Optimal Attenuation of Experimental Autoimmune Encephalomyelitis by Intravenous Immunoglobulin Requires an Intact Interleukin-11 Receptor

Background

Intravenous immunoglobulin (IVIg) has been used to treat a variety of autoimmune disorders including multiple sclerosis (MS); however its mechanism of action remains elusive. Recent work has shown that interleukin-11 (IL-11) mRNAs are upregulated by IVIg in MS patient T cells. Both IVIg and IL-11 have been shown to ameliorate experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The objective of this study was to determine whether the protective effects of IVIg in EAE occur through an IL-11 and IL-11 receptor (IL-11R)-dependent mechanism.

Methods

We measured IL-11 in the circulation of mice and IL-11 mRNA expression in various organs after IVIg treatment. We then followed with EAE studies to test the efficacy of IVIg in wild-type (WT) mice and in mice deficient for the IL-11 receptor (IL-11Rα^−/−). Furthermore, we evaluated myelin-specific Th1 and Th17 responses and assessed spinal cord inflammation and demyelination in WT and IL-11Rα^−/− mice, with and without IVIg treatment. We also examined the direct effects of mouse recombinant IL-11 on the production of IL-17 by lymph node mononuclear cells.

Results

IVIg treatment induced a dramatic surge (>1000-fold increase) in the levels of IL-11 in the circulation and a prominent increase of IL-11 mRNA expression in the liver. Furthermore, we found that IL-11Rα^−/− mice, unlike WT mice, although initially protected, were resistant to full protection by IVIg during EAE and developed disease with a similar incidence and severity as control-treated IL-11Rα^−/− mice, despite initially showing protection. We observed that Th17 cytokine production by myelin-reactive T cells in the draining lymph nodes was unaffected by IVIg in IL-11Rα^−/− mice, yet was downregulated in WT mice. Finally, IL-11 was shown to directly inhibit IL-17 production of lymph node cells in culture.

Conclusion

These results implicate IL-11 as an important immune effector of IVIg in the prevention of Th17-mediated autoimmune inflammation during EAE.     

Centrally Administered Pertussis Toxin Inhibits Microglia Migration to the Spinal Cord and Prevents Dissemination of Disease in an EAE Mouse Model

Background

Experimental autoimmune encephalomyelitis (EAE) models are important vehicles for studying the effect of infectious elements such as Pertussis toxin (PTx) on disease processes related to acute demyelinating encephalomyelitis (ADEM) or multiple sclerosis (MS). PTx has pleotropic effects on the immune system. This study was designed to investigate the effects of PTx administered intracerebroventricularly (icv) in preventing downstream immune cell infiltration and demyelination of the spinal cord.

Methods and Findings

EAE was induced in C57BL/6 mice with MOG_35–55. PTx icv at seven days post MOG immunization resulted in mitigation of clinical motor symptoms, minimal T cell infiltration, and the marked absence of axonal loss and demyelination of the spinal cord. Integrity of the blood brain barrier was compromised in the brain whereas spinal cord BBB integrity remained intact. PTx icv markedly increased microglia numbers in the brain preventing their migration to the spinal cord. An in vitro transwell study demonstrated that PTx inhibited migration of microglia.

Conclusion

Centrally administered PTx abrogated migration of microglia in EAE mice, limiting the inflammatory cytokine milieu to the brain and prevented dissemination of demyelination. The effects of PTx icv warrants further investigation and provides an attractive template for further study regarding the pleotropic effects of infectious elements such as PTx in the pathogenesis of autoimmune disorders.     

Generation of Human Antigen-Specific Monoclonal IgM Antibodies Using Vaccinated “Human Immune System” Mice

Background

Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the ‘humanization’ of murine monoclonal antibodies (mAbs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique.

Methodology/Principal Findings

After transplantation with CD34⁺CD38⁻ human hematopoietic progenitor cells, BALB/c Rag2^−/−IL-2Rγc^−/− mice acquire a human immune system and harbor B cells with a diverse IgM repertoire. “Human Immune System” mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis B surface antigen. Sorted human CD19⁺CD27⁺ B cells were retrovirally transduced with the human B cell lymphoma (BCL)-6 and BCL-XL genes, and subsequently cultured in the presence of CD40-ligand and IL-21. This procedure allows generating stable B cell receptor-positive B cells that secrete immunoglobulins. We recovered stable B cell clones that produced IgM specific for tetanus toxoid and the hepatitis B surface antigen, respectively.

Conclusion/Significance

This work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mAbs against a wide range of antigens.     

VR09 Cell Line: An EBV-Positive Lymphoblastoid Cell Line with In Vivo Characteristics of Diffuse Large B Cell Lymphoma of Activated B-Cell Type

Background

small B-cell neoplasms can show plasmacytic differentiation and may potentially progress to aggressive lymphoma (DLBCL). Epstein-Barr virus (EBV) infection may cause the transformation of malignant cells in vitro.

Design and Method

we established VR09 cell line with plasmacytic differentiation, obtained from a case of atypical, non-CLL B-cell chronic lymphoproliferative disease with plasmacytic features. We used flow cytometry, immunohistochemistry, polymerase chain reaction, cytogenetic analysis and florescence in situ hybridization in the attempt at thoroughly characterizing the cell line. We showed VR09 tumorigenic potential in vivo, leading to the development of activated DLBCL with plasmacytic features.

Results

VR09 cells displayed plasmacytic appearance and grew as spherical tumors when inoculated subcutaneously into immunodeficient Rag2^−/− γ-chain^−/− mice. VR09 cell line and tumors displayed the phenotype of activated stage of B cell maturation, with secretory differentiation (CD19+ CD20+ CD79a+ CD79b+/− CD138+ cyclin D1- Ki67 80% IgM+ IgD+ MUM1+ MNDA+ CD10- CD22+ CD23+ CD43+ K+, λ- Bcl2+ Bcl6-) and they presented episomal EBV genome, chromosome 12 trisomy, lack of c-MYC rearrangement and Myd88 gene mutation, presence of somatic hypermutation in the VH region, and wild-type p53.

Conclusion

This new EBV-positive cell line may be useful to further characterize in vivo activated DLBCL with plasmacytic features.     

Jak3 Is Involved in Dendritic Cell Maturation and CCR7-Dependent Migration

Background

CCR7-mediated signalling is important for dendritic cell maturation and homing to the lymph nodes. We have previously demonstrated that Jak3 participates in the signalling pathway of CCR7 in T lymphocytes.

Methodology and Principal Findings

Here, we used Jak3^−/− mice to analyze the role of Jak3 in CCR7-mediated dendritic cells migration and function. First, we found no differences in the generation of DCs from Jak3^−/− bone marrow progenitors, when compared to wild type cells. However, phenotypic analysis of the bone marrow derived DCs obtained from Jak3^−/− mice showed reduced expression of co-stimulatory molecules compared to wild type (Jak3^+/+). In addition, when we analyzed the migration of Jak3^−/− and Jak3^+/+ mature DCs in response to CCL19 and CCL21 chemokines, we found that the absence of Jak3 results in impaired chemotactic responses both in vitro and in vivo. Moreover, lymphocyte proliferation and contact hypersensitivity experiments showed that DC-mediated T lymphocyte activation is reduced in the absence of Jak3.

Conclusion/Significance

Altogether, our data provide strong evidence that Jak3 is important for DC maturation, migration and function, through a CCR7-mediated signalling pathway.     

BAFF promotes Th17 cells and aggravates experimental autoimmune encephalomyelitis

Background

BAFF, in addition to promoting B cell survival and differentiation, may affect T cells. The objective of this study was to determine the effect of BAFF on Th17 cell generation and its ramifications for the Th17 cell-driven disease, EAE.

Methodology/Principal Findings

Th17 cells were increased in BAFF-Tg B6 (B6.BTg) mice and decreased in B6.Baff^−/− mice. Th17 cells in B6.Baff^−/− mice bearing a BAFF Tg (B6.Baff^−/−.BTg mice) were identical to those in B6.BTg mice, indicating that membrane BAFF is dispensable for Th17 cell generation as long as soluble BAFF is plentiful. In T + non-T cell criss-cross co-cultures, Th17 cell generation was greatest in cultures containing B6.BTg T cells and lowest in cultures containing B6.Baff^−/− T cells, regardless of the source of non-T cells. In cultures containing only T cells, Th17 cell generation followed an identical pattern. CD4⁺ cell expression of CD126 (IL-6R α chain) was increased in B6.BTg mice and decreased in B6.Baff^−/− mice, and activation of STAT3 following stimulation with IL-6 + TGF-β was also greatest in B6.BTg cells and lowest in B6.Baff^−/− cells. EAE was clinically and pathologically most severe in B6.BTg mice and least severe in B6.Baff^−/− mice and correlated with MOG_35–55 peptide-induced Th17 cell responses.

Conclusions/Significance

Collectively, these findings document a contribution of BAFF to pathogenic Th17 cell responses and suggest that BAFF antagonism may be efficacious in Th17 cell-driven diseases.     

Interleukin-10 attenuation of collagen-induced arthritis is associated with suppression of interleukin-17 and retinoid-related orphan receptor γt production in macrophages and repression of classically activated macrophages

Introduction

Our objective in the present study was to determine the signaling pathway of interleukin 10 (IL-10) for modulating IL-17 expression in macrophages and the importance of this mediation in collagen-induced arthritis (CIA).

Methods

IL-10-knockout (IL-10^−/−) mice and wild-type (WT) mice were immunized with chicken type II collagen (CII) to induce arthritis. The expression levels of IL-17 and retinoid-related orphan receptor γt (RORγt) in macrophages and joint tissues of IL-10^−/− and WT mice were analyzed by enzyme-linked immunosorbent assay, quantitative RT-PCR (qRT-PCR) and Western blotting. The F4/80 macrophages and positive IL-17-producing macrophages in synovial tissues of the mice were determined by immunohistochemistry. The populations of classically activated macrophage (M1) and alternatively activated macrophage (M2) phenotypes were analyzed by flow cytometry. The expression of genes associated with M1 and M2 markers was analyzed by qRT-PCR.

Results

Compared to WT mice, IL-10^−/− mice had exacerbated CIA development, which was associated with increased production of T helper 17 cell (Th17)/Th1 proinflammatory cytokines and CII-specific immunoglobulin G2a antibody after CII immunization. Macrophages in IL-10^−/− mice had increased amounts of IL-17 and RORγt compared with the amounts in WT mice with CIA. Immunofluorescence microscopy showed that the number of IL-17-producing macrophages in synovial tissues was significantly higher in IL-10^−/− mice than in WT mice. IL-10 deficiency might promote macrophage polarization toward the proinflammatory M1 phenotype, which contributes to the rheumatoid arthritis inflammation response.

Conclusion

IL-10 inhibits IL-17 and RORγt expression in macrophages and suppresses macrophages toward the proinflammatory M1 phenotype, which is important for the role of IL-10 in mediating the pathogenesis of CIA.     

Differential and Site Specific Impact of B Cells in the Protective Immune Response to Mycobacterium tuberculosis in the Mouse

Cell-mediated immune responses are known to be critical for control of mycobacterial infections whereas the role of B cells and humoral immunity is unclear. B cells can modulate immune responses by secretion of immunoglobulin, production of cytokines and antigen-presentation. To define the impact of B cells in the absence of secreted immunoglobulin, we analyzed the progression of Mycobacterium tuberculosis (Mtb) infection in mice that have B cells but which lack secretory immunoglobulin (AID^−/−µS^−/−mice). AID^−/−µS^−/− mice accumulated a population of activated B cells in the lungs when infected and were more susceptible to aerosol Mtb when compared to wild type (C57BL/6) mice or indeed mice that totally lack B cells. The enhanced susceptibility of AID^−/−µS^−/− mice was not associated with defective T cell activation or expression of a type 1 immune response. While delivery of normal serum to AID^−/−µS^−/− mice did not reverse susceptibility, susceptibility in the spleen was dependent upon the presence of B cells and susceptibility in the lungs of AID^−/−µS^−/−mice was associated with elevated expression of the cytokines IL-6, GM-CSF, IL-10 and molecules made by alternatively activated macrophages. Blocking of IL-10 signaling resulted in reversal of susceptibility in the spleens and lungs of AID^−/−µS^−/− mice. These data support the hypothesis that B cells can modulate immunity to Mtb in an organ specific manner via the modulation of cytokine production and macrophage activation.     

Altered exosomal protein expression in the serum of NF-κB knockout mice following skeletal muscle ischemia-reperfusion injury

Background

The NF-κB signaling pathway plays a role in local and remote tissue damage following ischemia-reperfusion (I/R) injury to skeletal muscles. Evidence suggests that exosomes can act as intercellular communicators by transporting active proteins to remote cells and may play a role in regulating inflammatory processes. This study aimed to profile the exosomal protein expression in the serum of NF-κB knockout mice following skeletal muscle ischemia-reperfusion injury.

Results

To investigate the potential changes in protein expression mediated by NF-κB in secreted exosomes in the serum following I/R injury, the levels of circulating exosomal proteomes in C57BL/6 and NF-κB^−/− mice were compared using two dimensional differential in-gel electrophoresis (2-DE), liquid chromatography tandem mass spectrometry (LC-MS/MS), and proteomic analysis. In C57BL/6 mice, the levels of circulating exosomal proteins, including complement component C3 prepropeptide, PK-120 precursor, alpha-amylase one precursor, beta-enolase isoform 1, and adenylosuccinate synthetase isozyme 1, increased following I/R injury. However, in the NF-κB^−/− mice, the expression of the following was upregulated in the exosomes: protease, serine 1; glyceraldehyde-3-phosphate dehydrogenase-like isoform 1; glyceraldehyde-3-phosphate dehydrogenase; and pregnancy zone protein. In contrast, the expression of apolipoprotein B, complement component C3 prepropeptide, and immunoglobulin kappa light chain variable region was downregulated in NF-κB^−/− mice. Bioinformatic annotation using the Protein Analysis Through Evolutionary Relationships (PANTHER) database revealed that the expression of the exosomal proteins that participate in metabolic processes and in biological regulation was lower in NF-κB^−/− mice than in C57BL/6 mice, whereas the expression of proteins that participate in the response to stimuli, in cellular processes, and in the immune system was higher.

Conclusions

The data presented in this study suggest that NF-κB might regulate exosomal protein expression at a remote site via circulation following I/R injury.     

Treg Cell Resistance to Apoptosis in DNA Vaccination for Experimental Autoimmune Encephalomyelitis Treatment

Background

Regulatory T (Treg) cells can be induced with DNA vaccinations and protect mice from the development of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS). Tacrolimus (FK506) has been shown to have functions on inducing immunosuppression and augmenting apoptosis of pathologic T cells in autoimmune disease. Here we examined the therapeutic effect of DNA vaccine in conjunction with FK506 on EAE.

Methodology/Principal Findings

After EAE induction, C57BL/6 mice were treated with DNA vaccine in conjunction with FK506. Functional Treg cells were induced in treated EAE mice and suppressed Th1 and Th17 cell responses. Infiltrated CD4 T cells were reduced while Treg cells were induced in spinal cords of treated EAE mice. Remarkably, the activated CD4 T cells augmented apoptosis, but the induced Treg cells resisted apoptosis in treated EAE mice, resulting in alleviation of clinical EAE severity.

Conclusions/Significance

DNA vaccine in conjunction with FK506 treatment ameliorates EAE by enhancing apoptosis of CD4 T cells and resisting apoptosis of induced Treg cells. Our findings implicate the potential of tolerogenic DNA vaccines for treating MS.     

CD4+ and CD8+ T Cells Can Act Separately in Tumour Rejection after Immunization with Murine Pneumotropic Virus Chimeric Her2/neu Virus-Like Particles

Background

Immunization with murine pneumotropic virus virus-like particles carrying Her2/neu (Her2MPtVLPs) prevents tumour outgrowth in mice when given prophylactically, and therapeutically if combined with the adjuvant CpG. We investigated which components of the immune system are involved in tumour rejection, and whether long-term immunological memory can be obtained.

Methodology and Results

During the effector phase in BALB/c mice, only depletion of CD4⁺ and CD8⁺ in combination, with or without NK cells, completely abrogated tumour protection. Depletion of single CD4⁺, CD8⁺ or NK cell populations only had minor effects. During the immunization/induction phase, combined depletion of CD4⁺ and CD8⁺ cells abolished protection, while depletion of each individual subset had no or negligible effect. When tumour rejection was studied in knock-out mice with a C57Bl/6 background, protection was lost in CD4^−/−CD8^−/− and CD4^−/−, but not in CD8^−/− mice. In contrast, when normal C57Bl/6 mice were depleted of different cell types, protection was lost irrespective of whether only CD4⁺, only CD8⁺, or CD4⁺ and CD8⁺ cells in combination were eradicated. No anti-Her2/neu antibodies were detected but a Her2/neu-specific IFNγ response was seen. Studies of long-term memory showed that BALB/c mice could be protected against tumour development when immunized together with CpG as long as ten weeks before challenge.

Conclusion

Her2MPtVLP immunization is efficient in stimulating several compartments of the immune system, and induces an efficient immune response including long-term memory. In addition, when depleting mice of isolated cellular compartments, tumour protection is not as efficiently abolished as when depleting several immune compartments together.     

Haploinsufficiency of Activation-Induced Deaminase for Antibody Diversification and Chromosome Translocations both In Vitro and In Vivo

The humoral immune response critically relies on the secondary diversification of antibodies. This diversification takes places through somatic remodelling of the antibody genes by two molecular mechanisms, Class Switch Recombination (CSR) and Somatic Hypermutation (SHM). The enzyme Activation Induced Cytidine Deaminase (AID) initiates both SHM and CSR by deaminating cytosine residues on the DNA of immunoglobulin genes. While crucial for immunity, AID-catalysed deamination is also the triggering event for the generation of lymphomagenic chromosome translocations. To address whether restricting the levels of AID expression in vivo contributes to the regulation of its function, we analysed mice harbouring a single copy of the AID gene (AID^+/−). AID^+/− mice express roughly 50% of normal AID levels, and display a mild hyperplasia, reminiscent of AID deficient mice and humans. Moreover, we found that AID^+/− cells have an impaired competence for CSR and SHM, which indicates that AID gene dose is limiting for its physiologic function. We next evaluated the impact of AID reduction in AID^+/− mice on the generation of chromosome translocations. Our results show that the frequency of AID-promoted c-myc/IgH translocations is reduced in AID^+/− mice, both in vivo and in vitro. Therefore, AID is haploinsufficient for antibody diversification and chromosome translocations. These findings suggest that limiting the physiologic levels of AID expression can be a regulatory mechanism that ensures an optimal balance between immune proficiency and genome integrity.     

Passive Immunization with Hypochlorite-oxLDL Specific Antibodies Reduces Plaque Volume in LDL Receptor-Deficient Mice

Aims

New strategies to overcome complications of cardiovascular diseases are needed. Since it has been demonstrated that atherosclerosis is an inflammatory disease, modulation of the immune system may be a promising approach. Previously, it was suggested that antibodies may confer protective effects on the development of atherosclerosis. In this study, we hypothesised that passive immunization with anti-oxLDL IgM antibodies specific for hypochlorite (HOCl) may be athero-protective in mice.

Methods and Results

Monoclonal mouse IgM antibodies were produced and the antibody with specificity for hypochlorite-oxLDL (HOCl-oxLDL) (Moab A7S8) was selected. VH sequence determination revealed that Moab A7S8 is a natural IgM antibody. Atherosclerosis in LDLr^−/− mice was induced by a perivascular collar placement around the right carotid artery in combination with feeding a high-fat diet. Subsequently, the mice were treated every six days with 500 µg Moab A7S8, non-relevant IgM or with PBS and the carotid arteries and aortic roots were studied for atherosclerosis. Passive immunization with this Moab A7S8 resulted in a significant reduced plaque volume formation in LDLr^−/− mice when compared with PBS treatment (P = 0.002 and P = 0.035). Cholesterol levels decreased by 20% when mice were treated with Moab A7S8 compared to PBS. Furthermore, anti-oxLDL specific IgM and IgG antibody production increased significantly in the Moab A7S8 treated mice in comparison with PBS treated mice.

Conclusion

Our data show that passive immunization with a natural IgM antibody, directed to HOCl-oxLDL, can reduce atherosclerotic plaque development. We postulate that specific antibody therapy may be developed for use in human cardiovascular diseases.     

Preventive Effect of Dipeptidyl Peptidase-4 Inhibitor on Atherosclerosis Is Mainly Attributable to Incretin's Actions in Nondiabetic and Diabetic Apolipoprotein E-Null Mice

Aim

Several recent reports have revealed that dipeptidyl peptidase (DPP)-4 inhibitors have suppressive effects on atherosclerosis in apolipoprotein E-null (Apoe ^−/−) mice. It remains to be seen, however, whether this effect stems from increased levels of the two active incretins, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP).

Methods

Nontreated Apoe ^−/− mice, streptozotocin-induced diabetic Apoe ^−/− mice, and db/db diabetic mice were administered the DPP-4 inhibitor vildagliptin in drinking water and co-infused with either saline, the GLP-1 receptor blocker, exendin(9–39), the GIP receptor blocker, (Pro³)GIP, or both via osmotic minipumps for 4 weeks. Aortic atherosclerosis and oxidized low-density lipoprotein-induced foam cell formation in exudate peritoneal macrophages were determined.

Results

Vildagliptin increased plasma GLP-1 and GIP levels without affecting food intake, body weight, blood pressure, or plasma lipid profile in any of the animals tested, though it reduced HbA1c in the diabetic mice. Diabetic Apoe ^−/− mice exhibited further-progressed atherosclerotic lesions and foam cell formation compared with nondiabetic counterparts. Nondiabetic and diabetic Apoe ^−/− mice showed a comparable response to vildagliptin, namely, remarkable suppression of atherosclerotic lesions with macrophage accumulation and foam cell formation in peritoneal macrophages. Exendin(9–39) or (Pro³)GIP partially attenuated the vildagliptin-induced suppression of atherosclerosis. The two blockers in combination abolished the anti-atherosclerotic effect of vildagliptin in nondiabetic mice but only partly attenuated it in diabetic mice. Vildagliptin suppressed macrophage foam cell formation in nondiabetic and diabetic mice, and this suppressive effect was abolished by infusions with exendin(9–39)+(Pro³)GIP. Incubation of DPP-4 or vildagliptin in vitro had no effect on macrophage foam cell formation.

Conclusions

Vildagliptin confers a substantial anti-atherosclerotic effect in both nondiabetic and diabetic mice, mainly via the action of the two incretins. However, the partial attenuation of atherosclerotic lesions by the dual incretin receptor antagonists in diabetic mice implies that vildagliptin confers a partial anti-atherogenic effect beyond that from the incretins.     

Oxidative Stress Induced Age Dependent Meibomian Gland Dysfunction in Cu,Zn-Superoxide Dismutase-1 (Sod1) Knockout Mice

Purpose

The purpose of our study was to investigate alterations in the meibomian gland (MG) in Cu, Zn-Superoxide Dismutase-1 knockout (Sod1 ^−/−) mouse.

Methods

Tear function tests [Break up time (BUT) and cotton thread] and ocular vital staining test were performed on Sod1 ^−/− male mice (n = 24) aged 10 and 50 weeks, and age and sex matched wild–type (+/+) mice (n = 25). Tear and serum samples were collected at sacrifice for inflammatory cytokine assays. MG specimens underwent Hematoxylin and Eosin staining, Mallory staining for fibrosis, Oil Red O lipid staining, TUNEL staining, immunohistochemistry stainings for 4HNE, 8-OHdG and CD45. Transmission electron microscopic examination (TEM) was also performed.

Results

Corneal vital staining scores in the Sod1 ^−/− mice were significantly higher compared with the wild type mice throughout the follow-up. Tear and serum IL-6 and TNF-α levels also showed significant elevations in the 10 to 50 week Sod1 ^−/− mice. Oil Red O staining showed an accumulation of large lipid droplets in the Sod1 ^−/− mice at 50 weeks. Immunohistochemistry revealed both increased TUNEL and oxidative stress marker stainings of the MG acinar epithelium in the Sod1 ^−/− mice compared to the wild type mice. Immunohistochemistry staining for CD45 showed increasing inflammatory cell infiltrates from 10 to 50 weeks in the Sod1 ^−/− mice compared to the wild type mice. TEM revealed prominent mitochondrial changes in 50 week Sod1 ^−/− mice.

Conclusions

Our results suggest that reactive oxygen species might play a vital role in the pathogensis of meibomian gland dysfunction. The Sod1 ^−/− mouse appears to be a promising model for the study of reactive oxygen species associated MG alterations.     

Central Muscarinic Cholinergic Activation Alters Interaction between Splenic Dendritic Cell and CD4+CD25- T Cells in Experimental Colitis

Background

The cholinergic anti-inflammatory pathway (CAP) is based on vagus nerve (VN) activity that regulates macrophage and dendritic cell responses in the spleen through alpha-7 nicotinic acetylcholine receptor (a7nAChR) signaling. Inflammatory bowel disease (IBD) patients present dysautonomia with decreased vagus nerve activity, dendritic cell and T cell over-activation. The aim of this study was to investigate whether central activation of the CAP alters the function of dendritic cells (DCs) and sequential CD4⁺/CD25⁻T cell activation in the context of experimental colitis.

Methods

The dinitrobenzene sulfonic acid model of experimental colitis in C57BL/6 mice was used. Central, intracerebroventricular infusion of the M1 muscarinic acetylcholine receptor agonist McN-A-343 was used to activate CAP and vagus nerve and/or splenic nerve transection were performed. In addition, the role of α7nAChR signaling and the NF-kB pathway was studied. Serum amyloid protein (SAP)-A, colonic tissue cytokines, IL-12p70 and IL-23 in isolated splenic DCs, and cytokines levels in DC-CD4⁺CD25⁻T cell co-culture were determined.

Results

McN-A-343 treatment reduced colonic inflammation associated with decreased pro-inflammatory Th1/Th17 colonic and splenic cytokine secretion. Splenic DCs cytokine release was modulated through α7nAChR and the NF-kB signaling pathways. Cholinergic activation resulted in decreased CD4⁺CD25⁻T cell priming. The anti-inflammatory efficacy of central cholinergic activation was abolished in mice with vagotomy or splenic neurectomy.

Conclusions

Suppression of splenic immune cell activation and altered interaction between DCs and T cells are important aspects of the beneficial effect of brain activation of the CAP in experimental colitis. These findings may lead to improved therapeutic strategies in the treatment of IBD.     

Paradoxical Effect of Pertussis Toxin on the Delayed Hypersensitivity Response to Autoantigens in Mice

Background

Pertussis toxin (PTX), an exotoxin of Bordetella pertussis, enhances the development of experimental autoimmune diseases such as experimental autoimmune uveitis (EAU) and experimental autoimmune encephalomyelitis (EAE) in rodent models. The mechanisms of the promotion of experimental autoimmune diseases by PTX may be based upon PTX-induced disruption of the blood eye/brain barriers facilitating the infiltration of inflammatory cells, the modulation of inflammatory cell migration and the enhancement of the activation of inflammatory cells. We hypothesized that the facilitation of experimental autoimmunity by PTX suggests that its influence on the in vivo immune response to auto-antigen may differ from its influence on non-self antigens.

Methodology/Principal Findings

We have evaluated the effect of PTX on the simultaneous generation of delayed type hypersensitivity (DTH) responses and autoimmune responses to uveitogenic interphotoreceptor retinoid binding protein peptide (IRBP_161–180), encephalitogenic myelin oligodendrocyte glycoprotein peptide (MOG_35–55) or ovalbumin (OVA). PTX injection of mice immunized to IRBP peptide_161–180 led to (i) the development of EAU as shown by histopathology of the retina, (ii) pro-inflammatory cytokine production by splenocytes in response to IRBP peptide _161–180, and (iii) symptomatic EAE in mice immunized with encephalitogenic MOG peptide_35–55. However, mice that received PTX had a reduced DTH response to IRBP_161–180 peptide or MOG peptide_35–55 when challenged distal to the site affected by autoreactive T cells. Moreover, footpad challenge with MOG_35–55 peptide reduced EAE in mice immunized with MOG peptide. In contrast, the use of PTX when immunizing with OVA protein or an OVA immunogenic peptide did not affect the DTH response to OVA.

Conclusions/Significance

The results suggest that that the reduced DTH response in mice receiving PTX may be specific for autoantigens and autoantigen-reactive T cells are diverted away from ectopic sites that received the autoantigen and towards the tissue site of the autoantigen.     

Prokineticin Receptor 1 as a Novel Suppressor of Preadipocyte Proliferation and Differentiation to Control Obesity

Background

Adipocyte renewal from preadipocytes occurs throughout the lifetime and contributes to obesity. To date, little is known about the mechanisms that control preadipocyte proliferation and differentiation. Prokineticin-2 is an angiogenic and anorexigenic hormone that activate two G protein-coupled receptors (GPCRs): PKR1 and PKR2. Prokineticin-2 regulates food intake and energy metabolism via central mechanisms (PKR2). The peripheral effect of prokineticin-2 on adipocytes/preadipocytes has not been studied yet.

Methodology/Principal Findings

Since adipocytes and preadipocytes express mainly prokineticin receptor-1 (PKR1), here, we explored the role of PKR1 in adipose tissue expansion, generating PKR1-null (PKR1^−/−) and adipocyte-specific (PKR1^ad−/−) mutant mice, and using murine and human preadipocyte cell lines. Both PKR1^−/− and PKR1^ad−/− had excessive abdominal adipose tissue, but only PKR1^−/− mice showed severe obesity and diabetes-like syndrome. PKR1^ad−/−) mice had increased proliferating preadipocytes and newly formed adipocyte levels, leading to expansion of adipose tissue. Using PKR1-knockdown in 3T3-L1 preadipocytes, we show that PKR1 directly inhibits preadipocyte proliferation and differentiation. These PKR1 cell autonomous actions appear targeted at preadipocyte cell cycle regulatory pathways, through reducing cyclin D, E, cdk2, c-Myc levels.

Conclusions/Significance

These results suggest PKR1 to be a crucial player in the preadipocyte proliferation and differentiation. Our data should facilitate studies of both the pathogenesis and therapy of obesity in humans.