Chad A. Hudson

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.

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.

Susceptibility of Lupus-Prone Nzm Mouse Strains to Lead Exacerbation of Systemic Lupus Erythematosus Symptoms

It has been repeatedly shown that the heavy metal mercury can induce or exacerbate lupuslike autoimmunity in susceptible strains of rats and mice. A hallmark of such autoimmune induction is the accompaniment of an immune shift, in which there is usually an initial skewing toward a Th2-like immune environment. Another heavy metal, lead (Pb), has also been found to induce a Th2 shift in mice. However, exposure of normal mouse strains to Pb does not appear to induce autoimmunity. In order to investigate whether mice genetically predisposed to murine systemic lupus erythematosus (SLE) are susceptible to a Pb-induced exacerbation of lupus, males and females of four New Zealand mixed (NZM) mouse strains, along with BALB/c and C57Bl/6 controls, were administered three 100-µl intraperitoneal injections of either 1.31 mM lead or sodium acetate per week for 3 wk. The four NZM strains chosen, NZM391, NZM2328, NZM88, and NZM2758, have differential genetic penetrance for SLE with variances in certain manifestations of the disease, but all of these strains naturally develop glomerulonephritis and produce high titers of anti-nuclear autoantibodies. The mice were prebled for baseline values and were bled directly after the injection period (d 1) and monthly thereafter for 5 mo. Sera were assessed for anti-double-stranded DNA titers, urea nitrogen levels, and creatine kinase activity, as well as for total immunoglobulin (Ig) G2a and IgG1 levels. Mortality and morbidity of the mice were also recorded. All NZM strains showed an acute, non-gender-based, susceptibility to Pb at d 1, but the control strains were unaffected. Over time, it became apparent that the strains diverged: The NZM391 strain showed gender-independent susceptibility to Pb enhancement of lupus manifestations and mortality; the NZM2328 strain exhibited gender-independent Pb susceptibility to manifestations, although only females had increased mortality; the NZM2758 strain exhibited non-gender-based elevations in urea nitrogen and creatine kinase activity levels; and the NZM88 strain displayed male susceptibility to anti-DNA and life span. Surprisingly, Pb increased the longevity of NZM88 and NZM2758 females. These results indicate that Pb indeed can exacerbate SLE in lupus-prone mice; however, even among lupus-prone strains, genetic differences determine the degree of exacerbation. Using the known phenotype and genetic differences, one can identify and characterize possible traits and loci associated with Pb susceptibility.

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.

Acute cold/restraint stress inhibits host resistance to Listeria monocytogenes via β1-adrenergic receptors

We previously reported that acute cold/restraint stress (ACRS) significantly inhibits host resistance to Listeria monocytogenes (LM) in BALB/c mice and that the sympathetic nervous system plays a major role in this inhibition. Here, we have further investigated the involvement of adrenergic receptor (ADR) subtypes. beta-ADR antagonist propranolol, but not alpha-ADR antagonist phentolamine significantly enhanced host resistance of ACRS mice. Pro-inflammatory cytokine (IL-6, IL-1beta, and TNFalpha) and IFNgamma levels positively correlated with the LM levels in all groups of mice. Furthermore, beta1-ADR antagonist atenolol but not beta2-ADR antagonist ICI118,551 significantly decreased LM burden in ACRS mice. In addition, SCID mice on the same genetic background (BALB/c), which have no adaptive immune potential, were used to assess the immune responses targeted by ACRS. ACRS-induced suppression of host resistance was not observed in SCID mice, and propranolol pretreatment provided no further improvement of host resistance, indicating that ACRS mainly affects adaptive immunity, which is less critical in mice with greater innate than adaptive immunity. In summary, the data suggest that ACRS inhibition of host resistance to LM is mediated through beta1-ADR stimulation, which appears to directly or indirectly modify activation of T cells or subsequent T cell functions involved in adaptive immunity, thus inhibiting overall host resistance. Interestingly, with heightened innate immunity and the absence of adaptive immunity, as observed in the SCID mice, ACRS does not affect host resistance, which emphasizes the importance of innate immunity in defense against bacterial infection.

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.

Inverse regulation of inducible nitric oxide synthase (iNOS) and arginase I by the protein tyrosine phosphatase SHP-1 in CNS glia.

We have previously shown that the SH2 domain‐containing protein tyrosine phosphatase SHP‐1 plays a critical role in controlling virus infection in CNS glia in vivo and in vitro. The present study addressed whether increased virus replication in SHP‐1‐deficient glia in vitro may be a result of altered expression of inducible nitric oxide synthase (iNOS/NOS2). First, we observed a profound reduction in iNOS protein expression and production of nitric oxide (NO) in response to the viral mimic double‐stranded RNA (dsRNA), despite the induction of high levels of iNOS mRNA, in SHP‐1‐deficient motheaten mouse compared to wild type littermate mouse glia. Because both iNOS expression and NO production are suppressed by multiple pathways involving arginase I activity, it was important that we observed abnormally high constitutive expression of arginase I in cultured glia of SHP‐1‐deficient compared to wild type mice. Further, both constitutive and IL‐4/IL‐10‐induced expression of arginase I correlated with elevated STAT6 nuclear binding activity, decreased NO production, and increased virus replication in motheaten compared to wild type astrocytes. These findings provide the first evidence of an inverse relationship between NO and arginase I activity regulated by SHP‐1 in CNS glia that is relevant to modulation of innate anti‐viral responses. Thus, we propose that SHP‐1 is a critical regulator of innate immunity to virus infections in CNS cells.

Interferon-β treatment in multiple sclerosis attenuates inflammatory gene expression through inducible activity of the phosphatase SHP-1

Interferon-beta is a current treatment for multiple sclerosis (MS). Interferon-beta is thought to exert its therapeutic effects on MS by down-modulating the immune response by multiple potential pathways. Here, we document that treatment of MS patients with interferon beta-1a (Rebif) results in a significant increase in the levels and function of the protein tyrosine phosphatase SHP-1 in PBMCs. SHP-1 is a crucial negative regulator of cytokine signaling, inflammatory gene expression, and CNS demyelination as evidenced in mice deficient in SHP-1. In order to examine the functional significance of SHP-1 induction in MS PBMCs, we analyzed the activity of proinflammatory signaling molecules STAT1, STAT6, and NF-kappaB, which are known SHP-1 targets. Interferon-beta treatment in vivo resulted in decreased NF-kappaB and STAT6 activation and increased STAT1 activation. Further analysis in vitro showed that cultured PBMCs of MS patients and normal subjects had a significant SHP-1 induction following interferon-beta treatment that correlated with decreased NF-kappaB and STAT6 activation. Most importantly, experimental depletion of SHP-1 in cultured PBMCs abolished the anti-inflammatory effects of interferon-beta treatment, indicating that SHP-1 is a predominant mediator of interferon-beta activity. In conclusion, interferon-beta treatment upregulates SHP-1 expression resulting in decreased transcription factor activation and inflammatory gene expression important in MS pathogenesis.

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