Although IL-1α has long been recognized as a critical regulator of inflammation and immune responses (8), its specific functions in physiologic and pathologic inflammatory outcomes in health and disease remain unclear. IL-1α is subjected to complex regulation, and early genetic studies using different knockout mice produced conflicting observations (27, 30–32). To clarify the previously observed contradictory roles of IL-1α in IL-1β expression in Il1a-KO^line1 mice, we generated a new line of IL-1α knockout (KO) mice using CRISPR-Cas9 technology, referred to here as Il1a-KO^line2 ( Figure 1 ). Exons 2-5 of the Il1a gene were deleted by using simultaneous injection of two individual gRNAs with human codon optimized Cas9 mRNA ( Figure 1A ). We opted to use pronuclear-staged C57BL/6J zygotes for the injections to minimize the background-related genetic issues. Successful generation of the Il1a-deficient mice was assessed by targeted deep sequencing and further confirmed by PCR amplification of genomic DNA from the WT and mutant alleles ( Figure 1B ), and western blot analysis to confirm the loss of IL-1α protein production ( Figure 2A ). Additionally, because IL-1α is a multifaceted cytokine that we postulated may have a role in regulating immune cell phenotypes at basal levels, we evaluated the immune cellularity in the blood from the newly generated CRISPR Il1a ^–/– mice (Il1a-KO^line2). We found that these mice did not show any gross abnormalities in the immune cellularity ( Supplementary Figures 1A, B ). In sum, we generated a new line of IL-1α knockout mice, Il1a-KO^line2, and confirmed the loss of IL-1α expression with no defects in overall blood immune cellularity.

Both IL-1α and IL-1β are known to be highly induced in response to pathogenic insults. Therefore, we next sought to characterize the cytokine expression in cells from the newly generated Il1a-KO^line2 mice in response to PAMPs and pathogens. Treatment of BMDMs with lipopolysaccharide (LPS, a toll-like receptor 4 (TLR4) agonist from Gram-negative bacteria) induced robust and time-dependent expression of IL-1α protein in WT cells but not in Il1a-KO^line2 cells ( Figure 2A ). In addition, the induction of IL-1α protein expression was not affected by Il1b genetic deletion, and the expression of IL-1β in response to LPS was similar in the WT and Il1a-KO^line2 cells ( Figure 2A ). In contrast, we observed a delay and reduction in the production of IL-1β in the previously generated Il1a-KO^line1 cells in response to LPS ( Supplementary Figure 2A ).

We next sought to further understand the potential roles for IL-1α and IL-1β in NLRP3 inflammasome priming, which is known to produce mature IL-1β. We found that IL-1α was not required for upregulation of NLRP3 or IL-1β expression in response to the innate immune triggers LPS, LPS plus ATP, Pam3CSK4 (Pam3) plus ATP, or Gram-negative bacteria Escherichia coli or Citrobacter rodentium ( Figures 2A–C ). Moreover, the activation of canonical and non-canonical inflammasomes, as measured by cleavage of caspase-1 and gasdermin D (GSDMD), was not reduced by deletion of Il1a ( Figures 2B, C ). Consistently, IL-1β release was similar in WT and Il1a-KO^line2 BMDMs ( Figures 2D, E ). In contrast, using similar experimental approaches, we observed defects in IL-1β expression in macrophages from the earlier Il1a-KO^line1 line, with pronounced reductions in IL-1β expression at early time points in response to LPS, while the induction improved at later timepoints ( Supplementary Figure 2A ). We also observed reductions in IL-1β expression in response to NLRP3 inflammasome triggers LPS plus ATP and Pam3 plus ATP ( Supplementary Figure 2B ). We did not observe defects in NLRP3 production or caspase-1 and GSDMD activation in Il1a-KO^line1 cells ( Supplementary Figures 2A, B ). Together, these results show that while the previously generated Il1a-KO^line1 mice had defects in IL-1β production, Il1a-KO^line2 mice did not share these defects.

IL-1α is a pleiotropic cytokine and critical amplifier of inflammation in response to both infection and sterile cellular insults (8). IL-1α also plays key roles in regulating neutrophil-chemotactic factors such as the chemokine KC (CXCL1) in mice (33). Therefore, to further confirm the IL-1α deletion in the newly generated Il1a-KO^line2 mice and assess its functional effects, we evaluated expression of TNF and KC in response to innate immune triggers. We specifically used LPS plus ATP as the stimulation to mimic the inflammasome activation conditions used above to determine differences in IL-1β production, as this stimulus has been previously shown to be suitable for measuring inflammatory markers (34). We found that IL-1α specifically was required to produce KC, but not TNF, in response to both PAMP- and pathogen-induced signaling in macrophages; loss of IL-1α resulted in significant decreases in KC release, while loss of IL-1β did not decrease KC release ( Figures 3A–D ). Instead, we observed significantly increased levels of KC production in Il1b ^−⁄− cells in response to LPS plus ATP and Pam3 plus ATP treatments ( Figure 3A ), suggesting a competition between IL-1α and IL-1β for IL-1R binding in this context, where the increased availability of IL-1R molecules for binding by IL-1α may promote hyper-expression of select inflammatory factors in the absence of IL-1β. Together, these findings confirm the specific role of IL-1α for the release of KC, further supporting the functional relevance of the newly created Il1a-KO^line2 mice for the evaluation of IL-1α–mediated signaling and disease phenotypes.

Il1b ^−⁄− (51) and Il1a ^−⁄− (Il1a-KO^line1) (52) mice were both previously described. Il1a ^−⁄− (Il1a-KO^line2) mice were generated in the current study and are described below. All mice were generated on or extensively backcrossed to the C57BL/6 background. All mice were bred at the Animal Resources Center at St. Jude Children’s Research Hospital and maintained under specific pathogen-free conditions. Mice were maintained with a 12 h light/dark cycle and were fed standard chow. Animal studies were conducted under protocols approved by the St. Jude Children’s Research Hospital committee on the Use and Care of Animals.

The new Il1a-KO^line2 mouse was generated using CRISPR/Cas9 technology in collaboration with the St. Jude Transgenic/Gene Knockout Shared Resource facility. Pronuclear-staged C57BL/6J zygotes were injected with human codon-optimized Cas9 mRNA transcripts (50 ng/μl) combined with two guide RNAs (120 ng/μl each; sgRNA1 for the 5’ of exon 2: AAAAGCTTCTGACGTACCACagg, and sgRNA2 for the 3’ of exon 5: AAGTAACAGCGGAGCGCTTTtgg (pam sequences are underlined)) to generate a long deletion encompassing exons (E) 2–5 of the Il1a gene ( Figure 1A ). Zygotes were surgically transplanted into the oviducts of pseudo-pregnant CD1 females, and newborn mice carrying the desired deletion in the Il1a allele were identified by PCR agarose gel-electrophoresis ( Figure 1B ) and Sanger sequencing. The WT allele was PCR amplified by using the primers IL1a_F1 (5’-GGGCACACGAATTCACACTCACA-3’; primer P1) and IL1a_R1 (5’-GGAGAACTTGGTTCCTGTTAGGGTGA-3’; primer P2), and the KO allele was amplified by using IL1a_F1 and IL1a_R2 (5’- TGATTAGCTTCCTTTGGGCTTTGA-3’; primer P3) primer pairs. The details of the generation of the CRISPR reagents were described previously (53). The uniqueness of sgRNAs and the off-target sites with fewer than three mismatches were found using the Cas-OFFinder algorithm (54).

BMDMs were prepared as described previously (55). In short, bone marrow cells were cultured in IMDM supplemented with 30% L929 cell-conditioned medium, 10% FBS, 1% nonessential amino acids, and 1% penicillin-streptomycin for 6 days to differentiate into macrophages. On day 6, BMDMs were counted and seeded at 10⁶ cells per well in 12-well culture plates in DMEM containing 10% FBS, 1% nonessential amino acids, and 1% penicillin-streptomycin. iBMDMs (immortalized BMDMs from Il1a ^−⁄− (Il1a-KO^line1) mice) were maintained in DMEM supplemented with 5% L929 cell-conditioned medium, 10% FBS, 1% nonessential amino acid, and 1% penicillin-streptomycin. Stimulations were performed with LPS alone (100 ng/ml) for the indicated times, LPS (100 ng/ml) or Pam3 (1 µg/ml) for 3.5 h followed by the addition of ATP (5 mM final concentration) for 30 min, or E. coli (MOI, 20) or C. rodentium (MOI, 20) for 24 h.

The cellular phenotypes of immune cells in the blood were analyzed either by flow cytometry (for T cell subsets and B cells) or by using an automated hematology analyzer machine (for % lymphocytes, % neutrophils, % monocytes, red blood cell (RBC) counts, hemoglobin (HB) quantification, and platelet (PLT) quantification). The following antibodies were used for cell staining: anti-CD19 (APC, clone ID3), anti-CD45.2 (FITC, clone 104), and anti-TCRβ (PECy7, clone H57-597) from Biolegend, and anti-CD8a (efluor450, clone 53-6.7) from eBiosciences. Samples were assessed and data were acquired on LSR II Flow Cytometer from BD Biosciences and analyzed using the FlowJo software (Tree Star), version 10.2 (FlowJo LLC).

Samples for immunoblotting of caspase-1 were prepared by mixing the cell lysates with culture supernatants (lysis buffer: 5% NP-40 solution in water supplemented with 10 mM DTT and protease inhibitor solution at 1× final concentration); samples for all other protein immunoblotting were prepared without the supernatants in RIPA lysis buffer. Samples were mixed and denatured in loading buffer containing SDS and 100 mM DTT and boiled for 12 min. SDS-PAGE–separated proteins were transferred to PVDF membranes and immunoblotted with primary antibodies against IL-1α (503207, Biolegend), IL-1β (12426, Cell Signaling Technology), caspase-1 (AG-20B-0042, Adipogen), NLRP3 (AG-20B-0014, Adipogen), GSDMD (ab209845, Abcam), and β-Actin (sc-47778 HRP, Santa Cruz), Appropriate horseradish peroxidase (HRP)–conjugated secondary antibodies (anti-Armenian hamster [127-035-099], anti-mouse [315-035-047], and anti-rabbit [111-035-047], Jackson ImmunoResearch Laboratories) were used as described previously (56). Immunoblot images were acquired on an Amersham Imager using Immobilon^® Forte Western HRP Substrate (WBLUF0500, Millipore).

Cytokines and chemokines were measured by multiplex ELISA (Millipore), as per the manufacturer's instructions.

GraphPad Prism 9.0 software was used for data analysis. Data are presented as mean ± SEM. Statistical significance was determined by t tests (two-tailed) for two groups.