Role of Cytokines in Demyelinating Disease Studied in Transgenic Mice

The pathogenesis of inflammatory demyelinating diseases of the central nervous system (CNS) is complex and in part reflects the contribution of multiple cellular and molecular factors. Determining whether these factors are primary or secondary to lesion development or are pathogenic versus protective or reparative represents a major research challenge and will be critical in the development of effective therapeutic strategies. The recent development of experimental procedures that permit the stable germline transmission in mice (so-called transgenic mice) of specific genes with expression targeted to the intact CNS offers a powerful new approach for tackling this problem. In this paper we discuss our experience in the application of genetic engineering to develop transgenic mice with the astrocyte-targeted expression of the key mediators of the host response, the cytokines. Cytokines are a large family of pluripotent mediators with specific classes of these molecules being incriminated in the pathogenesis of inflammatory demyelinating diseases. Transgenic mice were developed in which the expression of the cytokines interleukin-6 or interleukin-3 was targeted to astrocytes using a glial fibrillary acidic protein genomic expression vector. Depending on which cytokine was expressed, these animals developed white matter disease with distinct patterns of demyelination associated with different pathologic features. Here we will describe the procedural details associated with the development of these transgenic models, focusing on three general themes: (i) production of transgenic animals, (ii) analysis of transgene expression, and (iii) characterization of the transgenic mouse phenotype.     

Physiological and pathological roles of interleukin-6 in the central nervous system

The cytokine interleukin-6 (IL-6) is an important mediator of inflammatory and immune responses in the periphery. IL-6 is produced in the periphery and acts systemically to induce growth and differentiation of cells in the immune and hematopoietic systems and to induce and coordinate the different elements of the acute-phase response. In addition to these peripheral actions, recent studies indicate that IL-6 is also produced within the central nervous system (CNS) and may play an important role in a variety of CNS functions such as cell-to-cell signaling, coordination of neuroimmune responses, protection of neurons from insult, as well as neuronal differentiation, growth, and survival. IL-6 may also contribute to the etiology of neuropathological disorders. Elevated levels of IL-6 in the CNS are found in several neurological disorders including AIDS dementia complex, Alzheimer's disease, multiple sclerosis, systemic lupus erythematosus, CNS trauma, and viral and bacterial meningitis. Moreover, several studies have shown that chronic overexpression of IL-6 in transgenic mice can lead to significant neuroanatomical and neurophysiological changes in the CNS similar to that commonly observed in various neurological diseases. Thus, it appears that IL-6 may play a role in both physiological and pathophysiological processes in the CNS.     

Type I interferon in neurological disease—The devil from within

The members of the type I interferon (IFN-I) family of cytokines are pleiotropic factors that have seminal roles in host defence, acting as antimicrobial and antitumor mediators as well as potent immunomodulatory factors that bridge the innate and adaptive immune responses. Despite these beneficial actions there is mounting evidence that link inappropriate or chronic production of IFN-I in the CNS to the development of a number of severe neuroinflammatory disorders. The most persuasive example is the genetically determined inflammatory encephalopathy, Aicardi–Goutières syndrome (AGS) in which patients have chronically elevated IFN-α production in the CNS. The presentation of AGS can often mimic congenital viral infection, however, molecular genetic studies have identified mutations in six genes that can cause AGS, most likely via dysregulated nucleic acid metabolism and activation of the innate immune response leading to increased intrathecal production of IFN-α. The role of IFN-α as a pathogenic factor in AGS and other neurological disorders has gained considerable support from experimental studies. In particular, a transgenic mouse model with CNS-restricted production of IFN-α replicates many of the cardinal neuropathologic features of AGS and reveal IFN-I to be the “devil from within”, mediating molecular and cellular damage within the CNS. Thus, targeting IFN-I may be an effective strategy for the treatment of AGS as well as some other autoimmune and infectious neurological “interferonopathies”.     

Analysis of the neuroinflammatory response to TLR7 stimulation in the brain: comparison of multiple TLR7 and/or TLR8 agonists

Activation of astrocytes and microglia and the production of proinflammatory cytokines and chemokines are often associated with virus infection in the CNS as well as a number of neurological diseases of unknown etiology. These inflammatory responses may be initiated by recognition of pathogen-associated molecular patterns (PAMPs) that stimulate TLRs. TLR7 and TLR8 were identified as eliciting antiviral effects when stimulated by viral ssRNA. In the present study, we examined the potential of TLR7 and/or TLR8 agonists to induce glial activation and neuroinflammation in the CNS by intracerebroventricular inoculation of TLR7 and/or TLR8 agonists in newborn mice. The TLR7 agonist imiquimod induced astrocyte activation and up-regulation of proinflammatory cytokines and chemokines, including IFN-beta, TNF, CCL2, and CXCL10. However, these responses were only of short duration when compared with responses induced by the TLR4 agonist LPS. Interestingly, some of the TLR7 and/or TLR8 agonists differed in their ability to activate glial cells as evidenced by their ability to induce cytokine and chemokine expression both in vivo and in vitro. Thus, TLR7 stimulation can induce neuroinflammatory responses in the brain, but individual TLR7 agonists may differ in their ability to stimulate cells of the CNS.     

Toll-like receptor 2 (TLR2) mediates astrocyte activation in response to the Gram-positive bacterium Staphylococcus aureus

Astrocytes play an important role in initiating and regulating CNS immune responses through the release of proinflammatory cytokines and chemokines. Here we demonstrate that primary astrocytes are capable of recognizing the Gram-positive bacterium Staphylococcus aureus and its cell wall product peptidoglycan (PGN) and respond by producing numerous proinflammatory mediators including interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1beta (MIP-1beta), MIP-2, and monocyte chemoattractant protein (MCP-1). Astrocytes have recently been shown to express Toll-like receptor 2 (TLR2), a pattern recognition receptor important for recognizing structural components of various Gram-positive bacteria, fungi, and protozoa. However, the functional significance of TLR2 in mediating astrocyte activation remains unknown. Primary astrocytes from TLR2 knockout mice were used to evaluate the role of TLR2 in astrocyte responses to S. aureus and PGN. The results demonstrate that TLR2 is essential for maximal proinflammatory cytokine and chemokine production, but not phagocytosis, in primary astrocytes following S. aureus and PGN exposure. In addition, both stimuli led to a significant increase in TLR2 mRNA expression in wild-type astrocytes as assessed by real-time quantitative RT-PCR. These findings suggest that astrocytes may play a key role in the initial antibacterial immune response in the CNS through engagement of TLR2.     

Complexity of the microglial activation pathways that drive innate host responses during lethal alphavirus encephalitis in mice

Microglia express multiple TLRs (Toll-like receptors) and provide important host defence against viruses that invade the CNS (central nervous system). Although prior studies show these cells become activated during experimental alphavirus encephalitis in mice to generate cytokines and chemokines that influence virus replication, tissue inflammation and neuronal survival, the specific PRRs (pattern recognition receptors) and signalling intermediates controlling microglial activation in this setting remain unknown. To investigate these questions directly in vivo, mice ablated of specific TLR signalling molecules were challenged with NSV (neuroadapted Sindbis virus) and CNS viral titres, inflammatory responses and clinical outcomes followed over time. To approach this problem specifically in microglia, the effects of NSV on primary cells derived from the brains of wild-type and mutant animals were characterized in vitro. From the standpoint of the virus, microglial activation required viral uncoating and an intact viral genome; inactivated virus particles did not elicit measurable microglial responses. At the level of the target cell, NSV triggered multiple PRRs in microglia to produce a broad range of inflammatory mediators via non-overlapping signalling pathways. In vivo, disease survival was surprisingly independent of TLR-driven responses, but still required production of type-I IFN (interferon) to control CNS virus replication. Interestingly, the ER (endoplasmic reticulum) protein UNC93b1 facilitated host survival independent of its known effects on endosomal TLR signalling. Taken together, these data show that alphaviruses activate microglia via multiple PRRs, highlighting the complexity of the signalling networks by which CNS host responses are elicited by these infections.     

Enterocyte expression of the eotaxin and interleukin-5 transgenes induces compartmentalized dysregulation of eosinophil trafficking.

Eosinophils accumulate in the gastrointestinal tract in a number of medical disorders, but the mechanisms involved are largely unknown. To understand the significance of cytokine expression by enterocytes, enterocyte transgenic mice that overexpressed the eosinophil-selective cytokines eotaxin and interleukin (IL)-5 were generated. Transgenic mice, generated by utilizing the rat intestinal fatty acid-binding protein promoter (Fabpi), overexpressed the mRNA for these cytokines in the small intestine. Overexpression of IL-5 resulted in marked increases of eosinophils in the bone marrow and blood, whereas eotaxin overexpression resulted in similar levels compared with nontransgenic control mice. In contrast, both IL-5 and eotaxin transgenic mice had significant accumulation of eosinophils in the gastrointestinal mucosa compared with control mice. Eotaxin-induced gastrointestinal eosinophilia was substantially higher than that induced by IL-5 and was especially prominent within the lamina propria of the villi. Interestingly, genetic rescue of eotaxin deficiency (by transgenic overexpression of eotaxin in eotaxin gene-targeted mice) resulted in significant restoration of gastrointestinal eosinophil levels. Finally, the intestinal eosinophilia induced by the eotaxin transgene was beta(7) integrin-dependent. Taken together, these results demonstrate that expression of eotaxin and IL-5 in intestinal epithelium induces compartmentalized dysregulation of eosinophil trafficking and the important role of the beta(7) integrin in gastrointestinal allergic responses.     

Paracellular permeability restricts airway epithelial responses to selectively allow activation by mediators at the basolateral surface

Respiratory pathogens and toxins often assault the lung from the airway lumen. Airway epithelia may initiate and amplify inflammation in response to these attacks, but under certain conditions confinement of inflammation to the airway lumen may be beneficial to the host. Accordingly, we hypothesized that airway epithelial polarity allows different responses to basolateral vs apical stimuli that may modulate inflammation. Using primary human airway epithelial cells differentiated at an air-liquid interface in culture, we found that responses to several cytokines required basolateral mediator application. In contrast, responses to Haemophilus influenzae occurred after either basolateral or apical interaction with airway epithelia. Experiments focused on IFN-gamma receptor polarity confirmed its predominant basolateral location in cultured airway epithelia as well as in normal human airway tissue. Furthermore, physical and pharmacologic disruption of barrier function in airway epithelia allowed responses to apical application of IFN-gamma and other cytokines. These in vitro studies directly correlated with experiments in mice in which an airway epithelial response to IFN-gamma injected into the airway lumen was seen only after disruption of barrier function. The results indicate that airway epithelia with intact barrier function restrict inflammatory responses by limitation of cell activation through requiring interaction of selected mediators with the basolateral surface. However, loss of barrier integrity allows epithelial responses to these mediators if located in the airway lumen to amplify airway defenses.     

Detection of autoantibodies to cytokines

Autoantibodies to various cytokines have been reported in normal individuals and in patients with various infectious and immunoinflammatory disorders, and similar antibodies (Ab) may be induced in patients receiving human recombinant cytokines. The clinical relevance of these Ab is often difficult to evaluate. Not only are in vitro neutralizing cytokine Ab not necessarily neutralizing in vivo, but assays for binding and neutralizing Ab to cytokines are often difficult to interpret. For example, denaturation of immobilized cytokines in immunoblotting techniques and immunometric assays may leave Ab to the native forms of the mediators unrecognized. On the other hand, Ab may bind nonspecifically and/or with biologically irrelevant low affinities, leading to erroneous interpretations. This article describes in detail the use of radioimmunoassays that we have optimized and used successfully for the detection of high-affinity (auto)Ab to IL-1α, IL-6, GM-CSF, and IFNα.     

Th2 polarization enhanced by oral administration of higher doses of antigen

Although oral administration of a soluble proteinantigen can induce various immune responses, theeffect of the dosage of oral antigen on thepredominance of Th2-type cytokine and antibodyresponses has not been well clarified yet. In thepresent study, we fed T cell receptor (TCR) transgenic(tg) mice various amounts of chicken ovalbumin (0.1,5, and 250 mg) and examined the resulting immuneresponses to this antigen. In these TCR tg mice, theresponses of antigen-specific T cells were greatlyamplified concomitantly with significantantigen-specific cytokine secretion. We found that ahigh dose (250 mg) of antigen significantlyupregulated the serum antigen-specific IgG1 and IgAantibody responses in mice later intraperitoneallyinjected with antigen plus adjuvant. The miceadministered the same oral dose but not immunizedshowed upregulation of Th2-type IL-4 and IL-5secretion and downregulation of Th1-type IL-2 andIFN-. This enhancement of Th2-type cytokineand antibody responses was more marked when largerdoses of antigen orally administered. These resultsdemonstrated that antigen feeding induces thedevelopment of T cells secreting Th2-type cytokines ina dose-dependent manner and that these T cells have ahelper function for the production of antibodies ofthe Th2-type isotypes. This experimental system shouldbe useful to screen foods and other substances thatcan modulate Th2-type responses relating to allergy.     

Overexpression of Bcl(XL) in B cells promotes Th1 response and exacerbates collagen-induced arthritis

B cells play a pathogenic or regulatory role in many autoimmune diseases through production of autoantibodies, cytokine production, and Ag presentation. However, the mechanisms that regulate these B cell functions under different autoimmune settings remain unclear. In the current study, we found that when B cells overexpress an antiapoptotic gene, Bcl(XL), they significantly increased production of IFN-gamma and enhanced Th1 response. Consistently, Bcl-x(L) transgenic mice developed more severe and sustained collagen-induced arthritis due to the enhanced Th1 response. The production of autoantibodies in Bcl(XL) transgenic mice was comparable to that in wild-type mice. Thus, our results indicate a novel role of Bcl(XL) in regulating B cell functions and immune responses. In patients with rheumatoid arthritis, arthritogenic B cells often up-regulate Bcl(XL) expression, which may not only render B cells resistant to apoptosis but also alter the ability of the autoreactive B cells to produce cytokines and modulate the inflammatory response. This may have therapeutic implications if Bcl(XL) expression can be down-regulated in autoreactive B cells.     

Innate immune responses to Rhodococcus equi

We examined innate immune responses to the intracellular bacterium Rhodococcus equi and show that infection of macrophages with intact bacteria induced the rapid translocation of NF-kappa B and the production of a variety of proinflammatory mediators, including TNF, IL-12, and NO. Macrophages from mice deficient in MyD88 failed to translocate NF-kappa B and produced virtually no cytokines in response to R. equi infection, implicating a TLR pathway. TLR4 was not involved in this response, because C3H/HeJ macrophages were fully capable of responding to R. equi infection, and because RAW-264 cells transfected with a dominant negative form of TLR4 responded normally to infection by R. equi. A central role for TLR2 was identified. A TLR2 reporter cell was activated by R. equi, and RAW-264 cells transfected with a dominant negative TLR2 exhibited markedly reduced cytokine responses to R. equi. Moreover, macrophages from TLR2(-/-) mice exhibited diminished cytokine responses to R. equi. The role of the surface-localized R. equi lipoprotein VapA (virulence-associated protein A), in TLR2 activation was examined. Purified rVapA activated a TLR2-specific reporter cell, and it induced the maturation of dendritic cells and the production of cytokines from macrophages. Importantly, TLR2(-/-)-deficient but not TLR4(-/-)-deficient mice were found to be compromised in their ability to clear a challenge with virulent R. equi. We conclude that the efficient activation of innate immunity by R. equi may account for the relative lack of virulence of this organism in immunocompetent adults.     

Genetically Modified Pig Models for Human Diseases

Genetically modified animal models are important for understanding the pathogenesis of human disease and developing therapeutic strategies.Although genetically modified mice have been widely used to model human diseases,some of these mouse models do not replicate important disease symptoms or pathology.Pigs are more similar to humans than mice in anatomy,physiology,and genome. Thus,pigs are considered to be better animal models to mimic some human diseases.This review describes genetically modified pigs that have been used to model various diseases including neurological,cardiovascular,and diabetic disorders.We also discuss the development in gene modification technology that can facilitate the generation of transgenic pig models for human diseases.     

CNS-Targeted Production of IL-17A Induces Glial Activation,Microvascular Pathology and Enhances the Neuroinflammatory Response to Systemic Endotoxemia

Interleukin-17A (IL-17A) is a key cytokine modulating the course of inflammatory diseases. Whereas effector functions of IL-17A like induction of antimicrobial peptides and leukocyte infiltration could clearly be demonstrated for peripheral organs, CNS specific effects are not well defined and appear controversial. To further clarify the functional significance of IL-17A in the CNS, we generated a transgenic mouse line with astrocyte-restricted expression of the IL-17A gene. GFAP/IL-17A transgenic mice develop normally and do not show any signs of neurological dysfunction. However, histological characterization revealed astrocytosis and activation of microglia. Demyelination, neurodegeneration or prominent tissue damage was not observed but a vascular pathology mimicking microangiopathic features was evident. Histological and flow cytometric analysis demonstrated the absence of parenchymal infiltration of immune cells into the CNS of GFAP/IL-17A transgenic mice. In GFAP/IL-17A mice, LPS-induced endotoxemia led to a more pronounced microglial activation with expansion of a distinct CD45^high/CD11b⁺ population and increased induction of proinflammatory cytokines compared with controls. Our data argues against a direct role of IL-17A in mediating tissue damage during neuroinflammation. More likely IL-17A acts as a modulating factor in the network of induced cytokines. This novel mouse model will be a very useful tool to further characterize the role of IL-17A in neuroinflammatory disease models.     

Lymphomagenesis, hydronephrosis, and autoantibodies result from dysregulation of IL-9 and are differentially dependent on Th2 cytokines

Interleukin-9 is an immunoregulatory cytokine implicated in the development of asthma and allergy. To investigate the role of IL-9 in vivo, we have generated transgenic mice in which IL-9 is expressed from its own promoter. Strikingly, overexpression of IL-9 resulted in premature mortality associated with a complex phenotype characterized by the development of autoantibodies, hydronephrosis, and T cell lymphoma. By intercrossing IL-9 transgenic mice with a panel of Th2 cytokine-deficient mice, we demonstrate that these disorders represent distinct phenotypes that can be dissociated by their differential dependence on Th2 cytokines. Autoantibody production was ablated in IL-9 transgenic animals with a combined absence of IL-4, IL-5, and IL-13, coincident with a reduction in peritoneal B-1 cells. Hydronephrosis arose in 75% of IL-9 transgenic animals and was dependent on the presence of IL-4 and IL-13. In contrast, T cell lymphomas developed independently of the other Th2 cytokines, with the generation of rapidly proliferating CD8(+) or CD4(+)CD8(+) T cell clones that arose in the thymus before infiltrating both lymphoid and nonlymphoid tissues. Our data highlight potentially important new roles for IL-9, through its regulation of downstream Th2 effector cytokines, in autoantibody production and in hydronephrosis.     

Faithful expression of the human 5q31 cytokine cluster in transgenic mice

Interleukins -4, -5, and -13, cardinal cytokines produced by Th2 cells, are coordinately expressed and clustered in 150-kb syntenic regions on mouse chromosome 11 and human chromosome 5q31. We analyzed two sets of human yeast artificial chromosome transgenic mice that contained the 5q31 cytokines to assess whether conserved sequences required for their coordinate and cell-specific regulation are contained within the cytokine cluster itself. Human IL-4, IL-13, and IL-5 were expressed under Th2, but not Th1, conditions in vitro. Each of these cytokines was produced during infection with Nippostrongylus brasiliensis, a Th2-inducing stimulus, and human IL-4 was generated after activation of NK T cells in vivo. Consistently fewer cells produced the endogenous mouse cytokines in transgenic than in control mice, suggesting competition for stable expression between the mouse and human genes. These data imply the existence of both conserved trans-activating factors and cis-regulatory elements that underlie the coordinate expression and lineage specificity of the type 2 cytokine genes in lymphocytes.     

Regulation of avoidant behaviors and pain by the anti-inflammatory tyrosine phosphatase SHP-1

The protein tyrosine phosphatase SHP-1 is a critical regulator of cytokine signaling and inflammation. Mice homozygous for a null allele at the SHP-1 locus have a phenotype of severe inflammation and are hyper-responsive to the TLR4 ligand LPS. TLR4 stimulation in the CNS has been linked to both neuropathic pain and sickness behaviors. To determine if reduction in SHP-1 expression affects LPS-induced behaviors, responses of heterozygous SHP-1-deficient (me/+) and wild-type (+/+) mice to LPS were measured. Chronic (4-week) treatment with LPS induced avoidant behaviors indicative of fear/anxiety in me/+, but not +/+, mice. These behaviors were correlated with a LPS-induced type 2 cytokine, cytokine receptor, and immune effector arginase profile in the brains of me/+ mice not found in +/+ mice. Me/+ mice also had a constitutively greater level of TLR4 in the CNS than +/+ mice. Additionally, me/+ mice displayed constitutively increased thermal sensitivity compared to +/+ mice, measured by the tail-flick test. Moreover, me/+ glial cultures were more responsive to LPS than +/+ glia. Therefore, the reduced expression of SHP-1 in me/+ imparts haploinsufficiency with respect to the control of CNS TLR4 and pain signaling. Furthermore, type 2 cytokines become prevalent during chronic TLR4 hyperstimulation in the CNS and are associated positively with behaviors that are usually linked to type 1 pro-inflammatory cytokines. These findings question the notion that type 2 immunity is solely anti-inflammatory in the CNS and indicate that type 2 immunity induces/potentiates CNS inflammatory processes.     

Sphingosine kinase 1 regulates pro-inflammatory responses triggered by TNFalpha in primary human monocytes

Monocytes play an important role in inflammation, angiogenesis, and atherosclerosis. During these processes monocytes release pre-formed proinflammatory mediators from granules, and synthesize de novo cytokines and chemokines important in the amplification of the inflammatory response. One of the most prominent triggers of inflammatory responses is the cytokine TNFalpha. However, the intracellular signaling cascades triggered by TNFalpha are not fully understood. In this study we investigated the roles of SPHK on the TNFalpha-triggered responses on human primary monocytes. We show that TNFalpha rapidly triggers S1P generation and activation of SPHK. Moreover, our data shows that SPHK1 is the isoform activated by TNFalpha, and plays an essential role on the TNFalpha-triggered intracellular Ca2+ signals, degranulation, cytokine production, and activation of NFkappaB, thus suggesting a pivotal role for SPHK1 on the proinflammatory responses triggered by TNFalpha.     

Differential sensitivity to Itk kinase signals for T helper 2 cytokine production and chemokine-mediated migration.

Allergic asthma is dependent on chemokine-mediated Th2 cell migration and Th2 cytokine secretion into the lungs. The inducible T cell tyrosine kinase Itk regulates the production of Th2 cytokines as well as migration in response to chemokine gradients. Mice lacking Itk are resistant to developing allergic asthma. However, the role of kinase activity of Itk in the development of this disease is unclear. In addition, whether distinct Itk-derived signals lead to T cell migration and secretion of Th2 cytokines is also unknown. Using transgenic mice specifically lacking Itk kinase activity, we show that active kinase signaling is required for control of Th2 responses and development of allergic asthma. Moreover, dominant suppression of kinase Itk activity led to normal Th2 responses, but significantly reduced chemokine-mediated migration, resulting in prevention of allergic asthma. These observations indicate that signals required for Th2 responses and migration are differentially sensitive to Itk activity. Manipulation of Itk's activity can thus provide a new strategy to treat allergic asthma by differentially affecting migration of T cells into the lungs, leaving Th2 responses intact.