Toll-Like Receptor 7 Is Required for Lacrimal Gland Autoimmunity and Type 1 Diabetes Development in Male Nonobese Diabetic Mice

Sjögren syndrome (SS) is an immunologically complex, chronic autoimmune disease targeting lacrimal and salivary glands. Nonobese diabetic (NOD) mice spontaneously develop inflammation of lacrimal and salivary glands with histopathological features similar to SS in humans including focal lymphocytic infiltrates in the affected glands. The innate immune signals driving lymphocytic infiltration of these glands are not well-defined. Here we evaluate the role of Toll-like receptor (TLR) 7 in the development of SS-like manifestations in NOD mice. We created a Tlr7 knockout NOD mouse strain and performed histological and gene expression studies to characterize the effects of TLR7 on autoimmunity development. TLR7 was required for male-specific lacrimal gland inflammation but not for female-specific salivary gland inflammation. Moreover, TLR7 was required for type 1 diabetes development in male but not female NOD mice. RNA sequencing demonstrated that TLR7 was associated with a type I interferon (IFN) response and a type I IFN-independent B cell response in the lacrimal glands. Together these studies identify a previously unappreciated pathogenic role for TLR7 in lacrimal gland autoimmunity and T1D development in male NOD mice adding to the growing body of evidence supporting sex differences in mechanisms of autoimmune disease in NOD mice.     

Salivary and Lacrimal Gland Alterations of the Epidermal Fatty Acid-Binding Protein (E-FABP) in Non-Obese Diabetic Mice

The purpose of this study was to investigate the changes in E-FABP in the salivary and lacrimal glands of the Sjögren syndrome (SS) model non-obese diabetic mice (NOD). Cotton thread and ocular vital staining tests were performed on 10-week NOD male mice (n = 24) and age- and sex-matched wild-type (WT) mice (n = 25). Tear and saliva samples were collected at sacrifice for E-FABP ELISA assays. Salivary and lacrimal gland specimens underwent immunohistochemistry stainings for E-FABP. Real-time RT-PCR was also performed for the quantification of mRNA expression levels in the salivary and lacrimal glands. Corneal vital staining scores in the NOD mice were significantly higher compared with those for the wild-type mice (p = 0.0001). The mean tear E-FABP level showed a significantly lower concentration in the NOD mice (p = 0.001). The mean saliva E-FABP level also showed a significantly lower concentration in the NOD mice (p = 0.04). Immunohistochemistry revealed intense E-FABP staining in the LG acinar epithelium and less intense staining in the acinar epitheliae of the SGs in the NOD mice compared to the WT mice. Real-time RT-PCR for the mRNA expression of E-FABP showed a significantly decreased expression in the SG and a significant increase in the LG of the NOD mice compared to the WT mice. In conclusion, the E-FABP showed marked alterations in the tear film, saliva, lacrimal, and salivary glands of the NOD mouse, which may help explain the ocular surface changes in relation to the dry eye disease in this SS model mouse and keratoconjunctivitis sicca in SS patients.     

Interleukin-22 Exerts Detrimental Effects on Salivary Gland Integrity and Function

Interleukin-22 (IL-22) affects epithelial tissue function and integrity in a context-dependent manner. IL-22 levels are elevated in salivary glands of Sjögren’s syndrome (SS) patients, but its role in the pathogenesis of this disease remains unclear. The objective of this study is to elucidate the impact of IL-22 on salivary gland tissue integrity and function in murine models. We showed that IL-22 levels in sera and salivary glands increased progressively in female non-obese diabetic (NOD) mice, accompanying the development of SS. Administration of IL-22 to the submandibular glands of NOD mice prior to the disease onset reduced salivary secretion and induced caspase-3 activation in salivary gland tissues, which were accompanied by alterations in multiple genes controlling tissue integrity and inflammation. Similarly, IL-22 administration to submandibular glands of C57BL/6 mice also induced hyposalivation and caspase-3 activation, whereas blockade of endogenous IL-22 in C57BL/6 mice treated with anti-CD3 antibody mitigated hyposalivation and caspase-3 activation. Finally, IL-22 treatment reduced the number of viable C57BL/6 mouse submandibular gland epithelial cells cultured in vitro, indicating a direct impact of this cytokine on these cells. We conclude that IL-22 exerts a detrimental impact on salivary gland tissues.     

Chemokine CXCL10 Modulates the Tumor Microenvironment of Fibrosis-Associated Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) constitutes a devastating health burden. Recently, tumor microenvironment-directed interventions have profoundly changed the landscape of HCC therapy. In the present study, the function of the chemokine CXCL10 during fibrosis-associated hepatocarcinogenesis was analyzed with specific focus on its impact in shaping the tumor microenvironment. C57BL/6J wild type (WT) and Cxcl10 knockout mice (Cxcl10^−/−) were treated with diethylnitrosamine (DEN) and tetrachloromethane (CCl₄) to induce fibrosis-associated HCCs. Cxcl10 deficiency attenuated hepatocarcinogenesis by decreasing tumor cell proliferation as well as tumor vascularization and modulated tumor-associated extracellular matrix composition. Furthermore, the genetic inactivation of Cxcl10 mediated an alteration of the tumor-associated immune response and modified chemokine/chemokine receptor networks. The DEN/CCl₄-treated Cxcl10^−/− mice presented with a pro-inflammatory tumor microenvironment and an accumulation of anti-tumoral immune cells in the tissue. The most striking alteration in the Cxcl10^−/− tumor immune microenvironment was a vast accumulation of anti-tumoral T cells in the invasive tumor margin. In summary, our results demonstrate that CXCL10 exerts a non-redundant impact on several hallmarks of the tumor microenvironment and especially modulates the infiltration of anti-tumorigenic immune cells in HCC. In the era of microenvironment-targeted HCC therapies, interfering with CXCL10 defines a novel asset for further improvement of therapeutic strategies.     

Alternate-Day Fasting Ameliorates Newly Established Sjögren’s Syndrome-like Sialadenitis in Non-Obese Diabetic Mice

Intermittent fasting confers protections to various diseases including autoimmune disorders, but the specific effects of intermittent fasting on Sjögren’s syndrome (SS) remains inconclusive. The present study was undertaken to determine the specific impact of alternate-day fasting (ADF) on newly established SS-like sialadenitis using non-obese diabetic (NOD) mice. Female NOD mice were deprived of food every other day from 10 to 13 weeks of age, the early stage of established SS, and then analyzed for the disease characteristics. Mice in the ADF group had higher salivary flow rate and attenuated submandibular gland (SMG) inflammation, compared to the control mice fed with standard chow ad libitum. The improvements were accompanied with a decrease in the total leukocytes, T and B lymphocytes and activated CD4 and CD8 T cells, and a down-regulation of pro-inflammatory cytokines IFN-γ and IL-17, chemokine receptor CXCR3 and its ligands CXCL9 and CXCL11 in the SMGs. ADF also led to elevated mRNA levels of water channel protein aquaporin 5 and tight junction protein claudin-1, two factors crucial for normal salivary secretion in the SMGs. In addition, ADF reduced the proportion of IFN-γ- and IL-17- expressing CD4 T cells and diminished mRNA levels of IFN-γ, TNF-α, and IL-17 in the total submandibular draining lymph node cells. Taken together, ADF is effective in ameliorating newly established SS-associated salivary gland exocrinopathy in NOD mice.     

Immunological Properties of Atopic Dermatitis-Associated Alopecia Areata

Alopecia areata (AA) is regarded as a tissue-specific and cell-mediated autoimmune disorder. Regarding the cytokine balance, AA has been considered a type 1 inflammatory disease. On the other hand, AA often complicates atopic dermatitis (AD) and AD is regarded as type 2 inflammatory disease. However, the immunological aspects of AA in relation to AD are still poorly understood. Therefore, we aim to clarify the immunological properties of AD-associated AA. In this study, we performed comparative analysis of the expression of intracytoplasmic cytokines (IFN-γ, IL-4, and IL-13), chemokine receptors (CXCR3 and CCR4) in peripheral blood which were taken from healthy controls, non-atopic AA patients, AA patients with extrinsic AD, and AA patients with intrinsic AD by flowcytometric analysis. We also compared the scalp skin samples taken from AA patients with extrinsic AD before and after treatment with dupilumab. In non-atopic AA patients, the ratios of CD4+IFN-γ+ cells to CD4⁺IL-4⁺ cells and CD4⁺IFN-γ⁺ cells to CD4⁺IL-13⁺ cells were higher than those in AA patients with extrinsic AD. Meanwhile, the ratio of CD8⁺IFN-γ⁺ cells to CD8+IL-13+ cells was significantly higher in the non-atopic AA than in the healthy controls. In AA patients with extrinsic AD, the skin AA lesion showed dense infiltration of not only CXCR3+ cells but also CCR4⁺ cells around hair bulb before dupilumab treatment. However, after the treatment, the number of CXCR3⁺ cells had no remarkable change while the number of CCR4⁺ cells significantly decreased. These results indicate that the immunological condition of AA may be different between atopic and non-atopic patients and between extrinsic and intrinsic AD patients. Our study provides an important notion that type 2 immunity may participate in the development of AA in extrinsic AD patients. It may be considered that the immunological state of non-atopic AA is different from that of atopic AA.     

Mouse Models of Sjögren’s Syndrome with Ocular Surface Disease

Sjögren’s syndrome (SS) is a systemic rheumatic disease that predominantly affects salivary and lacrimal glands resulting in oral and ocular dryness, respectively, referred to as sicca symptoms. The clinical presentation of ocular dryness includes keratoconjunctivitis sicca (KCS), resulting from the inflammatory damage to the ocular surface tissues of cornea and conjunctiva. The diagnostic evaluation of KCS is a critical component of the classification criteria used by clinicians worldwide to confirm SS diagnosis. Therapeutic management of SS requires both topical and systemic treatments. Several mouse models of SS have contributed to our current understanding of immunopathologic mechanisms underlying the disease. This information also helps develop novel therapeutic interventions. Although these models address glandular aspects of SS pathology, their impact on ocular surface tissues is addressed only in a few models such as thrombospondin (TSP)-1 deficient, C57BL/6.NOD.Aec1Aec2, NOD.H2^b, NOD.Aire KO, and IL-2Rα (CD25) KO mice. While corneal and/or conjunctival damage is reported in most of these models, the characteristic SS specific autoantibodies are only reported in the TSP-1 deficient mouse model, which is also validated as a preclinical model. This review summarizes valuable insights provided by investigations on the ocular spectrum of the SS pathology in these models.     

Distinct Expression Patterns of Cxcl12 in Mesenchymal Stem Cell Niches of Intact and Injured Rodent Teeth

Specific stem cell populations within dental mesenchymal tissues guarantee tooth homeostasis and regeneration throughout life. The decision between renewal and differentiation of stem cells is greatly influenced by interactions with stromal cells and extracellular matrix molecules that form the tissue specific stem cell niches. The Cxcl12 chemokine is a general marker of stromal cells and plays fundamental roles in the maintenance, mobilization and migration of stem cells. The aim of this study was to exploit Cxcl12-GFP transgenic mice to study the expression patterns of Cxcl12 in putative dental niches of intact and injured teeth. We showed that endothelial and stromal cells expressed Cxcl12 in the dental pulp tissue of both intact molars and incisors. Isolated non-endothelial Cxcl12⁺ dental pulp cells cultured in different conditions in vitro exhibited expression of both adipogenic and osteogenic markers, thus suggesting that these cells possess multipotent fates. Taken together, our results show that Cxcl12 is widely expressed in intact and injured teeth and highlight its importance as a key component of the various dental mesenchymal stem cell niches.     

Increased Indoleamine 2,3-Dioxygenase Levels at the Onset of Sjögren’s Syndrome in SATB1-Conditional Knockout Mice

Sjögren’s syndrome (SS) is a chronic autoimmune disease characterized by dysfunction of salivary and lacrimal glands, resulting in xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes). Autoantibodies, such as anti-SSA and anti-SSB antibodies, are hallmarks and important diagnostic factors for SS. In our previous study, we demonstrated that SS-like xerostomia was observed in SATB1 conditional knockout (SATB1cKO) mice, in which the floxed SATB1 gene was specifically deleted in hematopoietic cells as early as 4 weeks of age. In these mice, autoantibodies were not detected until 8 weeks of age in SATB1cKO mice, although exocrine gland function reached its lowest at this age. Therefore, other markers may be necessary for the diagnosis of SS in the early phase. Here, we found that mRNA expression of the interferon γ (IFN-γ) gene and the IFN-responsive indoleamine 2,3-dioxygenase (IDO) gene is upregulated in the salivary glands of SATB1cKO mice after 3 and 4 weeks of age, respectively. We detected l-kynurenine (l-KYN), an intermediate of l-tryptophan (l-Trp) metabolism mediated by IDO, in the serum of SATB1cKO mice after 4 weeks of age. In addition, the upregulation of IDO expression was significantly suppressed by the administration of IFN-γ neutralizing antibodies in SATB1cKO mice. These results suggest that the induction of IFN-dependent IDO expression is an initial event that occurs immediately after the onset of SS in SATB1cKO mice. These results also imply that serum l-KYN could be used as a marker for SS diagnosis in the early phases of the disease before autoantibodies are detectable.     

Reduced Colonic Mucosal Injury in 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Poly ADP-Ribose Polymerase (TIPARP/PARP7)-Deficient Mice

Poly-ADP-ribose polymerases (PARPs) are important regulators of the immune system, including TCDD-inducible poly-ADP-ribose polymerase (TIPARP), also known as poly-ADP-ribose polymerase 7 (PARP7). PARP7 negatively regulates aryl hydrocarbon receptor (AHR) and type I interferon (IFN-I) signaling, both of which have been implicated in intestinal homeostasis and immunity. Since the loss of PARP7 expression increases AHR and IFN-I signaling, we used a murine dextran sulfate sodium (DSS)-induced colitis model to investigate the effect of PARP7 loss on DSS-induced intestinal inflammation. DSS-exposed Parp7^−/− mice had less body weight loss, lower disease index scores, and reduced expression of several inflammation genes, including interleukin IL-6, C-x-c motif chemokine ligand 1 (Cxcl1), and lipocalin-2, when compared with wild-type mice. However, no significant difference was observed between genotypes in the colonic expression of the AHR target gene cytochrome P450 1A1 (Cyp1a1). Moreover, no significant differences in microbial composition were observed between the genotypes. Our findings demonstrate that the absence of PARP7 protein results in an impaired immune response to colonic inflammation and suggests that PARP7 may participate in the recruitment of immune cells to the inflammation site, which may be due to its role in IFN-I signaling rather than AHR signaling.     

Autoimmune Epithelitis and Chronic Inflammation in Sjögren’s Syndrome-Related Dry Eye Disease

Autoimmune epithelitis and chronic inflammation are one of the characteristic features of the immune pathogenesis of Sjögren’s syndrome (SS)-related dry eye disease. Autoimmune epithelitis can cause the dysfunction of the excretion of tear fluid and mucin from the lacrimal glands and conjunctival epithelia and meibum from the meibomian glands. The lacrimal gland and conjunctival epithelia express major histocompatibility complex class II or human leukocyte antigen-DR and costimulatory molecules, acting as nonprofessional antigen-presenting cells for T cell and B cell activation in SS. Ocular surface epithelium dysfunction can lead to dry eye disease in SS. Considering the mechanisms underlying SS-related dry eye disease, this review highlights autoimmune epithelitis of the ocular surface, chronic inflammation, and several other molecules in the tear film, cornea, conjunctiva, lacrimal glands, and meibomian glands that represent potential targets in the treatment of SS-related dry eye disease.     

Changes in the Expression and Functional Activities of C-X-C Motif Chemokine Ligand 13 (CXCL13) in Hyperplastic Prostate

Background: C-X-C motif chemokine ligand 13 (CXCL13), a member of the CXC subtype in chemokine superfamily, affects numerous biological processes of various types of cells and the progress of a great number of clinical diseases. The purpose of the current study was to reveal the internal mechanism between CXCL13 and benign prostatic hyperplasia (BPH). Methods: Human serum, prostate tissues and human prostate cell lines (BPH-1, WPMY-1) were utilized. The effect of recombinant human CXCL13 (rHuCXCL13) protein and the influences of the knockdown/overexpression of CXCL13 on two cell lines were studied. Rescue experiments by anti-CXCR5 were also conducted. In vivo, rHuCXCL13 was injected into the ventral prostate of rats. Additionally, a tissue microarray of hyperplastic prostate tissues was constructed to analyze the correlations between CXCL13 and clinical parameters. Results: CXCL13 was highly expressed in the prostate tissues and upregulated in the BPH group. It was observed that CXCL13 modulated cell proliferation, apoptosis, and the epithelial–mesenchymal transition (EMT) through CXCR5 via AKT and the ERK1/2 pathway in BPH-1, while it contributed to inflammation and fibrosis through CXCR5 via the STAT3 pathway in WPMY-1. In vivo, rHuCXCL13 induced the development of rat BPH. Additionally, CXCL13 was positively correlated with the prostate volume and total prostate specific antigen. Conclusions: Our novel data demonstrated that CXCL13 modulated cell proliferation, cell cycle, the EMT of epithelial cells, and induced the fibrosis of prostatic stromal cells via a variety of inflammatory factors, suggesting that CXCL13 might be rediscovered as a potential therapeutic target for the treatment of BPH.     

Role of the Innate Immunity Signaling Pathway in the Pathogenesis of Sjögren’s Syndrome

Sjögren’s syndrome (SS) is a systemic autoimmune disease characterized by chronic inflammation of the salivary and lacrimal glands and extra-glandular lesions. Adaptive immune response including T- and B-cell activation contributes to the development of SS. However, its pathogenesis has not yet been elucidated. In addition, several patients with SS present with the type I interferon (IFN) signature, which is the upregulation of the IFN-stimulated genes induced by type I IFN. Thus, innate immune responses including type I IFN activity are associated with SS pathogenesis. Recent studies have revealed the presence of activation pattern recognition receptors (PRRs) including Toll-like receptors, RNA sensor retinoic acid-inducible gene I and melanoma differentiation-associated gene 5, and inflammasomes in infiltrating and epithelial cells of the salivary glands among patients with SS. In addition, the activation of PRRs via the downstream pathway such as the type I IFN signature and nuclear factor kappa B can directly cause organ inflammation, and it is correlated with the activation of adaptive immune responses. Therefore, this study assessed the role of the innate immune signal pathway in the development of inflammation and immune abnormalities in SS.     

C-X-C Motif Chemokine Ligand 9 and Its CXCR3 Receptor Are the Salt and Pepper for T Cells Trafficking in a Mouse Model of Gaucher Disease

Gaucher disease is a lysosomal storage disease, which happens due to mutations in GBA1/Gba1 that encodes the enzyme termed as lysosomal acid β-glucosidase. The major function of this enzyme is to catalyze glucosylceramide (GC) into glucose and ceramide. The deficiency of this enzyme and resultant abnormal accumulation of GC cause altered function of several of the innate and adaptive immune cells. For example, augmented infiltration of T cells contributes to the increased production of pro-inflammatory cytokines, (e.g., IFNγ, TNFα, IL6, IL12p40, IL12p70, IL23, and IL17A/F). This leads to tissue damage in a genetic mouse model (Gba1^9V/−) of Gaucher disease. The cellular mechanism(s) by which increased tissue infiltration of T cells occurs in this disease is not fully understood. Here, we delineate role of the CXCR3 receptor and its exogenous C-X-C motif chemokine ligand 9 (CXCL9) in induction of increased tissue recruitment of CD4⁺ T and CD8⁺ T cells in Gaucher disease. Intracellular FACS staining of macrophages (Mϕs) and dendritic cells (DCs) from Gba1^9V/− mice showed elevated production of CXCL9. Purified CD4⁺ T cells and the CD8⁺ T cells from Gba1^9V/− mice showed increased expression of CXCR3. Ex vivo and in vivo chemotaxis experiments showed CXCL9 involvement in the recruitment of Gba1^9V/− T cells. Furthermore, antibody blockade of the CXCL9 receptor (CXCR3) on T cells caused marked reduction in CXCL9- mediated chemotaxis of T cells in Gba1^9V/− mice. These data implicate abnormalities of the CXCL9-CXCR3 axis leading to enhanced tissue recruitment of T cells in Gaucher disease. Such results provide a rationale for blockade of the CXCL9/CXCR3 axis as potential new therapeutic targets for the treatment of inflammation in Gaucher disease.     

Rab27a Contributes to Cathepsin S Secretion in Lacrimal Gland Acinar Cells

Altered lacrimal gland (LG) secretion is a feature of autoimmune dacryoadenitis in Sjögren’s syndrome (SS). Cathepsin S (CTSS) is increased in tears of SS patients, which may contribute to disease. Rab3D and Rab27a/b isoforms are effectors of exocytosis in LG, but Rab27a is poorly studied. To investigate whether Rab27a mediates CTSS secretion, we utilized quantitative confocal fluorescence microscopy of LG from SS-model male NOD and control male BALB/c mice, showing that Rab27a-enriched vesicles containing CTSS were increased in NOD mouse LG. Live-cell imaging of cultured lacrimal gland acinar cells (LGAC) transduced with adenovirus encoding wild-type (WT) mCFP-Rab27a revealed carbachol-stimulated fusion and depletion of mCFP-Rab27a-enriched vesicles. LGAC transduced with dominant-negative (DN) mCFP-Rab27a exhibited significantly reduced carbachol-stimulated CTSS secretion by 0.5-fold and β-hexosaminidase by 0.3-fold, relative to stimulated LGAC transduced with WT mCFP-Rab27a. Colocalization of Rab27a and endolysosomal markers (Rab7, Lamp2) with the apical membrane was increased in both stimulated BALB/c and NOD mouse LG, but the extent of colocalization was much greater in NOD mouse LG. Following stimulation, Rab27a colocalization with endolysosomal membranes was decreased. In conclusion, Rab27a participates in CTSS secretion in LGAC though the major regulated pathway, and through a novel endolysosomal pathway that is increased in SS.     

The Anti-TNF-α Antibody Infliximab Inhibits the Expression of Fat-Transporter-Protein FAT/CD36 in a Selective Hepatic-Radiation Mouse Model

Previously, we reported a radiation-induced inflammation triggering fat-accumulation through fatty-acid-translocase/cluster of differentiation protein 36 (FAT/CD36) in rat liver. Furthermore, inhibition of radiation-induced FAT/CD36-expression by anti-tumor necrosis factor-α (anti-TNF-α) (infliximab) was shown in vitro. The current study investigates fat-accumulation in a mouse-model of single-dose liver-irradiation (25-Gray) and the effect of anti-TNF-α-therapy on FAT/CD36 gene-expression. Mice livers were selectively irradiated in vivo in presence or absence of infliximab. Serum- and hepatic-triglycerides, mRNA, and protein were analyzed by colorimetric assays, RT-PCR, Immunofluorescence and Western-Blot, respectively. Sudan-staining was used demonstrating fat-accumulation in tissue. In mice livers, early (1–3 h) induction of TNF-α-expression, a pro-inflammatory cytokine, was observed. It was followed by elevated hepatic-triglyceride level (6–12 h), compared to sham-irradiated controls. In contrast, serum-triglyceride level was decreased at these time points. Similar to triglyceride level in mice livers, Sudan staining of liver cryosections showed a quick (6–12 h) increase of fat-droplets after irradiation. Furthermore, expression of fat-transporter-protein FAT/CD36 was increased at protein level caused by radiation or TNF-α. TNF-α-blockage by anti-TNF-α showed an early inhibition of radiation-induced FAT/CD36 expression in mice livers. Immunohistochemistry showed basolateral and cytoplasmic expression of FAT/CD36 in hepatocytes. Moreover, co-localization of FAT/CD36 was detected with α-smooth muscle actin (α-SMA⁺) cells and F4/80⁺ macrophages. In summary, hepatic-radiation triggers fat-accumulation in mice livers, involving acute-phase-processes. Accordingly, anti-TNF-α-therapy prevented early radiation-induced expression of FAT/CD36 in vivo.