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Background The role of cytokines in the pathogenesis of febrile seizures (FSs) is unclear, and information regarding cytokine production outside of FS episodes is scarce. Methods In our controlled follow-up study of patients with FSs, we compared the levels of 12 serum cytokines after the patients’ first FSs, during febrile episodes without FSs, after recurrent FSs, during healthy periods after FSs, and between patients and controls. Results Two-hundred fifty-one patients with first FS participated in the study, of whom 17 (mean age 1.6 years, SD 0.7) with recurrent FSs completed the protocol as required by the sample size calculations. The mean IL-1RA level was higher after the first FSs (2580 pg/mL, SD 1516) than during febrile episodes without FSs (1336 pg/mL, SD 1364, P = 0.006) and healthy periods after FSs (474 pg/mL, SD 901, P = 0.001). IL-1RA levels were also higher during first (2580 pg/mL) and recurrent FSs (2666 pg/mL, SD 1747) in comparison with febrile controls (746 pg/mL, SD 551) (P P = 0.001, respectively), but there was no difference in the IL-1RA between febrile episodes without FSs and febrile controls. Conclusions Patients with FSs produce stronger inflammatory reactions during febrile episodes with FSs compared with febrile episodes without FSs and febrile controls. Impact In patients with FSs, IL-1RA was higher following first FS than during febrile episodes without FSs and healthy periods after FSs. IL-1RA was higher in patients with FSs following first and recurrent FSs than in febrile controls. There was no significant difference in IL-1RA between febrile episodes of patients without FSs and febrile controls. Using IL-1RA as a surrogate marker of IL-1 axis activity, our results indicate that patients with FSs produced stronger inflammatory reactions during FS episodes but not during other febrile episodes or healthy periods after FSs. Cytokines may play a role in pathogenesis of FSs.

The EGFR adaptor protein, CIN85, has been shown to promote breast cancer malignancy and hypoxia-inducible factor (HIF) stability. However, the mechanisms underlying cancer promotion remain ill defined. Here we show that CIN85 is a novel binding partner of the main HIF-prolyl hydroxylase, PHD2, but not of PHD1 or PHD3. Mechanistically, the N-terminal SRC homology 3 domains of CIN85 interacted with the proline-arginine–rich region within the N-terminus of PHD2, thereby inhibiting PHD2 activity and HIF degradation. This activity is essential in vivo, as specific loss of the CIN85–PHD2 interaction in CRISPR/Cas9-edited cells affected growth and migration properties, as well as tumor growth in mice. Overall, we discovered a previously unrecognized tumor growth checkpoint that is regulated by CIN85-PHD2 and uncovered an essential survival function in tumor cells by linking growth factor adaptors with hypoxia signaling.Significance:This study provides unprecedented evidence for an oxygen-independent mechanism of PHD2 regulation that has important implications in cancer cell survival.

Plasmids and site directed mutagenesis, GST-pull down, Fluorescence microscopy, CRISPR/Cas9-mediated EGLN1 gene editing, Lentivirus-mediated expression of sgRNAs and Cas9, Genomic DNA extraction and genotyping, RNA extraction and qRT-PCR, Scanning electron microscopy, Monolayer colony formation assay, Live Cell Imaging Assays Surface plasmon resonance (SPR)

Supplementary figure 1. CIN85 interacts with PHD2 via three N-terminal SH3 domains. Supplementary figure 2. The amino acids 81-98 within the N-terminal region of PHD2 have a major impact on the interaction with CIN85. Supplementary figure 3. CRISPR/Cas9-mediated generation of MDA-MB-231 cells lacking the amino acids critical for interaction with CIN85. Supplementary figure 4. Loss of PHD2-CIN85 interaction does not influence HIF-1α and HIF-2α mRNA levels. Supplementary figure 5. Lack of the CIN85-PHD2 interaction in E10 and E12 cells mediates HIF-α resistance upon depletion of CIN85. Supplementary figure 6. Microscopy images of S, E10, and E12 cells. Supplementary figure 7. Live cell proliferation analysis of S, E10, and E12 cells. Supplementary figure 8. CRISPR/Cas9 mediated EGLN1 (PHD2) editing in MDA-MB-231 cells with EGLN1-sgRNA-219 and with EGLN1-sgRNA-292 did not show off-target effects.

Loss-of-function (LOF) mutations in NFKB1, coding for p105, may cause common variable immunodeficiency due to dysregulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κΒ) pathway. Monoallelic LOF variants of NFKB1 can predispose to uncontrolled inflammation including sterile necrotizing fasciitis or pyoderma gangrenosum. In this study, we explored the impact of a heterozygous NFKB1 c.C936T/p.R157X LOF variant on immunity in sterile fasciitis patients and their family members. The p50 or p105 protein levels were reduced in all variant carriers. Interleukin-1β (IL-1β) and interleukin-8 (IL-8) levels were elevated in vitro, potentially contributing to the very high neutrophil counts observed during fasciitis episodes. Phosphorylation of p65/RelA was reduced in p.R157X neutrophils suggesting defective activation of canonical NF-κB. Oxidative burst after NF-κB-independent phorbol 12-myristate 13-acetate (PMA) stimulation was similar in both p.R157X and control neutrophils. Comparable amounts of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex subunits were found in p.R157X and control neutrophils. However, a compromised oxidative burst was observed in p.R157X neutrophils following activation of NF-κB-dependent mechanisms following stimulation of toll-like receptor 2 (TLR2) and Dectin-1. Neutrophil extracellular trap formation was not affected by p.R157X. In summary, the NFKB1 c.C936T/p.R157X LOF variant has an impact on inflammation and neutrophil function and may play a role in the pathogenesis of sterile necrotizing fasciitis.

Objective: The aim of this study was to analyze the usefulness of myeloid-related protein 8/14 (MRP8/14) in the prediction of disease course in a real-life setting for patients with new-onset juvenile idiopathic arthritis (JIA), to identify the relationship between MRP8/14 and disease activity using the physician’s global assessment of disease activity (PGA), and determine whether the MRP8/14 levels measured in serum and plasma are equally useful.Methods: In this prospective follow-up study, 87 new-onset non-systemic JIA patients were studied. Blood and synovial fluid samples were collected prior to any antirheumatic medication use. MRP8/14 was measured from serum (S-MRP8/14), plasma (P-MRP8/14), and synovial fluid samples using ELISA. Results: The baseline MRP8/14 blood levels were significantly higher in patients using synthetic antirheumatic drugs than in patients with no systemic medications at one year after diagnosis in serum (mean 298 vs. 198 ng/ml, PConclusions: Blood MRP8/14 levels at baseline seem to predict disease course in new-onset JIA patients. P-MRP8/14 might be superior to S-MRP8/14 in assessing disease activity at the time of JIA diagnosis.

Dipeptidyl peptidase 4 inhibitors (DPP4is), commonly used drugs for treatment of type 2 diabetes, increase the risk for bullous pemphigoid (BP). Currently, the mechanism leading to the loss of immunological tolerance of the cutaneous adhesion molecule BP180 as well as similarities and differences in disease progression between DPP4i-associated BP (DPP4i-BP) and DPP4i-independent regular BP are largely unknown. We analyzed the expression of 32 cytokines and two proteases by Luminex and ELISA assays in samples taken from lesional and nonlesional skin of patients with regular BP or DPP4i-BP and healthy controls. Cytokines mediating B-cell survival and targeting such as BAFF, CCL4, CXCL12, and IL-6 were expressed at a higher level in the lesional regular BP skin than the levels in the lesional DPP4i-BP skin. The DPP4i-BP samples had increased levels of eosinophilic cytokines CCL1, CCL17, CCL26, and IL-5, which correlated with the serum level of anti-BP180 NC16A IgG autoantibodies. The mRNA expression of BAFF, IL6, CCL1, CCL17, CCL26, and IL5 measured by qPCR correlated with the protein levels. Taken together, the cutaneous cytokine profiles were found to provide distinctive molecular fingerprints between regular BP and DPP4i-BP.

The role of cytokines in the pathogenesis of febrile seizures (FSs) is unclear, and information regarding cytokine production outside of FS episodes is scarce.In our controlled follow-up study of patients with FSs, we compared the levels of 12 serum cytokines after the patients' first FSs, during febrile episodes without FSs, after recurrent FSs, during healthy periods after FSs, and between patients and controls.Two-hundred fifty-one patients with first FS participated in the study, of whom 17 (mean age 1.6 years, SD 0.7) with recurrent FSs completed the protocol as required by the sample size calculations. The mean IL-1RA level was higher after the first FSs (2580 pg/mL, SD 1516) than during febrile episodes without FSs (1336 pg/mL, SD 1364, P = 0.006) and healthy periods after FSs (474 pg/mL, SD 901, P = 0.001). IL-1RA levels were also higher during first (2580 pg/mL) and recurrent FSs (2666 pg/mL, SD 1747) in comparison with febrile controls (746 pg/mL, SD 551) (P 0.001 and P = 0.001, respectively), but there was no difference in the IL-1RA between febrile episodes without FSs and febrile controls.Patients with FSs produce stronger inflammatory reactions during febrile episodes with FSs compared with febrile episodes without FSs and febrile controls.In patients with FSs, IL-1RA was higher following first FS than during febrile episodes without FSs and healthy periods after FSs. IL-1RA was higher in patients with FSs following first and recurrent FSs than in febrile controls. There was no significant difference in IL-1RA between febrile episodes of patients without FSs and febrile controls. Using IL-1RA as a surrogate marker of IL-1 axis activity, our results indicate that patients with FSs produced stronger inflammatory reactions during FS episodes but not during other febrile episodes or healthy periods after FSs. Cytokines may play a role in pathogenesis of FSs.

The IKAROS family transcription factors regulate lymphocyte development. Loss-of-function variants in IKZF1 cause primary immunodeficiency, but IKAROS family members IKZF2 and IKZF3 have not yet been associated with immunodeficiency yet. Here, we describe a pedigree with a heterozygous truncating variant in IKZF2, encoding the translational activator and repressor HELIOS which is highly expressed in regulatory T cells and effector T cells, particularly of the CD8+ T cell lineage. Protein-protein interaction analysis revealed that the variant abolished HELIOS dimerizations as well as binding to members of the Mi-2/NuRD chromatin remodeling complex. Patients carrying the IKZF2 variant presented with a combined immunodeficiency phenotype characterized by recurrent upper respiratory infections, thrush and mucosal ulcers, as well as chronic lymphadenopathy. With extensive immunophenotyping, functional assays, and transcriptional analysis we show that reduced HELIOS expression was associated with chronic T cell activation and increased production of pro-inflammatory cytokines both in effector and regulatory T cells. Lymph node histology from patients indicated dysregulated germinal center reactions. Moreover, affected individuals displayed profoundly reduced circulating MAIT cell numbers. In summary, we show that this novel loss-of-function variant in HELIOS leads to an immunodeficiency with signs of immune overactivation.One sentence summaryTruncating variant of HELIOS causes immunodeficiency with signs of immune overactivation.

Objective There are limited data on the pathogen-related and host-related factors in the pathogenesis of febrile seizures (FS). We designed a controlled study to compare the role of different respiratory viruses and febrile response in FS. Methods In a prospective cohort study of 1899 pediatric emergency room patients aged 6 months-6 years with a positive respiratory virus multiplex PCR, we identified 225 patients with FSs. We first compared the distribution of respiratory viruses in age-stratified patients with FSs with that in other patients. In an embedded case-control study, we compared the febrile response in patients with FSs with that in the controls matched for age, season and the same respiratory virus. Results The relative risk for FS was the highest for coronavirus OC43, 229E, and NL63 infections [RR: 3.2, 95 % confidence interval (CI): 1.4–7.2) and influenza A and B [RR: 2.5, 95 % CI: 1.4–4.7] as compared to those with other respiratory viral infections. The patients with FSs had a stronger febrile response of 39.2 °C (difference: 0.8 °C, 95 % CI: 0.5–1.2) later during hospitalization after acute care than the controls matched for the same respiratory virus. Conclusions Influenza and coronaviruses caused relatively more FS-related emergency room visits than other respiratory viruses. Furthermore, the febrile response was stronger in the patients with FSs than in the controls matched for the same respiratory virus. The results suggest that the pathomechanism of FSs includes modifiable pathogen-related and host-related factors with possible potential in the prevention of FSs.

Background: Tumor necrosis factor α–induced protein 3 gene (TNFAIP3, also called A20) haploinsufficiency (HA20) leads to autoinflammation and autoimmunity. We have recently shown that a p.(Lys91*) mutation in A20 disrupts nuclear factor κB signaling, impairs protein-protein interactions of A20, and leads to inflammasome activation. Methods: We now describe the clinical presentations and drug responses in a family with HA20 p.(Lys91*) mutation, consistent with our previously reported diverse immunological and functional findings. Results: We report for the first time that inflammasome-mediated autoinflammatory lung reaction caused by HA20 can be treated with interleukin 1 antagonist anakinra. We also describe severe anemia related to HA20 successfully treated with mycophenolate. In addition, HA20 p.(Lys91*) was found to associate with autoimmune thyroid disease, juvenile idiopathic arthritis, psoriasis, liver disease, and immunodeficiency presenting with specific antibody deficiency and genital papillomatosis. Conclusions: We conclude that HA20 may lead to combination of inflammation, immunodeficiency, and autoimmunity. The condition may present with variable and unpredictable symptoms with atypical treatment responses.

Puumala hantavirus (PUUV) hemorrhagic fever with renal syndrome (HFRS) is common in Northern Europe; this infection is usually self-limited and severe complications are uncommon. PUUV and other hantaviruses, however, can rarely cause encephalitis. The pathogenesis of these rare and severe events is unknown. In this study, we explored the possibility that genetic defects in innate anti-viral immunity, as analogous to Toll-like receptor 3 (TLR3) mutations seen in HSV-1 encephalitis, may explain PUUV encephalitis. We completed exome sequencing of seven adult patients with encephalitis or encephalomyelitis during acute PUUV infection. We found heterozygosity for the TLR3 p.L742F novel variant in two of the seven unrelated patients (29%, p = 0.0195). TLR3-deficient P2.1 fibrosarcoma cell line and SV40-immortalized fibroblasts (SV40-fibroblasts) from patient skin expressing mutant or wild-type TLR3 were tested functionally. The TLR3 p.L742F allele displayed low poly(I:C)-stimulated cytokine induction when expressed in P2.1 cells. SV40-fibroblasts from three healthy controls produced increasing levels of IFN-λ and IL-6 after 24 h of stimulation with increasing concentrations of poly(I:C), whereas the production of the cytokines was impaired in TLR3 L742F/WT patient SV40-fibroblasts. Heterozygous TLR3 mutation may underlie not only HSV-1 encephalitis but also PUUV hantavirus encephalitis. Such possibility should be further explored in encephalitis caused by these and other hantaviruses. Electronic supplementary material The online version of this article (10.1007/s10875-020-00834-2) contains supplementary material, which is available to authorized users.

Background Rare tonsillar granulomas may be caused for example by infections, malignancies or sarcoidosis. Granulomas also occur in inborn errors of immunity (IEI) such as common variable immunodeficiency (CVID) with B cell maturation defects and hypogammaglobulinemia. CVID shares various features with sarcoidosis and drug-induced secondary hypogammaglobulinemia; careful consideration of differential diagnosis between these conditions is warranted. Case presentation A 29-year-old female with epilepsy developed dysphagia, dyspnea and impaired exercise tolerance. Obstruction caused by swollen lingual tonsil and edema in the epiglottis and arytenoid mucosa were found. Lingual tonsil and epiglottis biopsies displayed non-necrotizing granulomas. There was no evidence of viral, bacterial, mycobacterial or fungal infections. Chest X-ray, computerized tomography of chest and ultrasound of neck and abdomen remained unremarkable. Positron emission tomography/computed tomography (PET/CT) showed laryngeal enhancement. Empiric antimicrobials combined with prednisolone were insufficient to control her disease. In immunological evaluation, the patient had normal counts of B and T cells. Proportions of CD27+ memory B cells (30.3%) and IgD−IgM−CD27+ switched memory B cells (7.2%; normal range 6.5–29.2%) were normal. Percentage of activated CD21low B cells was high (6.6%; normal range 0.6–3.5%). IgG (3.5 g/L; normal range 6.77–15.0 g/l) and all IgG subclass concentrations were low. Anti-polysaccharide responses were impaired, with 3/10 serotypes reaching a level of 0.35 µg/ml after immunization with Pneumovax®. The findings were consistent with hypogammaglobulinemia resembling CVID, possibly secondary to antiepileptic medication. Her dyspnea and dysphagia responded favorably to subcutaneous IgG and rituximab. Conclusions Tonsillar granulomas can be the presenting and only clinical feature of B cell deficiency, highlighting the diversity of symptoms and findings in primary or secondary immunodeficiencies.

Susceptibility to herpes simplex virus type 1 (HSV-1) encephalitis (HSE-1) in otherwise healthy individuals, in the course of primary infection, can be caused by single-gene inborn errors of Toll-like receptor 3 (TLR3) dependent, interferon (IFN)-alpha/beta-mediated immunity,(1,2) or by single-gene inborn errors of snoRNA31.(3) These variations underlie infections of the forebrain, whereas mutations of DBR1 underlie infections of the brainstem.(3) HSV-2 encephalitis (HSE-2) is typically observed in neonates, albeit also rarely in older children and adults.(4) Its manifestations include altered level of consciousness, cranial neuropathies or more extensive brainstem encephalitis, hemiparesis, hemisensory loss, and permanent neurologic deficit.(4) MRI in HSE-2 may show normal findings, nonspecific white matter, orbitofrontal, mesial temporal lobe, or brainstem lesions. Inborn errors of immunity underlying HSE-2 have not been described. Non

The effects of topical calcipotriol/betamethasone combination therapy and betamethasone monotherapy on inflammatory T-cell numbers and molecular markers were compared in patients with psoriasis. Combination therapy down-regulated the expression of tumour necrosis factor (TNF)-α, interleukin (IL)-23A, IL-17A, S100A7, CCL-20 and interferon (IFN)-γ in skin and TNF-α, IL-6, IL-23A, T-bet and IFN-γ in peripheral blood mononuclear cells (PBMCs). Betamethasone monotherapy had less effect. Expression of FoxP3 in both skin and PBMCs was down-regulated by calcipotriol/betamethasone, but not by betamethasone. Immunohistochemical analysis revealed that calcipotriol/betamethasone reduced the numbers of CD4+ and CD8+ T cells and Tregs in psoriatic lesions more than betamethasone. Flow cytometric analyses demonstrated that calcipotriol/betamethasone decreased the numbers of circulating CD8+ T cells, Tregs, skin-homing Th17 memory cells and Th22 memory cells, while betamethasone had little or no effect. Glucocorticoid receptors GRα and GRß were expressed in psoriatic skin. In conclusion, calcipotriol increases the immunosuppressive power of betamethasone by suppressing the inflammatory TNF-α - IL-23 - IL-17 axis.

Mutations in several proteins functioning as endolysosomal components cause monogenic autoimmune diseases, of which pathogenesis is linked to increased endoplasmic reticulum stress, inefficient autophagy, and defective recycling of immune receptors. We report here a heterozygous TOM1 p.G307D missense mutation, detected by whole-exome sequencing, in two related patients presenting with early-onset autoimmunity, antibody deficiency, and features of combined immunodeficiency. The index patient suffered from recurrent respiratory tract infections and oligoarthritis since early teens, and later developed persistent low-copy EBV-viremia, as well as an antibody deficiency. Her infant son developed hypogammaglobulinemia, autoimmune enteropathy, interstitial lung disease, profound growth failure, and treatment-resistant psoriasis vulgaris. Consistent with previous knowledge on TOM1 protein function, we detected impaired autophagy and enhanced susceptibility to apoptosis in patient-derived cells. In addition, we noted diminished STAT and ERK1/2 signaling in patient fibroblasts, as well as poor IFN-γ and IL-17 secretion in T cells. The mutant TOM1 failed to interact with TOLLIP, a protein required for IL-1 recycling, PAMP signaling and autophagosome maturation, further strengthening the link between the candidate mutation and patient pathophysiology. In sum, we report here an identification of a novel gene, TOM1, associating with early-onset autoimmunity, antibody deficiency, and features of combined immunodeficiency. Other patient cases from unrelated families are needed to firmly establish a causal relationship between the genotype and the phenotype.

The EGFR adaptor protein, CIN85, has been shown to promote breast cancer malignancy and hypoxia-inducible factor (HIF) stability. However, the mechanisms underlying cancer promotion remain ill defined. Here we show that CIN85 is a novel binding partner of the main HIF-prolyl hydroxylase, PHD2, but not of PHD1 or PHD3. Mechanistically, the N-terminal SRC homology 3 domains of CIN85 interacted with the proline-arginine–rich region within the N-terminus of PHD2, thereby inhibiting PHD2 activity and HIF degradation. This activity is essential in vivo, as specific loss of the CIN85–PHD2 interaction in CRISPR/Cas9-edited cells affected growth and migration properties, as well as tumor growth in mice. Overall, we discovered a previously unrecognized tumor growth checkpoint that is regulated by CIN85-PHD2 and uncovered an essential survival function in tumor cells by linking growth factor adaptors with hypoxia signaling. Significance: This study provides unprecedented evidence for an oxygen-independent mechanism of PHD2 regulation that has important implications in cancer cell survival.

SummaryThe EGFR-adaptor protein CIN85 has been shown to promote breast cancer malignancy and hypoxia-inducible factor (HIF) stability. However, the mechanisms underlying cancer promotion remain ill-defined. Here, we show that CIN85 is a novel binding partner of the main HIF-prolyl hydroxylase PHD2, but not of PHD1 or PHD3. Mechanistically, the N-terminal SH3 domains of CIN85 interact with the proline-arginine rich region within the N-terminus of PHD2, thereby inhibiting PHD2 activity and HIF-degradation. This activity is essential in vivo, as specific loss of the CIN85-PHD2 interaction in CRISPR/Cas9 edited cells affected growth and migration properties as well as tumor growth in mice. Overall, we discovered a previously unrecognized tumor growth checkpoint that is regulated by CIN85-PHD2, and uncovered an essential survival function in tumor cells linking growth factor adaptors with hypoxia signaling.

Our aim was to study whether the aberrant amount or function of regulatory T cells is related to the development of type 1 diabetes (T1D) in children. We also set out to investigate the balance of different T cell subtype markers during the T1D autoimmune process. Treg cells were quantified with flow cytometric assay, and the suppression capacity was analysed with a carboxyfluorescein succinimidyl ester (CFSE)-based T cell suppression assay in children in various phases of T1D disease process and in healthy autoantibody-negative control children. The mRNA expression of different T cell subpopulation markers was analysed with real-time qPCR method. The proportion and suppression capacity of regulatory T cells were similar in seroconverted children at an early stage of beta cell autoimmunity and also in children with T1D when compared to healthy and autoantibody-negative children. Significant differences were observed in the mRNA expression of different T cell subpopulation markers in prediabetic children with multiple (≥2) autoantibodies and in children with newly diagnosed T1D when compared to the control children. In conclusion, there were no quantitative or functional differences in regulatory T cells between the case and control groups in any phase of the autoimmune process. Decreased mRNA expression levels of T cell subtype markers were observed in children with multiple islet autoantibodies and in those with newly diagnosed T1D, probably reflecting an exhaustion of the immune system after the strong immune activation during the autoimmune process or a generally aberrant immune response related to the progression of the disease.

ObjectivesTNFAIP3encodes A20 that negatively regulates nuclear factor kappa light chain enhancer of activated B cells (NF-κB), the major transcription factor coordinating inflammatory gene expression.TNFAIP3polymorphisms have been linked with a spectrum of inflammatory and autoimmune diseases and recently, loss-of-function mutations in A20 were found to cause a novel inflammatory disease ‘haploinsufficiency of A20’ (HA20). Here we describe a family with HA20 caused by a novelTNFAIP3loss-of-function mutation and elucidate the upstream molecular mechanisms linking HA20 to dysregulation of NF-κB and the related inflammasome pathway.MethodsNF-κB activation was studied in a mutation-expressing cell line using luciferase reporter assay. Physical and close-proximity protein-protein interactions of wild-type andTNFAIP3p.(Lys91*) mutant A20 were analyzed using mass spectrometry. NF-κB –dependent transcription, cytokine secretion, and inflammasome activation were compared in immune cells of the HA20 patients and control subjects.ResultsThe protein-protein interactome of p.(Lys91*) mutant A20 was severely impaired, including inter-actions with proteins regulating NF-κB activation, DNA repair responses, and the NLR family pyrin domain containing 3 (NLRP3) inflammasome. The p.(Lys91*) mutant A20 failed to suppress NF-κB signaling, which led to increased NF-κB –dependent proinflammatory cytokine transcription. Functional experiments in the HA20 patients’ immune cells uncovered a novel caspase-8-dependent mechanism of NLRP3 inflammasome hyperresponsiveness that mediated the excessive secretion of interleukin-1β and -18.ConclusionsThe current findings significantly deepen our understanding of the molecular mechanisms underlying HA20 and other diseases associated with reduced A20 expression or function, paving the way for future therapeutic targeting of the pathway.

Trans-membrane (or T cell) immunoglobulin and mucin (TIM) molecules are known regulators of immune response whose function in hematopoiesis is unknown. Earlier, we found that tim-1 and tim-4 are expressed by CD45 + cells in the para-aortic region of chicken embryo. Because the para-aortic region is a known site for hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) differentiation and expansion, we hypothesize that TIM molecules have a role in hematopoiesis. To study this role further, we analyzed TIM expression more precisely in chicken para-aortic region and mouse fetal liver hematopoietic cells. Additionally, we examined the hematopoietic potential of TIM-4 + mouse fetal liver cells with a colony-forming assay. tim-1 gene expression was detected in chicken and mouse embryos in the aorta-gonads-mesonephros-region at the time of HSC emergence, whereas tim-3 mRNA was widely expressed in different tissues. tim-4 expression was restricted to fetal liver CD45 + F4/80 + cells. Moreover, two TIM-4 + populations were distinguished: F4/80 hi TIM-4 hi and F4/80 lo TIM-4 lo . F4/80 hi TIM-4 hi cells had no hematopoietic potential and were morphologically similar to mature macrophages, suggesting that they are yolk sac–derived macrophages. Instead, many of the F4/80 lo TIM-4 lo cells were c-kit + and Sca-1 + and had primitive morphology and multilineage colony-forming ability. In addition, F4/80 lo TIM-4 lo cells included a considerable population expressing ER-MP12, a known marker for macrophage colony–forming cells and other myeloid progenitors. We conclude that TIM molecules are expressed in embryonic hematopoietic tissues in chicken and mouse and that in fetal liver, TIM-4 is expressed by myeloid progenitor cells.

First-line treatments of bullous pemphigoid (BP) are topical and systemic glucocorticoids (GC). The actions of GC are mediated by glucocorticoid receptors (GR), which exist in several isoforms, of which GRα and GRβ are the most important. In many inflammatory diseases, up-regulation of GRβ is associated with GC insensitivity. The aims of this study were to determine the expression of GRα and GRβ in patients with BP and to investigate the effect of prednisolone treatment on the expression of GR isoforms in BP. Quantitative real-time PCR (qPCR) analysis demonstrated that GR isoform mRNAs are expressed in peripheral blood mononuclear cells (PBMC) from patients with BP. Expression of GRα and GRβ protein was confirmed by immunohistochemical staining of BP skin biopsies and by Western blot analysis and flow cytometric analysis of PBMCs. During prednisolone treatment, GRα and GRβ expression varied markedly, but changes were not suitable as a clinical marker of GC sensitivity in patients with BP.

Background: The nuclear factor kappa light-chain enhancer of activated B cells (NF-kappa B) signaling pathway is a key regulator of immune responses. Accordingly, mutations in several NF-kappa B pathway genes cause immunodeficiency. Objective: We sought to identify the cause of disease in 3 unrelated Finnish kindreds with variable symptoms of immunodeficiency and autoinflammation. Methods: We applied genetic linkage analysis and next-generation sequencing and functional analyses of NFKB1 and its mutated alleles. Results: In all affected subjects we detected novel heterozygous variants in NFKB1, encoding for p50/p105. Symptoms in variant carriers differed depending on the mutation. Patients harboring a p.I553M variant presented with antibody deficiency, infection susceptibility, and multiorgan autoimmunity. Patients with a p.H67R substitution had antibody deficiency and experienced autoinflammatory episodes, including aphthae, gastrointestinal disease, febrile attacks, and small-vessel vasculitis characteristic of Behc, et disease. Patients with a p.R157X stop-gain experienced hyperinflammatory responses to surgery and showed enhanced inflammasome activation. In functional analyses the p.R157X variant caused proteasome-dependent degradation of both the truncated and wild-type proteins, leading to a dramatic loss of p50/p105. The p.H67R variant reduced nuclear entry of p50 and showed decreased transcriptional activity in luciferase reporter assays. The p.I553M mutation in turn showed no change in p50 function but exhibited reduced p105 phosphorylation and stability. Affinity purification mass spectrometry also demonstrated that both missense variants led to altered protein-protein interactions. Conclusion: Our findings broaden the scope of phenotypes caused by mutations in NFKB1 and suggest that a subset of autoinflammatory diseases, such as Behcet disease, can be caused by rare monogenic variants in genes of the NF-kappa B pathway.

Intra-amniotic lipopolysaccharide (LPS) causes an acute inflammatory response and cardiac dysfunction in fetal mice. We hypothesized that the placenta protects the fetus against maternally administered bacterial toxins, delaying the onset of a fetal inflammatory response and vascular compromise. At 14 to 15 days of gestation, DBA mice were randomized to receive LPS (2.4 mg/kg) or vehicle intraperitoneally. Doppler ultrasonography of fetal cardiovascular hemodynamics was performed before and 6 hours after maternal LPS. Six hours after the LPS, maternal serum concentrations of tumor necrosis factor-alpha and interleukin (IL)-6 (P0.05) were increased. Placenta showed severe maternal vascular dilatation and congestion. The expressions of tumor necrosis factor-alpha, IL-1alpha, and IL-6 (P0.05) were increased, and the expression of Toll-like receptor 4 was constitutive in placenta. The expression of Toll-like receptor 2 increased (P0.05) and was detected in labyrinthine macrophages. No inflammatory activation was found in fetal tissues, and amniotic fluid revealed no significant increase in cytokines. The ultrasonographic examination demonstrated increased fetal cardiac afterload after LPS, with 65% of the fetuses exhibiting atrioventricular valve regurgitation. In conclusion, maternal inflammatory insult activates placental labyrinthine macrophages leading to an acute increase in placental vascular resistance and fetal cardiac dysfunction without an inflammatory response in fetus.

A qualitative multiplex reverse transcription (RT)-PCR and liquid hybridization assay for the detection of human enteroviruses, rhinoviruses, parechoviruses, and Aichi virus was developed. Furthermore, a separate assay for the recognition of hepatitis A virus was established to complement the test pattern so that all human picornaviruses were covered. The amplicons, which represented the 5′ untranslated regions of the viral RNA genomes, were identified in liquid hybridization reactions with genus-specific digoxigenin-labeled oligonucleotide probes. The sensitivity of the multiplex RT-PCR and liquid hybridization assay was 10 to 100 picornavirus genome equivalents for representatives of each picornavirus genus. The hepatitis A virus assay exhibited a sensitivity of 10 genome copies. Both the uniplex and the multiplex tests were highly specific for the target viruses. Twenty-three clinical samples, including cerebrospinal fluid, serum, and nasopharyngeal swab specimens, were used for clinical evaluation of the multiplex RT-PCR assay. The results obtained were consistent with the results of routine virus diagnostic assays. Furthermore, the assay was used to screen 68 stool specimens for the presence of parechoviruses and Aichi virus. One sample was found to contain parechovirus RNA, whereas no Aichi virus was detected. The assay described here can be applied for the efficient identification of human enteroviruses and rhinoviruses in clinical specimens and simultaneously enables the collection of information on the epidemiology and clinical outcomes of infections caused by the currently poorly known human parechoviruses and Aichi virus.

Spontaneous preterm birth due to intrauterine infection is associated with increased concentrations of cytokines in amniotic fluid and in the airways at birth. Intra-amniotic IL-1 induces fetal lung maturity, consistent with the decrease in the incidence of respiratory distress syndrome (RDS) in intrauterine inflammation. On the other hand, antenatal corticosteroid decreases the incidence of RDS in infants born prematurely. The aim of the present study was to investigate the interaction between IL-1 and glucocorticoid in the expression of the surfactant proteins SP-A, -B, and -C. Lung explants from rabbit fetuses at 22 (immature), 27 (transitional), and 30 (mature) d of gestation (term, 30-31 d) and on d 1 after term birth were cultured with dexamethasone (Dx), IL-1alpha, or vehicle in the presence or absence of actinomycin D. According to the present results, IL-1alpha and Dx additively increased the expression of SP-A and SP-B on d 22. Later in gestation, SP-B and SP-C were suppressed by IL-1, whereas glucocorticoid tended to increase the expression of SP-B and SP-C and prevented the IL-1-induced suppression of SP. IL-1alpha and steroid interactively increased the stability of SP mRNA compared with the single agonist, possibly explaining the additive effects on the SP mRNA levels. The present results reveal beneficial additive effects of glucocorticoid and cytokine on lung surfactant. They may explain some of the acute beneficial effects of glucocorticoid therapy in chorioamnionitis before premature birth and in inflammatory lung disease after birth.

The signal transducer and activator of transcription (STAT) family of transcription factors orchestrate hematopoietic cell differentiation. Recently, mutations in STAT1, STAT5B, and STAT3 have been linked to development of immunodysregulation polyendocrinopathy enteropathy X-linked-like syndrome. Here, we immunologically characterized 3 patients with de novo activating mutations in the DNA binding or dimerization domains of STAT3 (p.K392R, p.M394T, and p.K658N, respectively). The patients displayed multiorgan autoimmunity, lymphoproliferation, and delayed-onset mycobacterial disease. Immunologically, we noted hypogammaglobulinemia with terminal B-cell maturation arrest, dendritic cell deficiency, peripheral eosinopenia, increased double-negative (CD4(-)CD8(-)) T cells, and decreased natural killer, T helper 17, and regulatory T-cell numbers. Notably, the patient harboring the K392R mutation developed T-cell large granular lymphocytic leukemia at age 14 years. Our results broaden the spectrum of phenotypes caused by activating STAT3 mutations, highlight the role of STAT3 in the development and differentiation of multiple immune cell lineages, and strengthen the link between the STAT family of transcription factors and autoimmunity.

Surfactant protein (SP) A and SP-D are collectins that have roles in host defense. The Eustachian tube (ET) maintains the patency between the upper airways and the middle ear. Dysfunction of local mucosal immunity in ET may predispose infants to recurrent otitis media. We recently described preliminary evidence of the expression of SP-A and SP-D in the ET. Our present aim was to establish the sites of SP-A and SP-D expression within the epithelium of the ET in vivo. With in situ hybridization, electron microscopy, and immunoelectron microscopy, the cells responsible for SP-A and SP-D expression and storage were identified. SP-A expression was localized within the ET epithelium, and the protein was found in the electron-dense granules of microvillar epithelial cells. Being concentrated in the epithelial lining, only a few cells revealed intracellular SP-D, and it was not associated with granules. The SP-A and SP-D immunoreactivities in ET lavage fluid, as shown by Western blot analyses, were similar to those in bronchoalveolar lavage fluid. We propose that there are specialized cells in the ET epithelium expressing and secreting SP-A and SP-D. SP-A and SP-D may be important for antibody-independent protection of the middle ear against infections.

Surfactant protein (SP) B is an essential component of the pulmonary surfactant complex, which participates in reducing the surface tension across the alveolar air-liquid interface. The Eustachian tube (ET) connects the upper respiratory tract to the middle ear, serving as an intermittent airway between the pharynx and the middle ear. Recently, we described the expression of SP-A and SP-D in the ET, suggesting their role in middle ear host defense. Our present aim was to detect whether the expression of SP-B is evident in the porcine ET. With Northern blot analysis, RT-PCR, and in situ hybridizations, SP-B mRNA was identified and localized in the ET epithelium. The cellular localization of SP-B was revealed with immunohistochemistry, electron microscopy, and immunoelectron microscopy. The protein was found in the secretory granules of epithelial cells and also attached to the microvilli at the luminal side of these cells. The SP-B immunoreactivity of aggregates isolated from ET lavage fluid was similar to that isolated from bronchoalveolar lavage fluid. We conclude that there are specialized cells in the ET epithelium expressing and secreting SP-B and propose that SP-B may facilitate normal opening of the tube and mucociliary transport.

Surfactant proteins A and D are collectins which are considered to play an important role in the innate immunity of lungs. Our aim was to investigate whether surfactant protein A or D is expressed in the porcine Eustachian tube originating from the upper airways. Both surfactant proteins A and D were present in the epithelial cells of the Eustachian tube, as shown by strong immunostaining. Using RT-PCR and Northern hybridization, these collectins were detected in the Eustachian tube. The present study is the first report demonstrating surfactant protein gene expression in the Eustachian tube. Surfactant proteins A and D may be important in the antibody-independent protection of the middle ear.

The NFkB signaling pathway is a master regulator of the immune response. Recently, dominant mutations in NFKB2 have shown to cause antibody deficiency with adrenal insufficiency. We investigated two families with antibody deficiency and autoinflammatory features, with onset in teenage years and early adulthood (Fig. 1 and Table 1). In family 1, all patients available to study presented with respiratory tract infections and B cell dysfunction marked by hypogammaglobulinemia, poor antibody response to vaccines, or low switched memory B cell counts. Several experienced recurrent episodes of aphthous mucositis in upper gastrointestinal and genital area, sometimes accompanied by abdominal pain, monoarthritis, or fever with elevated inflammatory markers (peripheral blood leukocytes >10x106 cells/ml, C reactive protein >100 mg/L). A lesional biopsy revealed lymphocytic small cell vasculitis reminiscent of Behcet's disease. In family 2, all patients presented with respiratory tract infections, hypogammaglobulinemia, and poor vaccine responses. Based on the pedigrees, we assumed autosomal dominant inheritance. For family 1, we performed genotyping and linkage analysis coupled with whole genome sequencing to pinpoint the causative variant. Linkage analysis showed a 15.4 Mb haplotype on chr4 segregating with the phenotype in all affected individuals. When focusing our search on novel, heterozygous variants negatively affecting conserved residues, we identified only one variant in this genomic location, localizing to NFKB1 gene (p.H67R). In family 2, we exome sequenced one affected individual and filtered the data for novel heterozygous variants negatively affecting conserved residues. This resulted in 17 variants, out of which the I553M NFKB1 variant was considered the best candidate. Several functional analyses showed that the mutations had a negative impact on NFKB1 function. The H67R variant increased NFKB1 affinity to NEMO and delayed protein entry to nucleus, when assessed by affinity purification mass spectrometry and immunofluorescence microscopy. The mutation also decreased NFKB1 transcriptional activity, when expressed together with an NFkB-responsive luciferase reporter, and showed a neomorphic DNA binding pattern on chromatin immunoprecipitation-sequencing. Furthermore, the cell growth was hampered when the mutant NFKB1 was overexpressed in cultured HEK293 cells. The I553M showed a similar negative effect on cultured cell proliferation. Furthermore, the I553M variant negatively affected serine S907 and S893 phosphorylation, leading to defective posttranslational processing of the full-length NFKB1 upon TNF stimulation. To conclude, we show that hypogammaglobulinemia and Behcet's disease-like autoinflammatory syndrome can be caused by mutations in NFKB1. This suggests that a subset of Behcet's disease cases may be of monogenic origin, and highlights the role of NFkB complex in B cell maturation and innate immune regulation. Table 1. Patient characteristics Patient 1 2 3 4 5 6 7 8 9 10 11 12 Age 55 43# 25 29 40 30 37 7 10 60# 61 32 Sex F M F F F F M F M M F M Mutation status H67R ND H67R H67R H67R H67R H67R H67R H67R ND I553M I553M Immunodeficiency Infections URTI RTI URTI - URTI URTI URTI URTI - RTI RTI RTI Antibody deficiency Hypogammaglobulinemia, SAD ND Hypogammaglobulinemia, SAD - IgG subclass deficiency, SAD Hypogammaglobulinemia, SAD Hypogammaglobulinemia, SAD Hypogammaglobulinemia Hypogammaglobulinemia Hypogammaglobulinemia Hypogammaglobulinemia, SAD SAD Immunoglobulin replacement + - + - - + + - - - + - Immune dysregulation Febrile attacks - + + + - - + + - - - - Complex aphtae Mouth, genitalia ND Mouth, genitalia Mouth, genitalia - Mouth Esophagus Mouth, esophagus - - - - Arthritis Monoarthritis - Monoarthritis Monoarthritis - - - - - - Oligoarthritis - Gut disease Periodic abdominal pain, chronic idiopathic diarrhea ND Periodic abdominal pain, microscopic colitis Periodic abdominal pain - - - Periodic abdominal pain - ND Chronic idiopathic diarrhea Coeliac disease Other - Hyperinflammatory response and death after gallbladder surgery - Iritis, Hyperinflammatory response to tooth excision Kidney tumor - Rudimentary left kidney - - - Astma, hypothyreosis - URTI, upper respiratory tract infections; RTI, respiratory tract infections including recurrent pneumonia; SAD, Specific antibody deficiency; ND, No data; #Death age Figure 1. Family pedigrees. Figure 1. Family pedigrees. Disclosures Mustjoki: Sigrid Juselius Foundation: Research Funding; Signe and Ane Gyllenberg Foundation: Research Funding; the Finnish Cancer Societies: Research Funding; Novartis: Honoraria, Research Funding; Academy of Finland: Research Funding; Finnish Cancer Institute: Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Saarela:Roche: Honoraria.

Genomic clones capable of complementing a previously isolated arginine auxotrophic mutant strain of the filamentous yeast Trichosporon cutaneum DSM 70698 have been identified by DNA-mediated transformation, and a complementing 4,082-bp subfragment was sequenced. This analysis revealed an intact gene (arg4) showing a high degree of homology with the Saccharomyces cerevisiae CPA2 gene encoding the large subunit of carbamoyl-phosphate synthetase (CPS-A). The inferred amino acid sequence of the T. cutaneum argA-encoded protein contains 1,168 residues showing 62% identity with the sequence of the S. cerevisiae CPA2 protein, and the comparison of the two sequences uncovered a putative intron sequence of 81 nucleotides close to the 5' end of the coding region of the T. cutaneum argA gene. The presence of this intron was confirmed by nuclease protection studies and by direct DNA sequence analysis of a cDNA fragment which had been obtained by PCR amplification. The T. cutaneum intron shares the general characteristics of introns found in yeasts and filamentous fungi. A major transcript of around 4 kb was found in Northern (RNA) blots. The T. cutaneum argA coding region was expressed in Escherichia coli under the control of the regulatable tac promoter. A roughly 130-kDa protein which was found to cross-react with an anti-rat CPS antibody in Western blots (immunoblots) was observed. Two putative ATP-binding domains were identified, one in the amino-terminal half of the argA-encoded protein and the other in the carboxy-terminal half. These domains are highly conserved among the known CPS-A sequences from S. cerevisiae, E. coli, and the rat. From these results we conclude that the T. cutaneum argA gene encodes the large subunit of CPS. This is the first gene to be identified and analyzed in the T. cutaneum DSM 70698 strain.

Toll-like receptors (TLR:) have recently been linked to the immunostimulatory function of microbial toxins in human and mice. TLR: signals activation of nuclear factor kappaB that leads to the production of a number of proinflammatory mediators. TLR:4 mediates the endotoxin-induced inflammatory response, whereas TLR:2 may be involved in the response to yeast and Gram-positive bacterial products. To better understand age-related changes in acute inflammatory response, we studied the ontogeny of TLR:2 and TLR:4 mRNA in murine fetal lung, liver, and placenta by quantitative reverse transcriptase-PCR. Different expression patterns were seen between the tissues and between the TLR: This is in accordance with the evidence that there are differences in the receptors for different microbial toxins and that the response is organ specific. We additionally show that the expression of TLR: was dependent on the stage of differentiation. In the liver, the levels of Tlr2 and Tlr4 were high regardless of the age. In the lung, Tlr2 and Tlr4 expression levels were barely detectable in immature fetus (d 14-15). Tlr2 and Tlr4 were increased several-fold during prenatal development and further increased after birth. The present results support the finding of a deficient inflammatory response of the immature lung to microbial toxins.

Puumala hantavirus (PUUV) causes a hemorrhagic fever with renal syndrome (HFRS) also called nephropathia epidemica (NE). Recent case reports and retrospective studies suggest that NE may damage the pituitary gland. Based on these observations, our goal was to explore the nature of this complication prospectively.A total of 58 hospitalized patients with acute NE volunteered to participate. Central nervous system (CNS) symptoms were recorded, cerebrospinal fluid (CSF) samples were collected, human leukocyte antigen (HLA) haplotype was analyzed, brain magnetic resonance imaging (MRI) was acquired, and electroencephalography (EEG) was recorded. Patients with abnormal pituitary MRI finding were examined by an endocrinologist.Most patients experienced CNS symptoms, and half of the CSF samples were positive for PUUV IgM, elevated protein level, or leukocyte count. CSF of patients negative for DR15(2)-DQ6 haplotype was less frequently affected. MRI revealed pituitary hemorrhage in two patients; these two patients suffered sudden loss of vision associated with headache, and they both developed hypopituitarism. Only one patient required long-term hormonal replacement therapy.CNS-related symptoms and inflammation in the CSF are common in acute NE. Genetic properties of the host may predispose to CNS involvement. It does seem that pituitary injury and subsequent hormonal insufficiency may complicate the recovery.

Recent evidence suggests a role for phosphatidylinositol (PI) 3-kinase in various inflammatory responses. In this study, the consequences of LPS-induced PI 3-kinase activation on cytokine and chemokine expression and the intracellular mechanisms of inflammatory activation were examined in mouse macrophages. LPS stimulation induced a complex formation between PI 3-kinase and myeloid differentiation factor 88 (MyD88), which was followed by an induction of IL-1beta, tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein (MIP)-2. The induction of IL-1beta, but not of MIP-2 or TNF-alpha, was blocked by the PI 3-kinase inhibitors LY294002 and wortmannin. The nuclear factor-kappaB (NF-kappaB) inhibitor pyrrolidinedithiocarbamate (PDTC) blocked the induction of IL-1beta and TNF-alpha, but had no effect on MIP-2 expression. Inhibition of PI 3-kinase decreased the LPS-induced transcriptional activity of NF-kappaB, but it had no effect on the nuclear DNA binding activity of NF-kappaB. These findings suggest that, while NF-kappaB nuclear localization and DNA binding are necessary, they are not sufficient for transcriptional activation of the IL-1beta gene in the absence of PI 3-kinase activity. Taken together, our results demonstrate that activation of Toll-like receptor (TLR)-4 results in PI 3-kinase-MyD88 complex formation, and that PI 3-kinase activity selectively leads to cytokine induction downstream of TLR4.

The main extracellular matrix components of the lung, type I and III collagens, were studied in chronic allograft rejection developing in a porcine heterotopic bronchial transplantation model. Specific porcine complementary DNA probes were constructed for detection of the expression of type I and III procollagen messenger RNAs in the bronchial wall structures and in the obliterative plug by in situ hybridization. In autografts, and in allografts immunosuppressed with 40-O-(2-hydroxyethyl)-rapamycin, cyclosporine A, and methylprednisolone, no histological changes of obliterative bronchiolitis (OB) developed, and the number of fibroblast-like cells expressing type I and III procollagen mRNA remained low. In nontreated allografts obliterating within 21 d, a preponderance of fibroblast-like cells showing positivity for type III procollagen mRNA existed in the obliterative plug and bronchial wall. This study shows for the first time the temporal and spatial activation of type I and III procollagen genes during the course of obliterative bronchiolitis. The number of cells expressing procollagen III mRNA increased parallel to developing obliteration and fibrosis in nontreated allografts, whereas autografts and immunosuppressed allografts exhibited no such trend. This finding suggests a positive association between type III collagen mRNA expression in fibroblast-like cells and development of obliterative bronchiolitis.