A Review of Neuroreceptors for Clinical and Experimental Neuropharmacology in Central Nervous System Disorders.

The neurobiology drug discovery landscape has transformed over the past decade or so by the discovery of allosteric modulators of receptor superfamilies. A wide range of physiological reactions can occur in response to a limited number of neurotransmitters. This review provides an update on physiological features of the receptors and the signaling pathways that are generated in response to neuroreceptor activation that allow the explanation of this vast array of neurotransmitter responses. Primarily based upon structure, receptors in the nervous system can be classified into four groups: Gprotein coupled receptors, ligand-gated receptors, enzyme-linked receptors, and nuclear receptors. With a particular emphasis on the central nervous system, i.e., brain, spinal cord, and optic nerves, we identify the neuroreceptors, their endogenous agonists, antagonists, sites of expression within the nervous system, current neuropharmacological clinical use, and potential for new drug discovery. New molecular approaches and advances in our knowledge of neuronal communication in processes involved in development, functioning and disorders of the nervous system combined with opportunities to re-purpose existing drugs for new indications continue to highlight the exciting opportunities to improve human health.

DPP4 inhibition mitigates ANG II-mediated kidney immune activation and injury in male mice.

Recent evidence suggests that dipeptidyl peptidase-4 (DPP4) inhibition with saxagliptin (Saxa) is renoprotective under comorbid conditions associated with activation of the renin-angiotensin-aldosterone system (RAAS), such as diabetes, obesity, and hypertension, which confer a high cardiovascular risk. Immune system activation is now recognized as a contributor to RAAS-mediated tissue injury, and, importantly, immunomodulatory effects of DPP4 have been reported. Accordingly, we examined the hypothesis that DPP4 inhibition with Saxa attenuates angiotensin II (ANG II)-induced kidney injury and albuminuria via attenuation of immune activation in the kidney. To this end, male mice were infused with either vehicle or ANG II (1,000 ng/kg/min, s.c.) for 3 wk and received either placebo or Saxa (10 mg/kg/day, p.o.) during the final 2 wk. ANG II infusion increased kidney, but not plasma, DPP4 activity in vivo as well as DPP4 activity in cultured proximal tubule cells. The latter was prevented by angiotensin receptor blockade with olmesartan. Further, ANG II induced hypertension and kidney injury characterized by mesangial expansion, mitochondrial damage, reduced brush border megalin expression, and albuminuria. Saxa inhibited DPP4 activity ∼50% in vivo and attenuated ANG II-mediated kidney injury, independent of blood pressure. Further mechanistic experiments revealed mitigation by Saxa of proinflammatory and profibrotic mediators activated by ANG II in the kidney, including CD8+ T cells, resident macrophages (CD11bhiF4/80loLy6C-), and neutrophils. In addition, Saxa improved ANG II suppressed anti-inflammatory regulatory T cell and T helper 2 lymphocyte activity. Taken together, these results demonstrate, for the first time, blood pressure-independent involvement of renal DPP4 activation contributing to RAAS-dependent kidney injury and immune activation.NEW & NOTEWORTHY This work highlights the role of dipeptidyl peptidase-4 (DPP4) in promoting ANG II-mediated kidney inflammation and injury. Specifically, ANG II infusion in mice led to increases in blood pressure and kidney DPP4 activity, which then led to activation of CD8+ T cells, Ly6C- macrophages, and neutrophils and suppression of anti-inflammatory T helper 2 lymphocytes and regulatory T cells. Collectively, this led to kidney injury, characterized by mesangial expansion, mitochondrial damage, and albuminuria, which were mitigated by DPP4 inhibition independent of blood pressure reduction.

Metabolomic Profiling in Neuromyelitis Optica Spectrum Disorder Biomarker Discovery.

There is no specific test for diagnosing neuromyelitis optica spectrum disorder (NMOSD), a disabling autoimmune disease of the central nervous system. Instead, diagnosis relies on ruling out other related disorders with overlapping clinical symptoms. An urgency for NMOSD biomarker discovery is underscored by adverse responses to treatment following misdiagnosis and poor prognosis following the delayed onset of treatment. Pathogenic autoantibiotics that target the water channel aquaporin-4 (AQP4) and myelin oligodendrocyte glycoprotein (MOG) contribute to NMOSD pathology. The importance of early diagnosis between AQP4-Ab+ NMOSD, MOG-Ab+ NMOSD, AQP4-Ab- MOG-Ab- NMOSD, and related disorders cannot be overemphasized. Here, we provide a comprehensive data collection and analysis of the currently known metabolomic perturbations and related proteomic outcomes of NMOSD. We highlight short chain fatty acids, lipoproteins, amino acids, and lactate as candidate diagnostic biomarkers. Although the application of metabolomic profiling to individual NMOSD patient care shows promise, more research is needed.

Act1 is a negative regulator in T and B cells via direct inhibition of STAT3.

Although Act1 (adaptor for IL-17 receptors) is necessary for IL-17-mediated inflammatory responses, Act1- (but not Il17ra-, Il17rc-, or Il17rb-) deficient mice develop spontaneous SLE- and Sjögren's-like diseases. Here, we show that Act1 functions as a negative regulator in T and B cells via direct inhibition of STAT3. Mass spectrometry analysis detected an Act1-STAT3 complex, deficiency of Act1 (but not Il17ra-, Il17rc-, or Il17rb) results in hyper IL-23- and IL-21-induced STAT3 activation in T and B cells, respectively. IL-23R deletion or blockade of IL-21 ameliorates SLE- and Sjögren's-like diseases in Act1-/- mice. Act1 deficiency results in hyperactivated follicular Th17 cells with elevated IL-21 expression, which promotes T-B cell interaction for B cell expansion and antibody production. Moreover, anti-IL-21 ameliorates the SLE- and Sjögren's-like diseases in Act1-deficient mice. Thus, IL-21 blocking antibody might be an effective therapy for treating SLE- and Sjögren's-like syndrome in patients containing Act1 mutation.

TNFR2 Deficiency Acts in Concert with Gut Microbiota To Precipitate Spontaneous Sex-Biased Central Nervous System Demyelinating Autoimmune Disease.

TNF-α antagonists provide benefit to patients with inflammatory autoimmune disorders such as Crohn's disease, rheumatoid arthritis, and ankylosing spondylitis. However, TNF antagonism unexplainably exacerbates CNS autoimmunity, including multiple sclerosis and neuromyelitis optica. The underlying mechanisms remain enigmatic. We demonstrate that TNFR2 deficiency results in female-biased spontaneous autoimmune CNS demyelination in myelin oligodendrocyte glycoprotein-specific 2D2 TCR transgenic mice. Disease in TNFR2(-/-) 2D2 mice was associated with CNS infiltration of T and B cells as well as increased production of myelin oligodendrocyte glycoprotein-specific IL-17, IFN-γ, and IgG2b. Attenuated disease in TNF(-/-) 2D2 mice relative to TNFR2(-/-) 2D2 mice identified distinctive roles for TNFR1 and TNFR2. Oral antibiotic treatment eliminated spontaneous autoimmunity in TNFR2(-/-) 2D2 mice to suggest role for gut microbiota. Illumina sequencing of fecal 16S rRNA identified a distinct microbiota profile in male TNFR2(-/-) 2D2 that was associated with disease protection. Akkermansia muciniphila, Sutterella sp., Oscillospira sp., Bacteroides acidifaciens, and Anaeroplasma sp. were selectively more abundant in male TNFR2(-/-) 2D2 mice. In contrast, Bacteroides sp., Bacteroides uniformis, and Parabacteroides sp. were more abundant in affected female TNFR2(-/-) 2D2 mice, suggesting a role in disease causation. Overall, TNFR2 blockade appears to disrupt commensal bacteria-host immune symbiosis to reveal autoimmune demyelination in genetically susceptible mice. Under this paradigm, microbes likely contribute to an individual's response to anti-TNF therapy. This model provides a foundation for host immune-microbiota-directed measures for the prevention and treatment of CNS-demyelinating autoimmune disorders.

Transmembrane TNF-TNFR2 Impairs Th17 Differentiation by Promoting Il2 Expression.

The double-edged sword nature by which IL-2 regulates autoimmunity and the unpredictable outcomes of anti-TNF therapy in autoimmunity highlight the importance for understanding how TNF regulates IL-2. Transmembrane TNF (tmTNF) preferentially binds TNFR2, whereas soluble TNF (sTNF) binds TNFR1. We previously showed reduced IL-2 production in TNFR1(-/-) TNFR2(-/-) CD4(+) T cells. In this study, we generated TNFR1(-/-), TNFR2(-/-), or TNFR1(-/-) TNFR2(-/-) 5C.C7 TCR Il2-GFP mice and report that CD4(+) T cell-intrinsic tmTNF/TNFR2 stimulates Il2 promoter activity and Il2 mRNA stability. We further used tmTNF Foxp3 gfp reporter mice and pharmacological TNF blockade in wild-type mice to report a tmTNF/TNFR2 interaction for Il2 expression. IL-17 is critical for host defense, but its overabundance promotes autoimmunity. IL-2 represses Th17 differentiation, but the role for TNFR2 in this process is not well understood. We report elevated expression of TNFR2 under Th17-polarization conditions. Genetic loss-of-function experimental models, as well as selective TNF blockade by etanercept and XPro1595 in wild-type mice, demonstrate that impaired tmTNF/TNFR2, but not sTNF/TNFR1, promotes Th17 differentiation in vivo and in vitro. Under Th17-polarizing conditions, elevated IL-17 production by TNFR2-knockout CD4(+) T cells was associated with increased STAT3 activity and decreased STAT5 activity. Increased IL-17 production in TNFR2-knockout T cells was prevented by adding exogenous IL-2. We conclude that CD4(+) T cell-intrinsic tmTNF/TNFR2 promotes IL-2 production that inhibits the generation of Th17 cells in a Foxp3-independent manner. Moreover, under Th17-polarizing conditions, selective blockade of CD4(+) T cell-intrinsic TNFR2 appears to be sufficient to promote Th17 differentiation.

Differential regulation of hepatic organic cation transporter 1, organic anion-transporting polypeptide 1a4, bile-salt export pump, and multidrug resistance-associated protein 2 transporter expression in lymphocyte-deficient mice associates with interleukin-6 production.

Cholestasis results from interrupted bile flow and is associated with immune-mediated liver diseases. It is unclear how inflammation contributes to cholestasis. The aim of this study was to determine whether T and B cells contribute to hepatic transporter expression under basal and inflammatory conditions. C57BL/6J wild-type mice or strains lacking T, B, or both T and B cells were exposed to lipopolysaccharide (LPS) or saline, and livers were collected 16 hours later. Branched DNA signal amplification was used to assess mRNA levels of organic anion-transporting polypeptides (Oatp) 1a1, 1a4, and 1b2; organic cation transporter (Oct) 1; canalicular bile-salt export pump (Bsep); multidrug resistance-associated proteins (Mrp) 2 and 3; and sodium-taurocholate cotransporting polypeptide (Ntcp). Real-time polymerase chain reaction analysis was used to correlate changes of transporter expression with interleukin-1b (IL-1b), IL-6, IL-17A, IL-17F, tumor necrosis factor-α (TNF-α), and interferon-γ expression in the liver. LPS treatment inhibited Bsep and Oct1 mRNA expression, and this was abrogated with a loss of T cells, but not B cells. In addition, the absence of T cells increased Mrp2 mRNA expression, whereas B cell deficiency attenuated Oatp1a4 mRNA in LPS-treated mice. Oatp1a1, Oatp1b2, Ntcp, and Mrp3 were largely unaffected by T or B cell deficiency. Lymphocyte deficiency altered basal and inflammatory IL-6, but not TNF-α or IL-1b, mRNA expression. Taken together, these data implicate lymphocytes as regulators of basal and inflammatory hepatic transporter expression and suggest that IL-6 signaling may play a critical role.

Immunotoxicology: fifty years of global scientific progress.

The Immunotoxicology Specialty Section of the Society of Toxicology (SOT) celebrated the 50(th) Anniversary of the SOT by constructing a poster to highlight the milestones of Immunotoxicology during that half-century period. This poster was assembled by an ad hoc committee and intertwines in words, citations, graphics, and photographs our attempts to capture a timeline reference of the development and progressive movement of immunotoxicology across the globe. This poster was displayed during the 50(th) Annual SOT Meeting in Washington DC in March, 2011. The poster can be accessed by any Reader at the SOT Website via the link http://www.toxicology.org/AI/MEET/AM2011/posters_rcsigss.asp#imss. We dedicate this poster to all of the founders and the scientists that followed them who have made the discipline of Immunotoxicology what it is today.

Biphasic regulation of Il2 transcription in CD4+ T cells: roles for TNF-alpha receptor signaling and chromatin structure.

We describe a novel biphasic regulation of Il2 transcription in naive CD4(+) T cells. Few ( approximately 5%) CD4(+) T cells transcribe Il2 within 6 h of anti-TCR-beta plus anti-CD28 stimulation (early phase). Most naive CD4(+) T cells do not initiate Il2 transcription until after an additional approximately 12 h of T cell stimulation (late phase). In comparison, essentially all previously activated (Pre-Ac) CD4(+) T cells that transcribe Il2 do so with an early-phase response. Late-phase Il2 expression mostly requires c-Rel, CD28, and TNFR signaling. In contrast, early-phase transcription is only partly c-Rel and CD28 dependent and TNFR independent. There was also increased stable DNA accessibility at the Il2 locus and elevated c-Rel expression in resting Pre-Ac CD4(+) cells. Upon T cell activation, a faster and greater increase in DNA accessibility as well as c-Rel nuclear expression were observed in Pre-Ac CD4(+) cells relative to naive CD4(+) T cells. In addition, both acetylated histone H3 and total H3 decreased at the Il2 locus upon rechallenge of Pre-Ac CD4(+) T cells, whereas increased acetylated histone H3 with no change in total H3 was observed following activation of naive CD4(+) T cells. We propose a model in which nucleosome disassembly facilitates rapid initiation of Il2 transcription in CD4(+) T cells, and suggest that a threshold level of c-Rel must be reached for Il2 promoter activity in both naive and Pre-Ac CD4(+) T cells. This is provided, at least partially, by TNFR signaling during priming, but not during recall.

Distinct effects of TGF-beta 1 on CD4+ and CD8+ T cell survival, division, and IL-2 production: a role for T cell intrinsic Smad3.

TGF-beta1 is critical for maintaining T cell homeostasis. Smad3 has been implicated in this regulatory process, yet the cellular targets and molecular details remain poorly understood. In this study, we report that TGF-beta1 impairs the entry of CD4+ and CD8+ T cells into the cell cycle as well as their progression through subsequent rounds of division, and show that Smad3 is essential for TGF-beta1 to inhibit TCR-induced division of only CD4+ and not CD8+ T cells. Both CD8+ and CD4+ T cells from Smad3-/- mice were refractory to TGF-beta1-induced inhibition of IL-2 production, thus demonstrating that not all CD8+ T cell responses to TGF-beta1 are Smad3 independent. These TGF-beta1 effects were all T cell intrinsic, as they were reproduced in purified CD4+ and CD8+ T cells. Finally, we found that Smad3 was critical for the survival of CD8+, but not CD4+ T cells following activation ex vivo. The TCR-induced death of Smad3-/- CD8+ T cells was not dependent upon TNF-alpha production. Exogenous TGF-beta1 partially rescued the CD8+ T cells by signaling through a Smad3-independent pathway. TGF-beta1 also enhanced survival of TCR-stimulated CD4+CD44high T cells in a Smad3-independent manner. Collectively, these findings firmly establish for the first time that TGF-beta1 discriminately regulates CD4+ and CD8+ T cell expansion by signaling through distinct intracellular pathways.

Smad3 is essential for TGF-beta 1 to suppress IL-2 production and TCR-induced proliferation, but not IL-2-induced proliferation.

Transforming growth factor-beta1 is essential to maintain T cell homeostasis, as illustrated by multiorgan inflammation in mice deficient in TGF-beta1 signaling. Despite the physiological importance, the mechanisms that TGF-beta1 uses to regulate T cell expansion remain poorly understood. TGF-beta1 signals through transmembrane receptor serine/threonine kinases to activate multiple intracellular effector molecules, including the cytosolic signaling transducers of the Smad protein family. We used Smad3(-/-) mice to investigate a role for Smad3 in IL-2 production and proliferation in T cells. Targeted disruption of Smad3 abrogated TGF-beta1-mediated inhibition of anti-CD3 plus anti-CD28-induced steady state IL-2 mRNA and IL-2 protein production. CFSE labeling demonstrated that TGF-beta1 inhibited entry of wild-type anti-CD3 plus anti-CD28-stimulated cells into cycle cell, and this inhibition was greatly attenuated in Smad3(-/-) T cells. In contrast, disruption of Smad3 did not affect TGF-beta1-mediated inhibition of IL-2-induced proliferation. These results demonstrate that TGF-beta1 signals through Smad3-dependent and -independent pathways to inhibit T cell proliferation. The inability of TGF-beta1 to inhibit TCR-induced proliferation of Smad3(-/-) T cells suggests that IL-2 is not the primary stimulus driving expansion of anti-CD3 plus anti-CD28-stimulated T cells. Thus, we establish that TGF-beta1 signals through multiple pathways to suppress T cell proliferation.

Concentration-dependent bifunctional effect of TGF-beta 1 on immunoglobulin production: a role for Smad3 in IgA production in vitro.

Injury to the liver results in rapid induction of transforming growth factor-beta1 (TGF-beta(1)) consistent with a role for TGF-beta(1) in repairing damaged tissue. In addition to its ubiquitous role in injury repair, TGF-beta(1) is also well established as a critical regulator of immune homeostasis; however, its mechanisms of action remain enigmatic. We have previously demonstrated that the hepatotoxic chlorinated hydrocarbon, carbon tetrachloride, suppresses helper T-lymphocyte function in a TGF-beta(1)-dependent manner. Here, we report that, in opposition to its immunosuppressive effects at picomolar concentrations, femtomolar concentrations of TGF-beta(1) augment T cell-dependent anti-sRBC IgM antibody forming cell (AFC) and T cell-independent DNP-Ficoll-induced AFC responses. These data support a concentration-dependent bifunctional effect by TGF-beta(1) on humoral immune responses in vitro. We further investigated a putative mechanistic role for Smad3, an intracellular mediator of TGF-beta(1) signaling, in propagating the inhibitory effects of TGF-beta(1) on humoral immune responses. Relative to wild type littermates, splenocytes from mice homologous for a null mutation in the gene encoding the TGF-beta receptor-activated Smad3 (Smad3(Exon8-/-)) were less sensitive to inhibition by TGF-beta(1) following anti-sRBC- and LPS-sensitization in vitro. In agreement, inhibition of IgM protein production by TGF-beta(1) was also dampened in LPS-sensitized Smad3(Exon8-/-) splenic B cells. Moreover, stimulation of IgA by TGF-beta(1) was abrogated in LPS-sensitized Smad3(Exon8-/-) splenocytes suggesting an additional role for Smad3 in regulating IgA production in vitro. Our results suggest that the effects of TGF-beta(1) on humoral immune responses fundamentally differ in a concentration-dependent manner and are mediated, in part, through Smad3 signaling.