Neutrophils Lose the Capacity to Suppress T Cell Proliferation Upon Migration Towards Inflamed Joints in Juvenile Idiopathic Arthritis

Neutrophils are highly abundant in synovial fluid of rheumatic inflamed joints. In oligoarticular juvenile idiopathic arthritis (JIA), synovial fluid neutrophils have impaired effector functions and altered phenotype. We hypothesized that these alterations might impact the immunoregulatory interplay between neutrophils and T cells. In this study we analyzed the suppressive effect of neutrophils, isolated from blood and synovial fluid of oligoarticular JIA patients, on CD4+ T cells activated by CD3/CD28 stimulation. JIA blood neutrophils suppressed T cell proliferation but synovial fluid neutrophils from several patients did not. The loss of T cell suppression was replicated in an in vitro transmigration assay, where healthy control neutrophils migrated into synovial fluid through transwell inserts with endothelial cells and synoviocytes. Non-migrated neutrophils suppressed proliferation of activated CD4+ T cells, but migrated neutrophils had no suppressive effect. Neutrophil suppression of T cells was partly dependent on reactive oxygen species (ROS), demonstrated by impaired suppression in presence of catalase. Migrated neutrophils had reduced ROS production compared to non-migrated neutrophils. A proteomic analysis of transwell-migrated neutrophils identified alterations in proteins related to neutrophil ROS production and degranulation, and biological processes involving protein transport, cell-cell contact and inflammation. In conclusion, neutrophils in synovial fluid of children with JIA have impaired capacity to suppress activated T cells, which may be due to reduced oxidative burst and alterations in proteins related to cell-cell contact and inflammation. The lack of T cell suppression by neutrophils in synovial fluid may contribute to local inflammation and autoimmune reactions in the JIA joint.

An ontogenic study of receptor mechanisms by which acute administration of low-doses of methamphetamine suppresses DOI-induced 5-HT2A-receptor mediated head-twitch response in mice

Background Methamphetamine (MA) is a non-selective monoamine releaser and thus releases serotonin (5-HT), norepinephrine (NE) and dopamine (DA) from corresponding nerve terminals into synapses. DOI ((±)-2, 5-dimethoxy-4-iodoamphetamine) is a direct-acting serotonergic 5-HT2A/C receptor agonist and induces the head-twitch response (HTR) via stimulation of 5-HT2A receptor in mice. While more selective serotonin releasers such as d-fenfluramine evoke the HTR, monoamine reuptake blockers (e.g., cocaine) suppress the DOI-evoked HTR via indirect stimulation of serotonergic 5-HT1A- and adrenergic ɑ2-receptors. Since the induction of HTR by DOI is age-dependent, we investigated whether: (1) during development MA can evoke the HTR by itself, and (2) acute pretreatment with either the selective 5-HT2A receptor antagonist EMD 281014 or low-doses of MA can: (i) modulate the DOI-induced HTR in mice across postnatal days 20, 30 and 60, and (ii) alter the DOI-induced c-fos expression in mice prefrontal cortex (PFC). To further explore the possible modulatory effect of MA on DOI-induced HTR, we investigated whether blockade of inhibitory serotonergic 5-HT1A- or adrenergic ɑ2-receptors by corresponding selective antagonists (WAY 100635 or RS 79948, respectively), can prevent the effect of MA on DOI-induced HTR during aging. Results Although neither EMD 281014 nor MA by themselves could evoke the HTR, acute pretreatment with either EMD 281014 (0.01, 0.05 and 0.1 mg/kg, i.p.) or MA (1, 2.5, 5 mg/kg, i.p.), dose-dependently suppressed the DOI-induced HTR across ages. While WAY 100635 significantly reversed the inhibitory effect of MA in 20- and 30-day old mice, RS 79948 failed to significantly counter MA’s inhibitory effect. Moreover, DOI significantly increased c-fos expressions in several PFC regions. EMD 281014 prevented the DOI-induced increases in c-fos expression. Despite the inhibitory effect of MA on DOI-induced HTR, MA alone or in combination with DOI, significantly increased c-fos expression in several regions of the PFC. Conclusion The suppressive effect of MA on the DOI-evoked HTR appears to be mainly due to functional interactions between the HTR-inducing 5-HT2A receptor and the inhibitory 5-HT1A receptor. The MA-induced increase in c-fos expression in different PFC regions may be due to MA-evoked increases in synaptic concentrations of 5-HT, NE and/or DA.

Circ_0078607 inhibits the progression of ovarian cancer via regulating the miR-32-5p/SIK1 network

Background Circular RNA (circRNA) has been shown to be involved in the regulation of human disease progression, including ovarian cancer (OC). Circ_0078607 was found to participate in OC progression. But its function and mechanism in OC deserve further exploration. Methods The expression levels of circ_0078607, salt-inducible kinase 1 (SIK1) and microRNA (miR)-32-5p were examined by qRT-PCR. And the protein expression levels of SIK1, metastasis marker and apoptosis marker were determined using western blot analysis. EDU staining, colony formation assay, transwell assay and flow cytometry were used to detect the proliferation, migration, invasion and apoptosis of cells. Moreover, dual-luciferase reporter assay was employed to verify the interaction between miR-32-5p and circ_0078607 or SIK1. Xenograft models were constructed to perform in vivo experiments. Results Circ_0078607 and SIK1 were downregulated in OC tissues and cells. Overexpressed circ_0078607 and SIK1 could inhibit OC cell proliferation, migration, invasion, and promote apoptosis. MiR-32-5p could be sponged by circ_0078607, and its overexpression could reverse the suppressive effect of circ_0078607 on OC progression. Furthermore, SIK1 was a target of miR-32-5p, and circ_0078607 could regulate SIK1 by sponging miR-32-5p. The inhibitory effect of circ_0078607 on OC progression also could be reversed by SIK1 silencing. In vivo experiments showed that circ_0078607 reduced OC tumorigenesis by regulating the miR-32-5p/SIK1 axis. Conclusion Circ_0078607 could serve as a sponge of miR-32-5p to regulate SIK1 expression, thereby inhibiting OC progression.

Prelimbic proBDNF facilitates memory destabilization by regulation of neuronal function in juveniles

Fear regulation changes as a function of age and adolescence is a key developmental period for the continued maturation of fear neural circuitry. The involvement of prelimbic proBDNF in fear memory extinction and its mediated signaling were reported previously. Given the inherent high level of proBDNF during juvenile period, we tested whether prelimbic proBDNF regulated synaptic and neuronal functions allowing to influencing retrieval-dependent memory processing. By examining freezing behavior of auditory fear conditioned rats, we found high levels of prelimbic proBDNF in juvenile rats enhanced destabilization of the retrieval-dependent weak but not strong fear memory through activating p75NTR-GluN2B signaling. This modification was attributed to the increment in proportion of thin type spine and promotion in synaptic function, as evidence by facilitation of NMDA-mediated EPSCs and GluN2B-dependent synaptic depression. The strong prelimbic theta- and gamma-oscillation coupling predicted the suppressive effect of proBDNF on the recall of post-retrieval memory. Our results critically emphasize the importance of developmental proBDNF for modification of retrieval-dependent memory and provide a potential critical targeting to inhibit threaten memories associated with neurodevelopment disorders.

Lactobacillus gallinarum modulates the gut microbiota and produces anti-cancer metabolites to protect against colorectal tumourigenesis

ObjectiveUsing faecal shotgun metagenomic sequencing, we identified the depletion of Lactobacillus gallinarum in patients with colorectal cancer (CRC). We aimed to determine the potential antitumourigenic role of L. gallinarum in colorectal tumourigenesis.DesignThe tumor-suppressive effect of L. gallinarum was assessed in murine models of CRC. CRC cell lines and organoids derived from patients with CRC were cultured with L. gallinarum or Escherichia coli MG1655 culture-supernatant to evaluate cell proliferation, apoptosis and cell cycle distribution. Gut microbiota was assessed by 16S ribosomal DNA sequencing. Antitumour molecule produced from L. gallinarum was identified by liquid chromatography mass spectrometry (LC-MS/MS) and targeted mass spectrometry.ResultsL. gallinarum significantly reduced intestinal tumour number and size compared with E. coli MG1655 and phosphate-buffered saline in both male and female murine intestinal tumourigenesis models. Faecal microbial profiling revealed enrichment of probiotics and depletion of pathogenic bacteria in L. gallinarum-treated mice. Culturing CRC cells with L. gallinarum culture-supernatant (5%, 10% and 20%) concentration-dependently suppressed cell proliferation and colony formation. L. gallinarum culture-supernatant significantly promoted apoptosis in CRC cells and patient-derived CRC organoids, but not in normal colon epithelial cells. Only L. gallinarum culture-supernatant with fraction size <3 kDa suppressed proliferation in CRC cells. Using LC-MS/MS, enrichments of indole-3-lactic acid (ILA) was identified in both L. gallinarum culture-supernatant and the gut of L. gallinarum-treated mice. ILA displayed anti-CRC growth in vitro and inhibited intestinal tumourigenesis in vivo.ConclusionL. gallinarum protects against intestinal tumourigenesis by producing protective metabolites that can promote apoptosis of CRC cells.

Different contra-sound effects between noise and music stimuli seen in N1m and psychophysical responses

Auditory-evoked responses can be affected by the sound presented to the contralateral ear. The different contra-sound effects between noise and music stimuli on N1m responses of auditory-evoked fields and those on psychophysical response were examined in 12 and 15 subjects, respectively. In the magnetoencephalographic study, the stimulus to elicit the N1m response was a tone burst of 500 ms duration at a frequency of 250 Hz, presented at a level of 70 dB, and white noise filtered with high-pass filter at 2000 Hz and music stimuli filtered with high-pass filter at 2000 Hz were used as contralateral noise. The contralateral stimuli (noise or music) were presented in 10 dB steps from 80 dB to 30 dB. Subjects were instructed to focus their attention to the left ear and to press the response button each time they heard burst stimuli presented to the left ear. In the psychophysical study, the effects of contralateral sound presentation on the response time for detection of the probe sound of a 250 Hz tone burst presented at a level of 70 dB were examined for the same contra-noise and contra-music used in the magnetoencephalographic study. The amplitude reduction and latency delay of N1m caused by contra-music stimuli were significantly larger than those by contra-noise stimuli in bilateral hemisphere, even for low level of contra-music near the psychophysical threshold. Moreover, this larger suppressive effect induced by contra-music effects was also observed psychophysically; i.e., the change in response time for detection of the probe sound was significantly longer by adding contralateral music stimuli than by adding contra-noise stimuli. Regarding differences in effect between contra-music and contra-noise, differences in the degree of saliency may be responsible for their different abilities to disturb auditory attention to the probe sound, but further investigation is required to confirm this hypothesis.

Rpl24Bst mutation suppresses colorectal cancer by promoting eEF2 phosphorylation via eEF2K

Increased protein synthesis supports the rapid cell proliferation associated with cancer. The Rpl24Bst mutant mouse reduces the expression of the ribosomal protein RPL24 and has been used to suppress translation and limit tumorigenesis in multiple mouse models of cancer. Here, we show that Rpl24Bst also suppresses tumorigenesis and proliferation in a model of colorectal cancer (CRC) with two common patient mutations, Apc and Kras. In contrast to previous reports, Rpl24Bst mutation has no effect on ribosomal subunit abundance but suppresses translation elongation through phosphorylation of eEF2, reducing protein synthesis by 40% in tumour cells. Ablating eEF2 phosphorylation in Rpl24Bst mutant mice by inactivating its kinase, eEF2K, completely restores the rates of elongation and protein synthesis. Furthermore, eEF2K activity is required for the Rpl24Bst mutant to suppress tumorigenesis. This work demonstrates that elevation of eEF2 phosphorylation is an effective means to suppress colorectal tumorigenesis with two driver mutations. This positions translation elongation as a therapeutic target in CRC, as well as in other cancers where the Rpl24Bst mutation has a tumour suppressive effect in mouse models.

A ceRNA network mediated by LINC00475 in papillary thyroid carcinoma

Papillary thyroid carcinoma (PTC) is the most frequent histological type of differentiated thyroid carcinoma. Long noncoding RNAs (lncRNAs) have been widely reported to play a key role in human malignancies, and PTC is included. This study aimed to find out the functions and mechanism of lncRNA LINC00475 in PTC. LINC00475 was upregulated in PTC cells and was mainly located in the cytoplasm according to reverse-transcription polymerase chain reaction analyses and subcellular fractionation assays. As shown by cell counting kit-8 assays, ethynyl deoxyuridine incorporation assays, wound healing assays, and transwell assays, LINC00475 knockdown suppressed cell viability, proliferation, migration, and invasion. Mechanistically, LINC00475 upregulated the expression of messenger RNA zinc finger CCHC-type containing 12 (ZCCHC12) by binding to miR-376c-3p. ZCCHC12 was a direct target gene of miR-376c-3p in PTC cells. The relationship between miR-376c-3p and LINC00475 (or ZCCHC12) in PTC cells was probed by luciferase reporter assays, RNA pulldown assays, and RNA immunoprecipitation assays. In addition, both mRNA and protein levels of ZCCHC12 were downregulated due to miR-376c-3p overexpression or LINC00475 silencing. ZCCHC12 overexpression partially reversed the suppressive effect of LINC00475 knockdown on malignant behaviors of PTC cells. In conclusion, LINC00475 promotes PTC cell proliferation, migration, and invasion by upregulating ZCCHC12 via the interaction with miR-376c-3p.