Interleukin-1 alpha (IL-1α) is a pleiotropic cytokine exerting its effects on various physiological processes [1]. IL-1α signaling is evoked by binding IL-1α to its cognate receptor [2].

IL-1α receptor family is composed of 11 members. Among them, IL-1 receptor type 1 (IL-1R1) plays intrinsic role in combination with type 3 receptor in signaling [2]. Both of them encompass the specific region called toll-IL-1 responsive (TIR) domain [3]. IL-1α binding to the type 1/type 3 heterodimer recruits its adaptor protein , myeloid

differentiation factor 88 (MyD88) and IL-1R-associated kinase (IRAK) and transduce IL-1α-binding information to the underlying signaling pathway [3]. IL-1α signal is not transduced with other receptor combinations. In this context, IL-1R1 is an intrinsic receptor for IL-1α signaling.

IL-1α is produced as a precursor (pIL-1α) form inside the cell [4, 5]. pIL-1α is enzymatically cleaved to generate C-terminal mature (mIL-1α) and N-terminal propiece (ppIL-1α). mIL-1α is released extracellulary and exerts its effect by binding to type1/type 3 receptor complex. On the other hand, ppIL-1α, having the nuclear localization sequence (NLS), is reported to contribute to the transcriptional control of its target gene in the nucleus [6-8]. pIL-1α is cleaved by several enzymes such as calpain, granzyme B, and thrombin [9-11]. Intriguingly, pIL-1α is released extracellulary by still unknown process [12] and expected to encounter the enzymes which can cleave it. Although the existence of extracellular ppIL-1α is not reported, these facts suggest the possible production of ppIL-1α in the extracellular space. If this is the case, ppIL-1α might exerts its effects through unidentified receptor. To elucidate the properties of ppIL-1α receptor, it is indispensable to know the difference with IL-1R1. For this purpose, establishment of the cell lines specifically lacking the IL-1R1 might be useful. In the present study, we report the establishment of the IL-1R1 knockout cell line with the CRISP/Cas9 system.

Cell Culture

Human fibroblastic cell line HeLa was used for the experiment. The cells were cultured with Dulbecco’s minimum essential medium (DMEM) supplemented with 10 % fetal calf serum, 50 mg/ml streptomycin and 50 U/ml penicillin (IWAKI, Tokyo, Japan)(10% FCS-DMEM) in a 5% CO2 incubator.

Vectors

The CRISP plasmid with Cas9 and single guided RNA (sgRNA) was obtained from the GeneCopoeia (Rockville, MD, USA). For IL-1R1 overexpression, IL-1R1 open reading frame was introduced to pMKIT-neo mammalian expression plasmid (pNKIT-IL-1R1) (a gift from Prof. Maruyama, Tokyo Medical and Dental University). The empty pMKIT-neo was used as a negative control. The structure of each plasmid was shown in Figure 1.

Figure 1: The structure of the plasmid.

a) The all-in-one vector was purchased from GeneCopoeia. The single guide RNA (sgRNA) is expressed under the control of U6 promotor. Cytomegalo virus promotor (CMV) allows the expression of Cas9. The green fluorescence protein (GFP) and neomycin resistant gene (neo) can be expressed under the control of T2A promotor.

b) The full length IL-1R1 cDNA was introduced to the mammalian expression vector pMKIT-neo vector. The expression of IL-1R1 is under the control of SRα promotor.

AmpR: ampicillin resistant gene, IRES2: internal ribosome entry site 2

Transfection and Establishment of the Knockout Clone

Transfection of the plasmids were conducted with Lipofectamin 3,000 reagent (Life technologies, MA, USA). The plasmid (125-500 ng) was dissolved in OPTI-MEM (25 L, Life technologies) and mixed with 0.75 μL of 3,000 reagent. The Lipofectamin 3,000 (0.75 μL) was dissolved in OPTI-MEM (25 μL). These two mixture was mixed and incubated at room temperature for 15 min. After incubation, the mixture was added to HeLa cells (5 x104/24 well plate) and cultured for 72 h. After transfection, the cells were maintained in the presence of G418 (250 μg/ml) for 7 days. The remaining cells were expanded and subjected to limiting dilution. The single cell clones were selected and used for the further experiments.

T7 Endonuclease Digestion

The genomic DNA was prepared by DNeasy Blood & Tissue Kit (Qiagen, Germany). The concentration of DAN solution was measured by NanoDrop (Thermo Scientific, Madison, WI, USA) and 100 to 500 ng of genomic DNA was subjected to PCR amplification. The sequence of the primer is as follows. forward primer 5’-gaaggcatgacctttgagct-3’ and reverse primer 5’- cagtttcccactctatatgg-3’. The amplification was confirmed by agarose gel electrophoresis. The amplified DNA fragment was cut out and purified by Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). DNA fragment was denatured and annealed as follows. 95° C, 5 min, 95° C to 85° C, - 2° C/sec, 85° C to 25° C, -0.1° C/sec. The DNA fragment was digested with T7 endonuclease (New England Biolabs, Tokyo, Japan, 10 U/μL) for 1 h at 37 ° C. The sample was loaded to 1.5% agarose gel to confirm digestion.

Enzyme-Linked Immunosolbent Assay (ELISA)

The cells (5 x10⁴/24 well plate) were stimulated with or without rhIL-1α (10 ng/ml), IL-1β (10 ng/ml) or phorbol 12-myristate 13-acetate (PMA, 1 μM) for 6 h. The culture supernatants were harvested and subjected to IL-8 ELISA (R&D systems, Minneapolis, MN, USA). The experiments were repeated at least five times and the results were expressed as mean ± standard deviation (SD).

Wester Blotting

The cell lysates were harvested and subjected to western blotting. After loading on 10% SDS-PAGE, the proteins were transferred to the nylon membrane (Merck Japan, Tokyo, Japan). The western blotting was performed as described previously [12]. For IL-1R1 detection, rabbit anti-human IL-1R1 antibody (Abcam, Tokyo, Japan) and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch, West Grove, PA, USA) were used.

Statistical Analysis

Statistically significant differences were determined by two-tailed Student’s t-tests and Tukey’s test. A p-value < 0.05 was considered significant. All data are plotted as means ± standard deviation (SD).

T7 Endonuclease Digestion

Following the selection with G418, we obtained 13 candidate clones. To confirm the genetic alteration of IL-1R1 gene by CRISP/Cas9 system, we first performed the T7 endonuclease digestion of the PCR products amplified using the genomic DNA prepared from the candidate cells. Figure 2 shows the representative data. The 733 bp band was generated by PCR in both parental HeLa and candidate cell (Figure 2a, arrows). The band was digested to 510 bp and 233 bp by T7 endonuclease in candidate, but not in HeLa cell (Figure 2a). The results indicated the possible establishment of IL-1R1 KO cell and we designated one of the candidate cell as CR-R1-4.

Figure 2: Establishment of IL-1R1 deficient HeLa cell.

a) The genomic DNA was extracted from HeLa and CR-R1-4 cells and PCR amplified with the primers describe in Materials and methods. The amplified fragments were purified and subjected to denature/anneal reaction. After the reaction, the fragments were digested with T7 endonuclease for 1 h. The samples were loaded to 2% agarose gel.

b) The whole cell lysates were obtained from both cells and subjected to western blot. The rabbit anti-human IL-1R1 antibody (x 1,000) or moue anti-human GAPDH antibody were used as the first antibody followed by HRP-conjugated goat anti-rabbit IgG (x5,000) or goat anti-mouse IgG antibody (x 5,000) as the secondary antibody, respectively.

c) Immunofluoresence staining of HeLa and CR-R1-4 cells. The cells were incubated with rabbit anti-human IL-1R1 antibody (x100) followed by FITC-conjugated goat anti-rabbit IgG (x100).

The representative of at least three independent experiments are shown.

Lack of IL-1R1 Protein

To further confirm the lack of IL-1R1 in a protein level, cell lysates were prepared and subjected to western blot. IL-1R1 was detected as 56 kDa single band in HeLa cell, however, the band was not detected in CR-R1-4 (Figure 2b). The lack of IL-1R1 was also confirmed by immunofluorescence staining. In HeLa cell, IL-1R1 was mainly localized in the nucleus (Figure 2c) but the fluorescence was not detected in CR-R1-4.

IL-1α Responsiveness

HeLa cell responds to IL-1α and induce the production of IL-8 [13]. We next examined whether this response was lacked in CR-R1-4. In steady state level, HeLa cell produces 150.1 ± 36.97 pg/ ml of IL-8 (Figure 3a). IL-8 production was significantly augmented to 1889.8 ± 82.91 pg/ml when HeLa cell was stimulated with IL-1α (Fig. 3a). On the other hand, IL-8 production was not augmented in CR-R1-4 (297.92 ± 46.98 vs 159.92 ± 11.07 pg/ml) (Fig. 3a). The responsiveness to IL-1β was further examined and CR-R1-4 did not respond to IL-1β neither (297.92 ± 46.98 vs 210.17 ± 28.22 pg/ml) (Figure 3b). CRISP/Cas9 system sometime shows off-target effects [14]. To exclude the non-specific IL-8 gene disruption, the cells were stimulated with or without PMA. As shown in Figure 3c, Both HeLa and CR-R1-4 cells produced IL-8 (HeLa; 2474.31 ± 10.12, CR-R1-4; 1845.30 ± 73.32 pg/ml) (Figure 3c).

Figure 3: Response of CR-R1-4 to IL-1α and IL-1β.

HeLa and CR-R1-4 cells were seeded to 24-well plate (5 x104/well) and stimulated with or without a) rhIL-1α (10 ng/ml), b) IL-1β (10 ng/ml) or c) 1 μM PMA for 6 h. The supernatants were collected and subjected to IL-8 ELISA. The data are the means ± SD of at least five independent experiments (n = 5).

Specific Disruption of IL-1R1

To confirm the specific IL-1R1 disruption, CR-R1-4 was transfected with IL-1R1 expression plasmid or pMKIT-neo (control) and stimulated with or without rhIL-1α. Basal IL-8 production of CR-R1-4 increased to 678.12 ± 151.85 pg/ml but CR-R1-4 did not respond to rhIL-1α (606.44 ± 138.77 pg/ml) (Figure 4). On the other hand, forced IL-1R1 expression to CR-R1-4 respond to rhIL-1α (1232.96 ± 219.66 pg/ml) and the basal IL-8 production was rather lower than that of control transfectant (453.32 ± 19.57 pg/ml) (Figure 4). The results suggested specific disruption of IL-1R1 gene by CRISP/Cas 9 system.

Figure 4: Forced expression of IL-1R1 rescued IL-1α responsiveness.

The pMKIT-IL-1R1 expression plasmid was transfected to CR-R1-4 cell. The transfectant was stimulated with or without rhIL-1α. The culture supernatants were collected after 6 h of incubation and subjected to IL-8 ELISA. The data are the means

± SD of at least five independent experiments (n = 5).

In the present study, we established IL-1R1 KO HeLa strain by transfecting the all-in-one vector. In addition to the transient expression of single-strand guide RNA for the proper gene alteration by Cas9 enzyme, this vector allows the expression of GFP and neomycin resistant gene under the control of T2A promotor. To utilize these selection tools, we attempted to measure the duration of GFP expression after transfection. To our surprise, the fluorescence of GFP can be observed until 7 days of transfection under the fluorescence microscope (data not shown). Based on this result, the cells were selected in the presence of 250 μg/ml of G418 for 7 days. As the clone selection without these tools was unsuccessful, these selection tools were extremely useful. By combining the fluorescence observation and G418, we successfully obtained the 13 candidates.

Using the specific primers flanking to the expected gene alteration site, PCR fragment was successfully be amplified from the genomic DNA. In the CRISP/Cas9 system, target DNA is cut by Cas9 and repaired by two different mechanisms, non-homology end joining (NHEJ) or homology-directed repair (HR) [14]. As the donor template was not included in our experiment, digested DNA is expected to be repaired by NHEJ. In this case, a few base insertion or deletion might be created with a certain probability. Thus created mismatch is recognized by T7 endonuclease and treatment of the PCR fragment resulted in the creation of the two fragments [14]. In our case, 733 bp fragment was obtained by PCR amplification and the T7 endonuclease treatment resulted in the creation of 510 and 223 bp fragments in 6 clones out of 13 clones (data not shown). These clones were tested for IL-1α-responsiveness. The CR-R1-4 clone reported in this study did not respond to IL-1α per se and the steady state IL-8 production was negligible. However, in other clones, sometimes baseline IL-8 production was relatively high. The reason for this is expected to be due to the off target effect of CRISP/Cas9 system. Taking these facts in account, functional selection of KO candidate might be indispensable.

By transfecting IL-1R1 expression plasmid to CR-R1-4, IL-1α reactivity was perfectly reconstituted indicating the specific alteration of IL-1R1 gene in our system. IL-1α is known as an alarmin [15, 16] and released from the dying cells [17]. The released IL-1α can bind to IL-1R1 on the neighboring healthy cells. These response sometimes skewes the results of some experiments. The CR-R1-4 established in our experiment is useful for these purposes.

Although the ppIL-1α, N-terminal fragment of pIL-1α, was reported to contribute to the gene transcription [18], its existence in the extracellular space is not reported. On the otherhand, pIL-1α is secreted to the extracellular space and might be able to encounter its processing enzymes. Based on these facts, it might be possible to speculate the extracellular function of ppIL-1α. However, the research regarding the function of ppIL-1α has been hampered due to the lack of specific antibody recognizing the ppIL-1α. If ppIL-1α exerts its function in extracellular space, it is indispensable to identify its specific receptor. As the three dimensional structure of ppIL-1α is expected to be totally different from that of p or mIL-1α, the CR-R1-4 lacking the IL-1R1 might contribute the identification of its specific receptor.

Acknowledgements

The present work was supported by a Grant-in-aid for Scientific Research (C) (2020-2022, 2022-2024) and a Grant-in-aid for Young Scientists (2019-2021). The Research grant from Scholarship Fund for Young/Women Researchers; a grant from the Dental Research Center and Sato Fund, Nihon University School of Dentistry. The authors do not have any conflict of interest.

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