Search

Supplemental material, sj-docx-2-otr-10.1177_22104917211056951 for Better immediate and early postoperative outcomes of unicompartmental knee replacement comparing with total knee replacement: A matched cohort of patients with medial knee osteoarthritis by Hiu-Kiu Yu, Bruce Yan-Ho Tang, Hok-Leung Wong, Sumire Sasaki and Tai-Fong Wong in Journal of Orthopaedics, Trauma and Rehabilitation

Supplemental material, sj-pdf-1-otr-10.1177_22104917211056951 for Better immediate and early postoperative outcomes of unicompartmental knee replacement comparing with total knee replacement: A matched cohort of patients with medial knee osteoarthritis by Hiu-Kiu Yu, Bruce Yan-Ho Tang, Hok-Leung Wong, Sumire Sasaki and Tai-Fong Wong in Journal of Orthopaedics, Trauma and Rehabilitation

Since its discovery two decades ago, the activation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway by numerous cytokines and growth factors has resulted in it becoming one of the most well-studied intracellular signalling networks. The field has progressed from the identification of the individual components to high-resolution crystal structures of both JAK and STAT, and an understanding of the complexities of the molecular activation and deactivation cycle which results in a diverse, yet highly specific and regulated pattern of transcriptional responses. While there is still more to learn, we now appreciate how disruption and deregulation of this pathway can result in clinical disease and look forward to adoption of the next generation of JAK inhibitors in routine clinical treatment.

The lack of expression of the suppressor of cytokine signalling-3 (SOCS3) or inactivation of the negative regulatory capacity of SOCS3 has been well documented in rheumatoid arthritis, viral hepatitis and cancer. The specific qualitative and quantitative consequences of SOCS3 deficiency on interleukin-6 (IL-6)-mediated pro- and anti-inflammatory responses remain controversial in vitro and unknown in vivo. Mice with a conditional deletion of SOCS3 in hematopoietic cells develop lethal inflammatory disease during adult life and develop gross histopathological changes during experimental arthritis, typified by elevated IL-6 levels. To clarify the nature of the IL-6 responses in vivo, we generated mice deficient in SOCS3 (SOCS3(-/Δvav)) or both SOCS3 and IL-6 (IL-6(-/-)/SOCS3(-/Δvav)), and examined responses in models of acute and chronic inflammation. Acute responses to IL-1β were lethal to SOCS3(-/Δvav) mice but not IL-6(-/-)/SOCS3(-/Δvav) mice, indicating that IL-6 was required for the lethal inflammation induced by IL-1β. Administration of IL-1β to SOCS3(-/Δvav) mice induced systemic apoptosis of lymphocytes in the thymus, spleen and lymph nodes that was dependent on the presence of IL-6. IL-6 deficiency prolonged survival of SOCS3(-/Δvav) mice and ameliorated spontaneous inflammatory disease developing during adult life. Infection of SOCS3(-/Δvav) mice with LCMV induced a lethal inflammatory response that was dependent on IL-6, despite SOCS3(-/Δvav) mice controlling viral replication. We conclude that SOCS3 is required for survival during inflammatory responses and is a critical regulator of IL-6 in vivo.

Suppressor of cytokine signalling (SOCS) 3 is an essential regulator of cytokine signalling, and in turn its expression is tightly regulated. Data from overexpression studies in cell lines suggest that SOCS2 regulates SOCS3 protein degradation, by forming a molecular bridge to an E3 ubiquitin-ligase complex. Whether this regulation is relevant in primary cells is unknown. In this study, we utilized Socs2( - / - ) mice to examine the role of SOCS2 in modulating SOCS3 expression and degradation, and its impact on interleukin-2 (IL-2) and IL-6 signalling in primary haemopoietic cells. Both biochemical and biological analyses demonstrated unperturbed SOCS3 expression and cytokine signalling in the absence of SOCS2. Our results suggest that SOCS2 is not a physiological regulator of SOCS3 expression and action in primary haemopoietic cells.

SOCS1 profoundly influences the development and peripheral homeostasis of CD8+ T cells but has less impact on CD4+ T cells. Despite the moderate influence of SOCS1 in the development of the total CD4 T-cell lineage, we show here that SOCS1 deficiency resulted in a 10-fold increase in Foxp3(+) CD4(+) T cells in the thymus. Increased numbers of Foxp3+ thymocytes occurred in mice with T-cell-specific ablation of SOCS1, suggesting that the effect is T-cell intrinsic. This increase in Foxp3+ CD4+cells in SOCS1-deficient mice also occurred in the absence of IFN-gamma or/and IL-7 signaling. Increase in CD25+CD4+ T cells in the absence of SOCS1 could be partly due to enhanced survival by CD25+CD4+cells, to a lesser degree CD25-CD4+ T cells, from SOCS1-deficient mice with or without T-cell growth factors.

Granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6) play key roles in regulating emergency granulopoiesis and inflammation, and are both negatively regulated by the inducible intracellular protein suppressor of cytokine signaling-3 (Socs3). Mice with Socs3 deleted specifically in hematopoietic cells succumb to severe neutrophil and macrophage-driven inflammation by 1 year of age, and responses to G-CSF are grossly exacerbated. In order to determine which elements of cellular responses to cytokines require Socs3, we have examined the differentiative and proliferative capacity of hematopoietic progenitor cells stimulated by G-CSF and IL-6. The differentiation of Socs3-deficient progenitor cells is skewed toward macrophage production in response to G-CSF or IL-6, whereas wild-type progenitor cells produce mainly neutrophils. The proliferative capacity of Socs3-deficient progenitor cells is greatly enhanced in response to G-CSF at all concentrations, but only at low concentrations for IL-6. Strikingly, synergistic responses to costimulation with stem cell factor and IL-6 (but not G-CSF) are lost at higher concentrations in Socs3-deficient progenitor cells. Cytokine-induced expression of transcriptional regulators including cebpb, Ets2, Bcl3, c-Myc, Jun, and Fosl2 are differentially regulated in Socs3-deficient cells. The tight regulation by Socs3 of signal transducer and activator of transcription 3 phosphorylation and gene transcription after cytokine receptor ligation significantly influences the fate of myeloid progenitor cells.

The Microbiota Makes for Good Therapy The gut microbiota has been implicated in the development of some cancers, such as colorectal cancer, but—given the important role our intestinal habitants play in metabolism—they may also modulate the efficacy of certain cancer therapeutics. Iida et al. (p. 967 ) evaluated the impact of the microbiota on the efficacy of an immunotherapy [CpG (the cytosine, guanosine, phosphodiester link) oligonucleotides] and oxaliplatin, a platinum compound used as a chemotherapeutic. Both therapies were reduced in efficacy in tumor-bearing mice that lacked microbiota, with the microbiota important for activating the innate immune response against the tumors. Viaud et al. (p. 971 ) found a similar effect of the microbiota on tumor-bearing mice treated with cyclophosphamide, but in this case it appeared that the microbiota promoted an adaptive immune response against the tumors.

The Suppressors of Cytokine Signalling (SOCS) proteins are negative regulators of cytokine signalling required to prevent excess cellular responses. SOCS1 and SOCS3 are essential to prevent inflammatory disease, SOCS1 by attenuating responses to IFNγ and gamma-common (γc) cytokines, and SOCS3 via regulation of G-CSF and IL-6 signalling. SOCS1 and SOCS3 show significant sequence homology and are the only SOCS proteins to possess a KIR domain. The possibility of overlapping or redundant functions was investigated in inflammatory disease via generation of mice lacking both SOCS1 and SOCS3 in hematopoietic cells. Loss of SOCS3 significantly accelerated the pathology and inflammatory disease characteristic of SOCS1 deficiency. We propose a model in which SOCS1 and SOCS3 operate independently to control specific cytokine responses and together modulate the proliferation and activation of lymphoid and myeloid cells to prevent rapid inflammatory disease.

The suppressor of cytokine signalling (SOCS) proteins were, as their name suggests, first described as inhibitors of cytokine signalling. While their actions clearly now extend to other intracellular pathways, they remain key negative regulators of cytokine and growth factor signalling. In this review we focus on the mechanics of SOCS action and the complexities of the mouse models that have underpinned our current understanding of SOCS biology.

Negative regulation of cytokine signaling is critical for the generation of the appropriate cellular outcome in response to signals, and can be modulated by other concomitant extracellular stimuli ("crosstalk"). Using both genetic and pharmacological manipulations we have investigated the mechanisms by which the pro-inflammatory stimuli, lipopolysaccharide (LPS) and Tumor necrosis factor alpha (TNFalpha), negatively regulate interleukin-6 (IL-6) signaling in primary mouse macrophages. Analysis of suppressor of cytokine signalling 3 (SOCS3)-deficient macrophages reveal that SOCS3 is necessary but surprisingly, not sufficient for the complete crosstalk inhibition of IL-6 signaling induced by LPS and TNFalpha. Analysis of macrophages from gp130 (Y757F) mutant mice suggest that SH2 domain-containing tyrosine phosphatase (SHP2) activity does not explain the residual inhibitory effect of these pro-inflammatory stimuli. In addition, p38 mitogen-activated protein kinase (p38) activation also negatively regulates IL-6 signaling independent of its parallel and necessary action to induce SOCS3 expression. Finally, we have identified an additional, novel mechanism of crosstalk inhibition: a reduction in total cellular levels of gp130 following stimulation with LPS and TNFalpha.

RA is an autoimmune disease characterized by sustained imbalance between pro- and antiinflammatory immune mechanisms. The SOCS proteins are negative regulators of cytokine signaling, but to date there has been little information on their function in disease. The generation of Socs3(-/Delta vav) mice, which lack SOCS-3 in the hematopoietic and endothelial cell compartment, allowed us to explore the role of endogenous SOCS-3 during acute inflammatory arthritis. Joint inflammation in Socs3(-/Delta vav) mice was particularly severe and was characterized by increased numbers of neutrophils in the inflamed synovium, bone marrow, peripheral blood, and spleen. These features were most likely due to increased production of and enhanced responsiveness to G-CSF and IL-6 during arthritis in these mice. Local osteoclast generation and bone destruction were also dramatically increased in the absence of SOCS-3, as was macrophage activation. Finally, SOCS-3 was found to negatively regulate CD4+ T lymphocyte activation, including production of the pleiotropic cytokine IL-17. The absence of SOCS-3 therefore had dramatic effects in this disease model, with a broader impact on cellular responses than SOCS-1 deficiency. These findings provide direct in vivo evidence that endogenous SOCS-3 is a critical negative regulator of multiple cell types orchestrating inflammatory joint disease.

The available evidence suggests that protective immunity to Leishmania is achieved by priming the CD4(+) Th1 response. Therefore, we utilised a reverse genetics strategy to generate influenza A viruses to deliver an immunogenic Leishmania peptide. The single, immunodominant Leishmania-specific LACK(158-173) CD4(+) peptide was engineered into the neuraminidase stalk of H1N1 and H3N2 influenza A viruses. These recombinant viruses were used to vaccinate susceptible BALB/c mice to determine whether the resultant LACK(158-173)-specific CD4(+) T cell responses protected against live L. major infection. We show that vaccination with influenza-LACK(158-173) triggers LACK(158-173)-specific Th1-biased CD4(+) T cell responses within an appropriate cytokine milieu (IFN-γ, IL-12), essential for the magnitude and quality of the Th1 response. A single intraperitoneal exposure (non-replicative route of immunisation) to recombinant influenza delivers immunogenic peptides, leading to a marked reduction (2-4 log) in parasite burden, albeit without reduction in lesion size. This correlated with increased numbers of IFN-γ-producing CD4(+) T cells in vaccinated mice compared to controls. Importantly, the subsequent prime-boost approach with a serologically distinct strain of influenza (H1N1->H3N2) expressing LACK(158-173) led to a marked reduction in both lesion size and parasite burdens in vaccination trials. This protection correlated with high levels of IFN-γ producing cells in the spleen, which were maintained for 6 weeks post-challenge indicating the longevity of this protective effector response. Thus, these experiments show that Leishmania-derived peptides delivered in the context of recombinant influenza viruses are immunogenic in vivo, and warrant investigation of similar vaccine strategies to generate parasite-specific immunity.

Studies using primary cells from mice with germline or tissue-specific deletion of SOCS3 indicate that SOCS3 is a negative regulator of IL-6, LIF, Leptin and G-CSF-induced cellular responses. Mice with selective deletion of SOCS3 gene in blood and endothelial cells develop a fatal inflammatory disease characterized by tissue infiltration with neutrophils and macrophages, and display hyper-responsiveness to G-CSF in vitro and in vivo (Immunity2004; 20:153–65). Previous structure-function studies in cell lines have suggested that SOCS3 negatively regulates cytokine signalling by targeting the activated receptor for ubiquination and proteasomal degradation. An alternative model proposes that SOCS3 selectively inactivates phosphorylated STAT3, or targets phosphorylated STAT3 for ubiquination and proteasomal degradation. To test in primary cells which model best fits physiology, we investigated signal transduction and cellular responses of SOCS3-deficient and wild-type lin- kit+ progenitor cells after stimulation with G-CSF, IL-6 and other haemopoietic cytokines. Activation of the STAT3, STAT5 and MAP kinase pathways was monitored by immunoblotting with phospho-specific antibodies, and quantified by flow cytometry after stimulating cells for between 5 min and 4 hours. Proliferation and differentiation were quantified in liquid and agar cultures stimulated with SCF, IL-3, GM-CSF, G-CSF, IL-6 and IL-11 alone, or in combination with SCF for 4 or 7 days. No differences in signalling, proliferation or differentiation between SOCS3-deficient and wild-type cells were observed after stimulation with SCF, IL-3, GM-CSF or combinations of these. However, marked differences were observed for G-CSF and IL-6, with increased cellular output and a skewing of differentiation away from purely neutrophil to a mixed neutrophil and macrophage output. Both cytokines induced prolonged phosphorylation of STAT3 in the absence of SOCS3 (consistent with previous observations in macrophages and neutrophils), but no perturbations in MAP Kinase activation. STAT5 phosphorylation was not detectable after stimulation with G-CSF or IL-6 in either genotype, but was robust and equal between genotypes after stimulation with IL-3. Stimulation with either G-CSF or IL-6 for 4 hours resulted in consistent differences between genotypes in transcriptional profiles, and increased transcription of CEBPβ was identified as a candidate regulator of the observed altered differentiation. Most intriguingly, in the absence of SOCS3, progenitor cells failed to show any synergy (or even additive effect) in proliferation when cultured in SCF plus IL-6, and the skewing of differentiation towards macrophages was magnified. As for individual cytokines, no differences in signalling pathway activation other than STAT3 were observed when progenitors were incubated with combinations of SCF and IL-6 or G-CSF. We conclude that SOCS3 primarily regulates STAT3 activation downstream of receptor activation by G-CSF or IL-6, rather than inhibiting all signal transduction from the activated receptors. These observations argue that further structure-function studies are required to define the relative importance of ubiquination and kinase inhibition as physiological mechanisms for SOCS3’s suppressor activities. They also provide new insight to the molecular regulation of synergy between SCF and IL-6.