Transcriptomic analysis reveals that RasGEF1b deletion alters basal and LPS-induced expression of genes involved in chemotaxis and cytokine responses in macrophages.

Ras guanine nucleotide exchange factor member 1b (RasGEF1b) of the RasGEF/CDC25 domain-containing family is preferentially expressed by macrophages. However, information is lacking about its role in macrophage function. In this study, we generated mice with ubiquitous deletion of Rasgef1b and used RNA-seq-based transcriptomics to compare the global gene expression in wild-type and knock-out primary bone-marrow-derived macrophages under basal conditions and after lipopolysaccharide (LPS) treatment. Transcriptional filtering identified several genes with significantly different transcript levels between wild-type and knock-out macrophages. In total, 49 and 37 differentially expressed genes were identified at baseline and in LPS-activated macrophages, respectively. Distinct biological processes were significantly linked to down-regulated genes at the basal condition only, and largely included chemotaxis, response to cytokines, and positive regulation of GTPase activity. Importantly, validation by RT-qPCR revealed that the expression of genes identified as down-regulated after LPS stimulation was also decreased in the knock-out cells under basal conditions. We used a luciferase-based reporter assay to showcase the capability of RasGEF1b in activating the Serpinb2 promoter. Notably, knockdown of RasGEF1b in RAW264.7 macrophages resulted in impaired transcriptional activation of the Serpinb2 promoter, both in constitutive and LPS-stimulated conditions. This study provides a small collection of genes that shows relative expression changes effected by the absence of RasGEF1b in macrophages. Thus, we present the first evidence that RasGEF1b mediates the regulation of both steady-state and signal-dependent expression of genes and propose that this GEF plays a role in the maintenance of the basal transcriptional level in macrophages.

Neuromuscular defects after infection with a beta coronavirus in mice.

COVID-19 affects primarily the lung. However, several other systemic alterations, including muscle weakness, fatigue and myalgia have been reported and may contribute to the disease outcome. We hypothesize that changes in the neuromuscular system may contribute to the latter symptoms observed in COVID-19 patients. Here, we showed that C57BL/6J mice inoculated intranasally with the murine betacoronavirus hepatitis coronavirus 3 (MHV-3), a model for studying COVID-19 in BSL-2 conditions that emulates severe COVID-19, developed robust motor alterations in muscle strength and locomotor activity. The latter changes were accompanied by degeneration and loss of motoneurons that were associated with the presence of virus-like particles inside the motoneuron. At the neuromuscular junction level, there were signs of atrophy and fragmentation in synaptic elements of MHV-3-infected mice. Furthermore, there was muscle atrophy and fiber type switch with alteration in myokines levels in muscles of MHV-3-infected mice. Collectively, our results show that acute infection with a betacoronavirus leads to robust motor impairment accompanied by neuromuscular system alteration.

TRPV1 modulation of contextual fear memory depends on stimulus intensity and endocannabinoid signalling in the dorsal hippocampus.

The transient receptor potential vanilloid type-1 (TRPV1) channels have been implicated in the modulation of aversive responses. The endocannabinoid anandamide acts as an endogenous TRPV1 agonist, exerting opposite functions at TRPV1 and type-1 cannabinoid receptors (CB1R). Here we tested the hypothesis that hippocampal TRPV1 modulates contextual fear memory retrieval and investigated the influence of the aversive stimulus intensity as well as the role of endocannabinoid signaling. Male C57BL/6J mice were tested for contextual fear memory after low-, moderate-, or high-intensity shock protocols. The selective TRPV1 blockers SB366791 (1-10 nmol) and 6-I-NC (2 nmol) were infused via intra-dorsal hippocampus before the retrieval test session. The local levels of endocannabinoids and Arc and Zif268 mRNAs, involved in synaptic plasticity and memory, were quantified. First, both TRPV1 blockers reduced memory retrieval in animals exposed to moderate or high (but not low) intensity training protocols. In the second series of results, the magnitude of the freezing responses positively correlated with the hippocampal anandamide levels; TRPV1 and CB1R were found co-localized in this brain region; and the CB1R antagonist, AM251, prevented the effects of SB366791. Thus, endocannabinoid signaling possibly mediates the effects of TRPV1 blockers. Finally, inhibition of memory retrieval by TRPV1 blockers increased Arc and Zif268 mRNAs and impaired fear memory reinstatement. In conclusion, the modulation of fear memories by dorsal hippocampal TRPV1 channels may depend on the aversive stimulus intensity and occur via anandamide/CB1 signaling. Moreover, TRPV1 blockers promote Arc and Zif268 transcription, with subsequent attenuation of aversive memory reinstatement.

Data in support of Rap2a GTPase expression, activation and effects in LPS-mediated innate immune response and NF-κB activation.

We present here the data to support the understanding of the implication of Rap2a GTPase in LPS-induced innate immune response and NF-κB activation. The data presented are related to molecular tools that were generated, acquired, optimized or validated to investigate Rap2a expression, activation and its effects in mammalian cells including RAW264.7 macrophages and THP-1 monocytes under inflammatory conditions. These data supplement important technical and biological information on immune function of Rap2a in macrophages activated by LPS, recently reported by us (Carvalho et al., 2019) [1].

Both knock-down and overexpression of Rap2a small GTPase in macrophages result in impairment of NF-κB activity and inflammatory gene expression.

Small Ras GTPases are key molecules that regulate a variety of cellular responses in different cell types. Rap1 plays important functions in the regulation of macrophage biology during inflammation triggered by toll-like receptors (TLRs). However, despite sharing a relatively high degree of similarity with Rap1, no studies concerning Rap2 in macrophages and innate immunity have been reported yet. In this work, we show that either way alterations in the levels of Rap2a hampers proper macrophages response to TLR stimulation. Rap2a is activated by LPS in macrophages, and although putative activator TLR-inducible Ras guanine exchange factor RasGEF1b was sufficient to induce, it was not fully required for Rap2a activation. Silencing of Rap2a impaired LPS-induced production of IL-6 cytokine and KC/Cxcl1 chemokine, and also NF-κB activity as measured by reporter gene studies. Surprisingly, overexpression of Rap2a did also lead to marked inhibition of NF-κB activation induced by LPS, Pam3CSK4 and downstream TLR signaling molecules. We also found that Rap2a can inhibit the LPS-induced phosphorylation of the NF-κB subunit p65 at serine 536. Collectively, our data suggest that expression levels of Rap2a in macrophages might be tightly regulated to avoid unbalanced immune response. Our results implicate Rap2a in TLR-mediated responses by contributing to balanced NF-κB activity status in macrophages.

Laticifer proteins from Plumeria pudica inhibit the inflammatory and nociceptive responses by decreasing the action of inflammatory mediators and pro-inflammatory cytokines

AbstractSome publications have described the pharmacological properties of latices proteins. Thus, in the present study proteins from Plumeria pudica Jacq., Apocynaceae, latex were evaluated for anti-inflammatory and antinociceptive activities. Obtained data showed that an intraperitoneal administration of different doses of latex was able to reduce the paw edema induced by carrageenan in a dose-dependent manner (better dose 40 mg/kg; 72.7% inhibition at 3rd and 78.7% at 4th hour) and the edema induced by dextran (40 mg/kg; 51.5% inhibition at 30 min and 93.0% at 1st hour). Inhibition of edema induced by carrageenan was accompanied by a reduction of myeloperoxidase activity. Pre-treating animals with latex (40 mg/kg) also inhibited the paw edema induced by histamine, serotonin, bradykinin, prostaglandin E2, compound 48/80. Additionally, the latex (40 mg/kg) reduced the leukocyte peritoneal migration induced by carrageenan and this event was followed by reduction of IL-1β and TNF-α in peritoneal fluid. The latex-treatment (40 mg/kg) reduced the animal abdominal constrictions induced by acetic acid and the first phase on paw licking model induced by formalin. When latex was treated with heat (at 100 °C for 30 min), anti-edematogenic and myeloperoxidase activities were significantly reduced, indicating the involvement of heat-sensitive proteins on anti-inflammatory effect. Our results evidence that latex fluids are a source of proteins with pharmacological properties.