Siva Karthik Varanasi

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.

Azacytidine treatment inhibits the progression of Herpes Stromal Keratitis by enhancing regulatory T cell function

ABSTRACT 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.

Frontline Science: Aspirin‐triggered resolvin D1 controls herpes simplex virus‐induced corneal immunopathology

Stromal keratitis (SK) is a chronic immunopathological lesion of the eye, caused by HSV‐1 infection, and a common cause of vision impairment in humans. The inflammatory lesions in the cornea are primarily caused by neutrophils with the active participation of CD4+ T cells. Therefore, the targeting of these immune cell types and their products represents a potentially valuable form of therapy to reduce the severity of disease. Resolvin D1 (RvD1) and its epimer aspirin‐triggered RvD1 (AT‐RvD1) are lipid mediators derived from docosahexaenoic acid (DHA) and were shown to promote resolution in several inflammatory disease models. In this report, we examined whether AT‐RvD1 administration, begun before infection or at a later stage after ocular infection of mice with HSV‐1, could control the severity of SK lesions. Treatment with AT‐RvD1 significantly diminished the extent of corneal neovascularization and the severity of SK lesions. AT‐RvD1‐treated mice had fewer numbers of inflammatory cells that included neutrophils as well as Th1 and Th17 cells in the infected cornea. The mechanisms by which AT‐RvD1 acts appear to be multiple. These include inhibitory effects on proinflammatory mediators, such as IL‐1β, IL‐6, IL‐12, CXCL1, MCP‐1, MIP‐2, vascular endothelial growth factor (VEGF)‐A, matrix metalloproteinase 9 (MMP‐9), and proinflammatory miRNA, such as miR‐155, miR‐132, and miR‐223, which are involved in SK pathogenesis and corneal neovascularization. In addition, AT‐RvD1 attenuated STAT1, which plays an important role in Th1 cell differentiation and IFN‐γ expression. These findings demonstrate that AT‐RvD1 treatment could represent a useful strategy for the management of virus‐induced immunopathological lesions.

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.

The inflammasome NLRP3 plays a protective role against a viral immunopathological lesion

Herpes simplex 1 infection of the eye can cause blindness with lesions in the corneal stroma largely attributable to inflammatory events that include components of both adaptive and innate immunity. Several innate immune responses are triggered by herpes simplex 1, but it is unclear how such innate events relate to the subsequent development of stromal keratitis. In this study, we compared the outcome of herpes simplex 1 ocular infection in mice unable to express NLRP3 because of gene knockout (NLRP3−/−) to that of wild‐type mice. The NLRP3−/− mice developed more‐severe and earlier stromal keratitis lesions and had higher angiogenesis scores than did infected wild‐type animals. In addition, NLRP3−/− mice generated an increased early immune response with heightened chemokines and cytokines, including interleukin‐1β and interleukin‐18, and elevated recruitment of neutrophils. Increased numbers of CD4+ T cells were seen at later stages of the disease in NLRP3−/− animals. Reduction in neutrophils prevented early onset of the disease in NLRP3−/− animals and lowered levels of bioactive interleukin‐1β but did not lower bioactive interleukin‐18. In conclusion, our results indicate that NLRP3 has a regulatory and beneficial role in herpetic stromal keratitis pathogenesis.

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.

Hexokinase II may be dispensable for CD4 T cell responses against a virus infection

Activation of CD4 T cells leads to their metabolic reprogramming which includes enhanced glycolysis, catalyzed through hexokinase enzymes. Studies in some systems indicate that the HK2 isoform is the most up regulated isoform in activated T cells and in this report the relevance of this finding is evaluated in an infectious disease model. Genetic ablation of HK2 was achieved in only T cells and the outcome was evaluated by measures of T cell function. Our results show that CD4 T cells from both HK2 depleted and WT animals displayed similar responses to in vitro stimulation and yielded similar levels of Th1, Treg or Th17 subsets when differentiated in vitro. A modest increase in the levels of proliferation was observed in CD4 T cells lacking HK2. Deletion of HK2 led to enhanced levels of HK1 indicative of a compensatory mechanism. Finally, CD4 T cell mediated immuno-inflammatory responses to a virus infection were similar between WT and HK2 KO animals. The observations that the expression of HK2 appears non-essential for CD4 T cell responses against virus infections is of interest since it suggests that targeting HK2 for cancer therapy may not have untoward effects on CD4 T cell mediated immune response against virus infections.

Role of IL-18 induced Amphiregulin expression on virus induced ocular lesions

This report deals with the possible mechanism by which IL-18 can contribute to the control and resolution of inflammatory lesions in the cornea caused by herpes simplex virus infection. Our results demonstrate that the expression of the IL-18R by both regulatory T cells (Treg) and effector T cells was a pivotal event that influenced lesion pathogenesis. The engagement of IL-18R on Treg with its cytokine ligand resulted in Amphiregulin expression a molecule associated with tissue repair. In support of this scheme of events, lesion severity became more severe in animals unable to express the IL-18R because of gene knockout and was reduced in severity when IL-18 was overexpressed in the cornea. These changes in lesion severity correlated with the frequency and number of both Treg and Teff that expressed Amphiregulin. Additional experiments indicated that IL-12 and IL-18 acted synergistically to enhance Amphiregulin expression in Treg, an event partly dependent on P38 MAPK activity. Finally, sub-conjunctival administration of Amphiregulin resulted in resolution of both developing and developed lesions. Thus, overall our results imply that IL-18 may participate in controlling the severity of SK and contribute to tissue repair by converting both Treg and effector T cells into those that produce Amphiregulin.

How host metabolism impacts on virus pathogenesis

The outcome of virus infections depends on multiple factors. This review deals with the role of host metabolism as one such factor. We describe how different cells in the immune system employ differential metabolic pathways and how this relates to the outcome of virus infections. We also discuss how nutritional and metabolic diseases can influence the nature of viral pathogenesis as well as how targeted therapies against metabolic processes can impact on the outcome of virus infections. The case is also made for metabolic profiling as a potential tool to predict the outcome of a virus infection and to guide therapies that enhance host resistance.