Fig 2: Effect of orally fed LGG on IL-22BP/IL-22/STAT3 expression. (A) Serum concentrations of IL-22 were determined by ELISA. Data are presented as the mean ± SEM for each time point (n = 6 per group). Mean values at the same time point without a common superscript (a, b) differ significantly (P < 0.05). Representative Western blot panels of (B) IL-22, (C) phosphorylation of STAT3 and total STAT3, and (E) IL-22BP in ileal tissues collected from pigs 10 days after S. Infantis challenge. Each band represents a single pig. Expression of GAPDH was measured as an internal control. Results are presented as the ratio of the intensity of the IL-22 or IL-22BP band to the intensity of the GAPDH band or the ratio of the intensity of the pSTAT3 band to the intensity of the STAT3 band. (D) Representative photomicrographs of immunostaining of IL-22BP and negative control using irrelevant mouse serum in jejunal tissues of pigs. IL-22BP-expressing cells in the jejunum were scattered predominantly throughout the lamina propria. Scale bars, 100 µm. Data are expressed in arbitrary units as the mean ± SEM for each tissue (n = 6 per group). *P < 0.05; **P < 0.01; ***P < 0.001 (Tukey's test).

Fig 3: IL-22BP protein and mRNA levels in monocyte-derived DCs. (A) (Upper left) Diagram showing exons with the primer-binding positions for IL22RA2 variant discrimination strategy. (Lower left) Expression of IL22RA2v1, v2, and v3 in moDC after 6 days of culture in differentiation medium (DM) ± AM580, a retinoic acid receptor agonist, by RT-PCR. IL22RA2v2 expression vector was used as positive control for IL22RA2v2 expression. (Upper right) Expression of IL22RA2 variants in moDC following 6 days of culture in DM ± AM580 by RT-qPCR using the isoform-specific primers indicated in the left diagram (mean ± SEM; n = 3) relative to the housekeeping gene GAPDH. (B) Effect of different maturation stimuli (CpG and LPS + IFN-? or Poly I:C) on IL22RA2, IL12B, and IL6 expression in moDC after 6 days of culture in DM by RT-qPCR using Taqman probes relative to the housekeeping gene ACTB. Extent of maturation is represented as percentage of CD83+ fluorescence by flow cytometry. Data showing representative experiment of 3 performed. (C) IL-22BP secretion by moDC cultured in DM detected by ELISA corresponds to IL22RA2 mRNA expression levels analyzed by RT-qPCR using Taqman probes and both increase over the cultivation period (mean ± SEM, n = 2). (D) Cell lysates (CL) of moDCs and conditioned media (CM) were harvested after 6 days in DM ± AM580 and immunoblotted for detection of IL-22BP (IL-22BP antibody 1) using tubulin as a loading control, IL22RA2 mRNA expression relative to the mean of the housekeeping genes GAPDH and ACTB was measured by RT-qPCR using pre-designed SYBR Green primers from the same cells. Representative experiment out of three performed. All primers used are listed in Supplementary Table 2.

Fig 4: Differential secretion, intracellular retention and degradation by the proteasome of IL-22BP isoforms. Data refer to HEK293 cells transiently transfected for 24 h unless otherwise specified. (A) HEK293 cells were transiently transfected with expression vectors encoding IL-22BPi1, BPi2, or BPi3. Intracellular and secreted IL-22BP protein levels were measured by WB (FLAG Ab) and ELISA, respectively. Transfection efficiency was measured by IL22RA2 RT-qPCR relative to the housekeeping gene GAPDH (mean ± SEM; n = 3). (B) Confocal microscopy of IL-22BP isoforms (green, IL-22BP Ab 4 with either ERp72 or trans-Golgi marker TGOLN2 (red) in transfected HEK293 cells; cells were counterstained with DAPI. (C) Secreted recombinant IL-22BPi2 is not detected by WB in unconcentrated conditioned media (CM). Various cytokine expression vectors, as indicated, were transiently transfected and CM were collected and immunoblotted for FLAG. (D) Detection of IL-22BP isoforms in cell lysates (CL) and acetone-precipitated CM (AP) by FLAG Ab. Secreted IL-22BPi2 was treated with PNGase F or Endo H. (E) Intracelullar isoforms of IL-22BP were treated with PNGase F or Endo H and detected by FLAG Ab. (F) HEK293 cells were transfected for 24 h with expression vectors for either IL-2 or IL-2EX4. The latter was generated by subcloning exon-4 from IL22RA2v1 into the open reading frame of IL-2 through its unique Xba1 position. Detection of resulting proteins in AP, CL, CM by FLAG Ab. (G) HEK293 cells were transiently transfected with the same expression plasmids as in (A) or IL-2EX4; 24 h later, cells were treated with a mix of proteasome inhibitors (PI) (5 µM lactacystin, 5 µM MG132, and 1 mM epoxomicin), and 18 h later cells were lysed and immunoblotted for FLAG using GAPDH as a loading control. All data are from at least 3 independent experiments.

Fig 5: IL-22BPi1 does not interact with IL-22 or IL-22BPi2. (A) A549 cells were exposed for 30 minutes to dilutions of IL-22-containing culture medium (CM) previously produced by IL22-transfected HEK293 cells. A549 cell lysates (CL) were immunoblotted for pSTAT3 and tubulin as loading control. The relative densitometry of pSTAT3 normalized to that of tubulin is also represented. (B) A549 cells were treated with the optimum IL-22 dilution from A (1/512) for different periods of time, lysed and immunoblotted for pSTAT3. An unspecific protein band (u.p.) was used as loading control. (C) IL-22BP concentration in conditioned medium (CM) of transfected HEK293 cells was measured by ELISA, and the indicated amounts in nanograms (ng) of IL-22BPi1 or IL-22BPi2 were pre-incubated for 1 h at 37°C with the selected IL-22 concentration from (A). A549 were exposed to the pre-incubated combinations for 20 min. An excess of IL-22BPi2 was used as phosphorylation blocking control (lane 3). Cells were lysed and immunoblotted for pSTAT3 and actin as loading control. (D) HeLa cells were co-transfected with the indicated expression plasmids, 24 h later cells were lysed and the CM was subjected to acetone precipitation (AP), and proteins were immunoblotted for FLAG. Intracellular IL-22BPi1 is indicated with dark purple arrows, intracellular and secreted IL-22BPi2 is indicated with green arrows, and co-expressed IL-22 and IL-17 are also indicated. (E) Conditioned media (CM) from 3 independent experiments, in which expression vectors for the three IL-22BP isoforms were individually transfected into HEK293 cells together with either IL-17 or IL-22 expression vectors or an empty vector control (EV), were analyzed 24 h after transfection by ELISA for IL-22BP (mean ± SEM; n = 3; **p < 0.01 by unpaired t-test). (F) IL-22BPi1 does not interact with IL-22BPi2. IL-22BPi1-MF expression plasmid containing Myc and FLAG tags was co-transfected with an inducible pTRE3G-based vector expressing IL-22BPi2 with only a FLAG tag. After 24 h, cells were induced for IL-22BPi2 production by adding Tet-Express activator to the medium for a further 24 h. Cells were lysed and immunoprecipitated with anti-Myc agarose, the flow-through fractions were then further subjected to FLAG immunoprecipitation. CL and eluted fractions were immunoblotted for FLAG.