Targeting IL-6 by engineered Lactococcus lactis via surface-displayed affibody

Abstract

Background: Dysregulated production of interleukin (IL)-6 is implicated in the pathology of inflammatory bowel disease (IBD). Neutralization of IL-6 in the gut by safe probiotic bacteria may help alleviate intestinal inflammation. Here, we developed Lactococcus lactis with potent and selective IL-6 binding activity by displaying IL-6-specific affibody on its surface.

Results: Anti-IL-6 affibody (designated as ZIL) was expressed in fusion with lactococcal secretion peptide Usp45 and anchoring protein AcmA. A high amount of ZIL fusion protein was detected on bacterial surface, and its functionality was validated by confocal microscopy and flow cytometry. Removal of IL-6 from the surrounding medium by the engineered L. lactis was evaluated using enzyme-linked immunosorbent assay. ZIL-displaying L. lactis sequestered recombinant human IL-6 from the solution in a concentration-dependent manner by up to 99% and showed no binding to other pro-inflammatory cytokines, thus proving to be highly specific for IL-6. The removal was equally efficient across different IL-6 concentrations (150-1200 pg/mL) that were found to be clinically relevant in IBD patients. The ability of engineered bacteria to capture IL-6 from cell culture supernatant was assessed using immunostimulated human monocytic cell lines (THP-1 and U-937) differentiated into macrophage-like cells. ZIL-displaying L. lactis reduced the content of IL-6 in the supernatants of both cell lines in a concentration-dependent manner by up to 94%. Dose response analysis showed that bacterial cell concentrations of 10⁷ and 10⁹ CFU/mL (colony forming units per mL) were required for half-maximal removal of recombinant and macrophage-derived IL-6, respectively.

Conclusion: The ability of ZIL-displaying L. lactis to bind pathological concentrations of IL-6 at common bacterial doses suggests physiological significance.

Keywords: Delivery system; IL-6; Inflammatory bowel disease; Lactococcus lactis; Microbiota.

Fig. 1

IL-6 binding affibody ZIL is expressed in L. lactis. a Gene constructs for expression of IL-6 binding affibody ZIL on the surface of L. lactis. USP, gene encoding Usp45 secretion signal (84 bp). ZIL, gene encoding IL-6-binding affibody (174 bp). Flag, epitope tag sequence. AcmA, gene encoding C-terminal domain of AcmA anchoring protein (642 bp). The arrow represents the nisin-inducible promoter. b Coomassie brilliant blue-stained SDS-PAGE gel (left) and Western blot analysis (right) of whole lysates of L. lactis harbouring plasmids pSD-ZIL or pSD-ZIL-flag. Cont., L. lactis containing empty plasmid pNZ8148. Bands representing untagged or flag-tagged ZIL fusion protein are indicated with arrows

Fig. 2

IL-6-binding affibody ZIL is displayed on L. lactis surface. a Representative confocal immunofluorescence microscopy images visualizing ZIL-flag at the bacterial surface and b flow cytometry analysis showing a large increase in mean fluorescence intensity and a shift of the population of ZIL-flag-expressing L. lactis compared to control bacterial cells. ZIL-flag, L. lactis harbouring pSD-ZIL-flag plasmid. Cont., L. lactis harbouring empty plasmid pNZ8148. Engineered bacteria were incubated with an anti-flag antibody and then probed with Alexa Fluor 488 or Alexa Fluor 555-conjugated secondary antibody. The results are expressed as mean ± standard deviation (SD) of three individual measurements. ***, P < 0.001 (unpaired t-test). a: Bright-field images (left) and the corresponding fluorescence images (right). Bar scale 20 µm. b: MFI, mean fluorescence intensity

Fig. 3

Surface-displayed IL-6-binding affibody ZIL is functional. Representative confocal immunofluorescence microscopy images (a) and flow cytometric analysis (b) showing binding of ZIL-flag-displaying L. lactis to human biotin-conjugated IL-6. ZIL-flag., L. lactis cells containing pSD-ZIL-flag plasmid. Cont., L. lactis control cells containing empty plasmid pNZ8148. Control or ZIL-flag-displaying L. lactis cells were incubated with IL-6-biotin and detected with an anti-biotin antibody, followed by a secondary antibody conjugated to Alexa Fluor 488. The results are presented as mean ± standard deviation (SD) of three individual measurements. ***, P < 0.001 (unpaired t-test). a: Bright-field images (left) and the corresponding fluorescence images (right). Bar scale 20 µm. b: MFI, mean fluorescence intensity

Fig. 4

ZIL-displaying L. lactis removes various amounts of recombinant IL-6 from the solution in a concentration-dependent manner. ELISA-determined concentrations of recombinant IL-6 that remained in the solution following incubation with ZIL-displaying L. lactis (3 × 10⁶–6 × 10⁹ CFU/mL) across four concentrations of recombinant IL-6 (150, 300, 600 and 1200 pg/mL) that were spiked into the PBS buffer (left). ZIL., L. lactis cells containing pSD-ZIL plasmid. Cont., L. lactis control cells containing empty plasmid pNZ8148. Dose response curves for calculating the concentration of ZIL-displaying bacterial cells necessary to remove a 50% of recombinant IL-6 from the solution (half-maximal effective concentration, EC₅₀) determined by curve fitting with four parameters logistic (4 PL) regression model in GraphPad Prism (right). The results are expressed as mean ± standard deviation (SD) of three individual measurements. **, P ≤ 0.006; ***, P < 0.001 (unpaired t-test)

Fig. 5

ZIL-displaying L. lactis does not bind TNF, IL-17, IL-23 or IL-8. ELISA-determined concentration of recombinant TNF (a), IL-17 (b), IL-23 (c), IL-8 (d) that remained in the solution following their incubation with increasing concentrations of ZIL-displaying L. lactis (ZIL). Cont., L. lactis control cells containing empty plasmid pNZ8148. The experiments are performed in triplicate. Data are means ± standard deviation (SD)

Fig. 6

ZIL-displaying L. lactis does not cross-react with mouse IL-6. ELISA-determined concentration of recombinant mouse IL-6 that remained in the solution following incubation with increasing concentrations of ZIL-displaying L. lactis (ZIL) and ZIL-flag-displaying L. lactis (ZIL-flag). Cont., L. lactis control cells containing empty plasmid pNZ8148. EVA-flag, L. lactis control cells containing pSD-EVA-flag plasmid. ZHER-flag, L. lactis control cells containing pSD-ZHER-flag plasmid. The experiment is performed in triplicate. Data are means ± standard deviation (SD)

Fig. 7

ZIL-displaying L. lactis removes IL-6 secreted by differentiated THP-1 and differentiated U-937 cells in proportion to the concentration of bacterial cells. ELISA-determined concentrations of IL-6 that remained in the supernatants of LPS-induced differentiated THP-1 cells (a) and differentiated U-937 cells (b) following incubation with ZIL-displaying L. lactis (ZIL). Cont., L. lactis control cells containing empty plasmid pNZ8148. Untr., untreated supernatants. Dose response curves for calculating the concentration of ZIL-displaying L. lactis cells necessary for removal of a 50% of IL-6 from the cell culture supernatants (EC_50, half-maximal effective concentration) was determined by curve fitting using four parameters logistic (4 PL) regression model in GraphPad Prism (c). The results are expressed as mean ± standard deviation (SD) of experiments performed in triplicate. **, P ≤ 0.007; ***, P < 0.001 (unpaired t-test)