Siddheshvar Bhela

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.

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.

Robo 4 Counteracts Angiogenesis in Herpetic Stromal Keratitis

The cornea is a complex tissue that must preserve its transparency to maintain optimal vision. However, in some circumstances, damage to the eye can result in neovascularization that impairs vision. This outcome can occur when herpes simplex virus type 1 (HSV-1) causes the immunoinflammatory lesion stromal keratitis (SK). Potentially useful measures to control the severity of SK are to target angiogenesis which with herpetic SK invariably involves VEGF. One such way to control angiogenesis involves the endothelial receptor Robo4 (R4), which upon interaction with another protein activates an antiangiogenic pathway that counteracts VEGF downstream signaling. In this study we show that mice unable to produce R4 because of gene knockout developed significantly higher angiogenesis after HSV-1 ocular infection than did infected wild type (WT) controls. Moreover, providing additional soluble R4 (sR4) protein by subconjunctival administration to R4 KO HSV-1 infected mice substantially rescued the WT phenotype. Finally, administration of sR4 to WT HSV-1 infected mice diminished the extent of corneal angiogenesis compared to WT control animals. Our results indicate that sR4 could represent a useful therapeutic tool to counteract corneal angiogenesis and help control the severity of SK.

Are miRNAs critical determinants in herpes simplex virus pathogenesis

miRNAs are small noncoding RNA that play a crucial role in gene regulation by inhibiting translation or promoting mRNA degradation. Viruses themselves express miRNAs that can target either the host or viral mRNA transcriptome. Moreover, viral infection of cells causes a drastic change in host miRNAs. This complex interaction between the host and viruses often favors the virus to evade immune elimination and favors the establishment and maintenance of latency. In this review we discuss the function of both host and viral miRNAs in regulating herpes simplex virus pathogenesis and also discuss the prospect of using miRNAs as biomarkers and therapeutic tools.

miR-31: a key player in CD8 T-cell exhaustion

Over the past decade, miRNAs have emerged as genetic factors that can influence immune functions.1 They act by post-transcriptionally downregulating gene expression either by degrading target mRNAs or by repressing their translation.2 Linking miRNA expression to disease was first accomplished by comparing outcomes in normal animals with those in miR-155 knockout animals.3 Our group also used a viral pathogen to show that knocking out miR-155 resulted in less severe inflammatory lesions caused by herpes simplex virus infection, yet animals without miR-155 became more susceptible to developing viral encephalitis.4,5 With infectious agents, miRNAs from the agent itself can also influence the outcome of events following infection.6 For example, herpes viruses express many miRNAs, and these, along with several host miRNAs, may influence the type of interaction the virus has with the host.6 These interactions can result in a productive infection, persistence in the form of latency, or, in the case of some herpes viruses, neoplasia.6 MiRNAs may also influence the nature of the inflammatory response to infectious agents that affect, for example, the functional type of lipid mediators produced.7 There have been many mechanistic studies to determine how various miRNAs influence individual immune components that react to infectious agents,1 but the present report of Moffett et al.8 adds an additional chapter to the story. The authors demonstrate that miR-31 production could be a key event in the expression of the immune exhaustion phenotype. Immune exhaustion has attracted much attention as it appears to explain the failure of the T-cell system to control some cancers and chronic infections.9 As the American public has learned from TV, immune exhaustion can be reversed, and some cancers can now be controlled with commercially available monoclonal antibodies.10 The immune exhaustion concept, as it applies to chronic infections, was first noted by Zinkernagel11 but was popularized by extensive studies of chronic lymphocytic meningitis virus (LCMV), mainly by the Ahmed laboratory.12,13 The present communication utilizes Ahmed’s LCMV system and makes the novel observation that miR-31 could be responsible for causing the exhaustion phenotype. Such studies may open an additional avenue for pursuing the therapeutic management of exhaustion during chronic infections. In the Moffett report, T-cell receptor (TCR) and CD28 co-stimulation of naive CD8 T cells was shown to induce the expression of miR-31, a process requiring calcium signaling and subsequent NFAT binding to the locus that encodes miR-31. Moreover, miR-31 production was sustained if the cells were stimulated with cytokines (IL-2 and IL-7), but the same cytokines could not induce miR-31 in naive CD8 T cells. These data clearly indicate that the TCR and downstream signaling stimulate the expression of miR-31 in CD8 T cells. To understand the in vivo consequences of producing miR-31, the effects of knocking out the miR-31 gene globally or specifically in T cells were evaluated in mice subsequently challenged with viral infections. To the authors’ disappointment, the outcome of acute infections with LCMV and influenza in knockout and wild-type (WT) animals was basically unchanged. The animals controlled the virus in much the same way and generated similar levels of immunity. However, to the authors’ delight, the responses of normal and knockout animals to chronic LCMV infection were considerably different. As previously observed by many groups, WT mice exhibited signs of chronic disease and wasting when infected with LCMV clone 13,9,13 but the pattern of responsiveness in the knockout animals was quite different. The knockouts had a better CD8 T-cell response in the blood and lymphoid organs and a phenotype that showed less evidence of exhaustion. They demonstrated that the CD8 T-cell population from the miR-31− /− mice had far fewer cells that expressed PD1, Mt1, Mt2 and Ptger2 (molecules that contribute to CD8 T-cell dysfunction) Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, Knoxville, TN 37996, USA