Dietary fat exacerbates postprandial hypothalamic inflammation involving glial fibrillary acidic protein-positive cells and microglia in male mice.

In humans, obesity is associated with brain inflammation, glial reactivity, and immune cells infiltration. Studies in rodents have shown that glial reactivity occurs within 24 hr of high-fat diet (HFD) consumption, long before obesity development, and takes place mainly in the hypothalamus (HT), a crucial brain structure for controlling body weight. Here, we sought to characterize the postprandial HT inflammatory response to 1, 3, and 6 hr of exposure to either a standard diet (SD) or HFD. HFD exposure increased gene expression of astrocyte and microglial markers (glial fibrillary acidic protein [GFAP] and Iba1, respectively) compared to SD-treated mice and induced morphological modifications of microglial cells in HT. This remodeling was associated with higher expression of inflammatory genes and differential regulation of hypothalamic neuropeptides involved in energy balance regulation. DREADD and PLX5622 technologies, used to modulate GFAP-positive or microglial cells activity, respectively, showed that both glial cell types are involved in hypothalamic postprandial inflammation, with their own specific kinetics and reactiveness to ingested foods. Thus, recurrent exacerbated postprandial inflammation in the brain might promote obesity and needs to be characterized to address this worldwide crisis.

Central CCL2 signaling onto MCH neurons mediates metabolic and behavioral adaptation to inflammation.

Sickness behavior defines the endocrine, autonomic, behavioral, and metabolic responses associated with infection. While inflammatory responses were suggested to be instrumental in the loss of appetite and body weight, the molecular underpinning remains unknown. Here, we show that systemic or central lipopolysaccharide (LPS) injection results in specific hypothalamic changes characterized by a precocious increase in the chemokine ligand 2 (CCL2) followed by an increase in pro-inflammatory cytokines and a decrease in the orexigenic neuropeptide melanin-concentrating hormone (MCH). We therefore hypothesized that CCL2 could be the central relay for the loss in body weight induced by the inflammatory signal LPS. We find that central delivery of CCL2 promotes neuroinflammation and the decrease in MCH and body weight. MCH neurons express CCL2 receptor and respond to CCL2 by decreasing both electrical activity and MCH release. Pharmacological or genetic inhibition of CCL2 signaling opposes the response to LPS at both molecular and physiologic levels. We conclude that CCL2 signaling onto MCH neurons represents a core mechanism that relays peripheral inflammation to sickness behavior.

Flexible stereospecific interactions and composition within nucleoprotein complexes assembled on the TCR alpha gene enhancer.

During thymocyte maturation, enhancers of genes encoding for TCRdelta (Tcrd) and TCRalpha (Tcra), Edelta(8), and Ealpha, work as a developmental switch controlling transition from Tcrd to Tcra activity at the Tcrad locus. Previous experiments revealed that an Ealpha fragment, Talpha1-Talpha2, which constitutes a well-characterized compact nucleoprotein structure led to premature activation of V(D)J recombination compared with that observed for the entire Ealpha or Talpha1-Talpha4. These experiments indicated that Talpha3-Talpha4 collaborates with factors bound to Talpha1-Talpha2 for the strict developmental regulation of Tcra rearrangement. The compact enhanceosome created on Talpha1-Talpha2 explained the molecular basis for requirement of intact Talpha2 TCF/LEF and ets sites for enhancer function. We have created a mutant version of Ealpha, EalphaMC, in which Edelta myb and runx sites have been substituted for Talpha2 runx and ets sites, that argues against the notion of a requirement for strict Ealpha enhanceosome structure for function. EalphaMC resulted in a very potent enhancer indicating that stereospecific interactions among proteins that form an Ealpha enhanceosome are rather flexible. Activation of V(D)J recombination by EalphaMC during thymocyte development resulted, however, to be premature and indistinguishable from that of Talpha1-Talpha2. These results indicate that Talpha3-Talpha4 itself is not sufficient to impart a developmental delay to a chimeric "early" enhancer, and indicate the need for functional collaboration between Talpha2 runx/ets sites binding proteins and proteins bound to Talpha3-Talpha4 for proper developmental activation. The possibility of assembly of distinct sets of proteins on Ealpha might represent a more flexible form of information processing during thymocyte development.

Effect of clotrimazole on the pump cycle of the Na,K-ATPase.

The effect of the antimycotic drug clotrimazole (CLT) on the Na,K-ATPase was investigated using fluorescence and electrical measurements. The results obtained by steady-state fluorescence experiments with the electrochromic styryl dye RH421 were combined with those achieved by a pre-steady-state method based on fast solution exchange on a solid supported membrane that adsorbs the protein. Both techniques are suitable for monitoring the electrogenic steps of the pump cycle and are in general complementary, yielding distinct kinetic information. The experiments show clearly that CLT affects specific partial reactions of the pump cycle of the Na,K-ATPase with an affinity in the low micromolar range and in a reversible manner. All results can be consistently explained by proposing the CLT-promoted formation of an ion-occluded-CLT-bound conformational E(2) state, E(2)(CLT)(X(2)) that acts as a "dead-end" side track of the pump cycle, where X stands for H+ or K+. Na+ binding, enzyme phosphorylation, and Na+ transport were not affected by CLT, and at high CLT concentrations approximately (1/3) of the enzyme remained active in the physiological transport mode. The presence of Na+ and K+ destabilized the inactivated form of the Na,K-ATPase.

A translocated bacterial protein protects vascular endothelial cells from apoptosis.

The modulation of host cell apoptosis by bacterial pathogens is of critical importance for the outcome of the infection process. The capacity of Bartonella henselae and B. quintana to cause vascular tumor formation in immunocompromised patients is linked to the inhibition of vascular endothelial cell (EC) apoptosis. Here, we show that translocation of BepA, a type IV secretion (T4S) substrate, is necessary and sufficient to inhibit EC apoptosis. Ectopic expression in ECs allowed mapping of the anti-apoptotic activity of BepA to the Bep intracellular delivery domain, which, as part of the signal for T4S, is conserved in other T4S substrates. The anti-apoptotic activity appeared to be limited to BepA orthologs of B. henselae and B. quintana and correlated with (i) protein localization to the host cell plasma membrane, (ii) elevated levels of intracellular cyclic adenosine monophosphate (cAMP), and (iii) increased expression of cAMP-responsive genes. The pharmacological elevation of cAMP levels protected ECs from apoptosis, indicating that BepA mediates anti-apoptosis by heightening cAMP levels by a plasma membrane-associated mechanism. Finally, we demonstrate that BepA mediates protection of ECs against apoptosis triggered by cytotoxic T lymphocytes, suggesting a physiological context in which the anti-apoptotic activity of BepA contributes to tumor formation in the chronically infected vascular endothelium.