G-CSF and Neutrophils Are Nonredundant Mediators of Murine Experimental Autoimmune Uveoretinitis.

Granulocyte colony-stimulating factor (G-CSF) is a regulator of neutrophil production, function, and survival. Herein, we investigated the role of G-CSF in a murine model of human uveitis-experimental autoimmune uveoretinitis. Experimental autoimmune uveoretinitis was dramatically reduced in G-CSF-deficient mice and in anti-G-CSF monoclonal antibody-treated, wild-type (WT) mice. Flow cytometric analysis of the ocular infiltrate in WT mice with experimental autoimmune uveoretinitis showed a mixed population, comprising neutrophils, macrophages, and T cells. The eyes of G-CSF-deficient and anti-G-CSF monoclonal antibody-treated WT mice had minimal neutrophil infiltrate, but no change in other myeloid-derived inflammatory cells. Antigen-specific T-cell responses were maintained, but the differentiation of pathogenic type 17 helper T cells in experimental autoimmune uveoretinitis was reduced with G-CSF deficiency. We show that G-CSF controls the ocular neutrophil infiltrate by modulating the expression of C-X-C chemokine receptors 2 and 4 on peripheral blood neutrophils, as well as actin polymerization and migration. These data reveal an integral role for G-CSF-driven neutrophil responses in ocular autoimmunity, operating within and outside of the bone marrow, and also identify G-CSF as a potential therapeutic target in the treatment of human uveoretinitis.

Regulation of interleukin-1beta by interferon-gamma is species specific, limited by suppressor of cytokine signalling 1 and influences interleukin-17 production.

Reports describing the effect of interferon-gamma (IFNgamma) on interleukin-1beta (IL-1beta) production are conflicting. We resolve this controversy by showing that IFNgamma potentiates IL-1beta release from human cells, but transiently inhibits the production of IL-1beta from mouse cells. Release from this inhibition is dependent on suppressor of cytokine signalling 1. IL-1beta and Th17 cells are pathogenic in mouse models for autoimmune disease, which use Mycobacterium tuberculosis (MTB), in which IFNgamma and IFNbeta are anti-inflammatory. We observed that these cytokines suppress IL-1beta production in response to MTB, resulting in a reduced number of IL-17-producing cells. In human cells, IFNgamma increased IL-1beta production, and this might explain why IFNgamma is detrimental for multiple sclerosis. In mice, IFNgamma decreased IL-1beta and subsequently IL-17, indicating that the adaptive immune response can provide a systemic, but transient, signal to limit inflammation.

G-CSF and GM-CSF as therapeutic targets in rheumatoid arthritis.

Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are well-recognized regulators of hematopoiesis and have an established role as growth factors in clinical practice. G-CSF and GM-CSF regulate myeloid cell production, differentiation and activation, and might also be important for driving inflammatory responses. Inappropriate engagement of this pathway could be a critical amplification mechanism when maladaptive immune responses predispose to autoimmunity and sterile tissue inflammation. We postulate that antagonism of G-CSF or GM-CSF could represent a novel therapeutic approach for a variety of autoimmune-mediated inflammatory diseases, including rheumatoid arthritis.

Suppressor of cytokine signaling 1 regulates the immune response to infection by a unique inhibition of type I interferon activity.

Suppressor of cytokine signaling 1 (SOCS1) is a critical regulator of cytokine signaling and immune responses. SOCS1-deficient mice develop severe inflammatory disease, but are very resistant to viral infections. Using neutralizing antibody to type I interferon (IFN-alpha and IFN-beta) and mice deficient in interferon-gamma or type I interferon receptor components (IFNAR1 or IFNAR2), we demonstrate here that SOCS1 deficiency amplified type I interferon antiviral and proinflammatory actions independently of interferon-gamma. The mechanism of the suppression of type I interferon responses by SOCS1 was distinct from that of other cytokines. SOCS1 associated with and regulated IFNAR1- but not IFNAR2-specific signals, abrogating tyrosine phosphorylation of transcription factor STAT1 and reducing the duration of antiviral gene expression. Thus, SOCS1 is an important in vivo inhibitor of type I interferon signaling and contributes to balancing its beneficial antiviral versus detrimental proinflammatory effects on innate immunity.

NKT cells are not critical for HSV-1 disease resolution.

NKT cells are a minor subset of T cells that have important roles in controlling immune responses in disease states including cancer, autoimmunity and pathogenic infections. In contrast to conventional T cells, NKT cells express an invariant TCR and respond to glycolipids presented by CD1d. In this study, we sought to investigate the role of NKT cells in regulating the response to infection with HSV-1, and the mechanism involved, in well-established mouse models. Previous studies of HSV-1 disease in mice have shown clear roles for CD4+ and CD8+ T cells. The role of NKT cells in the resolution of HSV-1 (KOS strain) infection was investigated through flank zosteriform or footpad infection in wild-type versus CD1d-deficient mice, by measurement of viral plaque-forming units at different sites after infection, lesion severity and HSV-1-specific T-cell responses. In contrast to a previous study using a more virulent strain of HSV-1 (SC16 strain), no differences were observed in disease magnitude or resolution, and furthermore, the T-cell response to HSV-1 (KOS strain) was unaltered in the absence of NKT cells. In conclusion, this study shows that NKT cells do not play a general role in controlling the resolution or severity of HSV-1 infection. Instead, the resolution or severity of the infection may depend on the HSV-1 strain under investigation.

SOCS-1 regulates IL-15-driven homeostatic proliferation of antigen-naive CD8 T cells, limiting their autoimmune potential.

Mice that are deficient in suppressor of cytokine signaling-1 (SOCS-1) succumb to neonatal mortality that is associated with extensive cellular infiltration of many tissues. T cells seem to be necessary for disease, which can be alleviated largely by neutralizing interferon-gamma. Examining T cell receptor (TCR) specificity shows that even monospecific T cells can mediate disease in SOCS-1-deficient mice, although disease onset is substantially faster with a polyclonal T cell repertoire. A major phenotype of SOCS-1-/- mice is the accumulation of CD44(high)CD8+ peripheral T cells. We show that SOCS-1-deficient CD8, but not CD4, T cells proliferate when transferred into normal (T cell-sufficient) mice, and that this is dependent on two signals: interleukin (IL)-15 and self-ligands that are usually only capable of stimulating homeostatic expansion in T cell-deficient mice. Our findings reveal that SOCS-1 normally down-regulates the capacity of IL-15 to drive activation and proliferation of naive CD8 T cells receiving TCR survival signals from self-ligands. We show that such dysregulated proliferation impairs the deletion of a highly autoreactive subset of CD8 T cells, and increases their potential for autoimmunity. Therefore, impaired deletion of highly autoreactive CD8 T cells, together with uncontrolled activation of naive CD8 T cells by homeostatic survival ligands, may provide a basis for the T cell-mediated disease of SOCS-1-/- mice.

Suppressor of cytokine signaling-1 regulates signaling in response to interleukin-2 and other gamma c-dependent cytokines in peripheral T cells.

Suppressor of cytokine signaling-1 (SOCS-1) is an essential regulator of cytokine signaling. SOCS-1-/- mice die before weaning with a complex disease characterized by fatty degeneration and necrosis of the liver. This disease is mediated by interferon (IFN) gamma as neonatal mortality fails to occur in SOCS-1-/-IFNgamma-/- mice. However, the immune system of healthy SOCS-1-/-IFNgamma-/- mice is dysregulated with a reduced ratio of CD4:CD8 T cells and increases in some aspects of T cell activation. SOCS-1-/-IFNgamma-/- mice also die before their wild type and IFNgamma-/- counterparts with a range of inflammatory conditions including pneumonia, gut infiltration, and skin ulceration, suggesting that SOCS-1 controls not only IFNgamma signaling, but also other immunoregulatory factors. This study shows that T cells from SOCS-1-deficient mice display hypersensitivity to cytokines that act through the gammac receptor. SOCS-1 expression is induced by interleukin (IL) 2, IL-4, IL-7, and IL-15, and SOCS-1-deficient T cells show increased proliferation and prolonged survival in response to IL-2 and IL-4. Furthermore, IL-2 induced increased STAT5 phosphorylation and CD44 expression in SOCS-1-deficient T cells compared with controls. Hypersensitivity to gammac-dependent cytokines may contribute to abnormal T cell function, as well as the pathology observed in mice lacking SOCS-1.

Suppressor of cytokine signaling-1 is a critical regulator of interleukin-7-dependent CD8+ T cell differentiation.

To determine the tissue-specific functions of SOCS-1, mice were generated in which the SOCS-1 gene could be deleted in individual tissues. A reporter gene of SOCS-1 promoter activity was also inserted. Using the reporter, high SOCS-1 expression was found at the CD4(+)CD8(+) stage in thymocyte development. To investigate the function of this expression, the SOCS-1 gene was specifically deleted throughout the thymocyte/T/NKT cell compartment. Unlike SOCS-1(-/-) mice, these mice did not develop lethal multiorgan inflammation but developed multiple lymphoid abnormalities, including enhanced differentiation of thymocytes toward CD8(+) T cells and very high percentages of peripheral CD8(+) T cells with a memory phenotype (CD44(hi)CD25(lo)CD69(lo)). These phenotypes were found to correlate with hypersensitivity to the gamma-common family of cytokines.

Suppressor of cytokine signaling-1 has IFN-gamma-independent actions in T cell homeostasis.

Suppressor of cytokine signaling (SOCS)-1 is a member of a family of proteins that negatively regulate cytokine signaling pathways. We have previously established that SOCS-1 is a key regulator of IFN-gamma signaling and that IFN-gamma is responsible for the complex inflammatory disease that leads to the death of SOCS-1-deficient mice. In this study, we provide evidence that SOCS-1 is also a critical regulator of IFN-gamma-independent immunoregulatory factors. Mice lacking both SOCS-1 and IFN-gamma, although outwardly healthy, have clear abnormalities in their immune system, including a reduced ratio of CD4:CD8 T cells in lymphoid tissues and increased expression of T cell activation markers. To examine the contribution of TCR Ag specificity to these immune defects, we have generated two lines of SOCS-1-deficient mice expressing a transgenic TCR specific for an exogenous Ag, OVA (OT-I and OT-II). Although TCR transgenic SOCS-1(-/-) mice have a longer lifespan than nontransgenic SOCS-1(-/-) mice, they still die as young adults with inflammatory disease and the TCR transgenic SOCS-1(-/-) T cells appear activated despite the absence of OVA. This suggests that both Ag-dependent and -independent mechanisms contribute to the disease in SOCS-1-deficient mice. Thus, SOCS-1 is a critical regulator of T cell activation and homeostasis, and its influence extends beyond regulating IFN-gamma signaling.