Interleukin-33 Signaling Controls the Development of Iron-Recycling Macrophages.

Splenic red pulp macrophages (RPMs) contribute to erythrocyte homeostasis and are required for iron recycling. Heme induces the expression of SPIC transcription factor in monocyte-derived macrophages and promotes their differentiation into RPM precursors, pre-RPMs. However, the requirements for differentiation into mature RPMs remain unknown. Here, we have demonstrated that interleukin (IL)-33 associated with erythrocytes and co-cooperated with heme to promote the generation of mature RPMs through activation of the MyD88 adaptor protein and ERK1/2 kinases downstream of the IL-33 receptor, IL1RL1. IL-33- and IL1RL1-deficient mice showed defective iron recycling and increased splenic iron deposition. Gene expression and chromatin accessibility studies revealed a role for GATA transcription factors downstream of IL-33 signaling during the development of pre-RPMs that retained full potential to differentiate into RPMs. Thus, IL-33 instructs the development of RPMs as a response to physiological erythrocyte damage with important implications to iron recycling and iron homeostasis.

IL-18 Binding Protein-Producing Cells Attenuate Anemia in Murine Macrophage Activation Syndrome.

IL-18 is a pleiotropic immunoregulatory cytokine of the IL-1 family. IL-18 has been identified as a potent IFN-γ inducer in synergy with IL-12 and IL-15 and thus as a powerful Th1 cell-polarizing cytokine. IL-18 activity is regulated by its naturally occurring soluble inhibitor IL-18 binding protein (IL-18BP), the production of which is stimulated by IFN-γ in a negative feedback loop. Circulating levels of IL-18BP are elevated, and unbound bioactive free IL-18 is thus not detectable in the circulation in physiologic conditions. However, emerging evidence indicates that the IL-18/IL-18BP balance could be dysregulated in macrophage activation syndrome (MAS), as mirrored by the presence of free IL-18 in the circulation of patients with MAS. Herein, we sought to identify IL-18BP-producing cells in a murine CpG-induced MAS model using IL-18BP knock-in tdTomato reporter mice. Endothelial cells, tissue-resident macrophages, and neutrophils appeared as major cellular sources of IL-18BP. We also identified extramedullary and medullary early erythroid progenitors as IL-18BP-producing cells in an IFN-γ-dependent manner. This finding suggests a novel regulation of IL-18 activity by erythroid precursors, which are likely involved in the prevention of the negative effects of IL-18 on erythropoiesis. Indeed, coherent in vivo and in vitro results indicate that IL-18 indirectly impairs erythropoiesis while favoring myelopoiesis and thus contributes to anemia associated with MAS and potentially with other IL-18-driven inflammatory diseases. In conclusion, IL-18BP production by endothelial cells, neutrophils, macrophages, and erythroid precursors attenuates the anemia associated with murine CpG-induced MAS.

IL-38 Ablation Reduces Local Inflammation and Disease Severity in Experimental Autoimmune Encephalomyelitis.

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.

Cell Intrinsic IL-38 Affects B Cell Differentiation and Antibody Production.

IL-38 is an IL-1 family receptor antagonist with an emerging role in chronic inflammatory diseases. IL-38 expression has been mainly observed not only in epithelia, but also in cells of the immune system, including macrophages and B cells. Given the association of both IL-38 and B cells with chronic inflammation, we explored if IL-38 affects B cell biology. IL-38-deficient mice showed higher amounts of plasma cells (PC) in lymphoid organs but, conversely, lower levels of plasmatic antibody titers. Exploring underlying mechanisms in human B cells revealed that exogenously added IL-38 did not significantly affect early B cell activation or differentiation into plasma cells, even though IL-38 suppressed upregulation of CD38. Instead, IL-38 mRNA expression was transiently upregulated during the differentiation of human B cells to plasma cells in vitro, and knocking down IL-38 during early B cell differentiation increased plasma cell generation, while reducing antibody production, thus reproducing the murine phenotype. Although this endogenous role of IL-38 in B cell differentiation and antibody production did not align with an immunosuppressive function, autoantibody production induced in mice by repeated IL-18 injections was enhanced in an IL-38-deficient background. Taken together, our data suggest that cell-intrinsic IL-38 promotes antibody production at baseline but suppresses the production of autoantibodies in an inflammatory context, which may partially explain its protective role during chronic inflammation.

Multifaceted roles of IL-38 in inflammation and cancer.

Interleukin (IL)-38 is the least well-understood cytokine of the IL-1 family. Since its discovery twenty years ago, numerous studies have linked IL-38 to diverse pathologies, especially in the context of autoimmune and inflammatory processes, while its role in cancer has been less explored. Broad anti-inflammatory effects have been reported for IL-38 in both in vitro and in vivo models, and, together with its homology to the IL-1 and IL-36 receptor antagonists, have raised expectations about its potential therapeutic utility. Data in human and mouse experimental systems support a negative regulatory role of IL-38 on the Th17 axis through effects on T cells and myeloid cells. Additional studies point to tolerogenic functions of IL-38, acting on dendritic cells and regulatory T cells, as well as to inhibition of pro-inflammatory macrophage activity. IL-38 further exhibits anti-inflammatory and tissue protective properties in epithelial and mesenchymal cells. However, published data also reveal variability and inconsistent dose-dependencies of these anti-inflammatory effects, as well as context-dependent pro-inflammatory properties of IL-38, and are difficult to interpret due to the high heterogeneity in the materials and experimental designs used across studies. In addition, it is still not clear which receptor(s) is/are fundamental for IL-38 signalling, and the biological impact of N-terminal processing of the protein remains to be clarified. In this review, we provide an overview of our current knowledge of IL-38 biology, discuss persistent controversies surrounding this cytokine, and highlight some questions to be addressed to facilitate progress towards a better understanding of its mechanisms of action.

IL-38 orchestrates proliferation and differentiation in human keratinocytes.

Interleukin (IL)-38 is a member of the IL-1 cytokine family with reported anti-inflammatory activity. The highest constitutive IL-38 expression is detected in the skin, where it is mainly produced by differentiating keratinocytes. However, little data are available regarding its biological functions. In this study, we investigated the role of IL-38 in skin physiology. We demonstrate here that dermal fibroblasts and epithelial cells of skin appendages, such as eccrine sweat glands and sebaceous glands, also express IL-38. Next, using two- and three-dimensional cell cultures, we show that endogenous expression of IL-38 correlates with keratinocyte differentiation and its ectopic overexpression inhibits keratinocyte proliferation and enhances differentiation. Accordingly, immunohistochemical analysis revealed downregulation of IL-38 in skin pathologies characterized by keratinocyte hyperproliferation, such as psoriasis and basal or squamous cell carcinoma. Finally, intracellular IL-38 can shuttle between the nucleus and the cytoplasm and its overexpression modulates the activity of the transcription regulators YAP and ID1. Our results indicate that IL-38 can act independently from immune system activation and suggest that it may affect the epidermis directly by decreasing proliferation and promoting differentiation of keratinocytes. These data suggest an important role of keratinocyte-derived IL-38 in skin homeostasis and pathologies characterized by epidermal alterations.

Production of IL-18 Binding Protein by Radiosensitive and Radioresistant Cells in CpG-Induced Macrophage Activation Syndrome.

IL-18 binding protein (IL-18BP) acts as a naturally occurring IL-18 decoy receptor. If the balance between IL-18 and IL-18BP is dysregulated, abnormal levels of free bioactive IL-18 are detected, such as in the sera of Il-18bp knockout (KO) mice with CpG-induced macrophage activation syndrome. To determine the cellular sources of Il-18bp in vivo, we selectively depleted Il-18bp expression in either radiosensitive or radioresistant cells using bone marrow transfer between wild-type (WT) and Il-18bp KO mice. Following repeated CpG injections, Il-18bp KO (donor)→ Il-18bp KO (recipient) chimeric mice exhibited more severe disease, with an enhanced Ifn-γ signature and circulating free Il-18 levels, in comparison with WT→WT chimeras. Interestingly, the phenotype of KO→WT and WT→KO mice did not differ from that of WT→WT mice. Consistent with this finding, serum Il-18bp levels were similar in these three groups of mice. The contribution of radioresistant and radiosensitive cells to Il-18bp production varied markedly according to the organ examined, with a major contribution of radiosensitive cells in the spleen as opposed to a major contribution of radioresistant cells in the lung. Finally, Ifn-γ blockade abrogated the CpG-induced but not the constitutive Il-18bp production. Our results demonstrate that circulating Il-18bp is induced in response to Ifn-γ during CpG-induced macrophage activation syndrome and is present at high levels in the circulation to prevent the deleterious systemic effects of Il-18.

Intracellular IL-1 Receptor Antagonist Isoform 1 Released from Keratinocytes upon Cell Death Acts as an Inhibitor for the Alarmin IL-1α.

The inflammatory effects of IL-1α/ß are controlled by IL-1R antagonist (IL-1Ra). One IL-1Ra isoform is secreted, whereas three other isoforms (intracellular IL-1Ra [icIL-1Ra] 1, 2, and 3) are supposed to remain intracellular because of the absence of a signal peptide. In contrast to the well-characterized function of the secreted isoform, the biological role of the intracellular isoforms remains largely unclear. icIL-1Ra1 represents the major isoform in keratinocytes. We created icIL-1Ra1-/- mice and investigated the role of icIL-1Ra1 in Aldara (5% imiquimod)-induced psoriasis-like skin inflammation. Naive icIL-1Ra1-/- mice bred habitually and exhibited a normal phenotype. icIL-1Ra1 deficiency aggravated Aldara-induced skin inflammation, as demonstrated by increased ear thickness and increased mRNA levels of key proinflammatory cytokines. No intracellular effect of icIL-1Ra1 could be detected in isolated keratinocytes using RNA-sequencing analysis; however, Aldara treatment led to caspase 1/11-, caspase 8-, and RIPK3-independent keratinocyte cell death accompanied by the release of both icIL-1Ra1 and IL-1α. Furthermore, blocking IL-1α attenuated the clinical severity of Aldara-induced ear thickening in icIL-1Ra1-/- mice. Our data suggest that upon keratinocyte damage icIL-1Ra1 acts extracellularly as an antagonist of the alarmin IL-1α to immediately counteract its inflammatory effects.

Detection of circulating highly expanded T-cell clones in at-risk individuals for rheumatoid arthritis before the clinical onset of the disease.

OBJECTIVES: To quantitatively profile the T-cell repertoire in the peripheral blood of individuals genetically at risk for RA, namely first-degree relatives of RA patients (RA-FDR) at different phases of disease development. METHODS: Next-generation sequencing of the TCR CDR3ß repertoire was performed on genomic DNA isolated from whole blood samples of RA-FDR selected at three different pre-clinical stages and of matched RA patients (n = 20/group). T-cell clones were identified by their unique sequence and their degree of expansion (frequency) within each sample was characterized. Clones with a frequency over 0.5% were considered highly expanded clones (HEC). RESULTS: The absolute number of HEC was significantly higher in established RA patients (mean 4.65) and tended to be higher in symptomatic RA-FDR (mean 3.4) compared with asymptomatic RA-FDR (mean 1.55, P =0.003 and P =0.07, respectively). Asymptomatic individuals with high levels of ACPA did not differ from asymptomatic RA-FDR in terms of absolute number and frequency of clones. The number of HEC tended to be slightly higher at the time of RA onset (P =0.055). Neither clones shared by several patients, nor clones previously associated with RA, were preferentially present within or between the different groups. Finally, a longitudinal analysis did not allow to uncover a kinetic expansion of RA-specific clones closely correlated with disease development. CONCLUSIONS: HEC were detected in the peripheral blood before the clinical onset of RA, in particular in the later pre-clinical phase of RA development, and their presence increased over time.

Unopposed IL-18 signaling leads to severe TLR9-induced macrophage activation syndrome in mice.

The term macrophage activation syndrome (MAS) defines a severe, potentially fatal disorder characterized by overwhelming inflammation and multiorgan involvement. Interleukin-18 (IL-18) is a proinflammatory cytokine belonging to the IL-1 family, the activity of which is regulated by its endogenous inhibitor IL-18 binding protein (IL-18BP). Elevated IL-18 levels have been reported in patients with MAS. Herein, we show that on repeated toll-like receptor 9 (TLR9) stimulation with unmethylated cytosine guanine dinucleotide containing single-stranded DNA (CpG), IL-18BP-/- mice display severe MAS manifestations, including increased weight loss, splenomegaly, anemia, thrombocytopenia, hyperferritinemia, and bone marrow hemophagocytosis as compared with wild-type mice. Serum-free IL-18 was detected in CpG-treated IL-18BP-/- mice only. Levels of interferon-γ (IFN-γ) and of IFN-γ signature genes, such as the chemokine Cxcl9 or the transcription factor CIIta, were significantly increased in IL-18BP-/- mice. Blocking IL-18 receptor signaling attenuated the severity of MAS and IFN-γ responses in IL-18BP-/- mice. Blocking IFN-γ had comparable effects to IL-18 inhibition on most MAS manifestations. Our data indicate that endogenous IL-18BP exerts a protective role in CpG-induced MAS and that IL-18, which acts upstream of IFN-γ, is involved in the severity of MAS.

Deficiency in IL-1 Receptor Type 2 Aggravates K/BxN Serum Transfer-Induced Arthritis in Mice but Has No Impact on Systemic Inflammatory Responses.

The biological activity of IL-1 is tightly regulated by the specific receptor antagonist (IL-1Ra) and the decoy receptor IL-1 receptor type 2 (IL-1R2). The role of IL-1Ra has been well demonstrated in IL-1Ra-deficient mice. In contrast, the role of endogenous IL-1R2 remains widely unknown. To define the functional role of endogenous IL-1R2 in the K/BxN serum transfer arthritis model and in IL-1ß- or LPS-induced systemic inflammation in vivo, IL-1R2-/- mice were created and compared with wild type mice. IL-1R2-/- mice bred habitually and exhibited a normal phenotype. IL-1R2 deficiency aggravated arthritis severity and increased mRNA levels for key cytokines and chemokines such as IL-6, IL-1ß, Cxcl-1, and Cxcl-2 significantly in ankles. There was no effect of IL-1R2 deficiency on the cell-autonomous cytokine response to IL-1ß in the tested cell types, i.e., neutrophils, macrophages, and fibroblasts, but IL-1R2 deficiency on neutrophils increased the IL-1-induced response of fibroblasts in trans. Furthermore, IL-1ß induced shedding of IL-1R2 in vivo. Inflammatory responses to IL-1ß and LPS-induced mortality were not different in IL-1R2-/- compared with wild type mice. Our data demonstrate that the decoy receptor IL-1R2 plays an important inhibitory role in local IL-1- and neutrophil-dependent tissue inflammation as shown in the K/BxN serum transfer arthritis model. In contrast to IL-1Ra, IL-1R2 appears to be less crucial for systemic responses to acute administration of IL-1 or LPS.

IL-36-Induced Toxicity in Neonatal Mice Involves TNF-α Production by Liver Myeloid Cells.

Human and mouse neonates exhibit limited vaccine responses characterized by predominant Th2 and limited Th1 responses. Because IL-36 exerts a synergic adjuvant effect with IL-12, enhancing Th1 polarization in adult (AD) mice, we administered IL-36ß to neonatal (1-wk old) and AD control mice at the time of immunization with tetanus toxoid adsorbed to aluminum hydroxide (TT/Alum). Unexpectedly, the combination of IL-36ß with TT/Alum, which was well tolerated in AD mice, proved toxic and even lethal in neonates. This neonatal toxicity was associated with high Il36r mRNA expression in neonatal liver, resulting in increased cytokine production. Liver Il36r mRNA expression decreased with the termination of fetal liver hematopoiesis, and this decrease correlated with a complete protection from TT/Alum/IL-36ß-induced mortality. The combination of IL-36ß and TT/Alum induced the rapid production of TNF-α and IFN-γ by liver myeloid and lymphoid cells, respectively. These responses were less marked when IL-36ß was used alone, with no adverse effect. The toxicity of IL-36ß + TT/Alum was abrogated by the administration of a neutralizing anti-TNF-α Ab, confirming causality. In conclusion, liver myeloid cells in neonatal mice are an important source of proinflammatory cytokines that may lead to TNF-α-mediated toxicity and even lethality.

IL-38 overexpression induces anti-inflammatory effects in mice arthritis models and in human macrophages in vitro.

OBJECTIVES: Interleukin (IL)-38 is a newly characterised cytokine that belongs to the IL-1 family. This cytokine is expressed in the rheumatoid arthritis (RA) synovial tissue and IL-38 deficient mice have exacerbated arthritis. Here, we analysed the effect of IL-38 overexpression in the joints of arthritic mice, in human macrophages and synovial fibroblasts in vitro. METHODS: Articular injections of an adeno-associated virus (AAV) 2/8 encoding IL-38 were performed in collagen-induced arthritis (CIA), K/BxN serum transfer-induced arthritis (STIA) and antigen-induced arthritis (AIA) in mice. The effect of IL-38 overexpression was evaluated through clinical scores, immunohistochemistry, microCT, Luminex and RT-qPCR analysis. THP-1 macrophages were transduced with a lentiviral vector to overexpress IL-38. RESULTS: Clinical inflammatory scores were significantly decreased after AAV IL-38 injection in joints of mice with CIA and STIA, but not AIA. This decrease was accompanied by reduced macrophage infiltration and a decreased expression of Th17 cytokines (IL-17, IL-23, IL-22) and TNFα. However, IL-38 overexpression had no effect on cartilage or bone destruction. In vitro, the THP-1 monocytic cell line expressed less IL-6, TNFα and IL-23 after IL-38 overexpression. Conditioned media from these cells, containing released IL-38, also exert an anti-inflammatory effect on human primary macrophages and synovial fibroblasts from patients with RA. CONCLUSIONS: This study shows for the first time that IL-38 overexpression attenuates the severity of experimental arthritis. IL-38 may exert its anti-inflammatory effects by decreasing the production of proinflammatory cytokines by macrophages and synovial fibroblasts. This effect can lead to the development of novel treatment strategies in arthritis.

Severe neutrophil-dominated inflammation and enhanced myelopoiesis in IL-33-overexpressing CMV/IL33 mice.

IL-33 is a cytokine of the IL-1 family, which signals through the ST2 receptor. Previous studies emphasized a role for IL-33 in shaping innate and adaptive immune responses. IL-33 was also reported to modulate myelopoiesis and myeloid cell activity. In this article, we describe IL-33-overexpressing CMV/IL33 and LysM/IL33 mice, which display an inflammatory phenotype associated with growth retardation and paw swelling. The phenotype of CMV/IL33 mice is dependent on activation of the ST2 receptor and is characterized by extensive neutrophil infiltration into different organs, including the paws. Local or systemic levels of proinflammatory mediators such as IL-1ß, Cxcl-1, G-CSF, and IL-6 are increased. CMV/IL-33 mice also suffer from anemia, thrombocytosis, and a marked dysregulation of myelopoiesis, leading to an important increase in myeloid cell production or accumulation in bone marrow (BM), spleen, and peripheral blood. Consistently, recombinant IL-33 induced proliferation of myeloid lineage cells in BM-derived granulocyte cultures, whereas IL-33 knockout mice exhibited minor deficiencies in spleen and BM myeloid cell populations. Our observations reveal a neutrophil-dominated inflammatory phenotype in IL-33-overexpressing CMV/IL33 and LysM/IL33 mice, and highlight important regulatory effects of IL-33 on myelopoiesis in vitro and in vivo, where excessive IL-33 signaling can translate into the occurrence of a myeloproliferative disorder.

Interleukin-36 potently stimulates human M2 macrophages, Langerhans cells and keratinocytes to produce pro-inflammatory cytokines.

Interleukin (IL)-36 cytokines belong to the IL-1 family and include three agonists, IL-36 α, ß and γ and one inhibitor, IL-36 receptor antagonist (IL-36Ra). IL-36 and IL-1 (α and ß) activate similar intracellular pathways via their related heterodimeric receptors, IL-36R/IL-1RAcP and IL-1R1/IL-1RAcP, respectively. However, excessive IL-36 versus IL-1 signaling induces different phenotypes in humans, which may be related to differential expression of their respective receptors. We examined the expression of IL-36R, IL-1R1 and IL-1RAcP mRNA in human peripheral blood, tonsil and skin immune cells by RT-qPCR. Monocyte-derived dendritic cells (MDDC), M0, M1 or M2-polarized macrophages, primary keratinocytes, dermal macrophages and Langerhans cells (LC) were stimulated with IL-1ß or IL-36ß. Cytokine production was assessed by RT-qPCR and immunoassays. The highest levels of IL-36R mRNA were found in skin-derived keratinocytes, LC, dermal macrophages and dermal CD1a(+) DC. In the blood and in tonsils, IL-36R mRNA was predominantly found in myeloid cells. By contrast, IL-1R1 mRNA was detected in almost all cell types with higher levels in tonsil and skin compared to peripheral blood immune cells. IL-36ß was as potent as IL-1ß in stimulating M2 macrophages, keratinocytes and LC, less potent than IL-1ß in stimulating M0 macrophages and MDDC, and exerted no effects in M1 and dermal macrophages. Levels of IL-1Ra diminished the ability of M2 macrophages to respond to IL-1. Taken together, these data are consistent with the association of excessive IL-36 signaling with an inflammatory skin phenotype and identify human LC and M2 macrophages as new IL-36 target cells.

The interleukin (IL)-1 cytokine family--Balance between agonists and antagonists in inflammatory diseases.

The interleukin (IL)-1 family of cytokines comprises 11 members, including 7 pro-inflammatory agonists (IL-1α, IL-1ß, IL-18, IL-33, IL-36α, IL-36ß, IL-36γ) and 4 defined or putative antagonists (IL-1R antagonist (IL-1Ra), IL-36Ra, IL-37, and IL-38) exerting anti-inflammatory activities. Except for IL-1Ra, IL-1 cytokines do not possess a leader sequence and are secreted via an unconventional pathway. In addition, IL-1ß and IL-18 are produced as biologically inert pro-peptides that require cleavage by caspase-1 in their N-terminal region to generate active proteins. N-terminal processing is also required for full activity of IL-36 cytokines. The IL-1 receptor (IL-1R) family comprises 10 members and includes cytokine-specific receptors, co-receptors and inhibitory receptors. The signaling IL-1Rs share a common structure with three extracellular immunoglobulin (Ig) domains and an intracellular Toll-like/IL-1R (TIR) domain. IL-1 cytokines bind to their specific receptor, which leads to the recruitment of a co-receptor and intracellular signaling. IL-1 cytokines induce potent inflammatory responses and their activity is tightly controlled at the level of production, protein processing and maturation, receptor binding and post-receptor signaling by naturally occurring inhibitors. Some of these inhibitors are IL-1 family antagonists, while others are IL-1R family members acting as membrane-bound or soluble decoy receptors. An imbalance between agonist and antagonist levels can lead to exaggerated inflammatory responses. Several genetic modifications or mutations associated with dysregulated IL-1 activity and autoinflammatory disorders were identified in mouse models and in patients. These findings paved the road to the successful use of IL-1 inhibitors in diseases that were previously considered as untreatable.

IL-36 signaling amplifies Th1 responses by enhancing proliferation and Th1 polarization of naive CD4+ T cells.

The interleukin-1 (IL-1) superfamily of cytokines comprises a set of pivotal mediators of inflammation. Among them, the action of IL-36 cytokines in immune responses has remained elusive. In a recent study, we demonstrated a direct effect of IL-36 on immune cells. Here we show that, among T cells, the IL-36 receptor is predominantly expressed on naive CD4(+) T cells and that IL-36 cytokines act directly on naive T cells by enhancing both cell proliferation and IL-2 secretion. IL-36ß acts in synergy with IL-12 to promote Th1 polarization and IL-36 signaling is also involved in mediating Th1 immune responses to Bacillus Calmette-Guerin infection in vivo. Our findings point toward a critical function of IL-36 in the priming of Th1 cell responses in vitro, and in adaptive immunity in a model of mycobacterial infection in vivo.