Th2-mediated Immune Responses Research Articles

Adjuvant and Formulation Considerations for a Subunit Blood-Stage Malaria Vaccine

Protection against malaria from a vaccine requires modulation of the immune response to malaria antigens. This modulation by pathogen-derived molecules is explored in this study. Human immune cells synergistically express signaling proteins when stimulated by certain combinations of pathogen-derived molecules. Some of these expression profiles indicate a Th1-mediated immune response, which may be the immune response needed for the next-generation malaria of vaccines, in terms of effectiveness.

A therapeutic vaccine prototype induces protective immunity and reduces cardiac fibrosis in a mouse model of chronic Trypanosoma cruzi infection

Chagas disease, caused by the parasite Trypanosoma cruzi, develops into chronic Chagas’ cardiomyopathy in ~30% of infected individuals, characterized by conduction disorders, arrhythmias, heart failure, and even sudden cardiac death. Current anti-parasitic treatments are plagued by significant side effects and poor efficacy in the chronic phase of disease; thus, there is a pressing need for new treatment options. A therapeutic vaccine could bolster the protective TH1-mediated immune response, thereby slowing or halting the progression of chronic Chagas’ cardiomyopathy. Prior work in mice has demonstrated therapeutic efficacy of a Tc24 recombinant protein vaccine in the acute phase of Chagas disease. However, it is anticipated that humans will be vaccinated therapeutically when in the chronic phase of disease. This study investigates the therapeutic efficacy of a vaccine prototype containing recombinant protein Tc24, formulated with an emulsion containing the Toll-like receptor 4 agonist E6020 as an immunomodulatory adjuvant in a mouse model of chronic T. cruzi infection. Among outbred ICR mice vaccinated during chronic T. cruzi infection, there is a significant increase in the number of animals with undetectable systemic parasitemia (60% of vaccinated mice compared to 0% in the sham vaccine control group), and a two-fold reduction in cardiac fibrosis over the control group. The vaccinated mice produce a robust protective TH1-biased immune response to the vaccine, as demonstrated by a significant increase in antigen-specific IFNγ-production, the number of antigen-specific IFNγ-producing cells, and IgG2a antibody titers. Importantly, therapeutic vaccination significantly reduced cardiac fibrosis in chronically infected mice. This is a first study demonstrating therapeutic efficacy of the prototype Tc24 recombinant protein and E6020 stable emulsion vaccine against cardiac fibrosis in a mouse model of chronic T. cruzi infection.

Pertussis vaccines and protective immunity.

Despite high vaccine coverage, reported cases of pertussis have increased steadily over the last twenty years. This resurgence has stimulated interest in host responses to pertussis infection and vaccination with the goal of developing more effective next-generation vaccines and vaccination strategies. Optimal protection against Bordetella pertussis appears to be multifactorial requiring both humoral and cellular responses. Natural infection and whole-cell pertussis vaccination induce Th1 and Th17-dominated responses. In contrast, acellular vaccines induce Th2-dominated responses. Available immunological data indicate that while antibodies provide protection against disease, Th1 and Th17-mediated immune responses are required for bacterial clearance and long-lasting protection. The nature of the priming in children appears to be important in modulating bias and durability of immune responses required to provide protection against B. pertussis. This review summarizes the current understanding of differences in immune responses and their role in protection against B. pertussis following infection or vaccination.

S90. Association of Autoimmunity With Dysfunctional Th2-Mediated Immune Response in Major Depressive Disorder

Dysfunctional T-helper 2 (Th2) cell mediated immune response, characterized by reduced interleukin 4 (IL-4), was reported recently in patients with major depressive disorder (MDD) who were enrolled in the Predicting Response to Depression Treatment study. Similarly, we found reduced levels of IL-4 in a separate sample of adolescents with MDD and recent suicide behavior as compared to healthy controls. However, the functional consequence of reduced IL-4 in patients with MDD remains unclear.

Intranasal immunization with recombinant outer membrane protein A induces protective immune response against Stenotrophomonas maltophilia infection.

Stenotrophomonas maltophilia (S. maltophilia), a multi-drug resistant opportunistic pathogen, is associated with nosocomial and community-acquired infections. Preventive and therapeutic strategies for such infections are greatly needed. In this study, sequence alignment analysis revealed that Outer membrane protein A (OmpA) was highly conserved among S. maltophilia strains but shared no significant similarity with human and mouse proteomes. In mice, intranasal immunization with S. maltophilia recombinant OmpA (rOmpA) without additional adjuvant induced sustained mucosal and systemic rOmpA-specific antibody responses. Treatment with rOmpA stimulated significantly higher levels of secretion of IFN-γ, IL-2, and IL-17A (All P<0.05) from the primary splenocytes isolated from rOmpA-immunized mice than from the primary splenocytes isolated from PBS-immunized mice. Furthermore, mice immunized with rOmpA showed significantly reduced bacterial burden in the lung and reduced levels of pro-inflammatory cytokines (TNF-α and IL-6) in bronchoalveolar lavage fluid (BALF) 24 hours after intranasal S. maltophilia infection, indicating that immunization with rOmpA may have protective effects against S. maltophilia challenge in mice. Our findings suggest that intranasal immunization with rOmpA may induce mucosal and systemic immune responses in mice, trigger Th1- and Th17-mediated cellular immune responses, and thus stimulate host immune defense against S. maltophilia infection. These results also demonstrate that intranasal vaccination may offer an alternative approach to current strategies since it induces a mucosal as well as a systemic immune response.

Inflammasome activation and Th17 responses

Immune sensing of exogenous molecules from microbes (e.g., pathogen-associated molecular patterns) and nonmicrobial molecules (e.g., asbestos, alum, and silica), as well as endogenous damage-associated molecular patterns (e.g., ATP, uric acid crystals, and amyloid A) activates innate immunity by inducing immune-related genes, including proinflammatory cytokines, which further facilitate the development of adaptive immunity. The roles of transcriptional responses downstream of immune sensing have been widely characterized in informing adaptive immunity; however, few studies focus on the effect of post-translational responses on the modulation of adaptive immune responses. Inflammasomes activated by the previously described endo- and exogenous stimuli autocatalytically induce intracellular pro-caspase-1, which cleaves the inactive precursors of interleukin-1β (IL-1β) and IL-18 into bioactive proinflammatory cytokines. IL-1β and IL-18 not only contribute to the host defense against infections by activating phagocytes, such as monocytes, macrophages, dendritic cells, and neutrophils, but also induce T-helper 17 (Th17)- and Th1-mediated adaptive immune responses. In synergy with IL-6 and IL-23, IL-1β activates IL-1 receptor (IL-1R) signaling to drive the differentiation of IL-17-producing Th17 cells, which not only play critical roles in host protective immunity to infections of bacteria, fungi, and certain viruses but also participate in the pathology of inflammatory disorders and tumorigenesis. Consequently, targeting inflammasomes and IL-1/IL-1R signaling may effectively improve the treatment of Th17-associated disorders, such as autoinflammatory diseases and cancers, thereby providing novel insights into drug development.

Neopterin Levels and Indoleamine 2,3-Dioxygenase Activity as Biomarkers of Immune System Activation and Childhood Allergic Diseases.

BackgroundAlthough Th2 immune activation is predominant in allergic diseases, neopterinlevels and indoleamine 2,3-dioxygenase (IDO)-1 activity (kynurenine:tryptophan ratio), which reflect Th1 immune activity, increase with interferon-gamma (IFN-γ) stimulation. We investigated neopterin, tryptophan, and kynurenine levels as biomarkersof the Th1 immune system activation and changes in IDO-1 activityin children with asthma, allergic rhinitis, and atopic dermatitis, as well as the relationship between these biomarkers and the total IgE level, age, and disease severity.MethodsWe divided 205 children (80 girls and 125 boys, four months to 17 years old) into four groups: controls, patients with asthma, patients with allergic rhinitis, and patients with atopic dermatitis. Peripheral venous blood samples were collected. Neopterin levels were determined by an enzyme immunoassay. Tryptophan and kynurenine levels were analyzed using HPLC. IDO-1 enzyme activity was calculated using tryptophan and kynurenine levels. IgE levels were measured. The Mann-Whitney U test, Kruskal-Wallis test, and Conover post-hoc method were used for statistical analysis.ResultsNeopterin, tryptophan, and kynurenine levels were higher and IgE levels and IDO-1 enzyme activity were lower in patients with asthma and allergic rhinitis than in controls (P<0.05). Patients with atopic dermatitis showed higher neopterin, tryptophan, and kynurenine levels, higher IDO-1 activity, and lower IgE levels thancontrols (P<0.05).ConclusionsThe Th1/Th2 balance is disrupted in children with allergic diseases, concomitant with increased Th1-mediated immune response activation and reduced IgEproduction, which is promoted by Th2-type cytokines.

Understanding of the Molecular Mechanisms of Allergy.

The prevalence of allergic disease has dramatically increased over the past 30years in Westernized countries. It is unlikely that the rapid increase in the prevalence of allergic disease is the result of genetic changes, which highlights the importance of environmental factors in the development of allergic disease. The "hygiene hypothesis" was put forward in 1989 and focused attention on the notion that exposure to microbes and their products in early life can modify the risk for development of allergic disease. Infections were thought to polarize the immunological response toward a Th2-mediated immune response causing allergic disease. However, it is likely that the Th1/Th2 imbalance is too simplistic to explain the increased prevalence of allergic disease. Current research is focusing on understanding the role of T-regulatory cells in inducing a state of tolerance and the resulting modified Th2 response observed in natural and induced tolerance.

F116USE OF THE INFINIUM SNP PSYCHARRAY SYSTEM IN A CRIMINAL TRIAL: THE FIRST CASE STUDY IN SUPPORT OF A CLINICAL DIAGNOSIS OF SCHIZOPHRENIA AND IMPLICATIONS FOR ITS FUTURE USE IN LEGAL PROCEEDINGS IN THE UNITED STATES

Elevated level of interleukin (IL)-17, predominantly produced by T helper (Th) 17 cells, has been implicated in diabetic retinopathy (DR), but it remains unclear whether IL-17 is involved in the pathogenesis of DR. Ins2Akita (Akita) mice spontaneously develop diabetes, and show early pathophysiological changes in diabetic complications. On the other hand, interferon-γ knock out (GKO) mice exhibit high differentiation and activation of Th2 and Th17 cells as a result of Th1 cell inhibition. In this study, Ins2Akita IFN-γ–deficient (Akita-GKO) mice were established by crossbreeding Akita mice with GKO mice, and Th17-mediated immune responses on DR were investigated. Blood glucose levels (BGL) of Akita mice and Akita-GKO mice were significantly higher than those of age-matched wild type (WT) or GKO mice, and there was no significant difference in BGL between Akita and Akita-GKO mice. Relative mRNA expression of ROR-γt that is a transcriptional factor of Th17 cells but not GATA-3 that is for Th2 cells was significantly upregulated only in Akita-GKO mice compared with WT mice, and the proportions of IL-17 and IL-22–producing splenic CD4+ cells were significantly higher in Akita-GKO mice than in wild type (WT), Akita, or GKO mice. In the retina, mRNA expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1) were increased in Akita-GKO mice more than in Akita or GKO mice, and statistically significant differences were observed between Akita-GKO mice and WT mice. Leukostasis in retinal vessels and ocular level of VEGF protein increased significantly in Akita-GKO mice compared with the other groups. Edematous change in the retinal surface layer, retinal exudative lesions depicted as areas of hyperfluorescence in fluorescein angiography (FA), and vascular basement membrane thickening in all layers of the retina were also observed in Akita-GKO mice at 9-week-old but not in age-matched Akita or GKO mice. These results suggested that Th17 cell-mediated immune responses might be involved in promotion of functional and morphological changes in the retina of mice spontaneously developing diabetes.

Inflammation, Immunity, and Vaccine Development for the Gastric Pathogen Helicobacter pylori.

It has been over 30years since a link was established between H. pylori infection of the gastric mucosa and the development of chronic gastric diseases. Research in rodent models supported by data from human tissue demonstrated that the host immune response to H. pylori is limited by host regulatory T cells. Immunization has been shown to induce a potent Th1- and Th17-mediated immune response capable of eradicating or at least significantly reducing the bacterial load of H. pylori in the stomach in small animal models. These results have not translated well to humans. Clinical trials employing many of the strategies used in rodents for oral immunization including the use of a mucosal adjuvant such as Escherichia coli LT or delivery by attenuated enteric bacteria have failed to limit H. pylori infection and have highlighted the potential toxicity of exotoxin-based mucosal adjuvants. A recent study, however, utilizing a recombinant fusion protein of H. pylori urease and the subunit B of E. coli LT, was performed on over 4000 children. Efficacy of over 70% was demonstrated against naturally acquired infection compared to control volunteers one year post-immunization. Efficacy was reduced, but still above 50% at three years. This study provided new insight into the strategies for developing an improved vaccine for widespread use in countries with high infection rates and where gastric cancer (GC) remains one of the most common causes of death due to cancer.

Sensing danger through a "finger".

In this issue of JEM, the study by Chen et al. (https://doi.org/10.1084/jem.20181031) reveals a previously unrecognized role of cellular nucleic acid-binding protein (Cnbp) as a novel transcriptional regulator of interleukin-12β (IL-12β) transcription and IL-12-driven, Th1-mediated immune responses, which has important implications for both host defense and inflammatory disease.

PERSISTENT LIP AND FACIAL SWELLING IN A 9 YEAR OLD BOY

Introduction Orofacial Granulomatosis (OFG) is a chronic granulomatous condition characterized by persistent or recurring swelling of soft tissues in the face. It is thought to be caused by a delayed hypersensitivity reaction involving a Th1-mediated immune response. There have been reports of cases associated with Sarcoidosis and Crohn's disease. Case Description A 9-year old male presented to clinic with 15 months of lip and facial swelling. Episodes last 2-3 weeks with resolution periods of 1-2 weeks. Antihistamines failed to improve the swelling. He has normal growth and development. He denies any fevers, headaches, eye redness/pain, oral/nasal ulcerations, difficulty swallowing, decrease in physical conditioning, nausea/vomiting/diarrhea, or bone/joint/muscle complaints. He was started on high dose steroids which improved his lip swelling but as the prednisone was tapered, the swelling returned. He had a mild elevation in Ig G4 and CRP, positive histone antibodies, and elevated C3/C4 and Ig G levels. His Hereditary Angioedema, Sarcoidosis, and Crohn's disease evaluation were negative. Eventually a biopsy of the vestibule of the anterior mandible was done which showed non-necrotizing granulomas. Discussion OFG symptoms can be indistinguishable to those caused by angioedema, contact dermatitis, melkersson-rosenthal syndrome, and lymphangioedema. OFG is made from a diagnosis of exclusion as biopsy cannot distinguish between OFG and Crohn's or Sarcoidosis. Treatment of this condition can be challenging and include steroids, NSAIDs, antibiotics, and TNF-alfa inhibitors. The goal of this case is to provide insight on this uncommon condition whose symptoms present as known sequela of frequent diseases that allergists encounter in their practice.

Hydrophilic Astragalin Galactoside Induces T Helper Type 1-Mediated Immune Responses via Dendritic Cells.

A flavonoid Astragalin (kaempferol-3-O-β-d-glucopyranoside, Ast) has several biological activities including anti-oxidant, anti-HIV, and anti-allergic effects. Nonetheless, its insolubility in hydrophilic solvents imposes restrictions on its therapeutic applications. In this study, we investigated the effects of water-soluble astragalin-galactoside (kaempferol-3-O-β-d-isomaltotrioside, Ast-Gal) on murine bone marrow-derived dendritic cell (DC) maturation and T helper (Th) cell-mediated immune responses. Ast-Gal significantly increased maturation and activation of DCs through the upregulation of surface markers, such as cluster of differentiation (CD)80, CD86, and Major histocompatibility complex (MHC) II in a dose-dependent manner, while Ast had little effects. Additionally, Ast-Gal-treated DCs markedly secreted immune-stimulating cytokines such as interleukin (IL)-1β, IL-6, and IL-12. Importantly, Ast-Gal strongly increased expression of IL-12, a polarizing cytokine of Th1 cells. In a co-culture system of DCs and CD4+ T cells, Ast-Gal-treated DCs preferentially differentiates naïve CD4+ T cells into Th1 cells. The addition of neutralizing IL-12 monoclonal antibody (mAb) to cultures of Ast-Gal-treated DCs and CD4+ T cells significantly decreased interferon (IFN)-γ production, thereby indicating that Ast-Gal-stimulated DCs enhance the Th1 response through IL-12 production by DCs. Injection with Ast-Gal-treated DCs in mice increased IFN-γ-secreting Th1 cell population. Collectively, these findings indicate that hydrophilically modified astragalin can enhance Th1-mediated immune responses via DCs and point to a possible application of water-soluble astragalin-galactoside as an immune adjuvant.

Dopamine regulates cytokine secretion during innate and adaptive immune responses.

Dopamine (DA) is synthesized by various immune cells. DA receptors (DARs), which comprise five isoforms, are expressed on the surface of these cells. Therefore, it is likely that DA plays a role in regulating innate and adaptive responses. However, the underlying molecular mechanism(s) is largely unknown. Here, we found that, during innate immune responses, DA suppressed secretion of IFN-γ, TNF-α and IL-1β, but promoted secretion of IL-10 and CXCL1 by lipopolysaccharide (LPS)-stimulated mouse splenocytes, suggesting that DA regulates cytokine secretion. Immune subset studies indicated that DA suppressed secretion of IFN-γ, TNF-α and IL-1β by NK cells, as well as secretion of TNF-α by neutrophils and monocytes; however, DA up-regulated IL-10 secretion by neutrophils, monocytes, B cells, macrophages (Mφs) and dendritic cells within the splenocyte population. In addition, DA up-regulated secretion of CXCL1 by LPS-stimulated NK cells and Mφs. Meanwhile, treatment with DAR agonists or antagonists suppressed secretion of inflammatory cytokines from LPS-stimulated splenocytes. Pre-treatment of LPS-stimulated splenocytes with the PI3K inhibitor wortmannin reversed DA-mediated suppression of IFN-γ secretion, indicating that DA regulates IFN-γ secretion via the inositol 1,4,5-trisphosphate signaling pathway in these cells. Administration of DA and LPS to mice immunized with chicken ovalbumin (OVA) increased secretion of IL-5 by mouse lung lymphocytes, suggesting that DA promotes OVA-specific Th2-mediated immune responses by these cells. Taken together, these findings indicate that DA regulates cytokine secretion during innate and adaptive immune responses.

Th17 activation by dendritic cells stimulated with gamma-irradiated Streptococcus pneumoniae

Dendritic cells (DCs) play an important role in antigen presentation, which is an essential step for the induction of antigen-specific adaptive immunity. Inactivated bacterial whole cell vaccines have been widely used to prevent many bacterial infections because they elicit good immunogenicity due to the presence of various antigens and are relatively inexpensive and easy to manufacture. Recently, gamma-irradiated whole cells of nonencapsulated Streptococcus pneumoniae were developed as a broad-spectrum and serotype-independent multivalent vaccine. In the present study, we generated gamma-irradiated S. pneumoniae (r-SP) and investigated its capacity to stimulate mouse bone marrow-derived DCs (BM-DCs) in comparison with heat-inactivated and formalin-inactivated S. pneumoniae (h-SP and f-SP, respectively). r-SP showed an attenuated binding and internalization level to BM-DCs when compared to h-SP or f-SP. r-SP weakly induced the expression of CD80, CD83, CD86, MHC class I, and PD-L2 compared with h-SP or f-SP. Furthermore, r-SP less potently induced IL-6, TNF-α, and IL-23 expression than h-SP or f-SP but more potently induced IL-1β expression than h-SP or f-SP in BM-DCs. Since Th17-mediated immune responses are known to be important for the protection against pneumococcal infections, r-SP-primed DCs were co-cultured with splenocytes or splenic CD4+ T cells. Interestingly, r-SP-sensitized BM-DCs markedly induced IL-17A+ CD4+ T cells whereas h-SP- or f-SP-sensitized BM-DCs weakly induced them. Collectively, these results suggest that r-SP could be an effective pneumococcal vaccine candidate eliciting Th17-mediated immune responses by stimulation of DCs.

Vaccination with Live Attenuated L. Major and TLR4 Agonist Promotes a Th1 Immune Response and Induces Protection against L. Major Infection in BALB/c Mice.

Toll like receptors play a major role in immune responses against Leishmania parasites. To evaluate the efficacy of vaccination with live attenuated L. major and TLR4 agonist in protection against L. major infection. Attenuated L. major was prepared by continuous sub-culturing of the parasite. A total of 90 mice were assigned to 9 groups including 6 groups of BALB/c (G1-6) and 3 groups (G7-9) of C57BL/6 mice. Group 1 was the control groups, group 2 received the wild-type L. major promastigotes, group 3 the attenuated line, group 4 the TLR4 agonist, group 5 the wild-type L. major and TLR4 agonist, and group 6 the attenuated line along with TLR4 agonist. Group 7 was control, group 8 received wild-type L. major and group 9 the wild-type along with TLR4 agonist. Vaccinated mice were then challenged with wild-type of L. major. Lesion size, parasite burden, and the expression levels of IL-4, IFN-γ, IL-2, 1L-17A, IL-10, TGF-β and TLR4 were evaluated before the challenge while parasite burden and lesion size were evaluated. Vaccinated mice with a TLR4 agonist or attenuated L. major plus TLR4 agonist produced the highest levels of IFN-γ, IL-2, and IL-17A. Post-challenge analysis revealed that mice vaccinated with the attenuated line along with TLR4 agonist displayed the lowest lesion size and parasite load. These mice developed a predominant Th1 immune response. Vaccination with the attenuated L. major along with TLR4 agonist promotes a Th1-mediated immune response which leads to the protection of BALB/c mice against L. major infection.

Effects of the Th2-dominant milieu on allergic responses in Der f 1-activated mouse basophils and mast cells

Although basophils and mast cells share similar phenotypic and functional properties, little is known about the difference in the initial Th2 immune responses of these cells following exposure to proteolytic allergens. Here, we investigated the mechanisms of Th2-mediated immune responses in mouse bone marrow-derived basophils (BMBs) and mast cells (BMMCs) via stimulation with the cysteine protease allergen Der f 1. Our results showed that Th2 cytokines were induced from BMBs by active recombinant Der f 1 (rDer f 1 independently with Toll-like receptor (TLR) 2 and TLR4. Although both BMBs and BMMCs expressed protease-activated receptors on their surfaces, PAR expression following exposure to rDer f 1 was altered only in basophils. G protein-coupled receptors in basophils were found to be associated with interleukin (IL)-4 and IL-13 production from BMBs upon Der f 1 treatment. Secretion of Th2 cytokines from rDer f 1-treated basophils was mediated by G protein βγ and phosphatidylinositol 3-kinase γ through the extracellular signal-regulated kinase and c-Jun N-terminal kinase pathways. These findings provide insights into the roles of cysteine proteases in Th2 immune responses, such as allergic diseases, and improve our understanding of the mechanisms of Th2 cytokine production.

Production and characterization of monoclonal antibodies specific for chicken interleukin-4

Abstract Interleukin-4 is an important Th2 type cytokine in mammals which regulates growth and differentiation of B Lymphocytes. On the mammalian counterparts, the function of IL-4 orthologue in chickens (chIL-4) is less understood. Although the source of chIL-4 is not well established, it has been shown to have an integral role in mediating Th2 type immune responses in chickens. Despite its significant role, no monoclonal antibodies (mAbs) against chIL-4 have been developed till date. Therefore, in the present study we used recombinant yeast-expressed chIL-4 as an immunogen to develop mAbs against chIL-4 to establish the basis for understanding the function of chIL-4 in regulating Th2 mediated immune responses in chickens. We developed two mAbs against chIL-4 and characterized their specificity by western blot, enzyme linked immunosorbent assay (ELISA) and intracellular staining (ICS). These antibodies displayed specific binding with the recombinant chl-IL-4 as well as endogenous chIL-4 by western blot, ELISA and ICS. Further, these antibodies neutralized the inhibitory effect of chIL-4 on lipopolysaccharide (LPS)-induced nitric oxide (NO) production and expression of inducible nitric oxide synthase (iNOS) in avian macrophage-like HD11 cells, thus confirming that these antibodies can block the chIL-4 bioactivity. In conclusion, the mAbs developed in the present study can be useful for understanding the role of chIL-4 in the basic concept of Th1 versus Th2 T cell subsets in chickens.

S-nitrosoglutathione reductase (GSNOR) inhibitor as an immune modulator in experimental autoimmune encephalomyelitis.

We previously reported that S-nitrosoglutathione (GSNO), an endogenous nitric oxide carrier, attenuated TH17-mediated immune responses in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Cellular GSNO homeostasis is regulated via its synthesis by reaction between nitric oxide and glutathione and its enzymatic catabolism by GSNO reductase (GSNOR). In this study, we evaluated potential of reversible inhibitor of GSNOR (N6022) in comparison with exogenous GSNO in immunopathogenesis of EAE. Daily treatment of EAE mice with N6022 or exogenous GSNO significantly attenuated the clinical disease of EAE, but N6022 treatment showed greater efficacy than GSNO. Both N6022 and exogenous GSNO treatments increased the spleen levels of GSNO, as documented by increased protein-associated S-nitrosothiols, and inhibited polarization and CNS effector function of proinflammatory TH17 cells while inducing the polarization and CNS effector function of anti-inflammatory CD4+ CD25+ FOXP3- regulatory T (Treg) cells. Moreover, N6022 further attenuated TH1 while inducing TH2 and CD4+ CD25+ FOXP3+ Treg in their polarization and CNS effector functions. Similar to GSNO, the N6022 treatment protected against the EAE disease induced demyelination. However, neither exogenous GSNO nor N6022 treatment did not cause significant systemic lymphopenic effect as compared to FTY720. Taken together, these data document that optimization of cellular GSNO homeostasis by GSNOR inhibitor (N6022) in NO metabolizing cells attenuates EAE disease via selective inhibition of pro-inflammatory subsets of CD4+ cells (TH1/TH17) while upregulating anti-inflammatory subsets of CD4+ cells (TH2/Treg) without causing lymphopenic effects and thus offers a potential treatment option for MS/EAE.

RKIP mediates autoimmune inflammation by positively regulating IL-17R signaling.

Th17 cells contribute to the development of autoimmune diseases by secreting interleukin-17 (IL-17), which activates its receptor (IL-17R) that is expressed on epithelial cells, macrophages, microglia, and resident neuroectodermal cells. However, the mechanisms through which IL-17R-mediated signaling contributes to the development of autoimmune disease have not been completely elucidated. Here, we demonstrate that Raf-1 kinase inhibitor protein (RKIP) deficiency in mice ameliorates the symptoms of experimental autoimmune encephalomyelitis (EAE). Adoptive T-cell-transfer experiments demonstrate that RKIP plays a predominant role in Th17-mediated, but not in Th1-mediated immune responses. RKIP deficiency has no effect on Th17-cell differentiation ex vivo, nor does it affect Th17-cell differentiation in EAE mice. However, RKIP significantly promotes IL-17R-induced proinflammatory cytokine and chemokine production. Mechanistically, RKIP directly interacts with IL-17RA and Act1 to promote the formation of an IL-17R-Act1 complex, resulting in enhanced MAPK- and P65-mediated NF-κB activation and downstream cytokine production. Together, these findings indicate that RKIP functions as an essential modulator of the IL-17R-Act1 axis in IL-17R signaling, which promotes IL-17-induced inflammation and autoimmune neuroinflammation.