Pannexin‐1‐dependent caspase‐1 activation and secretion of IL‐1β is regulated by zinc

Inflammatory processes induced by IL‐1β are critical for host defence responses, but are also implicated in disease. Zinc deficiency is a common consequence of, or contributor to, human inflammatory disease. However, the molecular mechanisms through which zinc contributes to inflammatory disease remain largely unknown. We report here that zinc metabolism regulates caspase‐1 activation and IL‐1β secretion. One of the endogenous mediators of IL‐1β secretion is adenosine triphosphate, acting via the P2X7‐receptor and caspase‐1 activation in cells primed with an inflammatory stimulus such as LPS. We show that this process is selectively abolished by a brief pre‐treatment with the zinc chelator N,N,N′,N′‐tetrakis‐(2‐pyridylmethyl) ethylene diamine (TPEN). These effects on IL‐1β secretion were independent of rapid changes in free zinc within the cell, not a direct effect on caspase‐1 activity, and upstream of caspase‐1 activation. TPEN did however inhibit the activity of pannexin‐1, a hemi‐channel critical for adenosine triphosphate and nigericin‐induced IL‐1β release. These data provide new insights into the mechanisms of caspase‐1 activation and how zinc metabolism contributes to inflammatory mechanisms.     

Myc binding protein 2 suppresses M2‐like phenotypes in macrophages during zymosan‐induced inflammation in mice

MYCBP2 is an E3 ubiquitin ligase, which is well characterized as a key element in the inhibition of neuronal growth, synapse formation and synaptic strength by regulating several signaling pathways. Although MYCBP2 was suspected to be expressed also in immune cells, to date nothing is known about its role in inflammation. We used Multi‐epitope ligand cartography (MELC), a method for multiple sequential immunohistology, to show that MYCBP2 is strongly expressed in monocyte‐derived macrophages during zymosan‐induced inflammation. We generated a myeloid‐specific knockout mouse and found that loss of MYCBP2 in myeloid cells reduced nociceptive (painful) behavior during the resolution phase (1–3 days after zymosan injection). Quantitative MELC analyses and flow cytometric analysis showed an increased number of CD206‐expressing macrophages in the inflamed paw tissue. Fittingly, CD206 and arginase 1 expression was upregulated in MYCBP2‐deficient bone marrow‐derived macrophages after polarization with IL10 or IL4. The regulation of protein expression in these macrophages by MYCBP2 varied depending on the polarization signal. The increased IL10‐induced CD206 expression in MYCBP2‐deficient macrophages was mediated by p38 MAPK, while IL4‐induced CD206 expression in MYCBP2‐deficient macrophages was mediated by protein kinase A.     

Protein tyrosine phosphatase PTPN22 regulates IL‐1β dependent Th17 responses by modulating dectin‐1 signaling in mice

A single nucleotide polymorphism within the PTPN22 gene is a strong genetic risk factor predisposing to the development of multiple autoimmune diseases. PTPN22 regulates Syk and Src family kinases downstream of immuno‐receptors. Fungal β‐glucan receptor dectin‐1 signals via Syk, and dectin‐1 stimulation induces arthritis in mouse models. We investigated whether PTPN22 regulates dectin‐1 dependent immune responses. Bone marrow derived dendritic cells (BMDCs) generated from C57BL/6 wild type (WT) and Ptpn22^?/? mutant mice, were pulsed with OVA_323‐339 and the dectin‐1 agonist curdlan and co‐cultured in vitro with OT‐II T‐cells or adoptively transferred into OT‐II mice, and T‐cell responses were determined by immunoassay. Dectin‐1 activated Ptpn22^?/? BMDCs enhanced T‐cell secretion of IL‐17 in vitro and in vivo in an IL‐1β dependent manner. Immunoblotting revealed that compared to WT, dectin‐1 activated Ptpn22^?/? BMDCs displayed enhanced Syk and Erk phosphorylation. Dectin‐1 activation of BMDCs expressing Ptpn22^R619W (the mouse orthologue of human PTPN22^R620W) also resulted in increased IL‐1β secretion and T‐cell dependent IL‐17 responses, indicating that in the context of dectin‐1 Ptpn22^R619W operates as a loss‐of‐function variant. These findings highlight PTPN22 as a novel regulator of dectin‐1 signals, providing a link between genetically conferred perturbations of innate receptor signaling and the risk of autoimmune disease.     

IL‐23 protection against Plasmodium berghei infection in mice is partially dependent on IL‐17 from macrophages

Although IL‐12 is believed to contribute to protective immune responses, the role played by IL‐23 (a member of the IL‐12 family) in malaria is elusive. Here, we show that IL‐23 is produced during infection with Plasmodium berghei NK65. Mice deficient in IL‐23 (p19KO) had higher parasitemia and died earlier than wild‐type (WT) controls. Interestingly, p19KO mice had lower numbers of IL‐17‐producing splenic cells than their WT counterparts. Furthermore, mice deficient in IL‐17 (17KO) suffered higher parasitemia than the WT controls, indicating that IL‐23‐mediated protection is dependent on induction of IL‐17 during infection. We found that macrophages were responsible for IL‐17 production in response to IL‐23. We observed a striking reduction in splenic macrophages in the p19KO and 17KO mice, both of which became highly susceptible to infection. Thus, IL‐17 appears to be crucial for maintenance of splenic macrophages. Adoptive transfer of macrophages into macrophage‐depleted mice confirmed that macrophage‐derived IL‐17 is required for macrophage accumulation and parasite eradication in the recipient mice. We also found that IL‐17 induces CCL2/7, which recruit macrophages. Our findings reveal a novel protective mechanism whereby IL‐23, IL‐17, and macrophages reduce the severity of infection with blood‐stage malaria parasites.     

Th1 cytokines are more effective than Th2 cytokines at licensing anti‐tumour functions in CD40‐activated human macrophages in vitro

CD40 agonists are showing activity in early clinical trials in patients with advanced cancer. In animal models, CD40 agonists synergise with T‐cell‐activating therapies to inhibit tumour growth by driving tumour macrophage repolarisation from an immunosuppressive to a Th1 immunostimulatory, tumouricidal phenotype. We therefore tested the hypothesis that T‐cell‐derived cytokines license anti‐tumour functions in CD40‐activated human macrophages. CD40 ligand (CD40L) alone activated macrophages to produce immunosuppressive IL‐10, in a similar fashion to bacterial LPS, but failed to promote anti‐tumour functions. The Th1 cytokine IFN‐γ optimally licensed CD40L‐induced macrophage anti‐tumour functions, inducing a switch from IL‐10 to IL‐12p70 production, promoting macrophage‐mediated Th1 T‐cell skewing and enhancing tumouricidal activity. We found that even the Th2 cytokines IL‐4 and IL‐13 promoted IL‐12p70 production (albeit without inhibiting IL‐10 production) and enhanced Th1 T‐cell skewing by CD40L‐activated macrophages. However, IL‐4 and IL‐13 did not enhance tumouricidal activity in CD40L‐activated macrophages. Thus, while both Th1 and Th2 cytokines biased macrophages to a Th1 immunostimulatory phenotype, only Th1 cytokines promoted tumouricidal activity in CD40L‐activated macrophages. The presence of tumour‐infiltrating Th1 or Th2 cells might therefore be predictive for patient response to CD40 agonism.     

IL‐34‐ and M‐CSF‐induced macrophages switch memory T cells into Th17 cells via membrane IL‐1α

Macrophages orchestrate the immune response via the polarization of CD4⁺ T helper (Th) cells. Different subsets of macrophages with distinct phenotypes, and sometimes opposite functions, have been described. M‐CSF and IL‐34 induce the differentiation of monocytes into IL‐10^high IL‐12^low immunoregulatory macrophages, which are similar to tumor‐associated macrophages (TAMs) in ovarian cancer. In this study, we evaluated the capacity of human macrophages induced in the presence of M‐CSF (M‐CSF macrophages) or IL‐34 (IL‐34 macrophages) and ovarian cancer TAMs to modulate the phenotype of human CD4⁺ T cells. Taken together, our results show that M‐CSF‐, IL‐34 macrophages, and TAMs switch non‐Th17 committed memory CD4⁺ T cells into conventional CCR4⁺ CCR6⁺ CD161⁺ Th17 cells, expressing or not IFN‐gamma. Contrary, the pro‐inflammatory GM‐CSF macrophages promote Th1 cells. The polarization of memory T cells into Th17 cells is mediated via membrane IL‐1α (mIL‐1α), which is constitutively expressed by M‐CSF‐, IL‐34 macrophages, and TAMs. This study elucidates a new mechanism that allows macrophages to maintain locally restrained and smoldering inflammation, which is required in angiogenesis and metastasis.     

The purinergic P2×7 receptor is expressed on monocytes in Behçet's disease and is modulated by TNF‐α

The P2×7 receptor (P2×7r) is expressed in innate immune cells (e.g. monocyte/macrophages), playing a key role in IL‐1β release. Since innate immune activation and IL‐1β release seem to be implicated in Behçet's disease (BD), a systemic immune‐inflammatory disorder of unknown origin, we hypothesized that P2×7r is involved in the pathogenesis of the disease. Monocytes were isolated from 18 BD patients and 17 healthy matched controls. In BD monocytes, an increased P2×7r expression and Ca²⁺ permeability induced by the selective P2×7r agonist 2′‐3′‐O‐(4‐benzoylbenzoyl)ATP (BzATP) was observed. Moreover, IL‐1β release from LPS‐primed monocytes stimulated with BzATP was markedly higher in BD patients than in controls. TNF‐α‐incubated monocytes from healthy subjects almost reproduced the findings observed in BD patients, as demonstrated by the increase in P2×7r expression and BzATP‐induced Ca²⁺ intake. Our results provide evidence that in BD monocytes both the expression and function of the P2×7r are increased compared with healthy controls, as the possible result, at least in part, of a positive modulating effect of TNF‐α on the receptor. These data indicate P2×7r as a new potential therapeutic target for the control of BD, further supporting the rationale for the use of anti‐TNF‐α drugs in the treatment of the disease.     

IL‐18‐induced expression of high‐affinity IL‐2R on murine NK cells is essential for NK‐cell IFN‐γ production during murine Plasmodium yoelii infection

Early production of pro‐inflammatory cytokines, including IFN‐γ, is essential for control of blood‐stage malaria infections. We have shown that IFN‐γ production can be induced among human natural killer (NK) cells by coculture with Plasmodium falciparum infected erythrocytes, but the importance of this response is unclear. To further explore the role of NK cells during malaria infection, we have characterized the NK‐cell response of C57BL/6 mice during lethal (PyYM) or nonlethal (Py17XNL) P. yoelii infection. Ex vivo flow cytometry revealed that NK cells are activated within 24 h of Py17XNL blood‐stage infection, expressing CD25 and producing IFN‐γ; this response was blunted and delayed during PyYM infection. CD25 expression and IFN‐γ production were highly correlated, suggesting a causal relationship between the two responses. Subsequent in vitro experiments revealed that IL‐18 signaling is essential for induction of CD25 and synergizes with IL‐12 to enhance CD25 expression on splenic NK cells. In accordance with this, Py17XNL‐infected erythrocytes induced NK‐cell CD25 expression and IFN‐γ production in a manner that is completely IL‐18‐ and partially IL‐12‐dependent, and IFN‐γ production is enhanced by IL‐2. These data suggest that IL‐2 signaling via CD25 amplifies IL‐18‐ and IL‐12‐mediated NK‐cell activation during malaria infection.     

Secreted IL‐1α promotes T‐cell activation and expansion of CD11b+Gr1+ cells in carbon tetrachloride‐induced liver injury in mice

Interleukin‐1α is mainly expressed on the cell membrane, but can also be secreted during inflammation. The roles of secreted and membrane IL‐1α in acute liver inflammation are still not known. Here, we examined the functions of secreted and membrane IL‐1α in a mouse model of carbon tetrachloride‐induced acute liver injury. We show that secreted IL‐1α aggravates liver damage and membrane IL‐1α slightly protects mice from liver injury. Further studies showed that secreted IL‐1α promotes T‐cell activation. It also increased the expansion of CD11b⁺Gr1⁺ myeloid cells, which may serve as a negative regulator of acute liver inflammation. Moreover, secreted IL‐1α induced IL‐6 production from hepatocytes. IL‐6 neutralization reduced the proliferation of CD11b⁺Gr1⁺ myeloid cells in vivo. CCL2 and CXCL5 expression was increased by secreted IL‐1α in vitro and in vivo. Antagonists of the chemokine receptors for CCL2 and CXCL5 significantly reduced the migration of CD11b⁺Gr1⁺ myeloid cells. These results demonstrate that secreted and membrane IL‐1α play different roles in acute liver injury. Secreted IL‐1α could promote T‐cell activation and the recruitment and expansion of CD11b⁺Gr1⁺ myeloid cells through induction of CCL2, CXCL5, and IL‐6. The controlled release of IL‐1α could be a critical regulator during acute liver inflammation.     

TGF‐β interactions with IL‐1 family members trigger IL‐4‐independent IL‐9 production by mouse CD4+ T cells

TGF‐β and IL‐4 were recently shown to selectively upregulate IL‐9 production by naïve CD4⁺ T cells. We report here that TGF‐β interactions with IL‐1α, IL‐1β, IL‐18, and IL‐33 have equivalent IL‐9‐stimulating activities that function even in IL‐4‐deficient animals. This was observed after in vitro antigenic stimulation of immunized or unprimed mice and after polyclonal T‐cell activation. Based on intracellular IL‐9 staining, all IL‐9‐producing cells were CD4⁺ and 80–90% had proliferated, as indicated by reduced CFSE staining. In contrast to IL‐9, IL‐13 and IL‐17 were strongly stimulated by IL‐1 and either inhibited (IL‐13) or were unaffected (IL‐17) by addition of TGF‐β. IL‐9 and IL‐17 production also differed in their dependence on IL‐2 and regulation by IL‐1/IL‐23. As IL‐9 levels were much lower in Th2 and Th17 cultures, our results identify TGF‐β/IL‐1 and TGF‐β/IL‐4 as the main control points of IL‐9 synthesis.     

The CDK domain of p21 is a suppressor of IL‐1β‐mediated inflammation in activated macrophages

Significant morbidity and mortality can be attributed to inflammatory diseases; therefore, a greater understanding of the mechanisms involved in the progression of inflammation is crucial. Here, we demonstrate that p21^(WAF1/CIP1), an established suppressor of cell cycle progression, is a inhibitor of IL‐1β synthesis in macrophages. Mice deficient in p21 (p21^?/?) display increased susceptibility to endotoxic shock, which is associated with increased serum levels of IL‐1β. Administration of IL‐1 receptor antagonist reduces LPS‐induced lethality in p21^?/? mice. Analysis of isolated macrophages, which are one of the central producers of IL‐1β, reveals that deficiency for p21 led to more IL‐1β mRNA and pro‐protein synthesis following TLR ligation. The increase in IL‐1β pro‐protein is associated with elevated secretion of active IL‐1β by p21^?/? macrophages. siRNA‐mediated knockdown of p21 in human macrophages results in increased IL‐1β secretion as well. A peptide mapping strategy shows that the cyclin‐dependent‐kinase (CDK)‐binding domain of p21 is sufficient to reduce the secretion of IL‐1β by p21^?/? macrophages. These data suggest a novel role for p21 and specifically for the CDK‐binding domain of p21^(WAF1/CIP1) in inhibiting inflammation.     

IL‐15 modulates the balance between Bcl‐2 and Bim via a Jak3/1‐PI3K‐Akt‐ERK pathway to promote CD8αα+ intestinal intraepithelial lymphocyte survival

IL‐15 is an essential survival factor for CD8αα⁺ intestinal intraepithelial lymphocytes (iIELs) in vitro and in vivo. However, the IL‐15‐induced survival signals in primary CD8αα⁺ iIELs remains elusive. Although Bcl‐2 level in CD8αα⁺ iIELs positively correlates with IL‐15Rα expression in the intestinal epithelial cells, overexpression of Bcl‐2 only moderately restores CD8αα⁺ γδ iIELs in Il15^?/? mice. Here, we found that IL‐15 promptly activated a Jak3‐Jak1‐PI3K‐Akt pathway that led to the upregulation of Bcl‐2 and Mcl‐1. This pathway also induced a delayed but sustained ERK1/2 activation, which not only was necessary for the maintenance of Bcl‐2 but also resulted in the phosphorylation of extra‐long Bim at Ser⁶⁵. The latter event facilitated the dissociation of Bim from Bcl‐2 without affecting Bim abundance in IL‐15‐treated CD8αα⁺ iIELs. Using an adoptive cell transfer approach, we found that either overexpression of Bcl‐2 or removal of Bim from CD8αα⁺ iIELs promoted their survival in Il15ra^?/? mice. Taken together, IL‐15 promotes CD8αα⁺ iIEL survival by both increasing Bcl‐2 levels and dissociating Bim from Bcl‐2 through activation of a Jak3‐Jak1‐PI3K‐Akt‐ERK1/2 pathway, which differs from a previously reported IL‐15‐induced survival signal.     

Hypoxia inducible factor‐1α‐induced interleukin‐33 expression in intestinal epithelia contributes to mucosal homeostasis in inflammatory bowel disease

Intestinal epithelial cells (IECs), an important barrier to gut microbiota, are subject to low oxygen tension, particularly during intestinal inflammation. Hypoxia inducible factor‐1α (HIF‐1α) is expressed highly in the inflamed mucosa of inflammatory bowel disease (IBD) and functions as a key regulator in maintenance of intestinal homeostasis. However, how IEC‐derived HIF‐1α regulates intestinal immune responses in IBD is still not understood completely. We report here that the expression of HIF‐1α and IL‐33 was increased significantly in the inflamed mucosa of IBD patients as well as mice with colitis induced by dextran sulphate sodium (DSS). The levels of interleukin (IL)?33 were correlated positively with that of HIF‐1α. A HIF‐1α‐interacting element was identified in the promoter region of IL‐33, indicating that HIF‐1α activity regulates IL‐33 expression. Furthermore, tumour necrosis factor (TNF) facilitated the HIF‐1α‐dependent IL‐33 expression in IEC. Our data thus demonstrate that HIF‐1α‐dependent IL‐33 in IEC functions as a regulatory cytokine in inflamed mucosa of IBD, thereby regulating the intestinal inflammation and maintaining mucosal homeostasis.     

Patients with inflammatory bowel disease may have a transforming growth factor‐β‐, interleukin (IL)‐2‐ or IL‐10‐deficient state induced by intrinsic neutralizing antibodies

Ulcerative colitis (UC) and Crohn's disease (CD) are considered to be immunologically mediated disorders that share certain features with murine models of colitis. Whether any of these models are physiologically relevant to the human condition remains controversial. The hypothesis is that increased amounts of antibodies neutralizing transforming growth factor (TGF)‐β, interleukin (IL)‐2 or IL‐10 create a relative immunodeficient state in inflammatory bowel disease (IBD) that predisposes to disease. To evaluate this, serum samples from patients with UC or CD and from normal healthy individuals were studied by enzyme‐linked immunosorbent assays. Antibodies recognizing TGF‐β were most prevalent in UC (P < 0·01); anti‐IL‐10 antibodies were elevated in CD (P < 0·05), while anti‐IL‐2 antibodies were the same for all three groups. Importantly, the percentage of IBD patients with at least one of the antibody levels greater than any control value was 30% for UC and 33% for CD. To verify the presence of these antibodies, immobilized TGF‐β was exposed to UC sera and the attached proteins identified by Western blot assay. The proteins proved to be exclusively immunoglobulin (Ig) G. To evaluate the neutralizing activity of these antibodies, cytokine‐specific IgG from subjects in each group of patients was incubated with TGF‐β, IL‐2 or IL‐10 before addition to a bioassay with changes in viability determined by a colorimetric analysis. Antibodies from most individuals in all three groups neutralized the action of each cytokine. This study shows that about one‐third of IBD patients may have a relative deficiency of TGF‐β, IL‐2 or IL‐10 due to an increase in neutralizing antibodies in their sera.     

Cathepsin X cleaves the β2 cytoplasmic tail of LFA‐1 inducing the intermediate affinity form of LFA‐1 and α‐actinin‐1 binding

The motility of T cells depends on the dynamic spatial regulation of integrin‐mediated adhesion and de‐adhesion. Cathepsin X, a cysteine protease, has been shown to regulate T‐cell migration by interaction with lymphocyte function associated antigen‐1 (LFA‐1). LFA‐1 adhesion to the ICAM‐1 is controlled by the association of actin‐binding proteins with the cytoplasmic tail of the β₂ chain of LFA‐1. Cleavage by cathepsin X of the amino acid residues S⁷⁶⁹, E⁷⁶⁸ and A⁷⁶⁷ from the C‐terminal of the β₂ cytoplasmic tail of LFA‐1 is shown to promote binding of the actin‐binding protein α‐actinin‐1. Furthermore, cathepsin X overexpression reduced LFA‐1 clustering and induced an intermediate affinity LFA‐1 conformation that is known to associate with α‐actinin‐1. Increased levels of intermediate affinity LFA‐1 resulted in augmented cell spreading due to reduced attachment of T cells to the ICAM‐1‐coated surface. Gradual cleavage of LFA‐1 by cathepsin X enables the transition between intermediate and high affinity LFA‐1, an event that is crucial for effective T‐cell migration.     

Modulation of the expression of M150 on macrophages by Th1/Th2 cytokines and co-stimulatory molecules CD40, B7-1, B7-2 and ICAM-1

M150 is an 150-kDa protein associated with the surface of macrophages and is responsible chiefly for the activation of Th1 cells. It is a unique subset of the lysosome-associated membrane protein-1 glycoprotein and its co-stimulatory activity depends on its post-translational modification, which has a distinct glycosylation pattern restricted to macrophages. In the present study, we have observed that M150 is expressed constitutively on peritoneal but not splenic macrophages isolated from mice of different genetic backgrounds: Balb/c, C57BL/6 and C3He. However, M150 was expressed not only on peritoneal but also on splenic macrophages of non-obese diabetic (NOD) mice. Expression on splenic macrophages was induced by culture with lipopolysaccharide (LPS). Expression could also be significantly up-regulated by interferon (IFN)-gamma and granulocyte-macrophage colony stimulating factor (GM-CSF) but was inhibited by interleukin (IL)-10; IL-4 exhibited no effect. Further, cross-linking of B7-2, CD40, ICAM-1 but not B7-1 enhanced the level of M150 significantly. IFN-gamma and GM-CSF acted synergistically with CD40. The significance of these findings is that cytokines IFN-gamma, GM-CSF and IL-10 and the co-stimulatory molecules B7-2, CD40 and ICAM-1 can regulate the expression of M150 on macrophages.     

Interleukin‐17A participates in podocyte injury by inducing IL‐1β secretion through ROS‐NLRP3 inflammasome‐caspase‐1 pathway

Studies show that the Th17/IL ‐17A axis plays an important role in the pathogenesis of kidney diseases. Previously, we also showed that IL ‐17A may play a role in the pathogenesis of primary nephrotic syndrome; however, the underlying mechanism(s) is unclear. The aim of this study was to explore the molecular mechanism of IL ‐17A‐inducing podocyte injury in vitro. In this study, the NLRP 3 inflammasome activation and the morphology of podocytes were detected by Western blot and immunofluorescence. The results showed that podocytes persistently expressed IL ‐17A receptor and that NLRP 3 inflammasome in these cells was activated upon exposure to IL ‐17A. Also, activity of caspase‐1 and secretion of IL ‐1β increased in the presence of IL ‐17A. In addition, IL ‐17A disrupted podocyte morphology by decreasing expression of podocin and increasing expression of desmin. Blockade of intracellular ROS or inhibition of caspase‐1 prevented activation of the NLRP 3 inflammasome, thereby restoring podocyte morphology. Taken together, the results suggest that IL ‐17A induces podocyte injury by activating the NLRP 3 inflammasome and IL ‐1β secretion and contributes to disruption of the kidney's filtration system.     

Pro‐migratory and TGF‐β‐activating functions of αvβ6 integrin in pancreatic cancer are differentially regulated via an Eps8‐dependent GTPase switch

The integrin αvβ6 is up‐regulated in numerous carcinomas, where expression commonly correlates with poor prognosis. αvβ6 promotes tumour invasion, partly through regulation of proteases and cell migration, and is also the principal mechanism by which epithelial cells activate TGF‐β1; this latter function complicates therapeutic targeting of αvβ6, since TGF‐β1 has both tumour‐promoting and ‐suppressive effects. It is unclear how these different αvβ6 functions are linked; both require actin cytoskeletal reorganization, and it is suggested that tractive forces generated during cell migration activate TGF‐β1 by exerting mechanical tension on the ECM‐bound latent complex. We examined the functional relationship between cell invasion and TGF‐β1 activation in pancreatic ductal adenocarcinoma (PDAC) cells, and confirmed that both processes are αvβ6‐dependent. Surprisingly, we found that cellular functions could be biased towards either motility or TGF‐β1 activation depending on the presence or absence of epidermal growth factor receptor pathway substrate 8 (Eps8), a regulator of actin remodelling, endocytosis, and GTPase activation. Similar to αvβ6, we found that Eps8 was up‐regulated in >70% of PDACs. In complex with Abi1/Sos1, Eps8 regulated αvβ6‐dependent cell migration through activation of Rac1. Down‐regulation of Eps8, Sos1 or Rac1 suppressed cell movement, while simultaneously increasing αvβ6‐dependent TGF‐β1 activation. This latter effect was modulated through increased cell tension, regulated by Rho activation. Thus, the Eps8/Abi1/Sos1 tricomplex acts as a key molecular switch altering the balance between Rac1 and Rho activation; its presence or absence in PDAC cells modulates αvβ6‐dependent functions, resulting in a pro‐migratory (Rac1‐dependent) or a pro‐TGF‐β1 activation (Rho‐dependent) functional phenotype, respectively. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

IL‐18 associates to microvesicles shed from human macrophages by a LPS/TLR‐4 independent mechanism in response to P2X receptor stimulation

Extracellular ATP, released upon microbial infection, cell damage, or inflammation, acts as an alert signal toward immune cells by activating P2 receptors. The nucleotide causes microvesicle (MV) shedding from immune and nonimmune cells. Here, we show that IL‐18 associates with MVs shed by human ex vivo macrophages upon P2X receptor stimulation. MV shedding was potently induced by ATP and by the P2X7 agonist 3′‐benzoylbenzoyl adenosine 5′‐triphosphate, while it was greatly reduced by P2X irreversible inhibitor‐oxidized ATP and by the specific P2X7 inhibitors KN‐62, A‐740003, and A‐438079. Peculiarly, the P2X7 subtype was highly present in the MVs, while on the contrary the P2X3 and P2X4 subtypes were almost absent. The Ca²⁺ ionophore A23187 mimicked the effect of 3′‐benzoylbenzoyl adenosine 5′‐triphosphate suggesting that an intracellular Ca²⁺ increase was sufficient to evoke MV shedding. Caspase inhibitors Ac‐YVAD‐CMK or Z‐YVAD‐CMK did not block the cleavage of MV‐associated pro‐IL‐18. Pro‐IL‐18 formation in macrophages did not require pretreatment of cells with LPS, as the procytokine was already present in unprimed macrophages and did not decrease by incubating cells with the LPS‐binding antibiotic polymyxin B nor with the TLR‐4 intracellular inhibitor CLI‐095. These data reveal a nucleotide‐based mechanism responsible for the shedding of MV to which IL‐18 is associated.