Viral sensing by epithelial cells involves PKR- and caspase-3-dependent generation of gasdermin E pores.

Viral sensing in myeloid cells involves inflammasome activation leading to gasdermin pore formation, cytokine release, and cell death. However, less is known about viral sensing in barrier epithelial cells, which are critical to the innate immune response to RNA viruses. Here, we show that poly(I:C), a mimic of viral dsRNA, is sensed by NLRP1 in human bronchial epithelial cells, leading to inflammasome-dependent gasdermin D (GSDMD) pore formation via caspase-1. DsRNA also stimulated a parallel sensing pathway via PKR which activated caspase-3 to cleave gasdermin E (GSDME) to form active pores. Influenza A virus (IAV) infection of cells caused GSDME activation, cytokine release, and cell death, in a PKR-dependent but NLRP1-independent manner, involving caspase-8 and caspase-3. Suppression of GSDMD and GSDME expression increased IAV replication. These data clarify mechanisms of gasdermin cleavage in response to viral sensing and reveal that gasdermin pore formation is intrinsically antiviral in human epithelial cells.

PYHIN protein IFI207 regulates cytokine transcription and IRF7 and contributes to the establishment of K. pneumoniae infection.

PYHIN proteins AIM2 and IFI204 sense pathogen DNA, while other PYHINs have been shown to regulate host gene expression through as-yet unclear mechanisms. We characterize mouse PYHIN IFI207, which we find is not involved in DNA sensing but rather is required for cytokine promoter induction in macrophages. IFI207 co-localizes with both active RNA polymerase II (RNA Pol II) and IRF7 in the nucleus and enhances IRF7-dependent gene promoter induction. Generation of Ifi207-/- mice shows no role for IFI207 in autoimmunity. Rather, IFI207 is required for the establishment of a Klebsiella pneumoniae lung infection and for Klebsiella macrophage phagocytosis. These insights into IFI207 function illustrate that PYHINs can have distinct roles in innate immunity independent of DNA sensing and highlight the need to better characterize the whole mouse locus, one gene at a time.

PYHIN1 regulates pro-inflammatory cytokine induction rather than innate immune DNA sensing in airway epithelial cells.

Animal cells use pattern-recognition receptors (PRRs) to detect specific pathogens. Pathogen detection mounts an appropriate immune response, including interferon and cytokine induction. The intracellular PRR-signaling pathways that detect DNA viruses have been characterized, particularly in myeloid cells. In these pathways, cGMP-AMP synthase (cGAS) and the pyrin and HIN domain family member (PYHIN) protein interferon-γ-inducible protein 16 (IFI16) detect DNA and signal via stimulator of interferon genes protein (STING). However, although airway epithelial cells are frontline sentinels in detecting pathogens, information on how they respond to DNA viruses is limited, and the roles of PYHIN proteins in these cells are unknown. Here, we examined expression and activities of cGAS, STING, and PYHINs in human lung epithelial cells. A549 epithelial cells, commonly used for RNA-sensing studies, failed to respond to DNA because they lacked STING expression, and ectopic STING expression restored a cGAS-dependent DNA response in these cells. In contrast, NuLi-1 immortalized human bronchial epithelial cells did express STING, which was activated after DNA stimulation and mediated DNA-dependent gene induction. PYHIN1, which like IFI16 has been proposed to be a viral DNA sensor, was the only PYHIN protein expressed in both airway epithelial cell types. However, rather than having a role in DNA sensing, PYHIN1 induced proinflammatory cytokines in response to interleukin-1 (IL-1) or tumor necrosis factor α (TNFα) stimulation. Of note, PYHIN1, via its HIN domain, directly induced IL-6 and TNFα transcription, revealing that PYHIN proteins play a role in proinflammatory gene induction in airway epithelial cells.

Different patterns of gelatinolytic activity in pituitary macro- and microadenomas.

OBJECTIVE: Gelatinases, Matrix MetalloProteinase(MMP)-2 and MMP-9, belong to zinc-dependent endopeptidases involved in several physiological and pathological processes including inflammation and tumor development. Because the information about the involvement of gelatinases in pituitary adenoma (PA) development are scant, our objective was the analysis of MMP-2 and MMP-9 activity in serum and tumor tissue of PA patients. PATIENTS AND METHODS: Twenty one patients with PA (macroadenoma n=18, microadenoma n=3), qualified to the endoscopic resection of tumors were enrolled. Venous blood samples were collected before the surgery and PA tissue was collected during the surgery. Tissue material was homogenized in a buffer containing 0.1M Tris-HCl pH 7.4 and centrifuged. The supernatant was set to the equal protein content 18µg/sample. Protein level in tissue samples was estimated with Bradford method. MMP-2 and MMP-9 analysis in serum and tissue was performed with gelatin zymography. RESULTS: The proteolytically activated forms of MMPs were not observed in the analyzed sera. Serum activities of MMP-2 and MMP-9 did not statistically differ between patients with micro and macroadenomas. The analysis of material obtained from tissue of microadenomas showed slightly lower activities of both forms of MMP-9 (pro-MMP-9 and MMP-9/lipokalin heterodimer). Simultaneously the increased activity of pro-MMP-2 in comparison to macroadenomas was observed. Although differences observed did not reach statistical significance, only in the case of microadenomas the presence of the active form of MMP-2 (molecular weight 65kDa band) was observed. CONCLUSION: In the course of PA growth the change the biochemical profile of the gelatinolytic activity within the tumor tissue is observed. Initially, the higher activity of MMP-2 in microadenomas and elevated activity of MMP-9 in macroadenomas were detected.

DNA sensors are expressed in astrocytes and microglia in vitro and are upregulated during gliosis in neurodegenerative disease.

The detection of nucleic acids by the innate immune system is an essential host response during viral infection. In recent years, a number of immune sensors capable of recognizing cytosolic DNA have been identified and include the PYHIN family members AIM2, IFI16, and p204 as well as the enzyme, cGAS. Activation of these receptors leads to the induction of antiviral genes including Type-1 interferons and chemokines such as CCL5. We have carried out extensive expression profiling of these DNA sensors and other members of the PYHIN family in highly purified primary astrocytes and microglia and have demonstrated that both cell types express the majority of these proteins at the mRNA level. In microglia, several family members are highly upregulated in response to IFN-ß treatment while both cell types induce robust proinflammatory and antiviral cytokine production (e.g., IL-6, CCL5, IFN-ß) in the presence of immune stimulatory DNA and RNA. The production of IL-6 is partially dependent on the interferon receptor as is IFN-ß itself. Furthermore, we have found that p204 and AIM2 are upregulated in a Type I IFN dependent fashion in vivo, in a murine model of chronic neurodegeneration. Given the propensity of inflammatory responses to cause neuronal damage, increased expression and activation of these receptors, not only during viral infection but also during sterile inflammatory responses, has the potential to exacerbate existing neuroinflammation leading to further damage and impaired neurogenesis.

Natriuretic peptides in the evaluation of syncope in children and adolescents.

INTRODUCTION: Natriuretic peptides have an increasing role in assessing cardiovascular conditions. The number of papers addressing their role in the evaluation of children with syncope of unclear etiology is sparse. The aim of this study was to determine whether measuring atrial natriuretic peptide (ANP) and the inactive form, N-terminal prohormone of brain natriuretic peptide (NT-proBNP) concentration in children admitted due to differential diagnosis of syncope can be helpful in establishing the most probable cause of this condition. METHODS: The study included 88 patients between 9 and 18 years of age hospitalized due to syncope. The control group comprised 25 healthy children. In order to identify the cause of syncope, children with this condition were subjected to cardiologic and neurologic evaluation, and ANP and NT-proBNP concentrations were determined. RESULTS: The syncope group and the controls did not differ significantly in terms of natriuretic peptides concentrations. Similarly, no significant intergroup differences in natriuretic peptide concentrations were documented between children representing various types of response to the tilt test, and between the subgroups of patients with syncope of various origins. CONCLUSION: Analysis of natriuretic peptides concentrations in children with syncope does not result in unambiguous findings that would enable establishing accurate diagnosis.

[Urolithiasis risk factors in obese and overweight children]. / Czynniki ryzyka tworzenia kamieni nerkowych u dzieci z otyloscia i nadwaga

INTRODUCTION: Childhood obesity is becoming a worldwide epidemic and its metabolic and cardiovascular complications may already be evident at a young age. Several epidemiologic studies in adults have clearly demonstrated that obesity and overweight increase the risk of kidney disease and urolithiasis. AIM OF THE STUDY: The purpose of this study was to evaluate the relationship between overweight and obesity and urolithiasis risk factors in children. MATERIALS AND METHODS: The main kidney stones risk factors in urine such as calcium concentration, oxalate concentration, citrate concentration, pH of urine as well as BRI (Bonn Risk Index) were analyzed in 249 overweight and obese children (study group) and in 281 children with normal weight (control) at the age of 3 to 18 years old. RESULTS: In the study group the mean oxalate concentration was significantly higher than in the control (0.52±0.48 vs. 0.26±0.12; p <0.05). The mean calcium concentration of overweight/obese patients was higher than that of normal body weight and the difference was close to statistically significant (3.23±2.55 vs 2.58±1.59; p=0.0537). The mean urine pH in the study group was 6.28±0.46 and was significantly lower (p <0.05) than the mean urine pH in the control, witch was 6.40±0.47. The mean citrate concentration among overweight/obese patients was significantly lower than in control (431,2±309,5 vs. 637,2±310,7; p <0.05). CONCLUSIONS: Our results suggest that obesity or overweight at a young age are associated with an increased risk of kidney stones. Weight loss might be explored as a potential treatment to prevent kidney stone formation.

Vaccinia virus protein C6 is a virulence factor that binds TBK-1 adaptor proteins and inhibits activation of IRF3 and IRF7.

Recognition of viruses by pattern recognition receptors (PRRs) causes interferon-ß (IFN-ß) induction, a key event in the anti-viral innate immune response, and also a target of viral immune evasion. Here the vaccinia virus (VACV) protein C6 is identified as an inhibitor of PRR-induced IFN-ß expression by a functional screen of select VACV open reading frames expressed individually in mammalian cells. C6 is a member of a family of Bcl-2-like poxvirus proteins, many of which have been shown to inhibit innate immune signalling pathways. PRRs activate both NF-κB and IFN regulatory factors (IRFs) to activate the IFN-ß promoter induction. Data presented here show that C6 inhibits IRF3 activation and translocation into the nucleus, but does not inhibit NF-κB activation. C6 inhibits IRF3 and IRF7 activation downstream of the kinases TANK binding kinase 1 (TBK1) and IκB kinase-ε (IKKε), which phosphorylate and activate these IRFs. However, C6 does not inhibit TBK1- and IKKε-independent IRF7 activation or the induction of promoters by constitutively active forms of IRF3 or IRF7, indicating that C6 acts at the level of the TBK1/IKKε complex. Consistent with this notion, C6 immunoprecipitated with the TBK1 complex scaffold proteins TANK, SINTBAD and NAP1. C6 is expressed early during infection and is present in both nucleus and cytoplasm. Mutant viruses in which the C6L gene is deleted, or mutated so that the C6 protein is not expressed, replicated normally in cell culture but were attenuated in two in vivo models of infection compared to wild type and revertant controls. Thus C6 contributes to VACV virulence and might do so via the inhibition of PRR-induced activation of IRF3 and IRF7.

Cytosolic DNA sensors regulating type I interferon induction.

Type I interferon (IFN) induction is a crucial anti-pathogen response mediated by innate immune stimulation. Although it has been appreciated for some time that the presence of pathogen DNA within a cell leads to a type I IFN response, it is only in the past few years that some of the key signalling proteins and DNA sensors that regulate this response have been uncovered. Here, we review the nature of these DNA sensors, which include a new family of pattern recognition receptors termed the AIM2-like receptors, and consider the implications of their discovery for understanding emerging principles of innate immune DNA sensing. Furthermore, we discuss how their discovery provides a rationale as to why accumulation of self-DNA mediates IFN-dependent autoimmunity.

IFI16 is an innate immune sensor for intracellular DNA.

The detection of intracellular microbial DNA is critical to appropriate innate immune responses; however, knowledge of how such DNA is sensed is limited. Here we identify IFI16, a PYHIN protein, as an intracellular DNA sensor that mediates the induction of interferon-ß (IFN-ß). IFI16 directly associated with IFN-ß-inducing viral DNA motifs. STING, a critical mediator of IFN-ß responses to DNA, was recruited to IFI16 after DNA stimulation. Lowering the expression of IFI16 or its mouse ortholog p204 by RNA-mediated interference inhibited gene induction and activation of the transcription factors IRF3 and NF-κB induced by DNA and herpes simplex virus type 1 (HSV-1). IFI16 (p204) is the first PYHIN protein to our knowledge shown to be involved in IFN-ß induction. Thus, the PYHIN proteins IFI16 and AIM2 form a new family of innate DNA sensors we call 'AIM2-like receptors' (ALRs).

Survivin is an essential mediator of arthritis interacting with urokinase signalling.

Proto-oncogene survivin has recently been identified as a prognostic marker distinguishing patients with destructive rheumatoid arthritis (RA). In the present material of 132 RA patients and 82 controls, the levels of survivin correlated to urokinase (uPA) (r= 0.46), a plasminogen activator over-expressed in inflamed joints and known to exhibit potent arthritogenic properties. Here we evaluate the functional relationship between these proteins using primary synovial fibroblasts and leucocytes of RA patients, human monocytic (THP-1) and fibroblast (MRC-5) cell lines. Using inhibitors of intracellular signalling, we show that uPA and survivin share common transduction pathways in synovial fibroblasts being dependent on the activity of tyrosine kinases, phosphatidylinositide 3 kinase and mitogen effector kinase. Moreover, uPA production is significantly reduced in fibroblasts if survivin synthesis has been silenced by siRNA. Importantly, silencing of survivin in fibroblasts prevented their invasive growth in knee joints of severe combined immune deficient mice. Interaction of uPA with receptor up-regulates survivin expression in leucocytes. In turn, survivin is required for the up-regulation of uPA receptor on the cell surface. These findings indicate that survivin is an essential mediator of arthritogenic properties of uPA regulating its synthesis in synovial fibroblasts and uPAR expression in leucocytes. Close correlation between survivin and uPA levels in patients with RA supports the importance of this connection for the pathogenesis of arthritis.

[Correlation between intima-media thickness in carotid artery and markers of epithelial cell dysfunction in patients with juvenile idiopathic arthritis]. / Ocena zaleznosci pomiedzy gruboscia warstwy wewnetrznej i sródblonka u dzieci z mlodzienczym idiopatycznym zapaleniem stawów (MIZS).

UNLABELLED: Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disease in children. Prolonged immunological inflammatory process leads in these patients to an early onset of atherosclerosis. That is why it seems to be important to find methods of detecting atherosclerosis in its very early stage to start appropriate treatment. AIM: The aim of this study was to determine whether there is any correlation between intima-media thickness (IMT) in carotid artery and concentrations of markers of epithelial cell dysfunction - C-reactive protein and Intercellular Adhesive Molecule -1 (sICAM-1). MATERIALS AND METHODS: 63 children were enrolled in the study (40 with JIA and 23 healthy as control group). C-reactive protein concentration was assayed by immunoturbidimetric method with Hitachi 912 Chemistry Analyzer and sICAM-1 was assayed by ELISA. IMT was measured as defined by Pignoli. RESULTS: Mean CRP concentration in the study group was 1.97 mg/dl and in the control group -0.08 mg//dl. sICAM-1 concentration was 333.4 mg/ml, and mean IMT in carotid artery in children with JIA was 0.43 mm (max=0.58 mm, min=0.36 mm). There was a correlation between IMT and sICAM-1 concentration (r=0.75, p<0.001). CONCLUSIONS: Assessment of IMT in carotid artery and concentrations of sICAM-1 and CRP may be used as a marker of preclinical arthrosclerosis in children with JIA.

Viral targeting of DEAD box protein 3 reveals its role in TBK1/IKKepsilon-mediated IRF activation.

Viruses are detected by different classes of pattern recognition receptors (PRRs), such as Toll-like receptors and RIG-like helicases. Engagement of PRRs leads to activation of interferon (IFN)-regulatory factor 3 (IRF3) and IRF7 through IKKepsilon and TBK1 and consequently IFN-beta induction. Vaccinia virus (VACV) encodes proteins that manipulate host signalling, sometimes by targeting uncharacterised proteins. Here, we describe a novel VACV protein, K7, which can inhibit PRR-induced IFN-beta induction by preventing TBK1/IKKepsilon-mediated IRF activation. We identified DEAD box protein 3 (DDX3) as a host target of K7. Expression of DDX3 enhanced Ifnb promoter induction by TBK1/IKKepsilon, whereas knockdown of DDX3 inhibited this, and virus- or dsRNA-induced IRF3 activation. Further, dominant-negative DDX3 inhibited virus-, dsRNA- and cytosolic DNA-stimulated Ccl5 promoter induction, which is also TBK1/IKKepsilon dependent. Both K7 binding and enhancement of Ifnb induction mapped to the N-terminus of DDX3. Furthermore, virus infection induced an association between DDX3 and IKKepsilon. Therefore, this study shows for the first time the involvement of a DEAD box helicase in TBK1/IKKepsilon-mediated IRF activation and Ifnb promoter induction.