CNS distribution, signalling properties and central effects of G-protein coupled receptor 4.

Information on the distribution and biology of the G-protein coupled receptor 4 (GPR4) in the brain is limited. It is currently thought that GPR4 couples to Gs proteins and may mediate central respiratory sensitivity to CO2. Using a knock-in mouse model, abundant GPR4 expression was detected in the cerebrovascular endothelium and neurones of dorsal raphe, retro-trapezoidal nucleus locus coeruleus and lateral septum. A similar distribution was confirmed using RNAscope in situ hybridisation. In HEK293 cells, overexpressing GPR4, it was highly constitutively active at neutral pH with little further increase in cAMP towards acidic pH. The GPR4 antagonist NE 52-QQ57 effectively blocked GPR4-mediated cAMP accumulation (IC50 26.8 nM in HEK293 cells). In HUVEC which natively express GPR4, physiological acidification (pH 7.4-7.0) resulted in a cAMP increase by ∼55% which was completely prevented by 1 µM NE 52-QQ57. The main extracellular organic acid, l-lactic acid (LL; 1-10 mM), suppressed pH dependent activation of GPR4 in HEK293 and HUVEC cells, suggesting allosteric negative modulation. In unanaesthetised mice and rats, NE 52-QQ57 (20 mg kg-1) reduced ventilatory response to 5 and 10% CO2. In anaesthetised rats, systemic administration of NE 52-QQ57 (up to 20 mg kg-1) had no effect on hemodynamics, cerebral blood flow and blood oxygen level dependent responses. Central administration of NE 52-QQ57 (1 mM) in vagotomised anaesthetised rats did not affect CO2-induced respiratory responses. Our results indicate that GPR4 is expressed by multiple neuronal populations and endothelium and that its pH sensitivity is affected by level of expression and LL. NE 52-QQ57 blunts hypercapnic response to CO2 but this effect is absent under anaesthesia, possibly due to the inhibitory effect of LL on GPR4.

The disruption of the rod-derived cone viability gene leads to photoreceptor dysfunction and susceptibility to oxidative stress.

Rod-derived cone viability factor (RdCVF) is a thioredoxin-like protein, which has therapeutic potential for rod-cone dystrophies such as retinitis pigmentosa (RP). Cone loss in rodent models of RP is effectively reduced by RdCVF treatment. In this study, we investigate the physiological role of RdCVF in the retina by analyzing the phenotype of the mouse lacking the RdCVF gene, Nxnl1. Although the mice do not show an obvious developmental defect, an age-related reduction of both cone and rod function and a delay in the dark-adaptation of the retina are recorded by electroretinogram (ERG). This functional change is accompanied by a 17% reduction in cone density and a 20% reduction in thickness of the outer nuclear layer. The transcriptome of the retina reveals early changes in the expression of genes involved in programmed cell death, stress-response and redox-signaling, which is followed by a generalized injury response with increased microglial activation, GFAP, FGF2 and lipid peroxidation levels. Furthermore, cones of the mice lacking Nxnl1 are more sensitive to oxidative stress with a reduction of 65% in the cone flicker ERG amplitude measured under hyperoxic conditions. We show here that the RdCVF gene, in addition to therapeutic properties, has an essential role in photoreceptor maintenance and resistance to retinal oxidative stress.

Solving the IRAK-4 enigma: application of kinase-dead knock-in mice.

Interleukin-1 receptor-associated kinase (IRAK-4) is an essential component of the signal transduction complex downstream of the interleukin (IL)-1- and Toll-like receptors. Though regarded as the first kinase in the signaling cascade, the role of IRAK-4 kinase activity versus its scaffold function has been controversial. In order to investigate the role of IRAK-4 kinase function in vivo, we generated "knock-in" mice where the wild-type IRAK-4 gene is replaced with a mutant gene encoding kinase-deficient IRAK-4 protein (IRAK-4 KD). IRAK-4 kinase is rendered inactive by mutating the conserved lysine residues in the ATP pocket essential for coordinating ATP. Analyses of embryonic fibroblasts and macrophages obtained from IRAK-4 KD mice demonstrated lack of cellular responsiveness to stimulation with IL-1beta or Toll-like receptor 4 (TLR4) and TLR7 agonists. IRAK-4 KD cells were severely impaired in NF-kappaB, JNK, and p38 activation in response to IL-1beta or TLR7 ligand. In addition, activation of JNK and p38 was affected in lipopolysaccharide (LPS)-stimulated IRAK-4 KD macrophages. As a consequence, IL-1 receptor/TLR4/TLR7-mediated production of cytokines and chemokines was largely absent in these cells. Additionally, microarray analysis identified IL-1beta response genes and revealed that the induction of IL-1beta-responsive mRNAs is largely ablated in IRAK-4 KD cells. In summary, our results suggest that IRAK-4 kinase activity plays a critical role in IL-1R-, TLR4-, and TLR7-mediated induction of inflammatory responses.

Activation of STAT proteins and cytokine genes in human Th1 and Th2 cells generated in the absence of IL-12 and IL-4.

We have shown previously that human CD4+ 45RO- T cells could be primed for a Th2 phenotype independent of IL-4 if they were activated by anti-CD28 mAb plus IL-2. If additional TCR signals were provided, the cells differentiated toward Th1 independent of IL-12. Here we show that anti-CD28/IL-2-primed Th2 cells expressed high levels of activated STAT6, but no cytokine mRNA. Moreover, both Th1 and Th2 cells expressed active STAT1 and -3, but not STAT2, -4, and -5. Restimulation of Th1 or Th2 cells via CD3 plus CD28 induced production of IFN-gamma or IL-4, respectively, but did not alter the activation status/DNA binding activity of STATs. Addition of IL-4 (or anti-IL-4 mAb) to restimulated Th2 cells did not modulate STAT6 activation or IL-4 expression, confirming the full commitment. However, Th2 cells remained responsive to IL-12, which repressed STAT6 DNA binding but activated STAT4, and this coincided with a suppression of IL-4/IL-5 and an induction of IFN-gamma. In Th1 cells, IL-12 activated both STAT6 and STAT4, and IL-4 activated STAT6, but in both cases the Th1 phenotype remained. Together the data show that CD28/IL-2-dependent Th2 priming activated STAT6 without inducing IL-4 expression. The primed Th cells resembled memory cells and produced IL-4 upon the first CD3/CD28 costimulus without detectable modulation of STATs. Th2 cells remained responsive to IL-12, which repressed STAT6 DNA binding and activated STAT4, and switched the cells to Th1. The effects of IL-12 may depend on the commitment of the cells, since IL-12 phosphorylated STAT6 in Th1 and dephosphorylated STAT6 in Th2 cells.

TCR-independent activation of human CD4+ 45RO- T cells by anti-CD28 plus IL-2: Induction of clonal expansion and priming for a Th2 phenotype.

In this study we show that uncommitted human CD4+ CD45RA+ RO- CD25- CD71- HLA-DR- T cells can be primed for a Th2 phenotype before they encounter TCR signals and before they are exposed to IL-4. We found that >99% of uncommitted T cells proliferated upon costimulation by immobilized anti-CD3 plus anti-CD28 mAbs and differentiated into pure Th1 cells. In contrast, uncommitted T cells did not respond to stimulation by either anti-CD3 or anti-CD28, or by IL-2 alone. Interestingly, 5% of uncommitted T cells proliferated efficiently in response to stimulation by immobilized anti-CD28 plus IL-2 (in the absence of TCR/CD3 signals) and differentiated into pure Th2 "precursor" cells. Like murine CD4+ NK1.1+ T cells, human Th2 precursors promptly expressed mRNA for Th2 cytokines upon stimulation via the TCR/CD3 complex by anti-CD3 mAb or staphylococcal enterotoxin B, and secreted up to 50 ng of IL-4, IL-5, and IL-13 per 10(6) cells. Th2 "precursors" developed only in the complete absence of IL-4, as addition of 0.1 U (5 pg) of exogenous IL-4 suppressed their clonal expansion by >90%, whereas addition of neutralizing anti-IL-4 mAb had no effect. Together these results suggest that, in vivo, a significant fraction of uncommitted T cells may be primed for a Th2 phenotype independent of Ag and IL-4 if they are exposed to Th1 cell-derived IL-2 and simultaneously interact with accessory cells bearing the natural CD28 ligands B7-1 and B7-2. When stimulated by specific Ag, such primed Th2 precursor cells may provide a source of IL-4 to promote Th2 immunity.