High Levels Of IFN-α Research Articles

Screening for autoimmune diseases in apparently healthy antinuclear antibody positive individuals.

Anti-nuclear antibodies (ANA) assessed by immunofluorescence (IF) microscopy are associated with systemic autoimmune rheumatic diseases (SARD) and can be detected years before onset of clinical symptoms. Recent data indicate dysregulation of the immune system with increased levels of proinflammatory cytokines, including type I interferons (IFN), in ANA-positive versus ANA-negative individuals. Herein, the aims were to investigate IF-ANA, ANA fine specificities, and IFN-α protein levels in relation to self-reported symptoms, as well as clinical signs, of SARD in a large group of healthy blood donors (HBD). Sera from 825 HBD (48.8% females) were included. IF-ANA was assessed, using HEp-2 cells, according to the routine at the accredited laboratory of Clinical Immunology, Linköping University Hospital. All samples were analyzed for IgG-ANA fine specificities using addressable laser bead assay (ALBIA) at the same laboratory. IFN-α was determined using ELISA. Antibody-positive individuals, and their sex- and age-matched antibody-negative controls, were asked to fill a questionnaire regarding symptoms associated with SARD. In total, 130 HBD (15.8%) were positive with IF-ANA and/or ALBIA. Anti-U1RNP was significantly more common among women. Generally, self-reported symptoms correlated poorly with IF-ANA and/or ALBIA results. Two females with high levels of Ro60/SSA, Ro52/SSA and IFN-α reported mild sicca symptoms and were diagnosed with Sjögren's disease after clinical evaluation. A considerable proportion of apparently HBD are autoantibody positive, but without clear association to self-reported symptoms. Nevertheless, the combination of autoantibodies, relevant symptoms and high IFN-α levels identified the small proportion of individuals with SARD in the study population.

Resistance to African swine fever virus among African domestic pigs appears to be associated with a distinct polymorphic signature in the RelA gene and upregulation of RelA transcription

African swine fever virus (ASFV) is a highly contagious and fatal hemorrhagic disease of domestic pigs, which poses a major threat to the swine industry worldwide. Studies have shown that indigenous African pigs tolerate ASFV infection better than European pigs. The porcine v-rel avian reticuloendotheliosis viral oncogene homolog A (RelA) encoding a p65 kD protein, a major subunit of the NF-kB transcription factor, plays important roles in controlling both innate and adaptive immunity during infection with ASFV. In the present study, RelA genes from ASFV-surviving and symptomatic pigs were sequenced and found to contain polymorphisms revealing two discrete RelA amino acid sequences. One was found in the surviving pigs, and the other in symptomatic pigs. In total, 16 nonsynonymous SNPs (nsSNPs) resulting in codon changes were identified using bioinformatics software (SIFT and Polyphen v2) and web-based tools (MutPre and PredictSNP). Seven nsSNPs (P374-S, T448-S, P462-R, V464-P, Q478-H, L495-E, and P499-Q) were predicted to alter RelA protein function and stability, while 5 of these (P374-S, T448-S, P462-R, L495-E, and Q499-P) were predicted as disease-related SNPs.Additionally, the inflammatory cytokine levels of IFN-α, IL-10, and TNF-α at both the protein and the mRNA transcript levels were measured using ELISA and Real-Time PCR, respectively. The resulting data was used in correlation analysis to assess the association between cytokine levels and the RelA gene expression. Higher levels of IFN-α and detectable levels of IL-10 protein and RelA mRNA were observed in surviving pigs compared to healthy (non-infected). A positive correlation of IFN-α cytokine levels with RelA mRNA expression was also obtained. In conclusion, 7 polymorphic events in the coding region of the RelA gene may contribute to the tolerance of ASFV in pigs.

Food Insecurity and Undernutrition Are Associated With Distinct Immunologic Profiles in People With Tuberculosis and Advanced HIV Starting Antiretroviral Therapy.

Food insecurity and undernutrition are related but distinct concepts contributing to poor HIV and tuberculosis outcomes. Pathways linking them with immunologic profile, which may relate to clinical outcomes, remain understudied. We analyzed data from a cohort study of 165 antiretroviral therapy (ART)-naïve adults with advanced HIV and newly diagnosed tuberculosis in Botswana from 2009 to 2013. Twenty-nine plasma biomarkers were measured pre-ART and 4 weeks post-ART initiation. We used principal components analysis (PCA) and multivariable linear regression models to assess relationships between immunological profiles and food insecurity (based on the Household Food Insecurity Access Scale), undernutrition (body mass index <18.5 kg/m 2 ), and clinical outcomes. PCA identified 5 principal components with eigenvalues >1. After adjustment, food insecurity was associated with PC3 pre-ART (0.19 per increased category of severity, 95% CI: 0.02 to 0.36) and post-ART (0.24, 95% CI: 0.07 to 0.41). PC3 was driven by higher levels of IFN-α, IFN-γ, interleukin (IL)-12p40, vascular endothelial growth factor, IL-1α, and IL-8 and decreased concentrations of IL-3. Undernutrition was associated with PC5 post-ART (0.49, 95% CI: 0.16 to 0.82). PC5 was driven by higher levels of IL-8, MIP-1α, IL-6, and IL-10 and decreased concentrations in IP-10 and IFN-α. Post-ART PC3 (4.3 percentage point increased risk per increased score of 1, 95% CI: 0.3 to 8.9) and post-ART PC5 (4.8, 95% CI: 0.6 to 8.9) were associated with death in adjusted models. We identified 2 distinct immunologic profiles associated with food insecurity, undernutrition, and clinical outcomes in patients with advanced HIV and tuberculosis. Different pathophysiologic processes may link food insecurity and undernutrition with poor outcomes in this vulnerable patient population. Future studies should assess the impact of improving food access and intake on immune function and clinical outcomes.

Imprinted immune abnormalities in liver transplant patients cured of hepatitis C with antiviral drugs.

Chronic HCV infection induces interferon and dysregulates immune responses through inflammation and chronic antigenic stimulation. Antiviral drugs can cure HCV, providing a unique opportunity to examine the immunological restoration that does and does not occur when a chronic viral infection is eradicated. We quantified blood cytokines levels and used mass cytometry to immunophenotype peripheral blood mononuclear cells before and after HCV cure in 2 groups of patients and controls. At baseline, serum interferon α and soluble CD163 (a macrophage product) were elevated in both liver transplant and nonliver transplant patients compared to controls; the frequencies of several peripheral blood mononuclear cell populations differed from controls; and programmed death protein 1-positivity was increased in nearly all T cell subsets. Many abnormalities persisted after HCV cure, including elevated programmed death protein 1 expression on CD4 naïve and central memory T cells, elevated soluble CD163, and expansion of the plasmablast/plasma cell compartment. Several myeloid-lineage subsets, including Ag-presenting dendritic cells, remained dysregulated. In mechanistic studies, interferon α treatment increased programmed death protein 1 on human T cells and increased T cell receptor signaling. The data identify immunological abnormalities that persist after curative HCV treatment. Before cure, high levels of interferon α may stimulate programmed death protein 1 expression on human T cells, causing persistent functional changes.

TREX1 531C/T Polymorphism and Autoantibodies Associated with the Immune Status of HIV-1-Infected Individuals.

Autoimmune diseases can develop during HIV-1 infection, mainly related to the individual's immune competence. The study investigated the association of the TREX1 531C/T polymorphism and antinuclear antibodies (ANA) in HIV-1 infection and the time of antiretroviral therapy (ART) used. Cross-sectional and longitudinal assessments were carried out in 150 individuals, divided into three groups: ART-naïve, 5 years and 10 years on ART; ART-naïve individuals were evaluated for 2 years after initiation of treatment. The individuals' blood samples were submitted to indirect immunofluorescence tests, real-time PCR and flow cytometry. The TREX1 531C/T polymorphism was associated with higher levels of TCD4+ lymphocytes and IFN-α in individuals with HIV-1. Individuals on ART had a higher frequency of ANA, higher levels of T CD4+ lymphocytes, a higher ratio of T CD4+/CD8+ lymphocytes and higher levels of IFN-α than therapy-naïve individuals (p < 0.05). The TREX1 531C/T polymorphism was associated with better maintenance of the immune status of individuals with HIV-1 and ANA with immune restoration in individuals on ART, indicating the need to identify individuals at risk of developing an autoimmune disease.

Macrophage phagocytosis of SARS-CoV-2-infected cells mediates potent plasmacytoid dendritic cell activation

Early and strong interferon type I (IFN-I) responses are usually associated with mild COVID-19 disease, whereas persistent or unregulated proinflammatory cytokine responses are associated with severe disease outcomes. Previous work suggested that monocyte-derived macrophages (MDMs) are resistant and unresponsive to SARS-CoV-2 infection. Here, we demonstrate that upon phagocytosis of SARS-CoV-2-infected cells, MDMs are activated and secrete IL-6 and TNF. Importantly, activated MDMs in turn mediate strong activation of plasmacytoid dendritic cells (pDCs), leading to the secretion of high levels of IFN-α and TNF. Furthermore, pDC activation promoted IL-6 production by MDMs. This kind of pDC activation was dependent on direct integrin-mediated cell‒cell contacts and involved stimulation of the TLR7 and STING signaling pathways. Overall, the present study describes a novel and potent pathway of pDC activation that is linked to the macrophage-mediated clearance of infected cells. These findings suggest that a high infection rate by SARS-CoV-2 may lead to exaggerated cytokine responses, which may contribute to tissue damage and severe disease.

A single, oral dose of the TLR7 agonist JNJ-64794964 induces transcriptomic and phenotypic changes in peripheral immune cells in healthy adults.

Chronic hepatitis B (CHB) is responsible for major disease burden worldwide. However, the number of available therapies is limited; cure remains an elusive goal. JNJ-64794964 (JNJ-4964) is an oral toll-like receptor-7 (TLR7) agonist being evaluated for the treatment of CHB. Here, we investigated the capacity of JNJ-4964 to induce transcriptomic and immune cell changes in peripheral blood in healthy volunteers. Peripheral blood was collected in the JNJ-4964 first-in-human phase 1 trial at multiple time points to assess transcriptomics and changes in frequency and phenotype of peripheral-blood mononuclear cells. Correlation of changes to JNJ-4964 exposure (Cmax) and changes in cytokine levels (C-X-C motif chemokine ligand 10 [CXCL10] and interferon alpha [IFN-α]) were evaluated. Fifty-nine genes, mainly interferon-stimulated genes, were up-regulated between 6hours and 5days after JNJ-4964 administration. JNJ-4964 increased frequencies of CD69, CD134, CD137, and/or CD253-expressing natural killer (NK) cells, indicative of NK cell activation. These changes correlated with Cmax, increase of CXCL10, and induction of IFN-α and were observed at IFN-α levels that are associated with no/acceptable flu-like adverse events. JNJ-4964 administration resulted in increased frequencies of CD86-expressing B cells, indicative of B-cell activation. These changes were predominantly observed at high IFN-α levels, which are associated with flu-like adverse events. JNJ-4964 administration led to changes in transcriptional profiles and immune cell activation phenotype, particularly for NK cells and B cells. Together, these changes could represent a set of biomarkers for the characterization of the immune response in CHB patients receiving TLR7 agonists.

Abstract CT096: Phase 1 study of RO7119929 (TLR7 agonist prodrug) in patients (pts) with advanced primary or metastatic liver cancers

Abstract Background Agonists for TLR7, a major regulator of the innate immune response, have been tested in oncology across several nonclinical and clinical investigations due to their ability to stimulate T-cell mediated cancer cell killing. The orally available TLR7 agonist prodrug RO7119929 is converted to active drug predominantly in the liver. It was hypothesized to effectively reprogram the local immune microenvironment and thus enhance anti-tumor activity in liver tumors. Methods In this phase 1, first-in-human, open-label, dose-escalation study (NCT04338685), eligible pts had histologically confirmed advanced or metastatic primary liver cancers or solid tumors with predominant liver involvement. Pts received oral RO7119929 weekly in 3-week cycles in Part A (dose-escalation: Flat dosing [FD][A1], step-up dosing [SUD][A2] or FD with tocilizumab pre-treatment [A3]) and Part B (FD dose expansion with paired biopsies). Participants received treatment until disease progression, unacceptable toxicity or withdrawal. Primary endpoints were maximum tolerated dose (MTD) and/or optimal biologic dose and safety. Results At data cut-off (22 April 2022), 55 pts were enrolled (A1: n=27; A2: n=9; A3: n=1; B1: n=18). Median age: 58 years; 76% male; 51% Asian; 52% received ≥3 prior lines of therapy; 31% HCC as primary tumor type. Most pts discontinued treatment due to disease progression (75%). Among 17 HCC pts, a durable complete response was observed in one pt and 10 (59%) pts had stable disease. Treatment-related AEs were reported in 91% of pts, most common event was CRS (87%). Fever following drug administration was generally reported as CRS, and most CRS events were Grade 1 (fever). With FD, Grade 2 CRS occurred in 20 (44%) pts and Grade 3 CRS in 6 (13%) pts. With SUD, Grade 2 CRS occurred in one (11%) pt and no Grade 3 CRS was reported. The incidence and severity of CRS events was dose-dependent. Events generally had a predictable onset within 12h post-dosing. All events resolved, the majority within 1-2 days. CRS events of Grade 2/3 commonly occurred in Cycle 1, with later events rarely exceeding Grade 1. Increases in the TLR7-related peripheral pharmacodynamic (PD) biomarkers IFNα, ISG15 and IL6 were associated with higher doses, with particularly IL6 correlating with CRS severity. With SUD, high levels of IFNα and ISG15 levels were maintained while IL6 levels were reduced compared with FD. Pharmacokinetics (PK) of the active TLR7 agonist drug appeared linear (Cmax, AUC) and time-independent with a short effective t1/2 of approximately 5h on average. PK, PD and safety modeling-informed dose decisions. Conclusion CRS was identified as a dose-limiting safety risk, the incidence and severity of CRS appeared dose-dependent, and events were predictable and manageable. SUD reduced the risk of CRS whilst maintaining mode of action relevant PD effects. Combination therapy with a checkpoint inhibitor may be needed to leverage the pro-inflammatory potential of RO7119929 and further increase anti-tumor activity. Citation Format: Changhoon Yoo, Helena Verdaguer, Chia-Chi Lin, Camilla Qvortrup, Thomas Yau, Do-Youn Oh, Felix Lichtenegger, Izolda Franjkovic, Nicole Kratochwil, Juliana Bessa, Eva Rossmann, Elia Hall, Congqi Dai, Oliver Krieter, Audrey Yeo Te-Ying, Michael Cannarile, Christina Schiff, Bruno Sangro. Phase 1 study of RO7119929 (TLR7 agonist prodrug) in patients (pts) with advanced primary or metastatic liver cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT096.

Role of interferons (IFNs) in the differentiation of T peripheral helper (Tph) cells.

Interleukin (IL)-21-producing T peripheral helper (Tph) cells are thought to contribute to extra-follicular B cell activation and play a pathogenic role in autoimmune diseases. In this study, we investigated the relationship between Tph cells and interferons (IFNs) in several autoimmune diseases because our previous study demonstrated that type I IFNs promote the differentiation of IL-21-producing Tph-like cells. The frequency of Tph cells in the blood as well as serum IFN-α2a and IFN-λ1 were markedly elevated in patients with active systemic lupus erythematosus (SLE) compared to other autoimmune diseases or healthy controls. Notably, the frequency of Tph cells was positively correlated with the SLE disease activity index, serum IFN-α and serum IFN-λ1 in SLE patients. Additionally, we found that type III IFNs (IFN-λ1, IFN-λ2 and IFN-λ3) promote the differentiation of programmed cell death-1 (PD-1)+ CXCR5 -CD4+ T cells and enhance the secretion of IL-21, IFN-γ and CXCL13. IFN-λ1, like IFN-α, up-regulated the mRNA expression of IL21, IFNG, CXCL13, CD244, SLAMF7, GZMB, PRF1, CCR5 and PRDM1, whereas it down-regulated that of CXCR5 and BCL6, reflecting a Tph-related gene expression pattern. IFN-α in combination with IFN-λ1, IFN-λ2 or IFN-λ3 significantly increased the differentiation of PD-1+CXCR5- Tph-like cells and the secretion of Tph-related cytokines as compared with each IFN alone, suggesting a cooperative interaction. From these findings, it is highly probable that type III IFNs in addition to type I IFNs play a key role in the differentiation of Tph cells and that high levels of IFN-α and IFN-λ1 trigger the differentiation and expansion of Tph cells in SLE.

Levels of epigenetic regulators TET2 and DNMT1 correlate with virus-stimulated pDC differentiation from IFN-α producing cells to antigen presenting cells

Abstract Plasmacytoid dendritic cells (pDC) are potent producers of IFN-α in response to DNA and RNA viruses and subsequently mature into antigen presenting cells. We investigated the role of DNA methyltransferase 1 (DNMT1) and Ten-eleven translocation methyl-cytosine dioxygenase 2 (TET2), which are generally known as gene silencers and activators, respectively, in pDC activation. PBMC from healthy donors were stimulated with Herpes Simplex Virus (HSV) or Influenza-A virus (IAV) for 6- and 24h. PBMC were surface-stained for pDC markers CD123, HLA-DR and CD11C and co-stimulatory markers CD80 and CD86, then intracellularly for DNMT1, TET2 and IFN-α, and acquired by flow cytometry. mRNA expression of DNMT1 in purified pDC was determined by qRT-PCR. At 6h, DNMT1 protein was expressed at equivalent levels in pDC with and without stimulation and in IFN-α+ and IFN-α-pDC, and in unstimulated controls. In contrast, TET2 proteins were elevated in IFN-α+ but not IFN-α-pDC and controls at 6h. At 24h, DNMT1 increased while TET2 was reduced in stimulated pDC, correlating with high IFN-α levels at 6h and low levels at 24h. There was no change in DNMT1 gene expression in virus-stimulated vs. unstimulated controls. CD80 and CD86 were elevated at 24h compared to 6h in stimulated pDC, confirming pDC differentiation from IFN-α producing to antigen presenting cells. At the point of pDC differentiation, TET2 levels were decreased and DNMT1 increased. These results suggest that TET2 is demethylating and causing activation of genes associated with pDC IFN-α production, while DNMT1 methylates and silences genes in this pathway. This results in an initial induction of TET2 and IFN-α production followed by upregulation of co-stimulatory markers, DNMT1 and loss of IFN-α. Supported by 1. AAI trainee fellowship Careers in Immunology Fellowship Program AAI EIN #: 52-2317193 2. R01Al106125

IRF5 risk variants contribute to pre-symptomatic SLE by enhancing the levels of circulating NET antigens

Abstract SLE is a complex multifactorial autoimmune disease characterized by high levels of autoantibodies that impact many organs. Neutrophil extracellular traps (NETs) are a potential source of antigen leading to the production of autoantibodies, as antibodies against NET components are detected in SLE patients. Higher level of NETs and decreased clearance of NETs are associated with SLE and other autoimmune diseases. Neutrophils play an key role in pediatric lupus and NETs from pediatric lupus patients activate plasmacytoid dendritic cells (pDCs) to produce high levels of IFN-α. Polymorphisms within and around the IRF5 gene associate with risk of developing SLE. We previously reported that healthy donor neutrophils from IRF5 homozygous risk carriers underwent increased spontaneous NETosis, as compared to non-risk donors, that resulted in increased IFN-α production, plasma cell differentiation and autoantibody production. Whether the observed increase in neutrophil-mediated ETosis is a systemic signature of IRF5-driven pre-symptomatic SLE is not known, nor is it known whether these IRF5 risk carriers have defects in NET clearance. We thus developed and optimized new assays to detect small quantities of NET remnants in plasma samples using a combined MPO-CitH3 antibody cocktail followed by detection of DNA by ELISA. Further, we designed a novel immunofluorescence technique to visualize and quantify plasma NETs in healthy donor risk and non-risk carriers, as compared to pediatric and adult SLE patients. In addition, plasma DNase 1 and DNase 1L3 levels were measured. This study will report on the mechanisms by which circulating NETs are increased in pediatric and adult SLE, as well as the contribution of IRF5 genetic risk to NET clearance. Supported by Lupus Research Alliance Department of Defense CDMRP LRP W81XWH-18-1-0674

Porcine Plasmacytoid Dendritic Cells Are Unique in Their Expression of a Functional NKp46 Receptor.

The activating receptor NKp46 shows a unique expression pattern on porcine leukocytes. We showed already that in swine not all NK cells express NKp46 and that CD3+NKp46+ lymphocytes form a T-cell subset with unique functional properties. Here we demonstrate the expression of NKp46 on CD4highCD14-CD172a+ porcine plasmacytoid dendritic cells (pDCs). Multicolor flow cytometry analyses revealed that the vast majority of porcine pDCs (94.2% ± 4) express NKp46 ex vivo and have an increased expression on the single-cell level compared to NK cells. FSC/SSChighCD4highNKp46+ cells produced high levels of IFN-α after CpG ODN 2216 stimulation, a hallmark of pDC function. Following receptor triggering with plate-bound monoclonal antibodies against NKp46, phosphorylation of signaling molecules downstream of NKp46 was analyzed in pDCs and NK cells. Comparable to NK cells, NKp46 triggering led to an upregulation of the phosphorylated ribosomal protein S6 (pS6) in pDCs, indicating an active signaling pathway of NKp46 in porcine pDCs. Nevertheless, a defined effector function of the NK-associated receptor on porcine pDCs could not be demonstrated yet. NKp46-mediated cytotoxicity, as shown for NK cells, does not seem to occur, as NKp46+ pDCs did not express perforin. Yet, NKp46 triggering seems to contribute to cytokine production in porcine pDCs, as induction of TNF-α was observed in a small pDC subset after NKp46 cross-linking. To our knowledge, this is the first report on NKp46 expression on pDCs in a mammalian species, showing that this receptor contributes to pDC activation and function.

Intranasal delivery of inactivated PRRSV loaded cationic nanoparticles coupled with enterotoxin subunit B induces PRRSV-specific immune responses in pigs

This study was conducted to evaluate the induction of systemic and mucosal immune responses and protective efficacy following the intranasal administration of inactivated porcine reproductive and respiratory syndrome virus (PRRSV) loaded in polylactic acid (PLA) nanoparticles coupled with heat-labile enterotoxin subunit B (LTB) and dimethyldioctadecylammonium bromide (DDA). Here, 42- to 3-week-old PRRSV-free pigs were randomly allocated into 7 groups of 6 pigs each. Two groups represented the negative (nonvaccinated pigs/nonchallenged pigs, NoVacNoChal) and challenge (nonvaccinated/challenged, NoVacChal) controls. The pigs in the other 5 groups, namely, PLA nanoparticles/challenged (blank NPs), LTB-DDA coupled with PLA nanoparticles/challenged (adjuvant-blank NPs), PLA nanoparticles-encapsulating inactivated PRRSV/challenged (KNPs), LTB-DDA coupled with PLA nanoparticles loaded with inactivated PRRSV/challenged pigs (adjuvant-KNPs) and inactivated PRRSV/challenged pigs (inactivated PRRSV), were intranasally vaccinated with previously described vaccines at 0, 7 and 14 days post-vaccination (DPV). Serum and nasal swab samples were collected weekly and assayed by ELISA to detect the presence of IgG and IgA, respectively. Viral neutralizing titer (VNT) in sera, IFN-γ-producing cells and IL-10 secretion in stimulated peripheral blood mononuclear cells (PBMCs) were also measured. The pigs were intranasally challenged with PRRSV-2 at 28 DPV and necropsied at 35 DPV, and then macro- and microscopic lung lesions were evaluated. The results demonstrated that following vaccination, adjuvant-KNP-vaccinated pigs had significantly higher levels of IFN-γ-producing cells, VNT and IgG in sera, and IgA in nasal swab samples and significantly lower IL-10 levels than the other vaccinated groups. Following challenge, the adjuvant-KNP-vaccinated pigs had significantly lower PRRSV RNA and macro- and microscopic lung lesions than the other vaccinated groups. In conclusion, the results of the study demonstrated that adjuvant-KNPs are effective in eliciting immune responses against PRRSV and protecting against PRRSV infections over KNPs and inactivated PRRSV and can be used as an adjuvant for intranasal PRRSV vaccines.

Enhanced type I interferon signature induces neutrophil extracellular traps enriched in mitochondrial DNA in adult-onset Still's disease

Adult-onset Still's disease (AOSD) is a rare but clinically well-known auto-inflammatory disorder. Cytokine storm, the hallmark of AOSD, is mediated by neutrophil hyperactivation and enhanced neutrophil extracellular trap (NET) formation. Type I interferons (IFNs), having a primary role in the initiation of proinflammation responses, can induce subsequent inflammatory cytokine production. However, the role of type I IFNs in AOSD is unclear. Indeed, high levels of IFN-α and IFN-β expression are presented by AOSD patients. In this investigation, hierarchical unsupervised clustering was performed on IFN-α and IFN-β data to identify a cluster of AOSD patients who had a serious condition. Neutrophils from treatment-naïve active AOSD patients showed very strong enrichment in their IFN-α response, as shown by RNA-seq and confirmed by the IFN score. Whether IFN-α stimulates NET formation was also tested. IFN-α had the ability to form NETs that contained oxidized mitochondrial DNA (ox-mtDNA). Moreover, the JAK inhibitor could be used to dampen type I IFN-induced NET formation and eventually control ox-mtDNA release. Our results demonstrated the important roles of type I IFNs in the pathogenesis of AOSD through their promotion of NET formation, as characterized by the enhanced level of ox-mtDNA. The findings open up new avenues of research into therapeutic approaches for AOSD.

Immunogenic characteristics of the outer membrane phosphoporin as a vaccine candidate against Klebsiella pneumoniae

In recent years, Klebsiella pneumoniae (KP) has caused disease outbreaks in different animals, resulting in serious economic losses and biosafety concerns. Considering the broad antibiotic resistance of KP, vaccines are the most effective tools against infection. However, there is still no KP vaccine available in the veterinary field. Our results indicate that the highly conserved outer membrane phosphoporin (PhoE) of KP is immunogenic in mice and elicits high titers of antibodies that were shown to be specific for PhoE by immunoblotting. Immunization with PhoE also induced robust cell-mediated immunity and elicited the secretion of high levels of IFN-γ and IL-4, suggesting the induction of mixed Th1 and Th2 responses. Sera from PhoE-immunized mice induced significantly higher complement-mediated lysis of KP cells than did sera from the PBS control mice. Finally, mice immunized with PhoE were significantly protected against KP challenge, with better survival and a reduced visceral bacterial load. Our data underscore the great potential of PhoE as a novel candidate antigen for a vaccine against KP infection.

Lupus susceptibility region containing CTLA4 rs17268364 functionally reduces CTLA4 expression by binding EWSR1 and correlates IFN-\u03b1 signature

BackgroundDysregulation of T cells mediated immune responses is a hallmark in the development of systemic lupus erythematosus (SLE). Recent genome wide association study (GWAS) revealed the genetic contribution of variants located in the cytotoxic T lymphocyte-associated protein-4 (CTLA4)-inducible T cell co-stimulator (ICOS) intergenic region to SLE susceptibility. Our aim is to find a functional variant in this region.MethodsThe genetic association results in the CTLA4-ICOS region from previous GWAS were adopted to select the potential variant which was further replicated in two independent cohorts (Henan cohort 2053 SLE patients and 1845 healthy controls, Beijing cohort 2303 SLE patients and 19,262 healthy). In order to explore the functional significance in SLE, bioinformatics with validation experiments (including electrophoretic mobility shift assay and luciferase reporter assay) and mRNA expression analysis were also performed.ResultsA variant located in the CTLA4-ICOS intergenic region, rs17268364, was associated with susceptibility to SLE patients in Chinese populations (risk allele, pmeta = 7.02×10−11, OR 1.19, 95%CI 1.13–1.26). The bioinformatics suggested that rs17268364 might affect the expression of CTLA4, not ICOS. The rs17268364 risk G allele containing sequence reduced the expression of the reporter gene by binding transcriptional repressor Ewing sarcoma breakpoint region 1 (EWSR1). Following genotype-mRNA expression, the analysis also showed the risk allele of rs17268364 was associated with low CTLA4 expression in lupus nephritis (LN) patients. Healthy individuals carrying rs17268364 risk G allele was significantly correlated with higher levels of IFN-α signature including increased lymphocyte antigen 6E (LY6E) (p=0.031), interferon-stimulated gene 15 (ISG15) (p=0.038), interferon regulatory factor 9 (IRF9) (p=0.028), and interferon regulatory factor 5 (IRF5) (p=0.040) mRNA expression.ConclusionsThe present study confirmed the functional role of rs17268364 in the CTLA4-ICOS intergenic region that increased SLE susceptibility in the Chinese population.

Alloantigen-activated (AAA) CD4+ T cells reinvigorate host endogenous T cell immunity to eliminate pre-established tumors in mice

BackgroundCancer vaccines that induce endogenous antitumor immunity represent an ideal strategy to overcome intractable cancers. However, doing this against a pre-established cancer using autologous immune cells has proven to be challenging. “Allogeneic effects” refers to the induction of an endogenous immune response upon adoptive transfer of allogeneic lymphocytes without utilizing hematopoietic stem cell transplantation. While allogeneic lymphocytes have a potent ability to activate host immunity as a cell adjuvant, novel strategies that can activate endogenous antitumor activity in cancer patients remain an unmet need. In this study, we established a new method to destroy pre-developed tumors and confer potent antitumor immunity in mice using alloantigen-activated CD4+ (named AAA-CD4+) T cells.MethodsAAA-CD4+ T cells were generated from CD4+ T cells isolated from BALB/c mice in cultures with dendritic cells (DCs) induced from C57BL/6 (B6) mice. In this culture, allogeneic CD4+ T cells that recognize and react to B6 mouse-derived alloantigens are preferentially activated. These AAA-CD4+ T cells were directly injected into the pre-established melanoma in B6 mice to assess their ability to elicit antitumor immunity in vivo.ResultsUpon intratumoral injection, these AAA-CD4+ T cells underwent a dramatic expansion in the tumor and secreted high levels of IFN-γ and IL-2. This was accompanied by markedly increased infiltration of host-derived CD8+ T cells, CD4+ T cells, natural killer (NK) cells, DCs, and type-1 like macrophages. Selective depletion of host CD8+ T cells, rather than NK cells, abrogated this therapeutic effect. Thus, intratumoral administration of AAA-CD4+ T cells results in a robust endogenous CD8+ T cell response that destroys pre-established melanoma. This locally induced antitumor immunity elicited systemic protection to eliminate tumors at distal sites, persisted over 6 months in vivo, and protected the animals from tumor re-challenge. Notably, the injected AAA-CD4+ T cells disappeared within 7 days and caused no adverse reactions.ConclusionsOur findings indicate that AAA-CD4+ T cells reinvigorate endogenous cytotoxic T cells to eradicate pre-established melanoma and induce long-term protective antitumor immunity. This approach can be immediately applied to patients with advanced melanoma and may have broad implications in the treatment of other types of solid tumors.

Intranasal immunization with inactivated chlamydial elementary bodies formulated in VCG-chitosan nanoparticles induces robust immunity against intranasal Chlamydia psittaci challenge

Vaccines based on live attenuated Chlamydia elementary bodies (EBs) can cause disease in vaccinated animals and the comparably safer inactivated whole EBs are only marginally protective. Recent studies show that a vaccine formulation comprising UV-inactivated EBs (EB) and appropriate mucosal delivery systems and/or adjuvants induced significant protective immunity. We tested the hypothesis that intranasal delivery of UV-inactivated C. psittaci EB formulated in Vibrio cholerae ghosts (VCG)-chitosan nanoparticles will induce protective immunity against intranasal challenge in SPF chickens. We first compared the impact of VCG and CpG adjuvants on protective immunity following IN mucosal and IM systemic delivery of EB formulated in chitosan hydrogel/microspheres. Immunologic analysis revealed that IN immunization in the presence of VCG induced higher levels of IFN-γ response than IM delivery or the CpG adjuvanted groups. Also, vaccine efficacy evaluation showed enhanced pharyngeal bacterial clearance and protection against lung lesions with the VCG adjuvanted vaccine formulation, thereby establishing the superior adjuvanticity of VCG over CpG. We next evaluated the impact of different concentrations of VCG on protective immunity following IN mucosal immunization. Interestingly, the adjuvanticity of VCG was concentration-dependent, since protective immunity induced following IN mucosal immunization showed dose-dependent immune responses and protection. These studies reveal that formulation of inactivated chlamydial antigens with adjuvants, such as VCG and chitosan increases their ability to induce protective immune responses against challenge.

The safety and efficacy of BCG encapsulated alginate particle (BEAP) against M.tb H37Rv infection in Macaca mulatta : A pilot study

Due to the limited utility of Bacillus Calmette–Guérin (BCG), the only approved vaccine available for tuberculosis, there is a need to develop a more effective and safe vaccine. We evaluated the safety and efficacy of a dry powder aerosol (DPA) formulation of BCG encapsulated alginate particle (BEAP) and the conventional intradermal BCG immunization in infant rhesus macaques (Macaca mulatta). The infant macaques were immunized intratracheally with DPA of BEAP into the lungs. Animals were monitored for their growth, behaviour, any adverse and allergic response. The protective efficacy of BEAP was estimated by the ex-vivo H37Rv infection method. Post-immunization with BEAP, granulocytes count, weight gain, chest radiography, levels of liver secreted enzymes, cytokines associated with inflammation like TNF and IL-6 established that BEAP is non-toxic and it does not elicit an allergic response. The T cells isolated from BEAP immunized animals’ blood, upon stimulation with M.tb antigen, secreted high levels of IFN-γ, TNF, IL-6 and IL-2. The activated T cells from BEAP group, when co-cultured with M.tb infected macrophages, eliminated largest number of infected macrophages compared to the BCG and control group. This study suggests the safety and efficacy of BEAP in Non-human primate model.

Identification of Critical Transcriptomic Signaling Pathways in Patients with H Syndrome and Rosai-Dorfman Disease

Biallelic mutations in SLC29A3 cause histiocytosis-lymphadenopathy plus syndrome, also known as H syndrome (HS). HS is a complex disorder, with ~ 25% of patients developing autoinflammatory complications consisting of unexplained fevers, persistently elevated inflammatory markers, and unusual lymphadenopathies, with infiltrating CD68+, S100+, and CD1a− histiocytes, resembling the immunophenotype found in Rosai-Dorfman disease (RDD). We investigated the transcriptomic profiles of monocytes, non-activated (M0), classically activated (M1), and alternatively activated macrophages (M2) in two patients with HS, one without autoinflammatory (HS1) and one with autoinflammatory complications (HS2). RNA sequencing revealed a dysregulated transcriptomic profile in both HS patients compared to healthy controls (HC). HS2, when compared to HS1, had several differentially expressed genes, including genes associated with lymphocytic-histiocytic predominance (e.g. NINL) and chronic immune activation (e.g. B2M). The transcriptomic and cytokine profiles of HS patients were comparable to patients with SAID with high levels of TNF. SERPINA1 gene expression was found to be upregulated in all patients studied. Moreover, higher levels of IFNγ were found in the serum of both HS patients when compared to HC. Gene ontology (GO) enrichment analysis of the DEGs in HS patients revealed the terms “type I IFN,” “IFNγ signaling pathway,” and “immune responses” as the top 3 most significant terms for monocytes. Gene expression analysis of lymph node biopsies from sporadic and H syndrome-associated RDD suggests common underlying pathological process. In conclusion, monocytes and macrophages from both HS patients showed transcriptomic profiles similar to SAIDs and also uniquely upregulated IFNγ signature. These findings may help find better therapeutic options for this rare disorder.