Role of Toll interleukin-1 receptor (IL-1R) 8, a negative regulator of IL-1R/Toll-like receptor signaling, in resistance to acute Pseudomonas aeruginosa lung infection

Toll interleukin-1 receptor (IL-1R) 8 (TIR8), also known as single Ig IL-1 receptor (IL-R)-related molecule, or SIGIRR, is a member of the IL-1R-like family, primarily expressed by epithelial cells. Current evidence suggests that TIR8 plays a nonredundant role as a negative regulator in vivo under different inflammatory conditions that are dependent on IL-R and Toll-like receptor (TLR) activation. In the present study, we examined the role of TIR8 in innate resistance to acute lung infections caused by Pseudomonas aeruginosa, a Gram-negative pathogen responsible for life-threatening infections in immunocompromised individuals and cystic fibrosis patients. We show that Tir8 deficiency in mice was associated with increased susceptibility to acute P. aeruginosa infection, in terms of mortality and bacterial load, and to exacerbated local and systemic production of proinflammatory cytokines (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], IL-1β, and IL-6) and chemokines (CXCL1, CXCL2, and CCL2). It has been reported that host defense against P. aeruginosa acute lung infection can be improved by blocking IL-1 since exaggerated IL-1β production may be harmful for the host in this infection. In agreement with these data, IL-1RI deficiency rescues the phenotype observed in Tir8-deficient mice: in Tir8-/- IL-1RI-/- double knockout mice we observed higher survival rates, enhanced bacterial clearance, and reduced levels of local and systemic cytokine and chemokine levels than in Tir8-deficient mice. These results suggest that TIR8 has a nonredundant effect in modulating the inflammation caused by P. aeruginosa, in particular, by negatively regulating IL-1RI signaling, which plays a major role in the pathogenesis of this infectious disease.

Microbiological composition associated with interleukin-1 gene polymorphism in subjects undergoing supportive periodontal therapy

BACKGROUND: Interleukin-1 gene polymorphism (IL-1 gene) has been associated with periodontitis. The present study examined the subgingival microbiota by IL-1 gene status in subjects undergoing supportive periodontal therapy (SPT). METHODS: A total of 151 subjects with known IL-1 gene status (IL-1A +4845/IL-1B -3954) (IL-1 gene) were included in this study. Clinical data and subgingival plaque samples (40 taxa) were collected. These taxa were determined by the checkerboard DNA-DNA hybridization method. RESULTS: Gender, smoking habits (n-par tests), age, and clinical periodontal conditions did not differ by IL-1 gene status. IL-1 gene-negative subjects had a higher total bacterial load (mean difference, 480.4 x 10(5); 95% confidence interval [CI], 77 to 884 x 10(5); P <0.02). The levels of Actinobacillus actinomycetemcomitans (mean difference, 30.7 x 10(5); 95% CI, 2.2 to 59.5 x 10(5); P <0.05), Eubacterium nodatum (mean difference, 4.2 x 10(5); 95% CI, 0.6 to 7.8 x 10(5); P <0.02), Porphyromonas gingivalis (mean difference, 17.9 x 10(5); 95% CI, 1.2 to 34.5 x 10(5); P <0.05), and Streptococcus anginosus (mean difference, 4.0 x 10(5); 95% CI, 0.2 to 7.2 x 10(5); P <0.05) were higher in IL-1 gene-negative subjects, an observation specifically found at sites with probing depths <5.0 mm. CONCLUSIONS: Bleeding on probing did not differ by IL gene status, reflecting clinical SPT efficacy. IL-1 gene-negative subjects had higher levels of periodontal pathogens. This may suggest that among subjects undergoing SPT, a lower bacterial load is required in IL-1 gene-positive subjects to develop the same level of periodontitis as in IL-1 gene-negative subjects.

Immunohistochemical demonstration of interleukin-1 beta induced changes in acute-phase proteins and albumin in rat liver

Interleukin-1 beta is a potent mediator of the acute-phase response. However, the effects of interleukin-1 beta administration on the topic in vivo production of acute-phase proteins and albumin are so far not well understood. Overnight fasted rats were subcutaneously injected with 0.2 mL 0.9% NaCl (control group) or 6.25 micrograms recombinant human interleukin-1 beta, and rectal temperature was measured at intervals up to 48 h. Livers were perfused-fixed in vivo prior to injection (base-line), and at 9, 24, and 48 h following the interleukin-1 beta injection. Fibrinogen, orosomucoid (alpha 1-acid glycoprotein) and albumin were immunostained using a streptavidin-biotin-immunoperoxidase technique. Rectal temperature peaked 5 h after the single interleukin-1 beta injection, and fell gradually to base-line values by 24 h. Prior to injection only a few hepatocytes, randomly scattered throughout the liver lobule, stained positive for fibrinogen and orosomucoid. In contrast, all hepatocytes stained uniformly positive for fibrinogen and orosomucoid 9 h after interleukin-1 beta injection, whereas at 24 h a predominant centrilobular staining pattern occurred. Due to fasting, albumin positive hepatocytes were already reduced at base-line in both groups. Interleukin-1 beta induced a further significant loss of albumin positive cells in the periportal zone (35 +/- 21%) at 9 h when compared with controls (58 +/- 11%, p = 0.037). In conclusion, subcutaneous interleukin-1 beta (probably by stimulation of interleukin-6) strongly induces fibrinogen and orosomucoid expression in rat liver, and suppresses immunohistochemically stainable albumin in a heterogenous way, mainly in the periportal zone.

MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responses

OBJECTIVE: MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue-specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA-140 (miR-140). METHODS: To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR-140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin-1beta (IL-1beta) on miR-140 expression. Double-stranded miR-140 (ds-miR-140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA. RESULTS: Microarray analysis showed that miR-140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR-140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR-140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL-1beta suppressed miR-140 expression. Transfection of chondrocytes with ds-miR-140 down-regulated IL-1beta-induced ADAMTS5 expression and rescued the IL-1beta-dependent repression of AGGRECAN gene expression. CONCLUSION: This study shows that miR-140 has a chondrocyte differentiation-related expression pattern. The reduction in miR-140 expression in OA cartilage and in response to IL-1beta may contribute to the abnormal gene expression pattern characteristic of OA.

Development of an Interleukin-1β Vaccine in Patients with Type 2 Diabetes

Interleukin-1β (IL-1β) is a key cytokine involved in inflammatory illnesses including rare hereditary diseases and common chronic inflammatory conditions as gout, rheumatoid arthritis, and type 2 diabetes mellitus, suggesting reduction of IL-1β activity as new treatment strategy. The objective of our study was to assess safety, antibody response, and preliminary efficacy of a novel vaccine against IL-1β. The vaccine hIL1bQb consisting of full-length, recombinant IL-1β coupled to virus-like particles was tested in a preclinical and clinical, randomized, placebo-controlled, double-blind study in patients with type 2 diabetes. The preclinical simian study showed prompt induction of IL-1β-specific antibodies upon vaccination, while neutralizing antibodies appeared with delay. In the clinical study with 48 type 2 diabetic patients, neutralizing IL-1β-specific antibody responses were detectable after six injections with doses of 900 µg. The development of neutralizing antibodies was associated with higher number of study drug injections, lower baseline body mass index, improvement of glycemia, and C-reactive protein (CRP). The vaccine hIL1bQb was safe and well-tolerated with no differences regarding adverse events between patients receiving hIL1bQb compared to placebo. This is the first description of a vaccine against IL-1β and represents a new treatment option for IL-1β-dependent diseases such as type 2 diabetes mellitus (ClinicalTrials.gov NCT00924105).Molecular Therapy (2016); doi:10.1038/mt.2015.227.

Fetuin-A and cystatin C are endogenous inhibitors of human meprin metalloproteases

Meprin and , zinc metalloproteinases, play significant roles in inflammation, including inflammatory bowel disease (IBD), possibly by activating cytokines, like interleukin 1 , interleukin 18, or tumor growth factor . Although a number of potential activators for meprins are known, no endogenous inhibitors have been identified. In this work, we analyzed the inhibitory potential of human plasma and identified bovine fetuin-A as an endogenous meprin inhibitor with a K(i) (inhibition constant) of 4.2 × 10(-5) M for meprin and a K(i) of 1.1 × 10(-6) M meprin . This correlated with data obtained for a fetuin-A homologue from carp (nephrosin inhibitor) that revealed a potent meprin and inhibition (residual activities of 27 and 22%, respectively) at a carp fetuin concentration of 1.5 × 10(-6) M. Human fetuin-A is a negative acute phase protein involved in inflammatory diseases, thus being a potential physiological regulator of meprin activity. We report kinetic studies of fetuin-A with the proteolytic enzymes astacin, LAST, LAST_MAM, trypsin, and chymotrypsin, indeed demonstrating that fetuin-A is a broad-range protease inhibitor. Fetuin-A inhibition of meprin activity was 40 times weaker than that of meprin activity. Therefore, we tested cystatin C, a protein structurally closely related to fetuin-A. Indeed, cystatin C was an inhibitor for human meprin (K(i) = 8.5 × 10(-6) M) but, interestingly, not for meprin . Thus, the identification of fetuin-A and cystatin C as endogenous proteolytic regulators of meprin activity broadens our understanding of the proteolytic network in plasma.

Brain-borne IL-1 adjusts glucoregulation and provides fuel support to astrocytes and neurons in an autocrine/paracrine manner.

It is still controversial which mediators regulate energy provision to activated neural cells, as insulin does in peripheral tissues. Interleukin-1β (IL-1β) may mediate this effect as it can affect glucoregulation, it is overexpressed in the 'healthy' brain during increased neuronal activity, and it supports high-energy demanding processes such as long-term potentiation, memory and learning. Furthermore, the absence of sustained neuroendocrine and behavioral counterregulation suggests that brain glucose-sensing neurons do not perceive IL-1β-induced hypoglycemia. Here, we show that IL-1β adjusts glucoregulation by inducing its own production in the brain, and that IL-1β-induced hypoglycemia is myeloid differentiation primary response 88 protein (MyD88)-dependent and only partially counteracted by Kir6.2-mediated sensing signaling. Furthermore, we found that, opposite to insulin, IL-1β stimulates brain metabolism. This effect is absent in MyD88-deficient mice, which have neurobehavioral alterations associated to disorders in glucose homeostasis, as during several psychiatric diseases. IL-1β effects on brain metabolism are most likely maintained by IL-1β auto-induction and may reflect a compensatory increase in fuel supply to neural cells. We explore this possibility by directly blocking IL-1 receptors in neural cells. The results showed that, in an activity-dependent and paracrine/autocrine manner, endogenous IL-1 produced by neurons and astrocytes facilitates glucose uptake by these cells. This effect is exacerbated following glutamatergic stimulation and can be passively transferred between cell types. We conclude that the capacity of IL-1β to provide fuel to neural cells underlies its physiological effects on glucoregulation, synaptic plasticity, learning and memory. However, deregulation of IL-1β production could contribute to the alterations in brain glucose metabolism that are detected in several neurologic and psychiatric diseases.Molecular Psychiatry advance online publication, 8 December 2015; doi:10.1038/mp.2015.174.

Dose-response effect of interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, and interferon-y on the in vitro production of epithelial neutrophil activating peptide-78 (ENA-78), IL-8, and IL-6 by human endometrial stromal cells

The production of epithelial neutrophil activating peptide-78 (NA-78) and the interleukins IL-8 and IL-6 by endometrial stromal cells is stimulated by pro-inflammatory interleukin-1 (IL-1) and tumour necrosis factor-α (TNF-α). IL-8 is suggested to play a role in the pathogenesis of endometriosis, and in these women the peritoneal fluid concentrations of ENA-78 and IL-8 are increased. TNF-α has been tested together with interferon-γ because of their cooperative stimulation of IL-6. The release of IL-8, however, is inhibited with increasing interferon levels. The aim of the study was the analysis of the production of ENA-78, IL-6 and IL-8 by cultured human endometrial stromal cells in the presence of varying concentrations of IL-1β, TNF-α, and interferon-γ.

The association of the composite IL-1 genotype with periodontitis progression and/or treatment outcomes: a systematic review

BACKGROUND: Genetically transmitted traits such as cytokine gene polymorphisms may accentuate the host inflammatory response to the bacterial challenge and influence susceptibility to periodontitis. OBJECTIVE: To systematically review the evidence of an association between the interleukin-1 (IL-1) composite genotype, i.e. presence of the allele 2 in the gene clusters IL-1A-889 and in IL-1B +3953, and periodontitis progression and/or treatment outcomes. Material and Methods: Based on the focused question, a search was conducted for longitudinal clinical trials comparing progression of periodontitis and/or treatment outcomes in IL-1 genotype-positive (carrying allele 2) and IL-1 genotype-negative (not carrying allele 2) subjects. A search in the National Library of Medicine computerized bibliographic database MEDLINE and a manual search were performed. Selection of publications, extraction of data and validity assessment were made independently by two reviewers. RESULTS: The search provided 122 titles of which 11 longitudinal publications were included. The heterogeneity of the data prevented the performance of a meta-analysis. While findings from some publications rejected a possible role of IL-1 composite genotype on progression of periodontitis after various therapies, other reported a prognostic value for disease progression of the positive IL-1 genotype status. When assessed on a multivariate risk assessment model, several publications concluded that the assessment of the IL-1 composite genotype in conjunction with other covariates (e.g. smoking and presence of specific bacteria) may provide additional information on disease progression. The small sample size of the available publications, however, requires caution in the interpretation of the results. CONCLUSION: Based on these findings, (i) there is insufficient evidence to establish if a positive IL-1 genotype status contributes to progression of periodontitis and/or treatment outcomes. Therefore, (ii) results obtained with commercially available tests should be interpreted with caution.

Association of the composite IL-1 genotype with peri-implantitis: a systematic review

OBJECTIVE: Cytokine gene polymorphisms may modulate the host response to the bacterial challenge and influence susceptibility to peri-implantitis. OBJECTIVE: To systematically review the evidence of an association between the interleukin-1 (IL-1) composite genotype, i.e. presence of the allele 2 in the gene clusters IL-1A (-889) and in IL-1B (+3953), and peri-implantitis. MATERIAL AND METHODS: An electronic search in the National Library of Medicine-computerized bibliographic database MEDLINE and a manual search were performed. The search was conducted for longitudinal clinical trials comparing progression of peri-implantitis in IL-1 genotype positive (carrying allele 2) with IL-1 genotype negative (not carrying allele 2) subjects. Selection of publications, extraction of data and validity assessment were made independently by two reviewers. RESULTS: The search provided 44 titles of which two longitudinal publications were included. CONCLUSION: Based on the findings from this study, there is not enough evidence to support or refute an association between the IL-1 genotype status and peri-implantitis. Systematic genetic testing for the assessment of the risk of peri-implantitis cannot be recommended as a standard of care at this time.

PU.1 binding to the p53 family of tumor suppressors impairs their transcriptional activity

The transcription factor PU.1 is essential for terminal myeloid differentiation, B- and T-cell development, erythropoiesis and hematopoietic stem cell maintenance. PU.1 functions as oncogene in Friend virus-induced erythroleukemia and as tumor suppressor in acute myeloid leukemias. Moreover, Friend virus-induced erythroleukemia requires maintenance of PU.1 expression and the disruption of p53 function greatly accelerates disease progression. It has been hypothesized that p53-mediated expression of the p21(Cip1) cell cycle inhibitor during differentiation of pre-erythroleukemia cells promotes selection against p53 function. In addition to the blockage of erythroblast differentiation provided by increased levels of PU.1, we propose that PU.1 alters p53 function. We demonstrate that PU.1 reduces the transcriptional activity of the p53 tumor suppressor family and thus inhibits activation of genes important for cell cycle regulation and apoptosis. Inhibition is mediated through binding of PU.1 to the DNA-binding and/or oligomerization domains of p53/p73 proteins. Lastly, knocking down endogenous PU.1 in p53 wild-type REH B-cell precursor leukemia cells leads to increased expression of the p53 target p21(Cip1).

Ischemia-induced up-regulation of heme oxygenase-1 protects from apoptotic cell death and tissue necrosis

BACKGROUND: Tissues are endowed with protective mechanisms to counteract chronic ischemia. Previous studies have demonstrated that endogenous heme oxygenase (HO)-1 may protect parenchymal tissue from inflammation- and reoxygenation-induced injury. Nothing is known, however, on whether endogenous HO-1 also plays a role in chronic ischemia to protect from development of tissue necrosis. The aim of this study is, therefore, to evaluate in vivo whether endogenous HO-1 exerts protection on chronically ischemic musculocutaneous tissue, and whether this protection is mediated by an attenuation of the microcirculatory dysfunction. MATERIALS AND METHODS: In C57BL/6-mice, a chronically ischemic flap was elevated and fixed into a dorsal skinfold chamber. In a second group, tin-protoporphyrin-IX was administrated to competitively block the action of HO-1. Animals without flap elevation served as controls. With the use of intravital fluorescence microscopy, microcirculation, apoptotic cell death, and tissue necrosis were analyzed over a 10-day observation period. The time course of HO-1 expression was determined by Western blotting. RESULTS: Chronic ischemia induced an increase of HO-1 expression, particularly at day 1 and 3. This was associated with arteriolar dilation and hyperperfusion, which was capable of maintaining an adequate capillary perfusion density in the critically perfused central part of the flap, demarcating the distal necrosis. Inhibition of endogenous HO-1 by tin-protoporphyrin-IX completely abrogated arteriolar dilation (44.6 +/- 6.2 microm versus untreated flaps: 71.3 +/- 7.3 microm; P < 0.05) and hyperperfusion (3.13 +/- 1.29 nL/s versus 8.55 +/- 3.56 nL/s; P < 0.05). This resulted in a dramatic decrease of functional capillary density (16 +/- 16 cm/cm(2)versus 84 +/- 31 cm/cm(2); P < 0.05) and a significant increase of apoptotic cell death (585 +/- 51 cells/mm(2)versus 365 +/- 53 cells/mm(2); P < 0.05), and tissue necrosis (73% +/- 5% versus 51% +/- 5%; P < 0.001). CONCLUSION: Thus, our results suggest that chronic ischemia-induced endogenous HO-1 protects ischemically endangered tissue, probably by the vasodilatory action of the HO-1-associated carbon monoxide.

Flotillins interact with PSGL-1 in neutrophils and, upon stimulation, rapidly organize into membrane domains subsequently accumulating in the uropod

BACKGROUND: Neutrophils polarize and migrate in response to chemokines. Different types of membrane microdomains (rafts) have been postulated to be present in rear and front of polarized leukocytes and disruption of rafts by cholesterol sequestration prevents leukocyte polarization. Reggie/flotillin-1 and -2 are two highly homologous proteins that are ubiquitously enriched in detergent resistant membranes and are thought to shape membrane microdomains by forming homo- and hetero-oligomers. It was the goal of this study to investigate dynamic membrane microdomain reorganization during neutrophil activation. METHODOLOGY/PRINCIPAL FINDINGS: We show now, using immunofluorescence staining and co-immunoprecipitation, that endogenous flotillin-1 and -2 colocalize and associate in resting spherical and polarized primary neutrophils. Flotillins redistribute very early after chemoattractant stimulation, and form distinct caps in more than 90% of the neutrophils. At later time points flotillins accumulate in the uropod of polarized cells. Chemotactic peptide-induced redistribution and capping of flotillins requires integrity and dynamics of the actin cytoskeleton, but does not involve Rho-kinase dependent signaling related to formation of the uropod. Both flotillin isoforms are involved in the formation of this membrane domain, as uropod location of exogenously expressed flotillins is dramatically enhanced by co-overexpression of tagged flotillin-1 and -2 in differentiated HL-60 cells as compared to cells expressing only one tagged isoform. Flotillin-1 and -2 associate with P-selectin glycoprotein ligand 1 (PSGL-1) in resting and in stimulated neutrophils as shown by colocalization and co-immunoprecipitation. Neutrophils isolated from PSGL-1-deficient mice exhibit flotillin caps to the same extent as cells isolated from wild type animals, implying that PSGL-1 is not required for the formation of the flotillin caps. Finally we show that stimulus-dependent redistribution of other uropod-located proteins, CD43 and ezrin/radixin/moesin, occurs much slower than that of flotillins and PSGL-1. CONCLUSIONS/SIGNIFICANCE: These results suggest that flotillin-rich actin-dependent membrane microdomains are importantly involved in neutrophil uropod formation and/or stabilization and organize uropod localization of PSGL-1.

Stimulation of MMP-1 and CCL2 by NAMPT in PDL cells

Periodontitis is an inflammatory disease caused by pathogenic microorganisms and characterized by the destruction of the periodontium. Obese individuals have an increased risk of periodontitis, and elevated circulating levels of adipokines, such as nicotinamide phosphoribosyltransferase (NAMPT), may be a pathomechanistic link between both diseases. The aim of this in vitro study was to examine the regulation of periodontal ligament (PDL) cells by NAMPT and its production under inflammatory and infectious conditions. NAMPT caused a significant upregulation of 9 genes and downregulation of 3 genes, as analyzed by microarray analysis. Eight of these genes could be confirmed by real-time PCR: NAMPT induced a significant upregulation of EGR1, MMP-1, SYT7, ITPKA, CCL2, NTM, IGF2BP3, and NRP1. NAMPT also increased significantly the MMP-1 and CCL2 protein synthesis. NAMPT was significantly induced by interleukin-1 β and the periodontal microorganism P. gingivalis. NAMPT may contribute to periodontitis through upregulation of MMP-1 and CCL2 in PDL cells. Increased NAMPT levels, as found in obesity, may therefore represent a mechanism whereby obesity could confer an increased risk of periodontitis. Furthermore, microbial and inflammatory signals may enhance the NAMPT synthesis in PDL cells and thereby contribute to the increased gingival and serum levels of this adipokine, as found in periodontitis.

Sphingosine 1-phosphate (S1P) induces COX-2 expression and PGE2 formation via S1P receptor 2 in renal mesangial cells

Understanding the mechanisms of sphingosine 1-phosphate (S1P)-induced cyclooxygenase (COX)-2 expression and prostaglandin E2 (PGE2) formation in renal mesangial cells may provide potential therapeutic targets to treat inflammatory glomerular diseases. Thus, we evaluated the S1P-dependent signaling mechanisms which are responsible for enhanced COX-2 expression and PGE2 formation in rat mesangial cells under basal conditions. Furthermore, we investigated whether these mechanisms are operative in the presence of angiotensin II (Ang II) and of the pro-inflammatory cytokine interleukin-1β (IL-1β). Treatment of rat and human mesangial cells with S1P led to concentration-dependent enhanced expression of COX-2. Pharmacological and molecular biology approaches revealed that the S1P-dependent increase of COX-2 mRNA and protein expression was mediated via activation of S1P receptor 2 (S1P2). Further, inhibition of Gi and p42/p44 MAPK signaling, both downstream of S1P2, abolished the S1P-induced COX-2 expression. In addition, S1P/S1P2-dependent upregulation of COX-2 led to significantly elevated PGE2 levels, which were further potentiated in the presence of Ang II and IL-1β. A functional consequence downstream of S1P/S1P2 signaling is mesangial cell migration that is stimulated by S1P. Interestingly, inhibition of COX-2 by celecoxib and SC-236 completely abolished the migratory response. Overall, our results demonstrate that extracellular S1P induces COX-2 expression via activation of S1P2 and subsequent Gi and p42/p44 MAPK-dependent signaling in renal mesangial cells leading to enhanced PGE2 formation and cell migration that essentially requires COX-2. Thus, targeting S1P/S1P2 signaling pathways might be a novel strategy to treat renal inflammatory diseases.