At the Centennial of the Bacteriophage: Reviving the Overlooked Contribution of a Forgotten Pioneer, Richard Bruynoghe (1881-1957).

The year 2015 marks the 100th anniversary of a publication by William Twort, in which he first described lysis of bacterial cultures by a filterable, self-replicating agent. In 1917, Félix d'Herelle, coined the name "bacteriophage" for the proposed agent. Two Belgian teams of microbiologists were among the few to critically examine the nature of the bacteriophage at that time. Although their experimental results agreed, their interpretations did not. Richard Bruynoghe (University of Louvain/Leuven) interpreted them as supportive of d'Herelle's notion of an ultramicroscopic microorganism. Jules Bordet (University of Brussels) found the proposal of a complex organism unnecessary and saw the bacteriophage as a simple endogenous bacterial enzyme endowed with capacity to induce its own secretion as well as ability to cause lysis of the bacteria. Two decades would elapse before bacteriophages were visualized and confirmed to be organized particles. However, by that time, Bruynoghe's work, that had only been published in short notes in society proceedings, was virtually forgotten. The present paper revives his original observations and arguments, while also recognizing that Bordet's alternative hypothesis had scientific merit.

Pulmonary inflammation in mice with collagen-induced arthritis is conditioned by complete Freund's adjuvant and regulated by endogenous IFN-γ.

Following immunization with collagen II (CII) in complete Freund's adjuvant (CFA), DBA/1 mice develop arthritis of major joints. This collagen-induced arthritis (CIA) is used as a model for rheumatoid arthritis (RA) in man. Inflammatory changes in lung tissue commonly occur in RA. However, evidence for pulmonary inflammation in CIA is scarce and ambiguous. Here, we demonstrate pulmonary inflammation accompanying CIA in wild-type DBA/1 mice. In IFN-γ receptor-deficient (IFN-γR KO) mice, inflammation was more frequent and more severe. Injection of CFA only (without CII) proved to be as efficient in eliciting pulmonary inflammation as immunization with CFA + CII, though being less effective in causing arthritis. Significant correlation in severity between joint and pulmonary involvement could not be demonstrated. Macroscopic, microscopic, and functional characteristics of pulmonary inflammation in the mice resembled those seen in human RA. Increased inflammation in IFN-γR KO mice was accompanied by augmented expression of various cytokines and chemokines, as measured by RT-PCR on affected tissue. Treatment with a TNF-α inhibitor ameliorated lung pathology. We conclude that CIA in DBA/1 mice is accompanied by pulmonary inflammation. Although both disease processes are kept in check by endogenous IFN-γ, lack of strict parallelism indicates that overlap in their pathogeneses is partial.

A tale of two interferon bioassays: how frustration with discrepant results from slightly dissimilar methods can engender discovery.

This introductory article describes an episode that took place in the mid-1980s when the first wave of cytokine discoveries took place. During studies aimed at complete purification of human interferon-γ from crude mitogen-stimulated lymphokine preparations, the use of two different antiviral bioassays for the cytokine yielded disparate results. Analysis revealed the presence of a "contaminant" IFN-like cytokine that was detectable with only one of the two assays. Superficially, the contaminant resembled IFN-ß. However, further analysis showed that it was not an IFN at all but an IFN-inducing cytokine identifiable as interleukin-1.

Collagen-induced arthritis and related animal models: how much of their pathogenesis is auto-immune, how much is auto-inflammatory?

In this review, we discuss our studies on the pathogenesis of collagen-induced arthritis (CIA) and related mouse models for rheumatoid arthritis. Of note, these models invariably rely on the use of complete Freund's adjuvant (CFA). Our analysis has focused on explaining the dichotomous - either protective or disease-promoting - role of endogenous IFN-γ. Induction of a myelopoietic burst by CFA was identified as an important and underestimated factor in mediating the role of IFN-γ and other cytokines (IL-6, IL-17, GCP-2, RANK-L). Myelopoiesis provides an excess in precursors for joint-infiltrating neutrophils and osteoclasts. We postulate that classical CIA is primarily an auto-inflammatory disease, in part because of a strong innate immune response to the adjuvant. Superimposed on this, collagen-specific auto-immunity reinforces inflammatory reactivity in joints.

Collagen-induced arthritis as an animal model for rheumatoid arthritis: focus on interferon-γ.

Rheumatoid arthritis (RA), an autoimmune disease causing inflammation, destruction, and deformity of the joints, affects around 1% of the world population. It is a systemic disease as patients exhibit extra-articular manifestations as well. Collagen-induced arthritis (CIA) in DBA/1 mice is one of the many animal models used to study possible pathogenic mechanisms of RA. It involves immunizing mice with collagen type II in complete Freund's adjuvant. Here we briefly review the general characteristics of RA and CIA and present an overview of data obtained by studying CIA in several gene knockout mice. In particular, detailed analysis of CIA in interferon-gamma (IFN-γ) receptor-deficient mice has pin-pointed IFN-γ as an important cytokine in the pathogenesis and has exposed new functions of IFN-γ in immunological processes. Pilot trials with exogenous IFN-γ in RA have been indicative of a beneficial effect. That improvement of the disease symptoms by IFN-γ treatment was not spectacular may be explained by the fact that RA is a heterogeneous disease in which the severity of the autoimmune disease is strongly determined by environmental factors.

Myeloid cells are tunable by a polyanionic polysaccharide derivative and co-determine host rescue from lethal virus infection.

Insight into molecular and cellular mechanisms of innate immunity is critical to understand viral pathogenesis and immunopathology and might be exploited for therapy. Whereas the molecular mechanisms of the IFN defense are well established, cellular mechanisms of antiviral immunity are only emerging, and their pharmacological triggering remains unknown. COAM is a polysaccharide derivative with antiviral activity but without comprehension about its mechanism of action. The COAM mixture was fractionated, and prophylactic treatment of mice with COAM polymers of high MW resulted in a conversion from 100% lethal mengovirus infection to an overall survival rate of 93% without obvious clinical sequelae. Differential and quantitative analysis of peritoneal leukocytes demonstrated that COAM induced a profound influx of neutrophils. Selective cell depletion experiments pointed toward neutrophils and macrophages as key effector cells in the rescue of mice from lethal mengovirus. COAM was able to induce mRNA and protein expression of the mouse neutrophil chemokine GCP-2. Binding of GCP-2 to COAM was demonstrated in solution and confirmed by SPR technology. Although COAM was not chemotactic for neutrophils, COAM-anchored muGCP-2 retained chemotactic activity for human and mouse neutrophils. In conclusion, this study established that COAM rescued mice from acute and lethal mengovirus infection by recruiting antiviral leukocytes to the site of infection, as proposed through the induction, binding, and concentration of endogenous chemokines. These findings reinforce the role of neutrophils and macrophages as critical cells that can be manipulated toward antiviral defense.

Interferon-gamma: a historical perspective.

This article reviews the main lines of thinking and exploration that have led to our current conception of the role of IFN-gamma in immune defense and autoimmunity. In 1965 the first report appeared describing production of an interferon-like virus inhibitor in cultured human leukocytes following exposure to the mitogen phytohemagglutinin. In the early 1970s the active principle became recognized as being distinct from classical virus-induced interferons, leading to its designation as immune interferon or Type II interferon, and eventually IFN-gamma. Up to that point interest in the factor had come almost exclusively from virologists, in particular those among them who were believers in interferon. Evidence first coming forward in the 1980s that IFN-gamma is indistinguishable from macrophage-activating factor (MAF), then a prototype lymphokine, was the signal for immunologists at large to become interested. Today IFN-gamma ranks among the most important endogenous regulators of immune responses.

How interferon-gamma keeps autoimmune diseases in check.

Interferon-gamma (IFN-gamma) is regarded traditionally as a proinflammatory factor and as the signature cytokine of Th1-dominated autoimmune processes. Early evidence indicative of an opposite, protective role has recently received further attention from reports revealing an increasing number of pathways by which IFN-gamma can counteract harmful inflammation in Th1-associated autoimmune diseases. Here, we review evidence for IFN-gamma's anti-inflammatory effects primarily from the perspective of one experimental model, collagen-induced arthritis (CIA), and question the classic proinflammatory role of IFN-gamma and also the Th1-Th2 paradigm as a basis for explaining the regulation of autoimmune diseases. We conclude that endogenous production of IFN-gamma during inflammatory and autoimmune diseases should be considered as a process with bidirectional immunoregulatory consequences, often resulting in moderation of pathology.

Interferons: the pathways of discovery. II. Immunomodulatory, in vivo and applied aspects.

This article analyzes the conceptual and technological context in which, over a period of 50 years, exploration of the biological and clinical significance of type I interferon has evolved. The elaboration of techniques for production and purification of mouse and human interferons and the establishment of laboratory-size production units have been of crucial importance in this process. Animal experiments have been invaluable for elucidation of mechanisms underlying the in vivo antiviral, anti-tumour and immunomodulatory potential of interferon, but have been of limited help to define the areas of clinical applicability. Proof of principle for applications as they are established today has come from clinical trials performed quite independently of evidence from animal experiments.

Protective role of IFN-gamma in collagen-induced arthritis conferred by inhibition of mycobacteria-induced granulocyte chemotactic protein-2 production.

Mice with a disrupted IFN-gamma system are remarkably susceptible to experimental autoimmune diseases, such as collagen-induced arthritis (CIA), which rely on the use of CFA. The inflammatory lesions of these IFN-gamma knockout (KO) mice are characterized by an excessive proportion of neutrophils. Here, we show that the increased severity of CIA in IFN-gammaR KO as compared with wild-type mice is accompanied by increased levels of the CXC chemokine granulocyte chemotactic protein-2 (GCP-2), a major neutrophil-attracting chemokine in mice. We demonstrated that the heat-killed mycobacteria present in CFA elicited production of GCP-2 in mouse embryo fibroblast cultures and that this production was inhibited by IFN-gamma. Inhibition of GCP-2 production by IFN-gamma was STAT-1-dependent. IFN-gamma receptor KO mice treated with neutralizing anti-GCP-2 antibodies were protected from CIA, indicating the in vivo importance of GCP-2 in the pathogenesis of CIA. Our data support the notion that one of the mechanisms whereby endogenous IFN-gamma mitigates the manifestations of CIA consists of inhibiting production of GCP-2, thereby limiting mobilization and infiltration of neutrophils, which are important actors in joint inflammation. These results may also be applicable to other experimental models of autoimmunity that rely on the use of CFA.

Interferon: the pathways of discovery I. Molecular and cellular aspects.

This historical account covers 50 years of seminal research work on interferon done since its discovery in 1957. Topics related to molecular structure, production and action of interferons are considered from the viewpoint of how our insights have expanded and deepened within the context of evolving tools and general knowledge in cellular and molecular biology. Lines of thought that linked each discovery to the next are expounded.

Anti-inflammatory properties of Type I interferons.

The notion that Type I interferons (interferon-alpha and -beta) possess anti-inflammatory potential is supported by data from clinical application in multiple sclerosis, by studies on cultured immune-competent cells and by investigation of experimental diseases in whole animals. These observations deserve the attention of virologists for their potential role in the pathogenesis and clinical management of virus infections.

Pro-inflammatory properties of stromal cell-derived factor-1 (CXCL12) in collagen-induced arthritis.

CXCL12 (stromal cell-derived factor 1) is a unique biological ligand for the chemokine receptor CXCR4. We previously reported that treatment with a specific CXCR4 antagonist, AMD3100, exerts a beneficial effect on the development of collagen-induced arthritis (CIA) in the highly susceptible IFN-gamma receptor-deficient (IFN-gammaR KO) mouse. We concluded that CXCL12 plays a central role in the pathogenesis of CIA in IFN-gammaR KO mice by promoting delayed type hypersensitivity against the auto-antigen and by interfering with chemotaxis of CXCR4+ cells to the inflamed joints. Here, we investigated whether AMD3100 can likewise inhibit CIA in wild-type mice and analysed the underlying mechanism. Parenteral treatment with the drug at the time of onset of arthritis reduced disease incidence and modestly inhibited severity in affected mice. This beneficial effect was associated with reduced serum concentrations of IL-6. AMD3100 did not affect anti-collagen type II antibodies and, in contrast with its action in IFN-gammaR KO mice, did not inhibit the delayed type hypersensitivity response against collagen type II, suggesting that the beneficial effect cannot be explained by inhibition of humoral or cellular autoimmune responses. AMD3100 inhibited the in vitro chemotactic effect of CXCL12 on splenocytes, as well as in vivo leukocyte infiltration in CXCL12-containing subcutaneous air pouches. We also demonstrate that, in addition to its effect on cell infiltration, CXCL12 potentiates receptor activator of NF-kappaB ligand-induced osteoclast differentiation from splenocytes and increases the calcium phosphate-resorbing capacity of these osteoclasts, both processes being potently counteracted by AMD3100. Our observations indicate that CXCL12 acts as a pro-inflammatory factor in the pathogenesis of autoimmune arthritis by attracting inflammatory cells to joints and by stimulating the differentiation and activation of osteoclasts.

Defective CD4+CD25+ regulatory T cell functioning in collagen-induced arthritis: an important factor in pathogenesis, counter-regulated by endogenous IFN-gamma.

Mice with a deficiency in IFN-gamma or IFN-gamma receptor (IFN-gammaR) are more susceptible to collagen-induced arthritis (CIA), an experimental autoimmune disease that relies on the use of complete Freund's adjuvant (CFA). Here we report that the heightened susceptibility of IFN-gammaR knock-out (KO) mice is associated with a functional impairment of CD4+CD25+ Treg cells. Treatment of wild-type mice with depleting anti-CD25 antibody after CFA-assisted immunisation with collagen type II (CII) significantly accelerated the onset of arthritis and increased the severity of CIA. This is an indication of a role of Treg cells in the effector phase of CIA. IFN-gammaR deficiency did not affect the number of CD4+CD25+ T cells in the central and peripheral lymphoid tissues. In addition, CD4+CD25+ T cells isolated from naive IFN-gammaR KO mice had a normal potential to suppress T cell proliferation in vitro. However, after immunisation with CII in CFA, the suppressive activity of CD4+CD25+ T cells became significantly more impaired in IFN-gammaR-deficient mice. Moreover, expression of the mRNA for Foxp3, a highly specific marker for Treg cells, was lower. We further demonstrated that the effect of endogenous IFN-gamma, which accounts for more suppressive activity in wild-type mice, concerns both Treg cells and accessory cells. Our results demonstrate that the decrease in Treg cell activity in CIA is counter-regulated by endogenous IFN-gamma.

Biologic role of interferon beta in multiple sclerosis.

The mode of action of interferon-beta (IFNbeta) as a therapeutic agent for multiple sclerosis is still insufficiently elucidated. Of the known immunomodulatory effects of IFNbeta, those that seem important are its [1] effects on the production of cytokines by T helper lymphocytes, [2] antagonistic effect against some of the proinflammatory and immunoregulatory effects of IFNgamma, [3] inhibitory effect on resistance of T lymphocytes to apoptotic signals, [4] inhibitory effect on the production of matrix metalloproteases, and [5] modulatory effect on the expression of adhesion molecules and chemokines. The relative importance of each of these actions is presently unknown, and still other actions of IFNbeta may be at play. The possibility that therapeutic IFNbeta compensates for a lack of endogenous IFNbeta production is discussed.

Enhanced osteoclast development in collagen-induced arthritis in interferon-gamma receptor knock-out mice as related to increased splenic CD11b+ myelopoiesis.

Collagen-induced arthritis (CIA) in mice is accompanied by splenomegaly due to the selective expansion of immature CD11b+ myeloblasts. Both disease manifestations are more pronounced in interferon-gamma receptor knock-out (IFN-gammaR KO) mice. We have taken advantage of this difference to test the hypothesis that the expanding CD11b+ splenic cell population constitutes a source from which osteoclast precursors are recruited to the joint synovia. We found larger numbers of osteoclasts and more severe bone destruction in joints of IFN-gammaR KO mice than in joints of wild-type mice. Osteoclast-like multinucleated cells appeared in splenocyte cultures established in the presence of macrophage colony-stimulating factor (M-CSF) and stimulated with the osteoclast-differentiating factor receptor activator of NF-kappaB ligand (RANKL) or with tumour necrosis factor-alpha (TNF-alpha). Significantly larger numbers of such cells could be generated from splenocytes of IFN-gammaR KO mice than from those of wild-type mice. This was not accompanied, as might have been expected, by increased concentrations of the intracellular adaptor protein TRAF6, known to be involved in signalling of RANKL- and TNF-alpha-induced osteoclast formation. Splenocyte cultures of IFN-gammaR KO mice also produced more TNF-alpha and more RANKL than those of wild-type mice. Finally, splenocytes isolated from immunised IFN-gammaR KO mice contained comparatively low levels of pro-interleukin-1beta (pro-IL-1beta) and pro-caspase-1, indicating more extensive conversion of pro-IL-1beta into secreted active IL-1beta. These observations provide evidence that all conditions are fulfilled for the expanding CD11b+ splenocytes to act as a source of osteoclasts and to be indirectly responsible for bone destruction in CIA. They also provide a plausible explanation for the higher susceptibility of IFN-gammaR KO mice to CIA.

Dependence on interferon-gamma for the spontaneous occurrence of arthritis in DBA/1 mice.

OBJECTIVE: Male DBA/1 mice are known to spontaneously develop arthritis in the hind legs. The present study was undertaken to investigate the role of endogenous interferon-gamma (IFNgamma) in the pathogenesis of this ankylosing enthesopathy. METHODS: The role of IFNgamma was studied by examining the development of arthritis in IFNgamma receptor-knockout (IFNgammaR-KO) DBA/1 mice as compared with wild-type mice, and by treatment of wild-type mice with monoclonal anti-IFNgamma antibody. IFNgamma-disrupted and wild-type mice were mixed and housed in the same cage, and clinical symptoms of arthritis were assessed weekly for at least 9 weeks. Histologic examination was performed at the end of the experiment. RESULTS: In DBA/1 wild-type mice, 70% of the animals developed clinical symptoms of spontaneous arthritis, such as redness and swelling of the proximal interphalangeal joints, toe stiffness, and ankylosis. As evident from microscopic evaluation, the arthritis was mainly characterized by formation of new cartilage and bone, originating at the entheses and leading to ankylosis. The incidence and severity of arthritis, both clinically and histologically, were significantly reduced in IFNgammaR-KO mice. In wild-type mice, neutralizing anti-IFNgamma antibody inhibited the occurrence of the disease for the duration of treatment. CONCLUSION: The results suggest that endogenous IFNgamma plays an important role in the initial stages of spontaneous arthritis, and that the inflammatory components in its pathogenesis are more prominent than has been believed. In view of the similarity between this disease and spondylarthropathies in humans, the data suggest that endogenous IFNgamma may also play a disease-promoting role in the human condition and thus may serve as a target for therapy.

New candidate loci for multiple sclerosis susceptibility revealed by a whole genome association screen in a Belgian population.

We have completed a whole genome screen for association with multiple sclerosis (MS) in a Belgian population. The 6000 microsatellite markers provided through the Genetic Association of Multiple Sclerosis in EuropeanS (GAMES) collaborative were genotyped in case-control and family-based samples. The 20 most promising markers included three markers (D6S1615, D6S2444 and TNFa) from the classically established HLA class II cluster and one (D6S265) from the recently re-emphasized HLA class I cluster. In other highlighted regions, preliminary candidate genes from the immune system have been identified: e.g. the integrin ligand EDIL3, the high-mobility group box protein TOX, neutral sphingomyelinase activating factor (NSMAF) and the B-cell specific transcription factor POU2AF1.

The conserved helix C region in the superfamily of interferon-gamma /interleukin-10-related cytokines corresponds to a high-affinity binding site for the HSP70 chaperone DnaK.

HSP70 chaperones mediate protein folding by ATP-dependent interaction with short linear peptide segments that are exposed on unfolded proteins. The mode of action of the Escherichia coli homolog DnaK is representative of all HSP70 chaperones, including the endoplasmic reticulum variant BiP/GRP78. DnaK has been shown to be effective in assisting refolding of a wide variety of prokaryotic and eukaryotic proteins, including the alpha-helical homodimeric secretory cytokine interferon-gamma (IFN-gamma). We screened solid-phase peptide libraries from human and mouse IFN-gamma to identify DnaK-binding sites. Conserved DnaK-binding sites were identified in the N-terminal half of helix B and in the C-terminal half of helix C, both of which are located at the IFN-gamma dimer interface. Soluble peptides derived from helices B and C bound DnaK with high affinity in competition assays. No DnaK-binding sites were found in the loops connecting the alpha-helices. The helix C DnaK-binding site appears to be conserved in most members of the superfamily of interleukin (IL)-10-related cytokines that comprises, apart from IL-10 and IFN-gamma, a series of recently discovered small secretory proteins, including IL-19, IL-20, IL-22/IL-TIF, IL-24/MDA-7 (melanoma differentiation-associated gene), IL-26/AK155, and a number of viral IL-10 homologs. These cytokines belong to a relatively small group of homodimeric proteins with highly interdigitated interfaces that exhibit the strongly hydrophobic character of the interior core of a single-chain folded domain. We propose that binding of DnaK to helix C in the superfamily of IL-10-related cytokines may constitute the hallmark of a novel conserved regulatory mechanism in which HSP70-like chaperones assist in the formation of a hydrophobic dimeric "folding" interface.

Immunomodulatory properties of interferon-gamma. An update.

During the early aspecific phase of host defense, production of interferon (IFN)-gamma by natural killer cells plays an important role in bringing about acute inflammation, mainly because of the activating effects of IFN-gamma on adhesive properties of endothelial cells and on mediator production by mononuclear phagocytes (MPCs). In the subsequent antigen-specific phase of the immune response, IFN-gamma acts as a regulator of antigen presentation and of proliferation and differentiation of lymphocyte populations. Immunosuppressive as well as immunostimulatory effects may result from these actions. High-level production of IFN-gamma during this phase of host defense is now classically seen as a hallmark of a T-helper 1 (TH1)-type reaction, characterized by activation of antimicrobial activity of macrophages and by inflammatory reactions with a DTH character. Development of TH1-type lymphocyte populations producing IFN-gamma is regulated by other cytokines including interleukin (IL)-12. In many systems IL-12 and IFN-gamma act in a similar fashion, and a current subject of debate is the question of whether all activities of IL-12 are mediated by IFN-gamma. Another question is whether IFN-gamma, by its ability to potentiate MPCs' ability to produce IL-12, plays a role in bringing about or stabilizing TH1 type responses. In two model systems of autoimmune disease, experimental autoimmune encephalomyelitis and collagen-induced arthritis, IL-12 and IFN-gamma were found to act independently.