FSP1 is a glutathione-independent ferroptosis suppressor.

Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10, CoQ10): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1-CoQ10-NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.

Exposure to Multiwall Carbon Nanotubes Promotes Fibrous Proliferation by Production of Matrix Metalloproteinase-12 via NF-κB Activation in Chronic Peritonitis.

The toxicologic effects of nanomaterials, such as carbon nanotubes (CNTs), on the immune system are understood well. However, the precise relationship between long-term exposure to CNTs and chronic inflammation remains unclear. In this study, a mouse model of chronic peritonitis was established using i.p. injection of multiwalled CNTs treated by the Taquann method with high dispersion efficiency. Chronic peritonitis with fibrosis was observed in Taquann-treated multiwalled CNT (T-CNT)-injected mice, but not in Taquann-treated titanium dioxide-injected mice. In vivo and in vitro experiments showed that matrix metalloproteinase-12 (MMP-12) of macrophages was up-regulated by T-CNT to enhance fibroblast activation and profibrotic molecule expression in fibroblasts. In addition, T-CNT-induced peritonitis reduced MMP-12 expression in Nfκb1-/- mice, suggesting that MMP-12-producing macrophages play a key role in chronic inflammation due to T-CNT exposure through NF-κB activation. The results of this study could be helpful in understanding the molecular toxicity of nanomaterial and chronic inflammation.

Inorganic polyphosphate potentiates lipopolysaccharide-induced macrophage inflammatory response.

Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate units that are linked by phosphoanhydride bonds and is involved in various pathophysiological processes. However, the role of polyP in immune cell dysfunction is not well-understood. In this study, using several biochemical and cell biology approaches, including cytokine assays, immunofluorescence microscopy, receptor-binding assays with quartz crystal microbalance, and dynamic light scanning, we investigated the effect of polyP on in vitro lipopolysaccharide (LPS)-induced macrophage inflammatory response. PolyP up-regulated LPS-induced production of the inflammatory cytokines, such as tumor necrosis factor α, interleukin-1ß, and interleukin-6, in macrophages, and the effect was polyP dose- and chain length-dependent. However, orthophosphate did not exhibit this effect. PolyP enhanced the LPS-induced intracellular macrophage inflammatory signals. Affinity analysis revealed that polyP interacts with LPS, inducing formation of small micelles, and the polyP-LPS complex enhanced the binding affinity of LPS to Toll-like receptor 4 (TLR4) on macrophages. These results suggest that inorganic polyP plays a critical role in promoting inflammatory response by enhancing the interaction between LPS and TLR4 in macrophages.

Selective and reversible modification of kinase cysteines with chlorofluoroacetamides.

Irreversible inhibition of disease-associated proteins with small molecules is a powerful approach for achieving increased and sustained pharmacological potency. Here, we introduce α-chlorofluoroacetamide (CFA) as a novel warhead of targeted covalent inhibitor (TCI). Despite weak intrinsic reactivity, CFA-appended quinazoline showed high reactivity toward Cys797 of epidermal growth factor receptor (EGFR). In cells, CFA-quinazoline showed higher target specificity for EGFR than the corresponding Michael acceptors in a wide concentration range (0.1-10 µM). The cysteine adduct of the CFA derivative was susceptible to hydrolysis and reversibly yielded intact thiol but was stable in solvent-sequestered ATP-binding pocket of EGFR. This environment-dependent hydrolysis can potentially reduce off-target protein modification by CFA-based drugs. Oral administration of CFA quinazoline NS-062 significantly suppressed tumor growth in a mouse xenograft model. Further, CFA-appended pyrazolopyrimidine irreversibly inhibited Bruton's tyrosine kinase with higher target specificity. These results demonstrate the utility of CFA as a new class warheads for TCI.

Formation of Autoimmune Lesions Is Independent of Antibiotic Treatment in NOD Mice.

The relationship between autoimmunity and changes in intestinal microbiota is not yet fully understood. In this study, the role of intestinal microbiota in the onset and progression of autoimmune lesions in non-obese diabetic (NOD) mice was evaluated by administering antibiotics to alter their intestinal microenvironment. Flow cytometric analysis of spleen cells showed that antibiotic administration did not change the proportion or number of T and B cells in NOD mice, and pathological analysis demonstrated that autoimmune lesions in the salivary glands and in the pancreas were also not affected by antibiotic administration. These results suggest that the onset and progression of autoimmunity may be independent of enteral microbiota changes. Our findings may be useful for determining the appropriate use of antibiotics in patients with autoimmune diseases who are prescribed drugs to maintain systemic immune function.

Bicyclobutane Carboxylic Amide as a Cysteine-Directed Strained Electrophile for Selective Targeting of Proteins.

Expanding the repertoire of electrophiles with unique reactivity features would facilitate the development of covalent inhibitors with desirable reactivity profiles. We herein introduce bicyclo[1.1.0]butane (BCB) carboxylic amide as a new class of thiol-reactive electrophiles for selective and irreversible inhibition of targeted proteins. We first streamlined the synthetic routes to generate a variety of BCB amides. The strain-driven nucleophilic addition to BCB amides proceeded chemoselectively with cysteine thiols under neutral aqueous conditions, the rate of which was significantly slower than that of acrylamide. This reactivity profile of BCB amide was successfully exploited to develop covalent ligands targeting Bruton's tyrosine kinase (BTK). By tuning BCB amide reactivity and optimizing its disposition on the ligand, we obtained a selective covalent inhibitor of BTK. The in-gel activity-based protein profiling and mass spectrometry-based chemical proteomics revealed that the selected BCB amide had a higher target selectivity for BTK in human cells than did a Michael acceptor probe. Further chemical proteomic study revealed that BTK probes bearing different classes of electrophiles exhibited distinct off-target profiles. This result suggests that incorporation of BCB amide as a cysteine-directed electrophile could expand the capability to develop covalent inhibitors with the desired proteome reactivity profile.

Loss of the cystine/glutamate antiporter in melanoma abrogates tumor metastasis and markedly increases survival rates of mice.

The cystine/glutamate antiporter, system xc- , is essential for the efficient uptake of cystine into cells. Interest in the mechanisms of system xc- function soared with the recognition that system xc- presents the most upstream node of ferroptosis, a recently described form of regulated necrosis relevant for degenerative diseases and cancer. Since targeting system xc- hold the great potential to efficiently combat tumor growth and metastasis of certain tumors, we disrupted the substrate-specific subunit of system xc- , xCT (SLC7A11) in the highly metastatic mouse B16F10 melanoma cell line and assessed the impact on tumor growth and metastasis. Subcutaneous injection of tumor cells into the syngeneic B16F10 mouse melanoma model uncovered a marked decrease in the tumor-forming ability and growth of KO cells compared to control cell lines. Strikingly, the metastatic potential of KO cells was markedly reduced as shown in several in vivo models of experimental and spontaneous metastasis. Accordingly, survival rates of KO tumor-bearing mice were significantly prolonged in contrast to those transplanted with control cells. Analyzing the in vitro ability of KO and control B16F10 cells in terms of endothelial cell adhesion and spheroid formation revealed that xCT expression indeed plays an important role during metastasis. Hence, system xc- emerges to be essential for tumor metastasis in mice, thus qualifying as a highly attractive anticancer drug target, particularly in light of its dispensable role for normal life in mice.

Molecular basis of triterpene-based chemophenetics in ferns.

MAIN CONCLUSION: A hypothesis that squalene cyclase genes are widely distributed throughout ferns was proposed. We successfully isolated a squalene cyclase pseudogene from a fern from which no triterpene hydrocarbons were detected Ferns are the most primitive vascular plants, with their locations ranging from tropical to cold temperate regions and from lowland to alpine zones. The triterpene hydrocarbons and their derivatives are characteristic fern metabolites, and are also chemophenetic markers. Recently, our biosynthetic study into fern squalene cyclases (SCs), the enzymes responsible for triterpene synthesis, gave an unexpected inconsistency between genotype (enzyme function) and chemotype (triterpene profile). This finding prompted us to propose a hypothesis that SC genes are widely distributed throughout ferns and lycophytes whether or not they produce triterpene hydrocarbons. To test this hypothesis, we employed a multifaceted approach based on phytochemical, biochemical, and phylogenetic analyses. As anticipated, we successfully isolated two SC pseudogenes from a fern from in which no or only one triterpene hydrocarbon was detected. Subsequent mutagenesis experiments resulted in the functional conversion of these pseudogenes into active SC genes. Given an auxiliary hypothesis regarding the inherent limit of the degenerate polymerase chain reaction (PCR) method, the overall dataset supported our hypothesis, although correction was required with respect to plant coverage. Not only did the corrected hypothesis outline the distribution of SC genes throughout ferns, it provided insight into the molecular basis of the triterpene-based chemophenetics in ferns, which is also discussed.

Achaete-Scute Homologue 2-Regulated Follicular Helper T Cells Promote Autoimmunity in a Murine Model for Sjögren Syndrome.

Follicular helper T (Tfh) cells contribute to various immune responses as well as to the pathogenesis of several immune diseases. However, the precise mechanism underlying the onset or development of autoimmunity via Tfh cells remains unclear. Herein, the detailed relationship between autoimmune disease and Tfh cells was analyzed using a murine model for Sjögren syndrome (SS) wherein the mice underwent neonatal thymectomy. Germinal center (GC) development was promoted in this SS model along with an increase of Tfh cells and GC B cells. The severity of the autoimmune lesions was correlated with the number of Tfh cells detected in the spleen of the SS model mice. In addition, treatment with an anti-CD20 monoclonal antibody effectively suppressed the autoimmune lesions with a reduction of Tfh cells and GC B cells. Comprehensive gene analysis revealed that several genes associated with Tfh cell differentiation, including achaete-scute homologue 2 (Ascl2), were up-regulated in peripheral CD25- CD4+ T cells in SS model mice compared with those in control mice. Moreover, an experiment using CD4CreBcl6fl/fl mice that received neonatal thymectomy treatment demonstrated that Ascl2 contributes to the Tfh cell differentiation associated with autoimmunity during the early stages, independent of Bcl6. In conclusion, our results indicate that abnormal Tfh cell differentiation via Ascl2 regulation might contribute to the pathogenesis of autoimmunity.

Adsorption of Protein-Bound Uremic Toxins Using Activated Carbon through Direct Hemoperfusion in vitro.

BACKGROUND/AIMS: Accumulation of protein-bound uremic toxins (PBUTs) is associated with mortality due to various systemic disorders in patients with chronic kidney disease (CKD), especially in those undergoing dialysis treatment. The clinical outcomes of such patients could be improved by removing sufficient amounts of PBUTs; however, conventional dialysis lacks this ability. We examined the efficacy of activated carbon in adsorbing circulating PBUTs through direct hemoperfusion (DHP) in vitro. METHODS: An in vitro blood circulating system was constructed with 8.5 mL blood circulating around a column containing activated carbon (50, 100, or 200 mg). Bovine blood containing a kind of PBUT (at the same concentration as that found in the blood of dialysis patients) and blood from hemodialysis patients (n = 8) were used. After circulation for the designated amount of time, sera were collected and the levels of PBUTs, including indoxyl sulfate (IS), p-cresyl sulfate, indole acetic acid (IAA), phenyl sulfate, and hippuric acid, were analyzed with mass spectrometry. RESULTS: Activated carbon decreased the PBUT level in bovine blood in a dose-dependent manner (e.g., reduction rate of IS: 67.9 ± 3.8, 83.3 ± 1.9, and 94.5 ± 1.1% after 60-min circulation in columns containing 50, 100, and 200 mg activated carbon respectively). IS, PCS, and IAA were dramatically adsorbed by activated carbon from the blood of patients undergoing hemodialysis (pre vs. post 240-min reaction: IS 2.835 ± 0.876 vs. 0.455 ± 0.108 mg/dL [p < 0.01], PCS 3.208 ± 2.876 vs. 0.768 ± 0.632 mg/dL [p < 0.01], IAA 0.082 ± 0.045 vs. 0.016 ± 0.005 mg/dL [p < 0.01]). CONCLUSION: Activated carbon effectively adsorbed blood PBUTs in vitro. DHP with activated carbon could be a promising strategy for removing circulating PBUTs from the blood of patients with CKD.

Cystine/glutamate transporter, system xc-, is involved in nitric oxide production in mouse peritoneal macrophages.

The amino acid transport system xc- is important for maintaining intracellular glutathione levels and extracellular redox balance. The main component of system xc-, xCT, is strongly induced by various stimuli, including oxidative stress and bacterial lipopolysaccharides (LPS) in macrophages. In the present study, we investigated the production of nitric oxide by LPS-stimulated mouse peritoneal macrophages isolated from both xCT-deficient and wild-type mice. After culturing macrophages in the presence of LPS for 24-48 h, nitrite levels in the medium of xCT-deficient macrophages were significantly decreased compared to that of wild-type cells. However, the transport activity of arginine, a precursor of nitric oxide, and the expression of nitric oxide synthase 2 in xCT-deficient macrophages were similar to those of wild-type cells. When wild-type macrophages were cultured in the medium that contained no cystine, nitric oxide production was decreased to the level similar to that of the xCT-deficient macrophages. When xCT-deficient macrophages were cultured with 2-mercaptoethanol, intracellular cysteine levels were increased and nitrite accumulation in the medium was significantly increased. On the other hand, when these cells were cultured with buthionine sulfoximine, an inhibitor of glutathione synthesis, nitrite accumulation in the medium was essentially unchanged, although intracellular glutathione levels were very low. Reactive oxygen species levels in xCT-deficient macrophages were higher than those of wild-type cells, and treatment with LPS caused an increase in oxidative stress in both cells. These results suggest that intracellular cysteine supplied by xCT contributes to nitric oxide production and the reduction of oxidative stress in macrophages.

Adsorption of Protein-Bound Uremic Toxins Through Direct Hemoperfusion With Hexadecyl-Immobilized Cellulose Beads in Patients Undergoing Hemodialysis.

An accumulation of protein-bound uremic toxins (PBUTs) is one of major reasons for development of uremia-related complications. We examined the PBUT removal ability of a hexadecyl-immobilized cellulose bead (HICB)-containing column for patients undergoing hemodialysis. Adsorption of indoxyl sulfate (IS), a representative PBUT, to HICBs was examined in vitro. The HICB column was used in patients undergoing hemodialysis for direct hemoperfusion with a regular hemodialyzer. The serum IS, indole acetic acid (IAA), phenyl sulfate (PhS), and p-cresyl sulfate (PCS) levels were measured before and after passing the column. HICBs adsorbed protein-free (free) IS in a dose- and time-dependent manner in vitro (55.4 ± 1.4% adsorption of 1 millimolar, 251 µg/mL, IS for 1 h). In clinical studies, passing the HICB-containing column decreased the serum level of free IS, IAA, PhS, and PCS levels significantly (by 34.4 ± 30.0%, 34.8 ± 25.4%, 28.4 ± 18.0%, and 34.9 ± 22.1%, respectively), but not protein-bound toxins in maintenance hemodialysis patients. HICBs absorbed some amount of free PBUTs, but the clinical trial to use HICB column did not show effect to reduce serum PBUTs level in hemodialysis patients. Adsorption treatment by means of direct hemoperfusion with regular hemodialysis may become an attractive blood purification treatment to increase PBUT removal when more effective materials to adsorb PBUTs selectively will be developed.

Cystathionine is a novel substrate of cystine/glutamate transporter: implications for immune function.

The cystine/glutamate transporter, designated as system xc(-), is important for maintaining intracellular glutathione levels and extracellular redox balance. The substrate-specific component of system xc(-), xCT, is strongly induced by various stimuli, including oxidative stress, whereas it is constitutively expressed only in specific brain regions and immune tissues, such as the thymus and spleen. Although cystine and glutamate are the well established substrates of system xc(-) and the knockout of xCT leads to alterations of extracellular redox balance, nothing is known about other potential substrates. We thus performed a comparative metabolite analysis of tissues from xCT-deficient and wild-type mice using capillary electrophoresis time-of-flight mass spectrometry. Although most of the analyzed metabolites did not show significant alterations between xCT-deficient and wild-type mice, cystathionine emerged as being absent specifically in the thymus and spleen of xCT-deficient mice. No expression of either cystathionine ß-synthase or cystathionine γ-lyase was observed in the thymus and spleen of mice. In embryonic fibroblasts derived from wild-type embryos, cystine uptake was significantly inhibited by cystathionine in a concentration-dependent manner. Wild-type cells showed an intracellular accumulation of cystathionine when incubated in cystathionine-containing buffer, which concomitantly stimulated an increased release of glutamate into the extracellular space. By contrast, none of these effects could be observed in xCT-deficient cells. Remarkably, unlike knock-out cells, wild-type cells could be rescued from cystine deprivation-induced cell death by cystathionine supplementation. We thus conclude that cystathionine is a novel physiological substrate of system xc(-) and that the accumulation of cystathionine in immune tissues is exclusively mediated by system xc(-).

Liesegang patterns engineered by a chemical reaction assisted by complex formation.

Liesegang rings based on a chemical reaction, not a conventional precipitation reaction, have been developed by appropriate design of the nucleation dynamics in a system involving complex formation in a matrix. The periodic and concentric rings consisted of well-dispersed Ag nanoparticles with diameters of a few nanometers. The approach modeled here could be applied to form novel micropatterns out of inorganic salts, metal nanoparticles, organic nanocrystals, or polymeric fibers, and it could also offer a scaffold for novel models of a wide variety of reaction-diffusion phenomena in nature.

Expression of ISG60 is induced by TLR3 signaling in BEAS­2B bronchial epithelial cells: Possible involvement in CXCL10 expression.

Viral infections in the respiratory tract are common, and, in recent years, severe acute respiratory syndrome coronavirus 2 outbreaks have highlighted the effect of viral infections on antiviral innate immune and inflammatory reactions. Specific treatments for numerous viral respiratory infections have not yet been established and they are mainly treated symptomatically. Therefore, understanding the details of the innate immune system underlying the airway epithelium is crucial for the development of new therapies. The present study aimed to investigate the function and expression of interferon (IFN)­stimulated gene (ISG)60 in non­cancerous bronchial epithelial BEAS­2B cells exposed to a Toll­like receptor 3 agonist. BEAS­2B cells were treated with a synthetic TLR3 ligand, polyinosinic­polycytidylic acid (poly IC). The mRNA and protein expression levels of ISG60 were analyzed using reverse transcription­quantitative PCR and western blotting, respectively. The levels of C­X­C motif chemokine ligand 10 (CXCL10) were examined using an enzyme­linked immunosorbent assay, and the effects of knockdown of IFN­ß, ISG60 and ISG56 were examined using specific small interfering RNAs. Notably, ISG60 expression was increased in proportion to poly IC concentration, and recombinant human IFN­ß also induced ISG60 expression. By contrast, knockdown of IFN­ß and ISG56 decreased ISG60 expression, and ISG60 knockdown reduced CXCL10 and ISG56 expression. These findings suggested that ISG60 is partly implicated in CXCL10 expression and that ISG60 may serve a role in the innate immune response of bronchial epithelial cells. The present study highlights ISG60 as a potential target for new therapeutic strategies against viral infections in the airway.

Mass spectrometry-based proteomic analysis of proteins adsorbed by hexadecyl-immobilized cellulose bead column for the treatment of dialysis-related amyloidosis.

BACKGROUND: Dialysis-related amyloidosis (DRA) is a severe complication in end-stage kidney disease (ESKD) patients undergoing long-term dialysis treatment, characterized by the deposition of ß2-microglobulin-related amyloids (Aß2M amyloid). To inhibit DRA progression, hexadecyl-immobilized cellulose bead (HICB) columns are employed to adsorb circulating ß2-microglobulin (ß2M). However, it is possible that the HICB also adsorbs other molecules involved in amyloidogenesis. METHODS: We enrolled 14 ESKD patients using HICB columns for DRA treatment; proteins were extracted from HICBs following treatment and identified using liquid chromatography-linked mass spectrometry. We measured the removal rate of these proteins and examined the effect of those molecules on Aß2M amyloid fibril formation in vitro. RESULTS: We identified 200 proteins adsorbed by HICBs. Of these, 21 were also detected in the amyloid deposits in the carpal tunnels of patients with DRA. After passing through the HICB column and hemodialyzer, the serum levels of proteins such as ß2M, lysozyme, angiogenin, complement factor D and matrix Gla protein were reduced. These proteins acted in the Aß2M amyloid fibril formation. CONCLUSIONS: HICBs adsorbed diverse proteins in ESKD patients with DRA, including those detected in amyloid lesions. Direct hemoperfusion utilizing HICBs may play a role in acting Aß2M amyloidogenesis by reducing the amyloid-related proteins.

Syndecan-4 prevents cardiac rupture and dysfunction after myocardial infarction.

RATIONALE: Syndecan-4 (Syn4), a cell-surface heparan sulfate proteoglycan, has been detected in the infarct region after myocardial infarction (MI), but its functional significance has not been elucidated. OBJECTIVE: We examined whether and how Syn4 regulates the cardiac healing process after MI. METHODS AND RESULTS: Although the heart in Syn4-deficient (Syn4(-/-)) mice was morphologically and functionally normal, Syn4(-/-) mice exhibited impaired heart function and increased mortality rate as a result of cardiac ruptures after MI. Cardiac ruptures in Syn4(-/-) mice were associated with reduced inflammatory reaction and impaired granulation tissue formation during the early phase of MI, as evidenced by reduced numbers of leukocytes, fibroblasts, myofibroblasts, macrophages, and capillary vessels, along with reduced extracellular matrix protein deposition in the infarct region after MI. Transforming growth factor-ß1-dependent cell signaling was preserved, whereas cell migration, fibronectin-induced cell signaling, and differentiation into myofibroblasts were defective in Syn4(-/-) cardiac fibroblasts. We also found that Syn4 was involved in basic fibroblast growth factor-dependent endothelial cell signaling, cell proliferation, and tube formation. Finally, overexpression of the shed form of Syn4 before MI creation led to an increase in mortality due to cardiac rupture via its action as a dominant-negative inhibitor of endogenous Syn4 signaling, which suggested a protective role of Syn4 signaling in MI. CONCLUSIONS: These results suggest that Syn4 plays an important role in the inflammatory response and granulation tissue formation, thereby preventing cardiac rupture and dysfunction after MI.

Microchannel-induced change of chemical wave propagation dynamics: importance of ratio between the inlet and the channel sizes.

The ability to control chemical wave propagation dynamics could stimulate the science and technology of artificial and biological spatiotemporal oscillating phenomena. In contrast to the conventional chemical approaches to control the wave front dynamics, here we report a physical approach to tune the propagation dynamics under the same chemical conditions. By using well-designed microchannels with different channel widths and depths, the propagation velocity was successfully controlled based on two independent effects: (i) a transition in the proton diffusion mode and (ii) the formation of a slanted wave front. Numerical analysis yielded a simple relationship between the propagation velocity and the microchannel configuration, which offers a simple and general way of controlling chemical wave propagation.

Virus Infections Play Crucial Roles in the Pathogenesis of Sjögren's Syndrome.

Sjögren's syndrome (SS) is an autoimmune disease especially targeting exocrine glands, such as the salivary and lacrimal glands. A radical therapy for SS based on its etiology has not been established because of the complex pathogenesis of the disease. Several studies have demonstrated a relationship between virus infection and SS pathogenesis. In particular, infection with the Epstein-Barr (EB) virus among others is a potent factor associated with the onset or development of SS. Specifically, virus infection in the target organs of SS triggers or promotes autoreactive responses involving the process of autoantigen formation, antigen-presenting function, or T-cell response. Our review of recent research highlights the crucial roles of virus infection in the pathogenesis of SS and discusses the critical association between virus infection and the etiology of autoimmunity in SS.

Disturbed natural killer cell homeostasis in the salivary gland enhances autoimmune pathology via IFN-γ in a mouse model of primary Sjögren's syndrome.

Objective: Innate lymphoid cells (ILCs), including natural killer (NK) cells, ILC1, ILC2, lymphoid tissue-inducer (LTi) cells, and ILC3 cell, play a key role in various immune responses. Primary Sjögren's syndrome (pSS) is an autoimmune disease characterized by chronic inflammation of exocrine glands, such as the lacrimal and salivary glands (SGs). The role of NK cells among ILCs in the pathogenesis of pSS is still unclear. In this study, the characteristics and subsets of NK cells in the salivary gland (SG) tissue were analyzed using a murine model of pSS. Methods: Multiple phenotypes and cytotoxic signature of the SG NK cells in control and pSS model mice were evaluated by flow cytometric analysis. Intracellular expression of interferon-γ (IFN-γ) among T cells and NK cells from the SG tissues was compared by in vitro experiments. In addition, pathological analysis was performed using anti-asialo-GM1 (ASGM1) antibody (Ab)-injected pSS model mice. Results: The number of conventional NK (cNK) cells in the SG of pSS model mice significantly increased compared with that in control mice at 6 weeks of age. The production level of IFN-γ was significantly higher in SG NK cells than in SG T cells. The depletion of NK cells by ASGM1 Ab altered the ratio of tissue resident NK (rNK) cells to cNK cells, which inhibited the injury to SG cells with the recovery of saliva secretion in pSS model mice. Conclusion: The results indicate that SG cNK cells may enhance the autoreactive response in the target organ by upregulating of IFN-γ, whereas SG rNK cells protect target cells against T cell cytotoxicity. Therefore, the activation process and multiple functions of NK cells in the target organ could be helpful to develop potential markers for determining autoimmune disease activity and target molecules for incurable immune disorders.