Contribution of microRNAs and Robo4 signaling after herpes simplex virus infection: Role for regulatory mechanisms

Herpes simplex virus (HSV) infection of the eye results in chronic immunopathological response in corneal stroma orchestrated by CD4 T cells. Due to its neurotrophic nature, HSV can also invade brain and leads to deadly encephalitis. HSV infection of the eyes leads to corneal neovascularization (CV), which is an important step in the pathogenesis of herpetic stromal keratitis (HSK), an important cause of human blindness. This study was undertaken to investigate the contribution of miRNAs and Robo4 signaling in HSK. The role of miR-132 and miR-155 was evaluated. In the first chapter, we review literature regarding the contribution of miRNAs in several human diseases and speculate about the role of those miRNAs in herpes viral latency and HSK. We also discuss involvement of Robo4 signaling in the context of pathological angiogenesis. In the second chapter, we show that HSV infection of the eyes of mice leads to appearance of Robo4+ blood vessel endothelial cells in corneas but levels of Slit2 (a ligand for Robo4 receptor) were minimal. The provision of exogenous Slit2 protein reduced CV following HSV infection. In the third chapter, we report the role of miR-132 in angiogenesis following ocular HSV infection. We observed VEGF and IL-17 driven upregulation of miR-132 during HSK. miR-132 was shown to augment VEGF induced angiogenic responses and blockade of miR-132 using antagomir nanoparticles reduced pathological angiogenesis in eyes after HSV infection. In the fourth chapter, we analyzed the involvement of miR-155 after ocular infection with HSV. The mice unable to produce miR-155 were found to be highly susceptible to encephalitis after HSV infection with the majority of mice dyeing by day 9. We further show that virus replication in brain was responsible for death in miR-155 null mice. When T cell responses were measured, miR-155 knockout animals demonstrated attenuated CD4 and virus specific CD8 T cell immunity. Additionally, we also evaluated the role of miR-155 in HSK. miR-155KO survivors from herpes simplex encephalitis were relatively resistant to HSK. In conclusion, we demonstrated that the miR-155 is a regulator of herpes simplex encephalitis but it promotes inflammation during HSK

Controlling viral inflammatory lesions by rebalancing immune response patterns

In this review, we discuss a variety of immune modulating approaches that could be used to counteract tissue-damaging viral immunoinflammatory lesions which typify many chronic viral infections. We make the point that in several viral infections the lesions can be largely the result of one or more aspects of the host response mediating the cell and tissue damage rather than the virus itself being directly responsible. However, within the reactive inflammatory lesions along with the pro-inflammatory participants there are also other aspects of the host response that may be acting to constrain the activity of the damaging components and are contributing to resolution. This scenario should provide the prospect of rebalancing the contributions of different host responses and hence diminish or even fully control the virus-induced lesions. We identify several aspects of the host reactions that influence the pattern of immune responsiveness and describe approaches that have been used successfully, mainly in model systems, to modulate the activity of damaging participants and which has led to lesion control. We emphasize examples where such therapies are, or could be, translated for practical use in the clinic to control inflammatory lesions caused by viral infections

Killer lymphocytes use granulysin, perforin and granzymes to kill intracellular parasites

Protozoan infections are a serious global health problem1, 2. Natural killer (NK) cells and cytolytic T lymphocytes (CTLs) eliminate pathogen-infected cells by releasing cytolytic granule contents—granzyme (Gzm) proteases and the pore-forming perforin (PFN)—into the infected cell3. However, these cytotoxic molecules do not kill intracellular parasites. CD8+ CTLs protect against parasite infections in mice primarily by secreting interferon (IFN)-γ4, 5, 6, 7, 8, 9, 10. However, human, but not rodent, cytotoxic granules contain the antimicrobial peptide granulysin (GNLY), which selectively destroys cholesterol-poor microbial membranes11, 12, 13, 14, and GNLY, PFN and Gzms rapidly kill intracellular bacteria15. Here we show that GNLY delivers Gzms into three protozoan parasites (Trypanosoma cruzi, Toxoplasma gondii and Leishmania major), in which the Gzms generate superoxide and inactivate oxidative defense enzymes to kill the parasite. PFN delivers GNLY and Gzms into infected cells, and GNLY then delivers Gzms to the intracellular parasites. Killer cell–mediated parasite death, which we term 'microbe-programmed cell death' or 'microptosis', is caspase independent but resembles mammalian apoptosis, causing mitochondrial swelling, transmembrane potential dissipation, membrane blebbing, phosphatidylserine exposure, DNA damage and chromatin condensation. GNLY-transgenic mice are protected against infection by T. cruzi and T. gondii, and survive infections that are lethal to wild-type mice. Thus, GNLY-, PFN- and Gzm-mediated elimination of intracellular protozoan parasites is an unappreciated immune defense mechanism

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

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

Memory like NK cells display stem cell like properties after Zika virus infection.

NK cells have been shown to display adaptive traits such as memory formation akin to T and B lymphocytes. Here we show that Zika virus infection induces memory like NK cells that express CD27. Strikingly, these cells exhibit stem-like features that include expansion capacity, self-renewal pathway, differentiation into effector cells, longer telomeres and gene signature associated with hematopoietic stem cell (HSC) progenitors. This subset shared transcriptional and epigenetic changes with memory CD8 T cells, stem cells and stem like T cells. These NK cells with memory and stem cell features, which we term "NK memory stem cells", demonstrated greater antiviral potential than CD27- or naïve CD27+ NK when adoptively transferred to Zika infected mice. Our results also suggest a role for the transcription factor TCF-1 in memory and stemness features of this NK subset. This study defines a unique TCF1hi CD27+ NK subset with memory capacity and stem cell features that play a role in antiviral immunity

Costimulatory CD226 Signaling Regulates Proliferation of Memory-like NK Cells in Healthy Individuals with Latent <i>Mycobacterium tuberculosis</i> Infection

It is now widely accepted that NK cells can acquire memory, and this makes them more effective to protect against some pathogens. Prior reports indicate memory-like NK cells (mlNKs) in murine model of Mycobacterium tuberculosis (Mtb) as well as in healthy individuals with latent TB infection (LTBI). The increased expression of CD226 was evident in mlNKs from LTBI+ people after stimulation with γ-irradiated Mtb (γ-Mtb). We thus evaluated the contribution of costimulatory CD226 signaling in the functionality of mlNKs in LTBI+ people. We found that blockade of CD226 signaling using the antibody- or CRISPR/Cas9-mediated deletion of the CD226 gene in NK cells diminished the proliferation of mlNKs from LTBI+ people. Blocking CD226 signaling also reduced the phosphorylation of FOXO1 and cMyc expression. Additionally, cMyc inhibition using a chemical inhibitor reduced proliferation by mlNKs from LTBI+ people. Moreover, blocking CD226 signaling reduced glycolysis in NK cells, and the inhibition of glycolysis led to reduced effector function of mlNKs from LTBI+ people. Overall, our results provide a role for CD226 signaling in mlNK responses to Mtb

Provision of sR4 reduces corneal inflammation and angiogenesis.

<p>WT mice were infected with HSV-1 RE and treated with sR4 of vehicle (PBS) from day 2 pi. to 14 pi. At day 15 pi. corneas were collected and pooled for analysis by flow cytometry or Q-RT-PCR. The frequency and total cell number per cornea for (A) endothelial cells (CD31+) gated on total CD45- cells infiltrate, (B) CD4+ T cells (CD4+) (gated on total CD45+ cells infiltrate) and (C) neutrophils (Ly6G+ CD11b+ gated on total CD45+ cells infiltrate) show significant decrease in sR4 treated mice compared to WT mice. Data are a combination of 3 independent experiments and show mean values ± SEM (n = 7 and each sample is representative of 2 corneas). ***<i>p</i> ≤ 0.001, **<i>p</i> ≤ 0.01, *<i>p</i> ≤ 0.05. Statistical levels of significance were analyzed by <i>t</i> test. (D) Relative fold change in mRNA expression of IL-1β, IL-6 and CXCL-1 was examined and compared between sR4 treated and control mice on day 15 pi. by Q-RT-PCR. Data represent means ± SEM from three different independent experiments (n = 2 and each sample is representative of 6 corneas). ***<i>p</i> ≤ 0.001, **<i>p</i> ≤ 0.01, *<i>p</i> ≤ 0.05. Statistical levels of significance were analyzed by <i>t</i> test.</p

Administration of soluble Robo4 (sR4) shifts the Robo4 knockout to the WT phenotype.

<p>WT and Robo4 knockout mice (R4 KO) were infected with HSV-1 RE. (A) R4 KO mice received either sR4 (R4 KO treated w/ sR4) or vehicle (R4 KO Control) from 2 to 14 days pi. WT mice were included and received vehicle (WT Control) under the same regimen previously stated. (B) SK lesions and angiogenesis severity was decreased in R4 KO mice treated with sR4. (C) Representative eye photos show that R4 KO mice treated with sR4 do not develop the severe phenotype that R4 KO control animals do (D) Hematoxylin and eosin staining was carried out on 6-μm sections, and pictures were taken 40 x magnification. Representative eye sections show decreased cellular infiltration in R4 KO treated w/ sR4 and WT control compared to R4 KO control mice. Data are representative of two independent experiments and show mean values ± SEM (n = 12 mice/group). ***<i>p</i> ≤ 0.001,**<i>p</i> ≤ 0.01,*<i>p</i> ≤ 0.05. Statistical levels of significance were analyzed by one-way ANOVA test with Tuckey’s post hoc test settings.</p

Robo4 deficient mice present more inflammation and vascularization.

<p>Robo4 knockout (R4 KO) and WT mice were infected with HSV-1 RE and at day 15 pi. corneas were collected and pooled for analysis by flow cytometry or Q-RT-PCR. The frequency and total cell number per cornea for (A) endothelial cells (CD31+) gated on total CD45- cells infiltrate, (B) CD4+ T cells (CD4+) (gated on total CD45+ cells infiltrate) and (C) neutrophils (Ly6G+ CD11b+ gated on total CD45+ cells infiltrate) show significant increase in R4 KO mice. Data are a combination of 3 independent experiments and show mean values ± SEM (n = 7 and each sample is representative of 2 corneas). ***<i>p</i> ≤ 0.001, **<i>p</i> ≤ 0.01, *<i>p</i> ≤ 0.05. Statistical levels of significance were analyzed by <i>t</i> test. (E) Relative fold change in mRNA expression of IL-1β, IL-6 and CXCL-1 was examined and compared between WT and Robo4 KO mice on day 15 pi. by Q-RT-PCR. Data represent means ± SEM from two different independent experiments (n = 3 and each sample is representative of 5 corneas). ***<i>p</i> ≤ 0.001, **<i>p</i> ≤ 0.01, *<i>p</i> ≤ 0.05. Statistical levels of significance were analyzed by <i>t</i> test.</p