The Cag pathogenicity island and interaction between TLR2/NOD2 and NLRP3 regulate IL‐1β production in Helicobacter pylori infected dendritic cells

Helicobacter pylori colonization of the stomach affects about half of the world population and is associated with the development of gastritis, ulcers, and cancer. Polymorphisms in the IL1B gene are linked to an increased risk of H. pylori associated cancer, but the bacterial and host factors that regulate interleukin (IL)‐1β production in response to H. pylori infection remain unknown. Using murine BM‐derived DCs, we show that the bacterial virulence factors cytotoxin‐associated genes pathogenicity island and CagL, but not vacuolating cytotoxin A or CagA, regulate the induction of pro‐IL‐1β and the production of mature IL‐1β in response to H. pylori infection. We further show that the host receptors, Toll‐like receptor 2 (TLR2) and nucleotide‐binding oligomerization domain 2 (NOD2), but not NOD1, are required for induction of pro‐IL‐1β and NOD‐like receptor pyrin domain containing 3 (NLRP3) in H. pylori infected DCs. In contrast, NLRP3 and the adaptor ASC were essential for the activation of caspase‐1, processing of pro‐IL‐1β into IL‐1β, and IL‐1β secretion. Finally, we show that mice deficient in caspase‐1, IL‐1β, and IL‐1 receptor, but not NLRP3, are impaired in the clearance of CagA‐positive H. pylori from the stomach when compared with WT mice. These studies identify bacterial cag pathogenicity island and the cooperative interaction among host innate receptors TLR2, NOD2, and NLRP3 as important regulators of IL‐1β production in H. pylori infected DCs.     

Impaired NLRP3 inflammasome activity during fetal development regulates IL‐1β production in human monocytes

Interleukin‐1β (IL‐1β) production is impaired in cord blood monocytes. However, the mechanism underlying this developmental attenuation remains unclear. Here, we analyzed the extent of variability within the Toll‐like receptor (TLR)/NLRP3 inflammasome pathways in human neonates. We show that immature low CD14 expressing/CD16^pos monocytes predominate before 33 weeks of gestation, and that these cells lack production of the pro‐IL‐1β precursor protein upon LPS stimulation. In contrast, high levels of pro‐IL‐1β are produced within high CD14 expressing monocytes, although these cells are unable to secrete mature IL‐1β. The lack of secreted IL‐1β in these monocytes parallels a reduction of NLRP3 induction following TLR stimulation resulting in a lack of caspase‐1 activity before 29 weeks of gestation, whereas expression of the apoptosis‐associated speck‐like protein containing a CARD and function of the P2×7 receptor are preserved. Our analyses also reveal a strong inhibitory effect of placental infection on LPS/ATP‐induced caspase‐1 activity in cord blood monocytes. Lastly, secretion of IL‐1β in preterm neonates is restored to adult levels during the neonatal period, indicating rapid maturation of these responses after birth. Collectively, our data highlight important developmental mechanisms regulating IL‐1β responses early in gestation, in part due to a downregulation of TLR‐mediated NLRP3 expression. Such mechanisms may serve to limit potentially damaging inflammatory responses in a developing fetus.     

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.     

IL‐10 interferes directly with TCR‐induced IFN‐γ but not IL‐17 production in memory T cells

IL‐10 is a potent immunoregulatory and anti‐inflammatory cytokine. However, therapeutic trials in chronic inflammation have been largely disappointing. It is well established that IL‐10 can inhibit Th1 and Th2 cytokine production via indirect effects on APC. Less data are available about the influence of IL‐10 on IL‐17 production, a cytokine which has been recently linked to chronic inflammation. Furthermore, there are only few reports about a direct effect of IL‐10 on T cells. We demonstrate here that IL‐10 can directly interfere with TCR‐induced IFN‐γ production in freshly isolated memory T cells in the absence of APC. This effect was independent of the previously described effects of IL‐10 on T cells, namely inhibition of IL‐2 production and inhibition of CD28 signaling. In contrast, IL‐10 did not affect anti‐CD3/anti‐CD28‐induced IL‐17 production from memory T cells even in the presence of APC. This might have implications for the interpretation of therapeutic trials in patients with chronic inflammation where Th17 cells contribute to pathogenesis.     

TSLP production by dendritic cells is modulated by IL‐1β and components of the endoplasmic reticulum stress response

Thymic stromal lymphopoietin (TSLP) produced by epithelial cells acts on dendritic cells (DCs) to drive differentiation of T_H2‐cells, and is therefore important in allergic disease pathogenesis. However, DCs themselves make significant amounts of TSLP in response to microbial products, but little is known about the key downstream signals that induce and modulate this TSLP secretion from human DCs. We show that human monocyte derived DC (mDC) secretion of TSLP in response to Candida albicans and β‐glucans requires dectin‐1, Syk, NF‐κB, and p38 MAPK signaling. In addition, TSLP production by mDCs is greatly enhanced by IL‐1β, but not TNF‐α, in contrast to epithelial cells. Furthermore, TSLP secretion is significantly increased by signals emanating from the endoplasmic reticulum (ER) stress response, specifically the unfolded protein response sensors, inositol‐requiring transmembrane kinase/endonuclease 1 and protein kinase R‐like ER kinase, which are activated by dectin‐1 stimulation. Thus, TSLP production by mDCs requires the integration of signals from dectin‐1, the IL‐1 receptor, and ER stress signaling pathways. Autocrine TSLP production is likely to play a role in mDC‐controlled immune responses at sites removed from epithelial cell production of the cytokine, such as lymphoid tissue.     

The Gasdermin‐D pore acts as a conduit for IL‐1β secretion in mice

The pro‐inflammatory cytokine IL‐1β is well known for its role in host defense and the initiation of potent inflammatory responses. It is processed from its inactive pro‐form by the inflammatory caspase‐1 into its mature bioactive form, which is then released from the cell via an unconventional secretion mechanism. Recently, gasdermin‐D has been identified as a new target of caspase‐1. After proteolytical cleavage of gasdermin‐D, the N‐terminal fragment induces pyroptosis, a lytic cell death, by forming large permeability pores in the plasma membrane. Here we show using the murine system that gasdermin‐D is required for IL‐1β secretion by macrophages, dendritic cells and partially in neutrophils, and that secretion is a cell‐lysis‐independent event. Liposome transport assays in vitro further demonstrate that gasdermin‐D pores are large enough to allow the direct release of IL‐1β. Moreover, IL‐18 and other small soluble cytosolic proteins can also be released in a lysis‐independent but gasdermin‐D‐dependent mode, suggesting that the gasdermin‐D pores allow passive the release of cytosolic proteins in a size‐dependent manner.     

IFN‐γ‐producing NKT cells exacerbate sepsis by enhancing C5a generation via IL‐10‐mediated inhibition of CD55 expression on neutrophils

A role for NKT cells has been implicated in sepsis, but the mechanism by which NKT cells contribute to sepsis remains unclear. Here, we examined WT and NKT‐cell‐deficient mice of C57BL/6 background during cecal ligation and puncture‐induced sepsis. The levels of C5a, IFN‐γ, and IL‐10 were higher in the serum and peritoneal fluid of WT mice than in those of CD1d^?/? mice, while the mortality rate was lower in CD1d^?/? mice than in WT mice. C5a blockade decreased mortality of WT mice during sepsis, whereas it did not alter that of CD1d^?/? mice. As assessed by intracellular staining, NKT cells expressed IFN‐γ, while neutrophils expressed IL‐10. Upon coculture, IL‐10‐deficient NKT cells enhanced IL‐10 production by WT, but not IFN‐γR‐deficient, neutrophils. Meanwhile, CD1d^?/? mice exhibited high CD55 expression on neutrophils during sepsis, whereas those cells from WT mice expressed minimal levels of CD55. Recombinant IL‐10 administration into CD1d^?/? mice reduced CD55 expression on neutrophils. Furthermore, adoptive transfer of sorted WT, but not IFN‐γ‐deficient, NKT cells into CD1d^?/? mice suppressed CD55 expression on neutrophils, but increased IL‐10 and C5a levels. Taken together, IFN‐γ‐producing NKT cells enhance C5a generation via IL‐10‐mediated inhibition of CD55 expression on neutrophils, thereby exacerbating sepsis.     

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.     

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.     

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.     

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.     

IFN‐λ‐mediated IL‐12 production in macrophages induces IFN‐γ production in human NK cells

With increasing interest in alternative options to interferon‐alpha‐based treatments, IFN‐λ has shown therapeutic promise in a variety of diseases. Although the antiviral activity of IFN‐λ has been extensively studied, there is limited knowledge regarding the immunological functions of IFN‐λ and how these differ from those of other classes of IFNs. In this study, we investigated the effects of IFN‐λ on primary human NK cells, both in a direct and indirect capacity. We demonstrate that in contrast to interferon‐alpha, IFN‐λ is unable to directly stimulate NK cells, due to the absence of IFN‐λ receptor chain 1 (IFN‐λR1) on NK cells. However, IFN‐λ, in combination with TLR4 challenge, is able to induce the production of select members of the IL‐12 family of cytokines in monocyte‐derived macrophages. We further show that through macrophage‐mediated IL‐12 production, IFN‐λ is able to indirectly affect NK cells and ultimately induce IFN‐γ production.     

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.     

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.     

IL‐1β activation in response to Staphylococcus aureus lung infection requires inflammasome‐dependent and independent mechanisms

Maintaining balanced levels of IL‐1β is extremely important to avoid host tissue damage during infection. Our goal was to understand the mechanisms behind the reduced pathology and decreased bacterial burdens in Ifnlr1^?/? mice during lung infection with Staphylococcus aureus. Intranasal infection of Ifnlr1^?/? mice with S. aureus led to significantly improved bacterial clearance, survival and decrease of proinflammatory cytokines in the airway including IL‐1β. Ifnlr1^?/? mice treated with recombinant IL‐1β displayed increased bacterial burdens in the airway and lung. IL‐1β levels in neutrophils from Ifnlr1^?/? infected mice lungs were decreased when compared to neutrophils from WT mice. Mice lacking NLRP3 and caspase‐1 had reduced IL‐1β levels 4 h after infection, due to reductions or absence of active caspase‐1 respectively, but levels at 24 h were comparable to WT infected mice. Ifnlr1^?/? infected mice had decreases in both active caspase‐1 and neutrophil elastase indicating an important role for the neutrophil serine protease in IL‐1β processing. By inhibiting neutrophil elastase, we were able to decrease IL‐1β levels by 39% in Nlrp3^?/? infected mice when compared to WT mice. These results highlight the crucial role of both proteases in IL‐1β processing, via inflammasome‐dependent and ‐independent mechanisms.