Endothelial NLRP3 Inflammasome Activation and Enhanced Neointima Formation in Mice by Adipokine Visfatin

Inflammasomes serve as an intracellular machinery to initiate inflammatory response to various danger signals. The present study tested whether an inflammasome centered on nucleotide oligomerization domain-like receptor protein 3 (NLRP3) triggers endothelial inflammatory response to adipokine visfatin, a major injurious adipokine during obesity. NLRP3 inflammasome components were abundantly expressed in cultured mouse microvascular endothelial cells, including NLRP3, apoptosis-associated speck-like protein, and caspase-1. These NLRP3 inflammasome molecules could be aggregated to form an inflammasome complex on stimulation of visfatin, as shown by fluorescence confocal microscopy and size exclusion chromatography. Correspondingly, visfatin significantly increased caspase-1 activity and IL-1β release in microvascular endothelial cells, indicating an activation of NLRP3 inflammasomes. In animal experiments, direct infusion of visfatin in mice with partially ligated left carotid artery were found to have significantly increased neointimal formation, which was correlated with increased NLRP3 inflammasome formation and IL-1β production in the intima. Further, visfatin-induced neointimal formation, endothelial inflammasome formation, and IL-1β production in mouse partially ligated left carotid artery were abolished by caspase-1 inhibition, local delivery of apoptosis-associated speck-like protein shRNA or deletion of the ASC gene. In conclusion, the formation and activation of NLRP3 inflammasomes by adipokine visfatin may be an important initiating mechanism to turn on the endothelial inflammatory response leading to arterial inflammation and endothelial dysfunction in mice during early stage obesity.Obesity is a major risk factor for cardiovascular disease and has been strongly associated with endothelial dysfunction and coronary atherosclerosis. Obese patients have significantly elevated morbidity and mortality due to coronary artery disease.¹ However, weight loss can decrease cardiovascular risk, improve endothelial function, and protect coronary arteries from atherosclerotic injury. However, mechanisms underlying obesity-associated coronary atherosclerotic injury and endothelial dysfunction are not fully understood. Numerous studies have reported a critical role of vascular inflammation in the development of coronary atherosclerosis, which has been characterized as an inflammatory disease.^2–6 To date, the precise mechanism that mediates the early inflammatory responses of endothelial cells (ECs) during obesity remains unknown.Recently, the inflammasome as an intracellular inflammatory machinery has been reported to switch on the inflammatory response of tissues or organs to various danger signals.^7,8 Among different types of inflammasomes, the nucleotide oligomerization domain (Nod)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is well characterized in a variety of mammalian cells, especially as a receptor for endogenous danger signals such as ATP, cholesterol crystal, β-amyloid, and monosodium urate.^2,9–14 The NLRP3 inflammasome is characteristic of a proteolytic complex mainly composed of NLRP3, the adaptor protein apoptosis-associated speck-like protein (ASC), and caspase-1. On stimulation, NLRP3 inflammasomes oligomerize to form large multimolecular complexes that control the caspase-1 activity and subsequent bioactive IL-1β production.^10,15–18 More recently, NLRP3 inflammasomes have been implicated in the development of obesity and insulin resistance.¹¹ For example, the consumption of a high-fat diet (HFD) has been considered as critical contributor to type 2 diabetes, and NLRP3 inflammasome might be an important pathway of HFD mediating insulin resistance leading to inflammation.¹⁹ These findings led us to wonder whether activation of NLRP3 inflammasome is an initiating mechanism for obesity-induced endothelial inflammatory responses.Adipose tissue as an active metabolic tissue secretes multiple metabolically important proteins known as ‘adipokines.''^20,21 Visfatin is a newly identified adipokine and a major injurious factor during obesity-associated diseases, including diabetes,²² carotid and coronary atherosclerosis,^23,24 and chronic kidney disease.^25,26 Visfatin has also been considered as a pro-inflammatory adipokine to promote endothelial inflammation and injury.^27,28 The present study was designed to test the hypothesis that activation of NLRP3 inflammasomes is one of the important mechanisms that mediate endothelial inflammatory response to visfatin during early-stage obesity. We used a series of molecular and physiological approaches both in vitro and in vivo to test this hypothesis.     

αB-Crystallin Regulates Subretinal Fibrosis by Modulation of Epithelial-Mesenchymal Transition

Subretinal fibrosis is an end stage of neovascular age-related macular degeneration, characterized by fibrous membrane formation after choroidal neovascularization. An initial step of the pathogenesis is an epithelial-mesenchymal transition (EMT) of retinal pigment epithelium cells. αB-crystallin plays multiple roles in age-related macular degeneration, including cytoprotection and angiogenesis. However, the role of αB-crystallin in subretinal EMT and fibrosis is unknown. Herein, we showed attenuation of subretinal fibrosis after regression of laser-induced choroidal neovascularization and a decrease in mesenchymal retinal pigment epithelium cells in αB-crystallin knockout mice compared with wild-type mice. αB-crystallin was prominently expressed in subretinal fibrotic lesions in mice. In vitro, overexpression of αB-crystallin induced EMT, whereas suppression of αB-crystallin induced a mesenchymal-epithelial transition. Transforming growth factor-β2–induced EMT was further enhanced by overexpression of αB-crystallin but was inhibited by suppression of αB-crystallin. Silencing of αB-crystallin inhibited multiple fibrotic processes, including cell proliferation, migration, and fibronectin production. Bone morphogenetic protein 4 up-regulated αB-crystallin, and its EMT induction was inhibited by knockdown of αB-crystallin. Furthermore, inhibition of αB-crystallin enhanced monotetraubiquitination of SMAD4, which can impair its nuclear localization. Overexpression of αB-crystallin enhanced nuclear translocation and accumulation of SMAD4 and SMAD5. Thus, αB-crystallin is an important regulator of EMT, acting as a molecular chaperone for SMAD4 and as its potential therapeutic target for preventing subretinal fibrosis development in neovascular age-related macular degeneration.Age-related macular degeneration (AMD) is a leading cause of blindness because of progressive degeneration of the macular region of the retina that is responsible for visual acuity and color vision. The natural history of AMD is a progression from its early stage to the two forms of late stage of AMD: geographic atrophy and neovascular AMD (nAMD).¹The visual prognosis for nAMD is poor; the condition progresses rapidly with the development of choroidal neovascularization (CNV) and subsequent subretinal fibrosis. Although the commonly used treatment with anti-vascular endothelial growth factor (VEGF) drugs improves visual acuity in nAMD patients, the subretinal scarring (fibrosis) that may develop in approximately half of all anti–VEGF-treated eyes within 2 years has been identified as a cause of unsuccessful outcomes.² Thus, therapeutic strategies for the inhibition of subretinal fibrosis are currently an active area of investigation. Among the critical growth factors involved in subretinal fibrosis, platelet-derived growth factor (PDGF) is a potential therapeutic target. Currently, several clinical trials for the treatment for nAMD have been evaluating the efficacy of dual VEGF/PDGF inhibitors, such as the following: {"type":"entrez-nucleotide","attrs":{"text":"E10030","term_id":"22026652","term_text":"E10030"}}E10030 (Ophthotech, New York, NY), an anti-PDGF pegylated aptamer as an adjunct to anti-VEGF therapy; sorafenib, an inhibitor of VEGF receptor, PDGF receptor, and Raf kinases; and pazopanib, an inhibitor of VEGF receptor, PDGF receptor, and c-kit.³ In addition, it has been shown that nucleotide-binding oligomerization domain-, leucine-rich repeat domain-, and pyrin domain–containing 3 inflammasome activation is implicated in the pathogenesis of nAMD.⁴ The recent implication of inflammasome activation in the pathogenesis of hepatic fibrosis⁵ suggests that the inflammasome should be further evaluated for its potential role in subretinal fibrosis. Important inflammasome effector cytokines, IL-1β and IL-18, can be potential therapeutic targets for the treatment of subretinal fibrosis in nAMD. In support of this contention, inhibition of IL-1β has been shown to inhibit the development of experimental CNV in mice.⁶Subretinal fibrosis results from an excessive wound healing response, characterized by fibrous membrane formation after CNV. In fibrous membranes, the main cellular components are myofibroblasts, the cells immunoreactive for α-smooth muscle actin (α-SMA). Previous histological studies imply that the source of myofibroblasts can be both bone marrow–derived cells and retinal pigment epithelium (RPE) cells.^{7, 8} After injury to RPE, the cells undergo epithelial-mesenchymal transition (EMT), which enables transdifferentiation, resulting in the conversion of epithelial cells to myofibroblasts. CNV induction can result in the recruitment of more inflammatory cells and fibroblasts, which can be a direct or indirect source of additional myofibroblasts. Myofibroblasts play important roles in the development of subretinal fibrosis, such as proliferation, migration, and extracellular matrix remodeling.⁸ Although previous studies have indicated the involvement of several growth factors and cytokines in EMT,⁸ the precise molecular mechanism and the critical regulators of this process remain to be determined.The soluble cytoplasmic protein αB-crystallin is a prominent member of the small heat shock protein family. The small heat shock proteins exert diverse biological activities in both normal and stressed cells. They can act as molecular chaperones by binding misfolded proteins to prevent their denaturation and aggregation.⁹ αB-crystallin can bind to and stabilize cytoskeleton proteins, such as desmin and actin, and help to maintain cytoskeletal integrity.¹⁰ The role of αB-crystallin in EMT in liver and lung fibrosis has been recently reported.^{11, 12}Our previous work has suggested an important role for αB-crystallin in both the early and late stages of AMD. The early stage of AMD is characterized by the accumulation of drusen between the RPE and Bruch''s membrane, accompanied by RPE cell death and synaptic dysfunction.¹³ Geographic atrophy is caused by extensive atrophy and loss of the RPE and the overlying photoreceptors that rely on the RPE for trophic support.¹ αB-crystallin can be seen in RPE, associated with drusen and identified as one of the components of drusen.^{14, 15, 16} Our laboratory has shown that RPE cells lacking αB-crystallin are more susceptible to oxidative and endoplasmic reticulum stress compared with normal RPE.^{17, 18, 19, 20} Furthermore, RPE-overexpressing αB-crystallin shows resistance to apoptosis.²¹ These findings suggest that αB-crystallin plays a cytoprotective role against multiple stress stimuli that can cause RPE cell death, resulting in drusen formation and geographic atrophy. In addition, we previously demonstrated that αB-crystallin plays a regulatory role by functioning as a chaperone for VEGF in ocular angiogenesis and may play a part in CNV formation in nAMD.²² However, the involvement of αB-crystallin in subretinal fibrosis in nAMD has not been studied.Herein, we examined the pathogenesis of subretinal fibrosis in αB-crystallin^−/− and wild-type (WT) mice; we further investigated the role of αB-crystallin in EMT and fibrotic process in cultured RPE cells.     

The Epithelial Danger Signal IL-1α Is a Potent Activator of Fibroblasts and Reactivator of Intestinal Inflammation

Intestinal epithelial cell (IEC) death is typical of inflammatory bowel disease (IBD). We investigated: i) whether IEC–released necrotic cell products (proinflammatory mediators) amplify mucosal inflammation, ii) the capacity of necrotic cell lysates from HT29 cells or human IECs to induce human intestinal fibroblasts'' (HIF) production of IL-6 and IL-8, and iii) whether IL-1α, released by injured colonocytes, exacerbated experimental IBD. Necrotic cell lysates potently induced HIF IL-6 and IL-8 production independent of Toll-like receptors 2 and 4, receptor for advanced glycation end-products, high-mobility group box 1, uric acid, IL-33, or inflammasome activation. IL-1α was the key IEC-derived necrotic cell product involved in HIF cytokine production. IL-1α–positive cells were identified in the epithelium in human IBD and dextran sulfate sodium (DSS)-induced colitis. IL-1α was detected in the stool of colitic mice before IL-1β. IL-1α enemas reactivated inflammation after DSS colitis recovery, induced IL-1 receptor expression in subepithelial fibroblasts, and activated de novo inflammation even in mice without overt colitis, after the administration of low-dose DSS. IL-1α amplifies gut inflammation by inducing cytokine production by mesenchymal cells. IL-1α–mediated IEC–fibroblast interaction may be involved in amplifying and perpetuating inflammation, even without obvious intestinal damage. IL-1α may be a target for treating early IBD or preventing the reactivation of IBD.Intestinal epithelial cell (IEC) injury and death are extremely common events but only recently have been recognized as drivers of inflammation.^1–3 Excessive cell death results in barrier defects and uncontrolled bacterial translocation that together induce and sustain gut inflammation.^1,4 In addition, epithelial cells contain a myriad of intracellular substances normally not recognized by the immune system but, during cell necrosis, they are passively released in the surrounding microenvironment and trigger inflammation. This response may represent a novel pathogenic component of inflammatory bowel disease (IBD), since epithelial damage is a typical feature of both ulcerative colitis and Crohn disease.^5,6 Cell products released by cells undergoing necrosis (passive, programed, or after apoptosis) are called damage-associated molecular patterns (DAMPs) or alarmins as they alert the immune system and trigger a sterile inflammatory response.⁷ These events give rise to the danger model of immunity, which suggests that inflammation is preferentially geared to defend against host damage rather than foreign signals.^8,9DAMPs represent the endogenous counterpart of exogenous pathogen-associated molecular patterns.¹⁰ DAMP signals are likely to converge with microbe-derived pathogen-associated molecular patterns to amplify inflammation, as they share various receptors and elicit similar responses.^11,12 This integration is especially important to the microbiota-rich gut microenvironment, where plentiful and diversified signals that mediate key cell interactions in IBD are elicited. Evidence of the involvement of DAMPs in IBD pathogenesis is limited. IBD tissue releases abundant S100A12, S100A8/S100A9 complexes (calprotectin)¹³ and high-mobility group box 1 (HMGB-1), which serve as fecal biomarkers of intestinal inflammation.¹⁴ Hmgb-1 levels are elevated in Il10^−/− and in dextran sulfate sodium (DSS)-induced murine colitis; blockade of Hmgb-1 ameliorates inflammation.^15,16IL-1α has been recognized as a major product of epidermal keratinocytes and enterocytes for some time^17,18 but has only recently emerged as a major DAMP and inducer of sterile inflammation.^19–22 This prototypical member of the IL-1 family is involved in the pathogenesis of several acute and chronic inflammatory diseases and is expressed in most cells, including IECs. However, unlike its other family member IL-1β, little information is available on the mechanisms by which IL-1α may function as a DAMP in intestinal inflammation.^23,24We report here that epithelial cell–derived DAMPs elicit a potent proinflammatory cytokine response from human intestinal fibroblasts (HIFs). These cells are active participants in intestinal inflammation,²⁵ acting as first responders to products of epithelial cell necrosis due to their anatomical proximity. Among a number of potential DAMPs, our data indicate that IL-1α appears to be the major alarmin involved in the induction of proinflammatory cytokine production by fibroblasts. We also present evidence that IL-1α is an early mediator and reactivator of intestinal injury in vivo in experimental colitis, suggesting a key role in the pathogenesis of IBD.     

Synaptic Amyloid-β Oligomers Precede p-Tau and Differentiate High Pathology Control Cases

Amyloid-β (Aβ) and hyperphosphorylated tau (p-tau) aggregates form the two discrete pathologies of Alzheimer disease (AD), and oligomeric assemblies of each protein are localized to synapses. To determine the sequence by which pathology appears in synapses, Aβ and p-tau were quantified across AD disease stages in parietal cortex. Nondemented cases with high levels of AD-related pathology were included to determine factors that confer protection from clinical symptoms. Flow cytometric analysis of synaptosome preparations was used to quantify Aβ and p-tau in large populations of individual synaptic terminals. Soluble Aβ oligomers were assayed by a single antibody sandwich enzyme-linked immunosorbent assay. Total in situ Aβ was elevated in patients with early- and late-stage AD dementia, but not in high pathology nondemented controls compared with age-matched normal controls. However, soluble Aβ oligomers were highest in early AD synapses, and this assay distinguished early AD cases from high pathology controls. Overall, synapse-associated p-tau did not increase until late-stage disease in human and transgenic rat cortex, and p-tau was elevated in individual Aβ-positive synaptosomes in early AD. These results suggest that soluble oligomers in surviving neocortical synaptic terminals are associated with dementia onset and suggest an amyloid cascade hypothesis in which oligomeric Aβ drives phosphorylated tau accumulation and synaptic spread. These results indicate that antiamyloid therapies will be less effective once p-tau pathology is developed.A large body of evidence indicates that soluble oligomers of amyloid-β (Aβ) are the primary toxic peptides that initiate downstream tau pathology in the amyloid cascade hypothesis of Alzheimer disease (AD).^{1, 2} However, the time course and severity of AD dementia have been generally found to correlate with neurofibrillary tangle development rather than plaque appearance,^{3, 4, 5, 6, 7, 8} although a few studies have linked plaques with early cognitive decline.^{9, 10, 11, 12} Soluble oligomeric Aβ has been highlighted as the primary toxin for loss of dendritic spines and synaptic function¹³ and has also been directly linked to downstream tau pathology. For example, suppression of a tau kinase pathway can prevent Aβ42 oligomer-induced dendritic spine loss,¹⁴ and injection of Aβ42 fibrils into mutant tau mice induces neurofibrillary tangles in cell bodies retrograde to the injections.¹⁵ In vivo, effects of Aβ oligomers versus fibrils are harder to separate; however, lowering soluble Aβ oligomers by halving β–site amyloid precursor protein (APP) cleaving enzyme reduces accumulation and phosphorylation of wild-type tau in a mouse model.¹⁶ Evidence for Aβ and tau association is particularly strong in the dendritic compartment, where tau was shown to mediate Aβ toxicity via linkage of fyn to downstream N-methyl-d-aspartate receptor toxicity.¹⁷The earliest cognitive losses in AD have long been thought to correlate with synapse loss.^{8, 18, 19, 20, 21} In humans, electron microscopic studies have documented synapse-associated Aβ and tau,^{22, 23} and much work documents activity-dependent release of synaptic Aβ into interstitial fluid, which drives local Aβ deposition in human subjects and in rodents.^{4, 24, 25} Of importance, most synapse-associated Aβ in cortical synapses of AD patients consists of soluble oligomeric species,²⁶ and synaptic tau pathology in AD also includes accumulations of SDS-stable tau oligomers.^{27, 28, 29, 30, 31} With the use of synaptosomes (resealed nerve terminals) from the cortex of postmortem human subjects and a transgenic rat model of AD, the present experiments were aimed at determining the sequence of appearance of Aβ and hyperphosphorylated tau (p-tau) pathology in synaptic terminals. In addition to early- and late-stage disease, the AD samples included nondemented high pathology controls (HPCs) with substantial AD-related pathology. Synaptic accumulation of Aβ occurred in the earliest plaque stages, before the appearance of synaptic p-tau, which did not appear until late-stage disease. Soluble Aβ oligomers in synaptic terminals were elevated in early AD cases compared with HPCs, indicating an association with the onset of a dementia diagnosis.     

Interleukin-11 in Endometrial Adenocarcinoma Is Regulated by Prostaglandin F2α-F-Prostanoid Receptor Interaction via the Calcium-Calcineurin-Nuclear Factor of Activated T Cells Pathway and Negatively Regulated by the Regulator of Calcineurin-1

Interleukin-11 (IL-11) up-regulates the proliferative and invasive capacity of many cancers. Coexpression of glycoprotein 130 (GP130) and IL-11 receptor α (IL-11Rα) is necessary for high-affinity binding of IL-11 to IL-11Rα. This study investigated the expression of IL-11 and role of prostaglandin F_2α-F-prostanoid receptor (FP receptor) signaling in the modulation of IL-11 expression in endometrial adenocarcinoma cells. Localization of IL-11, IL-11Rα, and GP130 expression was performed by immunohistochemistry. IL-11 and regulator of calcineurin 1 isoform 4 (RCAN1-4) mRNA and protein expression were determined by real-time RT-PCR and/or enzyme-linked immunosorbent assay/Western blot analysis using Ishikawa endometrial adenocarcinoma cells stably expressing the FP receptor (FPS cells) and endometrial adenocarcinoma explants. IL-11 mRNA expression was significantly elevated in endometrial adenocarcinoma samples compared with normal endometrium and increased with tumor grade. IL-11 protein expression localized with FP receptor, IL-11Rα, and GP130 in the neoplastic glandular epithelium of endometrial adenocarcinomas. Prostaglandin F_2α-FP receptor signaling significantly elevated the expression of IL-11 mRNA and protein in a Gq-protein kinase C-calcium-calcineurin-nuclear factor of activated T cells-dependent manner in FPS cells. The calcineurin signaling pathway is known to be controlled by the RCAN (RCAN1-4). Indeed, RCAN1-4 expression was significantly elevated in well-differentiated endometrial adenocarcinoma compared with normal endometrium and was found to decrease with tumor grade and negatively regulate IL-11 expression in vitro. This study has highlighted a new mechanism regulating IL-11 expression in endometrial adenocarcinoma cells by the FP receptor via the calcium-calcineurin-nuclear factor of activated T cells pathway.Endometrial cancer is the most common female gynecological malignancy in the Western world, ranking fourth in incidence among invasive tumors in women.^1–3 Most cases of endometrial carcinomas are sporadic estrogen-dependent disorders that occur in pre- or postmenopausal women as low-grade (well differentiated, type I) endometrioid adenocarcinomas.⁴ However, ∼20% of tumors in postmenopausal women are not estrogen dependent and have a poor prognosis.^1–4 In these patients, predominantly high-grade (poorly differentiated, type II) tumors arise either as endometrioid adenocarcinomas, uterine papillary serous carcinomas, or clear cell carcinomas with a high frequency of myometrial invasion and spread into the pelvic lymph nodes.⁵Although the mechanisms regulating endometrial adenocarcinomas are still poorly defined, there is much evidence for a role for cyclooxygenase (COX) enzymes and prostaglandins (PGs) in uterine pathology. We and others^6,7 have demonstrated elevated expression of COX-2, biosynthesis of PG, and elevated expression of nuclear⁶ and membrane-bound G protein-coupled receptors^6,8 like the F-prostanoid (FP) receptor⁸ in endometrial adenocarcinomas. Moreover, we have shown that elevated PGF_2α-FP receptor signaling, via the Gq activation of inositol-1,4,5-trisphosphate, leads to up-regulation of tumorigenic and angiogenic genes including COX-2,⁹ fibroblast growth factor 2,¹⁰ and vascular endothelial growth factor,¹¹ indicating that PGF_2α-FP receptor signaling can promote endometrial tumor growth by regulating vascular function. Furthermore, FP receptor can regulate the proliferation of endometrial epithelial cells and can alter their adhesiveness to extracellular matrix and motility via the reorganization of the actin cytoskeleton and activation of focal adhesion kinase.^8,12,13 These findings suggest that PGF_2α-FP receptor signaling plays a multifactorial role in regulating endometrial adenocarcinoma by promoting an environment for angiogenesis and tissue remodeling to facilitate tumor growth.In addition to the regulation of cell architecture and growth factors by the COX-PG axis, a link between PG and chemoattractive cytokines (chemokines) such as CXCL1 has been demonstrated in colorectal cancer,¹⁴ where PGE₂ signaling has been shown to induce CXCL1 expression in colorectal cancer cells to enhance tumor growth. Similarly in COX-2-overexpressing breast cancer cells that had metastasized to bone, Singh et al¹⁵ have shown recently that the pleiotropic cytokine interleukin-11 (IL-11) is significantly elevated.¹⁵ IL-11 mediates its function via the IL-11 receptor α (IL-11Rα). On ligand binding to IL-11Rα, the glycoprotein (GP) 130 subunit, critical for signal transduction of IL-11, is recruited to form a IL-11/IL-11Rα/GP130 complex.¹⁶ Once activated, the IL-11/IL-11Rα/GP130 complex can activate signal transduction pathways¹⁷ to modulate target gene expression. IL-11 and IL-11Rα expression has been shown to correlate with cellular growth, differentiation, invasiveness, tumor progression, and poor prognosis in breast and colorectal cancer;^18–20 however, the expression and regulation of IL-11 in endometrial cancer has yet to be reported.Here we investigated the expression profile of IL-11, IL-11Rα, and GP130 in endometrial adenocarcinomas compared with normal proliferative-phase endometrium and its regulation in an in vitro model of endometrial adenocarcinoma cells by PGF_2α via the FP receptor.     

Cross-Regulation of T Regulatory—Cell Response after Coxsackievirus B3 Infection by NKT and γδ T Cells in the Mouse

Coxsackievirus B3 (CVB3) variants H3 and H310A1 differ by a single nonconserved amino acid in the VP2 capsid region. C57Bl/6 mice infected with the H3 virus develop myocarditis correlating with activation of T cells expressing the Vγ4 T cell receptor chain. Infecting mice with H310A1 activates natural killer T (NKT; mCD1d-tetramer⁺ TCRβ⁺) cells, but not Vγ4 T cells, and fails to induce myocarditis. H310A1 infection preferentially activates M2 alternatively activated macrophage and CD4⁺FoxP3 (T regulatory) cells, whereas CD4⁺Th1 (IFN-γ⁺) cells are suppressed. By contrast, H3 virus infection activates M1 proinflammatory and CD4⁺Th1 cells, but not T regulatory cells. The M1 macrophage show significantly increased CD1d expression compared to M2 macrophage. The ability of NKT cells to suppress myocarditis was shown by adoptive transfer of purified NKT cells into H3-infected NKT knockout (Jα18 knockout) mice, which inhibited cardiac inflammation and increased T regulatory cell response. Cardiac virus titers were equivalent in all mouse strains indicating that neither Vγ4 nor NKT cells participate in control of virus infection. These data show that NKT and Vγ4 cells cross-regulate T regulatory cell responses during CVB3 infections and are the primary factor determining viral pathogenesis in this mouse model.Enteroviruses and adenoviruses cause approximately 80% of clinical viral myocarditis in all age groups.¹ Cardiac injury results from direct viral injury to infected myocytes and also from host immune responses triggered by the infection.² Host responses include: i) induction of proinflammatory cytokines [IL-6, IL-1β, and tumor necrosis factor-α (TNF-α)] that suppress myocardial cell contractility³; ii) lysis of infected cardiocytes⁴; and iii) humoral or cellular autoimmunity to heart antigens, leading to cardiocyte death or dysfunction.^5–7 T-cell depletion of mice infected with coxsackievirus B3 (CVB3) dramatically reduces animal mortality and cardiac inflammation,⁸ and heart-specific, autoimmune CD8⁺ T cells isolated from CVB3-infected mice⁹ transfer myocarditis into uninfected recipients. Furthermore, immunizing mice with cardiac myosin in adjuvant causes cardiac inflammation closely resembling the virus-induced disease.^7,10–12 Several studies demonstrate that induction of autoimmunity in myocarditis corresponds to a decrease in T regulatory cells,^13,14 and T regulatory 1 (Tr1) cells making IL-10 are the probable suppressive effectors causing myocarditis resistance in both myosin- and CVB3-induced disease.^12,15,16 Recently, studies have shown that γδ T cells activated during pathological CVB3 infections are primarily responsible for preventing T regulatory cell responses and directly kill differentiated CD4⁺CD25⁺FoxP3⁺ T regulatory cells through Fas-dependent mechanisms.^2,17Not all CVB3 variants cause myocarditis. Two CVB3 variants, H3 and H310A1, have been cloned and characterized. The H310A1 virus was isolated from the parental H3 virus using a monoclonal antibody to the viral receptor and has a single nonconserved mutation in the VP2 capsid protein in a puff region known for decay accelerating factor (DAF) binding.¹⁸ Unlike the highly myocarditic H3 virus, the H310A1 virus is amyocarditic and preferentially activates T regulatory cells¹⁶ due to an inability to stimulate γδ T cells during H310A1 virus infections.¹⁹ As shown here, although the γδ T cell response is defective in H310A1-infected mice, substantial numbers of natural killer T (NKT) cells are present in the hearts of H310A1-infected, but not H3-infected, animals. This raises the question whether NKT cells promote the generation of T regulatory cells in the myocarditis-resistant animals. This idea is supported by recent studies in which CVB3-infected mice given the NKT ligand, α-galactosylceramide (α-GalCer), develop significantly less myocarditis than untreated animals.²⁰ This study found alterations in cytokine environment in the α-GalCer–treated mice but did not investigate the role of T regulatory cells in causing the anti-inflammatory cytokine response.Although somewhat controversial, various reports indicate that NKT cells suppress autoimmunity or promote tolerance by their effect on T regulatory cell response. Interaction of antigen-presenting cells and NKT cells through CD1d during oral tolerance to nickel results in secretion of IL-4 and IL-10, and activation of T regulatory cells.^21–23 Similarly, systemic tolerance could not be established in a mouse model of anterior chamber–associated immune deviation in CD1d knockout (KO) mice unless the animals were transfused with NKT cells and CD1d⁺ antigen-presenting cells.²⁴ Other studies show that αGalCer, a well-known and specific NKT CD1d-restricted ligand, increases T regulatory cell numbers in vivo²⁵ and can suppress autoimmune diabetes in NOD mice.^26–28 Cytokines, such as transforming growth factor-β (TGF-β) and IL-10, can be produced by NKT cells,^29,30 which could affect dendritic cell cytokine (IL-10) and accessory molecule (CD40, CD80, and/or CD86) expression,^31–33 leading to T regulatory cell responses.^27,34 Here, studies show that NKT cells activated during H310A1 infection cause the increased T regulatory cell response seen in this model. These experiments show that the nature of the innate immune response following enterovirus infection is crucial in determining autoimmunity induction.     

Mesenchymal Deficiency of Notch1 Attenuates Bleomycin-Induced Pulmonary Fibrosis

Notch signaling pathway is involved in the regulation of cell fate, differentiation, proliferation, and apoptosis in development and disease. Previous studies suggest the importance of Notch1 in myofibroblast differentiation in lung alveogenesis and fibrosis. However, direct in vivo evidence of Notch1-mediated myofibroblast differentiation is lacking. In this study, we examined the effects of conditional mesenchymal-specific deletion of Notch1 on pulmonary fibrosis. Crossing of mice bearing the floxed Notch1 gene with α2(I) collagen enhancer-Cre-ER(T)–bearing mice successfully generated progeny with a conditional knockout (CKO) of Notch1 in collagen I–expressing (mesenchymal) cells on treatment with tamoxifen (Notch1 CKO). Because Notch signaling is known to be activated in the bleomycin model of pulmonary fibrosis, control and Notch1 CKO mice were analyzed for their responses to bleomycin treatment. The results showed significant attenuation of pulmonary fibrosis in CKO relative to control mice, as examined by collagen deposition, myofibroblast differentiation, and histopathology. However, there were no significant differences in inflammatory or immune cell influx between bleomycin-treated CKO and control mouse lungs. Analysis of isolated lung fibroblasts confirmed absence of Notch1 expression in cells from CKO mice, which contained fewer myofibroblasts and significantly diminished collagen I expression relative to those from control mice. These findings revealed an essential role for Notch1-mediated myofibroblast differentiation in the pathogenesis of pulmonary fibrosis.Notch signaling is known to play critical roles in development, tissue homeostasis, and disease.^{1, 2, 3, 4, 5, 6, 7, 8, 9, 10} Notch signaling is mediated via four known receptors, Notch 1, 2, 3, and 4, which serve as receptors for five membrane-bound ligands, Jagged 1 and 2 and Delta 1, 3, and 4.^{1, 11, 12, 13} The Notch receptors differ primarily in the number of epidermal growth factor-like repeats and C-terminal sequences.¹³ For instance, Notch 1 contains 36 of epidermal growth factor-like repeats, is composed of approximately 40 amino acids, and is defined largely by six conserved cysteine residues that form three conserved disulfide bonds.^{1, 13, 14, 15} These epidermal growth factor-like repeats can be modified by O-linked glycans at specific sites, which is important for their function.^{1, 14, 15} Modulation of Notch signaling by Fringe proteins,^{16, 17, 18} which are N-acetylglucosamine transferases, illustrates the importance of these carbohydrate residues.^{16, 18} Moreover, mutation of the GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase causes defective fucosylation of Notch1, resulting in impairment of the Notch1 signaling pathway and myofibroblast differentiation.^{19, 20, 21} Because myofibroblasts are important in both lung development and fibrosis, elucidation of the role of Notch signaling in their genesis in vivo will provide insight into the significance of this signaling pathway in either context.The importance of Notch signaling in tissue fibrosis is suggested in multiple studies.^{10, 21, 22, 23, 24} As in other organs or tissues, pulmonary fibrosis is characterized by fibroblast proliferation and de novo emergence of myofibroblasts, which is predominantly responsible for the increased extracellular matrix production and deposition.^{25, 26, 27, 28, 29, 30, 31} Animal models, such as bleomycin-induced pulmonary fibrosis, are characterized by both acute and chronic inflammation with subsequent myofibroblast differentiation that mainly originated from the mesenchymal compartment.^{21, 25, 26, 27, 28} In vitro studies of cultured cells implicate Notch signaling in myofibroblast differentiation,²¹ which is mediated by induction of the Notch1 ligand Jagged1 when lung fibroblasts are treated with found in inflammatory zone 1.²¹ Moreover, GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase knockout mice with defective fucosylation of Notch1 exhibit consequent impairment of Notch signaling and attenuated pulmonary fibrosis in studies using the bleomycin model.²¹ The in vivo importance of Notch signaling in myofibroblast differentiation during lung development has also been suggested by demonstration of impaired alveogenesis in mice deficient in lunatic fringe³² or Notch receptors.^{10, 33, 34, 35} These in vivo studies, however, do not pinpoint the cell type in which deficient Notch signaling is causing the observed impairment of myofibroblast differentiation. This is further complicated by the extensive evidence showing that, in addition to myofibroblast differentiation, Notch1 mediates multiple functional responses in diverse cell types, including inflammation and the immune system.^{21, 36, 37, 38} In the case of tissue injury and fibrosis, including the bleomycin model, the associated inflammation and immune response as well as parenchymal injury can affect myofibroblast differentiation via paracrine mechanisms.^{39, 40} Thus, although global impairment of Notch signaling can impair myofibroblast differentiation in vivo, it does not necessarily indicate a specific direct effect on the mesenchymal precursor cell. Furthermore, understanding the importance of Notch signaling in these different cell compartments is critical for future translational studies to develop effective drugs targeting this signaling pathway with minimal off-target or negative adverse effects.In this study, the effects of conditional selective Notch1 deficiency in the mesenchymal compartment on myofibroblast differentiation and bleomycin-induced pulmonary fibrosis were examined using a Cre-Lox strategy. The transgenic Cre mice bore the Cre-ER(T) gene composed of Cre recombinase and a ligand-binding domain of the estrogen receptor⁴¹ driven by a minimal promoter containing a far-upstream enhancer from the α2(I) collagen gene. When activated by tamoxifen, this enhancer enabled selective Cre expression only in type I collagen-expressing (mesenchymal) cells, such as fibroblasts and other mesenchymal cells,⁴² leading to excision of LoxP consensus sequence flanked target gene DNA fragment (floxed gene) of interest.^{41, 43, 44, 45, 46} To evaluate the importance of Notch1 in the mesenchymal compartment and discriminate its effects from those in the inflammatory and immune system and other compartments, the transgenic Cre-ER(T) mice [Col1α2-Cre-ER(T)^+/0] were crossed with mice harboring the floxed (containing loxP sites) Notch1 gene (Notch1^fl/fl). The resulting progeny mice [Notch1 conditional knockout (CKO)] that were homozygous for the floxed Notch1 allele and hemizygous for the Col1α2-Cre-ER(T) allele with genotype [Notch1^fl/fl,Col1α2-Cre-ER(T)^+/0] were Notch1 deficient in the mesenchymal compartment when injected with tamoxifen. Control Notch1 wild-type (WT) mice exhibited the expected pulmonary fibrosis along with induction of Jagged1 and Notch1 on treatment with bleomycin, consistent with previous observation of Notch signaling activation in this model.²¹ Isolated and cultured Notch1 CKO mouse lung fibroblasts were deficient in Notch1 and exhibited diminished myofibroblast differentiation compared with cells from the corresponding WT control mice. Most important, compared with WT control mice, the CKO mice exhibited diminished bleomycin-induced pulmonary fibrosis that was accompanied by significant reduction in α-smooth muscle actin (α-SMA) and type I collagen gene expression, consistent with defective myofibroblast differentiation. In contrast, enumeration of lung inflammatory and immune cells failed to show a significant difference in bleomycin-induced recruitment of these cells between control and CKO mice. Thus, selective Notch1 deficiency in mesenchymal cells caused impairment of fibrosis that is at least, in part, because of deficient myofibroblast differentiation, and without affecting the inflammatory and immune response in this animal model.     

Interferon-γ Protects First-Trimester Decidual Cells against Aberrant Matrix Metalloproteinases 1, 3, and 9 Expression in Preeclampsia

Human extravillous trophoblast (EVT) invades the decidua via integrin receptors and subsequently degrades extracellular matrix proteins. In preeclampsia (PE), shallow EVT invasion elicits incomplete spiral artery remodeling, causing reduced uteroplacental blood flow. Previous studies show that preeclamptic decidual cells, but not interstitial EVTs, display higher levels of extracellular matrix–degrading matrix metalloproteinase (MMP)-9, but not MMP-2. Herein, we extend our previous PE-related assessment of MMP-2 and MMP-9 to include MMP-1, which preferentially degrades fibrillar collagens, and MMP-3, which can initiate a local proteolytic cascade. In human first-trimester decidual cells incubated with estradiol, tumor necrosis factor-α (TNF-α) significantly enhanced MMP-1, MMP-3, and MMP-9 mRNA and protein levels and activity measured by real-time quantitative RT-PCR, ELISA, immunoblotting, and zymography, respectively. In contrast, interferon γ (IFN-γ) reversed these effects and medroxyprogesterone acetate elicited further reversal. Immunoblotting revealed that p38 mitogen-activated protein kinase signaling mediated TNF-α enhancement of MMP-1, MMP-3, and MMP-9, whereas IFN-γ inhibited p38 mitogen-activated protein kinase phosphorylation. Unlike highly regulated MMP-1, MMP-3, and MMP-9, MMP-2 mRNA and protein expression was constitutive in decidual cells. Because inflammation underlies PE-associated shallow EVT invasion, these results suggest that excess macrophage-derived TNF-α augments expression of MMP-1, MMP-3, and MMP-9 in decidual cells to interfere with normal stepwise EVT invasion of the decidua. In contrast, decidual natural killer cell–derived IFN-γ reverses such TNF-α–induced MMPs to protect against PE.Preeclampsia (PE) is a multifactorial disease that affects 6% to 8% of pregnancies in the United States, is responsible for nearly 8% of maternal deaths, and is a leading cause of perinatal morbidity and mortality. Severe PE is a major indication for early, medically indicated preterm birth.¹ The diagnosis of PE is usually made after 20 weeks by the appearance of hypertension and proteinuria (maternal syndrome).¹During the first 20 weeks of gestation, extravillous trophoblasts (EVTs) arise from cytotrophoblast at the tips of placental anchoring villi and invade the decidua and upper third of the myometrium. As they navigate through the decidua, EVTs enter and facilitate remodeling of spiral arteries and arterioles into large-bore, low-resistance vessels that increase uteroplacental blood flow to the intervillous space requisite for fetal growth and development.^2,3 The onset of PE is strongly associated with shallow decidual EVT invasion, which leads to incomplete vascular transformation and reduced uteroplacental blood flow. The resulting hypoxic placenta⁴ secretes several putative inducers of endothelial cell activation and angiogenesis (eg, soluble flt-1 and endoglin) into the maternal circulation that elicits vascular damage,^5,6 leading to the maternal syndrome.¹Invasion of the decidua by EVT involves sequential attachment to adhesion molecules, followed by their degradation. Relevant integrin (ITG) heterodimers include ITG-α1/ITG-β1 and ITG-α5/ITG-β1, which recognize laminin/collagen IV and fibronectin, respectively, in the decidual extracellular matrix (ECM),^7–9 as well as vascular endothelial cadherin, an endothelial cell receptor.¹⁰ In addition to newly synthesized basement membrane–type proteins, the decidual ECM also contains significant residual interstitial collagens.¹¹ Degradation of the ECM scaffolding structure is mediated principally by matrix metalloproteinases (MMPs), a family of zinc-requiring enzymes that includes collagenases, gelatinases, and stromelysins.¹² Tissue inhibitors of MMPs (TIMPs) regulate MMP catalytic activity.¹³ The MMPs act in concert with urokinase-type plasminogen activator (uPA) and its specific inhibitor, plasminogen activator inhibitor-1 (PAI-1).¹⁴Previously, our laboratory compared immunostaining of the decidua from women with PE versus gestational age–matched control decidua for the presence of the basement membrane–degrading gelatinases, MMP-2 and MMP-9, as well as their respective inhibitors, TIMP-1 and TIMP-2, and found that PE is accompanied by a significant increase in MMP-9 levels in decidual cells, but not in interstitial EVTs. Unlike MMP-9, no PE-related changes in immunostaining were observed for either MMP-2 or TIMP-1 or TIMP-2 in either decidual cells or interstitial EVTs.¹⁵ Significant subsets of PE are associated with underlying maternal infections and/or inflammation,¹⁶ accompanied by an excess of decidual macrophages^17–20 that are likely sources of elevated levels of the proinflammatory cytokines IL-1β and tumor necrosis factor-α (TNF-α).²¹Consistent with the in situ observations described above and strong evidence that the pathogenesis of most cases of PE are initiated in early pregnancy,¹ we found that incubation of primary leukocyte-free, first-trimester human decidual cells with either IL-1β or TNF-α markedly enhanced MMP-9 mRNA and protein expression, unaccompanied by significant changes in either MMP-2 or TIMP-1 or TIMP-2 mRNA and protein expression.¹⁵The current study extends our previous PE-related assessment of MMP-2 and MMP-9 to include MMP-1, which preferentially degrades fibrillar collagens, and MMP-3, which can initiate a local proteolytic cascade by degrading a wide array of ECM proteins and by activating the secreted zymogenic form of other MMPs, such as pro–MMP-1 and pro–MMP-9.^13,22 We found the following using a two-tiered approach of integrating in situ with in vitro observations: immunoreactive MMP-1 and MMP-3 levels were compared in decidual cells and interstitial EVTs of decidual placental sections from women with PE versus gestational age–matched controls: MMP-1, MMP-3, as well as MMP-2 and MMP-9, were measured in the conditioned medium of primary, leukocyte-free, first-trimester human decidual cells incubated in parallel with estradiol (E₂), which was used as the control incubation for E₂ + medroxyprogesterone acetate (MPA) to mimic the pregnant steroid milieu. The steroids were added alone or with either TNF-α or interferon γ (IFN-γ) or TNF-α + IFN-γ. Inclusion of IFN-γ, a primary decidual natural killer (dNK) cell product,²³ was prompted by our recent observations that co-incubation of first-trimester human decidual cells with IFN-γ and either IL-1β or TNF-α synergistically enhances expression of two chemokines, interferon gamma-induced protein 10 (IP-10; alias CXCL10) and interferon-inducible T cell alpha chemoattractant (ITAC; alias CXCL11), that can selectively recruit the peripheral C-X-C chemokine receptor 3–expressing CD56^bright CD16(−) NK cell population to the decidua,²⁴ where they mediate several pregnancy protective effects.^25–27     

Inhibition of Protein Kinase C δ Attenuates Blood-Retinal Barrier Breakdown in Diabetic Retinopathy

Vision loss in diabetic retinopathy is due to macular edema characterized by increased vascular permeability, which involves phosphorylation associated with activation of protein kinase C (PKC) isoforms. Herein, we demonstrated PKC δ inhibition could prevent blood-retinal barrier breakdown in diabetic retinopathy. Increased vascular permeability of diabetic retina was accompanied by a decrease of zonula occludens (ZO)-1 and ZO-2 expression. In diabetic retina and advanced glycation end product-treated human retinal microvascular endothelial cells, vascular leakage and loss of ZO-1 and ZO-2 on retinal vessels were effectively restored or prevented with treatment of rottlerin, transfection of PKC-δ-DN, or siRNA for PKC δ. Interestingly, PKC δ translocated from cytosol to membrane in advanced glycation end product-treated human retinal microvascular endothelial cells, which was blocked by PKC δ inhibition. Taken together, PKC δ activation, related to its subcellular translocation, is involved in vascular permeability in response to diabetes, and inhibition of PKC δ effectively restores loss of tight junction proteins in retinal vessels. Therefore, we suggest that inhibition of PKC δ could be an alternative treatment to blood-retinal barrier breakdown in diabetic retinopathy.Diabetic retinopathy (DR), a common and serious complication of diabetes, is one of the leading causes of blindness.¹ Clinically, DR can be classified into two stages: nonproliferative and proliferative. With progression of retinal ischemia, nonproliferative DR progresses to proliferative DR, which is characterized by the growth of new blood vessels on the surface of the retina or the optic disk. These abnormal vessels are fragile to bleeding, resulting in vitreous hemorrhage and tractional retinal detachment.² However, the principal cause of vision loss in diabetic patients is diabetic macular edema, which can occur at any stage of DR and is characterized by increased vascular permeability.³Diabetes alters the structure and function of most cell types in the retina including the vasculature and neural network,^4,5 which is closely related to the blood-retinal barrier (BRB) breakdown in the early stage of diabetic retinopathy.^6,7 Therefore, BRB breakdown characterizes early stages of vascular dysfunction in DR.⁸ As our previous reports, the cellular interactions, regulating blood neural barrier by modulating both brain angiogenesis and tight junction formation,⁹ also play the critical role in retinal barrier genesis,¹⁰ and the barrier function in retinal vessels is modulated by the retinal endothelial junction structure.^10,11 Specific junction molecules in retinal endothelial cells are requisite for the maintenance of barrier function. Recently, we have shown that zonula occludens (ZO)-1 and occludin are well-characterized components of the tight junction in retinal endothelial cells.^10,12,13 ZO-1 is a cytoplasmic protein which links occludin to the other intracellular junction structures. Particularly, the level of ZO-1 expression is inversely related to permeability in blood-retinal barrier^10,12–14 as well as that of occludin.¹⁵ However, the specific molecular pathogenesis for increased permeability has not been elucidated.Increased vascular permeability in diabetes involves phosphorylation and reorganization of specific junction proteins.¹⁶ In diabetes, excess glucose is metabolized by glycolysis, which increases synthesis of intracellular diacylglycerol (DAG), the main endogenous activator of protein kinase C (PKC). The PKC superfamily is composed of three subfamilies including classical PKC (cPKC; α, β1, β2, and γ), novel PKC (nPKC; δ, ε, η, θ, and μ), and atypical PKC (aPKC: ζ, λ/ι).¹⁷ cPKC is activated by both calcium and DAG, nPKC is regulated by DAG, but not by calcium, and aPKC responds to neither calcium nor DAG.¹⁷ Since PKC activation appears to be due to increase of DAG, all isoforms sensitive to DAG are likely to be activated in diabetes. Actually, ruboxistaurin, a selective inhibitor for β isoforms, has shown the efficacy to ameliorate the vascular dysfunction in diabetes.¹⁸ Besides PKC β, some specific isoforms, PKC α and δ, are also crucial in diabetic microvascular complications.¹⁹In the current study, we investigated that in diabetic retina, PKC δ activation is involved in decrease of tight junction proteins, particularly ZO-1 and ZO-2, which is followed by BRB breakdown. Moreover, our results suggest that PKC δ inhibition could prevent BRB breakdown in diabetic retinopathy.     

CXCL9 Is Important for Recruiting Immune T?Cells into the Brain and Inducing an Accumulation of the T Cells to the Areas of Tachyzoite Proliferation to Prevent Reactivation of Chronic Cerebral Infection with Toxoplasma gondii

T cells are required to maintain the latency of chronic infection with Toxoplasma gondii in the brain. Here, we examined the role of non–glutamic acid-leucine-arginine CXC chemokine CXCL9 for T-cell recruitment to prevent reactivation of infection with T. gondii. Severe combined immunodeficient (SCID) mice were infected and treated with sulfadiazine to establish a chronic infection. Immune T cells from infected wild-type mice were transferred into the SCID mice in combination with treatment with anti-CXCL9 or control sera. Three days later, sulfadiazine was discontinued to initiate reactivation of infection. Numbers of CD4⁺ and CD8⁺ T cells isolated from the brains were markedly less in mice treated with anti-CXCL9 serum than in mice treated with control serum at 3 days after sulfadiazine discontinuation. Amounts of tachyzoite (acute stage form of T. gondii)-specific SAG1 mRNA and numbers of foci associated with tachyzoites were significantly greater in the former than the latter at 5 days after sulfadiazine discontinuation. An accumulation of CD3⁺ T cells into the areas of tachyzoite growth was significantly less frequent in the SCID mice treated with anti-CXCL9 serum than in mice treated with control serum. These results indicate that CXCL9 is crucial for recruiting immune T cells into the brain and inducing an accumulation of the T cells into the areas where tachyzoites proliferate to prevent reactivation of chronic T. gondii infection.Toxoplasma gondii is an obligate intracellular parasite in humans and animals. Interferon (IFN)-γ–mediated immune responses^{1, 2} and, to a lesser degree, humoral immunity^{3, 4, 5} control the tachyzoite growth during the acute stage of infection, but the parasite establishes a chronic infection by forming cysts preferentially in the brain. Chronic infection with T. gondii is ubiquitous in humans, and 500 million to 2 billion people worldwide are estimated to be chronically infected with this parasite.^{6, 7} This chronic infection can be reactivated and develop life-threatening toxoplasmic encephalitis (TE) in immunocompromised persons such as those with AIDS, neoplastic diseases, and organ transplants.^{8, 9} This fact clearly indicates an importance of the protective immunity to maintain the latency of chronic infection with T. gondii in the brain. However, the mechanisms by which the immune responses prevent reactivation of the chronic infection are not well understood.Although T. gondii has three major genotypes (types I, II, and III), type II is predominant in the strains isolated from patients with TE in North America and Europe.^{10, 11, 12} Therefore, for investigating the mechanisms by which the immune system maintains the latency of chronic T. gondii infection and prevents TE, animals that establish a latent, chronic infection with type II parasite in their brains provide an excellent model. BALB/c mice are one of those animals.^{13, 14} IFN-γ is essential to maintain the latency of chronic cerebral infection with T. gondii.^{15, 16} This cytokine can activate microglia^{17, 18} and astrocytes^{19, 20, 21} to prevent tachyzoite proliferation. In addition, IFN-γ plays an important role in regulating recruitment of immune T cells into the brain of BALB/c mice during both the acute and chronic stages of infection.^{22, 23} Induction of vascular cell adhesion molecule 1 (VCAM-1) expression on the cerebral vessels during the chronic infection is largely mediated by IFN-γ,²² and the binding of α4β1 integrin expressed on the surface of T cells to VCAM-1 expressed on the cerebrovascular endothelial cells is important for inducing prompt recruitment of immune T cells into their brains during the early stage of reactivation of chronic T. gondii infection to prevent TE.²⁴Chemokines, in addition to adhesion molecules, are crucial for T-cell entry into various organs.^{25, 26} In BALB/c mice chronically infected with T. gondii, CXCL9, CXCL10, and CCL5 are the chemokines predominantly expressed in their brains.^{27, 28} In the present study, we examined the role of IFN-γ in cerebral expression of these three chemokines during reactivation of the chronic infection in BALB/c-background immunodeficient mice, and found that the CXCL9 expression requires IFN-γ. On the basis of this observation, we examined the role of CXCL9 in recruiting immune T cells into the brain for maintaining the latency of chronic infection with T. gondii with the use of a model of reactivation of the infection in severe combined immunodeficient (SCID) mice with adoptive transfer of immune T cells from infected wild-type animals. By applying anti-CXCL9 antiserum in this animal model, the present study revealed that CXCL9 is crucial for recruiting immune T cells into the brain and for inducing an accumulation of the T cells around the areas associated with tachyzoites to prevent reactivation of cerebral infection with T. gondii.