Meeting the Challenge of Controlling Viral Immunopathology.

The mission of this review is to identify immune-damaging participants involved in antiviral immunoinflammatory lesions. We argue these could be targeted and their activity changed selectively by maneuvers that, at the same time, may not diminish the impact of components that help resolve lesions. Ideally, we need to identify therapeutic approaches that can reverse ongoing lesions that lack unwanted side effects and are affordable to use. By understanding the delicate balance between immune responses that cause tissue damage and those that aid in resolution, novel strategies can be developed to target detrimental immune components while preserving the beneficial ones. Some strategies involve rebalancing the participation of immune components using various approaches, such as removing or blocking proinflammatory T cell products, expanding regulatory cells, restoring lost protective cell function, using monoclonal antibodies (moAb) to counteract inhibitory molecules, and exploiting metabolic differences between inflammatory and immuno-protective responses. These strategies can help reverse ongoing viral infections. We explain various approaches, from model studies and some clinical evidence, that achieve innate and adaptive immune rebalancing, offering insights into potential applications for controlling chronic viral-induced lesions.

Controlling Herpes Simplex Virus-Induced Immunoinflammatory Lesions Using Metabolic Therapy: a Comparison of 2-Deoxy-d-Glucose with Metformin.

Herpes simplex virus (HSV) infection of the eye can result in a blinding immunoinflammatory lesion in the cornea called herpetic stromal keratitis (HSK). This lesion is orchestrated by T cells and can be reduced in magnitude by anti-inflammatory drugs and procedures that change the balance of cellular participants in lesions. This report evaluates the effect of drugs that cause metabolic reprogramming on lesion expression using two drugs that affect glucose metabolism: 2-deoxy-d-glucose (2DG) and metformin. Both drugs could limit HSK severity, but 2DG therapy could result in herpes encephalitis if used when replicating virus was still present. The reason metformin was a safer therapy was its lack of marked inhibitory effects on inflammatory cells particularly interferon-γ (IFN-γ)-producing Th1 and CD8 T cells in the trigeminal ganglion (TG), in which HSV latency is established and sustained. Additionally, whereas 2DG in TG cultures with established latency accelerated the termination of latency, this did not occur in the presence of metformin, likely because the inflammatory cells remained functional. Our results support the value of metabolic reprogramming to control viral immunoinflammatory lesions, but the approach used should be chosen with caution. IMPORTANCE Herpes simplex virus (HSV) infection of the eye is an example where damaging lesions are in part the consequence of a host response to the infection. Moreover, it was shown that changing the representation of cellular participants in the inflammatory reaction can minimize lesion severity. This report explores the value of metabolic reprogramming using two drugs that affect glucose metabolism to achieve cellular rebalancing. It showed that two drugs, 2-deoxy-d-glucose (2DG) and metformin, effectively diminished ocular lesion expression, but only metformin avoided the complication of HSV spreading to the central nervous system (CNS) and causing herpetic encephalitis. The report provides some mechanistic explanations for the findings.

Inhibiting Glucose Metabolism Results in Herpes Simplex Encephalitis.

This report evaluates how HSV enters the brain to cause herpes simplex encephalitis following infection at a peripheral site. We demonstrate that encephalitis regularly occurred when BALB/c mice were infected with HSV and treated daily with 2-deoxy-d-glucose (2DG), which inhibits glucose use via the glycolysis pathway. The outcome of infection in the trigeminal ganglion (TG), the site to which the virus spreads, replicates, and establishes latency, showed marked differences in viral and cellular events between treated and untreated animals. In control-untreated mice, the replicating virus was present only during early time points, whereas in 2DG recipients, replicating virus remained for the 9-d observation period. This outcome correlated with significantly reduced numbers of innate inflammatory cells as well as T cells in 2DG-treated animals. Moreover, T cells in the TG of treated animals were less activated and contained a smaller fraction of expressed IFN-γ production compared with untreated controls. The breakdown of latency was accelerated when cultures of TG cells taken from mice with established HSV latency were cultured in the presence of 2DG. Taken together, the results of both in vivo and in vitro investigations demonstrate that the overall effects of 2DG therapy impaired the protective effects of one or more inflammatory cell types in the TG that normally function to control productive infection and prevent spread of virus to the brain.

Supplementing the Diet with Sodium Propionate Suppresses the Severity of Viral Immuno-inflammatory Lesions.

This report evaluates a dietary manipulation approach to suppress the severity of ocular infections caused by herpes simplex virus infection. The virus causes chronic damage to the cornea that results from a T-cell-orchestrated inflammatory reaction to the infection. Lesion severity can be limited if cells with regulatory activity predominate over proinflammatory T cells and nonlymphoid inflammatory cells. In this report, we show that this outcome can be achieved by including the short-chain fatty acid (SCFA) salt sodium propionate (SP) in the drinking water. Animals given the SP supplement developed significantly fewer ocular lesions than those receiving no supplement. Corneas and lymphoid organs contained fewer CD4 Th1 and Th17 T cells, neutrophils, and macrophages than those of controls, but a higher frequency of regulatory T cells (Treg) was present. The inclusion of SP in cultures to induce CD4 T cell subsets in vitro reduced the magnitude of Th1 and Th17 responses but expanded Treg induction. Dietary manipulation was an effective approach to limit the severity of viral immuno-inflammatory lesions and may be worth exploring as a means to reduce the impact of herpetic lesions in humans.IMPORTANCE Herpetic lesions are a significant problem, and they are difficult to control with therapeutics. Our studies show that the severity of herpetic lesions in a mouse model can be diminished by changing the diet to include increased levels of SCFA, which act to inhibit the involvement of inflammatory T cells. We suggest that changing the diet to include higher levels of SCFA might be a useful approach to reducing the impact of recurrent herpetic lesions in humans.

Host-Directed Antiviral Therapy.

Antiviral drugs have traditionally been developed by directly targeting essential viral components. However, this strategy often fails due to the rapid generation of drug-resistant viruses. Recent genome-wide approaches, such as those employing small interfering RNA (siRNA) or clustered regularly interspaced short palindromic repeats (CRISPR) or those using small molecule chemical inhibitors targeting the cellular "kinome," have been used successfully to identify cellular factors that can support virus replication. Since some of these cellular factors are critical for virus replication, but are dispensable for the host, they can serve as novel targets for antiviral drug development. In addition, potentiation of immune responses, regulation of cytokine storms, and modulation of epigenetic changes upon virus infections are also feasible approaches to control infections. Because it is less likely that viruses will mutate to replace missing cellular functions, the chance of generating drug-resistant mutants with host-targeted inhibitor approaches is minimized. However, drug resistance against some host-directed agents can, in fact, occur under certain circumstances, such as long-term selection pressure of a host-directed antiviral agent that can allow the virus the opportunity to adapt to use an alternate host factor or to alter its affinity toward the target that confers resistance. This review describes novel approaches for antiviral drug development with a focus on host-directed therapies and the potential mechanisms that may account for the acquisition of antiviral drug resistance against host-directed agents.

Modulating glutamine metabolism to control viral immuno-inflammatory lesions.

Infection of the cornea with HSV results in an immune-inflammatory reaction orchestrated by proinflammatory T cells that is a major cause of human vision impairment. The severity of lesions can be reduced if the representation of inflammatory T cells is changed to increase the presence of T cells with regulatory function. This report shows that inhibiting glutamine metabolism using 6-Diazo-5-oxo-l-norleucine (DON) administered via intraperitoneal (IP) starting 6 days after ocular infection and continued until day 15 significantly reduced the severity of herpetic stromal keratitis lesions. The therapy resulted in reduced neutrophils, macrophages as well proinflammatory CD4 Th1 and Th17 T cells in the cornea, but had no effect on levels of regulatory T cells. A similar change in the representation of inflammatory and regulatory T cells occurred in the trigeminal ganglion (TG) the site where HSV infection establishes latency. Glutamine metabolism was shown to be required for the in-vitro optimal induction of both Th1 and Th17 T cells but not for the induction of Treg that were increased when glutamine metabolism was inhibited. Inhibiting glutamine metabolism also changed the ability of latently infected TG cells from animals previously infected with HSV to reactivate and produce infectious virus.

Climate change: how it impacts the emergence, transmission, resistance and consequences of viral infections in animals and plants.

The ongoing COVID-19 pandemic has made us wonder what led to its occurrence and what can be done to avoid such events in the future. As we document, one changing circumstance that is resulting in the emergence and changing the expression of viral diseases in both plants and animals is climate change. Of note, the rapidly changing environment and weather conditions such as excessive flooding, droughts, and forest fires have raised concerns about the global ecosystem's security, sustainability, and balance. In this review, we discuss the main consequences of climate change and link these to how they impact the appearance of new viral pathogens, how they may facilitate transmission between usual and novel hosts, and how they may also affect the host's ability to manage the infection. We emphasize how changes in temperature and humidity and other events associated with climate change influence the reservoirs of viral infections, their transmission by insects and other intermediates, their survival outside the host as well the success of infection in plants and animals. We conclude that climate change has mainly detrimental consequences for the emergence, transmission, and outcome of viral infections and plead the case for halting and hopefully reversing this dangerous event.

Virological and Immunological Outcomes of Coinfections.

Coinfections involving viruses are being recognized to influence the disease pattern that occurs relative to that with single infection. Classically, we usually think of a clinical syndrome as the consequence of infection by a single virus that is isolated from clinical specimens. However, this biased laboratory approach omits detection of additional agents that could be contributing to the clinical outcome, including novel agents not usually considered pathogens. The presence of an additional agent may also interfere with the targeted isolation of a known virus. Viral interference, a phenomenon where one virus competitively suppresses replication of other coinfecting viruses, is the most common outcome of viral coinfections. In addition, coinfections can modulate virus virulence and cell death, thereby altering disease severity and epidemiology. Immunity to primary virus infection can also modulate immune responses to subsequent secondary infections. In this review, various virological mechanisms that determine viral persistence/exclusion during coinfections are discussed, and insights into the isolation/detection of multiple viruses are provided. We also discuss features of heterologous infections that impact the pattern of immune responsiveness that develops.

Gal power: the diverse roles of galectins in regulating viral infections.

Viruses, as a class of pathogenic microbe, remain a significant health burden globally. Viral infections result in significant morbidity and mortality annually and many remain in need of novel vaccine and anti-viral strategies. The development of effective novel anti-viral therapeutics, in particular, requires detailed understanding of the mechanism of viral infection, and the host response, including the innate and adaptive arms of the immune system. In recent years, the role of glycans and lectins in pathogen-host interactions has become an increasingly relevant issue. This review focuses on the interactions between a specific lectin family, galectins, and the broad range of viral infections in which they play a role. Discussed are the diverse activities that galectins play in interacting directly with virions or the cells they infect, to promote or inhibit viral infection. In addition we describe how galectin expression is regulated both transcriptionally and post-transcriptionally by viral infections. We also compare the contribution of known galectin-mediated immune modulation, across a range of innate and adaptive immune anti-viral responses, to the outcome of viral infections.

Manipulating Glucose Metabolism during Different Stages of Viral Pathogenesis Can Have either Detrimental or Beneficial Effects.

This report deals with physiological changes and their implication following ocular infection with HSV. This infection usually results in a blinding inflammatory reaction in the cornea, orchestrated mainly by proinflammatory CD4 T cells and constrained in severity by regulatory T cells. In the present report, we make the unexpected finding that blood glucose levels change significantly during the course of infection. Whereas levels remained normal during the early phase of infection when the virus was actively replicating in the cornea, they increased around 2-fold during the time when inflammatory responses to the virus was occurring. We could show that glucose levels influenced the extent of induction of the inflammatory T cell subset in vitro that mainly drives lesions, but not regulatory T cells. Additionally, if glucose utilization was limited in vivo as a consequence of therapy in the inflammatory phase with the drug 2-deoxy-glucose (2DG), lesions were diminished compared with untreated infected controls. In addition, lesions in 2DG-treated animals contained less proinflammatory effectors. Glucose metabolism also influenced the acute phase of infection when the replicating virus was present in the eye. Thus, therapy with 2DG to limit glucose utilization caused mice to become susceptible to the lethal effects of HSV infection, with the virus spreading to the brain causing encephalitis. Taken together, our results indicate that glucose metabolism changed during the course of HSV infection and that modulating glucose levels can influence the outcome of infection, being detrimental or beneficial according to the stage of viral pathogenesis.

The Plasticity and Stability of Regulatory T Cells during Viral-Induced Inflammatory Lesions.

Ocular infection with HSV causes a chronic T cell-mediated inflammatory lesion in the cornea. Lesion severity is affected by the balance of different CD4 T cell subsets, with greater severity occurring when the activity of regulatory T cells (Tregs) is compromised. In this study, fate-mapping mice were used to assess the stability of Treg function in ocular lesions. We show that cells that were once Foxp3+ functional Tregs may lose Foxp3 and become Th1 cells that could contribute to lesion expression. The instability primarily occurred with IL-2Rlo Tregs and was shown, in part, to be the consequence of exposure to IL-12. Lastly, in vitro-generated induced Tregs (iTregs) were shown to be highly plastic and capable of inducing stromal keratitis when adoptively transferred into Rag1-/- mice, with 95% of iTregs converting into ex-Tregs in the cornea. This plasticity of iTregs could be prevented when they were generated in the presence of vitamin C and retinoic acid. Importantly, adoptive transfer of these stabilized iTregs to HSV-1-infected mice prevented the development of stromal keratitis lesions more effectively than did control iTregs. Our results demonstrate that CD25lo Treg and iTreg instability occurs during a viral immunoinflammatory lesion and that its control may help to avoid lesion chronicity.

Azacytidine Treatment Inhibits the Progression of Herpes Stromal Keratitis by Enhancing Regulatory T Cell Function.

Ocular infection with herpes simplex virus 1 (HSV-1) sets off an inflammatory reaction in the cornea which leads to both virus clearance and chronic lesions that are orchestrated by CD4 T cells. Approaches that enhance the function of regulatory T cells (Treg) and dampen effector T cells can be effective to limit stromal keratitis (SK) lesion severity. In this report, we explore the novel approach of inhibiting DNA methyltransferase activity using 5-azacytidine (Aza; a cytosine analog) to limit HSV-1-induced ocular lesions. We show that therapy begun after infection when virus was no longer actively replicating resulted in a pronounced reduction in lesion severity, with markedly diminished numbers of T cells and nonlymphoid inflammatory cells, along with reduced cytokine mediators. The remaining inflammatory reactions had a change in the ratio of CD4 Foxp3+ Treg to effector Th1 CD4 T cells in ocular lesions and lymphoid tissues, with Treg becoming predominant over the effectors. In addition, compared to those from control mice, Treg from Aza-treated mice showed more suppressor activity in vitro and expressed higher levels of activation molecules. Additionally, cells induced in vitro in the presence of Aza showed epigenetic differences in the Treg-specific demethylated region (TSDR) of Foxp3 and were more stable when exposed to inflammatory cytokines. Our results show that therapy with Aza is an effective means of controlling a virus-induced inflammatory reaction and may act mainly by the effects on Treg.IMPORTANCE HSV-1 infection has been shown to initiate an inflammatory reaction in the cornea that leads to tissue damage and loss of vision. The inflammatory reaction is orchestrated by gamma interferon (IFN-γ)-secreting Th1 cells, and regulatory T cells play a protective role. Hence, novel therapeutics that can rebalance the ratio of regulatory T cells to effectors are a relevant issue. This study opens up a new avenue in treating HSV-induced SK lesions by increasing the stability and function of regulatory T cells using the DNA methyltransferase inhibitor 5-azacytidine (Aza). Aza increased the function of regulatory T cells, leading to enhanced suppressive activity and diminished lesions. Hence, therapy with Aza, which acts mainly by its effects on Treg, can be an effective means to control virus-induced inflammatory lesions.

DNA Vaccines - A Modern Gimmick or a Boon to Vaccinology?

The reports in 1993 that naked DNA encoding viral genes conferred protective immunity came as a surprise to most vaccinologists. This review analyses the expanding number of examples where plasmid DNA induces immune responses. Issues such as the type of immunity induced, mechanisms of immune protection, and how DNA vaccines compare with other approaches are emphasized. Additional issues discussed include the likely means by which DNA vaccines induce CTL, how the potency and type of immunity induced can be modified, and whether DNA vaccines represent a practical means of manipulating unwanted immune response occurring during immunoinflammatory diseases. It seems doubtful if DNA vaccines will replace currently effective vaccines, but they may prove useful for prophylactic use against some agents that at present lack an effective vaccine. DNA vaccines promise to be valuable to manipulate the immune response in situations where responses to agents are inappropriate or ineffective.

Role of regulatory T cells during virus infection.

The host response to viruses includes multiple cell types that have regulatory function. Most information focuses on CD4(+) regulatory T cells that express the transcription factor Foxp3(+) (Tregs), which are the topic of this review. We explain how viruses through specific and non-specific means can trigger the response of thymus-derived natural Tregs as well as induce Tregs. The latter derive under appropriate stimulation conditions either from uncommitted precursors or from differentiated cells that convert to become Tregs. We describe instances where Tregs appear to limit the efficacy of antiviral protective immunity and other, perhaps more common, immune-mediated inflammatory conditions, where the Tregs function to limit the extent of tissue damage that occurs during a virus infection. We discuss the controversial roles that Tregs may play in the pathogenesis of human immunodeficiency and hepatitis C virus infections. The issue of plasticity is discussed, as this may result in Tregs losing their protective function when present in inflammatory environments. Finally, we mention approaches used to manipulate Treg numbers and function and assess their current value and likely future success to manage the outcome of virus infection, especially those that are responsible for chronic tissue damage.

Role of miR-155 in the pathogenesis of herpetic stromal keratitis.

Ocular infection with herpes simplex virus 1 can result in a chronic immunoinflammatory stromal keratitis (SK) lesion that is a significant cause of human blindness. A key to controlling SK lesion severity is to identify cellular and molecular events responsible for tissue damage and to manipulate them therapeutically. Potential targets for therapy are miRNAs, but these are minimally explored especially in responses to infection. Here, we demonstrated that Mir155 expression was up-regulated after ocular herpes simplex virus 1 infection, with the increased Mir155 expression occurring mainly in macrophages and CD4(+) T cells and to a lesser extent in neutrophils. In vivo studies indicated that Mir155 knockout mice were more resistant to herpes SK with marked suppression of T helper cells type 1 and 17 responses both in the ocular lesions and the lymphoid organs. The reduced SK lesion severity was reflected by increased phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 1 and interferon-γ receptor α-chain levels in activated CD4(+) T cells in the lymph nodes. Finally, in vivo silencing of miR-155 by the provision of antagomir-155 nanoparticles to herpes simplex virus 1-infected mice led to diminished SK lesions and corneal vascularization. In conclusion, our results indicate that miR-155 contributes to the pathogenesis of SK and represents a promising target to control SK severity.

Critical role of microRNA-155 in herpes simplex encephalitis.

HSV infection of adult humans occasionally results in life-threatening herpes simplex encephalitis (HSE) for reasons that remain to be defined. An animal system that could prove useful to model HSE could be microRNA-155 knockout (miR-155KO) mice. Thus, we observe that mice with a deficiency of miR-155 are highly susceptible to HSE with a majority of animals (75-80%) experiencing development of HSE after ocular infection with HSV-1. The lesions appeared to primarily represent the destructive consequences of viral replication, and animals could be protected from HSE by acyclovir treatment provided 4 d after ocular infection. The miR-155KO animals were also more susceptible to development of zosteriform lesions, a reflection of viral replication and dissemination within the nervous system. One explanation for the heightened susceptibility to HSE and zosteriform lesions could be because miR-155KO animals develop diminished CD8 T cell responses when the numbers, functionality, and homing capacity of effector CD8 T cell responses were compared. Indeed, adoptive transfer of HSV-immune CD8 T cells to infected miR-155KO mice at 24 h postinfection provided protection from HSE. Deficiencies in CD8 T cell numbers and function also explained the observation that miR-155KO animals were less able than control animals to maintain HSV latency. To our knowledge, our observations may be the first to link miR-155 expression with increased susceptibility of the nervous system to virus infection.

IL-17A differentially regulates corneal vascular endothelial growth factor (VEGF)-A and soluble VEGF receptor 1 expression and promotes corneal angiogenesis after herpes simplex virus infection.

Ocular infection with HSV causes corneal neovascularization (CV), an essential step in the pathogenesis of the blinding immunoinflammatory lesion stromal keratitis. The infection results in IL-17A production, which contributes to CV in ways that together serve to shift the balance between corneal concentrations of vascular endothelial growth factor A (VEGF-A) and the soluble vascular endothelial growth factor receptor 1 molecule, which binds to VEGF-A and blocks its function (a so-called VEGF trap). Accordingly, animals lacking responses to IL-17A signaling, either because of IL-17 receptor A knockout or wild-type animals that received neutralizing mAb to IL-17A, had diminished CV, compared with controls. The procedures reduced VEGF-A protein levels but had no effect on the levels of soluble vascular endothelial growth factor receptor 1. Hence the VEGF trap was strengthened. IL-17A also caused increased CXCL1/KC synthesis, which attracts neutrophils to the inflammatory site. Neutrophils further influenced the extent of CV by acting as an additional source of VEGF-A, as did metalloproteinase enzymes that degrade the soluble receptor, inhibiting its VEGF-blocking activity. Our results indicate that suppressing the expression of IL-17A, or increasing the activity of the VEGF trap, represents a useful approach to inhibiting CV and the control of an ocular lesion that is an important cause of human blindness.

On the role of regulatory T cells during viral-induced inflammatory lesions.

Ocular HSV-1 infection can result in stromal keratitis, a blinding immunoinflammatory lesion that represents an immunopathological response to the infection. CD4(+) T cells are the main orchestrators, and lesions are more severe if the regulatory T cell (Treg) response is compromised from the onset of infection. Little is known about the role of Foxp3(+)CD4(+) Tregs during ongoing inflammatory reactions, which is the topic of this article. We used DEREG mice and depleted Tregs at different times postinfection. We show that lesions became more severe even when depletion was begun in the clinical phase of the disease. This outcome was explained both by Tregs' influence on the activity of inflammatory effector T cells at the lesion site and by an effect in lymphoid tissues that led to reduced numbers of effectors and less trafficking of T cells and neutrophils to the eye. Our results demonstrate that Tregs can beneficially influence the impact of ongoing tissue-damaging responses to a viral infection and imply that therapies boosting Treg function in the clinical phase hold promise for controlling a lesion that is an important cause of human blindness.

Galectin-1 reduces the severity of herpes simplex virus-induced ocular immunopathological lesions.

Stromal keratitis is a chronic immunopathological lesion of the eye caused by HSV-1 infection that can result in blindness. Because the inflammatory lesions are primarily orchestrated by Th1 cells, and to a lesser extent by Th17 cells, inhibiting their activity represents a useful form of therapy. In this study we evaluated the therapeutic potential of galectin-1 (gal-1), an endogenous lectin that in some autoimmune diseases was shown to suppress the functions of Th1 and Th17 cells. Treatment was begun at different times after ocular infection with HSV and the outcome was assessed clinically as well as for effects on various immune parameters. Treatment with recombinant gal-1 significantly diminished stromal keratitis lesion severity and the extent of corneal neovascularization. Treated mice had reduced numbers of IFN-γ- and IL-17-producing CD4(+) T cells, as well as neutrophil infiltration in the cornea. Furthermore, disease severity was greater in gal-1 knockout mice compared with their wild-type counterparts. The many effects of gal-1 treatment include reduction in the production of proinflammatory cytokines and chemokines, increased production of IL-10, and inhibitory effects on molecules involved in neovascularization. To our knowledge, our findings are the first to show that gal-1 treatment represents a useful approach to control lesion severity in a virally induced immunopathological disease.

T cell immunoglobulin and mucin protein-3 (Tim-3)/Galectin-9 interaction regulates influenza A virus-specific humoral and CD8 T-cell responses.

Reactions to pathogens are usually tuned to effect immunity and limit tissue damage. Several host counterinflammatory mechanisms inhibit tissue damage but these may also act to constrain the effectiveness of immunity to acute infections, as we demonstrate in mice acutely infected with influenza A virus (IAV). We show that compared with wild type (WT), galectin-9 knockout (G9KO) mice mounted a more robust acute phase virus-specific CD8 T-cell response as well as higher and more rapid virus-specific serum IgM, IgG, and IgA responses and also cleared virus more rapidly than did WT mice. Blocking galectin-9 signals to Tim-3-expressing cells using a Tim-3 fusion protein resulted in improved immune responses in WT mice. When IAV immune mice were challenged with a heterologous IAV, the secondary IAV-specific CD8 T-cell responses were four- to fivefold higher in G9KO compared with WT mice. Our results indicate that manipulating galectin signals may represent a convenient approach to improve immune responses to some vaccines.