NF-κB regulated expression of A20 controls IKK dependent repression of RIPK1 induced cell death in activated T cells.

IKK signalling is essential for survival of thymocytes by repressing RIPK1 induced cell death rather than its canonical function of activating NF-κB. The role of IKK signalling in activated T cells is unclear. To investigate this, we analysed activation of IKK2 deficient T cells. While TCR triggering was normal, proliferation and expansion was profoundly impaired. This was not due to defective cell cycle progression, rather dividing T cells became sensitised to TNF induced cell death, since inhibition of RIPK1 kinase activity rescued cell survival. Gene expression analysis of activated IKK2 deficient T cells revealed defective expression of Tnfaip3, that encodes A20, a negative regulator of NF-κB. To test whether A20 expression was required to protect IKK2 deficient T cells from cell death, we generated mice with T cells lacking both A20 and IKK2. Doing this resulted in near complete loss of peripheral T cells, in contrast to mice lacking one or other gene. Strikingly, this phenotype was completely reversed by inactivation of RIPK1 kinase activity in vivo. Together, our data show that IKK signalling in activated T cells protects against RIPK1 dependent death, both by direct phosphorylation of RIPK1 and through NF-κB mediated induction of A20, that we identify for the first time as a key modulator of RIPK1 activity in T cells.

Interleukin-33-activated basophils promote asthma by regulating Th2 cell entry into lung tissue.

Asthma is characterized by lung eosinophilia, remodeling, and mucus plugging, controlled by adaptive Th2 effector cells secreting IL-4, IL-5, and IL-13. Inhaled house dust mite (HDM) causes the release of barrier epithelial cytokines that activate various innate immune cells like DCs and basophils that can promote Th2 adaptive immunity directly or indirectly. Here, we show that basophils play a crucial role in the development of type 2 immunity and eosinophilic inflammation, mucus production, and bronchial hyperreactivity in response to HDM inhalation in C57Bl/6 mice. Interestingly, conditional depletion of basophils during sensitization did not reduce Th2 priming or asthma inception, whereas depletion during allergen challenge did. During the challenge of sensitized mice, basophil-intrinsic IL-33/ST2 signaling, and not FcεRI engagement, promoted basophil IL-4 production and subsequent Th2 cell recruitment to the lungs via vascular integrin expression. Basophil-intrinsic loss of the ubiquitin modifying molecule Tnfaip3, involved in dampening IL-33 signaling, enhanced key asthma features. Thus, IL-33-activated basophils are gatekeepers that boost allergic airway inflammation by controlling Th2 tissue entry.

CARD14 signalosome formation is associated with its endosomal relocation and mTORC1-induced keratinocyte proliferation.

Rare mutations in CARD14 promote psoriasis by inducing CARD14-BCL10-MALT1 complexes that activate NF-κB and MAP kinases. Here, the downstream signalling mechanism of the highly penetrant CARD14E138A alteration is described. In addition to BCL10 and MALT1, CARD14E138A associated with several proteins important in innate immune signalling. Interactions with M1-specific ubiquitin E3 ligase HOIP, and K63-specific ubiquitin E3 ligase TRAF6 promoted BCL10 ubiquitination and were essential for NF-κB and MAP kinase activation. In contrast, the ubiquitin binding proteins A20 and ABIN1, both genetically associated with psoriasis development, negatively regulated signalling by inducing CARD14E138A turnover. CARD14E138A localized to early endosomes and was associated with the AP2 adaptor complex. AP2 function was required for CARD14E138A activation of mTOR complex 1 (mTORC1), which stimulated keratinocyte metabolism, but not for NF-κB nor MAP kinase activation. Furthermore, rapamycin ameliorated CARD14E138A-induced keratinocyte proliferation and epidermal acanthosis in mice, suggesting that blocking mTORC1 may be therapeutically beneficial in CARD14-dependent psoriasis.

Polo-like kinase 1 (PLK1) is a novel CARD14-binding protein in keratinocytes.

Caspase recruitment domain (CARD)-containing protein 14 (CARD14) is an intracellular protein that mediates nuclear factor-kappa B (NF-ĸB) signaling and proinflammatory gene expression in skin keratinocytes. Several hyperactivating CARD14 mutations have been associated with psoriasis and other inflammatory skin diseases. CARD14-induced NF-ĸB signaling is dependent on the formation of a CARD14-BCL10-MALT1 (CBM) signaling complex, but upstream receptors and molecular mechanisms that activate and regulate CARD14 signaling are still largely unclear. Using unbiased affinity purification and mass spectrometry (AP-MS) screening, we discover polo-like kinase 1 (PLK1) as a novel CARD14-binding protein. CARD14-PLK1 binding is independent of the CARD14 CARD domain but involves a consensus phospho-dependent PLK1-binding motif in the CARD14 linker region (LR). Expression of the psoriasis-associated CARD14(E138A) variant in human keratinocytes induces the recruitment of PLK1 to CARD14-containing signalosomes in interphase cells, but does not affect the specific location of PLK1 in mitotic cells. Finally, disruption of the PLK1-binding motif in CARD14(E138A) increases CARD14-induced proinflammatory signaling and gene expression. Together, our data identify PLK1 as a novel CARD14-binding protein and indicate a negative regulatory role for PLK1 in CARD14 signaling.

Chimeric and mutant CARD9 constructs enable analyses of conserved and diverged autoinhibition mechanisms in the CARD-CC protein family.

Caspase recruitment domain-containing protein (CARD)9, CARD10, CARD11, and CARD14 all belong to the CARD-coiled coil (CC) protein family and originated from a single common ancestral protein early in vertebrate evolution. All four proteins form CARD-CC/BCL10/MALT1 (CBM) complexes leading to nuclear factor-kappa-B (NF-κB) activation after upstream phosphorylation by various protein kinase C (PKC) isoforms. CBM complex signaling is critical for innate and adaptive immunity, but aberrant activation can cause autoimmune or autoinflammatory diseases, or be oncogenic. CARD9 shows a superior auto-inhibition compared with other CARD-CC family proteins, with very low spontaneous activity when overexpressed in HEK293T cells. In contrast, the poor auto-inhibition of other CARD-CC family proteins, especially CARD10 (CARMA3) and CARD14 (CARMA2), is hampering characterization of upstream activators or activating mutations in overexpression studies. We grafted different domains from CARD10, 11, and 14 on CARD9 to generate chimeric CARD9 backbones for functional characterization of activating mutants using NF-κB reporter gene activation in HEK293T cells as readout. CARD11 (CARMA1) activity was not further reduced by grafting on CARD9 backbones. The chimeric CARD9 approach was subsequently validated by using several known disease-associated mutations in CARD10 and CARD14, and additional screening allowed us to identify several previously unknown activating natural variants in human CARD9 and CARD10. Using Genebass as a resource of exome-based disease association statistics, we found that activated alleles of CARD9 correlate with irritable bowel syndrome (IBS), constipation, osteoarthritis, fibromyalgia, insomnia, anxiety, and depression, which can occur as comorbidities.

Laboratory mice with a wild microbiota generate strong allergic immune responses.

Allergic disorders are caused by a combination of hereditary and environmental factors. The hygiene hypothesis postulates that early-life microbial exposures impede the development of subsequent allergic disease. Recently developed "wildling" mice are genetically identical to standard laboratory specific pathogen-free (SPF) mice but are housed under seminatural conditions and have rich microbial exposures from birth. Thus, by comparing conventional SPF mice with wildlings, we can uncouple the impact of lifelong microbial exposures from genetic factors on the allergic immune response. We found that wildlings developed larger populations of antigen-experienced T cells than conventional SPF mice, which included interleukin-10-producing CD4 T cells specific for commensal Lactobacilli strains and allergy-promoting T helper 2 (TH2) cells. In models of airway exposure to house dust mite (HDM), recombinant interleukin-33, or Alternaria alternata, wildlings developed strong allergic inflammation, characterized by eosinophil recruitment, goblet cell metaplasia, and antigen-specific immunoglobulin G1 (IgG1) and IgE responses. Wildlings developed robust de novo TH2 cell responses to incoming allergens, whereas preexisting TH2 cells could also be recruited into the allergic immune response in a cytokine-driven and TCR-independent fashion. Thus, wildling mice, which experience diverse and lifelong microbial exposures, were not protected from developing pathological allergic immune responses. Instead, wildlings mounted robust allergic responses to incoming allergens, shedding new light on the hygiene hypothesis.

Exploring Species-Specificity in TLR4/MD-2 Inhibition with Amphiphilic Lipid A Mimicking Glycolipids.

The Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) complex is a key receptor of the innate immune system and a major driver of inflammation that is responsible for the multifaceted defense response to Gram-negative infections. However, dysfunction in the tightly regulated mechanisms of TLR4-mediated signaling leads to the uncontrolled upregulation of local and systemic inflammation, often resulting in acute or chronic disease. Therefore, the TLR4/MD-2 receptor complex is an attractive target for the design and development of anti-inflammatory therapies which aim to control the unrestrained activation of TLR4-mediated signaling. Complex structure-activity relationships and species-specificity behind ligand recognition by the TLR4/MD-2 complex complicate the development of MD-2-specific TLR4 antagonists. The restriction of the conformational flexibility of the disaccharide polar head group is one of the key structural features of the newly developed lipid A-mimicking glycophospholipids, which are potential inhibitors of TLR4-mediated inflammation. Since phosphorylation has a crucial influence on MD-2-ligand interaction, glycolipids with variable numbers and positioning of phosphate groups were synthesized and evaluated for their ability to inhibit TLR4-mediated pro-inflammatory signaling in human and murine immune cells. A bis-phosphorylated glycolipid was found to have nanomolar antagonist activity on human TLR4 while acting as a partial agonist on murine TLR4. The glycolipid inhibited mTLR4/MD-2-mediated cytokine release, acting as an antagonist in the presence of lipopolysaccharide (LPS), but at the same time induced low-level cytokine production.

Proteolytic Activity of the Paracaspase MALT1 Is Involved in Epithelial Restitution and Mucosal Healing.

The paracaspase MALT1 is a crucial regulator of immune responses in various cellular contexts. Recently, there is increasing evidence suggesting that MALT1 might represent a novel key player in mucosal inflammation. However, the molecular mechanisms underlying this process and the targeted cell population remain unclear. In this study, we investigate the role of MALT1 proteolytic activity in the context of mucosal inflammation. We demonstrate a significant enrichment of MALT1 gene and protein expression in colonic epithelial cells of UC patients, as well as in the context of experimental colitis. Mechanistically we demonstrate that MALT1 protease function inhibits ferroptosis, a form of iron-dependent cell death, upstream of NF-κB signaling, which can promote inflammation and tissue damage in IBD. We further show that MALT1 activity contributes to STAT3 signaling, which is essential for the regeneration of the intestinal epithelium after injury. In summary, our data strongly suggests that the protease function of MALT1 plays a critical role in the regulation of immune and inflammatory responses, as well as mucosal healing. Understanding the mechanisms by which MALT1 protease function regulates these processes may offer novel therapeutic targets for the treatment of IBD and other inflammatory diseases.

The impact of hepatocyte-specific deletion of hypoxia-inducible factors on the development of polymicrobial sepsis with focus on GR and PPARα function.

Introduction: Polymicrobial sepsis causes acute anorexia (loss of appetite), leading to lipolysis in white adipose tissue and proteolysis in muscle, and thus release of free fatty acids (FFAs), glycerol and gluconeogenic amino acids. Since hepatic peroxisome proliferator-activated receptor alpha (PPARα) and glucocorticoid receptor (GR) quickly lose function in sepsis, these metabolites accumulate (causing toxicity) and fail to yield energy-rich molecules such as ketone bodies (KBs) and glucose. The mechanism of PPARα and GR dysfunction is not known. Methods & results: We investigated the hypothesis that hypoxia and/or activation of hypoxia inducible factors (HIFs) might play a role in these issues with PPARα and GR. After cecal ligation and puncture (CLP) in mice, leading to lethal polymicrobial sepsis, bulk liver RNA sequencing illustrated the induction of the genes encoding HIF1α and HIF2α, and an enrichment of HIF-dependent gene signatures. Therefore, we generated hepatocyte-specific knock-out mice for HIF1α, HIF2α or both, and a new HRE-luciferase reporter mouse line. After CLP, these HRE-luciferase reporter mice show signals in several tissues, including the liver. Hydrodynamic injection of an HRE-luciferase reporter plasmid also led to (liver-specific) signals in hypoxia and CLP. Despite these encouraging data, however, hepatocyte-specific HIF1α and/or HIF2α knock-out mice suggest that survival after CLP was not dependent on the hepatocyte-specific presence of HIF proteins, which was supported by measuring blood levels of glucose, FFAs, and KBs. The HIF proteins were also irrelevant in the CLP-induced glucocorticoid resistance, but we found indications that the absence of HIF1α in hepatocytes causes less inactivation of PPARα transcriptional function. Conclusion: We conclude that HIF1α and HIF2α are activated in hepatocytes in sepsis, but their contribution to the mechanisms leading to lethality are minimal.

A20 is a master switch of IL-33 signaling in macrophages and determines IL-33-induced lung immunity.

BACKGROUND: IL-33 plays a major role in the pathogenesis of allergic diseases such as asthma and atopic dermatitis. On its release from lung epithelial cells, IL-33 primarily drives type 2 immune responses, accompanied by eosinophilia and robust production of IL-4, IL-5, and IL-13. However, several studies show that IL-33 can also drive a type 1 immune response. OBJECTIVE: We sought to determine the role of A20 in the regulation of IL-33 signaling in macrophages and IL-33-induced lung immunity. METHODS: We studied the immunologic response in lungs of IL-33-treated mice that specifically lack A20 in myeloid cells. We also analyzed IL-33 signaling in A20-deficient bone marrow-derived macrophages. RESULTS: IL-33-induced lung innate lymphoid cell type 2 expansion, type 2 cytokine production, and eosinophilia were drastically reduced in the absence of macrophage A20 expression, whereas neutrophils and interstitial macrophages in lungs were increased. In vitro, IL-33-mediated nuclear factor kappa B activation was only weakly affected in A20-deficient macrophages. However, in the absence of A20, IL-33 gained the ability to activate signal transducer and activator of transcription 1 (STAT1) signaling and STAT1-dependent gene expression. Surprisingly, A20-deficient macrophages produced IFN-γ in response to IL-33, which was fully STAT1-dependent. Furthermore, STAT1 deficiency partially restored the ability of IL-33 to induce ILC2 expansion and eosinophilia in myeloid cell-specific A20 knockout mice. CONCLUSIONS: We reveal a novel role for A20 as a negative regulator of IL-33-induced STAT1 signaling and IFN-γ production in macrophages, which determines lung immune responses.

Normal lymphocyte homeostasis and function in MALT1 protease-resistant HOIL-1 knock-in mice.

The uniqueness of MALT1 protease activity in controlling several aspects of immunity in humans has made it a very attractive therapeutic target for multiple autoimmune diseases and lymphoid malignancies. Despite several encouraging preclinical studies with MALT1 inhibitors, severe reduction in regulatory T cells and immune-mediated pathology seen in MALT1 protease-dead (MALT1-PD) mice and some, but not all, studies analysing the effect of prolonged pharmacological MALT1 protease inhibition, indicates the need to further unravel the mechanism of MALT1 protease function. Notably, the contribution of individual MALT1 substrates to the immune defects seen in MALT1-PD mice is still unclear. Previous in vitro studies indicated a role for MALT1-mediated cleavage of the E3 ubiquitin ligase HOIL-1 in the modulation of nuclear factor-κB (NF-κB) signalling and inflammatory gene expression in lymphocytes. Here, we addressed the immunological consequences of inhibition of HOIL-1 cleavage by generating and immunophenotyping MALT1 cleavage-resistant HOIL-1 knock-in (KI) mice. HOIL-1 KI mice appear healthy and have no overt phenotype. NF-κB activation in T or B cells, as well as IL-2 production and in vitro T-cell proliferation, is comparable between control and HOIL-1 KI cells. Inhibition of HOIL-1 cleavage in mice has no effect on thymic T-cell development and conventional T-cell homeostasis. Likewise, B-cell development and humoral immune responses are not affected. Together, these data exclude an important role of MALT1-mediated HOIL-1 cleavage in T- and B-cell development and function in mice.

IL-5-producing CD4+ T cells and eosinophils cooperate to enhance response to immune checkpoint blockade in breast cancer.

Immune checkpoint blockade (ICB) has heralded a new era in cancer therapy. Research into the mechanisms underlying response to ICB has predominantly focused on T cells; however, effective immune responses require tightly regulated crosstalk between innate and adaptive immune cells. Here, we combine unbiased analysis of blood and tumors from metastatic breast cancer patients treated with ICB with mechanistic studies in mouse models of breast cancer. We observe an increase in systemic and intratumoral eosinophils in patients and mice responding to ICB treatment. Mechanistically, ICB increased IL-5 production by CD4+ T cells, stimulating elevated eosinophil production from the bone marrow, leading to systemic eosinophil expansion. Additional induction of IL-33 by ICB-cisplatin combination or recombinant IL-33 promotes intratumoral eosinophil infiltration and eosinophil-dependent CD8+ T cell activation to enhance ICB response. This work demonstrates the critical role of eosinophils in ICB response and provides proof-of-principle for eosinophil engagement to enhance ICB efficacy.

GTF3A mutations predispose to herpes simplex encephalitis by disrupting biogenesis of the host-derived RIG-I ligand RNA5SP141.

Herpes simplex virus 1 (HSV-1) infects several billion people worldwide and can cause life-threatening herpes simplex encephalitis (HSE) in some patients. Monogenic defects in components of the type I interferon system have been identified in patients with HSE, emphasizing the role of inborn errors of immunity underlying HSE pathogenesis. Here, we identify compound heterozygous loss-of-function mutations in the gene GTF3A encoding for transcription factor IIIA (TFIIIA), a component of the RNA polymerase III complex, in a patient with common variable immunodeficiency and HSE. Patient fibroblasts and GTF3A gene-edited cells displayed impaired HSV-1-induced innate immune responses and enhanced HSV-1 replication. Chromatin immunoprecipitation sequencing analysis identified the 5S ribosomal RNA pseudogene 141 (RNA5SP141), an endogenous ligand of the RNA sensor RIG-I, as a transcriptional target of TFIIIA. GTF3A mutant cells exhibited diminished RNA5SP141 expression and abrogated RIG-I activation upon HSV-1 infection. Our work unveils a crucial role for TFIIIA in transcriptional regulation of a cellular RIG-I agonist and shows that GTF3A genetic defects lead to impaired cell-intrinsic anti-HSV-1 responses and can predispose to HSE.

CARD14 Signalling Ensures Cell Survival and Cancer Associated Gene Expression in Prostate Cancer Cells.

Prostate cancer (PCa) is one of the most common cancer types in men and represents an increasing global problem due to the modern Western lifestyle. The signalling adapter protein CARD14 is specifically expressed in epithelial cells, where it has been shown to mediate NF-κB signalling, but a role for CARD14 in carcinoma has not yet been described. By analysing existing cancer databases, we found that CARD14 overexpression strongly correlates with aggressive PCa in human patients. Moreover, we showed that CARD14 is overexpressed in the LNCaP PCa cell line and that knockdown of CARD14 severely reduces LNCaP cell survival. Similarly, knockdown of BCL10 and MALT1, which are known to form a signalling complex with CARD14, also induced LNCaP cell death. MALT1 is a paracaspase that mediates downstream signalling by acting as a scaffold, as well as a protease. Recent studies have already indicated a role for the scaffold function of MALT1 in PCa cell growth. Here, we also demonstrated constitutive MALT1 proteolytic activity in several PCa cell lines, leading to cleavage of A20 and CYLD. Inhibition of MALT1 protease activity did not affect PCa cell survival nor activation of NF-κB and JNK signalling, but reduced expression of cancer-associated genes, including the cytokine IL-6. Taken together, our results revealed a novel role for CARD14-induced signalling in regulating PCa cell survival and gene expression. The epithelial cell type-specific expression of CARD14 may offer novel opportunities for more specific therapeutic targeting approaches in PCa.

Editor Profile: Rudi Beyaert.

In this special interview series, we profile members of The FEBS Journal editorial board to highlight their research focus, perspectives on the journal and future directions in their field. Rudi Beyaert is Full Professor in Molecular Biology at the Department of Biomedical Molecular Biology, Faculty of Sciences, of the University of Ghent (Belgium). He also serves as Vice-Science Director of the Center for Inflammation Research of the VIB in Ghent, where he is heading the Unit of Molecular Signal Transduction in Inflammation. He has served as an Editorial Board Member of The FEBS Journal since 2016.

Engineering a highly sensitive biosensor for abscisic acid in mammalian cells.

Abscisic acid (ABA) is a signalling molecule conserved in plants, bacteria, fungi, and animals. Recently, ABA has gained attention for its pharmacological activities and its potential as a biomarker for the severity of chronic obstructive pulmonary disease and glioma. This prompts the development of a reliable, sensitive, rapid, and cost-effective method to quantify ABA levels in mammalian cells and tissues. The previously described ABA biosensor system based on the ABA-dependent interaction between the plant ABA receptor PYL1 and co-receptor ABI1 is not sensitive enough for the low ABA levels seen in mammals. Therefore, we optimized this system by replacing PYL1 with other high-affinity plant PYL proteins. The optimized biosensor system engineered with the PYL8 receptor enabled the quantification of ABA at low concentrations in HEK293T cells.

Mutations in RNU7-1 Weaken Secondary RNA Structure, Induce MCP-1 and CXCL10 in CSF, and Result in Aicardi-Goutières Syndrome with Severe End-Organ Involvement.

BACKGROUND: Aicardi-Goutières syndrome (AGS) is a type I interferonopathy usually characterized by early-onset neurologic regression. Biallelic mutations in LSM11 and RNU7-1, components of the U7 small nuclear ribonucleoprotein (snRNP) complex, have been identified in a limited number of genetically unexplained AGS cases. Impairment of U7 snRNP function results in misprocessing of replication-dependent histone (RDH) pre-mRNA and disturbance of histone occupancy of nuclear DNA, ultimately driving cGAS-dependent type I interferon (IFN-I) release. OBJECTIVE: We performed a clinical, genetic, and immunological workup of 3 unrelated patients with uncharacterized AGS. METHODS: Whole exome sequencing (WES) and targeted Sanger sequencing of RNU7-1 were performed. Primary fibroblasts were used for mechanistic studies. IFN-I signature and STAT1/2 phosphorylation were assessed in peripheral blood. Cytokines were profiled on serum and cerebrospinal fluid (CSF). Histopathology was examined on brain and kidney tissue. RESULTS: Sequencing revealed compound heterozygous RNU7-1 mutations, resulting in impaired RDH pre-mRNA processing. The 3' stem-loop mutations reduced stability of the secondary U7 snRNA structure. A discrete IFN-I signature in peripheral blood was paralleled by MCP-1 (CCL2) and CXCL10 upregulation in CSF. Histopathological analysis of the kidney showed thrombotic microangiopathy. We observed dysregulated STAT phosphorylation upon cytokine stimulation. Clinical overview of all reported patients with RNU7-1-related disease revealed high mortality and high incidence of organ involvement compared to other AGS genotypes. CONCLUSIONS: Targeted RNU7-1 sequencing is recommended in genetically unexplained AGS cases. CSF cytokine profiling represents an additional diagnostic tool to identify aberrant IFN-I signaling. Clinical follow-up of RNU7-1-mutated patients should include screening for severe end-organ involvement including liver disease and nephropathy.

Tumor-educated Tregs drive organ-specific metastasis in breast cancer by impairing NK cells in the lymph node niche.

Breast cancer is accompanied by systemic immunosuppression, which facilitates metastasis formation, but how this shapes organotropism of metastasis is poorly understood. Here, we investigate the impact of mammary tumorigenesis on regulatory T cells (Tregs) in distant organs and how this affects multi-organ metastatic disease. Using a preclinical mouse mammary tumor model that recapitulates human metastatic breast cancer, we observe systemic accumulation of activated, highly immunosuppressive Tregs during primary tumor growth. Tumor-educated Tregs show tissue-specific transcriptional rewiring in response to mammary tumorigenesis. This has functional consequences for organotropism of metastasis, as Treg depletion reduces metastasis to tumor-draining lymph nodes, but not to lungs. Mechanistically, we find that Tregs control natural killer (NK) cell activation in lymph nodes, thereby facilitating lymph node metastasis. In line, an increased Treg/NK cell ratio is observed in sentinel lymph nodes of breast cancer patients compared with healthy controls. This study highlights that immune regulation of metastatic disease is highly organ dependent.

Inflammatory cell-derived CXCL3 promotes pancreatic cancer metastasis through a novel myofibroblast-hijacked cancer escape mechanism.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal malignancy and lacks effective treatment. We aimed to understand molecular mechanisms of the intertwined interactions between tumour stromal components in metastasis and to provide a new paradigm for PDAC therapy. DESIGN: Two unselected cohorts of 154 and 20 patients with PDAC were subjected to correlation between interleukin (IL)-33 and CXCL3 levels and survivals. Unbiased expression profiling, and genetic and pharmacological gain-of-function and loss-of-function approaches were employed to identify molecular signalling in tumour-associated macrophages (TAMs) and myofibroblastic cancer-associated fibroblasts (myoCAFs). The role of the IL-33-ST2-CXCL3-CXCR2 axis in PDAC metastasis was evaluated in three clinically relevant mouse PDAC models. RESULTS: IL-33 was specifically elevated in human PDACs and positively correlated with tumour inflammation in human patients with PDAC. CXCL3 was highly upregulated in IL-33-stimulated macrophages that were the primary source of CXCL3. CXCL3 was correlated with poor survival in human patients with PDAC. Mechanistically, activation of the IL-33-ST2-MYC pathway attributed to high CXCL3 production. The highest level of CXCL3 was found in PDAC relative to other cancer types and its receptor CXCR2 was almost exclusively expressed in CAFs. Activation of CXCR2 by CXCL3 induced a CAF-to-myoCAF transition and α-smooth muscle actin (α-SMA) was uniquely upregulated by the CXCL3-CXCR2 signalling. Type III collagen was identified as the CXCL3-CXCR2-targeted adhesive molecule responsible for myoCAF-driven PDAC metastasis. CONCLUSIONS: Our work provides novel mechanistic insights into understanding PDAC metastasis by the TAM-CAF interaction and targeting each of these signalling components would provide an attractive and new paradigm for treating pancreatic cancer.

TIM3+ TRBV11-2 T cells and IFNγ signature in patrolling monocytes and CD16+ NK cells delineate MIS-C.

In rare instances, pediatric SARS-CoV-2 infection results in a novel immunodysregulation syndrome termed multisystem inflammatory syndrome in children (MIS-C). We compared MIS-C immunopathology with severe COVID-19 in adults. MIS-C does not result in pneumocyte damage but is associated with vascular endotheliitis and gastrointestinal epithelial injury. In MIS-C, the cytokine release syndrome is characterized by IFNγ and not type I interferon. Persistence of patrolling monocytes differentiates MIS-C from severe COVID-19, which is dominated by HLA-DRlo classical monocytes. IFNγ levels correlate with granzyme B production in CD16+ NK cells and TIM3 expression on CD38+/HLA-DR+ T cells. Single-cell TCR profiling reveals a skewed TCRß repertoire enriched for TRBV11-2 and a superantigenic signature in TIM3+/CD38+/HLA-DR+ T cells. Using NicheNet, we confirm IFNγ as a central cytokine in the communication between TIM3+/CD38+/HLA-DR+ T cells, CD16+ NK cells, and patrolling monocytes. Normalization of IFNγ, loss of TIM3, quiescence of CD16+ NK cells, and contraction of patrolling monocytes upon clinical resolution highlight their potential role in MIS-C immunopathogenesis.