Use of type I interferon-inducible mRNAs as pharmacodynamic markers and potential diagnostic markers in trials with sifalimumab,an anti-IFNα antibody,in systemic lupus erythematosus

Type I interferons are implicated in the pathogenesis of systemic lupus erythematosus (SLE). Type I interferon-inducible mRNAs are widely and concordantly overexpressed in the periphery and involved tissues of a subset of SLE patients, and provide utility as pharmacodynamic biomarkers to aid dose selection, as well as potential indicators of patients who might respond favorably to anti-IFNα therapy in SLE. We implemented a three-tiered approach to identify a panel of type I interferon-inducible mRNAs to be used as potential pharmacodynamic biomarkers to aid dose selection in clinical trials of sifalimumab, an anti-IFNα monoclonal antibody under development for the treatment of SLE. In a single-dose escalation phase 1 trial, we observed a sifalimumab-specific and dose-dependent inhibition of the overexpression of type I interferon-inducible mRNAs in the blood of treated subjects. Inhibition of expression of type I interferon-inducible mRNAs and proteins was also observed in skin lesions of SLE subjects from the same trial. Inhibiting IFNα resulted in a profound downstream effect in these SLE subjects that included suppression of mRNAs of B-cell activating factor belonging to the TNF family and the signaling pathways of TNFα, IL-10, IL-1β, and granulocyte-macrophage colony-stimulating factor in both the periphery and skin lesions. A scoring method based on the expression of type I interferon-inducible mRNAs partitioned SLE patients into two distinct subpopulations, which suggests the possibility of using these type I interferon-inducible genes as predictive biomarkers to identify SLE patients who might respond more favorably to anti-type I interferon therapy.     

Human primary immunodeficiencies of type I interferons

Type I interferons (IFN-alpha/beta and related molecules) are essential for protective immunity to experimental infection by numerous viruses in the mouse model. In recent years, human primary immunodeficiencies affecting either the production of (UNC-93B deficiency) or the response to (STAT1 and TYK2 deficiencies) these IFNs have been reported. Affected patients are highly susceptible to certain viruses. Patients with STAT1 or TYK2 deficiency are susceptible to multiple viruses, including herpes simplex virus-1 (HSV-1), whereas UNC-93B-deficient patients present isolated HSV-1 encephalitis. However, these immunological defects are not limited to type I IFN-mediated immunity. Impaired type II IFN (IFN-gamma)-mediated immunity plays no more than a minor role in the pathogenesis of viral diseases in these patients, but the contribution of impaired type III IFN (IFN-lambda)-mediated immunity remains to be determined. These novel inherited disorders strongly suggest that type I IFN-mediated immunity is essential for protection against natural infections caused by several viruses in humans.     

Human immunodeficiency virus viremia induces plasmacytoid dendritic cell activation in vivo and diminished alpha interferon production in vitro

Human immunodeficiency virus type 1 (HIV-1) infection has been associated with perturbations of plasmacytoid dendritic cells (PDC), including diminished frequencies in the peripheral blood and reduced production of type I interferons (IFNs) in response to in vitro stimulation. However, recent data suggest a paradoxical increase in production of type 1 interferons in vivo in HIV-infected patients compared to uninfected controls. Using a flow cytometric assay to detect IFN-alpha-producing cells within unseparated peripheral blood mononuclear cells, we observed that short-term interruptions of antiretroviral therapy are sufficient to result in significantly reduced IFN-alpha production by PDC in vitro in response to CpG A ligands or inactivated HIV particles. The primary cause of diminished IFN-alpha production was reduced responsiveness of PDC to de novo stimulation, not diminished per cell IFN-alpha production or migration of cells to lymphoid organs. Real-time PCR analysis of purified PDC from patients prior to and during treatment interruptions revealed that active HIV-1 replication is associated with upregulation of type I IFN-stimulated gene expression. Treatment of hepatitis C virus-infected patients with IFN-alpha2b and ribavirin for hepatitis C virus infection resulted in a profound suppression of de novo IFN-alpha production in response to CpG A or inactivated HIV particles, similar to the response observed in HIV-infected patients. Together, these results suggest that diminished production of type I interferons in vitro by PDC from HIV-1-infected patients may not represent diminished interferon production in vivo. Rather, diminished function in vitro is likely a consequence of prior activation via type I interferons or HIV virions in vivo.     

Interferon Induction by Viruses

Interferons are proteins of cellular origin capable of conferring virus resistance to vertebrate cells. Most cells do not produce interferons except in response to proper stimulation. Clearly, the stimulation of interferon production encompasses two phenomena. When stimulated, some cell systems produce their interferons by synthesizing new proteins. Other cell systems do not require the synthesis of new proteins to produce interferons, and still other cell systems may produce interferons by both means. Before much can be learned from the detailed study of the nature of the molecules which stimulate interferons, the type of phenomenon which the stimulus induces must be identified. Chick embryo tissues apparently make interferons by synthesizing new proteins. Many viruses stimulate interferon production in chick embryo tissues. Data available suggest that neither the protein nor nucleic acid moieties of the added virions act as inducing molecules. Also, double-stranded replicative form is probably not responsible. It is suggested that the inducer molecule may be cellular in nature and may be produced in response to a wide variety of insults among which are viral infections.     

Modulation of the interferon antiviral response by the TBK1/IKKi adaptor protein TANK

Induction of type I interferons can be triggered by viral components through Toll-like receptors or intracellular viral receptors such as retinoic acid-inducible gene I. Here, we demonstrate that the TRAF (tumor necrosis factor receptor-associated factor) family member-associated NF-kappaB activator (TANK) plays an important role in interferon induction through both retinoic acid-inducible gene I- and Toll-like receptor-dependent pathways. TANK forms complexes with both upstream signal mediators, such as Cardif/MAVS/IPS-1/VISA, TRIF (Toll-interleukin-1 receptor domain-containing adaptor inducing interferon-beta), and TRAF3 and downstream mediators TANK-binding kinase 1, inducible IkappaB kinase, and interferon regulatory factor 3. In addition, it synergizes with these signaling components in interferon induction. Specific knockdown of TANK results in reduced type I interferon production, increased viral titers, and enhanced cell sensitivity to viral infection. Thus, TANK may be a critical adaptor that regulates the assembly of the TANK-binding kinase 1-inducible IkappaB kinase complex with upstream signaling molecules in multiple antiviral pathways.     

The role of Toll-like receptor signalling in the pathogenesis of arthritis

Recent evidence highlighted the role of Toll-like receptors (TLRs) as key recognition structures of the innate immune system. The activation of TLRs initiates the production of inflammatory cytokines, chemokines, tissue destructive enzymes, and type I interferons. In addition, TLR signalling plays an important role in the activation and direction of the adaptive immune system by the upregulation of costimulatory molecules of antigen presenting cells. Considering the important role of TLR signalling as a critical link between innate and adaptive immunity it has been proposed that a dysregulation in TLR signalling might be associated with autoimmunity. In this review, recent studies on TLR signal transduction pathways activated by corresponding ligands are summarized and evidence for a possible role of TLR signalling in the pathogenesis of rheumatoid arthritis is discussed.     

The influence of type I interferons on immune cells can be mediated through regulation of estrogen receptor alpha level

Autoimmune disorders are connected with the actions of sex hormones. Clinical observations have shown that especially estrogens are involved in these phenomena. In some cases the administration of estrogens can increase the pathological symptoms of a disorder, while in others they can cause disease remission. In multiple autoimmune diseases, type I interferons, a family of cytokines acting through the common receptor IFNAR1/IFNAR2, seem to have action convergent with that of estrogens. We hypothesize that this coincidence is not accidental and type I interferons can regulate the level of estrogen receptor alpha (ERα) and consequently change the sensitivity of immune cells to estrogen's action. There is evidence that ERα is responsible for the effects exerted by estrogens and that this phenomenon mainly involves antigen-presenting cells. On the other hand, research on IFN-tau, a type I interferon family members, showed that this cytokine can modulate ERα levels in ovine endometrium. Because of the common receptor for these interferons, we suspect that other type I interferons can act in this way not only in endometrial cells, but also in immune cells. If there is such a mechanism, it can be exploited in the therapy of immune disorders, especially autoimmune disease, for example through simultaneous administration of less toxic interferons and estrogens.     

Interferons as cell-regulatory molecules

Summary This review presents the current evidence for interferons as cell-regulatory molecules. Apart from inducing an antiviral state, interferon preparations are powerful inhibitors of cell growth and have selective effects on cellular protein synthesis. In addition, interferons are produced during most immune reactions and can exert positive and negative influences on these reactions. Thus interferon molecules are of interest to cell biologists, immunologists, and oncologists. Interferon as a cell regulator offers a unique approach to cancer therapy, but for its judicious use, more understanding of basic mechanisms of action is required.     

MicroRNAs in Lymphoma: Regulatory Role and Biomarker Potential

Although it is now evident that microRNAs (miRNAs) play a critical regulatory role in many, if not all, pathological and physiological processes, remarkably they have only formally been recognized for less than fifteen years. These endogenously produced short non-coding RNAs have created a new paradigm of gene control and have utility as both novel biomarkers of cancer and as potential therapeutics. In this review we consider the role of miRNAs in lymphoid biology both under physiological (i.e. lymphopoiesis) and malignant (i.e. lymphomagenesis) conditions. In addition to the functional significance of aberrant miRNA expression in lymphomas we discuss their use as novel biomarkers, both as a in situ tumour biomarker and as a non-invasive surrogate for the tumour by testing miRNAs in the blood of patients. Finally we consider the use of these molecules as potential therapeutic agents for lymphoma (and other cancer) patients and discuss some of the hurdles yet to be overcome in order to translate this potential into clinical practice     

Interferons, immunity and cancer immunoediting

A clear picture of the dynamic relationship between the host immune system and cancer is emerging as the cells and molecules that participate in naturally occurring antitumour immune responses are being identified. The interferons (IFNs) - that is, the type I IFNs (IFNalpha and IFNbeta) and type II IFN (IFNgamma) - have emerged as central coordinators of tumour-immune-system interactions. Indeed, the decade-old finding that IFNgamma has a pivotal role in promoting antitumour responses became the focus for a renewed interest in the largely abandoned concept of cancer immunosurveillance. More recently, type I IFNs have been found to have distinct functions in this process. In this Review, we discuss the roles of the IFNs, not only in cancer immunosurveillance but also in the broader process of cancer immunoediting.     

Orally administered interferons exert their white blood cell suppressive effects via a novel mechanism.

Interferons (IFN) have been approved for a number of clinical uses. The accepted routes of administration are intramuscular, subcutaneous, and intravenous. Recently, interferons administered by the oral route have been shown to exert a systemic effect. Oral administrations of IFN-alpha, IFN-beta, and IFN-gamma have been shown to cause a suppression of the peripheral white blood cell (WBC) count in mice. This study investigates the mechanism by which this suppression occurs. The results show that, in contrast to their intraperitoneal administration, oral administration of rHuIFN-alpha A/D or rMuIFN-gamma does not result in the presence of detectable levels of interferons in the blood. In addition, although the presence of circulating specific antibody to interferon blocks the peripheral WBC suppressive effects of intraperitoneally administered MuIFN-beta or rMuIFN-gamma, the presence of those antibodies does not block the peripheral WBC suppressive effects of the orally administered interferons. The peripheral WBC suppressive effect of orally administered rHuIFN-alpha A/D and rMuIFN-gamma can be transferred by injection of blood from oral interferon-treated donor mice to recipient mice. Recipient mice receiving plasma from donor mice showed no peripheral WBC suppression. Recipient mice receiving blood cells from donor mice showed significant peripheral WBC suppression. No effect of orally administered rHuIFN-alpha A/D on the relative percentages of lymphocytes, neutrophils, and monocytes was noted. These results indicate that the mechanism by which orally administered interferons exert their WBC suppressive effect differs from that of intraperitoneally administered interferons. WBC suppression resulting from orally administered interferons may involve cell to cell transfer of the interferons' effects, rather than the systemic distribution of the interferons in the blood. These studies further suggest that there may be a role for oral administration as a new route of interferon administration and provide a glimpse into the mechanism by which the orally administered interferons exert their systemic effects.     

Droplet digital PCR,a prospective technological approach to quantitative profiling of microRNA

MicroRNA is a special type of regulatory molecules modulating gene expression. Circulating microRNAs found in blood and other biological body fluids are now considered as potential biomarkers of human pathology. Quantitative changes of particular microRNAs have been recognized in many oncological diseases and other disorders. A recently developed method of droplet digital PCR (ddPCR) possesses a number of advantages making this method the most suitable for verification and validation of perspective microRNA markers of various human pathologies. These advantages include high accuracy and reproducibility of microRNA quantification as well as possibility of direct high-throughput determination of the absolute number of microRNA copies within a wide dynamic range. The present review considers microRNA biogenesis, the origin of circulating microRNAs, and methods used for their quantification. The special technical features of ddPCR, which make this method especially attractive for studying microRNAs as biomarkers of human pathologies and for basic research devoted to aspects of gene regulation by microRNA molecules, are also discussed.     

The role of proteomics in assessing beta-cell dysfunction and death in type 1 diabetes

Introduction: Type 1 diabetes (T1D) is characterized by autoimmune-induced dysfunction and destruction of the pancreatic beta cells. Unfortunately, this process is poorly understood, and the current best treatment for type 1 diabetes is the administration of exogenous insulin. To better understand these mechanisms and to develop new therapies, there is an urgent need for biomarkers that can reliably predict disease stage.

Areas covered: Mass spectrometry (MS)-based proteomics and complementary techniques play an important role in understanding the autoimmune response, inflammation and beta-cell death. MS is also a leading technology for the identification of biomarkers. This, and the technical difficulties and new technologies that provide opportunities to characterize small amounts of sample in great depth and to analyze large sample cohorts will be discussed in this review.

Expert opinion: Understanding disease mechanisms and the discovery of disease-associated biomarkers are highly interconnected goals. Ideal biomarkers would be molecules specific to the different stages of the disease process that are released from beta cells to the bloodstream. However, such molecules are likely to be present in trace amounts in the blood due to the small number of pancreatic beta cells in the human body and the heterogeneity of the target organ and disease process.     

Bovine plasmacytoid dendritic cells are the major source of type I interferon in response to foot-and-mouth disease virus in vitro and in vivo

Type I interferons (alpha/beta interferons [IFN-α/β]) are the main innate cytokines that are able to induce a cellular antiviral state, thereby limiting viral replication and disease pathology. Plasmacytoid dendritic cells (pDCs) play a crucial role in the control of viral infections, especially in response to viruses that have evolved mechanisms to block the type I IFN signal transduction pathway. Using density gradient separation and cell sorting, we have highly enriched a population of bovine cells capable of producing high levels of biologically active type I IFN. These cells represented less than 0.1% of the total lymphocyte population in blood, pseudoafferent lymph, and lymph nodes. Phenotypic analysis identified these cells as bovine pDCs (CD3(-) CD14(-) CD21(-) CD11c(-) NK(-) TCRδ(-) CD4(+) MHC II(+) CD45RB(+) CD172a(+) CD32(+)). High levels of type I IFN were generated by these cells in vitro in response to Toll-like receptor 9 (TLR-9) agonist CpG and foot-and-mouth disease virus (FMDV) immune complexes. In contrast, immune complexes formed with UV-inactivated FMDV or FMDV empty capsids failed to elicit a type I IFN response. Depletion of CD4 cells in vivo resulted in levels of type I IFN in serum early during FMDV infection that were significantly lower than those for control animals. In conclusion, pDCs interacting with immune-complexed virus are the major source of type I interferon production during acute FMDV infection in cattle.     

Interferon in pregnancy

The cells of the immune system exchange information by a complex network of molecules referred to as lymphokines: these include the interferons. The physiology of the interferons, both in terms of control and function, is poorly understood. However, there is ample evidence that production of alpha-interferon is characteristic of the fetoplacental unit in both the human and other species. Indeed, the major trophoblast protein in early pregnancy in the sheep is alpha-interferon and in this species the molecule appears to have an important anti-luteolytic effect. The function of the interferons in human pregnancy is not known but, by analogy with information from other experimental systems, it might reflect aspects of the immune relationship between the mother and the fetus.     

The non-canonical role of Atg family members as suppressors of innate antiviral immune signaling

Recent research on autophagy clearly demonstrates that the autophagosome-lysosome pathway plays essential roles in elimination of certain pathogens such as group A Streptococcus, Mycobacterium tuberculosis, Listeria monocytogenes, and herpesvirus. (1-4) We have recently found that a key regulator of the autophagic process, the Atg12-Atg5 conjugate, associates with the signaling molecules retinoic acid-inducible gene I (RIG-I) and interferon-beta promoter stimulator 1 (IPS-1), which are essential for recognition of RNA virus infection and which transmit signals to upregulate type I interferons (IFNs). Interestingly, the Atg12-Atg5 conjugate seemed to negatively regulate the type I IFN modulating pathway through direct interaction with caspase recruitment domains (CARDs) presented by RIG-1 and IPS-1.(5) Thus, in contrast to the bactericidal properties of autophagic processes, the autophagy regulator (the Atg12-Atg5 conjugate) appeared to promote RNA virus replication by inhibiting innate anti-virus immune responses. In this addendum, we discuss the non-canonical role of the Atg12-Atg5 conjugate as a suppressor of innate immune responses.     

Induction of type I interferons by bacteria

Type I interferons (IFNs) are secreted cytokines that orchestrate diverse immune responses to infection. Although typically considered to be most important in the response to viruses, type I IFNs are also induced by most, if not all, bacterial pathogens. Although diverse mechanisms have been described, bacterial induction of type I IFNs occurs upon stimulation of two main pathways: (i) Toll‐like receptor (TLR) recognition of bacterial molecules such as lipopolysaccharide (LPS); (ii) TLR‐independent recognition of molecules delivered to the host cell cytosol. Cytosolic responses can be activated by two general mechanisms. First, viable bacteria can secrete stimulatory ligands into the cytosol via specialized bacterial secretion systems. Second, ligands can be released from bacteria that lyse or are degraded. The bacterial ligands that induce the cytosolic pathways remain uncertain in many cases, but appear to include various nucleic acids. In this review, we discuss recent advances in our understanding of how bacteria induce type I interferons and the roles type I IFNs play in host immunity.     

Bacteria-derived human leukocyte interferons alter in vitro humoral and cellular immune responses

Cultures of gradient-purified human peripheral blood mononuclear cells (PBMC) have been employed to examine the effects of three bacteria-derived human leukocyte interferon subtypes on certain aspects of in vitro immune responses. The addition of highly purified IFN-alpha 1, -alpha 2, -alpha 2/alpha 1 to PMBC cultures stimulated with phytohemagglutinin (PHA) or pokeweed mitogen resulted in a significant suppression of the mitogenic response. This suppression required the presence of interferon in the cultures because pretreatment of cells and removal of interferon had no effect on their response to PHA. The presence of these interferons at 200 U/ml also caused a substantial reduction of human mixed-lymphocyte reactions (MLR) as measured by [3H]thymidine incorporation by responder cells. Interestingly, pretreatment of stimulator cells was sufficient for this reduction to occur whereas pretreatment of responder cells had no effect on their ability to respond to allogenic stimulation. In contrast to these suppressive effects, the three interferons enhanced human in vitro primary immune response to sheep red blood cells (SRBC). These data demonstrate that both purified interferon subtypes and genetic hybrids of human interferons produced by recombinant DNA technology have effects on in vitro immune responses.     

抗病毒感染固有免疫应答的信号转导

入侵病毒的探知和适应性免疫应答启动均依靠固有免疫系统。三种模式识别受体(PRRs)在宿主防御系统第一线占据极其重要地位:Toll样受体、维甲酸诱导基因I样受体、核苷酸结合寡聚化结构域样受体。PRRs识别病原相关分子模式(PAMP)或危险信号分子模式(DAMPs)启动和调节固有免疫和适应性免疫应答。每种PRR都有单独的识别配体和细胞定位。激活的PRRs将信号分子传递给其配体分子(MyD88,TRIF,IRAK,IPS-1),配体活化后作为信使激活信号途径下游激酶(IKK复合物,MAPKs,TBK1,RIP-1)和转录因子(NF-κB,AP-1,IRF3),最终产生细胞因子、趋化因子、促炎细胞因子和I型干扰素。本文重点讨论PRRs信号通路及该领域取得的成果,以期为人类健康和免疫疾病防治提供策略。