Decidual NK Cells Transfer Granulysin to Selectively Kill Bacteria in Trophoblasts.

Maternal decidual NK (dNK) cells promote placentation, but how they protect against placental infection while maintaining fetal tolerance is unclear. Here we show that human dNK cells highly express the antimicrobial peptide granulysin (GNLY) and selectively transfer it via nanotubes to extravillous trophoblasts to kill intracellular Listeria monocytogenes (Lm) without killing the trophoblast. Transfer of GNLY, but not other cell death-inducing cytotoxic granule proteins, strongly inhibits Lm in human placental cultures and in mouse and human trophoblast cell lines. Placental and fetal Lm loads are lower and pregnancy success is greatly improved in pregnant Lm-infected GNLY-transgenic mice than in wild-type mice that lack GNLY. This immune defense is not restricted to pregnancy; peripheral NK (pNK) cells also transfer GNLY to kill bacteria in macrophages and dendritic cells without killing the host cell. Nanotube transfer of GNLY allows dNK to protect against infection while leaving the maternal-fetal barrier intact.

Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes.

When killer lymphocytes recognize infected cells, perforin delivers cytotoxic proteases (granzymes) into the target cell to trigger apoptosis. What happens to intracellular bacteria during this process is unclear. Human, but not rodent, cytotoxic granules also contain granulysin, an antimicrobial peptide. Here, we show that granulysin delivers granzymes into bacteria to kill diverse bacterial strains. In Escherichia coli, granzymes cleave electron transport chain complex I and oxidative stress defense proteins, generating reactive oxygen species (ROS) that rapidly kill bacteria. ROS scavengers and bacterial antioxidant protein overexpression inhibit bacterial death. Bacteria overexpressing a GzmB-uncleavable mutant of the complex I subunit nuoF or strains that lack complex I still die, but more slowly, suggesting that granzymes disrupt multiple vital bacterial pathways. Mice expressing transgenic granulysin are better able to clear Listeria monocytogenes. Thus killer cells play an unexpected role in bacterial defense.

Decidual NK cells kill Zika virus-infected trophoblasts.

Zika virus (ZIKV) during pregnancy infects fetal trophoblasts and causes placental damage and birth defects including microcephaly. Little is known about the anti-ZIKV cellular immune response at the maternal-fetal interface. Decidual natural killer cells (dNK), which directly contact fetal trophoblasts, are the dominant maternal immune cells in the first-trimester placenta, when ZIKV infection is most hazardous. Although dNK express all the cytolytic molecules needed to kill, they usually do not kill infected fetal cells but promote placentation. Here, we show that dNK degranulate and kill ZIKV-infected placental trophoblasts. ZIKV infection of trophoblasts causes endoplasmic reticulum (ER) stress, which makes them dNK targets by down-regulating HLA-C/G, natural killer (NK) inhibitory receptor ligands that help maintain tolerance of the semiallogeneic fetus. ER stress also activates the NK activating receptor NKp46. ZIKV infection of Ifnar1 -/- pregnant mice results in high viral titers and severe intrauterine growth restriction, which are exacerbated by depletion of NK or CD8 T cells, indicating that killer lymphocytes, on balance, protect the fetus from ZIKV by eliminating infected cells and reducing the spread of infection.

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.

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 Mycobacterium tuberculosis 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.

Endothelial Cell Protein C Receptor Deficiency Attenuates Streptococcus pneumoniae-induced Pleural Fibrosis.

Streptococcus pneumoniae is the leading cause of hospital community-acquired pneumonia. Patients with pneumococcal pneumonia may develop complicated parapneumonic effusions or empyema that can lead to pleural organization and subsequent fibrosis. The pathogenesis of pleural organization and scarification involves complex interactions between the components of the immune system, coagulation, and fibrinolysis. EPCR (endothelial protein C receptor) is a critical component of the protein C anticoagulant pathway. The present study was performed to evaluate the role of EPCR in the pathogenesis of S. pneumoniae infection-induced pleural thickening and fibrosis. Our studies show that the pleural mesothelium expresses EPCR. Intrapleural instillation of S. pneumoniae impairs lung compliance and lung volume in wild-type and EPCR-overexpressing mice but not in EPCR-deficient mice. Intrapleural S. pneumoniae infection induces pleural thickening in wild-type mice. Pleural thickening is more pronounced in EPCR-overexpressing mice, whereas it is reduced in EPCR-deficient mice. Markers of mesomesenchymal transition are increased in the visceral pleura of S. pneumoniae-infected wild-type and EPCR-overexpressing mice but not in EPCR-deficient mice. The lungs of wild-type and EPCR-overexpressing mice administered intrapleural S. pneumoniae showed increased infiltration of macrophages and neutrophils, which was significantly reduced in EPCR-deficient mice. An analysis of bacterial burden in the pleural lavage, the lungs, and blood revealed a significantly lower bacterial burden in EPCR-deficient mice compared with wild-type and EPCR-overexpressing mice. Overall, our data provide strong evidence that EPCR deficiency protects against S. pneumoniae infection-induced impairment of lung function and pleural remodeling.

Bacterial extracellular vesicles isolated from organic dust induce neutrophilic inflammation in the lung.

Inhalation of organic dust is an occupational hazard leading to the development of respiratory symptoms and respiratory diseases. Bioaerosols from concentrated animal feeding operations are rich in bacteria and could carry bacterial extracellular vesicles (EVs) that could induce lung inflammation. It is not known if organic dust contains bacterial EVs and whether they modulate lung inflammation. Herein, we show that poultry organic dust contains bacterial EVs (dust EVs) that induce lung inflammation. Treatment of airway epithelial cells, THP-1-monocytes and -macrophages with dust EVs rapidly induced IL-8, IL-6, ICAM-1, proIL-1ß, and TNF-α levels. In airway epithelial cells, induction of inflammatory mediators was due to increased mRNA levels and NF-κB activation. Induction of inflammatory mediators by dust EVs was not inhibited by polymyxin B. Single and repeated treatments of mice with dust EVs increased lung KC, IL-6, and TNF-α levels without significantly altering IL-17A levels. Increases in cytokines were associated with enhanced neutrophil infiltration into the lung. Repeated treatments of mice with dust EVs increased lung mean linear intercept and increased collagen deposition around airways indicating lung remodeling. Peribronchial cell infiltrates and airway epithelial thickening were also observed in treated mice. Because bacterial EVs are nanometer-sized particles, they can reach and accumulate in the bronchiolar and alveolar regions causing lung injury leading to the development of respiratory diseases. Our studies have provided new evidence for the presence of bacterial EVs in organic dust and for their role as one of the causative agents of organic dust-induced lung inflammation and lung injury.

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.

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.

TNFRSF25 agonistic antibody and galectin-9 combination therapy controls herpes simplex virus-induced immunoinflammatory lesions.

Ocular infection with herpes simplex virus 1 (HSV-1) results in a chronic immunoinflamammtory reaction in the cornea, which is primarily orchestrated by CD4(+) T cells. Hence, targeting proinflammatory CD4(+) T cells or increasing the representation of cells that regulate their function is a relevant therapeutic strategy. In this report, we demonstrate that effective therapeutic control can be achieved using a combination of approaches under circumstances where monotherapy is ineffective. We use a convenient and highly effective monoclonal antibody (MAb) approach with MAbT25 to expand cells that express the tumor necrosis factor receptor superfamily member 25 (TNFRSF25). In naïve animals, these are predominantly cells that are Foxp3-positive regulatory T cells. MAbT25 treatment before or at the time of initial HSV infection was an effective means of reducing the severity of subsequent stromal keratitis lesions. However, MAbT25 treatment was not effective if given 6 days after infection since it expanded proinflammatory effector T cells, which also express TNFRSF25. Therefore, the MAbT25 procedure was combined with galectin-9 (Gal-9), an approach that compromises the activity of T cells involved in tissue damage. The combination therapy provided highly effective lesion control over that achieved by treatment with one of them. The beneficial outcome of the combination therapy was attributed to the expansion of the regulatory T cell population that additionally expressed activation markers such as CD103 needed to access inflammatory sites. Additionally, there was a marked reduction of CD4(+) gamma interferon-producing effector T cells responsible for orchestrating the tissue damage. The approach that we describe has potential application to control a wide range of inflammatory diseases, in addition to stromal keratitis, an important cause of human blindness.

Role of miR-132 in angiogenesis after ocular infection with herpes simplex virus.

MicroRNAs (miRNAs) are small regulatory molecules that control diverse biological processes that include angiogenesis. Herpes simplex virus (HSV) causes a chronic immuno-inflammatory response in the eye that may result in corneal neovascularization during blinding immunopathological lesion stromal keratitis (SK). miR-132 is a highly conserved miRNA that is induced in endothelial cells in response to growth factors, such as vascular endothelial growth factor (VEGF). In this study, we show that miR-132 expression was up-regulated (10- to 20-fold) after ocular infection with HSV, an event that involved the production of both VEGF-A and IL-17. Consequently, blockade of VEGF-A activity using soluble VEGF receptor 1 resulted in significantly lower levels of corneal miR-132 after HSV infection. In addition, low levels of corneal miR-132 were detected in IL-17 receptor knockout mice after HSV infection. In vivo silencing of miR-132 by the provision of anti-miR-132 (antagomir-132) nanoparticles to HSV-infected mice led to reduced corneal neovascularization and diminished SK lesions. The anti-angiogenic effect of antagomir-132 was reflected by a reduction in angiogenic Ras activity in corneal CD31-enriched cells (presumably blood vessel endothelial cells) during SK. To our knowledge, this is one of the first reports of miRNA involvement in an infectious ocular disease. Manipulating miRNA expression holds promise as a therapeutic approach to control an ocular lesion that is an important cause of human blindness.

Ocular neovascularization caused by herpes simplex virus type 1 infection results from breakdown of binding between vascular endothelial growth factor A and its soluble receptor.

The normal cornea is transparent, which is essential for normal vision, and although the angiogenic factor vascular endothelial growth factor A (VEGF-A) is present in the cornea, its angiogenic activity is impeded by being bound to a soluble form of the VEGF receptor-1 (sVR-1). This report investigates the effect on the balance between VEGF-A and sVR-1 that occurs after ocular infection with HSV, which causes prominent neovascularization, an essential step in the pathogenesis of the vision-impairing lesion, stromal keratitis. We demonstrate that HSV-1 infection causes increased production of VEGF-A but reduces sVR-1 levels, resulting in an imbalance of VEGF-A and sVR-1 levels in ocular tissues. Moreover, the sVR-1 protein made was degraded by the metalloproteinase (MMP) enzymes MMP-2, -7, and -9 produced by infiltrating inflammatory cells that were principally neutrophils. Inhibition of neutrophils, inhibition of sVR-1 breakdown with the MMP inhibitor marimastat, and the provision of exogenous recombinant sVR-1 protein all resulted in reduced angiogenesis. Our results make the novel observation that ocular neovascularization resulting from HSV infection involves a change in the balance between VEGF-A and its soluble inhibitory receptor. Future therapies aimed to increase the production and activity of sVR-1 protein could benefit the management of stromal keratitis, an important cause of human blindness.

Controlling herpes simplex virus-induced ocular inflammatory lesions with the lipid-derived mediator resolvin E1.

Stromal keratitis (SK) is a chronic immunopathological lesion of the eye caused by HSV-1 infection and a common cause of blindness in humans. The inflammatory lesions are primarily perpetuated by neutrophils with the active participation of CD4(+) T cells. Therefore, targeting these immune cell types represents a potentially valuable form of therapy to reduce the severity of disease. Resolvin E1 (RvE1), an endogenous lipid mediator, was shown to promote resolution in several inflammatory disease models. In the current report, we determined whether RvE1 administration begun at different times after ocular infection of mice with HSV could influence the severity of SK lesions. Treatment with RvE1 significantly reduced the extent of angiogenesis and SK lesions that occurred. RvE1-treated mice had fewer numbers of inflammatory cells that included Th1 and Th17 cells as well as neutrophils in the cornea. The mechanisms by which RvE1 acts appear to be multiple. These included reducing the influx of neutrophils and pathogenic CD4(+) T cells, increasing production of the anti-inflammatory cytokine IL-10, and inhibitory effects on the production of proinflammatory mediators and molecules, such as IL-6, IFN-γ, IL-17, KC, VEGF-A, MMP-2, and MMP-9, that are involved in corneal neovascularization and SK pathogenesis. These findings are, to our knowledge, the first to show that RvE1 treatment could represent a novel approach to control lesion severity in a virally induced immunopathological disease.

Influence of galectin-9/Tim-3 interaction on herpes simplex virus-1 latency.

After HSV-1 infection, CD8(+) T cells accumulate in the trigeminal ganglion (TG) and participate in the maintenance of latency. However, the mechanisms underlying intermittent virus reactivation are poorly understood. In this study, we demonstrate the role of an inhibitory interaction between T cell Ig and mucin domain-containing molecule 3 (Tim-3)-expressing CD8(+) T cells and galectin 9 (Gal-9) that could influence HSV-1 latency and reactivation. Accordingly, we show that most K(b)-gB tetramer-specific CD8(+) T cells in the TG of HSV-1-infected mice express Tim-3, a molecule that delivers negative signals to CD8(+) T cells upon engagement of its ligand Gal-9. Gal-9 was also upregulated in the TG when replicating virus was present as well during latency. This could set the stage for Gal-9/Tim-3 interaction, and this inhibitory interaction was responsible for reduced CD8(+) T cell effector function in wild-type mice. Additionally, TG cell cultures exposed to recombinant Gal-9 in the latent phase caused apoptosis of most CD8(+) T cells. Furthermore, Gal-9 knockout TG cultures showed delayed and reduced viral reactivation as compared with wild-type cultures, demonstrating the greater efficiency of CD8(+) T cells to inhibit virus reactivation in the absence of Gal-9. Moreover, the addition of recombinant Gal-9 to ex vivo TG cultures induced enhanced viral reactivation compared with untreated controls. Our results demonstrate that the host homeostatic mechanism mediated by Gal-9/Tim-3 interaction on CD8(+) T cells can influence the outcome of HSV-1 latent infection, and manipulating Gal-9 signals might represent therapeutic means to inhibit HSV-1 reactivation from latency.

Activation of endothelial roundabout receptor 4 reduces the severity of virus-induced keratitis.

Antiangiogenic molecules exert a feedback control to restrain pathological angiogenesis, which includes physical binding or inhibition of angiogenic signaling in blood vessel endothelial cells. The latter is the case in which Slit2 ligand-dependent activation of the blood vessel endothelial cell receptor roundabout 4 (Robo4) occurs. In this study, we demonstrate that Robo4 receptors are upregulated following HSV infection of the eye on the majority of the new blood vessel endothelial cells that occur in the corneal stroma. However, expression levels of the ligand for Robo4 receptors, Slit2, was not significantly increased during the disease process, and the knockdown of Slit2 gene expression using lentiviral short hairpin RNAs had no effect on the extent of pathological angiogenesis. In contrast, providing additional Slit2 protein by subconjunctival administration resulted in significantly reduced angiogenesis. The Slit2 binding to Robo4 was shown to block the downstream vascular endothelial growth factor signaling molecules Arf 6 and Rac 1 and reduce the antiapoptotic molecule Bcl-xL in blood vessel endothelial cells. Our results indicate that augmenting the host Robo4/Slit2 system could provide a useful therapeutic approach to control pathological angiogenesis associated with HSV induced stromal keratitis.

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.