Induction of IL-26 protein synthesis was achieved in Escherichia coli strain XL1blue, which is capable of producing IL-26 in inclusion bodies, by adding 1mM isopropyl β-D-1-thiogalactopyranoside and cultivation for 6h (Knappe et al., 2000; Hör et al., 2004). First, the bacteria were disrupted mechanically in 6 M guanidinium chloride with 0.1M NaH₂PO₄, 0.01M Tris, and 100mM β-mercaptoethanol. Bacterial debris was removed by centrifugation for 30min at 8,873×g and by sterile filtration. Nickel-chelate affinity chromatography was used for protein purification under denaturing conditions (Ni Sepharose 6 Fast Flow, GE Healthcare Life Sciences, Buckinghamshire, UK). Particle-bound IL-26 was eluted under increasing imidazole concentrations (40mM, 500mM, and 600mM). Eluate fractions were tested for 19kDa bands in Coomassie-stained protein gels. Renaturation of relevant eluate fractions was achieved by dialysis for 24h twice, at 4°C (20mM HEPES, 1mM MgCl₂, 20mM KCl, 0.1mM EDTA, 1% glycerol, 1mM oxidated glutathione, and 5mM reduced glutathione, pH 8.0).

The physiological STAT3 phosphorylation activity of the newly produced IL-26 was tested with the human colon carcinoma cell line COLO-205. IL-26 (5μg/ml) was added for 45min at 37°C. Cell lysates were separated on protein gels and Western blots were probed with anti-phospho-STAT3 and anti-STAT3 antibodies (anti-phospho-Tyr705 STAT3, #9131, rabbit; anti-STAT3, #9132, rabbit) and horseradish peroxidase-conjugated anti-rabbit IgG antibodies (#7074, goat, Cell Signaling Technology, Beverly, United States) as secondary reagent. Signals were detected by chemiluminescence (Figure 1) using a charged-coupled device camera (LAS-3000, Fujifilm, Tokio, Japan). If phospho-STAT3 bands were detectable, the respective IL-26 fraction was considered biologically active.

Bacteria and C. albicans were initially grown on Columbia sheep blood agar at 37°C overnight and then for 20h at 37°C in 10ml tryptic soy broth (TSB) medium with 10mM NaCl. Then, 50μl of the culture was added to 10ml of TSB/10mM NaCl medium and incubated for 3h at 37°C for reaching the logarithmic phase of growth and the optical density at 600nm was determined. We diluted the culture with 10mM NaCl to reach target concentrations of 10⁵CFU/ml. Next, 100μl of the microbial suspension was added to 100μl of different concentrated IL-26 solutions (100, 30, 10, 3, and 1μg/ml) to achieve final IL-26 concentrations in the wells of 50, 15, 5, 1.5, and 0.5μg/ml. We always ran negative controls with pure dialysis buffer instead of the IL-26 solution, as well as positive controls with bacteria that were previously shown to be highly sensitive to IL-26, when species with low or lacking IL-26 sensitivity were examined.

Immediately after the start of the treatment, and after 1, 2, 3, and 4h, 20μl from each culture was sampled and diluted in a 0.85% NaCl solution and 100μl of that dilution was pipetted on two lysogeny broth (LB) agar plates each. The plates were incubated overnight at 37°C and the colonies were counted. The numbers of colony forming units per milliliter (CFU/ml) were calculated after averaging of the counting results of the two separate plates in consideration of the dilution factor. Totally, 15,000 LB agar plates were plated by hand for 66 independent experiments.

Statistical evaluation was performed with GraphPad PRISM 8 (GraphPad Software, Inc., San Diego, CA, United States). Values of p were determined by using the independent two-sample t-test. We used linear regression for calculating the minimal concentration for 90% bactericidal or fungicidal effects (MBC₉₀/MFC₉₀), respectively, the lethal dose or concentration for 90% killing (LD₉₀/LC₉₀). All results of this study are given as MBC₉₀/MFC₉₀, even though the use of LD₉₀/LC₉₀ is also common in AMP research. The use of the terms MBC/MFC may be more precise from a microbiological point of view. Results with values of p ≤0.05 were rated significant. As far as not given in numbers, significances are labeled as followed: ^*p≤0.05; ^**p≤0.01; ^***p≤0.001; and ^****p≤0.0001, ns=not significant.

Directly after adding 50μg/ml IL-26 to Enterococcus faecalis ATCC 29212 cultures, an immediate and highly significant (p≤0.0001) reduction of the number of colony forming units (CFU) of approximately 98.4% was detected. After 1h, significant effects were found from 5μg/ml on. Complete eradication of all bacteria was reached at 50μg/ml IL-26, after incubation for at least 1h. The CFU numbers after 2, 3, and 4h of incubation were reduced by 97.3, 92.3, and 96.1% at 15μg ml IL-26 (Figure 2A).

Similar to the antibiotic-sensitive type strain, a vancomycin-resistant clinical isolate of E. faecalis showed an instant significant effect. After 1h, significant CFU number reductions were found starting from 0.5μg/ml, whereas the strongest results were observed at 15μg/ml (reduction of 72.5%) and 50μg/ml (99.6%). The reduction levels stayed constant over time. After 4h, the incubation with 50μg/ml IL-26 resulted in a highly significant decline of CFU numbers by 99.95% (Figure 2B).

Likewise, Enterococcus faecium ATCC 6057 showed a prompt significant drop of the CFU numbers at 15μg/ml by 70.6% and at 50μg/ml by 99.5%. This effect increased, when the incubation was performed for 1h. Reduction values were 81.3% for 5μg/ml, 99.96% for 15μg/ml, and a complete killing of all bacteria was achieved for 50μg/ml. After 2 and 4h, complete eradications were reached at 15μg/ml. At 3h, reductions amounted to 99.98% for 15μg/ml and 100% for 50μg/ml (Figure 2C).

Subsequently, we checked for IL-26 sensitivity of vancomycin-resistant E. faecium DSM 17050 (Deutsche Sammlung von Mikroorganismen, German Collection of Microorganisms, Hannover, Germany). Again, a significant immediate decline by 99.1% was achieved at 50μg/ml IL-26. After 1h, complete eradication of all bacteria was reached at 50μg/ml, whereas 99.89% were killed at 15μg/ml. These effects were stable (± 0.2% at 15μg/ml) for 2, 3, and 4h incubation time. After 3h, an additional significant but weaker effect was found at 5μg/ml, which lost its significance after 4h (Figures 2D, 3).

In the case of S. aureus ATCC 6538, we found an instant reduction of the CFU numbers of approximately 23.2% at 50μg/ml IL-26 (p=0.37), which increased after 1h to a highly significant reduction by 94.6% (p<0.0001). Further increments were achieved upon ongoing incubation. CFU reductions at 50μg/ml reached 95.6, 98.0, and 93.4% after 2, 3, and 4h. After 2 and 3h, significant but weak effects were detectable at 15μg/ml (Figure 2E).

In contrast to the antibiotic-sensitive S. aureus ATCC 6538, the MRSA strain ATCC 33593 showed highly significant CFU reductions immediately after adding IL-26 to the cultures at 15μg/ml with a decline by 90.1% and at 50μg/ml by 99.3%. These effects increased after 1h with CFU reductions by 53.3% at 1.5μg/ml, 81.4% at 5μg/ml, 99.8% at 15μg/ml, and a complete eradication of all bacteria at 50μg/ml. After 2h, the effects at 5μg/ml and 15μg/ml were stable (±0.1%) and complete killing at 50μg/ml was still achieved. When incubating for 3h, reductions were found at 0.5μg/ml (33.5%), 1.5μg/ml (36.5%), 5μg/ml (83.9%), 15μg/ml (99.8%), and 50μg/ml (100%), of which all were significant, besides at 1.5μg/ml. Last, after 4h, declines by 63.6% at 5μg/ml, 98.6% at 15μg/ml, and complete eradication at 50μg/ml were observed (Figure 2F).

The MBC₉₀ for immediate effects ranged from 38.5μg/ml (MRSA ATCC 33593) to 57.5μg/ml (vancomycin-resistant E. faecalis), except for the methicillin-sensitive S. aureus ATCC 6538 with an unusually high MBC₉₀ of 136μg/ml. Two groups of similar MBC₉₀ ranges were defined. In the first group, E. faecalis ATCC 29212, E. faecium ATCC 6057, vancomycin-resistant E. faecium ATCC 17050, and MRSA ATCC 33593 had average MBC₉₀ values between 12.5μg/ml and 13.4μg/ml IL-26 for 1 to 3h and 19.3μg/ml after 4h. In the other group, the average MBC₉₀ for 1 to 4h for vancomycin-resistant E. faecalis and methicillin-sensitive S. aureus ATCC 6538 ranged between 43.7μg/ml and 45.5μg/ml (Table 1). Hence, we were able to show for the first time that IL-26 functions as a highly active bactericidal agent against different Gram-positive bacteria. These effects are independent of the antibiotic resistance phenotypes since multi-resistant strains of S. aureus, E. faecalis, and E. faecium were as sensitive as or even more sensitive than their antibiotic-sensitive counterparts.

Concerning Gram-negative species, we first tested A. baumannii ATCC 19606. Similar to all tested Gram-positive bacteria, an initial albeit weak CFU reduction was observed. After 1h of incubation, a highly significant decline of 99.8% was detected at 50μg/ml IL-26 and stayed constant for 4h (±0.3%). Additionally, we found a 50% non-significant reduction at 15μg/ml (Figure 2G).

Moreover, two highly resistant A. baumannii CR isolates were analyzed, which solely were sensitive for colistin. The first one was isolated in 2014 from a patient from a local outbreak at the University Hospital Schleswig-Holstein in Kiel, Germany. An immediate reduction was not observed but, again, highly significant CFU reductions were measureable at 1, 2, 3, and 4h (99.4, 99.3, 99.7, and 99.7%) at IL-26 concentrations of 50μg/ml (Figure 2H). The other A. baumannii CR isolate resulted from a patient from 2018 with previous hospitalization in a country with high prevalence of colonization with CR A. baumannii. Here, we detected a significant immediate reduction of 31.7% at 50μg/ml IL-26. Furthermore, weaker and non-significant reductions were detectable at 0.5μg/ml, 5μg/ml, and 15μg/ml. After 1h, the decline of the CFU count at 50μg/ml IL-26 was 98.7% and increased in the course of the experiments to 99.4% (2h), 99.8% (3h), and 99.6% (4h; Figure 2I). The mean MBC₉₀ values for the three A. baumannii strains (ATCC 19606, 2014, 2018) were 374.6μg/ml for 0h, 46.91μg/ml for 1h, 48.09μg/ml for 2h, 46.8μg/ml for 3h, and 47.22μg/ml for 4h (Table 1).

A. baumannii was the only tested Gram-negative species with IL-26 sensitivity. In contrast, we were unable to detect any antimicrobial effects against E. coli ATCC 11775, K. pneumoniae ATCC 4352, or P. aeruginosa ATCC 27853 for IL-26 concentrations up to 50μg/ml (Figures 2J–L).

Last, the sensitivity of C. albicans ATCC 24433 against IL-26 was investigated. Immediate effects were not detected at up to 50μg/ml IL-26. The CFU reduction values at 50μg/ml IL-26 were 29% after 1h (non-significant), 67.5% after 2h, 58.2% after 3h, and 78.5% after 4h (all significant). The MFC₉₀ values ranged from 54.7 to 81.41μg/ml (Table 1). Hence, we were able to show a reproducible and significant, albeit weak fungicidal activity of IL-26 against C. albicans.

Thus, the cytokine IL-26 with its proinflammatory, bactericidal, antiviral, and fungicidal activities can be attributed to the group of kinocidins which was defined for cytokines with direct antimicrobial effects, such as human mammalian platelet factor 4 (hPF-4; Yount et al., 2004; Yeaman et al., 2007; Larochette et al., 2019).