Involvement of REG Iα protein in the regeneration of ductal epithelial cells in the minor salivary glands of patients with Sjögren's syndrome

The regenerating gene (Reg) was originally isolated from regenerating rat pancreatic islets and revealed recently to constitute a multi‐gene family in humans. REG Iα protein is known to be overexpressed not only in various human inflammatory diseases but also in various experimental models of inflammation in animal tissues. However, its involvement in pathophysiology of the minor salivary gland (MSG) is not clear. We investigated REG Iα expression in the MSG of patients with primary Sjögren's syndrome (SS) and assessed its role in ductal epithelial cell proliferation in such tissues. Lip biopsy specimens were obtained from 40 patients with primary SS and examined using immunohistochemistry for REG Iα protein, Ki67 and single‐strand DNA (ssDNA). The relationships among clinicopathological factors and expression of REG Iα protein, Ki67 and ssDNA in the MSG were then analysed. REG Iα protein was expressed rarely in ductal epithelial cells of the normal MSG but was apparently overexpressed in those of patients with SS. The labelling indices for both Ki67 and ssDNA in the ductal cells of the MSGs were significantly higher in SS patients than in controls. Moreover, these labelling indices were significantly higher in REG Iα‐positive than in negative SS patients. REG Iα protein may play a role in the regeneration of ductal epithelial cells in the MSGs of patients with SS.     

Interferon lambda1/IL‐29 and inorganic polyphosphate are novel regulators of neutrophil‐driven thromboinflammation

Neutrophils and neutrophil‐released meshwork structures termed neutrophil extracellular traps (NETs) are major mediators of thromboinflammation and emerging targets for therapy, yet the mechanisms and pathways that control the role of neutrophils in thromboinflammation remain poorly understood. Here, we explored the role of IFN‐λ1/IL‐29, a major antiviral cytokine recently shown to suppress the neutrophil migratory capacity, in prothrombotic and proNETotic functions of neutrophils. In an ex vivo human experimental setting of acute ST‐segment elevation myocardial infarction (STEMI), we show that IFN‐λ1/IL‐29 hinders NET release and diminishes the amount of cytoplasmic TF in neutrophils. Since platelet–neutrophil interaction plays a major role in NET‐induced thromboinflammation, we further studied how IFN‐λ1/IL‐29 may interrupt this interaction. In this context, we identified inorganic polyphosphate (polyP) as a platelet‐derived NET inducer in STEMI. In arterial STEMI thrombi, polyP was present in platelets and in close proximity to NET remnants. PolyP release from activated platelets was dependent on thrombin present in infarcted artery plasma, resulting in NET formation by promoting mTOR inhibition and autophagy induction. The effect of polyP on mTOR inhibition was counteracted by IFN‐λ1/IL‐29 treatment, leading to inhibition of NET formation. Consistently, we show in an in vivo model of FeCl₃‐induced arterial thrombosis that IFN‐λ2/IL‐28A exerts strong antithrombotic potential. Taken together, these findings reveal a novel function of IFN‐λ1/IL‐29 in the suppression of thromboinflammation. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

IFN‐λ‐mediated IL‐12 production in macrophages induces IFN‐γ production in human NK cells

With increasing interest in alternative options to interferon‐alpha‐based treatments, IFN‐λ has shown therapeutic promise in a variety of diseases. Although the antiviral activity of IFN‐λ has been extensively studied, there is limited knowledge regarding the immunological functions of IFN‐λ and how these differ from those of other classes of IFNs. In this study, we investigated the effects of IFN‐λ on primary human NK cells, both in a direct and indirect capacity. We demonstrate that in contrast to interferon‐alpha, IFN‐λ is unable to directly stimulate NK cells, due to the absence of IFN‐λ receptor chain 1 (IFN‐λR1) on NK cells. However, IFN‐λ, in combination with TLR4 challenge, is able to induce the production of select members of the IL‐12 family of cytokines in monocyte‐derived macrophages. We further show that through macrophage‐mediated IL‐12 production, IFN‐λ is able to indirectly affect NK cells and ultimately induce IFN‐γ production.     

Type III interferons are expressed by Coxsackievirus‐infected human primary hepatocytes and regulate hepatocyte permissiveness to infection

Hepatitis is a common and potentially fatal manifestation of severe Coxsackievirus infections, particularly in newborn children. Little is known of the immune‐mediated mechanisms regulating permissiveness to liver infection. It is well established that type I interferons (IFNs) play an important role in the host innate immune response to Coxsackievirus infections. Recent studies have highlighted a role for another IFN family, the type III IFNs (also called IFN‐λ), in anti‐viral defence. Whether type III IFNs are produced by hepatocytes during a Coxsackievirus infection remains unknown. Moreover, whether or not type III IFNs protects hepatocytes from a Coxsackievirus infection has not been addressed. In this study, we show that primary human hepatocytes respond to a Coxsackievirus B3 (CVB3) infection by up‐regulating the expression of type III IFNs. We also demonstrate that type III IFNs induce an anti‐viral state in hepatocytes characterized by the up‐regulated expression of IFN‐stimulated genes, including IFN‐stimulated gene (ISG15), 2′‐5′‐oligoadenylate synthetase 2 (OAS2), protein kinase regulated by dsRNA (PKR) and myxovirus resistance protein 1 (Mx1). Furthermore, our study reveals that type III IFNs attenuate CVB3 replication both in hepatocyte cell lines and primary human hepatocytes. Our studies suggest that human hepatocytes express type III IFNs in response to a Coxsackievirus infection and highlight a novel role for type III IFNs in regulating hepatocyte permissiveness to this clinically relevant type of virus.     

Type I interferon potentiates T‐cell receptor mediated induction of IL‐10‐producing CD4+ T cells

Type I interferons (IFNs) have the dual ability to promote the development of the immune response and exert an anti‐inflammatory activity. We analyzed the integrated effect of IFN‐α, TCR signal strength, and CD28 costimulation on human CD4⁺ T‐cell differentiation into cell subsets producing the anti‐ and proinflammatory cytokines IL‐10 and IFN‐γ. We show that IFN‐α boosted TCR‐induced IL‐10 expression in activated peripheral CD45RA⁺CD4⁺ T cells and in whole blood cultures. The functional cooperation between TCR and IFN‐α efficiently occurred at low engagement of receptors. Moreover, IFN‐α rapidly cooperated with anti‐CD3 stimulation alone. IFN‐α, but not IL‐10, drove the early development of type I regulatory T cells that were mostly IL‐10⁺ Foxp3^? IFN‐γ^? and favored IL‐10 expression in a fraction of Foxp3⁺ T cells. Our data support a model in which IFN‐α costimulates TCR toward the production of IL‐10 whose level can be amplified via an autocrine feedback loop.     

Subtypes of type I IFN differentially enhance cytokine expression by suboptimally stimulated CD4+ T cells

Human type I interferons (IFNs) include IFN‐β and 12 subtypes of IFN‐α. During viral infection, infiltrating memory CD4 ⁺ T cells are exposed to IFNs, but their impact on memory T‐cell function is poorly understood. To address this, we pretreated PBMCs with different IFNs for 16 h before stimulation with Staphylococcus aureus enterotoxin B and measured cytokine expression by flow cytometry. IFN‐α8 and ‐α10 most potently enhanced expression of IFN‐γ, IL‐2, and IL‐4. Potency among the subtypes differed most at doses between 10 and 100 U/mL. While enhancement of IL‐2 and IL‐4 correlated with the time of preincubation with type I IFN, IFN‐γ production was enhanced best when IFN‐α was added immediately preceding or simultaneously with T‐cell stimulation. Comparison of T‐cell responses to multiple doses of Staphylococcus aureus enterotoxin B and to peptide libraries from RSV or CMV demonstrated that IFN‐α best enhanced cytokine expression when CD4 ⁺ T cells were suboptimally stimulated. We conclude that type I IFNs enhance Th1 and Th2 function with dose dependency and subtype specificity, and best when T‐cell stimulation is suboptimal. While type I IFNs may beneficially enhance CD4 ⁺ T‐cell memory responses to vaccines or viral pathogens, they may also enhance the function of resident Th2 cells and exacerbate allergic inflammation.     

Epigenetic alterations in Sjögren’s syndrome patient saliva

Epigenetic mechanisms have been implicated in the pathogenesis of Sjögren’s syndrome (SS). Extensive alterations in DNA methylation have been described in minor salivary gland (MSG) epithelial cells and lymphocytes derived from SS patients compared to sicca controls. In an effort to identify novel potential epigenetic markers that could prove useful in diagnosis and disease monitoring, we explored whether DNA methylation differences can also be detected in saliva from SS patients compared to sicca controls. We performed DNA methylation analysis by methylation-sensitive restriction digestion followed by quantitative real-time polymerase chain reaction of selected genomic loci in saliva samples of 16 SS patients and 10 sicca controls with negative MSG biopsy. We identified reduced DNA methylation of the imprinting control region (ICR) of the H19 locus in SS patient saliva compared to sicca controls. Levels of saliva H19 ICR methylation were negatively correlated with C4 serum complement levels. Consistent with the reduced methylation of the ICR, H19 RNA levels were increased in SS patient peripheral blood mononuclear cells (PBMCs), while no significant change was observed in MSG H19 RNA levels compared to sicca controls. Our findings support that H19 ICR methylation could be a useful molecular epigenetic marker in monitoring patients with SS, highlighting saliva as a valuable biological sample in SS research and clinical practice. The role of H19 in SS pathogenesis remains to be addressed.     

Differential Cytokine Expression and Regulatory Cells in Patients with Primary and Secondary Sjögren's Syndrome

Sjögren's syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration of the salivary and lacrimal glands. The aim of the study was to characterize and compare the presence of diverse cytokines and regulatory T and B cells in lip minor salivary gland (MSG) biopsies from patients with primary Sjögren's syndrome (pSS), secondary SS (sSS), and patients with connective tissue disease (CTD) without (w/o) SS. We included samples of MSG from 15 pSS, 24 sSS (six scleroderma, nine rheumatoid arthritis and nine lupus patients) and 15 patients with CTD w/o SS. Tissues were examined by an indirect immunoperoxidase technique (goat polyclonal anti‐human IL‐19, goat polyclonal anti‐human IL‐22 or mouse monoclonal anti‐human IL‐24). To determine the subpopulation of CD4⁺/IL‐17A⁺‐, CD4⁺/IL‐4⁺‐, CD4⁺/IFN‐?⁺‐expressing T cells, CD25⁺/Foxp3⁺ Treg cells and CD20⁺/IL‐10⁺‐producing B cell subset, a double‐staining procedure was performed. We estimated the mean percentage of positively staining cells in two fields per sample. CD4⁺/IFN‐?⁺, CD4⁺/IL‐4⁺ and IL‐22⁺ cell percentages were elevated in both SS varieties; however, the cells were more prevalent in pSS. Patients with pSS had a high number of CD4⁺/IL‐17A⁺ and IL‐19⁺ T cells and a lower percentage of IL‐24⁺ cells (P < 0.05). The Treg and IL‐10‐producing B cells were increased in pSS (P < 0.05). Concluding, in our patients, a pro‐inflammatory and regulatory balance coexists in SS, being both responses more intense in pSS. The explanation of these differences may be related to disease activity, disease duration and treatment.     

Rhinovirus infection and house dust mite exposure synergize in inducing bronchial epithelial cell interleukin‐8 release

Background Human rhinoviruses (HRVs) and house dust mites (HDMs) are among the most common environmental factors able to induce airway inflammation in asthma. Although epidemiological studies suggest that they also synergize in inducing asthma exacerbations, there is no experimental evidence to support this, nor any information on the possible mechanisms involved. Objective To investigate their interaction on the induction of airway epithelial inflammatory responses in vitro. Methods BEAS‐2B cells were exposed to activated HDM Dermatophagoides pteronyssinus major allergen I (Der p I), HRVs (HRV1b or HRV16) or both in different sequences. IL‐8/CXCL8 release, intercellular adhesion molecule (ICAM)‐1 surface expression and nuclear factor κB (NF‐κB) translocation were evaluated. Complementary, primary human bronchial epithelial cells (HBECs) exposed to both Der p I and RVs and IL‐8, IL‐6, IFN‐γ‐induced protein (IP)‐10/CXCL10, IFN‐λ1/IL‐29, regulated upon activation normal T lymphocyte expressed and secreted (RANTES)/CCL5 release were measured. Results RV and Der p I up‐regulated IL‐8 release, ICAM‐1 expression and NF‐κB translocation in BEAS‐2B cells. Simultaneous exposure to both factors, as well as when cells were initially exposed to HRV and then to Der p I, resulted in further induction of IL‐8 in a synergistic manner. Synergism was not observed when cells were initially exposed to Der p I and then to HRV. This was the pattern in ICAM‐1 induction although the phenomenon was not synergistic. Concurrent exposure induced an early synergistic NF‐κB translocation induction, differentiating with time, partly explaining the above observation. In HBECs, both HRV and Der p I induced IL‐8, IL‐6, IL‐29 and IP‐10, while RANTES was induced only by HRV. Synergistic induction was observed only in IL‐8. Conclusion HRV and enzymatically active Der p I can act synergistically in the induction of bronchial epithelial IL‐8 release, when HRV infection precedes or is concurrent with Der p I exposure. Such a synergy may represent an important mechanism in virus‐induced asthma exacerbations.     

CD40 on salivary gland epithelial cells: high constitutive expression by cultured cells from Sjögren's syndrome patients indicating their intrinsic activation

CD40 has been identified in an expanding list of haematopoietic and non‐haematopoietic cells and has received an increased interest based on its role in a variety of cell‐mediated responses and its potential to participate in the pathogenesis of chronic inflammatory disorders. Sjögren’s syndrome (SS) is an autoimmune exocrinopathy, which is characterized by chronic lymphocytic infiltration of exocrine glands and aberrant activation of epithelial tissues. We studied the expression of CD40 protein in cultured non‐neoplastic salivary gland epithelial cell (SGEC) lines as well as in minor SG biopsies obtained from 17 SS patients and 12 controls. Immunocytochemical and flow cytometric analyses had revealed the occurrence of constitutively expressed CD40 molecules on the surface of long‐term cultured SGEC lines, which could be further induced by interferon‐gamma (IFN‐γ) and IL‐1β cytokines, but not tumour necrosis factor‐alpha (TNF‐α), IL‐4, IL‐6, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) or IFN‐α. Triggering of SGEC through CD40 enhanced the surface expression of the adhesion molecule intercellular adhesion molecule‐1 (ICAM‐1)/CD54, but not MHC class I and class II (HLA‐DR) molecules. Spontaneous CD40 expression was significantly higher in SGEC lines derived from SS patients, compared with controls (P < 0·001), which is suggestive of their intrinsically activated status. In SG biopsies, CD40 was constitutively expressed by lymphocytes, ductal epithelial cells and endothelial cells but not by other glandular cell types, such as acinar cells, myoepithelial cells and fibroblasts. In addition, CD40L staining was also detected in 30–50% of the infiltrating lymphocytes in the biopsies of SS patients. Our findings indicate the immunoregulatory potential of SGEC and lend further support to a model of intrinsic activation in salivary epithelia in SS, whereby these cells actively participate in the induction and maintenance of lymphocytic infiltrates of patients.     

Tyrosine kinase 2 is not limiting human antiviral type III interferon responses

Tyrosine kinase 2 (TYK2) associates with interferon (IFN) alpha receptor, IL‐10 receptor (IL‐10R) beta and other cytokine receptor subunits for signal transduction, in response to various cytokines, including type‐I and type‐III IFNs, IL‐6, IL‐10, IL‐12 and IL‐23. Data on TYK2 dependence on cytokine responses and in vivo consequences of TYK2 deficiency are inconsistent. We investigated a TYK2 deficient patient, presenting with eczema, skin abscesses, respiratory infections and IgE levels >1000 U/mL, without viral or mycobacterial infections and a corresponding cellular model to analyze the role of TYK2 in type‐III IFN mediated responses and NK‐cell function. We established a novel simple diagnostic monocyte assay to show that the mutation completely abolishes the IFN‐α mediated antiviral response. It also partly reduces IL‐10 but not IL‐6 mediated signaling associated with reduced IL‐10Rβ expression. However, we found almost normal type‐III IFN signaling associated with minimal impairment of virus control in a TYK2 deficient human cell line. Contrary to observations in TYK2 deficient mice, NK‐cell phenotype and function, including IL‐12/IL‐18 mediated responses, were normal in the patient. Thus, preserved type‐III IFN responses and normal NK‐cell function may contribute to antiviral protection in TYK2 deficiency leading to a surprisingly mild human phenotype.     

Listeria monocytogenes induces an interferon‐enhanced activation of the integrated stress response that is detrimental for resolution of infection in mice

Type I interferons (IFNs) induce a detrimental response during Listeria monocytogenes (L. monocytogenes) infection. We were interested in identifying mechanisms linking IFN signaling to negative host responses against L. monocytogenes infection. Herein, we found that infection of myeloid cells with L. monocytogenes led to a coordinated induction of type I IFNs and activation of the integrated stress response (ISR). Infected cells did not induce Xbp1 splicing or BiP upregulation, indicating that the unfolded protein response was not triggered. CHOP (Ddit3) gene expression was upregulated during the ISR activation induced by L. monocytogenes. Myeloid cells deficient in either type I IFN signaling or PKR activation had less upregulation of CHOP following infection. CHOP‐deficient mice showed lower expression of innate immune cytokines and were more resistant than wild‐type counterparts following L. monocytogenes infection. These findings indicate that L. monocytogenes infection induces type I IFNs, which activate the ISR through PKR, which contributes to a detrimental outcome in the infected host.     

Enterovirus 71 blocks selectively type I interferon production through the 3C viral protein in mice

Type I interferons (IFNs) represent an essential innate defense mechanism for controlling enterovirus 71 (EV 71) infection. Mice inoculated with EV 71 produced a significantly lower amount of type I IFNs than those inoculated with poly (I:C), adenovirus type V, or coxsackievirus B3 (CB3). EV 71 infection, however, mounted a proinflammatory response with a significant increase in the levels of serum and brain interleukin (IL)‐6, monocyte chemoattractant protein‐1, tumor necrosis factor, and IFN‐γ. EV 71 infection abolished both poly (I:C)‐ and CB3‐induced type I IFN production of mice. Such effect was not extended to other enteroviruses including coxsackievirus A24, B2, B3, and echovirus 9, as mice infected with these viruses retained type I IFN responsiveness upon poly (I:C) challenge. In addition, EV 71‐infected RAW264.7 cells produced significantly lower amount of type I IFNs than non‐infected cells upon poly (I:C) stimulation. The inhibitory effect of EV 71 on type I IFN production was attributed to the viral protein 3C, which was confirmed using over‐expression systems in both mice and RAW264.7 cells. The 3C over‐expression, however, did not interfere with poly (I:C)‐induced proinflammatory cytokine production. These findings indicate that EV 71 can hamper the host innate defense by blocking selectively type I IFN synthesis through the 3C viral protein. J. Med. Virol. 84:1779–1789, 2012. © 2012 Wiley Periodicals, Inc.     

Anti‐cytokine autoantibodies suggest pathogenetic links with autoimmune regulator deficiency in humans and mice

Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) is a recessive disorder resulting from mutations in the autoimmune regulator (AIRE). The patients' autoantibodies recognize not only multiple organ‐specific targets, but also many type I interferons (IFNs) and most T helper type 17 (Th17) cell‐associated cytokines, whose biological actions they neutralize in vitro. These anti‐cytokine autoantibodies are highly disease‐specific: otherwise, they have been found only in patients with thymomas, tumours of thymic epithelial cells that fail to express AIRE. Moreover, autoantibodies against Th17 cell‐associated cytokines correlate with chronic mucocutaneous candidiasis in both syndromes. Here, we demonstrate that the immunoglobulin (Ig)Gs but not the IgAs in APECED sera are responsible for neutralizing IFN‐ω, IFN‐α2a, interleukin (IL)‐17A and IL‐22. Their dominant subclasses proved to be IgG1 and, surprisingly, IgG4 without IgE, possibly implicating regulatory T cell responses and/or epithelia in their initiation in these AIRE‐deficiency states. The epitopes on IL‐22 and IFN‐α2a appeared mainly conformational. We also found mainly IgG1 neutralizing autoantibodies to IL‐17A in aged AIRE‐deficient BALB/c mice – the first report of any target shared by these human and murine AIRE‐deficiency states. We conclude that autoimmunization against cytokines in AIRE deficiency is not simply a mere side effect of chronic mucosal Candida infection, but appears to be related more closely to disease initiation.     

The effect of types I and III interferons on adrenocortical cells and its possible implications for autoimmune Addison's disease

Autoimmune Addison's disease (AAD) is caused by selective destruction of the hormone‐producing cells of the adrenal cortex. As yet, little is known about the potential role played by environmental factors in this process. Type I and/or type III interferons (IFNs) are signature responses to virus infections, and have also been implicated in the pathogenesis of autoimmune endocrine disorders such as type 1 diabetes and autoimmune thyroiditis. Transient development of AAD and exacerbation of established or subclinical disease, as well as the induction of autoantibodies associated with AAD, have been reported following therapeutic administration of type I IFNs. We therefore hypothesize that exposure to such IFNs could render the adrenal cortex susceptible to autoimmune attack in genetically predisposed individuals. In this study, we investigated possible immunopathological effects of type I and type III IFNs on adrenocortical cells in relation to AAD. Both types I and III IFNs exerted significant cytotoxicity on NCI‐H295R adrenocortical carcinoma cells and potentiated IFN‐γ‐ and polyinosine‐polycytidylic acid [poly (I : C)]‐induced chemokine secretion. Furthermore, we observed increased expression of human leucocyte antigen (HLA) class I molecules and up‐regulation of 21‐hydroxylase, the primary antigenic target in AAD. We propose that these combined effects could serve to initiate or aggravate an ongoing autoimmune response against the adrenal cortex in AAD.