Characterization of Selective and Potent JAK1 Inhibitors Intended for the Inhaled Treatment of Asthma.

Purpose: Janus kinase 1 (JAK1) is implicated in multiple inflammatory pathways that are critical for the pathogenesis of asthma, including the interleukin (IL)-4, IL-5, IL-13, and thymic stromal lymphopoietin cytokine signaling pathways, which have previously been targeted to treat allergic asthma. Here, we describe the development of AZD0449 and AZD4604, two novel and highly selective JAK1 inhibitors with promising properties for inhalation. Methods: The effects of AZD0449 and AZD4604 in JAK1 signaling pathways were assessed by measuring phosphorylation of signal transducer and activator of transcription (STAT) proteins and chemokine release using immunoassays of whole blood from healthy human volunteers and rats. Pharmacokinetic studies performed on rats evaluated AZD0449 at a lung deposited dose of 52 µg/kg and AZD4604 at 30 µg/kg. The efficacy of AZD0449 and AZD4604 was assessed by evaluating lung inflammation (cell count and cytokine levels) and the late asthmatic response (average enhanced pause [Penh]). Results: Both compounds inhibited JAK1-dependent cytokine signaling pathways in a dose-dependent manner in human and rat leukocytes. After intratracheal administration in rats, both compounds exhibited low systemic exposures and medium-to-long terminal lung half-lives (AZD0449, 34 hours; AZD4604, 5 hours). Both compounds inhibited STAT3 and STAT5 phosphorylation in lung tissue from ovalbumin (OVA)-challenged rats. AZD0449 and AZD4604 also inhibited eosinophilia in the lung and reduced the late asthmatic response, measured as Penh in the OVA rat model. Conclusion: AZD0449 and AZD4604 show potential as inhibitors of signaling pathways involved in asthmatic immune responses, with target engagement demonstrated locally in the lung. These findings support the clinical development of AZD0449 and AZD4604 for the treatment of patients with asthma.

Actionable druggable genome-wide Mendelian randomization identifies repurposing opportunities for COVID-19.

Drug repurposing provides a rapid approach to meet the urgent need for therapeutics to address COVID-19. To identify therapeutic targets relevant to COVID-19, we conducted Mendelian randomization analyses, deriving genetic instruments based on transcriptomic and proteomic data for 1,263 actionable proteins that are targeted by approved drugs or in clinical phase of drug development. Using summary statistics from the Host Genetics Initiative and the Million Veteran Program, we studied 7,554 patients hospitalized with COVID-19 and >1 million controls. We found significant Mendelian randomization results for three proteins (ACE2, P = 1.6 × 10-6; IFNAR2, P = 9.8 × 10-11 and IL-10RB, P = 2.3 × 10-14) using cis-expression quantitative trait loci genetic instruments that also had strong evidence for colocalization with COVID-19 hospitalization. To disentangle the shared expression quantitative trait loci signal for IL10RB and IFNAR2, we conducted phenome-wide association scans and pathway enrichment analysis, which suggested that IFNAR2 is more likely to play a role in COVID-19 hospitalization. Our findings prioritize trials of drugs targeting IFNAR2 and ACE2 for early management of COVID-19.

Interaction between the STAT4 rs11889341(T) risk allele and smoking confers increased risk of myocardial infarction and nephritis in patients with systemic lupus erythematosus.

OBJECTIVE: To investigate how genetics influence the risk of smoking-related systemic lupus erythematosus (SLE) manifestations. METHODS: Patients with SLE (ndiscovery cohort=776, nreplication cohort=836) were genotyped using the 200K Immunochip single nucleotide polymorphisms (SNP) Array (Illumina) and a custom array. Sixty SNPs with SLE association (p<5.0×10-8) were analysed. Signal transducer and activator of transcription 4 (STAT4) activation was assessed in in vitro stimulated peripheral blood mononuclear cells from healthy controls (n=45). RESULTS: In the discovery cohort, smoking was associated with myocardial infarction (MI) (OR 1.96 (95% CI 1.09 to 3.55)), with a greater effect in patients carrying any rs11889341 STAT4 risk allele (OR 2.72 (95% CI 1.24 to 6.00)) or two risk alleles (OR 8.27 (95% CI 1.48 to 46.27)).Smokers carrying the risk allele also displayed an increased risk of nephritis (OR 1.47 (95% CI 1.06 to 2.03)). In the replication cohort, the high risk of MI in smokers carrying the risk allele and the association between the STAT4 risk allele and nephritis in smokers were confirmed (OR 6.19 (95% CI 1.29 to 29.79) and 1.84 (95% CI 1.05 to 3.29), respectively).The interaction between smoking and the STAT4 risk allele resulted in further increase in the risk of MI (OR 2.14 (95% CI 1.01 to 4.62)) and nephritis (OR 1.53 (95% CI 1.08 to 2.17)), with 54% (MI) and 34% (nephritis) of the risk attributable to the interaction. Levels of interleukin-12-induced phosphorylation of STAT4 in CD8+ T cells were higher in smokers than in non-smokers (mean geometric fluorescence intensity 1063 vs 565, p=0.0063).Lastly, the IL12A rs564799 risk allele displayed association with MI in both cohorts (OR 1.53 (95% CI 1.01 to 2.31) and 2.15 (95% CI 1.08 to 4.26), respectively). CONCLUSIONS: Smoking in the presence of the STAT4 risk gene variant appears to increase the risk of MI and nephritis in SLE. Our results also highlight the role of the IL12-STAT4 pathway in SLE-cardiovascular morbidity.

Function of multiple sclerosis-protective HLA class I alleles revealed by genome-wide protein-quantitative trait loci mapping of interferon signalling.

Interferons (IFNs) are cytokines that are central to the host defence against viruses and other microorganisms. If not properly regulated, IFNs may contribute to the pathogenesis of inflammatory autoimmune, or infectious diseases. To identify genetic polymorphisms regulating the IFN system we performed an unbiased genome-wide protein-quantitative trait loci (pQTL) mapping of cell-type specific type I and type II IFN receptor levels and their responses in immune cells from 303 healthy individuals. Seven genome-wide significant (p < 5.0E-8) pQTLs were identified. Two independent SNPs that tagged the multiple sclerosis (MS)-protective HLA class I alleles A*02/A*68 and B*44, respectively, were associated with increased levels of IFNAR2 in B and T cells, with the most prominent effect in IgD-CD27+ memory B cells. The increased IFNAR2 levels in B cells were replicated in cells from an independent set of healthy individuals and in MS patients. Despite increased IFNAR2 levels, B and T cells carrying the MS-protective alleles displayed a reduced response to type I IFN stimulation. Expression and methylation-QTL analysis demonstrated increased mRNA expression of the pseudogene HLA-J in B cells carrying the MS-protective class I alleles, possibly driven via methylation-dependent transcriptional regulation. Together these data suggest that the MS-protective effects of HLA class I alleles are unrelated to their antigen-presenting function, and propose a previously unappreciated function of type I IFN signalling in B and T cells in MS immune-pathogenesis.

The regulation and pharmacological modulation of immune complex induced type III IFN production by plasmacytoid dendritic cells.

OBJECTIVE: Patients with systemic lupus erythematosus (SLE) have an ongoing interferon (IFN) production due to an activation of plasmacytoid dendritic cells (pDCs), which can be triggered to type I IFN synthesis by RNA containing immune complexes (RNA-IC). Considering emerging data suggesting a role of type III IFN in the SLE disease process, we asked if RNA-IC can induce type III IFN production in pDC and how this production can be regulated. METHODS: Peripheral blood mononuclear cells (PBMCs) or immune cell subsets were isolated from healthy blood donors or SLE patients and stimulated with IC containing U1 snRNP and SLE-IgG (RNA-IC). Hydroxychloroquine (HCQ) and an interleukin receptor 1-associated kinase 4 inhibitor (IRAK4i) were added to cell cultures. Cytokine mRNA levels were determined with a microarray and protein levels with immunoassays. Single-cell RNA sequencing of pDCs using ddSEQ technology was performed. RESULTS: Type III IFN mRNA and protein was induced in RNA-IC-stimulated pDC-NK and pDC-B cell co-cultures. A subset of activated pDCs (3%) expressed both type III and type I IFN mRNA. IFN-λ2, IFN-α2b, interleukin (IL)-3, IL-6, or granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced IFN-λ1/3 production 2-5-fold. HCQ and an IRAK4i blocked the RNA-IC-triggered IFN-λ1/3 production (p < 0.01). IFN-α2b and GM-CSF increased the proportion of SLE patients producing IFN-λ1/3 in response to RNA-IC from 11 to 33%. CONCLUSIONS: Type III IFN production is triggered by RNA-IC in pDCs in a TLR-MyD88-dependent manner, enhanced by NK and B cells as well as several pro-inflammatory cytokines. These results support a contributing role for both type I and type III IFNs in SLE, which needs to be considered when targeting the IFN system in this disease.

Immunogenetics in systemic lupus erythematosus: Transitioning from genetic associations to cellular effects.

Systemic lupus erythematosus (SLE) is a heterogeneous rheumatic autoimmune disease. Genetic studies have identified up to 100 SLE risk loci. Many of these encode proteins of importance in the immune system, but the cellular and molecular mechanisms underlying these associations are still elusive. In this review, we will highlight some of the SLE risk loci where mechanistic insights have been achieved recently by linking genetic risk polymorphisms to cellular or molecular phenotypes important for the disease process.

Autoantibodies to Killer Cell Immunoglobulin-Like Receptors in Patients With Systemic Lupus Erythematosus Induce Natural Killer Cell Hyporesponsiveness.

Natural killer (NK) cell cytotoxicity toward self-cells is restrained by the inhibitory HLA class I-binding receptors CD94/NKG2A and the killer cell immunoglobulin-like receptors (KIRs). CD94/NKG2A and KIRs are also essential for NK cell education, which is a dynamic functional maturation process where a constitutive binding of inhibitory receptors to cognate HLA class I molecules is required for NK cells to maintain their full cytotoxic capacity. Previously, we described autoantibodies to CD94/NKG2A in patients with systemic lupus erythematosus (SLE). In this study we analyzed sera from 191 patients with SLE, 119 patients with primary Sjögren's syndrome (pSS), 48 patients with systemic sclerosis (SSc), and 100 healthy donors (HD) for autoantibodies to eight different KIRs. Anti-KIR autoantibodies were identified in sera from 23.0% of patients with SLE, 10.9% of patients with pSS, 12.5% of patients with SSc, and 3.0% of HD. IgG from anti-KIR-positive SLE patients reduced the degranulation and cytotoxicity of NK cells toward K562 tumor cells. The presence of anti-KIR-autoantibodies reacting with >3 KIRs was associated with an increased disease activity (p < 0.0001), elevated serum levels of IFN-α (p < 0.0001), nephritis (p = 0.001), and the presence of anti-Sm (p = 0.007), and anti-RNP (p = 0.003) autoantibodies in serum. Together these findings suggest that anti-KIR autoantibodies may contribute to the reduced function of NK cells in SLE patients, and that a defective NK cell function may be a risk factor for the development of lupus nephritis.

Cytokine production by activated plasmacytoid dendritic cells and natural killer cells is suppressed by an IRAK4 inhibitor.

BACKGROUND: In systemic lupus erythematosus (SLE), immune complexes (ICs) containing self-derived nucleic acids trigger the synthesis of proinflammatory cytokines by immune cells. We asked how an interleukin (IL)-1 receptor-associated kinase 4 small molecule inhibitor (IRAK4i) affects RNA-IC-induced cytokine production compared with hydroxychloroquine (HCQ). METHODS: Plasmacytoid dendritic cells (pDCs) and natural killer (NK) cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy individuals. PBMCs from SLE patients and healthy individuals were depleted of monocytes. Cells were stimulated with RNA-containing IC (RNA-IC) in the presence or absence of IRAK4i I92 or HCQ, and cytokines were measured by immunoassay or flow cytometry. Transcriptome sequencing was performed on RNA-IC-stimulated pDCs from healthy individuals to assess the effect of IRAK4i and HCQ. RESULTS: In healthy individuals, RNA-IC induced interferon (IFN)-α, tumor necrosis factor (TNF)-α, IL-6, IL-8, IFN-γ, macrophage inflammatory protein (MIP)1-α, and MIP1-ß production in pDC and NK cell cocultures. IFN-α production was selective for pDCs, whereas both pDCs and NK cells produced TNF-α. IRAK4i reduced the pDC and NK cell-derived cytokine production by 74-95%. HCQ interfered with cytokine production in pDCs but not in NK cells. In monocyte-depleted PBMCs, IRAK4i blocked cytokine production more efficiently than HCQ. Following RNA-IC activation of pDCs, 975 differentially expressed genes were observed (false discovery rate (FDR) < 0.05), with many connected to cytokine pathways, cell regulation, and apoptosis. IRAK4i altered the expression of a larger number of RNA-IC-induced genes than did HCQ (492 versus 65 genes). CONCLUSIONS: The IRAK4i I92 exhibits a broader inhibitory effect than HCQ on proinflammatory pathways triggered by RNA-IC, suggesting IRAK4 inhibition as a therapeutic option in SLE.

The STAT4 SLE risk allele rs7574865[T] is associated with increased IL-12-induced IFN-γ production in T cells from patients with SLE.

OBJECTIVES: Genetic variants in the transcription factor STAT4 are associated with increased susceptibility to systemic lupus erythematosus (SLE) and a more severe disease phenotype. This study aimed to clarify how the SLE-associated intronic STAT4 risk allele rs7574865[T] affects the function of immune cells in SLE. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from 52 genotyped patients with SLE. Phosphorylation of STAT4 (pSTAT4) and STAT1 (pSTAT1) in response to interferon (IFN)-α, IFN-γ or interleukin (IL)-12, total levels of STAT4, STAT1 and T-bet, and frequency of IFN-γ+ cells on IL-12 stimulation were determined by flow cytometry in subsets of immune cells before and after preactivation of cells with phytohaemagglutinin (PHA) and IL-2. Cellular responses and phenotypes were correlated to STAT4 risk allele carriership. Janus kinase inhibitors (JAKi) selective for TYK2 (TYK2i) or JAK2 (JAK2i) were evaluated for inhibition of IL-12 or IFN-γ-induced activation of SLE PBMCs. RESULTS: In resting PBMCs, the STAT4 risk allele was neither associated with total levels of STAT4 or STAT1, nor cytokine-induced pSTAT4 or pSTAT1. Following PHA/IL-2 activation, CD8+ T cells from STAT4 risk allele carriers displayed increased levels of STAT4 resulting in increased pSTAT4 in response to IL-12 and IFN-α, and an augmented IL-12-induced IFN-γ production in CD8+ and CD4+ T cells. The TYK2i and the JAK2i efficiently blocked IL-12 and IFN-γ-induced activation of PBMCs from STAT4 risk patients, respectively. CONCLUSIONS: T cells from patients with SLE carrying the STAT4 risk allele rs7574865[T] display an augmented response to IL-12 and IFN-α. This subset of patients may benefit from JAKi treatment.

Plasmacytoid dendritic cells and RNA-containing immune complexes drive expansion of peripheral B cell subsets with an SLE-like phenotype.

BACKGROUND: Hyperactive B cells and a continuous interferon (IFN)-α production by plasmacytoid dendritic cells (pDCs) play a key role in systemic lupus erythematosus (SLE). We asked whether the interaction between B cells and pDCs stimulated with RNA-containing immune complexes affects peripheral B cell subsets. METHODS: B cells and pDCs were isolated from blood of healthy individuals and stimulated with immune complexes consisting of SLE-IgG and U1snRNP (RNA-IC). Expression of cell surface molecules as well as IL-6 and IL-10 production were determined by flow cytometry and immunoassays. Gene expression profiles were determined by a NanoString nCounter expression array. RESULTS: We found a remarkable increase of double negative CD27-IgD- B cells, from 7% within fresh CD19+ B cells to 37% in the RNA-IC-stimulated co-cultures of B cells and pDCs, comparable to the frequency of double negative B cells in SLE patients. Gene expression analysis of the double negative CD27-IgD- and the CD27+IgD- memory B cells revealed that twenty-one genes were differentially expressed between the two B cell subsets (≥ 2-fold, p<0.001). The, IL21R, IL4R, CCL4, CCL3, CD83 and the IKAROS Family Zinc Finger 2 (IKZ2) showed higher expression in the double negative CD27-IgD- B cells. CONCLUSION: The interactions between B cells and pDCs together with RNA-containing IC led to an expansion of B cells with similar phenotype as seen in SLE, suggesting that the pDC-B cell crosstalk contributes to the autoimmune feed-forward loop in SLE.

Activated T cells enhance interferon-α production by plasmacytoid dendritic cells stimulated with RNA-containing immune complexes.

OBJECTIVES: Patients with systemic lupus erythematosus (SLE) have an ongoing interferon-α (IFN-α) production by plasmacytoid dendritic cells (pDCs). We investigated whether T cells can promote IFN-α production by pDCs. METHODS: Human pDCs were stimulated with immune complexes (ICs) containing U1 small nuclear ribonucleic proteins particles and SLE-IgG (RNA-IC) in the presence of T cells or T cell supernatants. T cells were activated by anti-CD3/CD28 antibodies or in a mixed leucocyte reaction. IFN-α and other cytokines were determined in culture supernatants or patient sera with immunoassays. The effect of interleukin (IL) 3 and granulocyte-macrophage-colony-stimulating factor (GM-CSF) on pDCs was examined by the use of antibodies, and the expression of CD80/CD86 was determined using flow cytometry. RESULTS: Activated T cells and supernatants from activated T cells increased IFN-α production by >20-fold. The stimulatory effect of T cell supernatants was reduced after depletion of GM-CSF (81%) or by blocking the GM-CSF receptor (55%-81%). Supernatant from activated T cells, furthermore, increased the frequency of CD80 and CD86 expressing pDCs stimulated with RNA-IC from 6% to 35% (p<0.05) and from 10% to 26% (p<0.01), respectively. Activated SLE T cells enhanced IFN-α production to the same extent as T cells from healthy individuals and a subset of patients with SLE had increased serum levels of GM-CSF. CONCLUSIONS: Activated T cells enhance IFN-α production by RNA-IC stimulated pDCs via GM-CSF and induce pDC maturation. Given the increased serum levels of GM-CSF in a subset of patients with SLE, these findings suggest that activated T cells may upregulate type I IFN production in SLE.

Functional anti-CD94/NKG2A and anti-CD94/NKG2C autoantibodies in patients with systemic lupus erythematosus.

OBJECTIVE: Recently we serendipitously identified a patient with systemic lupus erythematosus (SLE) who was positive for autoantibodies to CD94/natural killer receptor group 2A (NKG2A). The present study was undertaken to investigate the occurrence and function of autoantibodies targeting lectin-like NK cell receptors in SLE. METHODS: Sera from 203 SLE patients and 90 healthy individuals were analyzed, by flow cytometry, for Ig binding to Ba/F3 cells transfected with CD94/NKG2A, CD94/NKG2C, or NKG2D. Autoantibodies identified were characterized with regard to interference with HLA-E binding, effect on NK cell activation in response to HLA-E-transfected K562 cells, and capacity to facilitate antibody-dependent cell-mediated cytotoxicity (ADCC). Levels of autoantibodies were determined in longitudinally sampled sera, and correlations with disease activity (SLE Disease Activity Index 2000) and severity (Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index) were investigated. RESULTS: Anti-CD94/NKG2A autoantibodies were identified in 7 SLE patients. The autoantibodies from 6 patients inhibited binding of HLA-E to CD94/NKG2A, whereas those from the seventh patient augmented this binding. Autoantibodies from 2 patients also reacted with the activating receptor CD94/NKG2C, with inhibition of the binding of HLA-E to CD94/NKG2C observed in 1 case and enhancement of this binding in the other. None of the sera contained anti-NKG2D autoantibodies. The levels of anti-CD94/NKG2A and anti-CD94/NKG2C autoantibodies correlated with disease activity and with a more severe SLE phenotype. Mechanistically, anti-CD94/NKG2A and anti-CD94/NKG2C autoantibodies both interfered with HLA-E-mediated regulation of NK cell activation and facilitated the elimination of target cells expressing CD94/NKG2A or CD94/NKG2C through ADCC. CONCLUSION: Anti-CD94/NKG2A and anti-CD94/NKG2C autoantibodies occur in a subset of patients with clinically active SLE. Given their capacity to deplete certain NK cell subsets and interfere with particular NK cell function, such autoantibodies may promote the pathogenesis of SLE.

Systemic lupus erythematosus immune complexes increase the expression of SLAM family members CD319 (CRACC) and CD229 (LY-9) on plasmacytoid dendritic cells and CD319 on CD56(dim) NK cells.

Patients with systemic lupus erythematosus (SLE) display an activated type I IFN system due to unceasing IFN-α release from plasmacytoid dendritic cells (pDCs) stimulated by nucleic acid-containing immune complexes (ICs). NK cells strongly promote the IFN-α production by pDCs; therefore, we investigated surface molecules that could be involved in the pDC-NK cell cross-talk. In human PBMCs stimulated with RNA-containing ICs (RNA-ICs), the expression of the signaling lymphocyte activation molecule (SLAM) family receptors CD319 and CD229 on pDCs and CD319 on CD56(dim) NK cells was selectively increased. Upregulation of CD319 and CD229 on RNA-IC-stimulated pDCs was induced by NK cells or cytokines (e.g., GM-CSF, IL-3). IFN-α-producing pDCs displayed a higher expression of SLAM molecules compared with IFN-α⁻ pDCs. With regard to signaling downstream of SLAM receptors, pDCs expressed SHIP-1, SHP-1, SHP-2, and CSK but lacked SLAM-associated protein (SAP) and Ewing's sarcoma-activated transcript 2 (EAT2), indicating that these receptors may act as inhibitory receptors on pDCs. Furthermore, pDCs from patients with SLE had decreased expression of CD319 on pDCs and CD229 on CD56(dim) NK cells, but RNA-IC stimulation increased CD319 and CD229 expression. In conclusion, this study reveals that the expression of the SLAM receptors CD319 and CD229 is regulated on pDCs and NK cells by lupus ICs and that the expression of these receptors is specifically altered in SLE. These results, together with the observed genetic association between the SLAM locus and SLE, suggest a role for CD319 and CD229 in the SLE disease process.

Anti-NKG2A autoantibodies in a patient with systemic lupus erythematosus.

OBJECTIVES: To characterize a novel anti-NKG2A autoantibody detected in a patient with SLE during a severe flare, and in a cross-sectional study investigate the occurrence of such autoantibodies in patients with SLE and primary SS (pSS). METHODS: Serum or IgG from patients with SLE, pSS and healthy volunteers were assayed for blocking of anti-NKG2A or HLA-E binding to peripheral blood mononuclear cells or CD94/NKG2A- and CD94/NKG2C-transfected Ba/F3 cells. The anti-NKG2A autoantibodies were evaluated for effect on NK cell degranulation in response to HLA-E-transfected K562 cells. IFN-α was determined by an immunoassay and disease activity by the SLEDAI score. RESULTS: Anti-NKG2A autoantibodies, which blocked binding of HLA-E tetramers to CD94/NKG2A-transfected cells and impaired NKG2A-mediated inhibition of NK cell activation, were observed in a patient with SLE. The presence of anti-NKG2A autoantibodies was associated with high SLE disease activity (SLEDAI score 14 and 16) and increased serum IFN-α. Of 94 SLE, 60 pSS and 30 healthy donor sera, only the index patient serum contained anti-NKG2A autoantibodies. CONCLUSION: The presence of autoantibodies targeting NKG2A is a rare event, but when such autoantibodies occur they may promote excessive NK cell function. This can contribute to the pathogenesis by increasing the killing of cells and the release of autoantigens. Our findings highlight the possible importance of NK cells in the SLE disease process.

Genome-wide profiling of target genes for the systemic lupus erythematosus-associated transcription factors IRF5 and STAT4.

BACKGROUND: The transcription factors interferon regulatory factor 5 (IRF5) and signal transducer and activator of transcription 4 (STAT4) are encoded by two of the strongest susceptibility genes for systemic lupus erythematosus (SLE). OBJECTIVE: To investigate the target genes and functional roles of IRF5 and STAT4 in human peripheral blood mononuclear cells (PBMCs). METHODS: Chromatin immunoprecipitation-sequencing (ChIP-seq) was performed in PBMCs stimulated to activate IRF5 and STAT4. The expression of the target genes of IRF5 and STAT4 was investigated in a publicly available dataset generated from PBMCs from patients with SLE and healthy controls. The genomic regions bound by the transcription complexes mediated by IRF5 and STAT4 were examined for transcription factor binding motifs and SLE-associated sequence variants. RESULTS: More than 7000 target genes for IRF5 and STAT4 were identified in stimulated PBMCs. These genes were enriched to functional pathways in the type I interferon system, and have key roles in the inflammatory response. The expression patterns of the target genes were characteristic for patients with SLE. The transcription factors high mobility group-I/Y, specificity protein 1, and paired box 4 may function cooperatively with IRF5 and STAT4 in transcriptional regulation. Eight of the target regions for IRF5 and STAT4 contain SLE-associated sequence variants. CONCLUSIONS: By participating in transcription complex with other co-factors, IRF5 and STAT4 harbour the potential of regulating a large number of target genes, which may contribute to their strong association with SLE.

B lymphocytes enhance interferon-α production by plasmacytoid dendritic cells.

OBJECTIVE: The type I interferon (IFN) system and B cells are activated in many autoimmune diseases, such as systemic lupus erythematosus (SLE). The IFNα produced by plasmacytoid dendritic cells (PDCs) stimulates several B cell functions, including autoantibody production. However, not much is known about how B cells influence PDC function. The aim of this study was to investigate the regulatory effect of B cells on IFNα production by PDCs. METHODS: PDCs and B cells isolated from peripheral blood mononuclear cells from healthy blood donors were stimulated with RNA-containing immune complexes (ICs) consisting of U1 small nuclear RNP and SLE IgG, herpes simplex virus, or oligonucleotide (ODN) 2216, alone or in cocultures. IFNα, several other cytokines, and PDC- or B cell-associated surface molecules were analyzed using immunoassays or flow cytometry. RESULTS: B cells enhanced IFNα production by PDCs up to 47-fold, and the effect was most pronounced for PDCs stimulated with RNA-containing ICs. Anti-CD31 antibody reduced RNA-containing IC-induced IFNα production by 80% but had no effect on IFNα production when ODN 2216 was used as an inducer. Supernatants from ODN 2216-stimulated B cells promoted IFNα production by PDCs, while supernatants from RNA-containing IC-stimulated B cells did not. CONCLUSION: Our results showed that a novel function of B cells is enhancement of type I IFN production by PDCs. Because B cells are activated by type I IFN, this PDC-B cell cross-talk might be of fundamental importance in the etiopathogenesis of SLE and contribute to long-term immune activation in SLE and other systemic rheumatic diseases.

IFN-α production by plasmacytoid dendritic cells stimulated with RNA-containing immune complexes is promoted by NK cells via MIP-1ß and LFA-1.

Several systemic autoimmune diseases display a prominent IFN signature. This is caused by a continuous IFN-α production by plasmacytoid dendritic cells (pDCs), which are activated by immune complexes (ICs) containing nucleic acid. The IFN-α production by pDCs stimulated with RNA-containing IC (RNA-IC) consisting of anti-RNP autoantibodies and U1 small nuclear ribonucleoprotein particles was recently shown to be inhibited by monocytes, but enhanced by NK cells. The inhibitory effect of monocytes was mediated by TNF-α, PGE(2), and reactive oxygen species, but the mechanisms for the NK cell-mediated increase in IFN-α production remained unclear. In this study, we investigated the mechanisms whereby NK cells increase the RNA-IC-induced IFN-α production by pDCs. Furthermore, NK cells from patients with systemic lupus erythematosus (SLE) were evaluated for their capacity to promote IFN-α production. We found that CD56(dim) NK cells could increase IFN-α production >1000-fold after RNA-IC activation, whereas CD56(bright) NK cells required costimulation by IL-12 and IL-18 to promote IFN-α production. NK cells produced MIP-1α, MIP-1ß, RANTES, IFN-γ, and TNF-α via RNA-IC-mediated FcγRIIIA activation. The IFN-α production in pDCs was promoted by NK cells via MIP-1ß secretion and LFA-mediated cell-cell contact. Moreover, NK cells from SLE patients displayed a reduced capacity to promote the RNA-IC-induced IFN-α production, which could be restored by exogenous IL-12 and IL-18. Thus, different molecular mechanisms can mediate the NK cell-dependent increase in IFN-α production by RNA-IC-stimulated pDCs, and our study suggests that the possibility to therapeutically target the NK-pDC axis in IFN-α-driven autoimmune diseases such as SLE should be investigated.