Distinct Licensing of IL-18 and IL-1β Secretion in Response to NLRP3 Inflammasome Activation

Inflammasome activation permits processing of interleukins (IL)-1β and 18 and elicits cell death (pyroptosis). Whether these responses are independently licensed or are “hard-wired” consequences of caspase-1 (casp1) activity has not been clear. Here, we show that that each of these responses is independently regulated following activation of NLRP3 inflammasomes by a “non-canonical” stimulus, the secreted Listeria monocytogenes (Lm) p60 protein. Primed murine dendritic cells (DCs) responded to p60 stimulation with reactive oxygen species (ROS) production and secretion of IL-1β and IL-18 but not pyroptosis. Inhibitors of ROS production inhibited secretion of IL-1β, but did not impair IL-18 secretion. Furthermore, DCs from caspase-11 (casp11)-deficient 129S6 mice failed to secrete IL-1β in response to p60 but were fully responsive for IL-18 secretion. These findings reveal that there are distinct licensing requirements for processing of IL-18 versus IL-1β by NLRP3 inflammasomes.     

The NLRP3 Inflammasome and IL-1β Accelerate Immunologically Mediated Pathology in Experimental Viral Fulminant Hepatitis

Viral fulminant hepatitis (FH) is a severe disease with high mortality resulting from excessive inflammation in the infected liver. Clinical interventions have been inefficient due to the lack of knowledge for inflammatory pathogenesis in the virus-infected liver. We show that wild-type mice infected with murine hepatitis virus strain-3 (MHV-3), a model for viral FH, manifest with severe disease and high mortality in association with a significant elevation in IL-1β expression in the serum and liver. Whereas, the viral infection in IL-1β receptor-I deficient (IL-1R1^-/-) or IL-1R antagonist (IL-1Ra) treated mice, show reductions in virus replication, disease progress and mortality. IL-1R1 deficiency appears to debilitate the virus-induced fibrinogen-like protein-2 (FGL2) production in macrophages and CD45⁺Gr-1^high neutrophil infiltration in the liver. The quick release of reactive oxygen species (ROS) by the infected macrophages suggests a plausible viral initiation of NLRP3 inflammasome activation. Further experiments show that mice deficient of p47^phox, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit that controls acute ROS production, present with reductions in NLRP3 inflammasome activation and subsequent IL-1β secretion during viral infection, which appears to be responsible for acquiring resilience to viral FH. Moreover, viral infected animals in deficiencies of NLRP3 and Caspase-1, two essential components of the inflammasome complex, also have reduced IL-1β induction along with ameliorated hepatitis. Our results demonstrate that the ROS/NLRP3/IL-1β axis institutes an essential signaling pathway, which is over activated and directly causes the severe liver disease during viral infection, which sheds light on development of efficient treatments for human viral FH and other severe inflammatory diseases.     

Enterohemorrhagic Escherichia coli Specific Enterohemolysin Induced IL-1β in Human Macrophages and EHEC-Induced IL-1β Required Activation of NLRP3 Inflammasome

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major foodborne pathogen causing hemorrhagic colitis and hemolytic-uremic syndrome. The role of EHEC O157:H7-enterohemolysin (Ehx) in the pathogenesis of infections remains poorly defined. In this study, we used gene deletion and complement methods to confirm its putative functions. Results demonstrated that, in THP-1 cells, EHEC O157:H7-Ehx is associated with greater production of extracellular interleukin (IL)-1β than other cytokines. The data also showed that EHEC O157:H7-Ehx contributed to cytotoxicity in THP-1 cells, causing the release of lactate dehydrogenase (LDH). Although we observed a positive correlation between IL-1β production and cytotoxicity in THP-1 cells infected with different EHEC O157:H7 strains, our immunoblot results showed that the majority of IL-1β in the supernatant was mature IL-1β and not the pro-IL-1β that can be released after cell death. However, EHEC O157:H7-Ehx had no detectable effect on biologically inactive pro-IL-1β at the mRNA or protein synthesis levels. Neither did it affect the expression of apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, or NOD-like receptor family pyrin domain containing 3 (NLRP3). RNA interference experiments showed that EHEC O157:H7-induced IL-1β production required the involvement of ASC, caspase-1, and NLRP3 expression in THP-1 cells. Our results demonstrate that Ehx plays a crucial role in EHEC O157:H7-induced IL-1β production and its cytotoxicity to THP-1 cells. NLRP3 inflammasome activation is also involved in EHEC O157:H7-stimulated IL-1β release.     

Neutrophil extracellular trap formation is associated with IL-1β and autophagy-related signaling in gout

Background

Gout is a prevalent inflammatory arthritis affecting 1–2% of adults characterized by activation of innate immune cells by monosodium urate (MSU) crystals resulting in the secretion of interleukin-1β (IL-1β). Since neutrophils play a major role in gout we sought to determine whether their activation may involve the formation of proinflammatory neutrophil extracellular traps (NETs) in relation to autophagy and IL-1β.

Methodology/Principal Findings

Synovial fluid neutrophils from six patients with gout crisis and peripheral blood neutrophils from six patients with acute gout and six control subjects were isolated. MSU crystals, as well as synovial fluid or serum obtained from patients with acute gout, were used for the treatment of control neutrophils. NET formation was assessed using immunofluorescence microscopy. MSU crystals or synovial fluid or serum from patients induced NET formation in control neutrophils. Importantly, NET production was observed in neutrophils isolated from synovial fluid or peripheral blood from patients with acute gout. NETs contained the alarmin high mobility group box 1 (HMGB1) supporting their pro-inflammatory potential. Inhibition of phosphatidylinositol 3-kinase signaling or phagolysosomal fusion prevented NET formation, implicating autophagy in this process. NET formation was driven at least in part by IL-1β as demonstrated by experiments involving IL-1β and its inhibitor anakinra.

Conclusions/Significance

These findings document for the first time that activation of neutrophils in gout is associated with the formation of proinflammatory NETs and links this process to both autophagy and IL-1β. Modulation of the autophagic machinery may represent an additional therapeutic study in crystalline arthritides.     

Unprenylated RhoA Contributes to IL-1β Hypersecretion in Mevalonate Kinase Deficiency Model through Stimulation of Rac1 Activity

Protein prenylation is a post-translational modification whereby non-sterol isoprenoid lipid chains are added, thereby modifying the molecular partners with which proteins interact. The autoinflammatory disease mevalonate kinase deficiency (MKD) is characterized by a severe reduction in protein prenylation. A major class of proteins that are affected are small GTPases, including Rac1 and RhoA. It is not clear how protein prenylation of small GTPases relates to GTP hydrolysis activity and downstream signaling. Here, we investigated the contribution of RhoA prenylation to the biochemical pathways that underlie MKD-associated IL-1β hypersecretion using human cell cultures, Rac1 and RhoA protein variants, and pharmacological inhibitors. We found that when unprenylated, the GTP-bound levels of RhoA decrease, causing a reduction in GTPase activity and increased protein kinase B (PKB) phosphorylation. Cells expressing unprenylated RhoA produce increased levels of interleukin 1β mRNA. Of other phenotypic cellular changes seen in MKD, increased mitochondrial potential and mitochondrial elongation, only mitochondrial elongation was observed. Finally, we show that pharmacological inactivation of RhoA boosts Rac1 activity, a small GTPase whose activity was earlier implied in MKD pathogenesis. Together, our data show that RhoA plays a pivotal role in MKD pathogenesis through Rac1/PKB signaling toward interleukin 1β production and elucidate the effects of protein prenylation in monocytes.     

Oleanolic acid analogs as NO,TNF-α and IL-1β inhibitors: Synthesis,biological evaluation and docking studies

A series of oleanolic acid analogs, characterized by structural modifications at position C-3 and C-28 of oleanane skeleton were synthesized and assessed for antiinflammatory potential towards lipopolysaccharide (LPS) induced nitric oxide (NO) production in macrophages. Results revealed that all the synthesized analogs of oleanolic acid inhibit NO production with an IC₅₀ of 2.66–41.7 μM as compared to the specific nitric oxide synthase (NOS) inhibitor, L-NAME (IC₅₀ = 69.21 and 73.18 μM on RAW 264.7 and J774A.1 cells, respectively) without affecting the cell viability when tested at their half maximal concentration. The most potent NO inhibitors (2, 8, 9 and 10) at a concentration of 20 μg/mL also demonstrated mild inhibition (27.9–51.9%) of LPS-induced tumor necrosis factor alpha (TNF-α) and weak inhibition (11.1–37.5%) towards interleukin 1-beta (IL-1β) production in both the cells. The present study paves a direction that analogs of oleanolic acid can be employed as a lead in the development of potent NO inhibitors. Molecular docking studies also showed that 10 (with top Goldscore docking pose 19.05) showed similar interaction as that of co-crystallized inhibitor and, thereby, helps to design the potent inhibitors of TNF-α.     

Synthesis and Cloning of a Gene Encoding Human Interleukin-1β (IL-1β)

Abstract

The design, synthesis and cloning of a 466 base-pair DNA duplex coding for IL-lp is described.     

Night work effects on salivary cytokines TNF,IL-1β and IL-6

Shift work is unavoidable in modern societies, but at the same time disrupts biological rhythms and contributes to social distress and disturbance of sleep, health and well-being of shift workers. Shift work has been associated with some chronic diseases in which a chronic inflammatory condition may play a role. However, few studies investigating the association of cytokine and other inflammation markers with shift workers have been published in recent years. In this study we evaluated the effects of permanent night work on the production of tumor necrosis factor (TNF), interleukin-1β (IL-1β), IL-6 and melatonin in saliva. Another aim was to demonstrate the benefit of the use of salivary cytokines for studies in chronobiology, since it is an easy and non-invasive method that allows for sampling at several times. Thirty-eight healthy male workers, being 21 day workers and 17 night workers, agreed to participate in this study. Sleep was evaluated by actigraphy and activity protocols. Saliva was collected during three workdays approximately at the middle of the work shift and at bed and wake times of the main sleep episode. Saliva samples were then analyzed by enzyme-linked immunosorbent assay to measure TNF, IL-1β, IL-6 and melatonin levels, and the results were submitted to non-parametric statistical analysis. The use of saliva instead of blood allowed for a greater number of samples from the same subjects, allowing identifying alterations in the daily production patterns of salivary cytokines TNF, IL-1β and IL-6 that probably are linked to night work. Salivary TNF and IL-1β levels were similar for day and night workers, with higher daily production after awakening, in the morning hours for day workers and in the afternoon for night workers. Both groups presented a significant daily variation pattern of these two cytokines. Day and night workers produced similar amounts of salivary IL-6. Nevertheless, the daily variation pattern observed among day workers, with a peak after awakening, was absent among night workers. Thus, in our study, night workers showed partially adjusted daily variation patterns for salivary TNF and IL-1β, not seen for salivary IL-6. Results for salivary IL-6 could be better explained as a consequence of circadian disruption due to permanent night work. Our results suggest that the whole circadian system, including clocks and pineal gland, is involved in regulating cytokine profile in shift workers and that a coordinated production of these cytokines, important for an adequate inflammatory response, could be disturbed by shift work. The distinct effects that shift work may have on different cytokines could give some cues about the mechanisms involved in this association.     

Eccrine Sweat Contains IL-1α, IL-1β and IL-31 and Activates Epidermal Keratinocytes as a Danger Signal

Eccrine sweat is secreted onto the skin''s surface and is not harmful to normal skin, but can exacerbate eczematous lesions in atopic dermatitis. Although eccrine sweat contains a number of minerals, proteins, and proteolytic enzymes, how it causes skin inflammation is not clear. We hypothesized that it stimulates keratinocytes directly, as a danger signal. Eccrine sweat was collected from the arms of healthy volunteers after exercise, and levels of proinflammatory cytokines in the sweat were quantified by ELISA. We detected the presence of IL-1α, IL-1β, and high levels of IL-31 in sweat samples. To investigate whether sweat activates keratinocytes, normal human keratinocytes were stimulated with concentrated sweat. Western blot analysis demonstrated the activation of NF-κB, ERK, and JNK signaling in sweat-stimulated keratinocytes. Real-time PCR using total RNA and ELISA analysis of supernatants showed the upregulation of IL-8 and IL-1β by sweat. Furthermore, pretreatment with IL-1R antagonist blocked sweat-stimulated cytokine production and signal activation, indicating that bioactive IL-1 is a major factor in the activation of keratinocytes by sweat. Moreover, IL-31 seems to be another sweat stimulator that activates keratinocytes to produce inflammatory cytokine, CCL2. Sweat is secreted onto the skin''s surface and does not come into contact with keratinocytes in normal skin. However, in skin with a defective cutaneous barrier, such as atopic dermatitis-affected skin, sweat cytokines can directly act on epidermal keratinocytes, resulting in their activation. In conclusion, eccrine sweat contains proinflammatory cytokines, IL-1 and IL-31, and activates epidermal keratinocytes as a danger signal.     

Neutrophils require SHP1 to regulate IL-1β production and prevent inflammatory skin disease

The regulation of neutrophil recruitment, activation, and disposal is pivotal for circumscribed inflammation. SHP1(Y208N/Y208N) mutant mice develop severe cutaneous inflammatory disease that is IL-1R dependent. Genetic reduction in neutrophil numbers and neutrophilic responses to infection is sufficient to prevent the spontaneous initiation of this disease. Neutrophils from SHP1(Y208N/Y208N) mice display increased pro-IL-1β production due to altered responses to MyD88-dependent and MyD88-independent signals. The IL-1R-dependent inflammatory disease in SHP1(Y208N/Y208N) mice develops independently of caspase 1 and proteinase 3 and neutrophil elastase. In response to Fas ligand, a caspase 1-independent inducer of IL-1β production, neutrophils from SHP1(Y208N/Y208N) mice produce elevated levels of IL-1β but display reduced caspase 3 and caspase 7 activation. In neutrophils deficient in SHP1, IL-1β induces high levels of pro-IL-1β suggesting the presence of a paracrine IL-1β loop. These data indicate that the neutrophil- and IL-1-dependent disease in SHP1(Y208N/Y208N) mice is a consequence of loss of negative regulation of TLR and IL-1R signaling.     

Commensal Bacteria and MAMPs Are Necessary for Stress-Induced Increases in IL-1β and IL-18 but Not IL-6, IL-10 or MCP-1

Regular interactions between commensal bacteria and the enteric mucosal immune environment are necessary for normal immunity. Alterations of the commensal bacterial communities or mucosal barrier can disrupt immune function. Chronic stress interferes with bacterial community structure (specifically, α-diversity) and the integrity of the intestinal barrier. These interferences can contribute to chronic stress-induced increases in systemic IL-6 and TNF-α. Chronic stress, however, produces many physiological changes that could indirectly influence immune activity. In addition to IL-6 and TNF-α, exposure to acute stressors upregulates a plethora of inflammatory proteins, each having unique synthesis and release mechanisms. We therefore tested the hypothesis that acute stress-induced inflammatory protein responses are dependent on the commensal bacteria, and more specifically, lipopolysaccharide (LPS) shed from Gram-negative intestinal commensal bacteria. We present evidence that both reducing commensal bacteria using antibiotics and neutralizing LPS using endotoxin inhibitor (EI) attenuates increases in some (inflammasome dependent, IL-1 and IL-18), but not all (inflammasome independent, IL-6, IL-10, and MCP-1) inflammatory proteins in the blood of male F344 rats exposed to an acute tail shock stressor. Acute stress did not impact α- or β- diversity measured using 16S rRNA diversity analyses, but selectively reduced the relative abundance of Prevotella. These findings indicate that commensal bacteria contribute to acute stress-induced inflammatory protein responses, and support the presence of LPS-mediated signaling in stress-evoked cytokine and chemokine production. The selectivity of the commensal bacteria in stress-evoked IL-1β and IL-18 responses may implicate the inflammasome in this response.     

The combination of early and rapid type I IFN, IL-1α, and IL-1β production are essential mediators of RNA-like adjuvant driven CD4+ Th1 responses

There is a growing need for novel vaccine adjuvants that can provide safe and potent T-helper type 1 (Th1) activity. RNA-like immune response modifiers (IRMs) are candidate T-cell adjuvants that skew acquired immune responses towards a Th1 phenotype. We set out to delineate the essential signaling pathways by which the RNA-like IRMs, resiquimod (R-848) and polyinosinic:polycytidylic acid (poly I:C), augment CD4+ T-helper 1 (Th1) responses. Highly purified murine conventional dendritic cells (cDCs) and conventional CD4+ T-cells were co-cultured in allogeneic and MHC congenic mixed leukocyte reactions. The activation of CD4+ Th1 cells was examined utilizing cells from mice deficient in specific RNA-sensing pattern recognition receptors and signaling mediators. R-848 and poly I:C stimulation of Type I interferon production and signaling in cDCs was essential but not sufficient for driving CD4+ Th1 responses. The early and rapid production of IL-1α and IL-1β was equally critical for the optimal activation of Th1 CD4+ T-cells. R-848 activation of Toll-like receptor 7/MyD88-dependent signaling in cDCs led to a rapid upregulation of pro-IL-1α and pro-IL-1β production compared to poly I:C activation of MyD88-independent signaling pathways. The in vitro data show that CD4+ T-cell adjuvant activity of RNA-like IRMs is mediated by a critical combination of early and rapid Type I interferon, IL-1α and IL-1β production. These results provide important insights into the key signaling pathways responsible for RNA-like IRM CD4+ Th1 activation. A better understanding of the critical signaling pathways by which RNA-like IRMs stimulate CD4+ Th1 responses is relevant to the rational design of improved vaccine adjuvants.     

Pro-prion Binds Filamin A,Facilitating Its Interaction with Integrin β1, and Contributes to Melanomagenesis

Filamin A (FLNA) is an integrator of cell mechanics and signaling. The spreading and migration observed in FLNA sufficient A7 melanoma cells but not in the parental FLNA deficient M2 cells have been attributed to FLNA. In A7 and M2 cells, the normal prion (PrP) exists as pro-PrP, retaining its glycosylphosphatidyl-inositol (GPI) anchor peptide signal sequence (GPI-PSS). The GPI-PSS of PrP has a FLNA binding motif and binds FLNA. Reducing PrP expression in A7 cells alters the spatial distribution of FLNA and organization of actin and diminishes cell spreading and migration. Integrin β1 also binds FLNA. In A7 cells, FLNA, PrP, and integrin β1 exist as two independent, yet functionally linked, complexes; they are FLNA with PrP or FLNA with integrin β1. Reducing PrP expression in A7 cells decreases the amount of integrin β1 bound to FLNA. A PrP GPI-PSS synthetic peptide that crosses the cell membrane inhibits A7 cell spreading and migration. Thus, in A7 cells FLNA does not act alone; the binding of pro-PrP enhances association between FLNA and integrin β1, which then promotes cell spreading and migration. Pro-PrP is detected in melanoma in situ but not in melanocyte. Invasive melanoma has more pro-PrP. The binding of pro-PrP to FLNA, therefore, contributes to melanomagenesis.