Inflammasome-derived IL-1β regulates the production of GM-CSF by CD4(+) T cells and γδ T cells

Recent findings have demonstrated an indispensable role for GM-CSF in the pathogenesis of experimental autoimmune encephalomyelitis. However, the signaling pathways and cell populations that regulate GM-CSF production in vivo remain to be elucidated. Our work demonstrates that IL-1R is required for GM-CSF production after both TCR- and cytokine-induced stimulation of immune cells in vitro. Conventional αβ and γδ T cells were both identified to be potent producers of GM-CSF. Moreover, secretion of GM-CSF was dependent on IL-1R under both IL-12- and IL-23-induced stimulatory conditions. Deficiency in IL-1R conferred significant protection from experimental autoimmune encephalomyelitis, and this correlated with reduced production of GM-CSF and attenuated infiltration of inflammatory cells into the CNS. We also find that GM-CSF production in vivo is not restricted to a defined CD4(+) T cell lineage but is rather heterogeneously expressed in the effector CD4(+) T cell population. In addition, inflammasome-derived IL-1β upstream of IL-1R is a critical regulator of GM-CSF production by T cells during priming, and the adapter protein, MyD88, promotes GM-CSF production in both αβ and γδ T cells. These findings highlight the importance of inflammasome-derived IL-1β and the IL-1R/MyD88 signaling axis in the regulation of GM-CSF production.     

GM-CSF plays a key role in zymosan-stimulated human dendritic cells for activation of Th1 and Th17 cells

In this study, we compared the effects of zymosan and LPS on human monocyte-derived dendritic cells. The specific effects of zymosan on the expression of several key cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukins (IL-1α, IL-1β and IL-12 p70) were quite distinct from the effects of LPS. Unlike activation with LPS, DCs activated by zymosan expressed little or no IL-12 p70 due to lack of expression of the p35 subunit. However, treatment with zymosan resulted in a substantial increase in Th1 and Th17 cell-polarizing capacity of DCs. Furthermore, the GM-CSF secreted by zymosan-activated DCs enhanced IL-23 production, resulting in activation of a Th17 response. GM-CSF and IL-27, rather than IL-12 p70, were both major direct contributors to the activation of a Th1 response. This signaling mechanism is distinct and yet complementary to LPS-mediated T-cell activation. We suggest that this novel zymosan-induced GM-CSF-mediated signaling network may play a key role in regulating specific immune cell type activities.     

Autophagy controls IL-1beta secretion by targeting pro-IL-1beta for degradation

Autophagy is a key regulator of cellular homeostasis that can be activated by pathogen-associated molecules and recently has been shown to influence IL-1β secretion by macrophages. However, the mechanisms behind this are unclear. Here, we describe a novel role for autophagy in regulating the production of IL-1β in antigen-presenting cells. After treatment of macrophages with Toll-like receptor ligands, pro-IL-1β was specifically sequestered into autophagosomes, whereas further activation of autophagy with rapamycin induced the degradation of pro-IL-1β and blocked secretion of the mature cytokine. Inhibition of autophagy promoted the processing and secretion of IL-1β by antigen-presenting cells in an NLRP3- and TRIF-dependent manner. This effect was reduced by inhibition of reactive oxygen species but was independent of NOX2. Induction of autophagy in mice in vivo with rapamycin reduced serum levels of IL-1β in response to challenge with LPS. These data demonstrate that autophagy controls the production of IL-1β through at least two separate mechanisms: by targeting pro-IL-1β for lysosomal degradation and by regulating activation of the NLRP3 inflammasome.     

Induction of IL-4 release and upregulated expression of protease activated receptors by GM-CSF in P815 cells

GM-CSF has been showed to be able to induce up-regulated receptor and cytokine expression in mast cells in inflammatory conditions. However, little is known of its effects on protease activated receptor (PAR) expression and Th2 cytokine secretion from mast cells. In the present study, we examined potential influence of GM-CSF on mast cell PAR expression and IL-4 and IL-10 release by using flow cytometry analysis, quantitative real time PCR, ELISA and cellular activation of signaling ELISA (CASE) techniques. The results showed that GM-CSF induced up to 3.0-fold increase in IL-4 release from P815 cells, and FSLLRY-NH₂ and trans-cinnamoyl (tc)-YPGKF-NH₂ did not affect GM-CSF induced IL-4 release. GM-CSF reduced tryptase and trypsin induced IL-4 release by up to approximately 55.8% and 70.3%, respectively. GM-CSF elicited the upregulated expression of PAR-1, PAR-2, PAR-3 and PAR-4 mRNAs, but enhanced only PAR-4 protein expression in P815 cells. U0126, PD98059 and LY204002 almost completely abolished GM-CSF induced IL-4 release when they were preincubated with P815 cells for 30 min, indicating ERK and Akt cell signaling pathways may be involved in the event. In conclusion, GM-CSF can stimulate IL-4 release from mast cells through an ERK and Akt cell signaling pathway dependent, but PAR independent mechanism. GM-CSF may serve as a regulator for IL-4 production in mast cells and through which participates in the mast cell related inflammation.     

Absence of Appl2 sensitizes endotoxin shock through activation of PI3K/Akt pathway

Background

The adapter proteins Appl1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine domain, and leucine zipper motif 1) and Appl2 are highly homologous and involved in several signaling pathways. While previous studies have shown that Appl1 plays a pivotal role in adiponectin signaling and insulin secretion, the physiological functions of Appl2 are largely unknown.

Results

In the present study, the role of Appl2 in sepsis shock was investigated by using Appl2 knockout (KO) mice. When challenged with lipopolysaccharides (LPS), Appl2 KO mice exhibited more severe symptoms of endotoxin shock, accompanied by increased production of proinflammatory cytokines. In comparison with the wild-type control, deletion of Appl2 led to higher levels of TNF-α and IL-1β in primary macrophages. In addition, phosphorylation of Akt and its downstream effector NF-κB was significantly enhanced. By co-immunoprecipitation, we found that Appl2 and Appl1 interacted with each other and formed a complex with PI3K regulatory subunit p85α, which is an upstream regulator of Akt. Consistent with these results, deletion of Appl1 in macrophages exhibited characteristics of reduced Akt activation and decreased the production of TNFα and IL-1β when challenged by LPS.

Conclusions

Results of the present study demonstrated that Appl2 is a critical negative regulator of innate immune response via inhibition of PI3K/Akt/NF-κB signaling pathway by forming a complex with Appl1 and PI3K.

     

Activation of the PI3K pathway increases TLR-induced TNF-α and IL-6 but reduces IL-1β production in mast cells

Recognition of bacterial constituents by mast cells (MCs) is dependent on the presence of pattern recognition receptors, such as Toll-like receptors (TLRs). The final cellular response, however, depends on the influence of multiple environmental factors. In the current study we tested the hypothesis that the PI3K-activating ligands insulin-like growth factor-1 (IGF-1), insulin, antigen, and Steel Factor (SF) are able to modulate the TLR4-mediated production of proinflammatory cytokines in murine MCs. Costimulation with any of these ligands caused increased LPS-triggered secretion of IL-6 and TNF-α, but attenuated the production of IL-1β, though all three cytokines were produced in an NFκB-dependent manner. The pan-specific PI3K-inhibitor Wortmannin reverted the altered production of these cytokines. In agreement, MCs deficient for SHIP1, a negative regulator of the PI3K pathway, showed augmented secretion of IL-6/TNF-α and reduced production of IL-1β in response to LPS alone. The differential effects of IGF-1 on TLR4-mediated cytokine production were also observed in the context of TLR2 and IL-33 receptor-mediated MC activation. Importantly, these effects were seen in both bone marrow-derived and peritoneal MCs, suggesting general relevance for MCs. Using pharmacological and genetic tools, we could show that the p110δ isoform of PI3K is strongly implicated in SF-triggered suppression of LPS-induced IL-1β production. Costimulation with antigen was affected to a lesser extent. In conclusion, NFκB-dependent production of proinflammatory cytokines in MCs is differentially controlled by PI3K-activating ligand/receptor systems.     

IFN-beta differentially regulates CD40-induced cytokine secretion by human dendritic cells

We have previously shown that IFN-beta, a key cytokine associated with the early phase of the innate host defense, can prevent the generation of human Th1 cells. Specifically, we demonstrated that IFN-beta prevents the in vitro monocyte-derived mature dendritic cell (DC)-dependent differentiation of naive Th cells into IFN-gamma-secreting Th cells, as a result of its ability to inhibit DC IL-12 secretion. The goal of the present study was to identify how IFN-beta negatively regulates IL-12 secretion by DC. We report that in our Th cell differentiation model, DC IL-12 secretion is dependent on the CD40L/CD40 accessory pathway, and, utilizing a Th cell-free system, we find that IFN-beta inhibits anti-CD40 mAb-induced DC secretion of the p40 chain of the IL-12 heterodimer. In addition, we show that IFN-beta-mediated inhibition of CD40 signaling does not interfere with all signaling pathways emanating from CD40, since anti-CD40 mAb-induced DC IL-6 secretion is augmented by IFN-beta. Thus, our results demonstrate that signaling from CD40 is differentially regulated by IFN-beta. A second critical element of innate immunity involves the response against components of bacterial membranes such as LPS. DC respond to LPS by secreting IL-6 and IL-12. In contrast to CD40-dependent IL-6 and IL-12 secretion, we find that LPS-induced DC secretion of p40 IL-12 and IL-6 is not affected by IFN-beta. Our findings show that IFN-beta influences the generation of acquired immune responses through its regulation of CD40-dependent DC functions.     

Distinct roles for Nod2 protein and autocrine interleukin-1beta in muramyl dipeptide-induced mitogen-activated protein kinase activation and cytokine secretion in human macrophages

Elucidating factors regulating Crohn's disease-associated nucleotide-binding oligomerization domain 2 (Nod2) responses is critical to understanding the mechanisms of intestinal immune homeostasis. Stimulation of primary monocyte-derived macrophages by muramyl dipeptide (MDP), a component of bacterial peptidoglycan and specific Nod2 ligand, produces cytokines, including IL-1β. We found that IL-1β blockade profoundly inhibits MDP-induced cytokine production in human monocyte-derived macrophages, demonstrating a key role for IL-1β autocrine secretion in Nod2-mediated responses. Importantly, although MAPK activation has previously been attributed directly to Nod2 signaling, we determined that the IL-1β autocrine loop is responsible for the majority of MDP-induced MAPK activation. Because the critical effects of IL-1β autocrine secretion on MAPK activation are observed as early as 10 min after Nod2 stimulation, we hypothesized that secretion of IL-1β from preexisting intracellular pro-IL-1β stores is necessary for optimal MDP-mediated cytokine induction. Consistently, we detected IL-1β secretion within 10 min of MDP treatment. Moreover, caspase-1 inhibition significantly attenuates MDP-mediated early MAPK activation. Importantly, selective JNK/p38 activation is sufficient to rescue the decreased cytokine secretion during Nod2 stimulation in the absence of autocrine IL-1β. Finally, we found that the IL-1β autocrine loop significantly enhances responses by a broad range of pattern recognition receptors. Taken together, MDP stimulation activates Nod2 to process and release preexisting pro-IL-1β stores in a caspase-1-dependent fashion; this secreted IL-1β, in turn, contributes to the majority of MDP-initiated MAPK activation and leads to subsequent cytokine secretion. Our findings clarify mechanisms of IL-1β contributions to Nod2 responses and elucidate the dominant role of IL-1β in MDP-initiated MAPK and cytokine secretion.     

Intracellular invasion of Orientia tsutsugamushi activates inflammasome in asc-dependent manner

Orientia tsutsugamushi, a causative agent of scrub typhus, is an obligate intracellular bacterium, which escapes from the endo/phagosome and replicates in the host cytoplasm. O. tsutsugamushi infection induces production of pro-inflammatory mediators including interleukin-1β (IL-1β), which is secreted mainly from macrophages upon cytosolic stimuli by activating cysteine protease caspase-1 within a complex called the inflammasome, and is a key player in initiating and maintaining the inflammatory response. However, the mechanism for IL-1β maturation upon O. tsutsugamushi infection has not been identified. In this study, we show that IL-1 receptor signaling is required for efficient host protection from O. tsutsugamushi infection. Live Orientia, but not heat- or UV-inactivated Orientia, activates the inflammasome through active bacterial uptake and endo/phagosomal maturation. Furthermore, Orientia-stimulated secretion of IL-1β and activation of caspase-1 are ASC- and caspase-1- dependent since IL-1β production was impaired in Asc- and caspase-1-deficient macrophages but not in Nlrp3-, Nlrc4- and Aim2-deficient macrophages. Therefore, live O. tsutsugamushi triggers ASC inflammasome activation leading to IL-1β production, which is a critical innate immune response for effective host defense.     

Cutting Edge: Suppression of GM-CSF Expression in Murine and Human T Cells by IL-27

GM-CSF is a potent proinflammatory cytokine that plays a pathogenic role in the CNS inflammatory disease experimental autoimmune encephalomyelitis. As IL-27 alleviates experimental autoimmune encephalomyelitis, we hypothesized that IL-27 suppresses GM-CSF expression by T cells. We found that IL-27 suppressed GM-CSF expression in CD4(+) and CD8(+) T cells in splenocyte and purified T cell cultures. IL-27 suppressed GM-CSF in Th1, but not Th17, cells. IL-27 also suppressed GM-CSF expression by human T cells in nonpolarized and Th1- but not Th17-polarized PBMC cultures. In vivo, IL-27p28 deficiency resulted in increased GM-CSF expression by CNS-infiltrating T cells during Toxoplasma gondii infection. Although in vitro suppression of GM-CSF by IL-27 was independent of IL-2 suppression, IL-10 upregulation, or SOCS3 signaling, we observed that IL-27-driven suppression of GM-CSF was STAT1 dependent. Our findings demonstrate that IL-27 is a robust negative regulator of GM-CSF expression in T cells, which likely inhibits T cell pathogenicity in CNS inflammation.     

The development and function of memory regulatory T cells after acute viral infections

Leptospira interrogans is responsible for a zoonotic disease known to induce severe kidney dysfunction and inflammation. In this work, we demonstrate that L. interrogans induces NLRP3 inflammasome-dependent secretion of IL-1β through the alteration of potassium transport in bone marrow-derived macrophages. Lysosome destabilization also contributed to the IL-1β production upon stimulation with live, but not dead, bacteria. Using bone marrow-derived macrophages from various TLRs and nucleotide-binding oligomerization domain-deficient mice, we further determined that IL-1β production was dependent on TLR2 and TLR4, suggesting a participation of the leptospiral LPS to this process. Hypokaliemia in leptospirosis has been linked to the presence of glycolipoprotein, a cell wall component of L. interrogans that is known to inhibit the expression and functions of the Na/K-ATPase pump. We show in this study that glycolipoprotein activates the inflammasome and synergizes with leptospiral LPS to produce IL-1β, mimicking the effect of whole bacteria. These results were confirmed in vivo, as wild-type mice expressed more IL-1β in the kidney than TLR2/4-deficient mice 3 d postinfection with L. interrogans. Collectively, these findings provide the first characterization, to our knowledge, of bacteria-induced activation of the NLRP3 inflammasome through the downregulation of a specific host potassium transporter.     

Secretion of proinflammatory cytokines by normal human melanocytes in response to lipopolysaccharide

A large body of evidence suggests that epidermal melanocytes are an integral part of the skin immune system and can be considered immunocompetent cells. Recently, it has been reported that human melanocytes constitutively express Toll-like receptors and may be involved in the induction of several inflammatory cytokines. In the study the secretion of IL-1β, IL-6 and TNF-α by cultured normal melanocytes was investigated after stimulation with lipopolysaccharide. LPS increased the secretion of IL-1β in a dose-dependent manner. IL-1β stimulated release of IL-6 and TNF-α by melanocytes, whereas LPS activated production of TNF-α, but not of IL-6. These observations indicate that LPS can participate in the regulation of cytokine activity in normal human melanocytes and suggest that cytokines released by melanocytes could affect melanocytes themselves or/and other cells of the epidermis.     

TLR2/MyD88/NF-κB pathway, reactive oxygen species, potassium efflux activates NLRP3/ASC inflammasome during respiratory syncytial virus infection

Human respiratory syncytial virus (RSV) constitute highly pathogenic virus that cause severe respiratory diseases in newborn, children, elderly and immuno-compromised individuals. Airway inflammation is a critical regulator of disease outcome in RSV infected hosts. Although "controlled" inflammation is required for virus clearance, aberrant and exaggerated inflammation during RSV infection results in development of inflammatory diseases like pneumonia and bronchiolitis. Interleukin-1β (IL-1β) plays an important role in inflammation by orchestrating the pro-inflammatory response. IL-1β is synthesized as an immature pro-IL-1β form. It is cleaved by activated caspase-1 to yield mature IL-1β that is secreted extracellularly. Activation of caspase-1 is mediated by a multi-protein complex known as the inflammasome. Although RSV infection results in IL-1β release, the mechanism is unknown. Here in, we have characterized the mechanism of IL-1β secretion following RSV infection. Our study revealed that NLRP3/ASC inflammasome activation is crucial for IL-1β production during RSV infection. Further studies illustrated that prior to inflammasome formation; the "first signal" constitutes activation of toll-like receptor-2 (TLR2)/MyD88/NF-κB pathway. TLR2/MyD88/NF-κB signaling is required for pro-IL-1β and NLRP3 gene expression during RSV infection. Following expression of these genes, two "second signals" are essential for triggering inflammasome activation. Intracellular reactive oxygen species (ROS) and potassium (K(+)) efflux due to stimulation of ATP-sensitive ion channel promote inflammasome activation following RSV infection. Thus, our studies have underscored the requirement of TLR2/MyD88/NF-κB pathway (first signal) and ROS/potassium efflux (second signal) for NLRP3/ASC inflammasome formation, leading to caspase-1 activation and subsequent IL-1β release during RSV infection.     

Nicotinamide phosphoribosyltransferase is essential for interleukin-1beta-mediated dedifferentiation of articular chondrocytes via SIRT1 and extracellular signal-regulated kinase (ERK) complex signaling

Although much is known about interleukin (IL)-1β and its role as a key mediator of cartilage destruction in osteoarthritis, only limited information is available on IL-1β signaling in chondrocyte dedifferentiation. Here, we have characterized the molecular mechanisms leading to the dedifferentiation of primary cultured articular chondrocytes by IL-1β treatment. IL-1β or lipopolysaccharide, but not phorbol 12-myristate 13-acetate, retinoic acid, or epidermal growth factor, induced nicotinamide phosphoribosyltransferase (NAMPT) expression, showing the association of inflammatory cytokines with NAMPT regulation. SIRT1, in turn, was activated NAMPT-dependently, without any alteration in the expression level. Activation or inhibition of SIRT1 oppositevely regulates IL-1β-mediated chondrocyte dedifferentiation, suggesting this protein as a key regulator of chondrocytes phenotype. SIRT1 activation promotes induction of ERK and p38 kinase activities, but not JNK, in response to IL-1β. Subsequently, ERK and p38 kinase activated by SIRT1 also induce SIRT1 activation, forming a positive feedback loop to sustain downstream signaling of these kinases. Moreover, we found that the SIRT1-ERK complex, but not SIRT1-p38, is engaged in IL-1β-induced chondrocyte dedifferentiation via a Sox-9-mediated mechanism. JNK is activated by IL-1β and modulates dedifferentiation of chondrocytes, but this pathway is independent on NAMPT-SIRT1 signaling. Based on these findings, we propose that IL-1β induces dedifferentiation of articular chondrocytes by up-regulation of SIRT1 activity enhanced by both NAMPT and ERK signaling.     

DIURNAL,AGE, AND IMMUNE REGULATION OF INTERLEUKIN-1β AND INTERLEUKIN-1 TYPE 1 RECEPTOR IN THE MOUSE SUPRACHIASMATIC NUCLEUS

Circadian clocks serve to impose a near-24-h temporal architecture on an organism's physiology, metabolism, and behavior. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus functions as the master circadian pacemaker. There is growing evidence that immunomodulators, such as cytokines, may impinge on circadian timekeeping. We examined whether there is endogenous expression of the proinflammatory cytokine interleukin-1β (IL-1β) and its signaling receptor IL-1R1 in the SCN of young and older mice across the diurnal cycle. We found expression of both IL-1β and IL-1R1 in the young SCN, although only IL-1R1 displayed temporal regulation. In the older SCN, levels of IL-1β were expressed at lower levels than in the young SCN, and IL-1R1 did not vary across the 24?h. We also report age-related day-night variation of IL-1β and IL-1R1 in the paraventricular nucleus (PVN) of the hypothalamus. Further, we examined the effect of peripheral immune challenge on IL-1β and IL-1R1 in the SCN. We found that IL-1β immunoreactivity was not altered 6 or 24?h after a septic dose of lipopolysaccharide (LPS; 5?mg/kg), whereas IL-1R1 was significantly up-regulated in the SCN both 6 and 24?h after LPS. We also demonstrate cellular activation in the SCN 24?h following LPS treatment, as evidenced by increased c-Fos and p65-NF-κB (nuclear factor kappa B) expression. Our results indicate that IL-1β and its associated signaling system may play a role in mediating the response of the circadian timing system to immune challenge as well as potentially contributing to the basal functioning of the SCN clock. (Author correspondence: andrew.coogan@nuim.ie)