Global virus outbreaks: Interferons as 1st responders.

Outbreaks of severe virus infections with the potential to cause global pandemics are increasing. In many instances these outbreaks have been newly emerging (SARS coronavirus), re-emerging (Ebola virus, Zika virus) or zoonotic (avian influenza H5N1) virus infections. In the absence of a targeted vaccine or a pathogen-specific antiviral, broad-spectrum antivirals would function to limit virus spread. Given the direct antiviral effects of type I interferons (IFNs) in inhibiting the replication of both DNA and RNA viruses at different stages of their replicative cycles, and the effects of type I IFNs on activating immune cell populations to clear virus infections, IFNs-α/ß present as ideal candidate broad-spectrum antivirals.

Identification and targeting of novel CDK9 complexes in acute myeloid leukemia.

Aberrant activation of mTOR signaling in acute myeloid leukemia (AML) results in a survival advantage that promotes the malignant phenotype. To improve our understanding of factors that contribute to mammalian target of rapamycin (mTOR) signaling activation and identify novel therapeutic targets, we searched for unique interactors of mTOR complexes through proteomics analyses. We identify cyclin dependent kinase 9 (CDK9) as a novel binding partner of the mTOR complex scaffold protein, mLST8. Our studies demonstrate that CDK9 is present in distinct mTOR-like (CTOR) complexes in the cytoplasm and nucleus. In the nucleus, CDK9 binds to RAPTOR and mLST8, forming CTORC1, to promote transcription of genes important for leukemogenesis. In the cytoplasm, CDK9 binds to RICTOR, SIN1, and mLST8, forming CTORC2, and controls messenger RNA (mRNA) translation through phosphorylation of LARP1 and rpS6. Pharmacological targeting of CTORC complexes results in suppression of growth of primitive human AML progenitors in vitro and elicits strong antileukemic responses in AML xenografts in vivo.

A cluster randomized trial of interferon ß-1a for the reduction of transmission of SARS-Cov-2: protocol for the Containing Coronavirus Disease 19 trial (ConCorD-19).

BACKGROUND: SARS-CoV-2 infection rapidly spreads in populations due to the high rates of community transmission. Interrupting the shedding of SARS-CoV-2 may reduce the incidence of Coronavirus Disease 19 (COVID-19). Herein we provide a protocol for a cluster randomized trial that will examine the effectiveness of treatment with interferon (IFN) ß-1a compared to standard of care in limiting the transmission of SARS-CoV-2. Co-primary objectives are to determine whether IFN therapy reduces (a) the proportion of infected cases shedding SARS-CoV-2 at day 11 post randomization and (b) the incidence of transmission of SARS-CoV-2 infection from index cases to treatment-eligible household post-exposure contacts at day 11 after randomization. Secondary objectives include assessing the impact of IFN treatment on duration of viral clearance, hospitalizations and fatalities, and evaluating the safety of IFN treatment. METHODS: Three hundred and ten households, each including an index case with a recent COVID-19 diagnosis and at least one asymptomatic treatment-eligible household contact, will be randomized to receive 3 doses of 125 µg IFN ß-1a by subcutaneous administration (days 1, 6, and 11), or standard of care. All participants will be followed until day 29. DISCUSSION: The results from this trial will identify whether IFN ß treatment of mild or moderate COVID-19 cases accelerates viral clearance and prevents disease progression and whether IFN ß treatment of post-exposure contacts of COVID-19 cases reduces transmission of infection. TRIAL REGISTRATION: This trial is registered at ClinicalTrials.gov NCT04552379; date of registration September 17, 2020.

Sirtuin 2-mediated deacetylation of cyclin-dependent kinase 9 promotes STAT1 signaling in type I interferon responses.

Type I interferons (IFNs) induce expression of multiple genes that control innate immune responses to invoke both antiviral and antineoplastic activities. Transcription of these interferon-stimulated genes (ISGs) occurs upon activation of the canonical Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathways. Phosphorylation and acetylation are both events crucial to tightly regulate expression of ISGs. Here, using mouse embryonic fibroblasts and an array of biochemical methods including immunoblotting and kinase assays, we show that sirtuin 2 (SIRT2), a member of the NAD-dependent protein deacetylase family, is involved in type I IFN signaling. We found that SIRT2 deacetylates cyclin-dependent kinase 9 (CDK9) in a type I IFN-dependent manner and that the CDK9 deacetylation is essential for STAT1 phosphorylation at Ser-727. We also found that SIRT2 is subsequently required for the transcription of ISGs and for IFN-driven antiproliferative responses in both normal and malignant cells. These findings establish the existence of a previously unreported signaling pathway whose function is essential for the control of JAK-STAT signaling and the regulation of IFN responses. Our findings suggest that targeting sirtuin activities may offer an avenue in the development of therapies for managing immune-related diseases and cancer.

Cutting Edge: Endogenous IFN-ß Regulates Survival and Development of Transitional B Cells.

The transitional stage of B cell development is a formative stage in the spleen where autoreactive specificities are censored as B cells gain immune competence, but the intrinsic and extrinsic factors regulating survival of transitional stage 1 (T1) B cells are unknown. We report that B cell expression of IFN-ß is required for optimal survival and TLR7 responses of transitional B cells in the spleen and was overexpressed in T1 B cells from BXD2 lupus-prone mice. Single-cell gene expression analysis of B6 Ifnb+/+ versus B6 Ifnb-/- T1 B cells revealed heterogeneous expression of Ifnb in wild-type B cells and distinct gene expression patterns associated with endogenous IFN-ß. Single-cell analysis of BXD2 T1 B cells revealed that Ifnb is expressed in early T1 B cell development with subsequent upregulation of Tlr7 and Ifna1 Together, these data suggest that T1 B cell expression of IFN-ß plays a key role in regulating responsiveness to external factors.

The X-files in immunity: sex-based differences predispose immune responses.

Despite accumulating evidence in support of sex-based differences in innate and adaptive immune responses, in the susceptibility to infectious diseases and in the prevalence of autoimmune diseases, health research and clinical practice do not address these distinctions, and most research studies of immune responses do not stratify by sex. X-linked genes, hormones and societal context are among the many factors that contribute to disparate immune responses in males and females. It is crucial to address sex-based differences in disease pathogenesis and in the pharmacokinetics and pharmacodynamics of therapeutic medications to provide optimal disease management for both sexes.

Central Regulatory Role for SIN1 in Interferon γ (IFNγ) Signaling and Generation of Biological Responses.

The precise signaling mechanisms by which type II IFN receptors control expression of unique genes to induce biological responses remain to be established. We provide evidence that Sin1, a known element of the mammalian target of rapamycin complex 2 (mTORC2), is required for IFNγ-induced phosphorylation and activation of AKT and that such activation mediates downstream regulation of mTORC1 and its effectors. These events play important roles in the assembly of the eukaryotic translation initiation factor 4F (eIF4F) and mRNA translation of IFN-stimulated genes. Interestingly, IFNγ-induced tyrosine phosphorylation of STAT1 is reduced in cells with targeted disruption of Sin1, leading to decreased transcription of several IFNγ-inducible genes in an mTORC2-independent manner. Additionally, our studies establish that Sin1 is essential for generation of type II IFN-dependent antiviral effects and antiproliferative responses in normal and malignant hematopoiesis. Together, our findings establish an important role for Sin1 in both transcription and translation of IFN-stimulated genes and type II IFN-mediated biological responses, involving both mTORC2-dependent and -independent functions.

Chemokines in breast cancer: Regulating metabolism.

Accumulating evidence indicates that chemokine-chemokine receptor interactions invoke biological responses beyond their originally described function of orchestrating leukocyte trafficking. In this review we will extend the findings that chemokines participate actively in the neoplastic process, and consider the contribution of CCL5 activation of CCR5 on breast cancer cells to upregulation of anabolic metabolic events that would support the energy demands of cell replication and proliferation.

Interferon γ (IFNγ) Signaling via Mechanistic Target of Rapamycin Complex 2 (mTORC2) and Regulatory Effects in the Generation of Type II Interferon Biological Responses.

We provide evidence for a unique pathway engaged by the type II IFN receptor, involving mTORC2/AKT-mediated downstream regulation of mTORC1 and effectors. These events are required for formation of the eukaryotic translation initiation factor 4F complex (eIF4F) and initiation of mRNA translation of type II interferon-stimulated genes. Our studies establish that Rictor is essential for the generation of type II IFN-dependent antiviral and antiproliferative responses and that it controls the generation of type II IFN-suppressive effects on normal and malignant hematopoiesis. Together, our findings establish a central role for mTORC2 in IFNγ signaling and type II IFN responses.

Interferon-ß regulates dendritic cell activation and migration in experimental autoimmune encephalomyelitis.

CD11c+ dendritic cells (DCs) exert a critical role as antigen-presenting cells in regulating pathogenic T cells in multiple sclerosis (MS). To determine whether the therapeutic benefit of interferon-ß (IFN-ß) treatment for MS is in part influenced by IFN regulation of DC function, we examined the immunophenotype of DCs derived from IFN-ß+/+ and IFN-ß-/- mice using a myelin oligodendrocyte glycoprotein (MOG) peptide-induced mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Our earlier work identified that IFN-ß-/- mice exhibit earlier onset and more rapid progression of neurological impairment compared with IFN-ß+/+ mice. In this study we show that lipopolysaccharide-/MOG peptide-stimulated IFN-ß-/- DCs secrete cytokines associated with pathological T helper type 17 rather than regulatory T-cell polarization and exhibit increased CD80 and MHCII expression when compared with stimulated IFN-ß+/+ DCs. IFN-ß-/- DCs from mice immunized to develop EAE induce greater proliferation of MOG-transgenic CD4+ T cells and promote interleukin-17 production by these T cells. Adoptive transfer of MOG peptide-primed IFN-ß-/- DCs into IFN-ß+/+ and IFN-ß-/- mice immunized to develop EAE resulted in their rapid migration into the central nervous system of recipient mice, before onset of disease, which we attribute to failed signal transducer and activator of transcription 1-mediated inhibition of CCR7. Taken together, our data support immunoregulatory roles for IFN-ß in the activation and migration of DCs during EAE.

CCL5-CCR5 interactions modulate metabolic events during tumor onset to promote tumorigenesis.

BACKGROUND: In earlier studies we have shown that CCL5 activation of CCR5 induces the proliferation and survival of breast cancer cells in a mechanistic target of rapamycin (mTOR)-dependent manner and that this is in part due to CCR5-mediated increases in glycolytic metabolism. METHODS: Using the MDA-MB-231 triple negative human breast cancer cell line and mouse mammary tumor virus - polyomavirus middle T-antigen (MMTV-PyMT) mouse primary breast cancer cells, we conducted in vivo tumor transplant experiments to examine the effects of CCL5-CCR5 interactions in the context of regulating tumor metabolism. Additionally, we employed Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry imaging (MALDI-FTICR-MSI) to evaluate tumor utilization of cellular metabolites. RESULTS: We provide evidence that, in the absence of CCR5, the early events associated with rapid tumor growth in the MMTV-PyMT mouse model of spontaneous breast cancer development, are diminished, as demonstrated by a delay in tumor onset. In tumor transplant studies into immunocompromised mice we identify a direct correlation between reduced tumor proliferation and decreased metabolic activity, specifically associated with tumor expression of CCR5. The reduction in tumorigenesis is accompanied by decreases in glucose uptake, glucose transporter-1 (GLUT-1) cell surface expression, intracellular ATP and lactate levels, as well as reduced CCL5 production. Using MALDI-FTICR-MS, we show that the rapid early tumor growth of CCR5+/+ triple negative breast cancer cells in vivo is attributable to increased levels of glycolytic intermediates required for anabolic processes, in contrast to the slower growth rate of their corresponding CCR5-/- cells, that exhibit reduced glycolytic metabolism. CONCLUSIONS: These findings suggest that CCL5-CCR5 interactions in the tumor microenvironment modulate metabolic events during tumor onset to promote tumorigenesis.

Regulatory effects of SKAR in interferon α signaling and its role in the generation of type I IFN responses.

We provide evidence that S6 kinase 1 (S6K1) Aly/REF-like target (SKAR) is engaged in IFN-α signaling and plays a key role in the generation of IFN responses. Our data demonstrate that IFN-α induces phosphorylation of SKAR, which is mediated by either the p90 ribosomal protein S6 kinase (RSK) or p70 S6 kinase (S6K1), in a cell type-specific manner. This type I IFN-inducible phosphorylation of SKAR results in enhanced interaction with the eukaryotic initiation factor (eIF)4G and recruitment of activated RSK1 to 5' cap mRNA. Our studies also establish that SKAR is present in cap-binding CBP80 immune complexes and that this interaction is mediated by eIF4G. We demonstrate that inducible protein expression of key IFN-α-regulated protein products such as ISG15 and p21(WAF1/CIP1) requires SKAR activity. Importantly, our studies define a requirement for SKAR in the generation of IFN-α-dependent inhibitory effects on malignant hematopoietic progenitors from patients with chronic myeloid leukemia or myeloproliferative neoplasms. Taken altogether, these findings establish critical and essential roles for SKAR in the regulation of mRNA translation of IFN-sensitive genes and induction of IFN-α biological responses.

Fibrocyte and T cell interactions promote disease pathogenesis in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease. We previously identified a circulating cell population, fibrocytes, which is activated early in disease. As RA is characterized by the formation of autoantibodies and autoreactive T cells, which often precede symptom onset, the objective of these studies was to characterize fibrocyte activation in the context of T cell activation. Multidimensional flow cytometry was used to characterize the activation status of peripheral blood (PB) fibrocytes and T cells derived from RA patients with different levels of disease activity. Compared to healthy controls, fibrocytes from RA patients exhibited increased activation, denoted as elevated levels of phosphorylation of STAT3 and NF-κB. RA patients had higher numbers of circulating activated Th17 cells and Tregs compared with healthy controls, Th17 cell numbers being higher in patients with moderate to high disease activity. Additionally, increased numbers of FOXP3+ RORγt+ double positive CD4+ T cells were observed in RA patients with more severe disease. Our data confirm that circulating fibrocytes are expanded in RA and that there is a direct correlation between the increase in number of activated fibrocytes and increased number of CD4+ T cells. Moreover, our data suggest that interactions between circulating fibrocytes and activated T cells may promote disease activity. Specifically, we provide in vitro evidence that mouse-derived CD4+ T cells produce GM-CSF which induces fibrocyte proliferation. In turn, activated fibrocytes produce IL-6, promoting Th17 polarization.

Critical roles for Rictor/Sin1 complexes in interferon-dependent gene transcription and generation of antiproliferative responses.

We provide evidence that type I IFN-induced STAT activation is diminished in cells with targeted disruption of the Rictor gene, whose protein product is a key element of mTOR complex 2. Our studies show that transient or stable knockdown of Rictor or Sin1 results in defects in activation of elements of the STAT pathway and reduced STAT-DNA binding complexes. This leads to decreased expression of several IFN-inducible genes that mediate important biological functions. Our studies also demonstrate that Rictor and Sin1 play essential roles in the generation of the suppressive effects of IFNα on malignant erythroid precursors from patients with myeloproliferative neoplasms. Altogether, these findings provide evidence for critical functions for Rictor/Sin1 complexes in type I IFN signaling and the generation of type I IFN antineoplastic responses.

The yin and yang of viruses and interferons.

Interferons (IFNs)-α/ß are critical effectors of the innate immune response to virus infections. Through activation of the IFN-α/ß receptor (IFNAR), they induce expression of IFN-stimulated genes (ISGs) that encode antiviral proteins capable of suppressing viral replication and promoting viral clearance. Many highly pathogenic viruses have evolved mechanisms to evade an IFN response and the balance between the robustness of the host immune response and viral antagonistic mechanisms determines whether or not the virus is cleared. Here, we discuss IFNs as broad-spectrum antivirals for treatment of acute virus infections. In particular, they are useful for treatment of re-emerging virus infections, where direct-acting antivirals (DAAs) have limited utility due to DAA-resistant mutations, and for newly emerging virus strains in which the time to vaccine availability precludes vaccination at the onset of an outbreak.

Regulatory effects of mTORC2 complexes in type I IFN signaling and in the generation of IFN responses.

IFNs transduce signals by binding to cell surface receptors and activating cellular pathways and regulatory networks that control transcription of IFN-stimulated genes (ISGs) and mRNA translation, leading to generation of protein products that mediate biological responses. Previous studies have shown that type I IFN receptor-engaged pathways downstream of AKT and mammalian target of rapamycin complex (mTORC) 1 play important roles in mRNA translation of ISGs and the generation of IFN responses, but the roles of mTORC2 complexes in IFN signaling are unknown. We provide evidence that mTORC2 complexes control IFN-induced phosphorylation of AKT on serine 473 and their function is ultimately required for IFN-dependent gene transcription via interferon-stimulated response elements. We also demonstrate that such complexes exhibit regulatory effects on other IFN-dependent mammalian target of rapamycin-mediated signaling events, likely via engagement of the AKT/mTORC1 axis, including IFN-induced phosphorylation of S6 kinase and its effector rpS6, as well as phosphorylation of the translational repressor 4E-binding protein 1. We also show that induction of ISG protein expression and the generation of antiviral responses are defective in Rictor and mLST8-KO cells. Together, our data provide evidence for unique functions of mTORC2 complexes in the induction of type I IFN responses and suggest a critical role for mTORC2-mediated signals in IFN signaling.

Essential role for the Mnk pathway in the inhibitory effects of type I interferons on myeloproliferative neoplasm (MPN) precursors.

The mechanisms of generation of the antineoplastic effects of interferons (IFNs) in malignant hematopoietic cells remain to be precisely defined. We examined the activation of type I IFN-dependent signaling pathways in malignant cells transformed by Jak2V617F, a critical pathogenic mutation in myeloproliferative neoplasms (MPNs). Our studies demonstrate that during engagement of the type I IFN receptor (IFNAR), there is activation of Jak-Stat pathways and also engagement of Mnk kinases. Activation of Mnk kinases is regulated by the Mek/Erk pathway and is required for the generation of IFN-induced growth inhibitory responses, but Mnk kinase activation does not modulate IFN-regulated Jak-Stat signals. We demonstrate that for type I IFNs to exert suppressive effects in malignant hematopoietic progenitors from patients with polycythemia vera, induction of Mnk kinase activity is required, as evidenced by studies involving pharmacological inhibition of Mnk or siRNA-mediated Mnk knockdown. Altogether, these findings provide evidence for key and essential roles of the Mnk kinase pathway in the generation of the antineoplastic effects of type I IFNs in Jak2V617F-dependent MPNs.

Small molecule mimetics of an interferon-α receptor interacting domain.

Small molecules that mimic IFN-α epitopes that interact with the cell surface receptor, IFNAR, would be useful therapeutics. One such 8-amino acid region in IFN-α2, designated IRRP-1, was used to derive 11 chemical compounds that belong to 5 distinct chemotypes, containing the molecular features represented by the key residues Leu30, Arg33, and Asp35 in IRRP-1. Three of these compounds exhibited potential mimicry to IRRP-1 and, in cell based assays, as predicted, effectively inhibited IFNAR activation by IFN-α. Of these, compound 3 did not display cell toxicity and reduced IFN-α-inducible STAT1 phosphorylation and STAT-DNA binding. Based on physicochemical properties' analyses, our data suggest that moieties with acidic pKa on the small molecule may be a necessary element for mimicking the carboxyl group of Asp35 in IRRP-1. Our data confirm the relevance of this strategy of molecular mimicry of ligand-receptor interaction domains of protein partners for small molecule drug discovery.