IFN-regulated Gene Expression Research Articles

An Immune Gene Expression Risk Score for Distant Metastases after Radiotherapy for Cervical Cancer.

To develop an immune-based gene expression risk score to identify patients with cervical cancer at increased risk of distant metastases (DM). Tumor biopsies were obtained from 81 patients prior to chemoradiotherapy. Whole-transcriptome RNA sequencing was performed (Illumina NextSeq500). Beginning with 4,723 immune-related genes, a 55-gene risk score for DM was derived using Cox modeling and principal component analysis. It was validated in independent cohorts of 274 patients treated at the Norwegian Radium Hospital (NRH) and 206 patients from The Cancer Genome Atlas (TCGA). The risk score was predictive of DM (HR, 2.7; P < 0.0001) and lower cause-specific survival (CSS) by univariate analysis (HR, 2.0; P = 0.0003) and multivariate analysis adjusted for clinical factors (DM HR, 3.0; P < 0.0001; CSS HR, 2.2; P = 0.0004). The risk score predicted DM (HR, 1.4; P = 0.05) and CSS (HR, 1.48; P = 0.013) in the NRH cohort and CSS (HR, 1.4; P = 0.03) in TCGA cohort. Higher risk scores were associated with lower CIBERSORT estimates of tumor-infiltrating immune cells, including CD8 T cells and M1 and M2 macrophages (all P < 0.001). Higher risk scores were associated with lower expression (all P < 0.001) of important chemokines (CXCL12, CXCR4), IFN-regulated genes (IRF1, STAT1, IDO1), and immune checkpoint regulators (PD-1, PD-L1, CTLA-4). The immune metastatic risk score addresses important challenges in the treatment of cervical cancer-identifying patients at high risk of DM after radiotherapy. The findings of this study indicate that high tumor mutational burden and a "cold," immune-excluded tumor microenvironment influence distant metastatic recurrence. Further validation of the risk score is needed.

Genome Wide siRNA Screen Identifies CGAS-Sting Pathway As a Pharmacological Target That Promotes Survival of Hematopoietic Stem Cells Deficient in Fanconi Genes

Introduction Fanconi anemia (FA) is a homozygous recessive genetic disorder due to mutation in the Fanconi family of genes. The disease is characterized by early onset bone marrow failure and a predisposition to cancer. The FA genes are involved in DNA damage sensing and repair. Mutation in these genes leads to heightened DNA damage and extreme sensitivity to cross-linking agents. Hematopoietic stem cells of FA patients are defective in differentiation and self-renewal, leading to stem cell exhaustion and bone marrow failure early in life. The current treatment for bone marrow failure is allogeneic stem cell transplant from healthy donors. Due to difficulties in finding matching donors and the inherent problems associated with allogeneic transplants, pharmacological therapies that delay or prevent bone marrow failure are of clinical importance. Other proposed therapies including TGF-b, metformin, and resveratrol have met with limited clinical success. Methodology: To identify genes that promote survival of FA cells we performed a genome wide siRNA screen using isogenic pair of cell line with FANCD2 deletions and its corrected counterpart using transfection of siRNA libraries targeting 18,119 genes from Human Drug Targets, Human Druggable Genome, and Human Genome libraries at 2000 cells/well in 384-well plates followed by cell viability measurement after 72 hrs. We measured IRF3 phosphorylation and expression of immune related genes to analyze the CGAS-STING pathway. Using peripheral blood and bone marrow derived CD34+ cells transduced with lentivirus expressing shRNA against FANCD2 we tested the effect of CGAS inhibitor in promoting CFU-GM and CFU-E. Results: A genome wide siRNA screen identified IRF7 as one of the top 5 hits whose diminished expression resulted in increased viability of F-D2 mutant PD20 cells. IRF7 along with IRF3 serve as downstream transcription factors including various Toll-like receptors, CGAS-STING pathways, and other pattern recognition innate immune receptors. Using pIRF3 as an activation readout for CGAS-STING pathway we identified that both FANCD2 and FANCA mutant cells have elevated pIRF3 levels. The elevated pIRF3 levels were further increased upon addition of mitomycin C (MMC) in FA mutant cells as compared to gene corrected cells. The pIRF3 levels decreased upon both genetic KD and small molecule inhibition of both CGAS and STING. In line with increased pIRF3 FA mutant cells showed increased gene expression of various interferon-regulated genes, which were then also reduced by CGAS inhibitor. Overexpression of RNAseH1 partially reduced pIRF3 levels in FA mutant cells indicating that the RNA:DNA hybrids whose levels are elevated in FA mutant cells is one of the ligands of CGAS. Most importantly 10µM of CGAS inhibitor RU.51 was able to rescue FA-A mutant cell line 6914 cells from low dose MMC-induced cell death showing that the sensitivity of FA mutant cells to DNA damaging agents was at least partially driven by CGAS-STING pathway. We then tested if the protective effect of CGAS inhibitor extended to human CD34+ cells deficient in FA genes. 20µM of RU.51 was able to rescue the CFU-GM and CFU-E defects of CD34+ cells carrying lentiviral integrated shRNA against FANCD2 to a level to that in cells expressing non-silencing shRNAs. Interferon-regulated gene expression was also elevated in the FANCD2-deficient CD34+ cells, which was reversed by treatment with cGAS inhibitor. Conclusions: It has previously been shown that FA patients have elevated TNF-alpha, IFN-gamma levels, and NFkbactivation, and interferon alpha administration hastens the onset of bone marrow failure in FA patients. Our results show that FA mutant cells have elevated CGAS-STING signaling resulting in heightened interferon gene expression. Elevated R-loop levels in FA cells act as trigger for the CGAS-STING pathway. CGAS inhibitor is able to rescue low dose MMC-induced cell death in FA mutant fibroblasts and also restores the CFU-GM and CFU-E forming ability of CD34+ cells. These results indicate that CGAS-STING pathway is a pharmacologically tractable target to reverse the hematopoietic stem cell exhaustion seen in FA patients.

KDM6A Modulates Anti-Tumor Immune Response By Integrating Immunogenic Cell Death in Human Acute Myeloid Leukemia

Identifying tumor cell-epigenetic regulation of immune activation in acute myeloid leukemia (AML) would be instrumental in developing next-generation of targeted therapy. AML cells have low mutational burden; inefficient ‘cross-presentation’ through conventional dendritic cells (DC) and immune activation are the major challenges for AML immunomodulation. ‘Immunogenic cell death’ (ICD) represents changes in the composition of the tumor cell surface as well as the release of ‘damage associated molecular patterns’ (DAMPs) in the tumor microenvironment that stimulates the dysfunctional immune system. DAMPs in turn operate on a series of receptors expressed by DCs and natural killer (NK) cells and contribute to long-lasting, protective anti-tumor immunity. For example, plasma membrane exposure of endoplasmic-reticulum (ER)-translocated calreticulin (ecto-CRT), which functions as an ‘eat me’ signal, is an important hallmark of ICD. Previous study suggested that calreticulin (CALR) promotes immunity and type I interferon-dependent survival in mice with AML (Fucikova, Blood 2016; Chen, Oncoimmunology 2017). In addition, ecto-CRT on malignant blasts correlated with improved NK cell-mediated cytotoxicity in AML patients with superior overall survival (Truxova, Haematologica 2020). Ecto-CRT has also been identified as an endogenous sterile ligand for NK cells (Sen Santara, Nature 2023). Nevertheless, molecular regulation of ICD in the context of epigenetic derangements and inflammation and its implication in AML immunotherapeutic modulation is poorly understood. KDM6A is a histone 3 lysine 27 demethylase that plays tumor suppressor function in AML (Gozdecka, Nat Genet 2018; Sera, Blood 2021). KDM6A escapes X-chromosome inactivation, and loss-of-function deletion or point mutations associate with resistance to ‘3+7‘ chemotherapy (Stief, Leukemia 2020). We have identified that in comparison to control AML cells, deficiency of KDM6A ( KDM6A-kd) associated with daunorubicin/etoposide-induced ICD, which was manifested by significant increase (2-3 fold , P&amp;lt;0.05) in ecto-CRT, plasma membrane exposure of pE1F2α, HSP70 and HSP90 with extracellular release of HMGB1 and ATP. Similarly, pharmacological inhibition of JmjC catalytic function using GSK-J4 sensitized human AML lines as well as primary AML patient bone marrow-derived CD34 + hematopoietic stem/progenitor cells to ICD. Induction in ICD was accompanied by an elevated type I interferon production that was functionally active in promoting interferon regulated gene expression. Interestingly, treatment with acute recombinant IFN-β caused an increase in activated STAT1 and inflammatory gene expression in KDM6A-kd AML cells compared to control AML. Corroborating these results, gene set enrichment analysis revealed a significant enrichment (NES=1.708, P=0.007) of upregulated type I interferon signatures (n=40) in Kdm6a -/- primary leukemia cells. Re-analysis of ChIP-seq results indicated 1647 Kdm6a occupied genes, which were upregulated upon Kdm6a loss. Gene ontology (GO) analysis of the 1647 genes further suggested an enrichment ( P&amp;lt;0.001) of type I interferon-responsive GO terms, indicating Kdm6a role in interferon signaling. Ecto-CRT level relies on premortem ER stress. In OHSU AML, KDM6A expression correlates with ER stress/unfolded protein response. Mechanistically, quantitative-ChIP and promoter-reporter assays indicated that KDM6A-kd de-repressed ICD induced expression of ATF4 and HSPA5 through chromatin and transcriptional remodeling. In agreement with these findings, AML blasts-healthy immune cells ex vivo co-culture experiments revealed that increase in ICD in KDM6A-kd AML was associated with activation (~2-foldincrease inCD80 +CD86 +, P&amp;lt;0.05) and phagocytic function of DCs. Loss of KDM6A was shown to bestow ‘BRCAness’ (Boila, Leukemia 2023), and PARP inhibition itself can cause ICD and augment tumor immunity. We observed that there was a significant induction in ICD and expression of NKG2D ligands ULBP1, ULBP5 in KDM6A-kd AML cells treated with olaparib. In addition, olaparib and GSK-J4 treatment showed additive effects in ICD and immune activation using primary AML CD34 + cells. Together, we illustrate that KDM6A regulates ICD through epigenetic mechanisms, while KDM6A deficient AML subtypes could be sensitized with immunomodulatory targeted therapy.

PA24 Abnormal interferon signature in a patient with chilblain lupus

Abstract A 5-year-old girl of Sri Lankan ancestry with no history of parental relatedness presented with the clinical features of chilblain lupus. This was particularly florid over her dorsal fingers, toes and the soles of her feet with areas in keeping with panniculitis on her calves and an epidermal naevus on the right side of her trunk. There was no ulceration or necrosis. Her parent reported occasional fevers and palpable lymph nodes in her neck. She also has bilateral astigmatism and mild valvular and supravalvular pulmonary stenosis. Blood tests showed a persisting mild transaminitis, an antinuclear antibody of 1 in 160 and a negative double-stranded DNA. She had a skin biopsy that showed supporting features for chilblain lupus. A lymph node biopsy showed nonspecific changes. The clinical suspicion was of a mild form of Aicardi–Goutières syndrome (AGS), and a genetic panel was performed that included TREX1, RNASEH2B, TMEM173 and SAMHD1 (all negative). The common mechanism behind these conditions is a type I interferonopathy. We sent blood on a research basis for RNA extraction and testing of her interferon (IFN) gene signature (IGS), which came back as abnormal (11.895; cutoff score of 2.758); she had normal expression of neutrophil-associated genes. This IFN score is on par with patients who have confirmed AGS or STING-associated vasculopathy with onset in infancy. An IGS is derived from the expression of IFN-regulated genes. A raised IGS has not only been reported to be useful as a biomarker in many rheumatic diseases, but is also used to help identify type I interferonopathies where genetic testing has not identified any clear pathogenic mutations (Crow YJ, Stetson DB. The type I interferonopathies: 10 years on. Nat Rev Immunol 2022; 22:471–83). National Institute for Health and Care Excellence guidelines allow for treatment of interferonopathies with baricitinib in patients with two documented high IFN gene signatures, which is our current plan for this patient.

#4234 ADAM17 MODULATES IFN-MEDIATED INFLAMMATION IN RENAL TUBULAR CELLS

Abstract Background and Aims In diabetic patients, hyperglycemia has been associated with a hypoxic state that contributes to an increased rate of morbidity due to Acute Kidney Failure (AKI). For this reason, we are interested in studying the molecular mechanisms involved in the development of AKI to contribute to the search for effective therapies that can help prevent these episodes. We focused on ADAM17, a disintegrin and metalloproteinase that sheds the ectodomains of cell surface proteins, among which inflammatory cytokines stand out. In an animal model, we showed that the absence of ADAM17 in the renal tubule reduces protein expression related to fibrosis and inflammation induced by diabetes. In this context, we aimed to study the role of ADAM17 in modifying the expression of inflammatory molecules regulated by interferon (IFN) in renal proximal tubule cells subjected to hyperglycemic stress. In this study, we analyzed the effect of ADAM17 deletion in human renal tubular cells (HKC-8) incubated in high-glucose medium and subjected to hypoxia. Method ADAM17 was depleted using CRISPR/Cas9 (ADAM17-KO). Cells were incubated for 6 h or 24 h with high (35 mM HG) or normal (5.5 mM NG) glucose concentrations and were subjected to hypoxia plus 2h re-oxygenation (HIPX). Gene expression of several IFN-regulated inflammatory markers (IL6, IL8, CXCL10, ISG15, IL1a, IL1b, IL12A, CCL5, and CCL2) was determined in wild-type (WT) and ADAM17-KO cells. Results The absence of ADAM17 in HKC-8 cells induced changes in the mRNA expression of genes controlled by IFN, which is accentuated by the hyperglycemic stimulus. Under hypoxic and re-oxygenation conditions, these changes were in the same line, with more discrete values compared to control cells. TNFa, CXCL10 and LNC2 were the most relevant genes reduced in injury conditions in the ADAM17-KO cell line. Conclusion ADAM17 deletion modulates the expression of most IFN-regulated genes that could undergo post-transcriptional changes and subsequently act as an acute mechanism of protection from injury. The results show that ADAM17 plays a major role in the underlying inflammatory response to acute kidney injury, indicating its potential as a therapeutic target. The analysis of circulating proteins will provide information about this process to better understand the mechanism of action of ADAM17 in the protection of renal tubular damage.

Subviral Dense Bodies of Human Cytomegalovirus Enhance Interferon-Beta Responses in Infected Cells and Impair Progeny Production.

(1) Background: Infection with human cytomegalovirus (HCMV) leads to the production and release of subviral particles, termed Dense Bodies (DB). They are enclosed by a membrane resembling the viral envelope. This membrane mediates the entrance of DBs into cells in a way that is comparable to virus infection. HCMV attachment and entry trigger the induction of interferon synthesis and secretion, and the subsequent expression of interferon-regulated genes (IRGs) that might inhibit replication of the virus. Recently, we demonstrated that DBs induce a robust interferon response in the absence of infection. Little is known thus far, including how DBs influence HCMV infection and virus-host interaction. (2) Methods: Purified DBs were used to study the impact on virus replication and on the innate defense mechanisms of the cell. (3) Results: The incubation of cells with DBs at the time of infection had little effect on viral genome replication. Preincubation of DBs, however, led to a marked reduction in viral release from infected cells. These cells showed an enhancement of the cytopathic effect, associated with a moderate increase in early apoptosis. Despite virus-induced mechanisms to limit the interferon response, the induction of interferon-regulated genes (IRGs) was upregulated by DB treatment. (4) Conclusions: DBs sensitize cells against viral infection, comparable to the effects of interferons. The activities of these particles need to be considered when studying viral-host interaction.

Single cell analysis of docosahexaenoic acid suppression of sequential LPS-induced proinflammatory and interferon-regulated gene expression in the macrophage.

Preclinical and clinical studies suggest that consumption of long chain omega-3 polyunsaturated fatty acids (PUFAs) reduces severity of chronic inflammatory and autoimmune diseases. While these ameliorative effects are conventionally associated with downregulated expression of proinflammatory cytokine and chemokine genes, our laboratory has recently identified Type 1 interferon (IFN1)-regulated gene expression to be another key target of omega-3 PUFAs. Here we used single cell RNA sequencing (scRNAseq) to gain new mechanistic perspectives on how the omega-3 PUFA docosahexaenoic acid (DHA) influences TLR4-driven proinflammatory and IFN1-regulated gene expression in a novel self-renewing murine fetal liver-derived macrophage (FLM) model. FLMs were cultured with 25 µM DHA or vehicle for 24h, treated with modest concentration of LPS (20 ng/ml) for 1 and 4h, and then subjected to scRNAseq using the 10X Chromium System. At 0h (i.e., in the absence of LPS), DHA increased expression of genes associated with the NRF2 antioxidant response (e.g. Sqstm1, Hmox1, Chchd10) and metal homeostasis (e.g.Mt1, Mt2, Ftl1, Fth1), both of which are consistent with DHA-induced polarization of FLMs to a more anti-inflammatory phenotype. At 1h post-LPS treatment, DHA inhibited LPS-induced cholesterol synthesis genes (e.g. Scd1, Scd2, Pmvk, Cyp51, Hmgcs1, and Fdps) which potentially could contribute to interference with TLR4-mediated inflammatory signaling. At 4h post-LPS treatment, LPS-treated FLMs reflected a more robust inflammatory response including upregulation of proinflammatory cytokine (e.g. Il1a, Il1b, Tnf) and chemokine (e.g.Ccl2, Ccl3, Ccl4, Ccl7) genes as well as IFN1-regulated genes (e.g. Irf7, Mx1, Oasl1, Ifit1), many of which were suppressed by DHA. Using single-cell regulatory network inference and clustering (SCENIC) to identify gene expression networks, we found DHA modestly downregulated LPS-induced expression of NF-κB-target genes. Importantly, LPS induced a subset of FLMs simultaneously expressing NF-κB- and IRF7/STAT1/STAT2-target genes that were conspicuously absent in DHA-pretreated FLMs. Thus, DHA potently targeted both the NF-κB and the IFN1 responses. Altogether, scRNAseq generated a valuable dataset that provides new insights into multiple overlapping mechanisms by which DHA may transcriptionally or post-transcriptionally regulate LPS-induced proinflammatory and IFN1-driven responses in macrophages.

Genome engineering for estrogen receptor mutations reveals differential responses to anti-estrogens and new prognostic gene signatures for breast cancer

Mutations in the estrogen receptor (ESR1) gene are common in ER-positive breast cancer patients who progress on endocrine therapies. Most mutations localise to just three residues at, or near, the C-terminal helix 12 of the hormone binding domain, at leucine-536, tyrosine-537 and aspartate-538. To investigate these mutations, we have used CRISPR-Cas9 mediated genome engineering to generate a comprehensive set of isogenic mutant breast cancer cell lines. Our results confirm that L536R, Y537C, Y537N, Y537S and D538G mutations confer estrogen-independent growth in breast cancer cells. Growth assays show mutation-specific reductions in sensitivities to drugs representing three classes of clinical anti-estrogens. These differential mutation- and drug-selectivity profiles have implications for treatment choices following clinical emergence of ER mutations. Our results further suggest that mutant expression levels may be determinants of the degree of resistance to some anti-estrogens. Differential gene expression analysis demonstrates up-regulation of estrogen-responsive genes, as expected, but also reveals that enrichment for interferon-regulated gene expression is a common feature of all mutations. Finally, a new gene signature developed from the gene expression profiles in ER mutant cells predicts clinical response in breast cancer patients with ER mutations.

Type 1 interferon activation in systemic sclerosis: a biomarker, a target or the culprit.

Purpose of reviewActivation of the type 1 interferon (T1 IFN) pathway has been implicated in the pathogenesis of systemic sclerosis (SSc) by an increasing number of studies, most of which share key findings with similar studies in systemic lupus erythematosus (SLE). Here we will focus on the evidence for T1 IFN activation and dysregulation in SSc, and the rationale behind targeting the pathway going forward.Recent findingsAn increased expression and activation of T1 IFN-regulated genes has been shown to be present in a significant proportion of SSc patients. TI IFN activation markers have been found to predict and correlate with response to immunosuppressive treatment as well as severity of organ involvement. As inhibition of the IFN-α receptor has been proven to be effective in active SLE, benefit may be seen in targeting the IFN pathway in SSc.SummaryThe role played by T1 IFN and its regulatory genes in SSc is becoming increasingly evident and strikingly similar to the role observed in SLE. This observation, together with the benefit of type 1 IFN targeting in SLE, supports the notion of a potential therapeutic benefit in targeting T1 IFN in SSc.

C3aR dampens intracellular cAMP to control TRIF-mediated inflammation in activated macrophages

Abstract Recent studies from our lab and others have shown that complement components can enhance cell-intrinsic inflammatory signaling pathways. Specifically, we have found that activation of complement receptor C3aR enhances TLR4 signaling in lipopolysaccharide (LPS)-treated macrophages via enhanced p-38 phosphorylation. We found C3aR−/− mice treated with systemic LPS have decreased inflammatory cytokines (IL-6 and TNF) compared to wild-type (WT) mice. Our results corroborate previous studies showing C3aR−/− mice display decreased systemic cytokines (IL-6 and TNF) and increased bacterial burden during Gram-negative (GN) infection compared to WT mice. These results suggest C3aR is a critical regulator of TLR4 signaling. TLR4-TRIF signaling leads to activation of IRF3 and expression of interferon-regulated genes. We find that primary bone marrow-derived macrophages (BMDMs) from C3aR−/− mice show significantly reduced TRIF-dependent ifnb expression and IFNb secretion following 24h LPS exposure. Additionally, WT BMDMs treated with a C3aR antagonist (C3aRi) show a similar reduction in ifnb expression after LPS-treatment. These data demonstrate C3aR is critical for TRIF-dependent signaling downstream of TLR4 activation. Here we find WT BMDMs treated with C3aRi and LPS show increased intracellular cAMP. These findings demonstrate C3aR activation leads to depleted cAMP, which is essential for TRIF-mediated signaling downstream of TLR4. We hypothesize that C3aR is working as an inhibitory GPCR to regulate inflammation via intracellular cAMP and enhancing TRIF signaling in LPS-treated macrophages. Our findings are the first to suggest C3aR reduces cAMP to control TRIF-mediated inflammation in activated macrophages.

The interferon gene signature as a clinically relevant biomarker in autoimmune rheumatic disease.

The interferon gene signature (IGS) is derived from the expression of interferon-regulated genes and is classically increased in response to type I interferon exposure. A raised whole blood IGS has increasingly been reported in rheumatic diseases as sequencing technology has advanced. Although its role remains unclear, we explore how a raised IGS can function as a clinically relevant biomarker, independent of whether it is a bystander effect or a key pathological process. For example, a raised IGS can act as a diagnostic biomarker when predicting rheumatoid arthritis in patients with arthralgia and anti-citrullinated protein antibodies, or predicting systemic lupus erythematous (SLE) in those with antinuclear antibodies; a theragnostic biomarker when predicting response for patients receiving disease modifying therapy, such as rituximab in rheumatoid arthritis; a biomarker of disease activity (early rheumatoid arthritis, dermatomyositis, systemic sclerosis, SLE); or finally a predictor of clinical characteristics, such as lupus nephritis in SLE or disease burden in primary Sjögren's syndrome. A high IGS does not uniformly predict worse clinical phenotypes across all diseases, as demonstrated by a reduced disease burden in primary Sjögren's syndrome, nor does it predict a universally poorer response to all therapies, as shown in rheumatoid arthritis. This dichotomy highlights both the complexity of type I interferon signalling in vivo and the current lack of standardisation when calculating the IGS. The IGS as a biomarker warrants further exploration, with beneficial clinical applications anticipated in multiple rheumatic diseases.

Inhibition of PI3Kδ Differentially Regulates Poly I:C- and Human Metapneumovirus-Induced PD-L1 and PD-L2 Expression in Human Bronchial Epithelial Cells.

Bronchial epithelial cells are front sentinels eliciting innate and adaptive immunity to respiratory viral pathogens. Recognition of viral double-stranded RNA induces antiviral interferon (IFN) responses in bronchial epithelial cells. Co-inhibitory molecules programmed cell death 1 ligand 1 (PD-L1) and ligand 2 (PD-L2) were also induced on bronchial epithelial cells, which bind programmed cell death 1 on T cell and inhibit the function of virus-specific cytotoxic T lymphocyte. A previous study showed that antiviral type I IFN increased PD-L1 and PD-L2 expression in cultured melanoma cells. However, it remains unknown whether antiviral IFNs affect PD-L1 and PD-L2 expression in bronchial epithelial cells. In addition, we previously reported that inhibition of PI3Kδ signaling enhanced antiviral IFN responses in human primary bronchial epithelial cells (PBECs). Here we assessed the effect of exogenous IFNs or a selective PI3Kδ inhibitor IC87114 on PD-L1 and PD-L2 in PBECs stimulated with a synthetic double-stranded RNA poly I:C or human metapneumovirus. Treatment with IFNβ or IFNλ increased PD-L1 and PD-L2, and IFNβ or IFNλ treatment plus poly I:C further increased both expressions. Treatment with IC87114 or transfection with siRNA targeting PI3K p110δ enhanced poly I:C–induced gene and protein expression of PD-L2, whereas IC87114 suppressed poly I:C–induced PD-L1. IC87114 enhanced poly I:C–induced gene expression of IFNβ, IFNλ, and IFN-regulated genes via increased TBK1 and IRF3 phosphorylation. Transfection with siIRF3 counteracted the enhancement of poly I:C–induced PD-L2 by IC87114, whereas IC87114 suppressed poly I:C–induced PD-L1 regardless of transfection with siNC or siIRF3. Similar effects of IC87114 on PD-L1 and PD-L2 expression were observed in human metapneumovirus–infected PBECs. We showed for the first time that type I and type III IFNs induced the expression of PD-L1 and PD-L2 in PBECs. Our findings suggest that during viral infections, inhibition of PI3Kδ differentially regulates PD-L1 and PD-L2 expression in bronchial epithelial cells.

Hematopoietic Stem Cells Undergo Immune Training and Constitute a Long-Term Reservoir for Hyper-Inflammatory Macrophages in a Mouse Model of Chronic Autoimmunity

Auto-immune diseases (AD) are characterized by repeated flares of disease activity separated by periods of remission. Cycles of AD remission and relapse can occur even with therapeutic intervention and contribute to AD morbidity. Paradoxically, during remission myeloid cells retain increased expression of genes related to interferon signaling and antigen presentation. Given the relatively short lifespan of myeloid cells, these observations imply the existence of a clonal reservoir fueling AD relapse. Recent literature describes hematopoietic stem cells (HSC) as a cellular source for trained myeloid cells in response to immune stimuli. Thus, we hypothesize that HSC may also retain a long-term memory of chronic autoimmune inflammation, thereby providing a continuous supply of myeloid cells that promote AD pathogenesis.To test this hypothesis, we have combined high-throughput molecular and cytokine profiling approaches with functional assays to address heritable changes in immune function using the pristane-induced mouse model of systemic lupus erythematosus (SLE). Eight weeks after pristane injection, we observe significant myeloid lineage expansion in the bone marrow (BM), including mature myeloid cells, granulocyte/monocyte progenitors (GMP) and multipotent progenitor (MPP) populations. To understand the impact of SLE-like disease on the molecular programming of the hematopoietic system, we performed RNA-seq analyses of BM Mon and HSC. As anticipated, BM Mon activated inflammatory programs and antigen presentation genes, which overlapped with gene signatures of human monocytes from SLE patients. HSC also activated innate defense gene programs resembling signatures of trained immunity, thus establishing the potential for autoimmune inflammation to induce immune training in HSC.To establish whether these molecular programs potentiate myeloid cell function, we generated BM-derived macrophages (BMDM) from control and pristane-induced mice. BMDMs from pristane-induced mice exhibited significantly increased capacity to kill Mycobacterium avium. Further, co-culture of T cells with BMDMs from pristane-induced mice significantly boosted T cell proliferation, indicative of enhanced antigen presentation. To establish whether HSC from pristane-induced mice propagate molecular memory of SLE-like disease to myeloid progeny, we transplanted stringently enriched (LSK/SLAM/CD34 -/EPCR +) long-term (LT)-HSC into lethally irradiated recipient mice. 18 weeks post transplant, mice transplanted with LT-HSC from pristane-induced donors had a small but significant reduction in donor BM HSC chimerism but did not exhibit overt changes in lineage output. Strikingly, BMDMs from pristane-induced donors showed increased bacterial killing and inflammatory cytokine generation following M. avium challenge, as well as increased capacity to induce antigen-specific T cell proliferation. Thus, LT-HSC retain and pass on altered functional properties to myeloid cells, even in the absence of AD activity.To characterize the molecular mechanisms underlying HSC-propagated alterations in myeloid cell function, we ran RNA-seq on donor-derived GMP and BMDMs. Notably, GMP derived from pristane-induced donor LT-HSC had increased expression of Fos and Jun/b/d, key molecular drivers of immune training in stem cells. Likewise, pristane-induced donor BMDMs maintained increased expression of IFN-regulated genes including MHC-I, a gene also overexpressed in PBMCs from human SLE patients. Consistent with these findings, re-stimulation of recipient mice with pristane led to a significant increase in cytokine-producing lymph node T cells versus recipient mice transplanted with control HSC, further supporting a model of immune training.Overall, these data show that chronic autoimmune inflammation can induce in HSC a heritable trained immunity phenotype that is transmitted to myeloid progeny, enhancing their functional activity. Ongoing studies are establishing the capacity for this phenotype to exacerbate AD. We are also testing the importance of molecular players identified above in establishing AD-related immune training and assessing the potential for therapeutic interventions to disrupt HSC memory in this setting. Our data thus stand to establish a new paradigm for trained immunity in HSC as a key contributor to AD pathology and relapse. DisclosuresNo relevant conflicts of interest to declare.

TAK-981, a First-in-Class SUMO-Activating Enzyme Inhibitor, Combined with Rituximab in Adult Patients (Pts) with CD20-Positive Relapsed/Refractory (R/R) Non-Hodgkin Lymphoma (NHL): Phase 1 Data

TAK-981, a First-in-Class SUMO-Activating Enzyme Inhibitor, Combined with Rituximab in Adult Patients (Pts) with CD20-Positive Relapsed/Refractory (R/R) Non-Hodgkin Lymphoma (NHL): Phase 1 Data

Centrality of Myeloid-Lineage Phagocytes in Particle-Triggered Inflammation and Autoimmunity.

Exposure to exogenous particles found as airborne contaminants or endogenous particles that form by crystallization of certain nutrients can activate inflammatory pathways and potentially accelerate autoimmunity onset and progression in genetically predisposed individuals. The first line of innate immunological defense against particles are myeloid-lineage phagocytes, namely macrophages and neutrophils, which recognize/internalize the particles, release inflammatory mediators, undergo programmed/unprogrammed death, and recruit/activate other leukocytes to clear the particles and resolve inflammation. However, immunogenic cell death and release of damage-associated molecules, collectively referred to as “danger signals,” coupled with failure to efficiently clear dead/dying cells, can elicit unresolved inflammation, accumulation of self-antigens, and adaptive leukocyte recruitment/activation. Collectively, these events can promote loss of immunological self-tolerance and onset/progression of autoimmunity. This review discusses critical molecular mechanisms by which exogenous particles (i.e., silica, asbestos, carbon nanotubes, titanium dioxide, aluminum-containing salts) and endogenous particles (i.e., monosodium urate, cholesterol crystals, calcium-containing salts) may promote unresolved inflammation and autoimmunity by inducing toxic responses in myeloid-lineage phagocytes with emphases on inflammasome activation and necrotic and programmed cell death pathways. A prototypical example is occupational exposure to respirable crystalline silica, which is etiologically linked to systemic lupus erythematosus (SLE) and other human autoimmune diseases. Importantly, airway instillation of SLE-prone mice with crystalline silica elicits severe pulmonary pathology involving accumulation of particle-laden alveolar macrophages, dying and dead cells, nuclear and cytoplasmic debris, and neutrophilic inflammation that drive cytokine, chemokine, and interferon-regulated gene expression. Silica-induced immunogenic cell death and danger signal release triggers accumulation of T and B cells, along with IgG-secreting plasma cells, indicative of ectopic lymphoid tissue neogenesis, and broad-spectrum autoantibody production in the lung. These events drive early autoimmunity onset and accelerate end-stage autoimmune glomerulonephritis. Intriguingly, dietary supplementation with ω-3 fatty acids have been demonstrated to be an intervention against silica-triggered murine autoimmunity. Taken together, further insight into how particles drive immunogenic cell death and danger signaling in myeloid-lineage phagocytes and how these responses are influenced by the genome will be essential for identification of novel interventions for preventing and treating inflammatory and autoimmune diseases associated with these agents.

Interferon Genes Are Influenced by 17β-Estradiol in SLE.

Recent evidence suggests the existence of a nexus between inflammatory pathways and the female sex hormone 17β-estradiol, resulting in increased interferon-stimulated genes (ISGs), autoantibodies, and dysregulation of immune cells in SLE. However, the molecular mechanisms and the effect of estradiol on candidate target genes and their pathways remains poorly understood. Our previous work suggests that female SLE patients have increased estradiol levels compared to healthy controls. In the present study, we explored the effects of 17β-estradiol treatment on expression of IFN (interferons)-stimulated genes and pro-inflammatory cytokines/chemokines. We found significantly increased (5-10-fold) expression of IFN-regulated genes in healthy females. Furthermore, we found significantly increased plasma levels of IL-6, IL-12, IL-17, IL-18, stem cell factor (SCF), and IL-21/IL-23 in SLE patients compared to healthy controls, and those levels positively correlated with the plasma levels of 17β-estradiol. In addition, levels of IL-21 positively correlated with the SLE disease activity index (SLEDAI) score of SLE patients. In vitro treatment of PBMCs from either SLE patients or healthy controls with 17β-estradiol at physiological concentration (~50 pg/ml) also significantly increased secretion of many pro-inflammatory cytokines and chemokines (IL-6, IL-12, IL-17, IL-8, IFN-γ; MIP1α, and MIP1β) in both groups. Further our data revealed that 17β-estradiol significantly increased the percentage of CD3+CD69+ and CD3+IFNγ+ T cells; whereas, simultaneous addition of 17β-estradiol and an ERα inhibitor prevented this effect. Collectively, our findings indicate that 17β-estradiol participates in the induction of pro-inflammatory cytokines and chemokines and further influences interferon genes and pathways.

Updates on interferon in juvenile dermatomyositis: pathogenesis and therapy.

This review provides updates regarding the role of interferon (IFN) in juvenile dermatomyositis (JDM), including comparison to interferonopathies and therapeutic implications. Transcriptomic and protein-based studies in different tissues and peripheral IFN-α assessment have demonstrated the importance of the dysregulated IFN pathway in JDM. Additional studies have validated IFN-regulated gene and protein expression correlation with disease activity in blood and muscle, with potential to predict flares. Type I and II IFN both are dysregulated in peripheral blood and muscle, with more type I IFN in skin. Muscle studies connects hypoxia to IFN production and IFN to vascular dysfunction and muscle atrophy. JDM overlaps with interferonopathy phenotype and IFN signature. There are multiple case reports and case series noting decreased IFN markers and clinical improvement in refractory JDM with Janus kinase (JAK) inhibitors. Studies confirm IFN, particularly type I and II IFN, is an important part of JDM pathogenesis by the level of dysregulation and correlation with disease activity, as well as IFN recapitulating key JDM muscle pathology. Smaller studies indicate there may be differences by myositis-specific autoantibody group, but validation is needed. JAK inhibitors are a promising therapy as they can inhibit IFN signaling, but further study is needed regarding which patients will benefit, dosing, and safety monitoring.

Modulation of Early Host Innate Immune Response by an Avipox Vaccine Virus' Lateral Body Protein.

The avian pathogen fowlpox virus (FWPV) has been successfully used as a vaccine vector in poultry and humans, but relatively little is known about its ability to modulate host antiviral immune responses in these hosts, which are replication-permissive and nonpermissive, respectively. FWPV is highly resistant to avian type I interferon (IFN) and able to completely block the host IFN-response. Microarray screening of host IFN-regulated gene expression in cells infected with 59 different, nonessential FWPV gene knockout mutants revealed that FPV184 confers immunomodulatory capacity. We report that the FPV184-knockout virus (FWPVΔ184) induces the cellular IFN response as early as 2 h postinfection. The wild-type, uninduced phenotype can be rescued by transient expression of FPV184 in FWPVΔ184-infected cells. Ectopic expression of FPV184 inhibited polyI:C activation of the chicken IFN-β promoter and IFN-α activation of the chicken Mx1 promoter. Confocal and correlative super-resolution light and electron microscopy demonstrated that FPV184 has a functional nuclear localisation signal domain and is packaged in the lateral bodies of the virions. Taken together, these results provide a paradigm for a late poxvirus structural protein packaged in the lateral bodies, capable of suppressing IFN induction early during the next round of infection.

Omega-3 fatty acid intake suppresses induction of diverse autoantibody repertoire by crystalline silica in lupus-prone mice

Inhalation of crystalline silica (cSiO2) in the workplace is etiologically linked to lupus and other autoimmune diseases. Exposing lupus-prone NZBWF1 mice to respirable cSiO2 unleashes a vicious cycle of inflammation and cell death in the lung that triggers interferon-regulated gene expression, ectopic lymphoid structure (ELS) development, elevation of local and systemic autoantibodies (AAbs), and glomerulonephritis. However, cSiO2-induced inflammation and onset of autoimmunity can be prevented by inclusion of the ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) into the diet of these mice. Since cSiO2 both causes cell death and interferes with efferocytosis, secondary necrosis of residual cell corpses might provide a rich and varied autoantigen (AAg) source in the lung. While it is known that the particle induces anti-nuclear and anti-dsDNA AAbs in NZBWF1 mice, the full extent of the cSiO2-induced AAb response relative to specificity and isotype is not yet understood. The purpose of this study was to test the hypotheses that cSiO2 exposure induces a wide spectrum of AAbs in the pulmonary and systemic compartments, and that dietary DHA intervention prevents these changes. Archived tissue fluid samples were obtained from a prior study in which NZBWF1 mice were fed purified isocaloric diets containing no DHA (control) or DHA corresponding calorically to human doses of 2 and 5 g/day. Mice were intranasally instilled with 1 mg cSiO2 or saline vehicle weekly for 4 weeks, then groups euthanized 1, 5, 9, or 13 weeks post-instillation (PI) of the last cSiO2 dose. Bronchoalveolar lavage fluid (BALF) and plasma from each time point were subjected to AAb profiling using a microarray containing 122 AAgs. cSiO2 triggered robust IgG and IgM AAb responses against lupus-associated AAgs, including DNA, histones, ribonucleoprotein, Smith antigen, Ro/SSA, La/SSB, and complement as early as 1 week PI in BALF and 5 weeks PI in plasma, peaking at 9 and 13 weeks PI, respectively. Importantly, cSiO2 also induced AAbs to AAgs associated with rheumatoid arthritis (collagen II, fibrinogen IV, fibrinogen S, fibronectin, and vimentin), Sjögren’s syndrome (α-fodrin), systemic sclerosis (topoisomerase I), vasculitis (MPO and PR3), myositis (Mi-2, TIF1-γ, MDA5), autoimmune hepatitis (LC-1), and celiac disease (TTG). cSiO2 elicited comparable but more modest IgA AAb responses in BALF and plasma. cSiO2-induced AAb production was strongly associated with time dependent inflammatory/autoimmune gene expression, ELS development, and glomerulonephritis. AAb responses were dose-dependently suppressed by DHA supplementation and negatively correlated with the ω-3 index, an erythrocyte biomarker of ω-3 content in tissue phospholipids. Taken together, these findings suggest that cSiO2 exposure elicits a diverse multi-isotype repertoire of AAbs, many of which have been reported in individuals with lupus and other autoimmune diseases. Furthermore, induction of this broad AAb spectrum could be impeded by increasing ω-3 tissue content via dietary DHA supplementation.

Expression of interferon-regulated genes in juvenile dermatomyositis versus Mendelian autoinflammatory interferonopathies

BackgroundJuvenile dermatomyositis (JDM) is a systemic autoimmune disease with a prominent interferon (IFN) signature, but the pathogenesis of JDM and the etiology of its IFN signature remain unknown. The Mendelian autoinflammatory interferonopathies, Chronic Atypical Neutrophilic Dermatosis with Lipodystrophy and Elevated temperature (CANDLE) and STING-Associated Vasculopathy with onset in Infancy (SAVI), are caused by genetic mutations and have extremely elevated IFN signatures thought to drive pathology. The phenotypic overlap of some clinical features of CANDLE and SAVI with JDM led to the comparison of a standardized interferon-regulated gene score (IRG-S) in JDM and myositis-specific autoantibody (MSA) JDM subgroups, with CANDLE and SAVI.MethodsA peripheral 28-component IRG-S assessed by NanoString™ in 57 JDM patients subtyped by MSA was compared with IRG-S in healthy controls (HC) and CANDLE/SAVI patients. Principal component analysis (PCA) was performed, and individual genes were evaluated for their contribution to the score. IRG-S were correlated with disease assessments and patient characteristics.ResultsIRG-S in JDM patients were significantly higher than in HC but lower than in CANDLE or SAVI. JDM IRG-S overlapped more with SAVI than CANDLE by PCA. Among MSA groups, anti-MDA5 autoantibody-positive patients’ IRG-S overlapped most with SAVI. The IFI27 proportion was significantly higher in SAVI and CANDLE than JDM, but IFIT1 contributed more to IRG-S in JDM. Overall, the contribution of individual interferon-regulated genes (IRG) in JDM was more similar to SAVI. IRG-S correlated moderately with JDM disease activity measures (rs = 0.33–0.47) and more strongly with skin activity (rs = 0.58–0.79) in anti-TIF1 autoantibody-positive patients. Weakness and joint disease activity (multinomial OR 0.91 and 3.3) were the best predictors of high IRG-S.ConclusionsOur findings demonstrate peripheral IRG expression in JDM overlaps with monogenic interferonopathies, particularly SAVI, and correlates with disease activity. Anti-MDA5 autoantibody-positive JDM IRG-S were notably more similar to SAVI. This may reflect both a shared IFN signature, which is driven by IFN-β and STING pathways in SAVI, as well as the shared phenotype of vasculopathy in SAVI and JDM, particularly in anti-MDA5 autoantibody-positive JDM, and indicate potential therapeutic targets for JDM.