Ross C. Gruber

Induction of IL-33 expression and activity in central nervous system glia.

IL‐33 is a novel member of the IL‐1 cytokine family and a potent inducer of type 2 immunity, as mast cells and Th2 CD4+ T cells respond to IL‐33 with the induction of type 2 cytokines such as IL‐13. IL‐33 mRNA levels are extremely high in the CNS, and CNS glia possess both subunits of the IL‐33R, yet whether IL‐33 is produced by and affects CNS glia has not been studied. Here, we demonstrate that pathogen‐associated molecular patterns (PAMPs) significantly increase IL‐33 mRNA and protein expression in CNS glia. Interestingly, IL‐33 was localized to the nucleus of astrocytes. Further, CNS glial and astrocyte‐enriched cultures treated with a PAMP followed by an ATP pulse had significantly higher levels of supernatant IL‐1β and IL‐33 than cultures receiving any single treatment (PAMP or ATP). Supernatants from PAMP + ATP‐treated glia induced the secretion of IL‐6, IL‐13, and MCP‐1 from the MC/9 mast cell line in a manner similar to exogenous recombinant IL‐33. Further, IL‐33 levels and activity were increased in the brains of mice infected with the neurotropic virus Theilers murine encephalomyelitis virus. IL‐33 also had direct effects on CNS glia, as IL‐33 induced various innate immune effectors in CNS glia, and this induction was greatly amplified by IL‐33‐stimulated mast cells. In conclusion, these results implicate IL‐33‐producing astrocytes as a potentially critical regulator of innate immune responses in the CNS.

Interleukin-33 upregulation in peripheral leukocytes and CNS of multiple sclerosis patients.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS). Here we document for the first time that the cytokine IL-33 is upregulated in both the periphery and the CNS of MS patients. Plasma IL-33 was elevated in MS patients compared to normal subjects and a three-month treatment of MS patients with interferon β-1a resulted in a significant decrease of IL-33 levels. Similarly, stimulated cultured lymphocytes and macrophages from MS patients had elevated IL-33 levels compared to normal subjects. In parallel, the transcription factor NF-κB that mediates IL-33 transcription was also elevated in leukocytes of MS patients. IL-33 was elevated in normal-appearing white matter and plaque areas from MS brains and astrocytes were identified as an important source of IL-33 expression in the CNS. In summary, IL-33 levels are elevated in the periphery and CNS of MS patients, implicating IL-33 in the pathogenesis of MS.

Macrophages of multiple sclerosis patients display deficient SHP-1 expression and enhanced inflammatory phenotype.

Recent studies in mice have demonstrated that the protein tyrosine phosphatase SHP-1 is a crucial negative regulator of proinflammatory cytokine signaling, TLR signaling, and inflammatory gene expression. Furthermore, mice genetically lacking SHP-1 (me/me) display a profound susceptibility to inflammatory CNS demyelination relative to wild-type mice. In particular, SHP-1 deficiency may act predominantly in inflammatory macrophages to increase CNS demyelination as SHP-1-deficient macrophages display coexpression of inflammatory effector molecules and increased demyelinating activity in me/me mice. Recently, we reported that PBMCs of multiple sclerosis (MS) patients have a deficiency in SHP-1 expression relative to normal control subjects indicating that SHP-1 deficiency may play a similar role in MS as to that seen in mice. Therefore, it became essential to examine the specific expression and function of SHP-1 in macrophages from MS patients. Herein, we document that macrophages of MS patients have deficient SHP-1 protein and mRNA expression relative to those of normal control subjects. To examine functional consequences of the lower SHP-1, the activation of STAT6, STAT1, and NF-κB was quantified and macrophages of MS patients showed increased activation of these transcription factors. In accordance with this observation, several STAT6-, STAT1-, and NF-κB-responsive genes that mediate inflammatory demyelination were increased in macrophages of MS patients following cytokine and TLR agonist stimulation. Supporting a direct role of SHP-1 deficiency in altered macrophage function, experimental depletion of SHP-1 in normal subject macrophages resulted in an increased STAT/NF-κB activation and increased inflammatory gene expression to levels seen in macrophages of MS patients. In conclusion, macrophages of MS patients display a deficiency of SHP-1 expression, heightened activation of STAT6, STAT1, and NF-κB and a corresponding inflammatory profile that may be important in controlling macrophage-mediated demyelination in MS.

SHP-1 deficiency and increased inflammatory gene expression in PBMCs of multiple sclerosis patients

Recent studies in mice have demonstrated that the protein tyrosine phosphatase SHP-1 is a crucial negative regulator of cytokine signaling, inflammatory gene expression, and demyelination in central nervous system. The present study investigates a possible similar role for SHP-1 in the human disease multiple sclerosis (MS). The levels of SHP-1 protein and mRNA in PBMCs of MS patients were significantly lower compared to normal subjects. Moreover, promoter II transcripts, expressed from one of two known promoters, were selectively deficient in MS patients. To examine functional consequences of the lower SHP-1 in PBMCs of MS patients, we measured the intracellular levels of phosphorylated STAT6 (pSTAT6). As expected, MS patients had significantly higher levels of pSTAT6. Accordingly, siRNA to SHP-1 effectively increased the levels of pSTAT6 in PBMCs of controls to levels equal to MS patients. Additionally, transduction of PBMCs with a lentiviral vector expressing SHP-1 lowered pSTAT6 levels. Finally, multiple STAT6-responsive inflammatory genes were increased in PBMCs of MS patients relative to PBMCs of normal subjects. Thus, PBMCs of MS patients display a stable deficiency of SHP-1 expression, heightened STAT6 phosphorylation, and an enhanced state of activation relevant to the mechanisms of inflammatory demyelination.

Modulation of Macrophage Infiltration and Inflammatory Activity by the Phosphatase SHP-1 in Virus-Induced Demyelinating Disease

ABSTRACT The protein tyrosine phosphatase SHP-1 is a crucial negative regulator of cytokine signaling and inflammatory gene expression, both in the immune system and in the central nervous system (CNS). Mice genetically lacking SHP-1 (me/me) display severe inflammatory demyelinating disease following inoculation with the Theilers murine encephalomyelitis virus (TMEV) compared to infected wild-type mice. Therefore, it became essential to investigate the mechanisms of TMEV-induced inflammation in the CNS of SHP-1-deficient mice. Herein, we show that the expression of several genes relevant to inflammatory demyelination in the CNS of infected me/me mice is elevated compared to that in wild-type mice. Furthermore, SHP-1 deficiency led to an abundant and exclusive increase in the infiltration of high-level-CD45-expressing (CD45hi) CD11b+ Ly-6Chi macrophages into the CNS of me/me mice, in concert with the development of paralysis. Histological analyses of spinal cords revealed the localization of these macrophages to extensive inflammatory demyelinating lesions in infected SHP-1-deficient mice. Sorted populations of CNS-infiltrating macrophages from infected me/me mice showed increased amounts of viral RNA and an enhanced inflammatory profile compared to wild-type macrophages. Importantly, the application of clodronate liposomes effectively depleted splenic and CNS-infiltrating macrophages and significantly delayed the onset of TMEV-induced paralysis. Furthermore, macrophage depletion resulted in lower viral loads and lower levels of inflammatory gene expression and demyelination in the spinal cords of me/me mice. Finally, me/me macrophages were more responsive than wild-type macrophages to chemoattractive stimuli secreted by me/me glial cells, indicating a mechanism for the increased numbers of infiltrating macrophages seen in the CNS of me/me mice. Taken together, these findings demonstrate that infiltrating macrophages in SHP-1-deficient mice play a crucial role in promoting viral replication by providing abundant viral targets and contribute to increased proinflammatory gene expression relevant to the effector mechanisms of macrophage-mediated demyelination.

Suppression of Inflammatory Responses during Myelin Oligodendrocyte Glycoprotein–Induced Experimental Autoimmune Encephalomyelitis Is Regulated by AKT3 Signaling

AKT3, a member of the serine/threonine kinase AKT family, is involved in a variety of biologic processes. AKT3 is expressed in immune cells and is the major AKT isoform in the CNS representing 30% of the total AKT expressed in spinal cord, and 50% in the brain. Myelin-oligodendrocyte glycoprotein–induced experimental autoimmune encephalomyelitis (EAE) is a mouse model in which lymphocytes and monocytes enter the CNS, resulting in inflammation, demyelination, and axonal injury. We hypothesized that during EAE, deletion of AKT3 would negatively affect the CNS of AKT3−/− mice, making them more susceptible to CNS damage. During acute EAE, AKT3−/−mice were more severely affected than wild type (WT) mice. Evaluation of spinal cords showed that during acute and chronic disease, AKT3−/− spinal cords had more demyelination compared with WT spinal cords. Quantitative RT-PCR determined higher levels of IL-2, IL-17, and IFN-γ mRNA in spinal cords from AKT3−/− mice than WT. Experiments using bone marrow chimeras demonstrated that AKT3−/− mice receiving AKT3-deficient bone marrow cells had elevated clinical scores relative to control WT mice reconstituted with WT cells, indicating that altered function of both CNS cells and bone marrow–derived immune cells contributed to the phenotype. Immunohistochemical analysis revealed decreased numbers of Foxp3+ regulatory T cells in the spinal cord of AKT3−/− mice compared with WT mice, whereas in vitro suppression assays showed that AKT3-deficient Th cells were less susceptible to regulatory T cell–mediated suppression than their WT counterparts. These results indicate that AKT3 signaling contributes to the protection of mice against EAE.

Targeted GAS6 Delivery to the CNS Protects Axons from Damage during Experimental Autoimmune Encephalomyelitis

Growth arrest-specific protein 6 (GAS6) is a soluble agonist of the TYRO3, AXL, MERTK (TAM) family of receptor tyrosine kinases identified to have anti-inflammatory, neuroprotective, and promyelinating properties. During experimental autoimmune encephalomyelitis (EAE), wild-type (WT) mice demonstrate a significant induction of Gas6, Axl, and Mertk but not Pros1 or Tyro3 mRNA. We tested the hypothesis that intracerebroventricular delivery of GAS6 directly into the CNS of WT mice during myelin oligodendrocyte glycoprotein (MOG)-induced EAE would improve the clinical course of disease relative to artificial CSF (ACSF)-treated mice. GAS6 did not delay disease onset, but significantly reduced the clinical scores during peak and chronic EAE. Mice receiving GAS6 for 28 d had preserved SMI31+ neurofilament immunoreactivity, significantly fewer SMI32+ axonal swellings and spheroids and less demyelination relative to ACSF-treated mice. Alternate-day subcutaneous IFNβ injection did not enhance GAS6 treatment effectiveness. Gas6−/− mice sensitized with MOG35-55 peptide exhibit higher clinical scores during late peak to early chronic disease, with significantly increased SMI32+ axonal swellings and Iba1+ microglia/macrophages, enhanced expression of several proinflammatory mRNA molecules, and decreased expression of early oligodendrocyte maturation markers relative to WT mouse spinal cords with scores for 8 consecutive days. During acute EAE, flow cytometry showed significantly more macrophages but not T-cell infiltrates in Gas6−/− spinal cords than WT spinal cords. Our data are consistent with GAS6 being protective during EAE by dampening the inflammatory response, thereby preserving axonal integrity and myelination.

Promoter-specific induction of the phosphatase SHP-1 by viral infection and cytokines in CNS glia.

We have previously shown that the protein tyrosine phosphatase SHP‐1 is highly expressed in CNS glia and is an important modulator of cytokine signaling. As such, mice genetically lacking SHP‐1 display constitutive myelin abnormalities, severe virus‐induced demyelinating disease, and defects in innate anti‐viral responses in the CNS. In this study, we show the differential distribution of the SHP‐1 promoter‐specific transcripts and demonstrate that several cytokines significantly induce SHP‐1 expression in CNS glia. Consistent with these cytokine effects, infection with a neurotropic virus both in vitro and in vivo up‐regulates SHP‐1 transcripts and protein in CNS cells. Using CNS glial cultures of gene knockout mice, we show that interferons‐β and interferons‐γ act through STAT‐1 and interferon regulatory factor‐1 to induce the SHP‐1 promoter I transcripts. Conversely, interferons‐β and IL‐6 act through STAT‐3 to induce SHP‐1 promoter II transcripts. This study demonstrates that interferons and other cytokines associated with virus infections in the CNS can significantly induce the expression of SHP‐1 through STAT‐1/3 activity and provides a better understanding of the molecular mechanisms regulating cytokine‐induced expression important for multiple homeostatic functions of SHP‐1 in the CNS.

The control of reactive oxygen species production by SHP-1 in oligodendrocytes

We have previously described reduced myelination and corresponding myelin basic protein (MBP) expression in the central nervous system of Src homology 2 domain‐containing protein tyrosine phosphatase 1 (SHP‐1) deficient motheaten (me/me) mice compared with normal littermate controls. Deficiency in myelin and MBP expression in both brains and spinal cords of motheaten mice correlated with reduced MBP mRNA expression levels in vivo and in purified oligodendrocytes in vitro. Therefore, SHP‐1 activity seems to be a critical regulator of oligodendrocyte gene expression and function. Consistent with this role, this study demonstrates that oligodendrocytes of motheaten mice and SHP‐1‐depleted N20.1 cells produce higher levels of reactive oxygen species (ROS) and exhibit corresponding markers of increased oxidative stress. In agreement with these findings, we demonstrate that increased production of ROS coincides with ROS‐induced signaling pathways known to affect myelin gene expression in oligodendrocytes. Antioxidant treatment of SHP‐1‐deficient oligodendrocytes reversed the pathological changes in these cells, with increased myelin protein gene expression and decreased expression of nuclear factor (erythroid‐2)‐related factor 2 (Nrf2) responsive gene, heme oxygenase‐1 (HO‐1). Furthermore, we demonstrate that SHP‐1 is expressed in human white matter oligodendrocytes, and there is a subset of multiple sclerosis subjects that demonstrate a deficiency of SHP‐1 in normal‐appearing white matter. These studies reveal critical pathways controlled by SHP‐1 in oligodendrocytes that relate to susceptibility of SHP‐1‐deficient mice to both developmental defects in myelination and to inflammatory demyelinating diseases. GLIA 2015;63:1753–1771

Regulation of avoidant behaviors and pain by the anti-inflammatory tyrosine phosphatase SHP-1

The protein tyrosine phosphatase SHP-1 is a critical regulator of cytokine signaling and inflammation. Mice homozygous for a null allele at the SHP-1 locus have a phenotype of severe inflammation and are hyper-responsive to the TLR4 ligand LPS. TLR4 stimulation in the CNS has been linked to both neuropathic pain and sickness behaviors. To determine if reduction in SHP-1 expression affects LPS-induced behaviors, responses of heterozygous SHP-1-deficient (me/+) and wild-type (+/+) mice to LPS were measured. Chronic (4-week) treatment with LPS induced avoidant behaviors indicative of fear/anxiety in me/+, but not +/+, mice. These behaviors were correlated with a LPS-induced type 2 cytokine, cytokine receptor, and immune effector arginase profile in the brains of me/+ mice not found in +/+ mice. Me/+ mice also had a constitutively greater level of TLR4 in the CNS than +/+ mice. Additionally, me/+ mice displayed constitutively increased thermal sensitivity compared to +/+ mice, measured by the tail-flick test. Moreover, me/+ glial cultures were more responsive to LPS than +/+ glia. Therefore, the reduced expression of SHP-1 in me/+ imparts haploinsufficiency with respect to the control of CNS TLR4 and pain signaling. Furthermore, type 2 cytokines become prevalent during chronic TLR4 hyperstimulation in the CNS and are associated positively with behaviors that are usually linked to type 1 pro-inflammatory cytokines. These findings question the notion that type 2 immunity is solely anti-inflammatory in the CNS and indicate that type 2 immunity induces/potentiates CNS inflammatory processes.