Marieke A. Hoeve

Divergent effects of IL-12 and IL-23 on the production of IL-17 by human T cells.

IL‐23 is regarded as a major pro‐inflammatory mediator in autoimmune disease, a role which until recently was ascribed to its related cytokine IL‐12. IL‐23, an IL‐12p40/p19 heterodimeric protein, binds to IL‐12Rβ1/IL‐23R receptor complexes. Mice deficient for p19, p40 or IL‐12Rβ1 are resistant to experimental autoimmune encephalomyelitis or collagen‐induced arthritis. Paradoxically, however, IL‐12Rβ2‐ and IL‐12p35‐deficient mice show remarkable increases in disease susceptibility, suggesting divergent roles of IL‐23 and IL‐12 in modulating inflammatory processes. IL‐23 induces IL‐17, which mediates inflammation and tissue remodeling, but the role of IL‐12 in this respect remains unidentified. We investigated the roles of exogenous (recombinant) and endogenous (macrophage‐derived) IL‐12 and IL‐23, on IL‐17‐induction in human T‐cells. IL‐23 enhanced IL‐17 secretion, as did IL‐2, IL‐15, IL‐18 and IL‐21. In contrast, IL‐12 mediated specific inhibition of IL‐17 production. These data support the role of IL‐23 in inflammation through stimulating IL‐17 production by T lymphocytes, and importantly indicate a novel regulatory function for IL‐12 by specifically suppressing IL‐17 secretion. These data therefore extend previous reports that had indicated unique functions for IL‐23 and IL‐12 due to distinct receptor expression and signal transduction complexes, and provide novel insights into the regulation of immunity, inflammation and immunopathology.

The Same IκBα Mutation in Two Related Individuals Leads to Completely Different Clinical Syndromes

Both innate and adaptive immune responses are dependent on activation of nuclear factor κB (NF-κB), induced upon binding of pathogen-associated molecular patterns to Toll-like receptors (TLRs). In murine models, defects in NF-κB pathway are often lethal and viable knockout mice have severe immune defects. Similarly, defects in the human NF-κB pathway described to date lead to severe clinical disease. Here, we describe a patient with a hyper immunoglobulin M–like immunodeficiency syndrome and ectodermal dysplasia. Monocytes did not produce interleukin 12p40 upon stimulation with various TLR stimuli and nuclear translocation of NF-κB was impaired. T cell receptor–mediated proliferation was also impaired. A heterozygous mutation was found at serine 32 in IκBα. Interestingly, his father has the same mutation but displays complex mosaicism. He does not display features of ectodermal dysplasia and did not suffer from serious infections with the exception of a relapsing Salmonella typhimurium infection. His monocyte function was impaired, whereas T cell function was relatively normal. Consistent with this, his T cells almost exclusively displayed the wild-type allele, whereas both alleles were present in his monocytes. We propose that the T and B cell compartment of the mosaic father arose as a result of selection of wild-type cells and that this underlies the widely different clinical phenotype.

A thermoresponsive and chemically defined hydrogel for long-term culture of human embryonic stem cells

Cultures of human embryonic stem cell typically rely on protein matrices or feeder cells to support attachment and growth, while mechanical, enzymatic or chemical cell dissociation methods are used for cellular passaging. However, these methods are ill defined, thus introducing variability into the system, and may damage cells. They also exert selective pressures favouring cell aneuploidy and loss of differentiation potential. Here we report the identification of a family of chemically defined thermoresponsive synthetic hydrogels based on 2-(diethylamino)ethyl acrylate, which support long-term human embryonic stem cell growth and pluripotency over a period of 2–6 months. The hydrogels permitted gentle, reagent-free cell passaging by virtue of transient modulation of the ambient temperature from 37 to 15 °C for 30 min. These chemically defined alternatives to currently used, undefined biological substrates represent a flexible and scalable approach for improving the definition, efficacy and safety of human embryonic stem cell culture systems for research, industrial and clinical applications.

Murine gammaherpesvirus-induced fibrosis is associated with the development of alternatively activated macrophages

Murine gammaherpesvirus 68 (MHV‐68) is a natural pathogen of rodents closely related to the human γherpesviruses Kaposi’s sarcoma‐associated herpesvirus and EBV. Following intranasal infection, the virus replicates in the lung epithelium prior to establishing latent infection in lymphoid tissue. Infection of mice deficient in IFN‐γR signaling (IFN‐γR−/−) results in a multiple organ fibrosis, in which the spleen is severely affected. We show here that by Day 12 postinfection, prior to development of fibrosis in the spleens of IFN‐γR−/− mice, different subsets of splenic macrophages (Mϕs) are morphologically activated and enter latently infected germinal centers (GCs). Mϕs coexpressing arginase I (ARG1), a marker of alternative activation of Mϕs, and murine Mϕ markers F4/80, ER‐TR9, and MOMA‐1 are found in GCs of IFN‐γR−/− mice but not of wild‐type mice. Quantitative RT‐PCR of spleen RNA confirms induction of ARG1 and in addition, shows up‐regulation of found in inflammatory zone 1/resistin‐like molecule‐α, tissue inhibitor of metalloproteinase‐1, matrix metalloproteinase‐12, fibronectin, and factor XIIIA in IFN‐γR−/− mice. In contrast, inducible NO synthase, associated with classical Mϕ activation, is up‐regulated following infection of wild‐type mice but not IFN‐γR−/− mice. Concomitant with the aaMϕs, transcription of the Th2 cytokines IL‐13, IL‐21, and IL‐5 is up‐regulated. Thus, in the absence of IFN‐γR signaling, MHV‐68 initiates a Th2 immune response, leading to alternative activation of macrophages and induction of fibrosis. This system provides an important model for studying the pathogenesis of fibrosis initiated by a latent herpesvirus infection.

Influenza Virus A Infection of Human Monocyte and Macrophage Subpopulations Reveals Increased Susceptibility Associated with Cell Differentiation

Influenza virus infection accounts for significant morbidity and mortality world-wide. Interactions of the virus with host cells, particularly those of the macrophage lineage, are thought to contribute to various pathological changes associated with poor patient outcome. Development of new strategies to treat disease therefore requires a detailed understanding of the impact of virus infection upon cellular responses. Here we report that human blood-derived monocytes could be readily infected with the H3N2 influenza virus A/Udorn/72 (Udorn), irrespective of their phenotype (CD14++/CD16−, CD14++/CD16+ or CD14dimCD16++), as determined by multi-colour flow cytometry for viral haemagglutinin (HA) expression and cell surface markers 8–16 hours post infection. Monocytes are relatively resistant to influenza-induced cell death early in infection, as approximately 20% of cells showed influenza-induced caspase-dependent apoptosis. Infection of monocytes with Udorn also induced the release of IL-6, IL-8, TNFα and IP-10, suggesting that NS1 protein of Udorn does not (effectively) inhibit this host defence response in human monocytes. Comparative analysis of human monocyte-derived macrophages (Mph) demonstrated greater susceptibility to human influenza virus than monocytes, with the majority of both pro-inflammatory Mph1 and anti-inflammatory/regulatory Mph2 cells expressing viral HA after infection with Udorn. Influenza infection of macrophages also induced cytokine and chemokine production. However, both Mph1 and Mph2 phenotypes released comparable amounts of TNFα, IL-12p40 and IP-10 after infection with H3N2, in marked contrast to differential responses to LPS-stimulation. In addition, we found that influenza virus infection augmented the capacity of poorly phagocytic Mph1 cells to phagocytose apoptotic cells by a mechanism that was independent of either IL-10 or the Mer receptor tyrosine kinase/Protein S pathway. In summary, our data reveal that influenza virus infection of human macrophages causes functional alterations that may impact on the process of resolution of inflammation, with implications for viral clearance and lung pathology.

Plasmodium chabaudi limits early Nippostrongylus brasiliensis-induced pulmonary immune activation and Th2 polarization in co-infected mice

BackgroundLarvae of several common species of parasitic nematodes obligately migrate through, and often damage, host lungs. The larvae induce strong pulmonary Type 2 immune responses, including T-helper (Th)2 cells as well as alternatively activated macrophages (AAMφ) and associated chitinase and Fizz/resistin family members (ChaFFs), which are thought to promote tissue repair processes. Given the prevalence of systemic or lung-resident Type 1-inducing pathogens in geographical areas in which nematodes are endemic, we wished to investigate the impact of concurrent Type 1 responses on the development of these Type 2 responses to nematode larval migration. We therefore infected BALB/c mice with the nematode Nippostrongylus brasiliensis, in the presence or absence of Plasmodium chabaudi chabaudi malaria parasites. Co-infected animals received both infections on the same day, and disease was assessed daily before immunological measurements were taken at 3, 5, 7 or 20 days post-infection.ResultsWe observed that the nematodes themselves caused transient loss of body mass and red blood cell density, but co-infection then slightly ameliorated the severity of malarial anaemia. We also tracked the development of immune responses in the lung and thoracic lymph node. By the time of onset of the adaptive immune response around 7 days post-infection, malaria co-infection had reduced pulmonary expression of ChaFFs. Assessment of the T cell response demonstrated that the Th2 response to the nematode was also significantly impaired by malaria co-infection.ConclusionP. c. chabaudi co-infection altered both local and lymph node Type 2 immune activation due to migration of N. brasiliensis larvae. Given recent work from other laboratories showing that N. brasiliensis-induced ChaFFs correlate to the extent of long-term lung damage, our results raise the possibility that co-infection with malaria might alter pulmonary repair processes following nematode migration. Further experimentation in the co-infection model developed here will reveal the longer-term consequences of the presence of both malaria and helminths in the lung.

Over expression of IL-10 by macrophages overcomes resistance to murine filariasis

Individuals infected with parasitic helminths are able to tolerate the presence of parasites for considerable time without clinical pathology. Immunosuppressive responses induced by the filarial parasite are considered responsible for this long-lasting relationship, inuring to the benefit of both parasite and host. In order to directly link IL-10 with parasite survival, we infected mice, in which over expression of IL-10 was restricted to macrophages under control of the CD68 promoter (macIL-10tg), with Litomosoides sigmodontis. IL-10 overexpression by macrophages led to increased susceptibility with a significantly higher number of adult worms. Most profound, IL-10 overexpression was sufficient to convert resistant FVB wild-type mice towards a patent phenotype, since microfilariae were exclusively found in macIL-10tg mice. These findings were associated with reduced Th2 cytokine production in macIL-10tg mice. Expression of arginase-1, Ym1 and Fizz1, genes that are found strongly expressed in murine alternatively activated macrophages, were detected in macIL-10tg mice. Thus, IL-10 produced by macrophages with characteristics of alternative activation can overcome resistance and allow full patency in murine filariasis.

Raman spectroscopy and regenerative medicine : a review

The field of regenerative medicine spans a wide area of the biomedical landscape—from single cell culture in laboratories to human whole-organ transplantation. To ensure that research is transferrable from bench to bedside, it is critical that we are able to assess regenerative processes in cells, tissues, organs and patients at a biochemical level. Regeneration relies on a large number of biological factors, which can be perturbed using conventional bioanalytical techniques. A versatile, non-invasive, non-destructive technique for biochemical analysis would be invaluable for the study of regeneration; and Raman spectroscopy is a potential solution. Raman spectroscopy is an analytical method by which chemical data are obtained through the inelastic scattering of light. Since its discovery in the 1920s, physicists and chemists have used Raman scattering to investigate the chemical composition of a vast range of both liquid and solid materials. However, only in the last two decades has this form of spectroscopy been employed in biomedical research. Particularly relevant to regenerative medicine are recent studies illustrating its ability to characterise and discriminate between healthy and disease states in cells, tissue biopsies and in patients. This review will briefly outline the principles behind Raman spectroscopy and its variants, describe key examples of its applications to biomedicine, and consider areas of regenerative medicine that would benefit from this non-invasive bioanalytical tool.

Clearance of apo Nph induces an immunosuppressive response in pro-inflammatory type-1 and anti-inflammatory type-2 MΦ

Nph are crucial for proper host defence. Paradoxically, they also contribute to pathology in various inflammatory diseases. Hence, apo of Nph and subsequent removal from inflamed sites is critical for resolution of inflammation. Apo Nph are recognised and cleared by MΦ, supposedly in a “silent” fashion. MΦ show large heterogeneity, comprising various subsets with different functional and biochemical properties. The contribution of these distinct populations to clearance of apo Nph is as yet unknown. Here, we investigated phagocytosis and subsequent functional responses of in vitro generated pro-inflammatory MΦ1 and anti-inflammatory MΦ2. Although only MΦ2 were capable of efficient Nph phagocytosis, we found that contact with apo Nph excerts immunosuppressive effects on both subsets, skewing them towards an anti-inflammatory state.

Alternative activation of macrophages by filarial nematodes is MyD88-independent

Alternative macrophage activation is largely defined by IL-4Rα stimulation but the contribution of Toll-like receptor (TLR) signaling to this phenotype is not currently known. We have investigated macrophage activation status under Th2 conditions in the absence of the core TLR adaptor molecule, MyD88. No impairment was observed in the ability of MyD88-deficient bone marrow derived macrophages to produce or express alternative activation markers, including arginase, RELM-α or Ym1, in response to IL-4 treatment in vitro. Further, we observed no difference in the ability of peritoneal exudate cells from nematode implanted wild type (WT) or MyD88-deficient mice to produce arginase or express the alternative activation markers RELM-α or Ym1. Therefore, MyD88 is not a fundamental requirement for Th2-driven macrophage alternative activation, either in vitro or in vivo.