Martin J. Richer

Toll-Like Receptor 3 Signaling on Macrophages Is Required for Survival Following Coxsackievirus B4 Infection

Toll-like receptor 3 (TLR3) has been proposed to play a central role in the early recognition of viruses by sensing double stranded RNA, a common intermediate of viral replication. However, several reports have demonstrated that TLR3 signaling is either dispensable or even harmful following infection with certain viruses. Here, we asked whether TLR3 plays a role in the response to coxsackievirus B4 (CB4), a prevalent human pathogen that has been associated with pancreatitis, myocarditis and diabetes. We demonstrate that TLR3 signaling on macrophages is critical to establish protective immunity to CB4. TLR3 deficient mice produced reduced pro-inflammatory mediators and are unable to control viral replication at the early stages of infection resulting in severe cardiac damage. Intriguingly, the absence of TLR3 did not affect the activation of several key innate and adaptive cellular effectors. This suggests that in the absence of TLR3 signaling on macrophages, viral replication outpaces the developing adaptive immune response. We further demonstrate that the MyD88-dependent signaling pathways are not only unable to compensate for the loss of TLR3, they are also dispensable in the response to this RNA virus. Our results demonstrate that TLR3 is not simply part of a redundant system of viral recognition, but rather TLR3 plays an essential role in recognizing the molecular signatures associated with specific viruses including CB4.

Coxsackievirus infection as an environmental factor in the etiology of type 1 diabetes

Susceptibility to type 1 diabetes (T1D) is dictated by a complex interplay between genetic determinants and environmental influences. Accumulating evidence strongly supports viral infection as an important factor in the etiology of T1D. To this effect, several viruses have been associated with the capacity to induce or exacerbate T1D in both humans and mice. The most convincing evidence linking viral infection and autoimmunity comes from studies on enteroviruses, particularly coxsackievirus. In this review we will discuss the evidence associating coxsackievirus infection to T1D and present the current state of knowledge on the potential mechanism of coxsackievirus-mediated T1D.

Regulatory T-Cells Protect From Type 1 Diabetes After Induction by Coxsackievirus Infection in the Context of Transforming Growth Factor-β

OBJECTIVE—Coxsackievirus infections have long been associated with the induction of type 1 diabetes. Infection with coxsackievirus B4 (CB4) enhances type 1 diabetes onset in NOD mice by accelerating the presentation of β-cell antigen to autoreactive T-cells. It has been reported that a progressive defect in regulatory T-cell (Treg) function is, in part, responsible for type 1 diabetes onset in NOD mice. This defect may contribute to susceptibility to viral-induced type 1 diabetes. We asked whether the immune response after CB4 infection could be manipulated to reestablish peripheral tolerance while maintaining the immune response to virus. RESEARCH DESIGN AND METHODS—NOD mice expressing transforming growth factor-β (TGF-β) specifically in the β-cells were infected with CB4, and the functional role of Tregs in disease protection was measured. Systemic treatments with TGF-β were used to assess its therapeutic potential. RESULTS—Here, we report that Tregs induced after CB4 infection in the presence of TGF-β prevented type 1 diabetes. The capacity to directly infect pancreatic β-cells correlated with increased numbers of pancreatic Tregs, suggesting that presentation of β-cell antigen is integral to induction of diabetogenic protective Tregs. Furthermore, the presence of these viral induced Tregs correlated with protection from type 1 diabetes without altering the antiviral response. Finally, when TGF-β was administered systemically to NOD mice after infection, the incidence of type 1 diabetes was reduced, thereby signifying a potential therapeutic role for TGF-β. CONCLUSIONS—We demonstrate manipulations of the immune response that result in Treg-mediated protection from type 1 diabetes without concomitant loss of the capacity to control viral infection.

Selective pharmacological inhibition of phosphoinositide 3-kinase p110delta opposes the progression of autoimmune diabetes in non-obese diabetic (NOD) mice

During the progression of autoimmune (type 1) diabetes, T cells and macrophages infiltrate the pancreas, disrupt islet function, and destroy insulin-producing beta cells. B-lymphocytes, particularly innate like B-cell populations such as marginal zone B cells and B-1 cells, have been implicated in many autoimmune diseases, and non-obese diabetic (NOD) mice that lack B cells do not develop spontaneous autoimmune diabetes. Hence, inhibitors of B cell signaling pathways could be useful for limiting the autoimmune processes that contribute to type 1 diabetes. Signaling via phosphoinositide 3-kinase (PI3K) regulates many cellular processes. The p110δ isoform of PI3K is expressed primarily in cells of hematopoietic origin and the catalytic activity of p110δ is important for B cell migration, activation, proliferation, and antigen presentation. Because innate-like B cells are particularly sensitive to inhibition of p110δ activity, and p110δ inhibitors also suppress pro-inflammatory functions of other cell types that contribute to autoimmunity, we tested whether a p110δ inhibitor could delay the onset or reduce the incidence of autoimmune diabetes in NOD mice. We found that long-term preventative treatment of pre-diabetic NOD mice with IC87114, a highly selective small molecule inhibitor of p110δ, reduced the infiltration of inflammatory cells into the pancreatic islets and, accordingly, delayed and reduced the loss of glucose homeostasis. Moreover in a therapeutic treatment mode, IC87114 treatment conferred prolonged protection from progression to overt diabetes in a number of animals. These findings suggest that PI3Kδ inhibitors could be useful for managing type 1 diabetes.

Development of a red-shifted fluorescence-based assay for SARS-coronavirus 3CL protease: identification of a novel class of anti-SARS agents from the tropical marine sponge Axinella corrugata

Abstract SARS-coronavirus (SARS-CoV) encodes a main protease, 3CLpro, which plays an essential role in the viral life cycle and is currently the prime target for discovering new anti-coronavirus agents. In this article, we report our success in developing a novel red-shifted (RS) fluorescence-based assay for 3CLpro and its application for identifying small-molecule anti-SARS agents from marine organisms. We have synthesised and characterised the first generation of a red-shifted internally quenched fluorogenic substrate (RS-IQFS) for 3CLpro based on resonance energy transfer between the donor and acceptor pair CAL Fluor Red 610 and Black Hole Quencher-1 (K m and k cat values of 14 μM and 0.65 min-1). The RS-IQFS primary sequence was selected based on the results of our screening analysis of 3CLpro performed using a series of blue-shifted (BS)-IQFSs corresponding to the 3CLpro-mediated cleavage junctions of the SARS-CoV polyproteins. In contrast to BS-IQFSs, the RS-IQFS was not susceptible to fluorescence interference from coloured samples and allowed for successful screening of marine natural products and identification of a coumarin derivative, esculetin-4-carboxylic acid ethyl ester, a novel 3CLpro inhibitor (IC50=46 μM) and anti-SARS agent (EC50=112 μM; median toxic concentration >800 μM) from the tropical marine sponge Axinella corrugata.

The Innate Immune Response: An Important Partner in Shaping Coxsackievirus-Mediated Autoimmunity

To protect against viral infection, the immune response is critically dependent on innate sensing mechanisms to provide rapid detection of pathogens and allow for the development of an appropriate adaptive immune response. Mounting evidence suggests that mechanistic differences in the sensing of viruses by the innate immune response can contribute to the development of autoimmunity. Coxsackieviruses are common human pathogens that have been linked to the induction of autoimmune diseases such as chronic autoimmune myocarditis and type 1 diabetes. In this review, we will discuss the current knowledge of the interactions between coxsackievirus and the innate immune system and how these interactions can potentially lead to the induction of autoimmune diseases.

Immunomodulation of antigen presenting cells promotes natural regulatory T cells that prevent autoimmune diabetes in NOD mice.

Progression towards type 1 diabetes (T1D) in susceptible patients is linked to a progressive decline in the capacity of regulatory T cells (Treg) to maintain tolerance. As such, therapies aimed at redressing the failing Treg compartment have been the subject of intense investigation. Treg dysfunction in T1D has recently been linked to a reduced capacity of antigen presenting cells (APCs) to maintain Treg function rather than Treg intrinsic defects. This suggests that therapies aimed simply at addressing the failing Treg compartment are unlikely to provide long-term protection. Here, we demonstrate that modulation of the inflammatory status of CD11b+CD11c− APCs favors the upregulation of protective Tregs in a mouse model of T1D. We further demonstrate that reduced expression of the costimulatory molecule CD40 plays a role in this increased immunoregulatory capacity. Strikingly, Treg upregulation resulted exclusively from an increase in natural Tregs rather than the peripheral conversion of conventional T cells. This suggests that modulation of CD11b+ CD11c− APCs inflammatory properties favors the establishment of natural Treg responses that, unlike adaptive Treg responses, are likely to maintain tolerance to a broad range of antigens. As such, modulation of this APC subset represents a potential therapeutic avenue to reestablish peripheral tolerance and protect from autoimmune diseases such as T1D.

Early inflammatory responses direct chronic autoimmunity development in the heart following coxsackievirus infection

Coxsackievirus infections are a major cause of chronic autoimmune myocarditis, a known precursor to dilated cardiomyopathy. Dilated cardiomyopathy leads to heart failure and is responsible for nearly half of all heart transplant cases. The induction of chronic autoimmunity following coxsackievirus infection is governed by the interplay of several genetic and immunological factors. In this review, we will focus on how the innate immune response to viral infection directs a cascade of events that ultimately results in chronic autoimmune heart disease.

Preventing viral-induced type 1 diabetes: are regulatory T cells the answer?

The development of type 1 diabetes is, in part, dictated by the failure of regulatory T cells to maintain peripheral tolerance. As such, therapies aimed at correcting the declining function of regulatory T cells have been the subject of intense investigation. In this review, we discuss the potential of regulatory T cells to suppress autoreactive responses and prevent type 1 diabetes induced by viral infection while maintaining protective antiviral immunity.

Probing the Substrate Specificity of Hepatitis C Virus Nonstructural 3 Protein Serine Protease by Intramolecularly Quenched Fluorogenic Peptide Substrates

Recent information about the incidence of Hepatitis C (HCV) around the world clearly indicates that the virus is a major public health issue: the estimated worldwide prevalence is approximately 1% [1]. The need to rapidly identify new therapeutic approaches has resulted in intensive study of the molecular properties of this virus, yet thus far no efficient therapy or vaccine exists. Currently, one of the most promising approaches to anti-HCV therapy is the development of inhibitors of the virally encoded serine protease, nonstructural 3 (NS3) [1]. Elucidating the substrate specificity of the HCV NS3 protease is important for the development of high-throughput assays (HTA) for random screening of HCV protease inhibitors and for rational design of HCV protease-specific inhibitors [2]. As a first step towards the realization of these aims, we report here our progress in probing the substrate specificity of two HCV NS3 variants, ANS3 [3] and full-length (FL) NS3 [4], with intramolecularly-quenched fluorogenic peptide substrates (IQFS) [5], based on fluorescence resonance energy transfer (FRET) between the donor/acceptor couple (Abz, EDDnp; Figure 1A and [6]). Cleavage of a FRET substrate by an enzyme separates the fluorophore from the quenching group, which results in the generation of a fluorescence signal (Figure 1 and [5,6]).