ cells (T cells).(E) Proportion of GM-CSF-producing cells (n = 3). Vertical bars indicate SD. Symbols represent individual samples. Horizontal bars indicate the means.∗p < 0.05, ∗∗p < 0.01.

Fig 5: CD64+LPMF co-localize with AChE+fibers. (A) Immunofluorescence of epithelial region in cryosections of representative fiber-high and fiber-low patients. Anti-AChE (red); anti-CD64 or anti-CD3 (green); DAPI (blue). Scale bar: 10 µm (original magnification, ×63 images) and 30 µm (original magnification, ×20 images). (B) Confocal immunofluorescence image of an intercrypt region from a fiber-high patient. Overlapping pixels (yellow, arrows) were visualized on a Z-projection image. Scale bar: 7 µm. See Supplementary Video 1. (C) Quantification of pixel intensity of CD64+ MF and AChE or CD3+ T cells and AChE using a Fiji software macro. Confocal images from rectosigmoid colon regions of fiber-low (n = 4 for CD64; n = 3 for CD3) and fiber-high (n = 5 for CD64; n = 3 for CD3) patients as well as from colonic muscle regions of HSCR patients (n = 2) were taken. The following negative staining controls were used: CD64-A488 or CD3-A488 with biotin + streptavidin-Cy3 and IgG1-A488 + biotin/streptavidin-Cy3 (n = 2). Per patient (dot), 200–300 cells were analyzed. Scatter plots with bar show mean raw integrated density (RawIntDen) ± standard error of the mean. Significance between CD64/AChE and CD3/AChE within a group was determined using an unpaired nonparametric Mann-Whitney test. (D) CD64+ MF from 3 fiber-high patients were investigated with respect to bipolar (n = 170) or round/stellate (n = 152) morphologic phenotype. Raw integrated density of co-localized pixels between CD64 and AChE are shown. Significance was determined using an unpaired nonparametric Mann-Whitney test (****P = .0001).