PD-L1/B7-H1 Inhibits Viral Clearance by Macrophages in HSV-1-Infected Corneas.

Immune privilege helps protect the cornea from damaging inflammation but can also impair pathogen clearance from this mucosal surface. Programmed death-ligand 1 (PD-L1 or B7-H1) contributes to corneal immune privilege by inhibiting the function of a variety of immune cells. We asked whether programmed death-1 (PD-1)/PD-L1 interaction regulates HSV-1 clearance from infected corneas. We show that PD-L1 is constitutively expressed in the corneal epithelium and is upregulated upon HSV-1 corneal infection, with peak expression on CD45+ cells NK cells, dendritic cells, neutrophils, and macrophages and CD45- corneal epithelial cells at 4 d postinfection (dpi). As early as 1 dpi, HSV-1-infected corneas of B7-H1-/- mice as compared with wild-type mice showed increased chemokine expression and this correlated with increased migration of inflammatory cells into the viral lesions and decreased HSV-1 corneal titers. Local PD-L1 blockade caused a similar increase in viral clearance, suggesting a local effect of PD-1/PD-L1 in the cornea. The enhanced HSV-1 clearance at 2 dpi resulting from PD-1/PD-L1 blockade is mediated primarily by a monocyte/macrophage population. Studies in bone marrow chimeras demonstrated enhanced viral clearance when PD-L1 was absent only from nonhematopoietic cells. We conclude that PD-L1 expression on corneal cells negatively impacts the ability of the innate immune system to clear HSV-1 from infected corneas.

Liver-specific deletion of augmenter of liver regeneration accelerates development of steatohepatitis and hepatocellular carcinoma in mice.

BACKGROUND & AIMS: Augmenter of liver regeneration (ALR, encoded by GFER) is a widely distributed pleiotropic protein originally identified as a hepatic growth factor. However, little is known about its roles in hepatic physiology and pathology. We created mice with liver-specific deletion of ALR to study its function. METHODS: We developed mice with liver-specific deletion of ALR (ALR-L-KO) using the albumin-Cre/LoxP system. Liver tissues were collected from ALR-L-KO mice and ALR(floxed/floxed) mice (controls) and analyzed by histology, reverse-transcription polymerase chain reaction, immunohistochemistry, electron microscopy, and techniques to measure fibrosis and lipids. Liver tissues from patients with and without advanced liver disease were determined by immunoblot analysis. RESULTS: Two weeks after birth, livers of ALR-L-KO mice contained low levels of ALR and adenosine triphosphate (ATP); they had reduced mitochondrial respiratory function and increased oxidative stress, compared with livers from control mice, and had excessive steatosis, and hepatocyte apoptosis. Levels of carbamyl-palmitoyl transferase 1a and ATP synthase subunit ATP5G1 were reduced in livers of ALR-L-KO mice, indicating defects in mitochondrial fatty acid transport and ATP synthesis. Electron microscopy showed mitochondrial swelling with abnormalities in shapes and numbers of cristae. From weeks 2-4 after birth, levels of steatosis and apoptosis decreased in ALR-L-KO mice, and numbers of ALR-expressing cells increased, along with ATP levels. However, at weeks 4-8 after birth, livers became inflamed, with hepatocellular necrosis, ductular proliferation, and fibrosis; hepatocellular carcinoma developed by 1 year after birth in nearly 60% of the mice. Hepatic levels of ALR were also low in ob/ob mice and alcohol-fed mice with liver steatosis, compared with controls. Levels of ALR were lower in liver tissues from patients with advanced alcoholic liver disease and nonalcoholic steatohepatitis than in control liver tissues. CONCLUSIONS: We developed mice with liver-specific deletion of ALR, and showed that it is required for mitochondrial function and lipid homeostasis in the liver. ALR-L-KO mice provide a useful model for investigating the pathogenesis of steatohepatitis and its complications.

Reversible nerve damage and corneal pathology in murine herpes simplex stromal keratitis.

Herpes simplex virus type 1 (HSV-1) shedding from sensory neurons can trigger recurrent bouts of herpes stromal keratitis (HSK), an inflammatory response that leads to progressive corneal scarring and blindness. A mouse model of HSK is often used to delineate immunopathogenic mechanisms and bears many of the characteristics of human disease, but it tends to be more chronic and severe than human HSK. Loss of blink reflex (BR) in human HSK is common and due to a dramatic retraction of corneal sensory nerve termini in the epithelium and the nerve plexus at the epithelial/stromal interface. However, the relationship between loss of BR due to nerve damage and corneal pathology associated with HSK remains largely unexplored. Here, we show a similar retraction of corneal nerves in mice with HSK. Indeed, we show that much of the HSK-associated corneal inflammation in mice is actually attributable to damage to the corneal nerves and accompanying loss of BR and can be prevented or ameliorated by tarsorrhaphy (suturing eyelids closed), a clinical procedure commonly used to prevent corneal exposure and desiccation. In addition, we show that HSK-associated nerve retraction, loss of BR, and severe pathology all are reversible and regulated by CD4(+) T cells. Thus, defining immunopathogenic mechanisms of HSK in the mouse model will necessitate distinguishing mechanisms associated with the immunopathologic response to the virus from those associated with loss of corneal sensation. Based on our findings, investigation of a possible contribution of nerve damage and BR loss to human HSK also appears warranted. Importance: HSK in humans is a potentially blinding disease characterized by recurrent inflammation and progressive scarring triggered by viral release from corneal nerves. Corneal nerve damage is a known component of HSK, but the causes and consequences of HSK-associated nerve damage remain obscure. We show that desiccation of the corneal surface due to nerve damage and associated loss of BR severely exacerbates and prolongs inflammation-induced pathology in mice. Preventing corneal desiccation results in a milder and more transient HSK with variable scarring that mirrors HSK seen in most humans. We further show that nerve damage is reversible and regulated by CD4(+) T cells. Thus, we provide a mouse model that more closely resembles typical human HSK and suggest nerve damage is an important but largely overlooked factor in human disease.

Early responding dendritic cells direct the local NK response to control herpes simplex virus 1 infection within the cornea.

Dendritic cells (DCs) regulate both innate and adaptive immune responses. In this article, we exploit the unique avascularity of the cornea to examine a role for local or very early infiltrating DCs in regulating the migration of blood-derived innate immune cells toward HSV-1 lesions. A single systemic diphtheria toxin treatment 2 d before HSV-1 corneal infection transiently depleted CD11c(+) DCs from both the cornea and lymphoid organs of CD11c-DTR bone marrow chimeric mice for up to 24 h postinfection. Transient DC depletion significantly delayed HSV-1 clearance from the cornea through 6 d postinfection. No further compromise of viral clearance was observed when DCs were continuously depleted throughout the first week of infection. DC depletion did not influence extravasation of NK cells, inflammatory monocytes, or neutrophils into the peripheral cornea, but it did significantly reduce migration of NK cells and inflammatory monocytes, but not neutrophils, toward the HSV-1 lesion in the central cornea. Depletion of NK cells resulted in similar loss of viral control to transient DC ablation. Our findings demonstrate that resident corneal DCs and/or those that infiltrate the cornea during the first 24 h after HSV-1 infection contribute to the migration of NK cells and inflammatory monocytes into the central cornea, and are consistent with a role for NK cells and possibly inflammatory monocytes, but not polymorphonuclear neutrophils, in clearing HSV-1 from the infected cornea.

Dendritic cell activation and memory cell development are impaired among mice administered medroxyprogesterone acetate prior to mucosal herpes simplex virus type 1 infection.

Epidemiological studies indicate that the exogenous sex steroid medroxyprogesterone acetate (MPA) can impair cell-mediated immunity, but mechanisms responsible for this observation are not well defined. In this study, MPA administered to mice 1 wk prior to HSV type 1 (HSV-1) infection of their corneal mucosa impaired initial expansion of viral-specific effector and memory precursor T cells and reduced the number of viral-specific memory T cells found in latently infected mice. MPA treatment also dampened expression of the costimulatory molecules CD40, CD70, and CD80 by dendritic cells (DC) in lymph nodes draining acute infection, whereas coculture of such DC with T cells from uninfected mice dramatically impaired ex vivo T cell proliferation compared with the use of DC from mice that did not receive MPA prior to HSV-1 infection. In addition, T cell expansion was comparable to that seen in untreated controls if MPA-treated mice were administered recombinant soluble CD154 (CD40L) concomitant with their mucosal infection. In contrast, the immunomodulatory effects of MPA were infection site dependent, because MPA-treated mice exhibited normal expansion of virus-specific T cells when infection was systemic rather than mucosal. Taken together, our results reveal that the administration of MPA prior to viral infection of mucosal tissue impairs DC activation, virus-specific T cell expansion, and development of virus-specific immunological memory.

Regulation of mouse lens maturation and gene expression by Krüppel-like factor 4.

Conditional disruption of Klf4 in the surface ectoderm-derived tissues of the eye results in defective cornea, conjunctiva and the lens. This report describes the effects of disruption of Klf4 in the lens in greater detail. Expression of Klf4, first detected in the embryonic day-12 (E12) mouse lens, peaked at E16 and was decreased in later stages. Early embryonic disruption of Klf4 resulted in a smaller lens with cortical vacuolation and nuclear opacity. Microarray comparison of Klf4CN and WT lens transcriptomes revealed fewer changes in the E16.5 (59 increases, 20 decreases of >1.5-fold) than the PN56 Klf4CN lens (239 increases, 182 decreases of >2-fold). Klf4-target genes in the lens were distinct from those previously identified in the cornea, suggesting disparate functions for Klf4 in these functionally related tissues. Transcripts encoding different crystallins were down-regulated in the Klf4CN lens. Shsp/αB-crystallin promoter activity was stimulated upon co-transfection with pCI-Klf4. Mitochondrial density was significantly higher in the Klf4CN lens epithelial cells, consistent with mitochondrial dysfunction being the most significantly affected pathway within the PN56 Klf4CN lens. The Klf4CN lens contained elevated levels of Alox12 and Alox15 transcripts, less reduced glutathione (GSH) and more oxidized glutathione (GSSG) than the WT, suggesting that it is oxidatively stressed. Although the expression of 2087 genes was modulated during WT lens maturation, transcripts encoding crystallins were abundant at E16.5 and remained stable at PN56. Among the 1065 genes whose expression increased during WT lens maturation, there were 104 Klf4-target genes (9.8%) with decreased expression in the PN56 Klf4CN lens. Taken together, these results demonstrate that Klf4 expression is developmentally regulated in the mouse lens, where it controls the expression of genes associated with lens maturation and redox homeostasis.

17-beta estradiol promotion of herpes simplex virus type 1 reactivation is estrogen receptor dependent.

Correlations between estrogen and herpes simplex virus (HSV) reactivation from latency have been suggested by numerous clinical reports, but causal associations are not well delineated. In a murine HSV-1 corneal infection model, we establish 17-beta estradiol (17-betaE) treatment of latently infected ovariectomized mice induces viral reactivation, as demonstrated by increased viral load and increased immediate-early viral gene expression in the latently infected trigeminal ganglia (TG). Interestingly, the increased HSV reactivation occurred in the absence of inhibition of viral specific CD8(+) T-cell effector function. 17-betaE administration increased HSV reactivation in CD45(+) cell-depleted TG explant cultures, providing further support that leukocyte-independent effects on latently infected neurons were responsible for the increased reactivation. The drug-induced increases in HSV copy number were not recapitulated upon in vivo treatment of latently infected estrogen receptor alpha-deficient mice, evidence that HSV reactivation promoted by 17-betaE was estrogen receptor dependent. These findings provide additional framework for the emerging conceptualization of HSV latency as a dynamic process maintained by complex interactions among multiple cooperative and competing host, viral, and environmental forces. Additional research is needed to confirm whether pregnancy or hormonal contraceptives containing 17-betaE also promote HSV reactivation from latency in an estrogen receptor-dependent manner.

Transcription Factor EepR Is Required for Serratia marcescens Host Proinflammatory Response by Corneal Epithelial Cells.

Relatively little is known about how the corneal epithelium responds to vision-threatening bacteria from the Enterobacterales order. This study investigates the impact of Serratia marcescens on corneal epithelial cell host responses. We also investigate the role of a bacterial transcription factor EepR, which is a positive regulator of S. marcescens secretion of cytotoxic proteases and a hemolytic surfactant. We treated transcriptomic and metabolomic analysis of human corneal limbal epithelial cells with wild-type bacterial secretomes. Our results show increased expression of proinflammatory and lipid signaling molecules, while this is greatly altered in eepR mutant-treated corneal cells. Together, these data support the model that the S. marcescens transcription factor EepR is a key regulator of host-pathogen interactions, and is necessary to induce proinflammatory chemokines, cytokines, and lipids.

Constitutive release of powerful antioxidant-scavenging activity by hepatic stellate cells: protection of hepatocytes from ischemia/reperfusion injury.

Within the liver, reactive oxygen species produced by infiltrating blood cells and Kupffer cells (resident macrophages) can injure hepatocytes. We hypothesized that hepatocyte survival is influenced by the relatively small juxtaposed population of hepatic stellate cells (HSCs). We used cultures of primary rat hepatocytes as targets for superoxide-induced damage, which was determined by crystal violet assay and lactate dehydrogenase release. An HSC-conditioned medium prevented the superoxide-induced death of hepatocytes, and the protective factor released by HSCs was a protein or proteins (apparent molecular weight > 100 kDa) resistant to heat (70°C) and pH (4.5-8.5). The protein or proteins were partially purified on DE52 cellulose, and the active fraction contained no detectable levels of superoxide dismutase: after separation by Sephadex G-100 gel filtration, the antioxidant activity could be reconstituted by the combination of 2 protein peaks, and this reconstituted activity was protective both in vitro and against liver ischemia/reperfusion injury in intact rats. Mass spectrometry proteomic studies confirmed that this activity could not be attributed to any previously identified antioxidant protein. Thus, HSCs protect hepatocytes against oxidative damage through the production of a novel protein, the further purification of which may lead to the isolation of a powerful oxygen radical scavenger with clinical applications.

Medroxyprogesterone acetate inhibits CD8+ T cell viral-specific effector function and induces herpes simplex virus type 1 reactivation.

Clinical research suggests hormonal contraceptive use is associated with increased frequencies of HSV reactivation and shedding. We examined the effects of medroxyprogesterone acetate (MPA), the compound most commonly used for injectable hormonal contraception, on HSV type 1 (HSV-1) reactivation and CD8(+) T cell function in murine trigeminal ganglia (TG). In ex vivo TG cultures, MPA dramatically inhibited canonical CD8(+) T cell effector functions, including IFN-gamma production and lytic granule release, and increased HSV-1 reactivation from latency. In vivo, MPA treatment of latently infected ovariectomized mice inhibited IFN-gamma production and lytic granule release by TG resident CD8(+) T cells stimulated directly ex vivo. RNA specific for the essential immediate early viral gene ICP4 as well as viral genome DNA copy number were increased in mice that received MPA during latency, suggesting that treatment increased in vivo reactivation. The increase in HSV-1 copy number appeared to be the result of a two-tine effect, as MPA induced higher reactivation frequencies from latently infected explanted TG neurons in the presence or absence of CD45(+) cells. Our data suggest hormonal contraceptives that contain MPA may promote increased frequency of HSV reactivation from latency through the combinatory effects of inhibiting protective CD8(+) T cell responses and by a leukocyte-independent effect on infected neurons.

Gene expression profiling of Epstein-Barr virus-positive and -negative monomorphic B-cell posttransplant lymphoproliferative disorders.

Although most posttransplant lymphoproliferative disorders (PTLD) are related to Epstein-Barr virus (EBV) infection, approximately 20% lack detectable EBV (EBV-). It is uncertain whether the latter cases are truly distinct from EBV+ PTLD or possibly relate to another infectious agent. This study used gene expression profiling to further investigate the relationship between EBV+ and EBV- monomorphic B-cell PTLD, and to search for clues to their pathogenesis. Affymetrix HU133A GeneChips were used to compare 4 EBV+ and 4 EBV- cases of monomorphic B-cell PTLD. Hierarchical clustering successfully distinguished the EBV+ and EBV- groups. Relative to EBV- PTLD, 54 transcripts were over-expressed in EBV+ PTLD. The transcripts identified included IRF7 (a known regulator of EBV LMP1 expression), EBI2 (EBV-induced gene 2), and 3 that are interferon induced (MX1, IFITM1, and IFITM3). In addition, the EBV+ group contained 232 transcripts decreased relative to the EBV- group, including changes concordant with those previously reported after EBV infection of cultured B-cell lines. In summary, in a small group of monomorphic B-cell PTLD, EBV+ cases demonstrated a subset of gene expression changes associated with EBV infection of B cells. By contrast, EBV- PTLD lacked viral-associated changes suggesting that they are biologically distinct.

Role of hippocampal CA3 mu-opioid receptors in spatial learning and memory.

The dorsal CA3 region of the hippocampus is unique in its connectivity, sensitivity to neurotoxic lesions, and its ability to encode and retrieve episodic memories. Computational models of the CA3 region predict that blocking mossy-fiber and/or perforant path activity to CA3 would cause impairments in learning and recall of spatial memory, respectively. Because the CA3 region contains micro-opioid receptors and receives inputs from the mossy-fiber and lateral perforant pathways, both of which contain and release opioid peptides, we tested the hypothesis that inactivating micro-opioid receptors in the CA3 region would cause spatial learning and memory impairments and retrieval deficits. In this study, male Sprague Dawley rats were trained in a Morris water maze after a single bilateral intrahippocampal injection of either saline or the selective and irreversible micro-opioid receptor antagonist beta-funaltrexamine (beta-FNA) into area CA3. We found that micro-opioid receptor binding decreased 24 hr after beta-FNA injection and returned to control levels 11 d after injection. Injections of beta-FNA into the CA3 region, but not into the ventricles, caused a significant impairment in the acquisition of spatial learning without causing sensory or motor deficits. New learning was not affected once micro-opioid receptor levels replenished (>11 d after injection). In pretrained animals, beta-FNA significantly impaired spatial memory retrieval and new (reversal) learning. These data are consistent with theoretical models of CA3 function and suggest that CA3 micro-opioid receptors play an important role in the acquisition and retrieval of spatial memory.

Adenovirus-directed ocular innate immunity: the role of conjunctival defensin-like chemokines (IP-10, I-TAC) and phagocytic human defensin-alpha.

PURPOSE: Adenovirus (Ad), a major cause of viral conjunctivitis worldwide, is controlled initially by the innate immune response on the ocular surface. In the present study, cultured human conjunctival epithelial cells were used to identify host genes responsive to infection by a clinical isolate of Ad5, and the antiviral activity of some of their peptide products was investigated. METHODS: Primary human conjunctival epithelial cells in culture were infected with Ad5 at high (1.0), low (0.1), or zero (0.0) multiplicities of infection (MOI) and harvested at different times after infection (1.5, 6, and 16 hours). Host gene expression was profiled with oligonucleotide microarrays (GeneChips; Affymetrix, Santa Clara, CA). Peptide products of interest were tested for antiviral activity in direct inhibition assays against respiratory serotypes (Ad3, Ad5), ocular serotypes (Ad8, Ad19), and HSV-1. RESULTS: At high MOI, viral infection suppressed the interferon (IFN)-mediated host antiviral response seen at low MOI. At 16 hours after infection, 63 unique characterized transcripts were identified that were robustly and significantly suppressed by high MOI, (i.e., MOI [0.1]/MOI [0.1] > or = 2, P < 0.006). Of these, 29 (46%) transcripts are involved in IFN signaling or are IFN or virus induced. This subset included CXCL10 and CXCL11, encoding IP-10 and I-TAC, respectively. These defensin-like chemokines and human alpha-defensin-1 directly inhibited Ad3 and Ad5 but not Ad8 or Ad19. CONCLUSIONS: In response to low-level Ad5 infection, conjunctival epithelial cells showed upregulation of IFN-associated genes. The peptide products of two of these, IP-10 and I-TAC, are directly active against Ad3, and IP-10 is active against Ad5. The ocular tropism of Ad8 and Ad19 may be due in part to their resistance to these defensin-like chemokines.

Corneal Expression of SLURP-1 by Age, Sex, Genetic Strain, and Ocular Surface Health.

PURPOSE: Although secreted Ly6/urokinase-type plasminogen activator receptor-related protein-1 (Slurp1) transcript is highly abundant in the mouse cornea, corresponding protein expression remains uncharacterized. Also, SLURP1 was undetected in previous tear proteomics studies, resulting in ambiguity about its baseline levels. Here, we examine mouse corneal Slurp1 expression in different sexes, age groups, strains, and health conditions, and quantify SLURP1 in human tears from healthy or inflamed ocular surfaces. METHODS: Expression of Slurp1 in embryonic day-13 (E13), E16, postnatal day-1 (PN1), PN10, PN20, and PN70 Balb/C, FVBN, C57Bl/6, and DBA/2J mouse corneas, Klf4Δ/ΔCE corneas with corneal epithelial-specific ablation of Klf4, migrating cells in wild-type corneal epithelial wound edge, and in corneas exposed to pathogen-associated molecular patterns (PAMPs) poly(I:C), zymosan-A, or Pam3Csk4 was examined by QPCR, immunoblots, and immunofluorescent staining. Human SLURP1 levels were quantified by ELISA in tears from 34 men and women aged 18 to 80 years. RESULTS: Expression of Slurp1, comparable in different strains and sexes, was low in E13, E16, PN1, and PN10 mouse corneas, and increased rapidly after eyelid opening in a Klf4-dependent manner. We found Slurp1 was downregulated in corneas exposed to PAMPs, and in migrating cells at the wound edge. Human SLURP1 expression, comparable in different sexes and age groups, was significantly decreased in tears from inflamed ocular surfaces (0.34%) than those from healthy individuals (0.77%). CONCLUSIONS: These data describe the influence of age, sex, genetic background, and ocular surface health on mouse corneal expression of Slurp1, establish the baseline for human tear SLURP1 expression, and identify SLURP1 as a useful diagnostic and/or therapeutic target for inflammatory ocular surface disorders.

Neuronal changes induced by Varicella Zoster Virus in a rat model of postherpetic neuralgia.

A significant fraction of patients with herpes zoster, caused by Varicella Zoster Virus (VZV), experience chronic pain termed postherpetic neuralgia (PHN). VZV-inoculated rats develop prolonged nocifensive behaviors and serve as a model of PHN. We demonstrate that primary rat cultures show a post-entry block for VZV replication, suggesting the rat is not fully permissive. However, footpads of VZV infected animals show reduced peripheral innervation and innervating dorsal root ganglia (DRG) contained VZV DNA and transcripts of candidate immediate early and early genes. The VZV-infected DRG showed changes in host gene expression patterns, with 84 up-regulated and 116 down-regulated genes seen in gene array studies. qRT-PCR validated the modulation of nociception-associated genes Ntrk2, Trpv1, and Calca (CGRP). The data suggests that VZV inoculation of the rat results in a single round, incomplete infection that is sufficient to induce pain behaviors, and this involves infection of and changes induced in neuronal populations.

Downstream effects of ROCK signaling in cultured human corneal stromal cells: microarray analysis of gene expression.

PURPOSE: Rho-associated coiled-coil-containing protein kinase (ROCK) is a downstream target of Rho GTPase signaling and regulates the assembly of stress fibers. Previous reports indicate that Rho/ROCK signaling is involved in the regulation of several cellular processes, some of which may be cell-type specific and are probably critical to corneal stromal cell activation. The present study identified ROCK-regulated gene expression in corneal stromal cells. METHODS: Corneal stromal cells derived from eyes of three different donors were cultured to yield the following designated phenotypes: baseline fibroblasts (DMEM with 10% serum), activated fibroblasts (10% serum+bFGF+heparin), and myofibroblasts (1% serum+TGF-beta 1). Cells were exposed to the ROCK inhibitor Y-27632 or vehicle for 12 hours, and transcript levels altered by ROCK inhibition were identified with oligonucleotide microarrays (GeneChips; Affymetrix, Santa Clara, CA). RESULTS: In these phenotypes, Y-27632 caused marked (twofold or more) increases or decreases in 14/4, 12/3, and 15/10 transcripts. In both fibroblast groups Y-27632-treatment increased expression of endothelin receptors and of parathyroid hormone-like hormone. The upregulation of alpha-smooth muscle actin in myofibroblasts was attenuated by Y-27632. Combining data from all groups identified ROCK-supported (Y-27632 inhibitable) expression of 10 transcripts, including ribonucleotide reductase M2, the cyclin B1-CDC2-CKS2 system, and four mitotic spindle-associated proteins. CONCLUSIONS: ROCK inhibition causes broad inhibition of DNA synthesis and mitosis and causes changes that are different between (bFGF-activated) fibroblasts and (TGF-beta 1-induced) myofibroblasts. Thus, Rho/ROCK signaling regulates both common and distinct downstream events in corneal stromal cells activated (differentiated) to fibroblast or myofibroblast phenotype.

Splenectomy promotes indirect elimination of intraocular tumors by CD8+ T cells that is associated with IFNγ- and Fas/FasL-dependent activation of intratumoral macrophages.

Ocular immune privilege (IP) limits the immune surveillance of intraocular tumors as certain immunogenic tumor cell lines (P815, E.G7-OVA) that are rejected when transplanted in the skin grow progressively when placed in the anterior chamber of the eye. As splenectomy (SPLNX) is known to terminate ocular IP, we characterized the immune mechanisms responsible for rejection of intraocular tumors in SPLNX mice as a first step toward identifying how to restore tumoricidal activity within the eye. CD8(+) T cells, IFNγ, and FasL, but not perforin, or TNFα were required for the elimination of intraocular E.G7-OVA tumors that culminated in destruction of the eye (ocular phthisis). IFNγ and FasL did not target tumor cells directly as the majority of SPLNX IFNγR1(-/-) mice and Fas-defective lpr mice failed to eliminate intraocular E.G7-OVA tumors that expressed Fas and IFNγR1. Bone marrow chimeras revealed that IFNγR1 and Fas expression on immune cells was most critical for rejection, and SPLNX increased the frequency of activated macrophages (Mϕ) within intraocular tumors in an IFNγ- and Fas/FasL-dependent manner, suggesting an immune cell target of IFNγ and Fas. As depletion of Mϕs limited CD8 T cell-mediated rejection of intraocular tumors in SPLNX mice, our data support a model in which IFNγ- and Fas/FasL-dependent activation of intratumoral Mϕs by CD8(+) T cells promotes severe intraocular inflammation that indirectly eliminates intraocular tumors by inducing phthisis, and suggests that immunosuppressive mechanisms that maintain ocular IP interfere with the interaction between CD8(+) T cells and Mϕs to limit the immunosurveillance of intraocular tumors.

The transcriptomic response of rat hepatic stellate cells to endotoxin: implications for hepatic inflammation and immune regulation.

With their location in the perisinusoidal space of Disse, hepatic stellate cells (HSCs) communicate with all of the liver cell types both by physical association (cell body as well as cytosolic processes penetrating into sinusoids through the endothelial fenestrations) and by producing several cytokines and chemokines. Bacterial lipopolysaccharide (LPS), circulating levels of which are elevated in liver diseases and transplantation, stimulates HSCs to produce increased amounts of cytokines and chemokines. Although recent research provides strong evidence for the role of HSCs in hepatic inflammation and immune regulation, the number of HSC-elaborated inflammatory and immune regulatory molecules may be much greater then known at the present time. Here we report time-dependent changes in the gene expression profile of inflammatory and immune-regulatory molecules in LPS-stimulated rat HSCs, and their validation by biochemical analyses. LPS strongly up-regulated LPS-response elements (TLR2 and TLR7) but did not affect TLR4 and down-regulated TLR9. LPS also up-regulated genes in the MAPK, NFκB, STAT, SOCS, IRAK and interferon signaling pathways, numerous CC and CXC chemokines and IL17F. Interestingly, LPS modulated genes related to TGFß and HSC activation in a manner that would limit their activation and fibrogenic activity. The data indicate that LPS-stimulated HSCs become a major cell type in regulating hepatic inflammatory and immunological responses by altering expression of numerous relevant genes, and thus play a prominent role in hepatic pathophysiology including liver diseases and transplantation.

Human female genital tract infection by the obligate intracellular bacterium Chlamydia trachomatis elicits robust Type 2 immunity.

While Chlamydia trachomatis infections are frequently asymptomatic, mechanisms that regulate host response to this intracellular Gram-negative bacterium remain undefined. This investigation thus used peripheral blood mononuclear cells and endometrial tissue from women with or without Chlamydia genital tract infection to better define this response. Initial genome-wide microarray analysis revealed highly elevated expression of matrix metalloproteinase 10 and other molecules characteristic of Type 2 immunity (e.g., fibrosis and wound repair) in Chlamydia-infected tissue. This result was corroborated in flow cytometry and immunohistochemistry studies that showed extant upper genital tract Chlamydia infection was associated with increased co-expression of CD200 receptor and CD206 (markers of alternative macrophage activation) by endometrial macrophages as well as increased expression of GATA-3 (the transcription factor regulating TH2 differentiation) by endometrial CD4(+) T cells. Also among women with genital tract Chlamydia infection, peripheral CD3(+) CD4(+) and CD3(+) CD4(-) cells that proliferated in response to ex vivo stimulation with inactivated chlamydial antigen secreted significantly more interleukin (IL)-4 than tumor necrosis factor, interferon-γ, or IL-17; findings that repeated in T cells isolated from these same women 1 and 4 months after infection had been eradicated. Our results thus newly reveal that genital infection by an obligate intracellular bacterium induces polarization towards Type 2 immunity, including Chlamydia-specific TH2 development. Based on these findings, we now speculate that Type 2 immunity was selected by evolution as the host response to C. trachomatis in the human female genital tract to control infection and minimize immunopathological damage to vital reproductive structures.

Use of transcriptional profiling to delineate the initial response of mice to intravaginal herpes simplex virus type 2 infection.

Intravaginal (ivag) infection of mice with herpes simplex virus type 2 (HSV-2) causes genital tissue damage, quickly followed by development of fatal encephalopathy. To delineate initial host responses generated by HSV-2 infection, here oligonucleotide microarrays compared gene expression in vaginal tissue from uninfected mice and mice 1, 2, 3, 4, 5, 6, or 7 days after ivag infection with 10(4) pfu HSV-2. While comparison of mRNA expression in uninfected and HSV-infected vaginal tissue detected few changes during the first 2 days post infection (dpi), there were 156 genes whose expression was first significantly altered 3 dpi that remained significantly modified at all later time points examined. These 156 genes were significantly enriched in canonical pathways associated with interferon (IFN) signaling, activation of IFN elements by intracellular pattern recognition receptors, and antiviral immunity induced by cytosolic RIG-like receptors. Evaluation of this gene set with the National Center for Biotechnology Information Gene and INTERFEROME databases corroborated pathway analysis, as function of most (53%) were linked to IFN-mediated host immunity. In the final set of experiments, ivag administration of the Toll-like receptor 3 agonist polyinosinic: polycytidylic acid (poly I:C) 24 h before ivag HSV-2 infection reduced the incidence of genital pathology and encephalopathy, while these poly I:C-treated mice were subsequently protected from ocular HSV-2 challenge lethal to uninfected controls. The latter results imply that the exuberant antiviral immunity produced in our experimental model is simply formed too late to prevent viral replication and dissemination, and that poly I:C-induced formation of an antiviral state protecting against primary ivag infection also permits development of HSV-specific protective immunity.