IL-38 is an IL-1 family receptor antagonist that restricts IL-17–driven inflammation by limiting cytokine production from macrophages and T cells. In the current study, we aimed to explore its role in experimental autoimmune encephalomyelitis in mice, which is, among others, driven by IL-17. Unexpectedly, IL-38–deficient mice showed strongly reduced clinical scores and histological markers of experimental autoimmune encephalomyelitis. This was accompanied by reduced inflammatory cell infiltrates, including macrophages and T cells, as well as reduced expression of inflammatory markers in the spinal cord. IL-38 was highly expressed by infiltrating macrophages in the spinal cord, and in vitro activated IL-38–deficient bone marrow–derived macrophages showed reduced expression of inflammatory markers, accompanied by altered cellular metabolism. These data suggest an alternative cell-intrinsic role of IL-38 to promote inflammation in the CNS.

Multiple sclerosis (MS) is a demyelinating disease of the CNS caused by an autoimmune response to self-antigens present in the myelin sheath of axons. MS is characterized by progressive neuronal degeneration and the invasion of inflammatory cells from the circulation across the blood–brain barrier (BBB) into the CNS. Depending on the affected CNS areas, clinical symptoms vary from changes in vision to paralysis (1).

A specific cause for this pathology is still unknown, MS being a multifactorial genetically as well as environmentally influenced disease. However, inflammation is a key driver of tissue damage in MS, which requires peripheral immune cell infiltration into the CNS (2). The CNS is an immune-privileged site, where entry of external immune cells is tightly controlled by the BBB (24). When the BBB is disturbed, immune cells enter the CNS and cause neuronal damage. Both innate and adaptive immune cells are involved in the initiation and progression of MS. APCs, such as dendritic cells and macrophages, trigger activation and proliferation of T cells and, consequently, B cells (5). Th cells were early identified as drivers of the disease, depending on their polarization. Whereas Th1 and Th17 cells promote disease progression, Th2 responses promote recovery (6). Additionally, regulatory T cells are impaired during MS. With lower levels of regulatory T cells, immunosuppression of pathological cells fails, consequently leading to disease aggravation (7). Cytotoxic T cells participate in disease progression by killing oligodendrocytes and other glial cells and by promoting vascular cell death (8), enhancing BBB disruption. B cells produce Abs against autoantigens, such as the myelin oligodendrocyte glycoprotein (MOG). Ab binding to autoantigens drives site-directed activation of local (microglia) and recruited macrophages to aggravate disease (9). Moreover, B cells also contribute to pathological T cell activation by acting as APC and producing inflammatory cytokines, thus promoting IL-17 production (10, 11). Local macrophage activation further enhances recruitment of inflammatory cells, such as neutrophils and T cells. In addition, activated macrophages create a proinflammatory environment by releasing mediators such as TNF-α, IL-6, reactive oxygen species, and others (12), which directly promote axonal damage.

Among important inflammatory mediators involved in MS is IL-17, which is produced by Th17 cells and γδ T cells. IL-17 is induced by myeloid cytokines, including members of the IL-1 cytokine family (13). Among these, IL-1β, IL-18, and IL-36 promote IL-17 production by T cells (1315). In contrast, IL-1 family antagonists, including IL-1Ra and IL36Ra, have the capacity to block IL-17 production by T cells (15). IL-38, a new member of the IL-1 family, is described as an antagonist for more than one IL-1 family receptor, including IL-1 receptor, IL-36R, and IL-1R accessory protein-like 1 (IL-1RAPL1) (1517). IL-38 is constitutively expressed in the skin, predominantly in the epidermis, as well as in lymphatic tissue, particularly in B cells and macrophages (18). IL-38 polymorphism is associated with increased susceptibility for autoinflammatory and autoimmune diseases, such as psoriasis, spondyloarthritis, rheumatoid arthritis (19), psoriatic arthritis, and systemic lupus erythematosus (20). Accordingly, by serving as an IL-1 family receptor antagonist, IL-38 has been shown to limit IL-17 production in multiple disease models (17, 21, 22). Indeed, IL-38 has been shown to regulate activation of IL-17–producing γδ T cells by antagonizing IL-1RAPL1 in imiquimod-induced psoriasis (17). Furthermore, IL-38 reduced inflammation in several arthritis models at least partly by acting on macrophages (22). The ability of IL-38 to limit IL-17–driven diseases and high expression of one of its receptors, IL-1RAPL1, in the CNS (23) suggested a disease-restricting role for this cytokine during MS. In the current study, we thus aimed to elucidate the role of IL-38 in the experimental autoimmune encephalomyelitis (EAE) model of MS (24).

The IL-38 knockout (KO) mouse strain, B6:129S5-Il1f10tm1Lex/Mmucd, identification number 032391-UCD, was obtained from the Mutant Mouse Regional Resource Centre, a National Institutes of Health–funded strain repository, and was donated to the Mutant Mouse Resource & Research Centers by Genentech. They were backcrossed into the C57/BL6 background for at least 10 generations. Littermates were used to generate colonies, and mice in these colonies were used maximally until the F3 generation. After their arrival at the experimental facility at the age of 10 wk, the animals were chipped with s.c. transponders to facilitate identification. Furthermore, additional genotyping via PCR analysis was carried out to ensure the adequate genotype of each animal. The genotype was verified using the protocol provided by the supplier of the mice. Their allocation to the home cages happened in a randomized order, but genotypes were kept separately. All animals were housed in groups of two mice per cage (Green Line; Tecniplast, Hohenpeissenberg, Germany) with free access to food and tap water. In the colony room, constant temperature (20–24°C) and humidity (45–65%) conditions as well as a 12/12 h dark/light cycle (on 07.00/off 19.00) were provided. The pathogen-free status of the maintenance room was regularly monitored using sentinel mice. All experimental procedures were carried out in accordance with the Principles of Laboratory Animal Care (National Institutes of Health publication no. 86-23, revised 1985), the Directive 2010/63/EU, and the regulations of Gesellschaft für Versuchstierkunde/Society of Laboratory Animal Science and were approved by the local Ethics Committee for Animal Research (FU/1201; Darmstadt, Germany).

EAE was induced in wild-type (WT) (n = 17) and IL-38 KO (n = 17) female mice, in two independent cohorts of at least five animals of each genotype. Mice were acclimated for at least 7 d prior to immunization. The s.c. immunization with MOG35–55 peptide emulsified with CFA, followed by i.p. administration of Pertussis Toxin 2 and 22 h after immunization (Hooke Kit MOG35–55/CFA Emulsion Pertussis Toxin [EK-0115]) was performed. From 1 wk after immunization, mice were examined daily for body weight, and clinical scores were assigned as follows: 0, no paralysis; 0.5, distal end of the tail is paralyzed; 1, full tail paralysis; 1.5, mild weakness of one of the hind limbs; 2, clear weakness of one of the hind limbs; 2.5, mild incomplete paralysis of both hind limbs; 3, full paralysis of both hind limbs; 3.5, additional incomplete paralysis of one forelimb; 4, hind limb and forelimb paralysis.

Single-cell suspensions were generated from spinal cords using collagenase D (2 mg/ml) and DNase I (40 U/ml) after 30-min digestion at 37°C and dissociation using the gentleMACS Dissociator (Miltenyi). Blood cells were pelleted by centrifugation, followed by RBC lysis and fixation with BD CellFIX (BD). Single-cell suspensions from spinal cord and blood were blocked with FcR blocking reagent (Miltenyi Biotec) in 0.5% PBS-BSA; stained with the fluorochrome-conjugated Abs anti-CD3-PE-CF594 (145-2C11; BD), anti-CD4-BV711 (GK15; BD), anti-CD11b-BV605 (M170; BioLegend), anti-CD19-allophycocyanin-Fire750 (6D5; BioLegend), anti-CD38-BV510 (90; BD), anti-CD45-Vioblue (30F11; Miltenyi), anti-CD138-PE (281-2; BD), anti-MerTK-PE-Cy7 (DS5MMER; eBiosciences), anti-F4/80-PE-Cy7 (BM8; BioLegend), anti-HLA-DR-allophycocyanin (M5/114.15.2; Miltenyi), anti-Ly6C-PerCP-Cy5.5 (HK1.4; BioLegend), and anti-Ly6G-allophycocyanin-Cy7 (1A8; BioLegend); and analyzed on an LSR II/Fortessa flow cytometer. Data were analyzed using FlowJo V10 (TreeStar). All Abs and secondary reagents were titrated to determine optimal concentrations. Comp-Beads (BD) were used for single-color compensation to create multicolor compensation matrices. For gating, fluorescence minus one controls were used. The instrument calibration was controlled daily using Cytometer Setup and Tracking Beads (BD).

Four-micrometer-thick spinal cord sections were deparaffinized and rehydrated. The Opal 7-Color Fluorescent IHC Kit (Perkin-Elmer/Akoya) was used according to the manufacturer’s instructions. Slides were stained with primary Abs targeting F4/80 (1:200, A3-1; BioRad), Iba1 (1:500, polyclonal; Fujifilm), NeuN (1:500, polyclonal; Thermo Fisher Scientific), MBP (1:500, MBP101; Abcam), CD206 (1:50, polyclonal; R&D systems), and TNF-α (1:200, polyclonal; Abcam). The Vectra Polaris automated quantitative pathology imaging system (Perkin-Elmer/Akoya) was used for image acquisition at ×20 and ×40, and images were analyzed using inForm 2.0 Software (Perkin-Elmer/Akoya). In situ hybridization by the RNAscope technique was performed according to the manufacturer’s instructions (ACDbio), as previously described (17). Demyelination was assessed using Luxol Fast Blue (LFB) staining (Polysciences). Sections were incubated overnight in LFB at 56°C, and the signal was obtained after development in three successive baths of lithium carbonate (ScyTek), 70% ethanol, and distilled water.

To assess cytokine levels in spinal cord, blood, and cell culture supernatant, murine IL-1β, IL-6, IL-17A, TNF-α, and IFN-γ Cytometric Bead Array Flex Sets (BD) were used (17). Samples were acquired with an LSR Fortessa II flow cytometer (BD), and data were analyzed using BD FCAP software (V3.0).

Levels of TGF-β1 in cell culture supernatants were measured using an optimized ELISA kit (Thermo Fisher Scientific), according to the manufacturer’s protocol. Levels of anti-MOG autoantibodies in plasma were measured using an optimized ELISA kit (Sensolyte; Anaspec) according to the manufacturer’s protocol.

Total RNA from spinal cord, human PBMCs, and murine bone marrow–derived macrophages (BMDM) was extracted using pegGOLD RNAPure (Peqlab Biotechnologie), and 1 μg of mRNA was used for reverse transcription with the Maxima First Strand cDNA Synthesis kit (Thermo Fisher Scientific). Quantitative real-time PCR reactions were performed with the SYBR Select Master Mix, and the QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific). Relative mRNA expression was analyzed based on the ΔΔcycle threshold method and normalized to Rps27a as housekeeping gene. All primers were purchased from Biomers (Ulm, Germany) (Table I). Quantitect primer assays for mPdk4 were purchased from Qiagen.

Table I.
Primer sequences
GeneSpeciesForward 5′→3′Reverse 5′→3′
Il38 Mouse 5′-AGAGTGAACCCTCCACCCAT-3′ 5′-AAGATCTCAGACTGGGGGCA-3′ 
Rps27a Mouse 5′-GACCCTTACGGGGAAAACCAT-3′ 5′-AGACAAAGTCCGGCCATCTTC-3′ 
Tgfb1 Mouse 5′-GGACTCTCCACCTGCAAGAC-3′ 5′-GACTGGCGAGCCTTAGTTTG-3′ 
Mertk Mouse 5′-TGCGTTTAATCACACCATTGGA-3′ 5′-TGCCCCGAGCAATTCCTTTC-3′ 
Tnfa Mouse 5′-CTGAACTTCGGGGTGATCGG-3′ 5′-GGCTTGTCACTCGAATTTTGAGA-3′ 
Ptgs2 Mouse 5′-CGTGGAGTCCGCTTTACAGA-3′ 5′-CCTTCGTGAGCAGAGTCCTG-3′ 
Tgfb2 Mouse 5′-TCGACATGGATCAGTTTATGCG-3′ 5′-CCCTGGTACTGTTGTAGATGGA-3′ 
Mrc1 Mouse 5′-GGAGTGATGGAACCCCAGTG-3′ 5′-CTGTCCGCCCAGTATCCATC-3′ 
GeneSpeciesForward 5′→3′Reverse 5′→3′
Il38 Mouse 5′-AGAGTGAACCCTCCACCCAT-3′ 5′-AAGATCTCAGACTGGGGGCA-3′ 
Rps27a Mouse 5′-GACCCTTACGGGGAAAACCAT-3′ 5′-AGACAAAGTCCGGCCATCTTC-3′ 
Tgfb1 Mouse 5′-GGACTCTCCACCTGCAAGAC-3′ 5′-GACTGGCGAGCCTTAGTTTG-3′ 
Mertk Mouse 5′-TGCGTTTAATCACACCATTGGA-3′ 5′-TGCCCCGAGCAATTCCTTTC-3′ 
Tnfa Mouse 5′-CTGAACTTCGGGGTGATCGG-3′ 5′-GGCTTGTCACTCGAATTTTGAGA-3′ 
Ptgs2 Mouse 5′-CGTGGAGTCCGCTTTACAGA-3′ 5′-CCTTCGTGAGCAGAGTCCTG-3′ 
Tgfb2 Mouse 5′-TCGACATGGATCAGTTTATGCG-3′ 5′-CCCTGGTACTGTTGTAGATGGA-3′ 
Mrc1 Mouse 5′-GGAGTGATGGAACCCCAGTG-3′ 5′-CTGTCCGCCCAGTATCCATC-3′ 

BMDM were generated from WT and IL-38 KO bone marrow by differentiation with 20 ng/ml of both M-CSF and GM-CSF for 7 d. Afterwards, BMDM were stimulated with 1 μg/ml LPS and 10 ng/ml murine IFN-γ for 3 or 24 h.

BMDM were seeded on Seahorse XF96 cell culture microplates (Agilent) and differentiated for 7 d. On day of measurement, cells were treated with LPS/IFN-γ for 3 h, and medium was replaced by Seahorse XF DMEM (Agilent), supplemented with 2 mM glutamine and 1 mM pyruvate. Glycolysis stress test was performed on a Seahorse XF96 extracellular flux analyzer (Agilent) by injecting 10 mM glucose to induce glycolysis, 2.5 μM oligomycin to inhibit ATP-dependent respiration, and 50 mM 2-deoxy-d-glucose to inhibit glycolysis. Glycolytic rates are expressed as the difference between nonglycolytic acidification and glucose-mediated acidification. ATP-dependent respiration was calculated by subtracting the oxygen consumption rate after oligomycin treatment from the rate measured with glucose.

The in vivo EAE score and bodyweight data were statistically analyzed using IBM SPSS Statistics 26 (Ehningen, Germany). Shapiro–Wilk tests were used to test for Gaussian distribution. Nonparametric Mann–Whitney U tests were used to analyze in vivo data from WT versus IL-38 KO mice. EAE area under the curve (AUC) and peak scores are expressed as median ± interquartile range. The ex vivo data are presented as means ± SEM. Statistical analyses were performed with GraphPad Prism version 8. Means between two groups were compared with two-tailed, unpaired Student t test; comparisons of means from multiple groups were done with one-way ANOVA and Dunnett or Tukey post hoc test. A p value < 0.05 was considered statistically significant.

To test the involvement of IL-38 in MS, EAE was induced in WT and IL-38 KO mice, and clinical scores indicating disease development were monitored. Whereas WT mice developed clear EAE symptoms, disease development in IL-38 KO mice was markedly decreased (Fig. 1A). In addition to lower clinical scores at the peak of disease (Fig. 1B) and smaller EAE AUC values (Fig. 1C), IL-38 KO mice showed a delay in disease development as well as enhanced recovery after the disease peak, indicated by increasing body weight (Fig. 1D). To validate attenuated EAE development in IL-38 KO mice, we investigated spinal cord structure by multiplex histology (PhenOptics) at the experimental end point (day 20 after immunization) (Fig. 2A). Reduced EAE severity in IL-38 KO mice was confirmed by higher numbers of intact neurons (Fig. 2A, 2B) and lower numbers of Iba1+ phagocytes (Fig. 2A, 2C) in PhenOptics analyses compared with WT mice. Moreover, spinal cord sections from WT mice showed lower myelin content (Fig. 2D, 2E) accompanied by increased demyelination areas, which was less pronounced in IL-38 KO mice (Fig. 2D, 2F). Higher numbers of intact neurons accompanied by lower myelin sheath degradation thus correlated with impaired EAE development in IL-38 KO mice.

FIGURE 1.

Attenuated EAE in IL-38 KO mice. EAE was induced in WT (n = 17) and IL-38 KO (n = 17) mice, and animals were monitored for 20 d. (A) Clinical disease scores and (D) body weight were evaluated daily after 7 d. (B) The peak of the clinical disease score and (C) EAE AUC value were determined. EAE was performed in two independent cohorts of at least five animals of each genotype. *p < 0.05, **p < 0.01, ***p < 0.001. The p values were calculated using a Mann–Whitney U test (A–D).

FIGURE 1.

Attenuated EAE in IL-38 KO mice. EAE was induced in WT (n = 17) and IL-38 KO (n = 17) mice, and animals were monitored for 20 d. (A) Clinical disease scores and (D) body weight were evaluated daily after 7 d. (B) The peak of the clinical disease score and (C) EAE AUC value were determined. EAE was performed in two independent cohorts of at least five animals of each genotype. *p < 0.05, **p < 0.01, ***p < 0.001. The p values were calculated using a Mann–Whitney U test (A–D).

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FIGURE 2.

Improved spinal cord structure in IL-38 KO mice after EAE. EAE was induced in WT and IL-38 KO mice, and animals were monitored for 20 d. (A and D) Spinal cord sections from 20-d EAE-treated mice were stained for neurons (NeuN; yellow), microglia (Iba1; green), myelin (MBP; red) and nuclei (DAPI; white). Scale bars, 100 μm. (B and C) Quantification of histology sections from 12 WT and seven IL-38 KO mice. (D) Spinal cord sections were stained for MBP. Blue arrows indicate demyelination areas. (E) The mean fluorescence intensity (MFI)/area size of MBP in the white matter is shown. (F) Spinal cord sections were stained using LFB, and red arrows show demyelination. Scale bars, 100 μm. Each data point corresponds to one animal. *p < 0.05, ***p < 0.001. The p values were calculated using a Mann–Whitney U test (B, C, and E).

FIGURE 2.

Improved spinal cord structure in IL-38 KO mice after EAE. EAE was induced in WT and IL-38 KO mice, and animals were monitored for 20 d. (A and D) Spinal cord sections from 20-d EAE-treated mice were stained for neurons (NeuN; yellow), microglia (Iba1; green), myelin (MBP; red) and nuclei (DAPI; white). Scale bars, 100 μm. (B and C) Quantification of histology sections from 12 WT and seven IL-38 KO mice. (D) Spinal cord sections were stained for MBP. Blue arrows indicate demyelination areas. (E) The mean fluorescence intensity (MFI)/area size of MBP in the white matter is shown. (F) Spinal cord sections were stained using LFB, and red arrows show demyelination. Scale bars, 100 μm. Each data point corresponds to one animal. *p < 0.05, ***p < 0.001. The p values were calculated using a Mann–Whitney U test (B, C, and E).

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At steady state, IL-38 is only expressed in the epidermis and at low levels in B cells, which are responsible for autoantibody production and promote the Th17 response in EAE in an Ab-independent manner (9, 10). Flow cytometric analysis revealed lower levels of peripheral B cells and plasma cells but higher levels of B cells within the spleen in IL-38 KO compared with WT mice (Fig. 3A, 3B, Supplemental Fig. 1), indicating an impact of IL-38 on B cell homeostasis. However, histological analysis showed no difference in B cell levels in the spinal cord of WT and IL-38 KO mice (Fig. 3D, 3E). Moreover, assessment of anti-MOG35-55 IgG levels by ELISA rather suggested a tendency for higher production of anti-MOG35-55 autoantibodies in IL-38 KO compared with WT mice (Fig. 3C). Furthermore, there was no difference in the expression of IL-35 subunits, Ebi3 and Il12p35, which are important determinants of the Ab-independent B cell response in EAE (25) (Supplemental Fig. 3). Because these findings did not align with reduced disease symptoms in IL-38 KO mice, we concluded that B cell responses may not be major determinants of reduced EAE symptoms in IL-38 KO mice.

FIGURE 3.

Altered B cell homeostasis and autoantibody production in IL-38 KO mice. EAE was induced in WT and IL-38 KO mice for 20 d, and samples were acquired at the end point. (A) Percentage of peripheral CD19+ B cells and CD138+ plasma cells in blood and (B) percentage of CD19+ B cells in spleens of WT and IL-38 KO mice at day 20. (C) Quantification of total plasmatic IgG anti-MOG Abs measured by ELISA. (D) Quantification of total B cell numbers in the spinal cord of WT and IL-38 KO mice at day 20 by immunohistochemistry and (E) representative picture of a B220 staining (arrows indicate B220+ B cells). Each data point corresponds to one animal. *p < 0.05, **p < 0.01, ***p < 0.001. The p values were calculated using a Mann–Whitney U test (A–D).

FIGURE 3.

Altered B cell homeostasis and autoantibody production in IL-38 KO mice. EAE was induced in WT and IL-38 KO mice for 20 d, and samples were acquired at the end point. (A) Percentage of peripheral CD19+ B cells and CD138+ plasma cells in blood and (B) percentage of CD19+ B cells in spleens of WT and IL-38 KO mice at day 20. (C) Quantification of total plasmatic IgG anti-MOG Abs measured by ELISA. (D) Quantification of total B cell numbers in the spinal cord of WT and IL-38 KO mice at day 20 by immunohistochemistry and (E) representative picture of a B220 staining (arrows indicate B220+ B cells). Each data point corresponds to one animal. *p < 0.05, **p < 0.01, ***p < 0.001. The p values were calculated using a Mann–Whitney U test (A–D).

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Next, we investigated the local immune cell profile in the spinal cord to understand if IL-38 affected CNS inflammation. Analysis of single-cell suspensions from spinal cord by flow cytometry revealed lower relative abundance of monocytes, neutrophils, CD4+ T cells, and macrophages in IL-38 KO compared with WT spinal cords (Fig. 4A, 4B, Supplemental Fig. 2), the latter being consistent with the reduced numbers of Iba1+ phagocytes observed by histology (Fig. 2C). Lower infiltration of phagocytes in IL-38 KO spinal cords was again confirmed by immunofluorescence analysis of F4/80 expression (Fig. 4C, 4D). These data indicate attenuated immune cell infiltration into IL-38 KO spinal cords during EAE. Moreover, there was a strong tendency for CD4+ T cells and a weak tendency for monocytes to accumulate in the blood of IL-38 KO mice, corresponding to changes observed in the immune cell infiltrate in the spinal cord (Fig. 4E, 4F). Next, we investigated the inflammatory status at a molecular level in spinal cords and in the periphery in both genotypes. Analyzing cytokine levels with Cytometric Bead Arrays revealed higher IL-17A levels, but unaltered levels of other cytokines, in the blood of IL-38 KO mice (Fig. 5B), which supports data on IL-38 function from previous mouse models of inflammation, in which IL-38 restricted peripheral IL-17 production (1517). Nevertheless, lower TNF-α levels were noticed in the spinal cord of IL-38 KO mice (Fig. 5A). Lower expression of Tnfa in spinal cords of IL-38 KO mice was confirmed at the mRNA level, which was also observed for other markers of inflammation, including the macrophage marker Mertk as well as Ptgs2, Tgfb1, and Tgfb2 (Fig. 5C). Moreover, IL-38 KO phagocytes present in the spinal cord showed an overall reduced activation, demonstrated by lower CD206 and TNF-α expression (Fig. 5D, 5E). Thus, local but not peripheral inflammation was reduced in IL-38 KO mice subjected to EAE.

FIGURE 4.

IL-38 KO mice show attenuated immune cell infiltration. EAE was induced in WT and IL-38 KO mice for 20 d, and spinal cord (AD) as well as blood (E and F) samples were acquired at the end point. (A) Spinal cord–infiltrating immune cell subsets within total immune cells were determined by flow cytometry in WT and IL-38 KO mice. (B) Representative FACS plot of macrophages in the spinal cord. (C) Representative staining and (D) quantification of F4/80+ macrophage infiltration in the spinal cord. Scale bar, 50 μm. (E) Immune cell subsets within total blood immune cells and (F) CD4+ T cells within total blood T cells were determined by flow cytometry in WT and IL-38 KO mice. *p < 0.05, **p < 0.01. The p values were calculated using a Mann–Whitney U test (A and D) or two-way ANOVA (E).

FIGURE 4.

IL-38 KO mice show attenuated immune cell infiltration. EAE was induced in WT and IL-38 KO mice for 20 d, and spinal cord (AD) as well as blood (E and F) samples were acquired at the end point. (A) Spinal cord–infiltrating immune cell subsets within total immune cells were determined by flow cytometry in WT and IL-38 KO mice. (B) Representative FACS plot of macrophages in the spinal cord. (C) Representative staining and (D) quantification of F4/80+ macrophage infiltration in the spinal cord. Scale bar, 50 μm. (E) Immune cell subsets within total blood immune cells and (F) CD4+ T cells within total blood T cells were determined by flow cytometry in WT and IL-38 KO mice. *p < 0.05, **p < 0.01. The p values were calculated using a Mann–Whitney U test (A and D) or two-way ANOVA (E).

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FIGURE 5.

Absence of IL-38 attenuates local inflammation. EAE was induced in WT and IL-38 KO mice for 20 d, and samples were acquired at the end point. (A) Inflammatory cytokines from spinal cord and (B) blood were quantified via Cytometric Beads Array. (C) Mertk, Tnfa, Ptgs2, Tgfb1, and Tgfb2 mRNA expression in the spinal cord was quantified using qPCR, normalized to Rps27a. (D and E) Spinal cord sections from 20-d EAE-treated mice were stained for TNF-α, CD206, and Iba1 expression. (D) The quantification of TNF-α+ and CD206+ phagocytes in the spinal cord and (E) representative PhenOptics images are shown. Scale bar, 50 μm. Each data point corresponds to one animal. *p < 0.05, **p < 0.01. The p values were calculated using a Mann–Whitney U test (A–C).

FIGURE 5.

Absence of IL-38 attenuates local inflammation. EAE was induced in WT and IL-38 KO mice for 20 d, and samples were acquired at the end point. (A) Inflammatory cytokines from spinal cord and (B) blood were quantified via Cytometric Beads Array. (C) Mertk, Tnfa, Ptgs2, Tgfb1, and Tgfb2 mRNA expression in the spinal cord was quantified using qPCR, normalized to Rps27a. (D and E) Spinal cord sections from 20-d EAE-treated mice were stained for TNF-α, CD206, and Iba1 expression. (D) The quantification of TNF-α+ and CD206+ phagocytes in the spinal cord and (E) representative PhenOptics images are shown. Scale bar, 50 μm. Each data point corresponds to one animal. *p < 0.05, **p < 0.01. The p values were calculated using a Mann–Whitney U test (A–C).

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The data so far suggested reduced local inflammation as a reason for reduced EAE symptoms in IL-38 KO mice. We observed a prominent reduction of local and recruited macrophage levels and reduced production of inflammatory mediators, which are often attributed to these cells (26, 27). To investigate the hypothesis that reduced EAE symptoms in IL-38 KO mice are linked to decreased macrophage activation, we first investigated if and where IL-38 is expressed in the spinal cord in EAE. Analysis of IL-38 transcript abundance using quantitative PCR (qPCR) and RNAscope in situ hybridization showed that IL-38 is indeed expressed in the spinal cord at the experimental end point (Fig. 6A, 6B). Interestingly, 46% of the IL-38–expressing cells coexpressed the macrophage marker Mertk, whereas 23% coexpressed Iba1, suggesting local and recruited macrophages as major sources of IL-38 in the CNS during EAE (Fig. 6C, 6D).

FIGURE 6.

IL-38 is mainly expressed by macrophages within the spinal cord. (AD) EAE was induced in WT and IL-38 KO mice for 20 d, and samples were acquired at the end point. (A) qPCR quantification of Il38 mRNA expression in spinal cords of WT and IL-38 KO mice. (B–D) In situ hybridization by RNAscope indicates the expression of Il38 (red), Mertk (yellow), and Iba1 (yellow) in spinal cords at day 20 of EAE. Nuclei were counterstained with DAPI (white). (B and C) Representative images from three independent experiments are shown. Scale bars, 100 μm. Red arrows show single Il38-expressing cells, and yellow arrows indicate coexpression. (D) Quantification of in situ hybridization data. Each data point corresponds to one animal. *p < 0.05. The p values were calculated using a Mann–Whitney U test (A and D).

FIGURE 6.

IL-38 is mainly expressed by macrophages within the spinal cord. (AD) EAE was induced in WT and IL-38 KO mice for 20 d, and samples were acquired at the end point. (A) qPCR quantification of Il38 mRNA expression in spinal cords of WT and IL-38 KO mice. (B–D) In situ hybridization by RNAscope indicates the expression of Il38 (red), Mertk (yellow), and Iba1 (yellow) in spinal cords at day 20 of EAE. Nuclei were counterstained with DAPI (white). (B and C) Representative images from three independent experiments are shown. Scale bars, 100 μm. Red arrows show single Il38-expressing cells, and yellow arrows indicate coexpression. (D) Quantification of in situ hybridization data. Each data point corresponds to one animal. *p < 0.05. The p values were calculated using a Mann–Whitney U test (A and D).

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Based on expression data, ablation of IL-38 in macrophages may have contributed to modify EAE development. To test this, BMDM from WT and IL-38 KO mice were activated with LPS and IFN-γ for 24 h, and inflammatory gene expression was investigated. LPS/IFN-γ upregulated IL-38 expression in WT BMDM (Fig. 7A), which was required for full induction of inflammatory mediators such as Tnfa, Tgfb1, Mrc1, and pyruvate dehydrogenase kinase 4 (Pdk4) (Fig. 7B). However, other inflammatory mediators were not affected by the absence of IL-38 (Supplemental Fig. 4A). Moreover, lower production of TNF-α by IL-38 KO BMDM compared with WT at protein level was observed, in accordance with the mRNA data (Fig. 7C). In contrast, no differences were observed for TGF-β1 protein levels (Fig. 7C), likely because of high baseline production of TGF-β1 by both WT and IL-38 KO BMDM. Nevertheless, gene expression changes in BMDM were consistent with gene expression changes in spinal cords of IL-38 KO mice, which showed reduced Tnfa and Tgfb1 levels as well (Fig. 5C). Furthermore, human macrophages also upregulated IL-38 expression upon LPS/IFN-γ stimulation, in which we observed a diverse pattern of regulation either at early or later time points following LPS/IFN-γ stimulation (Supplemental Fig. 4B). To analyze if stimulation with LPS/IFN-γ altered the production of extracellular IL-38, we measured IL-38 in the supernatant of LPS/IFN-γ–stimulated human macrophages. As expected, IL-38 was not found in the supernatant (data not shown), suggesting that intracellular IL-38 may promote LPS/IFN-γ–stimulated expression of inflammatory mediators in macrophages. To approach a mechanism, we analyzed the metabolic capacity of LPS/IFN-γ–stimulated WT and IL-38 KO macrophages based on a reduced transcriptional upregulation of Pdk4 in IL-38 KO BMDM. Whereas WT BMDM, as expected, increased glycolysis and decreased ATP-dependent respiration upon LPS/IFN-γ stimulation, macrophages lacking IL-38 showed no metabolic change after LPS/IFN-γ stimulation (Fig. 7D). These data support an impact of cell-intrinsic production of IL-38 in inflammatory macrophages in reduced CNS inflammation in IL-38 KO mice.

FIGURE 7.

IL-38 is required for inflammatory macrophage activation. (AD) BMDM from WT and IL-38 KO mice were stimulated with LPS and IFN-γ for 24 h (A–C) or 3 h (D). (A and B) qPCR quantification of Il38 (A), Tnfa, Tgfb1, Mrc1, and Pdk4 (B) mRNA expression in BMDM, relative to Rps27a. (C) Protein levels of TNF-α quantified by cytometric bead array and of TGF-β1 quantified by ELISA are shown. (D) Glycolysis and ATP-dependent respiration were measured in WT and IL-38 KO BMDM after stimulation with LPS and IFN-γ for 3 h using Seahorse. Each data point corresponds to one animal. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The p values were calculated using a one-way ANOVA with Tukey correction (A–D).

FIGURE 7.

IL-38 is required for inflammatory macrophage activation. (AD) BMDM from WT and IL-38 KO mice were stimulated with LPS and IFN-γ for 24 h (A–C) or 3 h (D). (A and B) qPCR quantification of Il38 (A), Tnfa, Tgfb1, Mrc1, and Pdk4 (B) mRNA expression in BMDM, relative to Rps27a. (C) Protein levels of TNF-α quantified by cytometric bead array and of TGF-β1 quantified by ELISA are shown. (D) Glycolysis and ATP-dependent respiration were measured in WT and IL-38 KO BMDM after stimulation with LPS and IFN-γ for 3 h using Seahorse. Each data point corresponds to one animal. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The p values were calculated using a one-way ANOVA with Tukey correction (A–D).

Close modal

IL-38, an antagonist cytokine of the IL-1 family, has been described as an anti-inflammatory and proresolving mediator in IL-17–driven inflammatory diseases. Although similar effects of IL-38 were anticipated during EAE, which is highly IL-17–driven, ablation of IL-38 led to reduced disease development. This was contrary to expectations because anti-inflammatory properties of IL-38, particularly suppression of IL-17–dependent inflammation, are the current consensus in the literature (15, 17). Specifically, IL-38 was shown to control IL-17–driven inflammation in models of psoriasis, rheumatoid arthritis, psoriatic arthritis, and liver injury (17, 19, 21, 22). Even though ablation of IL-38 seemed to affect the production of autoantibodies, as well as B cell homeostasis in the periphery, higher levels of anti-MOG IgG in the IL-38 KO mice could not be the reason for the attenuated phenotype observed in those mice. Nevertheless, a potential impact of IL-38 on autoantibody production is of interest and needs to be further evaluated in other relevant models. Moreover, absence of IL-38 seemed to retain B cells within the secondary lymphoid organs and may suggest a role of IL-38 in the initiation of the humoral response, although this was not of relevance in this model. These findings are supported by recent data reporting that plasmatic levels of IL-38 positively correlate with the number of several B cell subsets in the circulation (28). Furthermore, B cells were described to play an Ab-independent role in the development of EAE by secreting cytokines such as IL-10 or IL-35 (25). This study shows that ablation of IL-38 was not affecting this response in the spinal cord, suggesting that IL-38 may act on B cells in the periphery rather than in the spinal cord.

It would be important to investigate if IL-38 is present extracellularly in the CNS at steady state and during disease, both in mice and humans, because extracellular IL-38 was previously described by ourselves and by others to downregulate the production of inflammatory cytokines when added to human and mouse macrophages, by acting as an IL-1 family receptor antagonist. Indeed, it was shown that IL-38 can bind to IL-1RAPL1 or IL-36R when added exogenously, thereby inhibiting proinflammatory activation of macrophages or γδ T cells (16, 17, 22). Consequently, exogenously added rIL-38 prevented microglial activation and inflammatory mediator production in vitro (29). Inhibition of inflammation by exogenous IL-38 could, thus, contribute to containing MS development. The data presented in this article suggest that, on the contrary, IL-38 has the capacity to promote production of inflammatory mediators, such as TNF-α or TGF-β1, in a cell-intrinsic manner in vitro, correlating with the inflammatory profile of the spinal cord in vivo. Thus, IL-38 may have intracellular effects in macrophages that are opposed to its extracellular function as a receptor antagonist. Indeed, although IL-38 was shown to decrease inflammatory markers when added exogenously in a number of models, we show in this study that BMDM and human macrophages upregulate IL-38 expression in response to a proinflammatory stimulus, and IL-38 was expressed mainly by macrophages and microglia in the spinal cord. Moreover, we did not detect IL-38 in the supernatant of human macrophages following LPS/IFN-γ stimulation, suggesting that IL-38 is not secreted under these conditions. This supports recent data, in which no changes in plasma IL-38 levels were detected in patients receiving a bolus injection of LPS, whereas other cytokines such as TNF-α, IL-6, or IL-1Ra were increased (30). Intracellular containment of IL-38 was shown in human keratinocytes, where it interacted with a couple of intracellular proteins (31). Distinct intra- and extracellular functions were previously reported for other IL-1 family cytokines, such as IL-1α, IL-33, or IL-37 that may act as transcription regulators intracellularly (3234). In the case of IL-1α, it has been shown that its nuclear translocation was implicated in several inflammatory chronic diseases. Unfortunately, the analysis of intra- versus extracellular IL-38 levels in our mouse models is hampered by the unavailability of a specific ELISA/anti–IL-38 Ab because commercially available mouse IL-38 ELISAs produced a high nonspecific signal in IL-38 KO mice.

IL-38 ablation limited TNF-α production, both in the CNS and in primary macrophages. BMDM lacking IL-38 astonishingly showed impaired upregulation of Pdk4. Pdk4 was recently shown to be a key metabolic checkpoint for inflammatory macrophage polarization (35). Accordingly, ablation of IL-38 in BMDM prevented a metabolic switch toward glycolysis upon LPS/IFN-γ stimulation, which likely results in altered proinflammatory activation of stimulated macrophages. During EAE, TNF-α is produced by infiltrating macrophages as well as by proinflammatory microglia, and its blockade allowed to decrease BBB permeability (36). TNF-α is thus part of the inflammatory microenvironment triggered by peripheral immune cells penetrating the CNS through the BBB. This promotes a feed-forward increase of BBB permeability, allowing an increased influx of cells and inflammatory mediators (4, 36). Changes in local inflammation in IL-38 KO mice resulting from decreased macrophage activation, as indicated by reduced numbers of microglia and recruited macrophages, and reduced expression of inflammatory mediators may therefore have stabilized BBB integrity, limiting further infiltration of inflammatory cells. Along this line, anti-inflammatory macrophages protect the CNS from immune cell invasion, whereas inflammatory macrophages disrupt the BBB through cytokine production (5). TGF-β1 triggers Th17 cell generation but also promotes EAE independently of Th17 cells (37). These findings indicate that ablation of IL-38 attenuates local expression of critical disease-promoting factors in EAE, suggesting that IL-38 may play different roles during inflammation in the CNS versus in the periphery.

Overall, our study demonstrates that a global KO of IL-38 protects mice from EAE development. In EAE, IL-38 appears to act differently compared with other autoinflammatory diseases, potentially because of IL-38 expression in macrophages as opposed to IL-38 from external sources acting on macrophages (17, 22). Investigating the mechanisms through which intracellular IL-38 regulates Pdk4 transcription and, consequently, energy metabolism in macrophages and possibly other immune cells appears as an important line of future research.

We thank Margarethe Mijatovic and Praveen Mathoor for excellent technical assistance.

This work was supported by the Deutsche Forschungsgemeinschaft (Grant GRK 2336 TP1 and TP6) and the Landesoffensive zur Entwicklung wissenschaftlich-ökonomischer Exzellenz Center for Translational Medicine and Pharmacology.

The online version of this article contains supplemental material.

Abbreviations used in this article:

AUC

area under the curve

BBB

blood–brain barrier

BMDM

bone marrow–derived macrophage

EAE

experimental autoimmune encephalomyelitis

IL-1RAPL1

IL-1R accessory protein-like 1

KO

knockout

LFB

Luxol Fast Blue

MOG

myelin oligodendrocyte glycoprotein

MS

multiple sclerosis

Pdk4

pyruvate dehydrogenase kinase 4

qPCR

quantitative PCR

WT

wild-type.

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The authors have no financial conflicts of interest.

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