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 CD14(dim)CD16(++)), 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.
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.
Macrophages (Mφ) play a central role as effector cells in immunity to intracellular pathogens such as Mycobacterium. Paradoxically, they also provide a habitat for intracellular bacterial survival. ...This paradoxical role of Mφ remains poorly understood. Here we report that this dual role may emanate from the functional plasticity of Mφ: Whereas Mφ-1 polarized in the presence of granulocyte-Mφ colony-stimulating factor promoted type 1 immunity, Mφ-2 polarized with Mφ colony-stimulating factor subverted type 1 immunity and thus may promote immune escape and chronic infection. Importantly, Mφ-1 secreted high levels of IL-23 (p40/p19) but no IL-12 (p40/p35) after (myco)bacterial activation. In contrast, activated Mφ-2 produced neither IL-23 nor IL-12 but predominantly secreted IL-10. Mφ-1 required IFN-γ as a secondary signal to induce IL-12p35 gene transcription and IL-12 secretion. Activated dendritic cells produced both IL-12 and IL-23, but unlike Mφ-1 they slightly reduced their IL-23 secretion after addition of IFN-γ. Binding, uptake, and outgrowth of a mycobacterial reporter strain was supported by both Mφ subsets, but more efficiently by Mφ-2 than Mφ-1. Whereas Mφ-1 efficiently stimulated type 1 helper cells, Mφ-2 only poorly supported type 1 helper function. Accordingly, activated Mφ-2 but not Mφ-1 down-modulated their antigen-presenting and costimulatory molecules (HLA-DR, CD86, and CD40). These findings indicate that (i) Mφ-1 and Mφ-2 play opposing roles in cellular immunity and (ii) IL-23 rather than IL-12 is the primary type 1 cytokine produced by activated proinflammatory Mφ-1. Mφ heterogeneity thus may be an important determinant of immunity and disease outcome in intracellular bacterial infection.
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.
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 ability to develop adequate immunity to intracellular bacterial pathogens is unequally distributed among human beings. In the case of tuberculosis, for example, infection with Mycobacterium ...tuberculosisresults in disease in 5–10% of exposed individuals, whereas the remainder control infection effectively. Similar interindividual differences in disease susceptibility are characteristic features of leprosy, typhoid fever, leishmaniasis, and other chronic infectious diseases, including viral infections. The outcome of infection is influenced by many factors, such as nutritional status, co-infections, exposure to environmental microbes, and previous vaccinations. It is clear, however, that genetic host factors also play an important part in controlling disease susceptibility to intracellular pathogens. Recently, patients with severe infections due to otherwise poorly pathogenic mycobacteria (non-tuberculous mycobacteria or Mycobacterium bovis BCG) or Salmonella spp have been identified. Many of these patients were unable to produce or respond to interferon γ, due to deleterious mutations in genes that encode major proteins in the type 1 cytokine (interleukin 12/interleukin 23/interferon γ) axis (interleukin 12p40/interleukin 23p40, IL12 receptor β1/IL23 receptor β1, interferon γ receptors 1 and 2, or signal transducer and activator of transcription 1). This axis is a major immunoregulatory system that bridges innate and adaptive immunity. Unusual mycobacterial infections were also reported in several patients with genetic defects in inhibitor of NFκB kinase γ, a key regulatory molecule in the nuclear factor κB pathway. New findings discussed in this review provide further and sometimes surprising insights into the role of type 1 cytokines, and into the unexpected heterogeneity seen in these syndromes.
Host genetic factors are important in determining the outcome of infections caused by intracellular pathogens, including mycobacteria and salmonellae, but until now have been poorly characterized. ...Recently, some individuals with severe infections due to otherwise weakly pathogenic mycobacteria (non-tuberculous mycobacteria or Mycobacterium bovis bacille Calmette-Guérin) or Salmonella species have been shown to be unable to produce or respond to interferon-gamma. This inability results from mutations in any of five genes encoding essential proteins of the type 1 cytokine cascade: interleukin-12p40, interleukin-12R beta 1, interferon-gamma R1, interferon-gamma R2 or STAT1. Ten syndromes have thus far been identified. Recent insights in genetically controlled host defense and susceptibility to mycobacterial disease are discussed.
Control of human host immunity to mycobacteria Ottenhoff, Tom H.M.; Verreck, Frank A.W.; Hoeve, Marieke A. ...
Tuberculosis (Edinburgh, Scotland),
01/2005, Letnik:
85, Številka:
1
Journal Article
Recenzirano
Infection with
Mycobacterium tuberculosis results in disease in 5–10% of exposed individuals, whereas the remainder controls infection effectively. Similar inter-individual differences in disease ...susceptibility are characteristic features of leprosy, typhoid fever, leishmaniasis and other chronic infectious diseases, including viral infections. Although the outcome of infection is influenced by many factors, it is clear that genetic host factors play an important role in controlling disease susceptibility to intracellular pathogens. Knowledge of the genes involved and their downstream cellular pathways will provide new insights for the design of improved and rationalized strategies to enhance host-resistance, e.g. by vaccination. In addition, this knowledge will aid in identifying better biomarkers of protection and disease, which are essential tools for the monitoring of vaccination and other intervention trials.
The recent identification of patients with deleterious mutations in genes that encode major proteins in the type-1 cytokine (IL-12/IL23-IFN-
γ) axis, that suffered from severe infections due to otherwise poorly pathogenic mycobacteria (non-tuberculous mycobacteria (NTM) or
M. bovis Bacille Calmette-Guérin (BCG)) or
Salmonella species has revealed the major role of this system in innate and adaptive immunity to mycobacteria and salmonellae. Clinical tuberculosis has now been described in a number of patients with IL-12/IL23-IFN-
γ system defects. Moreover, unusual mycobacterial infections were reported in several patients with genetic defects in NEMO, a key regulatory molecule in the NFκB pathway. These new findings will be discussed since they provide further insights into the role of type-1 cytokines in immunity to mycobacteria, including
M. tuberculosis.
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.
Larvae 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 (AAMphi) 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.
We 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.
P. 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.
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