< Back to previous page
Project
Macrophage activation syndrome in IFN-gamma-deficient mice: key role of natural killer cells and role of defective IFN-gamma signaling.
Whereas robust inflammation is required in immune responses to efficiently combat exogenous or endogenous pathogenic agents, regulatory feedback is needed to control inflammation and drive appropriate subsidence. Accordingly, insufficient immune regulation may lead to persistent systemic inflammatory responses and unwarranted tissue damage. The fine-tuned balance between stimulatory and regulatory immune pathways is strongly influenced by a complex interplay of cytokines. Although some cytokines are typically considered as pro- or anti-inflammatory, evidence emerges that any given cytokine can be involved in both circuits. Interferon-gamma(IFN-γ) constitutes a perfect example of this statement. Although historically considered as a typical proinflammatory cytokine, some important regulatory functions have been ascribed to IFN-γ since its discovery.
In systemic autoinflammatory syndromes, the exquisite balance between pro- and anti-inflammatory responses tips towards progressive inflammation, due to inappropriate production of cytokines. Recentadvances in the understanding of systemic juvenile idiopathic arthritis(sJIA) have likewise underlined the role of cytokines in the disease process. sJIA is a severe autoinflammatory disorder typically encountered during childhood. A defective decline of immune reactions underlies the pathogenesis of sJIA, as proinflammatory pathways are overrepresented inthe patients. sJIA is classified as a subtype of juvenile idiopathic arthritis (JIA), but the disease differs from other subtypes by the presentation of systemic symptoms that accompany the arthritis, i.e. spiking fever, skin rash, lymphadenopathy, hepatosplenomegaly, granulocytosis andthrombocytosis. A defective natural killer (NK) cell cytotoxicity and atypical cytokine profile, with high levels of interleukin (IL)-6 and IL-18, are distinctive features of sJIA compared to other subtypes. In this dissertation, we hypothesized that the latter two features go hand in hand. To justify this point of view, we summarized the cytokine profile of sJIA patients and discussed the influence of cytokines on NK cell function, with special attention to IL-18. In addition to IL-18, IL-1β and IL-6 have proven to exert a disease-promoting role in sJIA, given the effectiveness of cytokine-restricting treatment strategies. The well-established reputation of IFN-γ as a macrophage activating factor likewise led to the consideration of the cytokine as being proinflammatory in sJIA. However, recent investigations revealed a defective IFN-γ production by NK cells of sJIA patients and an absence of IFN-γ-induced gene expression in microarrays of patients peripheral bloodmononuclear cells. Taken together, the role of IFN-γ in sJIA remained unclear and required further investigations.
In this PhD project,we intended to investigate the protective nature of endogenous IFN-γ in mice by mimicking a chronic immune activation. To that purpose, weadministered a relatively innocent immune stimulus, i.e. heat-killed mycobacteria formulated in complete Freunds adjuvant (CFA), to BALB/c micewith a disrupted IFN-γ system. Mice of the BALB/c background are exquisitely susceptible for mycobacterial triggering given their difficulties in setting up a potent T helper 1 response. The deficiency in IFN-γ further contributed to the strong and long-lasting inflammatory reaction to CFA, eventually leading to symptoms reflecting the clinical, biological and histopathological picture of sJIA. Indeed, 10 to 20 days after CFA injection, IFN-γ knock-out (KO) mice developed cachexia, lymphadenopathy, splenomegaly, neutrophilia and thrombocytosis. A subset of the mice further demonstrated arthritis, rash or anemia. Immune pathological examinations revealed an increased amount of immature blood cells, hemophagocytosis, high plasma levels of IL-6 and elevated IL-17 producing capacity in lymph node cells. Also, the IL-17-producing γδ T cells subset was demonstrated to be impressively increased and neutralization of IL-12/IL-23p40 and IL‑17 rescued mice from the majority of symptoms. Defects in NK cell cytotoxicity were shown to be owed to the IFN-γ-deficient nature of the mice. Although wild‑type (WT) mice also developed certain symptoms, the inflammation remainedmore subtle, indicating that IFN-γ is a key player in preventing the escalation of a strong inflammatory reaction to CFA. New insights into the regulatory function of IFN‑γ constitute the prevention of anemia and overall systemic inflammation in this IL-17-mediated model.
In the second and third part of this study, we attempted to elucidate the mode of action of IFN‑γ in protecting against fulminant sJIA-like symptoms. To achieve that, we tried to uncover causative agents in CFA-challenged IFN-γ KO mice, via depletion and neutralization of specific cells and cytokines respectively. Furthermore, we looked for cells and cytokines that exerted a protective function by in-depth analysis of CFA-injected WT mice. As stated above, IFN-γ KO mice were rescued from the majority of symptoms by neutralization of IL-12/IL-23p40 and IL‑17. Depletion of CD4+ cells partly improved the disease status in the mice, indicating that other cell types contributed to the harmful IL-17 production. Symptoms remained unaffected afterneutralization of IL-1, IL-6, IL-18 or granulocyte macrophage-colony stimulating factor (GM‑CSF). In WT mice, a fulminant sJIA-like syndrome was driven by IFN-γ neutralization, indicating that a temporarydefect in IFN-γ suffices to annihilate the control that WT mice have over CFA. The depletion of CD8+ T cells didnt result in sJIA-like symptoms, whereas NK cell reduction led to a sJIA-like disease in subsets of mice. The latter may result from the reduced amount of cells with a powerful cytotoxic capacity, leading to insufficient killing of activatedimmune cells. Defects in the cytotoxic machinery, peculiar to IFN‑γ KO mice, may partly explain the increased susceptibility ofthese mice to CFA. Finally, an almost complete picture of sJIA emerged when inhibiting IL-10 in WT mice, strongly suggesting that this cytokineis protective in the model. In general, the findings indicate that IL-17 and CD4+ cells have a disease-promoting role in the mouse model of sJIA, while NK cells, IFN-γ and IL‑10 impede the CFA-induced systemic inflammation.
While investigating the role of cytokines in the model, we performed a detailed analysis of cytokine-producing organs and cell types. Cytokine expression in CFA-injected IFN-γ KO confirmed the overall proinflammatory nature of the model system, since especially IL-1β, IL-6, IL-17 and IL-22 were elevated compared to non-injected mice. Among bone marrow, spleen, draining lymph nodes, liver and lungs, the lymph nodes and lungs were found to produce the highest amount of cytokines. In purified cells, an important role was granted to innate immune cells, i.e. NK cells and γδ T cells, as key players in the cytokine production of the sJIA‑like syndrome.
To conclude, CFA induces a severe systemic inflammatory disease in IFN‑γ-deficient mice, which can be explained by loss of control over an IL-17-dominated innate and adaptive immune inflammatory response. Defects in NK cell function and in regulatory pathways, e.g. sufficient IL-10 production, may account for the symptoms. We suggest that IFN-γ has evolved the function to provide non‑pathogenic inflammation with the means to stimulate effective NK cell cytotoxicity and to mitigate the severe consequences of uncontrolled IL‑17 stimulation. The inflammatory condition seen in the IFN-γ KO mice ishighly reminiscent of clinical and immunological features seen in sJIA,and may therefore serve as a model for this perplexing childhood disease. Even though murine models are a reflection of the human situation, the need to understand sJIA pathology and the great challenge to correctlydiagnose patients explain the necessity of translational research.
In systemic autoinflammatory syndromes, the exquisite balance between pro- and anti-inflammatory responses tips towards progressive inflammation, due to inappropriate production of cytokines. Recentadvances in the understanding of systemic juvenile idiopathic arthritis(sJIA) have likewise underlined the role of cytokines in the disease process. sJIA is a severe autoinflammatory disorder typically encountered during childhood. A defective decline of immune reactions underlies the pathogenesis of sJIA, as proinflammatory pathways are overrepresented inthe patients. sJIA is classified as a subtype of juvenile idiopathic arthritis (JIA), but the disease differs from other subtypes by the presentation of systemic symptoms that accompany the arthritis, i.e. spiking fever, skin rash, lymphadenopathy, hepatosplenomegaly, granulocytosis andthrombocytosis. A defective natural killer (NK) cell cytotoxicity and atypical cytokine profile, with high levels of interleukin (IL)-6 and IL-18, are distinctive features of sJIA compared to other subtypes. In this dissertation, we hypothesized that the latter two features go hand in hand. To justify this point of view, we summarized the cytokine profile of sJIA patients and discussed the influence of cytokines on NK cell function, with special attention to IL-18. In addition to IL-18, IL-1β and IL-6 have proven to exert a disease-promoting role in sJIA, given the effectiveness of cytokine-restricting treatment strategies. The well-established reputation of IFN-γ as a macrophage activating factor likewise led to the consideration of the cytokine as being proinflammatory in sJIA. However, recent investigations revealed a defective IFN-γ production by NK cells of sJIA patients and an absence of IFN-γ-induced gene expression in microarrays of patients peripheral bloodmononuclear cells. Taken together, the role of IFN-γ in sJIA remained unclear and required further investigations.
In this PhD project,we intended to investigate the protective nature of endogenous IFN-γ in mice by mimicking a chronic immune activation. To that purpose, weadministered a relatively innocent immune stimulus, i.e. heat-killed mycobacteria formulated in complete Freunds adjuvant (CFA), to BALB/c micewith a disrupted IFN-γ system. Mice of the BALB/c background are exquisitely susceptible for mycobacterial triggering given their difficulties in setting up a potent T helper 1 response. The deficiency in IFN-γ further contributed to the strong and long-lasting inflammatory reaction to CFA, eventually leading to symptoms reflecting the clinical, biological and histopathological picture of sJIA. Indeed, 10 to 20 days after CFA injection, IFN-γ knock-out (KO) mice developed cachexia, lymphadenopathy, splenomegaly, neutrophilia and thrombocytosis. A subset of the mice further demonstrated arthritis, rash or anemia. Immune pathological examinations revealed an increased amount of immature blood cells, hemophagocytosis, high plasma levels of IL-6 and elevated IL-17 producing capacity in lymph node cells. Also, the IL-17-producing γδ T cells subset was demonstrated to be impressively increased and neutralization of IL-12/IL-23p40 and IL‑17 rescued mice from the majority of symptoms. Defects in NK cell cytotoxicity were shown to be owed to the IFN-γ-deficient nature of the mice. Although wild‑type (WT) mice also developed certain symptoms, the inflammation remainedmore subtle, indicating that IFN-γ is a key player in preventing the escalation of a strong inflammatory reaction to CFA. New insights into the regulatory function of IFN‑γ constitute the prevention of anemia and overall systemic inflammation in this IL-17-mediated model.
In the second and third part of this study, we attempted to elucidate the mode of action of IFN‑γ in protecting against fulminant sJIA-like symptoms. To achieve that, we tried to uncover causative agents in CFA-challenged IFN-γ KO mice, via depletion and neutralization of specific cells and cytokines respectively. Furthermore, we looked for cells and cytokines that exerted a protective function by in-depth analysis of CFA-injected WT mice. As stated above, IFN-γ KO mice were rescued from the majority of symptoms by neutralization of IL-12/IL-23p40 and IL‑17. Depletion of CD4+ cells partly improved the disease status in the mice, indicating that other cell types contributed to the harmful IL-17 production. Symptoms remained unaffected afterneutralization of IL-1, IL-6, IL-18 or granulocyte macrophage-colony stimulating factor (GM‑CSF). In WT mice, a fulminant sJIA-like syndrome was driven by IFN-γ neutralization, indicating that a temporarydefect in IFN-γ suffices to annihilate the control that WT mice have over CFA. The depletion of CD8+ T cells didnt result in sJIA-like symptoms, whereas NK cell reduction led to a sJIA-like disease in subsets of mice. The latter may result from the reduced amount of cells with a powerful cytotoxic capacity, leading to insufficient killing of activatedimmune cells. Defects in the cytotoxic machinery, peculiar to IFN‑γ KO mice, may partly explain the increased susceptibility ofthese mice to CFA. Finally, an almost complete picture of sJIA emerged when inhibiting IL-10 in WT mice, strongly suggesting that this cytokineis protective in the model. In general, the findings indicate that IL-17 and CD4+ cells have a disease-promoting role in the mouse model of sJIA, while NK cells, IFN-γ and IL‑10 impede the CFA-induced systemic inflammation.
While investigating the role of cytokines in the model, we performed a detailed analysis of cytokine-producing organs and cell types. Cytokine expression in CFA-injected IFN-γ KO confirmed the overall proinflammatory nature of the model system, since especially IL-1β, IL-6, IL-17 and IL-22 were elevated compared to non-injected mice. Among bone marrow, spleen, draining lymph nodes, liver and lungs, the lymph nodes and lungs were found to produce the highest amount of cytokines. In purified cells, an important role was granted to innate immune cells, i.e. NK cells and γδ T cells, as key players in the cytokine production of the sJIA‑like syndrome.
To conclude, CFA induces a severe systemic inflammatory disease in IFN‑γ-deficient mice, which can be explained by loss of control over an IL-17-dominated innate and adaptive immune inflammatory response. Defects in NK cell function and in regulatory pathways, e.g. sufficient IL-10 production, may account for the symptoms. We suggest that IFN-γ has evolved the function to provide non‑pathogenic inflammation with the means to stimulate effective NK cell cytotoxicity and to mitigate the severe consequences of uncontrolled IL‑17 stimulation. The inflammatory condition seen in the IFN-γ KO mice ishighly reminiscent of clinical and immunological features seen in sJIA,and may therefore serve as a model for this perplexing childhood disease. Even though murine models are a reflection of the human situation, the need to understand sJIA pathology and the great challenge to correctlydiagnose patients explain the necessity of translational research.
Date:1 Oct 2010 → 30 Sep 2014
Keywords:Myeloid cells, Autoimmunity, Animal models, Cytokines, Immunology
Disciplines:Immunology
Project type:PhD project