Fig 2: IL-22BPi1 and IL-2EX4 induce unfolded protein response (UPR) genes. HEK293 cells were transiently transfected with IL-22BPi1, IL-22BPi2, IL-22BPi3, IL-2, IL-2EX4 or empty vector (EV) as control. Cells were collected at the indicated hours after transfection. (A) Expression of different genes related to ER function or UPR were analyzed by RT-qPCR. Each gene expression value is represented as fold change relative to the same time-point expression value of the EV condition and relative to the housekeeping gene GAPDH. Mean ± SEM of three independent experiments. All primers are listed in Supplementary Table 2. (B) GRP78 and GRP94 protein levels correlate with mRNA levels observed in (A). Cell lysates (CL) were immunoblotted for FLAG, GRP94, KDEL and tubulin as loading control. (C) IL-22BPi1 and IL-2EX4 cause XPB1 splicing. XBP1 splicing was detected with conventional PCR for the indicated conditions and times. Un-spliced and spliced XBP1 are indicated as XBP1-u or XBP-1s respectively. (D) IL-22BPi2 secretion was not increased when co-expressed with different ratios of IL-22BPi1. HEK293 cells were co-expressed with different ratios of EV:IL-22BPi1:IL-22BPi2 expression plasmids. 48 h later, secreted IL-22BP in conditioned media (CM) was quantified by ELISA (mean ± SEM; n = 3). (E) Cell viability measured with alamarBlue was not compromised by any of the conditions in two different cell lines. Reduction of alamarBlue was measured after 48 h of transfection and assayed for the indicated times and cell lines. Values are represented as percentage of reduction in each condition relative to EV (mean ± SEM; n = 3).

Fig 3: Silencing of IL22RA2 in immature moDCs reduces GRP78 expression. (A) CD14+ monocytes were stained with DAPI (blue), trans-GOLGI (red) and IL-22BP (green, IL-22BP Ab 4). (B) Overall (best coverage, BC) and isoform-specific IL22RA2 expression levels in classical, intermediate and non-classical monocyte subpopulations (top) from four healthy donors (average ± SEM). Percentages of IL-22BP+ monocytes found in each subpopulation by immunofluorescence microscopy (bottom) (mean ± SEM; n = 4; *p < 0.05, Kruskal-Wallis test for comparison of differences in IL22RA2 mRNA levels or IL-22BP+ counts in the three subpopulations). (C) IL22RA2 expression levels in classical and intermediate immature moDCs at day 6 of cultivation. moDCs derived from non-classical monocytes showed high levels of cell death and had not experienced any changes in the shape of the cells. High Ct values of HK gene HPRT1 in qPCR compromised correct interpretation of IL22RA2 levels in this subgroup, which was therefore excluded from the graph. (D) Fold change relative to non-targeting siRNA control of IL22RA2 expression in CD14+ monocyte.-derived immature DCs on day 5 of cultivation following IL22RA2 silencing with Viromer Green at two concentrations of siRNA. (E) Silencing as described in (D) was not variant specific and both IL22RA2v1 and v2 were silenced to similar extent. (F) Scheme comparing efficiencies of four distinct silencing strategies on suppression of IL22RA2 mRNA (qPCR) and intracellular IL-22BP (ELISA) in immature CD14+ moDCs using 100 nM IL22RA2 siRNA or non-targeting siRNA in combination with Viromer Green. S, silencing, H, harvest. Bar diagrams represent fold change relative to non-targeting siRNA. (G) Effect of IL22RA2 silencing on GRP78 and ERp44 mRNA levels in CD14+ monocyte-derived immature DCs from healthy donors using silencing strategy B in (F). CD14+ monocytes were cultivated in DM medium in the absence or presence of AM580 starting on day 0 up to the day of harvest. Average of fold change (±SEM) relative to non-silencing control over 6 (control) and 5 (AM580) independent measurements relative to the housekeeping gene HPRT1. *p < 0.03 by Wilcoxon test.