EBNA2 driven enhancer switching at the CIITA-DEXI locus suppresses HLA class II gene expression during EBV infection of B-lymphocytes.

Viruses suppress immune recognition through diverse mechanisms. Epstein-Barr Virus (EBV) establishes latent infection in memory B-lymphocytes and B-cell malignancies where it impacts B-cell immune function. We show here that EBV primary infection of naïve B-cells results in a robust down-regulation of HLA genes. We found that the viral encoded transcriptional regulatory factor EBNA2 bound to multiple regulatory regions in the HLA locus. Conditional expression of EBNA2 correlated with the down regulation of HLA class II transcription. EBNA2 down-regulation of HLA transcription was found to be dependent on CIITA, the major transcriptional activator of HLA class II gene transcription. We identified a major EBNA2 binding site downstream of the CIITA gene and upstream of DEXI, a dexamethasone inducible gene that is oriented head-to-head with CIITA gene transcripts. CRISPR/Cas9 deletion of the EBNA2 site upstream of DEXI attenuated CIITA transcriptional repression. EBNA2 caused an increase in DEXI transcription and a graded change in histone modifications with activation mark H3K27ac near the DEXI locus, and a loss of activation marks at the CIITA locus. A prominent CTCF binding site between CIITA and DEXI enhancers was mutated and further diminished the effects of EBNA2 on CIITA. Analysis of HiC data indicate that DEXI and CIITA enhancers are situated in different chromosome topological associated domains (TADs). These findings suggest that EBNA2 down regulates HLA-II genes through the down regulation of CIITA, and that this down regulation is an indirect consequence of EBNA2 enhancer formation at a neighboring TAD. We propose that enhancer competition between these neighboring chromosome domains represents a novel mechanism for gene regulation demonstrated by EBNA2.

Postoperative abdominal sepsis induces selective and persistent changes in CTCF binding within the MHC-II region of human monocytes.

BACKGROUND: Postoperative abdominal infections belong to the most common triggers of sepsis and septic shock in intensive care units worldwide. While monocytes play a central role in mediating the initial host response to infections, sepsis-induced immune dysregulation is characterized by a defective antigen presentation to T-cells via loss of Major Histocompatibility Complex Class II DR (HLA-DR) surface expression. Here, we hypothesized a sepsis-induced differential occupancy of the CCCTC-Binding Factor (CTCF), an architectural protein and superordinate regulator of transcription, inside the Major Histocompatibility Complex Class II (MHC-II) region in patients with postoperative sepsis, contributing to an altered monocytic transcriptional response during critical illness. RESULTS: Compared to a matched surgical control cohort, postoperative sepsis was associated with selective and enduring increase in CTCF binding within the MHC-II. In detail, increased CTCF binding was detected at four sites adjacent to classical HLA class II genes coding for proteins expressed on monocyte surface. Gene expression analysis revealed a sepsis-associated decreased transcription of (i) the classical HLA genes HLA-DRA, HLA-DRB1, HLA-DPA1 and HLA-DPB1 and (ii) the gene of the MHC-II master regulator, CIITA (Class II Major Histocompatibility Complex Transactivator). Increased CTCF binding persisted in all sepsis patients, while transcriptional recovery CIITA was exclusively found in long-term survivors. CONCLUSION: Our experiments demonstrate differential and persisting alterations of CTCF occupancy within the MHC-II, accompanied by selective changes in the expression of spatially related HLA class II genes, indicating an important role of CTCF in modulating the transcriptional response of immunocompromised human monocytes during critical illness.

Expression of HLA class I and class II genes in patients with multiple skin warts.

Human leukocyte antigens (HLAs), which are genetic markers that have critical roles in the immune response against pathogens, vary greatly among individuals. The aim of the study is to investigate the frequency of HLA class I (HLA-A, HLA-B and HLAC) and class II (HLA-DRB1, HLA-DQB1 and HLA-DQA1) genes in patients with multiple skin warts and to elucidate the role of these genes in the genetic susceptibility to skin warts. Peripheral venous blood samples were collected from 100 patients with multiple skin warts and 300 healthy individuals (controls). HLA typing was performed after DNA isolation from the blood samples. The HLA-A*02 (odds ratio [OR]: 0.12; p = 0.0019), HLA-DQA1*03:01 (OR: 0.45; p = 0.0017) and DQA1*05:01 (OR: 0.17; p < 0.0001) genes were significantly more prevalent in the patients than in the healthy individuals and were thus identified as risk genes. The HLA-DQA1*01:01 (OR: 0.17; p < 0.0001), HLA-DQA1*01:02 (OR: 0.17; p < 0.0001), HLA-DQA1*01:03 (OR: 0.11; p < 0.0001), HLA-DQA1*02:01 (OR:027; p<0.0001) and HLA-DQA1*05:05 (OR:0.16; p<0.0001) genes were classified as protective genes because of their low frequencies in the patients. The limitation of the study is that Human papillomavirus typing could not be performed while investigating the relationship between skin warts and HLA class I and class II genes. Our data suggest the role of HLA genes in the development of skin warts. However, other components of the major histocompatibility complex system and acquired factors of the immune system could also be involved and should be further investigated.

Recognition of Class II MHC Peptide Ligands That Contain ß-Amino Acids.

Proteins are composed of α-amino acid residues. This consistency in backbone structure likely serves an important role in the display of an enormous diversity of peptides by class II MHC (MHC-II) products, which make contacts with main chain atoms of their peptide cargo. Peptides that contain residues with an extra carbon in the backbone (derived from ß-amino acids) have biological properties that differ starkly from those of their conventional counterparts. How changes in the structure of the peptide backbone affect the loading of peptides onto MHC-II or recognition of the resulting complexes by TCRs has not been widely explored. We prepared a library of analogues of MHC-II-binding peptides derived from OVA, in which at least one α-amino acid residue was replaced with a homologous ß-amino acid residue. The latter contain an extra methylene unit in the peptide backbone but retain the original side chain. We show that several of these α/ß-peptides retain the ability to bind tightly to MHC-II, activate TCR signaling, and induce responses from T cells in mice. One α/ß-peptide exhibited enhanced stability in the presence of an endosomal protease relative to the index peptide. Conjugation of this backbone-modified peptide to a camelid single-domain Ab fragment specific for MHC-II enhanced its biological activity. Our results suggest that backbone modification offers a method to modulate MHC binding and selectivity, T cell stimulatory capacity, and susceptibility to processing by proteases such as those found within endosomes where Ag processing occurs.

Plumage microbiota covaries with the major histocompatibility complex in blue petrels.

To increase fitness, a wide range of vertebrates preferentially mate with partners that are dissimilar at the major histocompatibility complex (MHC) or that have high MHC diversity. Although MHC often can be assessed through olfactory cues, the mechanism by which MHC genes influence odour remains largely unclear. MHC class IIB molecules, which enable recognition and elimination of extracellular bacteria, have been suggested to influence odour indirectly by shaping odour-producing microbiota, i.e. bacterial communities. However, there is little evidence of the predicted covariation between an animal's MHC genotype and its bacterial communities in scent-producing body surfaces. Here, using high-throughput sequencing, we tested the covariation between MHC class IIB genotypes and feather microbiota in the blue petrel (Halobaena caerulea), a seabird with highly developed olfaction that has been suggested to rely on oduor cues during an MHC-based mate choice. First, we show that individuals with similar MHC class IIB profiles also have similar bacterial assemblages in their feathers. Then, we show that individuals with high MHC diversity have less diverse feather microbiota and also a reduced abundance of a bacterium of the genus Arsenophonus, a genus in which some species are symbionts of avian ectoparasites. Our results, showing that feather microbiota covary with MHC, are consistent with the hypothesis that individual MHC genotype may shape the semiochemical-producing microbiota in birds.

Immune Escape of Relapsed AML Cells after Allogeneic Transplantation.

BACKGROUND: As consolidation therapy for acute myeloid leukemia (AML), allogeneic hematopoietic stem-cell transplantation provides a benefit in part by means of an immune-mediated graft-versus-leukemia effect. We hypothesized that the immune-mediated selective pressure imposed by allogeneic transplantation may cause distinct patterns of tumor evolution in relapsed disease. METHODS: We performed enhanced exome sequencing on paired samples obtained at initial presentation with AML and at relapse from 15 patients who had a relapse after hematopoietic stem-cell transplantation (with transplants from an HLA-matched sibling, HLA-matched unrelated donor, or HLA-mismatched unrelated donor) and from 20 patients who had a relapse after chemotherapy. We performed RNA sequencing and flow cytometry on a subgroup of these samples and on additional samples for validation. RESULTS: On exome sequencing, the spectrum of gained and lost mutations observed with relapse after transplantation was similar to the spectrum observed with relapse after chemotherapy. Specifically, relapse after transplantation was not associated with the acquisition of previously unknown AML-specific mutations or structural variations in immune-related genes. In contrast, RNA sequencing of samples obtained at relapse after transplantation revealed dysregulation of pathways involved in adaptive and innate immunity, including down-regulation of major histocompatibility complex (MHC) class II genes ( HLA-DPA1, HLA-DPB1, HLA-DQB1, and HLA-DRB1) to levels that were 3 to 12 times lower than the levels seen in paired samples obtained at presentation. Flow cytometry and immunohistochemical analysis confirmed decreased expression of MHC class II at relapse in 17 of 34 patients who had a relapse after transplantation. Evidence suggested that interferon-γ treatment could rapidly reverse this phenotype in AML blasts in vitro. CONCLUSIONS: AML relapse after transplantation was not associated with the acquisition of relapse-specific mutations in immune-related genes. However, it was associated with dysregulation of pathways that may influence immune function, including down-regulation of MHC class II genes, which are involved in antigen presentation. These epigenetic changes may be reversible with appropriate therapy. (Funded by the National Cancer Institute and others.).

Analysis of peptide-SLA binding by establishing immortalized porcine alveolar macrophage cells with different SLA class II haplotypes.

Primary porcine alveolar macrophages (PAM) are useful for studying viral infections and immune response in pigs; however, long-term use of these cells is limited by the cells' short lifespan. We immortalized primary PAMs by transfecting them with both hTERT and SV40LT and established two immortalized cell lines (iPAMs) actively proliferating even after 35 passages. These cells possessed the characteristics of primary PAMs, including strong expression of swine leukocyte antigen (SLA) class II genes and the inability to grow anchorage-independently. We characterized their SLA genes and subsequently performed peptide-SLA binding assays using a peptide from porcine circovirus type 2 open reading frame 2 to experimentally measure the binding affinity of the peptide to SLA class II. The number of peptides bound to cells measured by fluorescence was very low for PK15 cells (7.0% ± 1.5), which are not antigen-presenting cells, unlike iPAM61 (33.7% ± 3.4; SLA-DQA*0201/0303, DQB1*0201/0901, DRB1*0201/1301) and iPAM303 (73.3% ± 5.4; SLA DQA*0106/0201, DQB1*0202/0701, DRB1*0402/0602). The difference in peptide binding between the two iPAMs was likely due to the allelic differences between the SLA class II molecules that were expressed. The development of an immortal PAM cell panel harboring diverse SLA haplotypes and the use of an established method in this study can become a valuable tool for evaluating the interaction between antigenic peptides and SLA molecules and is important for many applications in veterinary medicine including vaccine development.

Outer membrane protein A: An immunogenic protein induces highly protective efficacy against Vibrio ichthyoenteri.

Vibrio ichthyoenteri was an important causative agent of bacterial enteritis in flounder (Paralichthys olivaceus). Outer membrane protein A (OmpA) of Gram-negative pathogen was a major cell surface antigen. In the present study, OmpA of V. ichthyoenteri was recombinantly expressed in Escherichia coli, and the immunogenicity of OmpA was identified by western blotting using flounder anti-rOmpA and anti-V. ichthyoenteri antibodies. The vaccine potential of rOmpA was tested in a flounder model, and a high relative percentage of survival rate was obtained with 73.1% after challenge with V. ichthyoenteri. Meanwhile, the immune response of flounder induced by rOmpA was also investigated, and the results showed that the sIg + lymphocytes in blood, spleen, and pronephros significantly proliferated, and the peak levels occurred at week 4 after immunization. Moreover, rOmpA could induce higher levels of specific serum antibodies than the control group after immunization, and the peak level occurred at week 5 after immunization. Meanwhile, qRT-PCR analysis showed that the expressions of CD4-1, CD8α, IL-1ß, IFN-γ, MHCIα and MHCIIα genes were significantly up-regulated after immunization with rOmpA. Taking together, these results demonstrated that rOmpA could evoke highly protective effects against V. ichthyoenteri challenge and induce strong immune response of flounder, which indicated that OmpA was a promising vaccine candidate.

Fraction of MHCII and EpCAM expression characterizes distal lung epithelial cells for alveolar type 2 cell isolation.

BACKGOUND: Alveolar type 2 (AT2) cells play important roles in maintaining adult lung homeostasis. AT2 cells isolated from the lung have revealed the cell-specific functions of AT2 cells. Comprehensive molecular and transcriptional profiling of purified AT2 cells would be helpful for elucidating the underlying mechanisms of their cell-specific functions. To enable the further purification of AT2 cells, we aimed to discriminate AT2 cells from non-AT2 lung epithelial cells based on surface antigen expression via fluorescence activated cell sorting (FACS). METHODS: Single-cell suspensions obtained from enzymatically digested murine lungs were labeled for surface antigens (CD45/CD31/epithelial cell adhesion molecule (EpCAM)/ major histocompatibility complex class II (MHCII)) and for pro-surfactant protein C (proSP-C), followed by FACS analysis for surface antigen expression on AT2 cells. AT2 cells were sorted, and purity was evaluated by immunofluorescence and FACS. This newly developed strategy for AT2 cell isolation was validated in different strains and ages of mice, as well as in a lung injury model. RESULTS: FACS analysis revealed that EpCAM+ epithelial cells existed in 3 subpopulations based on EpCAM and MHCII expression: EpCAMmedMHCII+ cells (Population1:P1), EpCAMhiMHCII- cells (P2), and EpCAMlowMHCII- cells (P3). proSP-C+ cells were enriched in P1 cells, and the purity values of the sorted AT2 cells in P1 were 99.0% by immunofluorescence analysis and 98.0% by FACS analysis. P2 cells were mainly composed of ciliated cells and P3 cells were composed of AT1 cells, respectively, based on the gene expression analysis and immunofluorescence. EpCAM and MHCII expression levels were not significantly altered in different strains or ages of mice or following lipopolysaccharide (LPS)-induced lung injury. CONCLUSIONS: We successfully classified murine distal lung epithelial cells based on EpCAM and MHCII expression. The discrimination of AT2 cells from non-AT2 epithelial cells resulted in the isolation of pure AT2 cells. Highly pure AT2 cells will provide accurate and deeper insights into the cell-specific mechanisms of alveolar homeostasis.

The roles of MHC class II genes and post-translational modification in celiac disease.

Our increasing understanding of the etiology of celiac disease, previously considered a simple food hypersensitivity disorder caused by an immune response to cereal gluten proteins, challenges established concepts of autoimmunity. HLA is a chief genetic determinant, and certain HLA-DQ allotypes predispose to the disease by presenting posttranslationally modified (deamidated) gluten peptides to CD4+ T cells. The deamidation of gluten peptides is mediated by transglutaminase 2. Strikingly, celiac disease patients generate highly disease-specific autoantibodies to the transglutaminase 2 enzyme. The dual role of transglutaminase 2 in celiac disease is hardly coincidental. This paper reviews the genetic mapping and involvement of MHC class II genes in disease pathogenesis, and discusses the evidence that MHC class II genes, via the involvement of transglutaminase 2, influence the generation of celiac disease-specific autoantibodies.

MHC class II expression and potential antigen-presenting cells in the retina during experimental autoimmune uveitis.

BACKGROUND: Controversy exists regarding which cell types are responsible for autoantigen presentation in the retina during experimental autoimmune uveitis (EAU) development. In this study, we aimed to identify and characterize the retinal resident and infiltrating cells susceptible to express major histocompatibility complex (MHC) class II during EAU. METHODS: EAU was induced in C57BL/6 mice by adoptive transfer of autoreactive lymphocytes from IRBP1-20-immunized animals. MHC class II expression was studied by immunostainings on eye cryosections. For flow cytometry (FC) analysis, retinas were dissected and enzymatically digested into single-cell suspensions. Three MHC class II+ retinal cell populations were sorted by FC, and their RNA processed for RNA-Seq. RESULTS: Immunostainings demonstrate strong induction of MHC class II expression in EAU, especially in the inner retina at the level of inflamed vessels, extending to the outer retinal layers and the subretinal space in severely inflamed eyes. Most MHC class II+ cells express the hematopoietic marker IBA1. FC quantitative analyses demonstrate that MHC class II induction significantly correlates with disease severity and is associated with upregulation of co-stimulatory molecule expression. In particular, most MHC class IIhi cells express co-stimulatory molecules during EAU. Further phenotyping identified three MHC class II+ retinal cell populations: CD45-CD11b- non-hematopoietic cells with low MHC class II expression and CD45+CD11b+ hematopoietic cells with higher MHC class II expression, which can be further separated into Ly6C+ and Ly6C- cells, possibly corresponding to infiltrating macrophages and resident microglia. Transcriptome analysis of the three sorted populations leads to a clear sample clustering with some enrichment in macrophage markers and microglial cell markers in Ly6C+ and Ly6C- cells, respectively. Functional annotation analysis reveals that both hematopoietic cell populations are more competent in MHC class II-associated antigen presentation and in T cell activation than non-hematopoietic cells. CONCLUSION: Our results highlight the potential of cells of hematopoietic origin in local antigen presentation, whatever their Ly6C expression. Our work further provides a first transcriptomic study of MHC class II-expressing retinal cells during EAU and delivers a series of new candidate genes possibly implicated in the pathogenesis of retinal autoimmunity.

Cells expressing CD4, CD8, MHCII and endoglin in the canine corpus luteum of pregnancy, and prepartum activation of the luteal TNFα system.

In the dog, knowledge about involvement of the immune system in controlling luteal function is restricted to observations showing a time-dependent invasion of immune cells into the corpus luteum (CL) of non-pregnant bitches. Therefore, this study investigated the presence of CD4-, CD8-, MHCII- and endoglin-expressing cells in CL collected throughout pregnancy from pre-implantation until prepartum luteolysis. Immunohistochemistry and semi-quantitative RT-PCR were applied. The time-dependent expression of CD4, CD8 and endoglin was more strongly related to formation of the CL, whereas MHCII was induced during luteolysis. Next, the luteal expression of TNFα and its receptors, TNFR1 and TNFR2, was analyzed in non-pregnant dogs between days 5-65 after ovulation and during pregnancy. Moreover, the effects of progesterone withdrawal were investigated in mid-pregnant dogs treated with an antigestagen aglepristone. The TNFα system was induced in the early CL of non-pregnant dogs. In pregnant dogs, expression of TNFα did not vary much, contrasting with increased expression of both receptors in the post-implantation period and significantly decreased expression at mid-gestation; prepartum luteolysis was characterized by increased TNFR2 expression. Apart from the downregulated expression of TNFR1, the changes observed following antigestagen treatment resembled those observed during normal prepartum luteolysis. A modulatory function of the TNFα system during formation of the canine CL is suggested, possibly related to the strong accompanying vascularization and luteal infiltration with activated macrophages. Contrasting with the slow luteal regression in non-pregnant dogs, in pregnant animals the upregulation of TNFR2 expression during prepartum luteolysis implies functional involvement of the TNFα system during that time.

RhoB induces the production of proinflammatory cytokines in TLR-triggered macrophages.

Toll-like receptors (TLRs) are the primary sensors detecting conserved molecular patterns on microorganisms, thus acting as important components of innate immunity against invading pathogens. Many positive and negative regulators of TLR-triggered signaling have been identified. The Rho GTPase RhoB plays a key role in cell migration, division and polarity; however, the function and regulatory mechanisms of RhoB in TLR ligand-triggered innate immune responses remain to be investigated. Here, we report that the expression of RhoB is induced by TLR agonists (lipopolysaccharide (LPS), CpG, poly(I:C)) in macrophages. Knockdown of RhoB expression markedly decreased TLR ligand-induced activation of mitogen activated protein kinases and nuclear factor-κB (NF-κB), and the production of tumor necrosis factor α (TNFα), interleukin (IL)-6 and IL-1ß in macrophages stimulated with TLR ligands. Furthermore, we demonstrated that RhoB interacts with major histocompatibility complex class II (MHCII) α chain, but not ß chain, in endosomes of macrophages. Knockdown of MHCII expression greatly reduced the interaction of RhoB with Btk, and attenuated the induction of NF-κB and interferon ß activity by RhoB upon LPS stimulation. These findings suggest that RhoB is a positive physiological regulator of TLRs signaling via binding to MHCII in macrophages, and therefore RhoB may be a potential therapeutic target in inflammatory diseases.

Sex-dependent treatment of chronic EAE with partial MHC class II constructs.

BACKGROUND: One of the main challenges in treating multiple sclerosis (MS) is reversing the effects of accumulated damage in the central nervous system (CNS) of progressive MS subjects. While most of the available drugs for MS subjects are anti-inflammatory and thus are limited to relapsing-remitting MS subjects, it is not clear to what extent their effects are capable of inducing axonal repair and remyelination in subjects with chronic MS. METHODS: A chronic model of experimental autoimmune encephalomyelitis (EAE) was used to evaluate the potency of partial MHC (pMHC) class II constructs in treating progressive EAE. RESULTS: We demonstrated an estrogen receptor alpha (ERα)-dependent increased dose requirement for effective treatment of female vs. male mice using pMHC. Such treatment using 100-µg doses of RTL342M or DRα1-mMOG-35-55 constructs significantly reversed clinical severity and showed a clear trend for inhibiting ongoing CNS damage, demyelination, and infiltration of inflammatory cells into the CNS in male mice. In contrast, WT female mice required larger 1-mg doses for effective treatment, although lower 100-µg doses were effective in ovariectomized or ERα-deficient mice with EAE. CONCLUSIONS: These findings will assist in the design of future clinical trials using pMHC for treatment of progressive MS.

MHC Class II Risk Alleles and Amino Acid Residues in Idiopathic Membranous Nephropathy.

Epitopes of phospholipase A2 receptor (PLA2R), the target antigen in idiopathic membranous nephropathy (iMN), must be presented by the HLA-encoded MHC class II molecules to stimulate autoantibody production. A genome-wide association study identified risk alleles at HLA and PLA2R loci, with the top variant rs2187668 within HLA-DQA1 showing a risk effect greater than that of the top variant rs4664308 within PLA2R1. How the HLA risk alleles affect epitope presentation by MHC class II molecules in iMN is unknown. Here, we genotyped 261 patients with iMN and 599 healthy controls at the HLA-DRB1, HLA-DQA1, HLA-DQB1, and HLA-DPB1 loci with four-digit resolution and extracted the encoded amino acid sequences from the IMGT/HLA database. We predicted T cell epitopes of PLA2R and constructed MHC-DR molecule-PLA2R peptide-T cell receptor structures using Modeler. We identified DRB1*1501 (odds ratio, 4.65; 95% confidence interval [95% CI], 3.39 to 6.41; P<0.001) and DRB1*0301 (odds ratio, 3.96; 95% CI, 2.61 to 6.05; P<0.001) as independent risk alleles for iMN and associated with circulating anti-PLA2R antibodies. Strong gene-gene interaction was noted between rs4664308(AA) and HLA-DRB1*1501/DRB1*0301. Amino acid positions 13 (P<0.001) and 71 (P<0.001) in the MHC-DRß1 chain independently associated with iMN. Structural models showed that arginine13 and alanine71, encoded by DRB1*1501, and lysine71, encoded by DRB1*0301, facilitate interactions with T cell epitopes of PLA2R. In conclusion, we identified two risk alleles of HLA class II genes and three amino acid residues on positions 13 and 71 of the MHC-DRß1 chain that may confer susceptibility to iMN by presenting T cell epitopes on PLA2R.

AAV-mediated gene delivery attenuates neuroinflammation in feline Sandhoff disease.

Sandhoff disease (SD) is a lysosomal storage disorder characterized by the absence of hydrolytic enzyme ß-N-acetylhexosaminidase (Hex), which results in storage of GM2 ganglioside in neurons and unremitting neurodegeneration. Neuron loss initially affects fine motor skills, but rapidly progresses to loss of all body faculties, a vegetative state, and death by five years of age in humans. A well-established feline model of SD allows characterization of the disease in a large animal model and provides a means to test the safety and efficacy of therapeutic interventions before initiating clinical trials. In this study, we demonstrate a robust central nervous system (CNS) inflammatory response in feline SD, primarily marked by expansion and activation of the microglial cell population. Quantification of major histocompatibility complex II (MHC-II) labeling revealed significant up-regulation throughout the CNS with areas rich in white matter most severely affected. Expression of the leukocyte chemokine macrophage inflammatory protein-1 alpha (MIP-1α) was also up-regulated in the brain. SD cats were treated with intracranial delivery of adeno-associated viral (AAV) vectors expressing feline Hex, with a study endpoint 16weeks post treatment. AAV-mediated gene delivery repressed the expansion and activation of microglia and normalized MHC-II and MIP-1α levels. These data reiterate the profound inflammatory response in SD and show that neuroinflammation is abrogated after AAV-mediated restoration of enzymatic activity.

Adrenomedullin 2 Improves Early Obesity-Induced Adipose Insulin Resistance by Inhibiting the Class II MHC in Adipocytes.

MHC class II (MHCII) antigen presentation in adipocytes was reported to trigger early adipose inflammation and insulin resistance. However, the benefits of MHCII inhibition in adipocytes remain largely unknown. Here, we showed that human plasma polypeptide adrenomedullin 2 (ADM2) levels were negatively correlated with HOMA of insulin resistance in obese human. Adipose-specific human ADM2 transgenic (aADM2-tg) mice were generated. The aADM2-tg mice displayed improvements in high-fat diet-induced early adipose insulin resistance. This was associated with increased insulin signaling and decreased systemic inflammation. ADM2 dose-dependently inhibited CIITA-induced MHCII expression by increasing Blimp1 expression in a CRLR/RAMP1-cAMP-dependent manner in cultured adipocytes. Furthermore, ADM2 treatment restored the high-fat diet-induced early insulin resistance in adipose tissue, mainly via inhibition of adipocyte MHCII antigen presentation and CD4(+) T-cell activation. This study demonstrates that ADM2 is a promising candidate for the treatment of early obesity-induced insulin resistance.

Mycobacterium leprae-Infected Macrophages Preferentially Primed Regulatory T Cell Responses and Was Associated with Lepromatous Leprosy.

BACKGROUND: The persistence of Mycobacterium leprae (M. leprae) infection is largely dependent on the types of host immune responses being induced. Macrophage, a crucial modulator of innate and adaptive immune responses, could be directly infected by M. leprae. We therefore postulated that M. leprae-infected macrophages might have altered immune functions. METHODOLOGY/PRINCIPAL FINDINGS: Here, we treated monocyte-derived macrophages with live or killed M. leprae, and examined their activation status and antigen presentation. We found that macrophages treated with live M. leprae showed committed M2-like function, with decreased interleukin 1 beta (IL-1beta), IL-6, tumor necrosis factor alpha (TNF-alpha) and MHC class II molecule expression and elevated IL-10 and CD163 expression. When incubating with naive T cells, macrophages treated with live M. leprae preferentially primed regulatory T (Treg) cell responses with elevated FoxP3 and IL-10 expression, while interferon gamma (IFN-gamma) expression and CD8+ T cell cytotoxicity were reduced. Chromium release assay also found that live M. leprae-treated macrophages were more resistant to CD8+ T cell-mediated cytotoxicity than sonicated M. leprae-treated monocytes. Ex vivo studies showed that the phenotype and function of monocytes and macrophages had clear differences between L-lep and T-lep patients, consistent with the in vitro findings. CONCLUSIONS/SIGNIFICANCE: Together, our data demonstrate that M. leprae could utilize infected macrophages by two mechanisms: firstly, M. leprae-infected macrophages preferentially primed Treg but not Th1 or cytotoxic T cell responses; secondly, M. leprae-infected macrophages were more effective at evading CD8+ T cell-mediated cytotoxicity.

MyD88 Mediates Instructive Signaling in Dendritic Cells and Protective Inflammatory Response during Rickettsial Infection.

Spotted fever group rickettsiae cause potentially life-threatening infections throughout the world. Several members of the Toll-like receptor (TLR) family are involved in host response to rickettsiae, and yet the mechanisms by which these TLRs mediate host immunity remain incompletely understood. In the present study, we found that host susceptibility of MyD88(-/-)mice to infection with Rickettsia conorii or Rickettsia australis was significantly greater than in wild-type (WT) mice, in association with severely impaired bacterial clearance in vivo R. australis-infected MyD88(-/-)mice showed significantly lower expression levels of gamma interferon (IFN-γ), interleukin-6 (IL-6), and IL-1ß, accompanied by significantly fewer inflammatory infiltrates of macrophages and neutrophils in infected tissues, than WT mice. The serum levels of IFN-γ, IL-12, IL-6, and granulocyte colony-stimulating factor were significantly reduced, while monocyte chemoattractant protein 1, macrophage inflammatory protein 1α, and RANTES were significantly increased in infected MyD88(-/-)mice compared to WT mice. Strikingly, R. australis infection was incapable of promoting increased expression of MHC-II(high)and production of IL-12p40 in MyD88(-/-)bone marrow-derived dendritic cells (BMDCs) compared to WT BMDCs, although costimulatory molecules were upregulated in both types of BMDCs. Furthermore, the secretion levels of IL-1ß by Rickettsia-infected BMDCs and in the sera of infected mice were significantly reduced in MyD88(-/-)mice compared to WT controls, suggesting that in vitro and in vivo production of IL-1ß is MyD88 dependent. Taken together, our results suggest that MyD88 signaling mediates instructive signals in DCs and secretion of IL-1ß and type 1 immune cytokines, which may account for the protective inflammatory response during rickettsial infection.