Paul T. Massa

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.

Novel NEMO/IκB Kinase and NF-κB Target Genes at the Pre-B to Immature B Cell Transition

The IκB kinase (IKK) signaling complex is responsible for activating NF-κB-dependent gene expression programs. Even though NF-κB-responsive genes are known to orchestrate stress-like responses, critical gaps in our knowledge remain about the global effects of NF-κB activation on cellular physiology. DNA microarrays were used to compare gene expression programs in a model system of 70Z/3 murine pre-B cellsversus their IKK signaling-defective 1.3E2 variant with lipopolysaccharide (LPS), interleukin-1 (IL-1), or a combination of LPS + phorbol 12-myristate 13-acetate under brief (2 h) or long term (12 h) stimulation. 70Z/3-1.3E2 cells lack expression of NEMO/IKKγ/IKKAP-1/FIP-3, an essential positive effector of the IKK complex. Some stimulated hits were known NF-κB target genes, but remarkably, the vast majority of the up-modulated genes and an unexpected class of repressed genes were all novel targets of this signaling pathway, encoding transcription factors, receptors, extracellular ligands, and intracellular signaling factors. Thirteen stimulated (B-ATF, Pim-2, MyD118,Pea-15/MAT1, CD82, CD40L,Wnt10a, Notch 1, R-ras,Rgs-16, PAC-1, ISG15, andCD36) and five repressed (CCR2,VpreB, λ5, SLPI, andCMAP/Cystatin7) genes, respectively, were bona fide NF-κB targets by virtue of their response to a transdominant IκBαSR (super repressor). MyD118 andISG15, although directly induced by LPS stimulation, were unaffected by IL-1, revealing the existence of direct NF-κB target genes, which are not co-induced by the LPS and IL-1 Toll-like receptors.

Novel NEMO/IKKγ and NF-κB target genes at the Pre-B to immature B cell transition

The IκB kinase (IKK) signaling complex is responsible for activating NF-κB-dependent gene expression programs. Even though NF-κB-responsive genes are known to orchestrate stress-like responses, critical gaps in our knowledge remain about the global effects of NF-κB activation on cellular physiology. DNA microarrays were used to compare gene expression programs in a model system of 70Z/3 murine pre-B cellsversus their IKK signaling-defective 1.3E2 variant with lipopolysaccharide (LPS), interleukin-1 (IL-1), or a combination of LPS + phorbol 12-myristate 13-acetate under brief (2 h) or long term (12 h) stimulation. 70Z/3-1.3E2 cells lack expression of NEMO/IKKγ/IKKAP-1/FIP-3, an essential positive effector of the IKK complex. Some stimulated hits were known NF-κB target genes, but remarkably, the vast majority of the up-modulated genes and an unexpected class of repressed genes were all novel targets of this signaling pathway, encoding transcription factors, receptors, extracellular ligands, and intracellular signaling factors. Thirteen stimulated (B-ATF, Pim-2, MyD118,Pea-15/MAT1, CD82, CD40L,Wnt10a, Notch 1, R-ras,Rgs-16, PAC-1, ISG15, andCD36) and five repressed (CCR2,VpreB, λ5, SLPI, andCMAP/Cystatin7) genes, respectively, were bona fide NF-κB targets by virtue of their response to a transdominant IκBαSR (super repressor). MyD118 andISG15, although directly induced by LPS stimulation, were unaffected by IL-1, revealing the existence of direct NF-κB target genes, which are not co-induced by the LPS and IL-1 Toll-like receptors.

Immune markers and differential signaling networks in ulcerative colitis and Crohn's disease†

Background: Cytokine signaling pathways play a central role in the pathogenesis of inflammatory bowel disease (IBD). Ulcerative colitis (UC) and Crohns disease (CD) have unique as well as overlapping phenotypes, susceptibility genes, and gene expression profiles. This study aimed to delineate patterns within cytokine signaling pathways in colonic mucosa of UC and CD patients, explore molecular diagnostic markers, and identify novel immune mediators in IBD pathogenesis. Methods: We quantified 70 selected immune genes that are important in IBD signaling from formalin‐fixed, paraffin‐embedded (FFPE) colon biopsy samples from normal control subjects and UC and CD patients having either severe colitis or quiescent disease (n = 98 subjects). We utilized and validated a new modified real‐time reverse‐transcription polymerase chain reaction (RT‐PCR) technique for gene quantification. Results: Expression levels of signaling molecules including IL‐6/10/12/13/17/23/33, STAT1/3/6, T‐bet, GATA3, Foxp3, SOCS1/3, and downstream inflammatory mediators such as chemokines CCL‐2/11/17/20, oxidative stress inducers, proteases, and mucosal genes were differentially regulated between UC and CD and between active and quiescent disease. We also document the possible role of novel genes in IBD, including SHP‐1, IRF‐1,TARC, Eotaxin, NOX2, arginase I, and ADAM 8. Conclusions: This comprehensive approach to quantifying gene expression provides insights into the pathogenesis of IBD by elucidating distinct immune signaling networks in CD and UC. Furthermore, this is the first study demonstrating that gene expression profiling in FFPE colon biopsies might be a practical and effective tool in the diagnosis and prognosis of IBD and may help identify molecular markers that can predict and monitor response to individualized therapeutic treatments. (Inflamm Bowel Dis 2012;)

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.

Expression and function of the protein tyrosine phosphatase SHP-1 in oligodendrocytes

Previous studies in this laboratory have shown that the SH‐2 domain‐containing protein tyrosine phosphatase SHP‐1 is expressed in CNS glia and functions to modulate cytokine activities in these cells. The present study demonstrates that SHP‐1 is expressed within multiple regions of the CNS in vivo, especially in white matter. Interestingly, we show that mice genetically lacking in SHP‐1 (motheaten mice) in the CNS displayed dysmyelination. We therefore examined the expression of SHP‐1 in the myelin‐forming oligodendrocytes. Oligodendrocytes present in either mixed glial cultures or pure cultures expressed high levels of SHP‐1 in the cytoplasm of cell bodies and processes. Oligodendrocytes isolated from motheaten mice did not express SHP‐1. To test possible functions for SHP‐1 in oligodendrocytes in controlling cytokine signaling, we compared the responsiveness of oligodendrocytes isolated from either motheaten or normal littermate mice with IL‐6. IL‐6 induced higher levels of STAT3 phosphorylation and STAT3‐responsive c‐fos gene expression in pure oligodendrocyte cultures of motheaten compared with normal littermate mice. These studies demonstrate that oligodendrocytes express SHP‐1 and that SHP‐1 functions to control IL‐6 signaling. SHP‐1 may therefore be a critical regulator of oligodendrocyte differentiation in response to IL‐6 family cytokines. Further, these findings may relate to dysmyelination in mice lacking SHP‐1. GLIA 29:376–385, 2000.

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.

NFκB in Neurons? The Uncertainty Principle in Neurobiology

Nuclear factor κB (NFκB) is a dynamically modulated transcription factor with an extensive literature pertaining to widespread actions across species, cell types and developmental stages. Analysis of NFκB in a complex environment such as neural tissue suffers from a difficulty in simultaneously establishing both activity and location. Much of the available data indicate a profound recalcitrance of NFκB activation in neurons, as compared with most other cell types. Few studies to date have sought to distinguish between the various combinatorial dimers of NFκB family members. Recent research has illuminated the importance of these problems, as well as opportunities to move past them to the nuances manifest through variable activation pathways, subunit complexity and target sequence preferences.

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.