STAT3 activation in circulating immune cells is related to neovascular age-related macular degeneration

Purpose:The Signal Transducer and Activator of Transcription 3 (STAT3) pathway is known to play an important role in inflammation and angiogenesis. STAT3 can be activated by IL-6 family cytokines through the receptor IL-6R/gp130. Increased IL-6 has been detected in the plasma and vitreous in neovascular age-related macular degeneration (nAMD) patients. The aim of this study was to investigate the role of the STAT3 pathway in the pathogenesis of nAMD.

Methods:Blood cells from nAMD patients (n = 11) and age-, gender-matched healthy controls (n = 13) were stimulated with IL-6 for 20 minutes. The expression of the activated form of STAT3 (p-STAT3) was examined by flow cytometry. The mRNA levels of gp130, IL-6R and the suppressor of cytokine signalling 3 (SOCS3, a negative regulator of p-STAT3) were evaluated by real-time RT-PCR. Laser-induced choroidal neovascularisation (CNV) was performed in WT C57BL/6J mice as well as in the myeloid cell specific SOCS3 deficiency mice i.e., the LysMCre-SOCS3fl/fl mice. STAT3 activation in CNV lesions was examined by western blot. The size of CNV at different times after laser treatment was measured by confocal microscopy of RPE/choroidal flatmounts.

Results:The expression of p-STAT3 in CD11b+ monocytes was significantly increased in nAMD patients compared to healthy controls, although mRNA expression of gp130, IL-6R and SOCS3 did not differ between patients and controls. The expression of p-STAT3 in the retinal and RPE/choroidal tissues was increased at 1 and 3 days after laser treatment. The administration of a STAT3 inhibitor LLL12 significantly suppressed CNV. CD11b+ monocytes from LysMCre-SOCS3fl/fl mice expressed higher levels of p-STAT3 compared to the cells from WT mice. Laser induced CNV developed earlier and were larger in LysMCre-SOCS3fl/fl mice compared to WT C57BL/6J mice.

Conclusions:Our results suggest that STAT3 activation in circulating monocytes may contribute to the development of choroidal neovascularisation in AMD, and targeting the STAT3 pathway may have therapeutic potential in nAMD.

Genetic pleiotropy between multiple sclerosis and schizophrenia but not bipolar disorder: differential involvement of immune-related gene loci

Converging evidence implicates immune abnormalities in schizophrenia (SCZ), and recent genome-wide association studies (GWAS) have identified immune-related single-nucleotide polymorphisms (SNPs) associated with SCZ. Using the conditional false discovery rate (FDR) approach, we evaluated pleiotropy in SNPs associated with SCZ (n=21 856) and multiple sclerosis (MS) (n=43 879), an inflammatory, demyelinating disease of the central nervous system. Because SCZ and bipolar disorder (BD) show substantial clinical and genetic overlap, we also investigated pleiotropy between BD (n=16 731) and MS. We found significant genetic overlap between SCZ and MS and identified 21 independent loci associated with SCZ, conditioned on association with MS. This enrichment was driven by the major histocompatibility complex (MHC). Importantly, we detected the involvement of the same human leukocyte antigen (HLA) alleles in both SCZ and MS, but with an opposite directionality of effect of associated HLA alleles (that is, MS risk alleles were associated with decreased SCZ risk). In contrast, we found no genetic overlap between BD and MS. Considered together, our findings demonstrate genetic pleiotropy between SCZ and MS and suggest that the MHC signals may differentiate SCZ from BD susceptibility.Molecular Psychiatry advance online publication, 28 January 2014; doi:10.1038/mp.2013.195.

The role of immune-related myeloid cells in angiogenesis

Macrophage function is not restricted to the innate and adaptive immune responses, but also includes host defence, wound healing, angiogenesis and homeostatic processes. Within the spectrum of macrophage activation there are two extremes: M1 classically activated macrophages which have a pro-inflammatory phenotype, and M2 alternatively activated macrophages which are pro-angiogenic and anti-inflammatory. An important property of macrophages is their plasticity to switch from one phenotype to the other and they can be defined in their polarisation state at any point between the two extremes. In order to determine what stage of activation macrophages are in, it is essential to profile various phenotypic markers for their identification. This review describes the angiogenic role for myeloid cells: circulating monocytes, Tie-2 expressing monocytes (TEMs), myeloid-derived suppressor cells (MDSCs), tumour associated macrophages (TAMs), and neutrophils. Each cell type is discussed by phenotype, roles within angiogenesis and possible targets as a cell therapy. In addition, we also refer to our own research on myeloid angiogenic cells (MACs), outlining their ability to induce angiogenesis and their similarities to alternatively activated M2 macrophages. MACs significantly contribute to vascular repair through paracrine mechanisms as they lack the capacity to differentiate into endothelial cells. Since MACs also retain plasticity, phenotypic changes can occur according to disease states and the surrounding microenvironment. This pro-angiogenic potential of MACs could be harnessed as a novel cellular therapy for the treatment of ischaemic diseases, such as diabetic retinopathy, hind limb ischaemia and myocardial infarction; however, caution needs to be taken when MACs are delivered into an inflammatory milieu.

Dissolving Microneedle Delivery of Nanoparticle Encapsulated Antigen Elicits Efficient Cross-Priming and Th1 Immune Responses by Murine Langerhans Cells

Dendritic cells (DCs) of the skin play an important role in skin-mediated immunity capable of promoting potent immune responses. We availed of polymeric dissolving microneedle (MN) arrays laden with nano-encapsulated antigen to specifically target skin DC networks. This modality of immunization represents an economic, efficient and potent means of antigen delivery directly to skin DCs, which are inefficiently targeted by more conventional immunization routes. Following MN immunization, Langerhans cells (LCs) constituted the major skin DC subset capable of cross-priming antigen-specific CD8(+) T cells ex-vivo. While all DC subsets were equally efficient in priming CD4(+) T cells, LCs were largely responsible for orchestrating the differentiation of CD4(+) IFN-γ and IL-17 producing effectors. Importantly, depletion of LCs prior to immunization had a profound effect on CD8(+) CTL responses in vivo, and vaccinated animals displayed reduced protective anti-tumour and viral immunity. Interestingly, this cross-priming bias was lost following MN immunization with soluble antigen, suggesting that processing and cross-presentation of nano-particulate antigen is favoured by LCs. Therefore, these studies highlight the importance of LCs in skin immunization strategies and that targeting of nano-particulate immunogens through dissolvable polymeric MNs potentially provides a promising technological platform for improved vaccination strategies.Journal of Investigative Dermatology accepted article preview online, 22 September 2014. doi:10.1038/jid.2014.415.

Macrophage migration inhibitory factor (MIF) expression in human malignant gliomas contributes to immune escape and tumour progression

Macrophage migration inhibitory factor (MIF), which inhibits apoptosis and promotes angiogenesis, is expressed in cancers suppressing immune surveillance. Its biological role in human glioblastoma is, however, only poorly understood. We examined in-vivo expression of MIF in 166 gliomas and 23 normal control brains by immunohistochemistry. MIF immunoreactivity was enhanced in neoplastic astrocytes in WHO grade II glioma and increased significantly in higher tumour grades (III-IV). MIF expression was further assessed in 12 glioma cell lines in vitro. Quantitative RT-PCR showed that MIF mRNA expression was elevated up to 800-fold in malignant glioma cells compared with normal brain. This translated into high protein levels as assessed by immunoblotting of total cell lysates and by ELISA-based measurement of secreted MIF. Wild-type p53-retaining glioma cell lines expressed higher levels of MIF, which may be connected with the previously described role of MIF as a negative regulator of wild-type p53 signalling in tumour cells. Stable knockdown of MIF by shRNA in glioma cells significantly increased tumour cell susceptibility towards NK cell-mediated cytotoxicity. Furthermore, supernatant from mock-transfected cells, but not from MIF knockdown cells, induced downregulation of the activating immune receptor NKG2D on NK and CD8+ T cells. We thus propose that human glioma cell-derived MIF contributes to the immune escape of malignant gliomas by counteracting NK and cytotoxic T-cell-mediated tumour immune surveillance. Considering its further cell-intrinsic and extrinsic tumour-promoting effects and the availability of small molecule inhibitors, MIF seems to be a promising candidate for future glioma therapy.

GDF-15 contributes to proliferation and immune escape of malignant gliomas

Growth and differentiation factor (GDF)-15 is a member of the transforming growth factor (TGF)-beta family. GDF-15 is necessary for the maintenance of pregnancy but has also been linked to other physiologic and pathologic conditions.

Macrophage migration inhibitory factor contributes to the immune escape of ovarian cancer by down-regulating NKG2D

The proinflammatory cytokine macrophage migration inhibitory factor (MIF) stimulates tumor cell proliferation, migration, and metastasis; promotes tumor angiogenesis; suppresses p53-mediated apoptosis; and inhibits antitumor immunity by largely unknown mechanisms. We here describe an overexpression of MIF in ovarian cancer that correlates with malignancy and the presence of ascites. Functionally, we find that MIF may contribute to the immune escape of ovarian carcinoma by transcriptionally down-regulating NKG2D in vitro and in vivo which impairs NK cell cytotoxicity toward tumor cells. Together with the additional tumorigenic properties of MIF, this finding provides a rationale for novel small-molecule inhibitors of MIF to be used for the treatment of MIF-secreting cancers.

Measurements of immune responses for establishing correlates of vaccine protection against HIV

Well-defined correlates of protective immunity are an essential component of rational vaccine development. Despite years of basic science and three HIV vaccine efficacy trials, correlates of immunological protection from HIV infection remain undefined. In December 2010, a meeting of scientists engaged in basic and translational work toward developing HIV-1 vaccines was convened. The goal of this meeting was to discuss current opportunities and optimal approaches for defining correlates of protection, both for ongoing and future HIV-1 vaccine candidates; specific efforts were made to engage young scientists. We discuss here the highlights from the meeting regarding the progress made and the way forward for a protective HIV-1 vaccine.

In vitro studies on the infection and replication of porcine circovirus type 2 in cells of the porcine immune system

Porcine circovirus type 2 (PCV2) nucleic acid and/or antigens are consistently observed in cells of monocytic morphology in lesions of pigs affected by post-weaning multisystemic wasting syndrome (PMWS). In this study, PCV2 antigen was detected in the cytoplasm of monocytes, pulmonary macrophages (PMs) and monocyte-derived macrophages exposed to the virus in vitro, by immunofluorescence analysis (IFA) and the phenotype of these cells confirmed by detection of monocytic cell surface markers using flow cytometry. Viral antigen was not observed in lymphocytic cells. Replication of the virus in PMs was investigated further by comparison to that observed in the continuous pig kidney cell line (PK15A) using quantitative virus titration, quantitative PCR and by the detection of double stranded DNA intermediates of viral replication by Southern blotting analyses. Although increases in viral DNA and levels of infectious virus progeny and the presence of replicative intermediates, indicative of viral replication, were observed in PK15A cells, no such changes were observed in PMs in spite of the fact that infectious virus, viral antigen and viral DNA persisted in the cells for at least the duration of the experiment. These results suggest that in vivo, monocytic cells may not represent the primary target for PCV2 replication. (C) 2003 Elsevier B.V. All rights reserved.

Identification of systemic immune response markers through metabolomic profiling of plasma from calves given an intra-nasally delivered respiratory vaccine

Vaccination procedures within the cattle industry are important disease control tools to minimize economic and welfare burdens associated with respiratory pathogens. However, new vaccine, antigen and carrier technologies are required to combat emerging viral strains and enhance the efficacy of respiratory vaccines, particularly at the point of pathogen entry. New technologies, specifically metabolomic profiling, could be applied to identify metabolite immune-correlates representative of immune protection following vaccination aiding in the design and screening of vaccine candidates. This study for the first time demonstrates the ability of untargeted UPLC-MS metabolomic profiling to identify metabolite immune correlates characteristic of immune responses following mucosal vaccination in calves. Male Holstein Friesian calves were vaccinated with Pfizer Rispoval® PI3 + RSV intranasal vaccine and metabolomic profiling of post-vaccination plasma revealed 12 metabolites whose peak intensities differed significantly from controls. Plasma levels of glycocholic acid, N-[(3α,5β,12α)-3,12-Dihydroxy-7,24-dioxocholan-24-yl]glycine, uric acid and biliverdin were found to be significantly elevated in vaccinated animals following secondary vaccine administration, whereas hippuric acid significantly decreased. In contrast, significant upregulation of taurodeoxycholic acid and propionylcarnitine levels were confined to primary vaccine administration. Assessment of such metabolite markers may provide greater information on the immune pathways stimulated from vaccine formulations and benchmarking early metabolomic responses to highly immunogenic vaccine formulations could provide a means for rapidly assessing new vaccine formulations. Furthermore, the identification of metabolic systemic immune response markers which relate to specific cell signaling pathways of the immune system could allow for targeted vaccine design to stimulate key pathways which can be assessed at the metabolic level.

Ageing and the immune system: focus on macrophages

A fully functioning immune system is essential in order to maintain good health. However, the immune system deteriorates with advancing age, and this contributes to increased susceptibility to infection, autoimmunity, and cancer in the older population. Progress has been made in identifying age-related defects in the adaptive immune system. In contrast, relatively little research has been carried out on the impact of ageing on the innate immune response. This area requires further research as the innate immune system plays a crucial role in protection against infection and represents a first line of defence. Macrophages are central effector cells of the innate immune system and have many diverse functions. As a result, age-related impairments in macrophage function are likely to have important consequences for the health of the older population. It has been reported that ageing in macrophages impacts on many processes including toll-like receptor signalling, polarisation, phagocytosis, and wound repair. A detailed understanding of the impact of ageing on macrophages is required in order to develop therapeutics that will boost immune responses in the older population.

An unbiased genetic screen reveals the polygenic nature of the influenza virus anti-interferon response

UNLABELLED: Influenza A viruses counteract the cellular innate immune response at several steps, including blocking RIG I-dependent activation of interferon (IFN) transcription, interferon (IFN)-dependent upregulation of IFN-stimulated genes (ISGs), and the activity of various ISG products; the multifunctional NS1 protein is responsible for most of these activities. To determine the importance of other viral genes in the interplay between the virus and the host IFN response, we characterized populations and selected mutants of wild-type viruses selected by passage through non-IFN-responsive cells. We reasoned that, by allowing replication to occur in the absence of the selection pressure exerted by IFN, the virus could mutate at positions that would normally be restricted and could thus find new optimal sequence solutions. Deep sequencing of selected virus populations and individual virus mutants indicated that nonsynonymous mutations occurred at many phylogenetically conserved positions in nearly all virus genes. Most individual mutants selected for further characterization induced IFN and ISGs and were unable to counteract the effects of exogenous IFN, yet only one contained a mutation in NS1. The relevance of these mutations for the virus phenotype was verified by reverse genetics. Of note, several virus mutants expressing intact NS1 proteins exhibited alterations in the M1/M2 proteins and accumulated large amounts of deleted genomic RNAs but nonetheless replicated to high titers. This suggests that the overproduction of IFN inducers by these viruses can override NS1-mediated IFN modulation. Altogether, the results suggest that influenza viruses replicating in IFN-competent cells have tuned their complete genomes to evade the cellular innate immune system and that serial replication in non-IFN-responsive cells allows the virus to relax from these constraints and find a new genome consensus within its sequence space.

IMPORTANCE: In natural virus infections, the production of interferons leads to an antiviral state in cells that effectively limits virus replication. The interferon response places considerable selection pressure on viruses, and they have evolved a variety of ways to evade it. Although the influenza virus NS1 protein is a powerful interferon antagonist, the contributions of other viral genes to interferon evasion have not been well characterized. Here, we examined the effects of alleviating the selection pressure exerted by interferon by serially passaging influenza viruses in cells unable to respond to interferon. Viruses that grew to high titers had mutations at many normally conserved positions in nearly all genes and were not restricted to the NS1 gene. Our results demonstrate that influenza viruses have fine-tuned their entire genomes to evade the interferon response, and by removing interferon-mediated constraints, viruses can mutate at genome positions normally restricted by the interferon response.

Burkholderia cenocepacia Lipopolysaccharide Modification and Flagellin Glycosylation Affect Virulence but Not Innate Immune Recognition in Plants

Burkholderia cenocepacia causes opportunistic infections in plants, insects, animals, and humans, suggesting that “virulence” depends on the host and its innate susceptibility to infection. We hypothesized that modifications in key bacterial molecules recognized by the innate immune system modulate host responses to B. cenocepacia. Indeed, modification of lipo- polysaccharide (LPS) with 4-amino-4-deoxy-L-arabinose and flagellin glycosylation attenuates B. cenocepacia infection in Arabi- dopsis thaliana and Galleria mellonella insect larvae. However, B. cenocepacia LPS and flagellin triggered rapid bursts of nitric oxide and reactive oxygen species in A. thaliana leading to activation of the PR-1 defense gene. These responses were drastically reduced in plants with fls2 (flagellin FLS2 host receptor kinase), Atnoa1 (nitric oxide-associated protein 1), and dnd1-1 (reduced production of nitric oxide) null mutations. Together, our results indicate that LPS modification and flagellin glycosylation do not affect recognition by plant receptors but are required for bacteria to establish overt infection.