Interferon Responsive Research Articles

Tissue kallikrein suppresses interferon responses and reduces depressive-like behavior in lupus-prone mice.

Abstract Recent studies show that systemic immune responses, including Type I IFNs, affect neuroinflammation and neuropsychiatric responses. Major depression is the most common psychiatric presentation of NPSLE. Reports indicate that neuropsychiatric symptoms, especially depression and anxiety, may be common co-morbidities of childhood-onset of SLE and clinical studies have demonstrated that IFNα can cause depressive symptoms. The management of patients with neuropsychiatric lupus (NPSLE) continues to be a major therapeutic challenge. We have shown that the kallikrein-kinin system (KKS) [comprised of kallikreins (klks), bradykinins (BKs)] and angiotensin converting enzyme inhibitors (ACEi’s), suppressed Type I IFN responses in murine dendritic cells (DCs) from normal and lupus-prone mice and human peripheral blood mononuclear cells (PBMC). Other studies have shown that klk genes are decreased in lupus patients; giving exogenous klk1 ameliorated kidney pathology in mice. We administered (retro-orbitally) adenovirus carrying the kallikrein gene (klk1- a widely expressed tissue klk) in the MRL/lpr lupus-prone mice (at early disease onset) and analyzed immune responses and depressive-like behavior response. Klk1 improved depressive-like behavior, suppressed Interferon Responsive Genes, decreased splenic plasmacytoid DCs, and reduced plasma IFNa levels. Together, these findings suggest that kallikreins and other KKS molecules may be used to control IFNa production/responses and other inflammatory responses in SLE and NPSLE. This work was supported by NIH Grant R21AR071679 and funds from the Department of Pathology and Laboratory Medicine.

Prior activation state shapes the microglia response to antihuman TREM2 in a mouse model of Alzheimer’s disease

Triggering receptor expressed on myeloid cells 2 (TREM2) sustains microglia response to brain injury stimuli including apoptotic cells, myelin damage, and amyloid β (Aβ). Alzheimer's disease (AD) risk is associated with the TREM2R47H variant, which impairs ligand binding and consequently microglia responses to Aβ pathology. Here, we show that TREM2 engagement by the mAb hT2AB as surrogate ligand activates microglia in 5XFAD transgenic mice that accumulate Aβ and express either the common TREM2 variant (TREM2CV) or TREM2R47H scRNA-seq of microglia from TREM2CV-5XFAD mice treated once with control hIgG1 exposed four distinct trajectories of microglia activation leading to disease-associated (DAM), interferon-responsive (IFN-R), cycling (Cyc-M), and MHC-II expressing (MHC-II) microglia types. All of these were underrepresented in TREM2R47H-5XFAD mice, suggesting that TREM2 ligand engagement is required for microglia activation trajectories. Moreover, Cyc-M and IFN-R microglia were more abundant in female than male TREM2CV-5XFAD mice, likely due to greater Aβ load in female 5XFAD mice. A single systemic injection of hT2AB replenished Cyc-M, IFN-R, and MHC-II pools in TREM2R47H-5XFAD mice. In TREM2CV-5XFAD mice, however, hT2AB brought the representation of male Cyc-M and IFN-R microglia closer to that of females, in which these trajectories had already reached maximum capacity. Moreover, hT2AB induced shifts in gene expression patterns in all microglial pools without affecting representation. Repeated treatment with a murinized hT2AB version over 10 d increased chemokines brain content in TREM2R47H-5XFAD mice, consistent with microglia expansion. Thus, the impact of hT2AB on microglia is shaped by the extent of TREM2 endogenous ligand engagement and basal microglia activation.

Synthetic rewiring and boosting type I interferon responses for visualization and counteracting viral infections.

Mammalian first line of defense against viruses is accomplished by the interferon (IFN) system. Viruses have evolved numerous mechanisms to reduce the IFN action allowing them to invade the host and/or to establish latency. We generated an IFN responsive intracellular hub by integrating the synthetic transactivator tTA into the chromosomal Mx2 locus for IFN-based activation of tTA dependent expression modules. The additional implementation of a synthetic amplifier module with positive feedback even allowed for monitoring and reacting to infections of viruses that can antagonize the IFN system. Low and transient IFN amounts are sufficient to trigger these amplifier cells. This gives rise to higher and sustained—but optionally de-activatable—expression even when the initial stimulus has faded out. Amplification of the IFN response induced by IFN suppressing viruses is sufficient to protect cells from infection. Together, this interfaced sensor/actuator system provides a toolbox for robust sensing and counteracting viral infections.

Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity.

The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread1,2. PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses3–5. Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-κB pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity.

All-Trans Retinoic Acid Stimulates Viral Mimicry, Interferon Responses and Antigen Presentation in Breast-Cancer Cells.

All-trans retinoic acid (ATRA), a recognized differentiating agent, has significant potential in the personalized/stratified treatment of breast cancer. The present study reports on the molecular mechanisms underlying the anti-tumor activity of ATRA in breast cancer. The work is based on transcriptomic experiments performed on ATRA-treated breast cancer cell-lines, short-term tissue cultures of patient-derived mammary-tumors and a xenograft model. ATRA upregulates gene networks involved in interferon-responses, immune-modulation and antigen-presentation in retinoid-sensitive cells and tumors characterized by poor immunogenicity. ATRA-dependent upregulation of these gene networks is caused by a viral mimicry process, involving the activation of endogenous retroviruses. ATRA induces a non-canonical type of viral mimicry, which results in increased expression of the IRF1 (Interferon Responsive Factor 1) transcription factor and the DTX3L (Deltex-E3-Ubiquitin-Ligase-3L) downstream effector. Functional knockdown studies indicate that IRF1 and DTX3L are part of a negative feedback loop controlling ATRA-dependent growth inhibition of breast cancer cells. The study is of relevance from a clinical/therapeutic perspective. In fact, ATRA stimulates processes controlling the sensitivity to immuno-modulatory drugs, such as immune-checkpoint-inhibitors. This suggests that ATRA and immunotherapeutic agents represent rational combinations for the personalized treatment of breast cancer. Remarkably, ATRA-sensitivity seems to be relatively high in immune-cold mammary tumors, which are generally resistant to immunotherapy.

Decreased Immune Activation in Cystic Fibrosis Airway Epithelium Following Lung Transplant

End-stage cystic fibrosis (CF) can be managed by lung transplantation (LT), but chronic allograft dysfunction (CLAD) limits long-term survival. While Pseudomonas aeruginosa airway infection (PsA) is a risk factor for CLAD for most lung allograft recipients, this association does not hold for CF recipients. We hypothesized that differences in host epithelial immune responses and the composition of airway microbial communities could contribute to protection from CLAD in CF recipients with PsA. We compared transcriptomes from clinical airway brushes following LT for 28 subjects, grouped by CF status and PsA culture result (CF+PsA+, n=8; CF+PsA-, n=6; CF-PsA+, n=5; and CF-PsA-, n=9) using DEseq2. For in vitro analysis of immune responses, airway epithelial cells (AEC) were isolated from lung explants (pre-LT) or surveillance airway brushes (post-LT) and grown at air-liquid interface until polarized and differentiated. Gene expression differences and DNA methylation were compared by CF status using quantitative PCR and DNA methylation array. For subjects with a positive PsA culture following LT, there were 193 differentially expressed genes between CF and non-CF airways. Pathway analysis demonstrated suppression of interferon response in CF airways. Relative to non-CF referents, CF AEC in vitro transitioned from increased expression of neutrophil-associated genes (Cxcl8 and Lcn2) pre-LT, to decreased expression of interferon-associated genes (Mx1 and Oas1) post-LT. DNA methylation analysis showed hypermethylation of promoters in type I interferon responsive genes (p = 0.002). All CF+PsA+ subjects had mucoid PsA strains while 72% of non-CF subjects had non-mucoid PsA (p <0.001). The combination of attenuated interferon responses and prevalence of mucoid strains may explain the differential outcomes in CF LT recipients with PsA.

Abstract 4473: DNMT and PARP inhibitor combination therapy induces an interferon-driven homologous recombination defect in triple-negative breast cancers and acute myeloid leukemia

Abstract Poly (ADP-ribose) polymerase inhibitors (PARPis) are effective in a subset of triple negative breast cancers (TNBC) with BRCA gene mutations, which generates homologous recombination deficiencies (HRD), through synthetic lethality. However, PARPis fail for the majority of sporadic TNBC with intact BRCA1/2 genes and other BRCA-proficient cancers, including acute myeloid leukemia (AML). PARPi, Talazoparib (TAL), has potent PARP trapping ability, in both BRCA mutant and proficient TNBCs and AML. We reported that a combination of TAL with low doses of DNA methyl transferase inhibitor (DNMTi) azacytidine (AZA) or decitabine (DAC), increased PARP trapping even further, leading to increased levels of cytotoxic double strand breaks in vitro and decreased tumor burden in vivo. Low doses of DNMTis have been shown previously to reprogram genome wide cancer signaling, including immune and DNA damage/repair pathways. We now tie these two signaling pathways together for events through which DNMTis plus TAL drive HRD. BRCA proficient TNBC (N=2) and AML (cell lines (N=2) and primary cells (N=4)) treated with low doses of DNMTis (DAC 10nM and AZA 250-500nM), significantly downregulate expression of genes in HR and Fanconi Anemia (FA) pathways, decreasing HR activity, thus generating HRD. HR is further decreased when DNMTis are combined with TAL. We now link DNMTi/TAL induced HRD to a coupled interferon (IFN) response in both TNBC and AML. In these systems, we elucidate DNMTi/TAL potentiated cGAS-Sting and type 1 IFN signaling, culminating in the induction of IFN responsive genes and inflammatory cytokine production. Importantly, in DNMTi/TAL treated cells, increased expression of IFN signaling genes is strongly linked to downregulation of FA/HR genes through protein interaction network mapping. This inverse association between IFN and HR related genes, was further validated in TCGA data sets for AML and invasive BC, suggesting broad applicability of this observed transcriptional program. The acquisition of HRD from IFN signaling specifically, is verified by the observation of reduced HR gene expression and activity in the presence of exogenous double stranded RNA (Poly(I:C)) or IFNβ treatment in both TNBC and AML cell lines. Finally, knockdown of a key connectivity node between IFN and HR related genes, interferon sensitive gene 15 (ISG15), abrogates downregulation of FA gene expression when treated with IFNβ, suggesting mediation of HRD through ISG15. In conclusion, we have identified a novel mechanism through which IFN-sensitive genes can create an HRD phenotype in BRCA proficient TNBC and AML, suggesting that innate immunity and inflammatory processes may be intrinsically linked to DNA repair in cancer. These studies suggest that immune therapies and/or epigenetic therapy are likely to enhance responses to PARPis in BRCA proficient cancers. Citation Format: Lena J. McLaughlin, Aksinija A. Kogan, Eun Yong Choi, Rena S. Lapidus, Ying Zou, Huili Li, Stephen B. Baylin, Michael J. Topper, Feyruz V. Rassool. DNMT and PARP inhibitor combination therapy induces an interferon-driven homologous recombination defect in triple-negative breast cancers and acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4473.

MiR-155 Promotes FLT3-ITD-Induced Myeloproliferative Disease through Inhibition of Interferon Signaling

Introduction & Background: MicroRNA expression is dysregulated in many human cancers, including hematologic malignancies. Among hematologic malignancies, acute myeloid leukemia (AML) carries a particularly poor prognosis, leading to over 10,000 deaths each year. The most common genetic aberration in AML is a gain-of-function mutation in the FMS-like tyrosine kinase 3 (FLT3) receptor. FLT3 internal tandem duplication (ITD) occurs in ~25% of all AML diagnoses, and confers a negative prognosis. MicroRNA expression has been shown to be dysregulated in FLT3-ITD+ AML, and miR-155 has been identified as the most highly overexpressed microRNA in this disease. MiR-155 is critical for the growth of FLT3-ITD+ cells in vitro, but the functional relevance of miR-155 in FLT3-ITD-mediated disease in vivo, and the downstream effects of miR-155 expression remain unclear.Methods and Design: In this study, we crossed mice homozygous for the FLT3-ITD mutation with miR-155 knockout mice to determine the specific role of miR-155 in the context of FLT3-ITD. The FLT3-ITD mice develop a chronic myeloid malignancy that we evaluated between 4-6 months of age, and compared this phenotype between groups (FLT3-ITD 155+/+ vs FLT3-ITD 155-/-).Results: FLT3-ITD miR-155-/- mice exhibited decreased myeloid expansion in the bone marrow, reduced splenomegaly, and decreased peripheral blood monocytosis and neutrophilia compared to their FLT3-ITD miR-155+/+ counterparts, indicating that miR-155 was playing a crucial role in promoting FLT3-ITD-mediated myeloproliferation. When examining the stem cell compartment of these animals, we found that miR-155 deficient animals had a reduced number of myeloid progenitors compared to their FLT3-ITD 155+/+ counterparts, suggesting miR-155 was critical for maintaining a robust myeloid progenitor pool in this disease. This phenotype was attributed to miR-155's role in promoting proliferation, but not survival, of the hematopoietic stem and progenitor cell (HSPC) and myeloid progenitor cell compartments in the bone marrow. RNA sequencing of the HSPC population in FLT3-ITD 155+/+ mice and FLT3-ITD 155-/- mice revealed that mice lacking miR-155 had an increased response to interferon, known to have a growth-suppressive effect on hematopoietic cells. We confirmed this finding through qPCR, where we found a number of interferon responsive genes were elevated in the HSPC and myeloid progenitor compartments of FLT3-ITD 155-/- mice compared to FLT3-ITD 155+/+ mice. We also observed increased protein levels of STAT1, a key transcription factor that activates interferon signaling, in the HSPCs and myeloid progenitors of FLT3-ITD 155-/- mice. These findings corresponded with human AML data from The Cancer Genome Atlas, where we found that FLT3-ITD+ AML samples had a decreased interferon signature compared to FLT3-WT AML samples. A number of putative miR-155 targets in the HSPCs are upregulated in the absence of miR-155, including PU.1, SHIP1, and CEBPB, all of which have previously been implicated as critical miR-155 targets in FLT3-ITD+ AML. Interestingly, CEBPB is known to regulate interferon responses.Conclusions and Future Directions: Our study establishes miR-155 as a critical promoter of FLT3-ITD-mediated myeloproliferative disease in vivo, a finding we attributed to miR-155's inhibition of the interferon response in this context. Further work will determine which of miR-155's targets could be potentiating this effect. We will also focus on the importance of miR-155 in FLT3-ITD+ leukemogenesis, as FLT3-ITD mice need collaborating mutations to undergo leukemic transformation. These findings suggest that miR-155 inhibitors may warrant clinical consideration as therapeutics in FLT3-ITD+ AML. DisclosuresNo relevant conflicts of interest to declare.

TREX1 Knockdown Induces an Interferon Response to HIV that Delays Viral Infection in Humanized Mice.

Despite their antiviral effect, the invivo effect of interferons on HIV transmission is difficult to predict, because interferons also activate and recruit HIV-susceptible cells to sites of infection. HIV does not normally induce type I interferons in infected cells, but does if TREX1 is knocked down. Here, we investigated the effect of topical TREX1 knockdown and local interferon production on HIV transmission in human cervicovaginal explants and humanized mice. Inexplants in which TREX1 was knocked down, HIV induced interferons, which blocked infection. In humanized mice, even though TREX1 knockdown increased infiltrating immune cells, it delayed viral replication for 3-4weeks. Similarly intravaginal application of type I interferons the day before HIV infection induced interferon responsive genes, reduced inflammation, and decreased viral replication. However, intravenous interferon enhanced inflammation and infection. Thus, in models of human sexual transmission, a localized interferon response inhibits HIV transmission but systemic interferons do not.

Boceprevir dosing for late responders and null responders: The role of bridging data between treatment-naïve and -experienced subjects

Boceprevir dosing for late responders and null responders: The role of bridging data between treatment-naïve and -experienced subjects

Extracellular Matrix Protein CCN1 Limits Oncolytic Efficacy in Glioma

Oncolytic viral therapy has been explored widely as an option for glioma treatment but its effectiveness has remained limited. Cysteine rich 61 (CCN1) is an extracellular matrix (ECM) protein elevated in cancer cells that modulates their adhesion and migration by binding cell surface receptors. In this study, we examined a hypothesized role for CCN1 in limiting the efficacy of oncolytic viral therapy for glioma, based on evidence of CCN1 induction that occurs in this setting. Strikingly, we found that exogenous CCN1 in glioma ECM orchestrated a cellular antiviral response that reduced viral replication and limited cytolytic efficacy. Gene expression profiling and real-time PCR analysis revealed a significant induction of type-I interferon responsive genes in response to CCN1 exposure. This induction was accompanied by activation of the Jak/Stat signaling pathway, consistent with induction of an innate antiviral cellular response. Both effects were mediated by the binding of CCN1 to the cell surface integrin α6β1, activating its signaling and leading to rapid secretion of interferon-α, which was essential for the innate antiviral effect. Together, our findings reveal how an integrin signaling pathway mediates activation of a type-I antiviral interferon response that can limit the efficacy of oncolytic viral therapy. Furthermore, they suggest therapeutic interventions to inhibit CCN1-integrin α6 interactions to sensitize gliomas to viral oncolysis.

Abstract 1173: A genomics approach to understanding DICER1's role in tumorigenesis

Abstract DICER1 is required for the generation of mature microRNAs (22-24 nt miRNAs) and other short noncoding RNAs. Several lines of evidence point to DICER1's role as a tumor suppressor. In particular, reduced DICER1 levels are associated with adverse outcomes in a variety of human and mouse cancers. To date the effects of reduced DICER1 on mRNA transcript abundance in tumor cells remain largely unknown. Furthermore, DICER1 may have non-canonical (unrelated to miRNA activity) functions in tumorigenesis. In model organisms, small RNAs initiate transcriptional silencing. DICER1 may have a similar role in transcriptional silencing and DNA methylation in mammalian cells. To begin to determine how reduced DICER1 levels contribute to tumor phenotypes we used shRNA to stably knock down DICER1 in endometrial cancer cell lines for extended periods (up to 25 passages). Reduced DICER1 levels (measured by Western blot) had no effect on cell proliferation but were associated with enhanced cell migration. We performed microRNA, mRNA, and DNA methylation profiling experiments in the KLE endometrial cancer cell line. MiRNA levels were decreased overall and the fold reductions for individual miRNAs were highly correlated in two different DICER1 shRNA knockdowns. RNAseq analysis (Illumina) revealed a 4-fold increase in the TARBP2 (TRBP) and MOV-10 transcripts. TARBP2 and MOV-10 are integral components of the DICER1 complex and the observed upregulation of their transcripts could reflect selection to stabilize DICER1 in the knockdowns. Furthermore, miR-103 and miR-107, which target DICER1 mRNA, were downregulated in the knockdown cells, possibility reflecting selection to maintain DICER1 activity. Consistent with decreases in miRNA levels, many mRNA transcripts were upregulated with DICER1 knockdown (average 1.1 fold increase across 8077 informative transcripts). Most striking was the number of interferon responsive genes that showed upregulation. Among the 8077 informative transcripts, 28 were classified as interferon responsive, and for those the average and median fold increase was 3.1 and 2.1 respectively (range 0.9 – 12.9). The majority of those interferon responsive genes with increased transcript levels lack miRNA target sequences. Previous work has shown that shRNAs do not induce a general antiviral interferon response. We are currently performing studies to confirm that the effect observed is DICER1 dependent. Technical and biologic validations for those interferon responsive genes that show upregulation based on RNAseq are underway. Increases in IFI6 and IFI44L transcript levels have been confirmed by qPCR. We hypothesize a transcription-based mechanism of DICER1 control of interferon responsive transcripts and are attempting to determine if there is such an effect and whether it is a direct or indirect effect. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1173. doi:10.1158/1538-7445.AM2011-1173

Hepatic Gene Expression Discriminates Responders and Nonresponders in Treatment of Chronic Hepatitis C Viral Infection

Pegylated interferon (IFN)-alpha plus ribavirin is the most effective treatment of chronic hepatitis C but has unpleasant side effects and high costs. A large proportion of patients do not respond to therapy for reasons that are unclear. We used gene expression profiling to investigate the molecular basis for treatment failure. Expression profiling was performed on percutaneous needle liver biopsy specimens taken before therapy. Gene expression levels were compared among 15 nonresponder, 16 responder, and 20 normal liver biopsy specimens. Differential gene expression was confirmed using real-time polymerase chain reaction. We identified 18 genes whose expression differed significantly between all responders and all nonresponders (P < .005). Many of these 18 genes are IFN sensitive and 2 (ISG15/USP18) are linked in a novel IFN-regulatory pathway, suggesting a possible rationale for treatment resistance. Using a number of independent classifier analyses, an 8-gene subset accurately predicted treatment response for 30 of 31 patients. The classifier analyses were applicable to patients with genotype 1 infection and were not correlated with viral load, disease activity, or fibrosis. Hepatic gene expression profiling identified consistent differences in patients who subsequently fail treatment with pegylated IFN-alpha plus ribavirin: up-regulation of a specific set of IFN-responsive genes predicts nonresponse to exogenous therapy. These data may be of use in predicting clinical responses to treatment.