Identification and Functional Characterization of lncRNAs involved in Human Monocyte-to-Macrophage Differentiation.

Long noncoding RNAs (lncRNAs) make up the largest portion of RNA produced from the human genome, but only a small fraction have any ascribed functions. Although the role of protein-coding genes in macrophage biology has been studied extensively, our understanding of the role played by lncRNAs in this context is still in its early stages. There are over 20,000 lncRNAs in the human genome therefore, attempting to select a lncRNA to characterize functionally can be a challenge. Here we describe two approaches to identify and functionally characterize lncRNAs involved in monocyte-to-macrophage differentiation. The first involves the use of RNA-seq to infer possible functions and the second involves a high throughput functional screen. We examine the advantages and disadvantages of these methodologies and the pipelines for validation that assist in determining functional lncRNAs.

CRISPRi screens identify the lncRNA, LOUP, as a multifunctional locus regulating macrophage differentiation and inflammatory signaling.

Long noncoding RNAs (lncRNAs) account for the largest portion of RNA from the transcriptome, yet most of their functions remain unknown. Here, we performed two independent high-throughput CRISPRi screens to understand the role of lncRNAs in monocyte function and differentiation. The first was a reporter-based screen to identify lncRNAs that regulate TLR4-NFkB signaling in human monocytes and the second screen identified lncRNAs involved in monocyte to macrophage differentiation. We successfully identified numerous noncoding and protein-coding genes that can positively or negatively regulate inflammation and differentiation. To understand the functional roles of lncRNAs in both processes, we chose to further study the lncRNA LOUP [lncRNA originating from upstream regulatory element of SPI1 (also known as PU.1)], as it emerged as a top hit in both screens. Not only does LOUP regulate its neighboring gene, the myeloid fate-determining factor SPI1, thereby affecting monocyte to macrophage differentiation, but knockdown of LOUP leads to a broad upregulation of NFkB-targeted genes at baseline and upon TLR4-NFkB activation. LOUP also harbors three small open reading frames capable of being translated and are responsible for LOUP's ability to negatively regulate TLR4/NFkB signaling. This work emphasizes the value of high-throughput screening to rapidly identify functional lncRNAs in the innate immune system.

Human long noncoding RNA, VILMIR, is induced by major respiratory viral infections and modulates the host interferon response.

Long noncoding RNAs (lncRNAs) are a newer class of noncoding transcripts identified as key regulators of biological processes. Here we aimed to identify novel lncRNA targets that play critical roles in major human respiratory viral infections by systematically mining large-scale transcriptomic datasets. Using bulk RNA-sequencing (RNA-seq) analysis, we identified a previously uncharacterized lncRNA, named virus inducible lncRNA modulator of interferon response (VILMIR), that was consistently upregulated after in vitro influenza infection across multiple human epithelial cell lines and influenza A virus subtypes. VILMIR was also upregulated after SARS-CoV-2 and RSV infections in vitro. We experimentally confirmed the response of VILMIR to influenza infection and interferon-beta (IFN-ß) treatment in the A549 human epithelial cell line and found the expression of VILMIR was robustly induced by IFN-ß treatment in a dose and time-specific manner. Single cell RNA-seq analysis of bronchoalveolar lavage fluid (BALF) samples from COVID-19 patients uncovered that VILMIR was upregulated across various cell types including at least five immune cells. The upregulation of VILMIR in immune cells was further confirmed in the human T cell and monocyte cell lines, SUP-T1 and THP-1, after IFN-ß treatment. Finally, we found that knockdown of VILMIR expression reduced the magnitude of host transcriptional responses to IFN-ß treatment in A549 cells. Together, our results show that VILMIR is a novel interferon-stimulated gene (ISG) that regulates the host interferon response and may be a potential therapeutic target for human respiratory viral infections upon further mechanistic investigation.

New Genetic Variants of RUNX2 in Mexican Families Cause Cleidocranial Dysplasia.

Cleidocranial dysplasia (CCD) is an autosomal dominant skeletal dysplasia characterized by persistent open skull sutures with bulging calvaria, hypoplasia, or aplasia of clavicles permitting abnormal opposition of the shoulders; wide public symphysis; short middle phalanx of the fifth fingers; and vertebral, craniofacial, and dental anomalies. It is a rare disease, with a prevalence of 1-9/1,000,000, high penetrance, and variable expression. The gene responsible for CCD is the Runt-related transcription factor 2 (RUNX2) gene. We characterize the clinical, genetic, and bioinformatic results of four CCD cases: two cases within Mexican families with six affected members, nine asymptomatic individuals, and two sporadic cases with CCD, with one hundred healthy controls. Genomic DNA analyses of the RUNX2 gene were performed for Sanger sequencing. Bioinformatics tools were used to predict the function, stability, and structural changes of the mutated RUNX2 proteins. Three novel heterozygous mutations (c.651_652delTA; c.538_539delinsCA; c.662T>A) and a previously reported mutation (c.674G>A) were detected. In silico analysis showed that all mutations had functional, stability-related, and structural alterations in the RUNX2 protein. Our results show novel mutations that enrich the pool of RUNX2 gene mutations with CCD. Moreover, the proband 1 presented clinical data not previously reported that could represent an expanded phenotype of severe expression.

Duplication of 12q24.21q24.33 in a Girl with Epilepsy, Expanding the Phenotype.

Introduction: Duplication of 12q is characterized by craniofacial dysmorphia, growth failure, occasional brain malformations, abnormalities of the extremities, skeletal and thoracic malformations, cardiovascular defects, anogenital abnormalities like cryptorchidism, psychomotor delay, and intellectual disability. Case presentation: We describe a female patient with typical manifestations of duplication 12q and epilepsy. She had a normal 46,XX karyotype. The microarray assay exhibited a 19.35-Mb gain at 12q24.21q24.33 due to ins(21;12)(p11.2;q24.21q24.33)mat. Discussion and Conclusion: The duplicated region in the patient encompasses 219 genes, 24 considered as pathological. No relation between epilepsy and the genes reported as pathological has been reported.

Soil Fungal Diversity of the Aguarongo Andean Forest (Ecuador).

Fungi represent an essential component of ecosystems, functioning as decomposers and biotrophs, and they are one of the most diverse groups of Eukarya. In the tropics, many species are unknown. In this work, high-throughput DNA sequencing was used to discover the biodiversity of soil fungi in the Aguarongo forest reserve, one of the richest biodiversity hotspots in Ecuador. The rDNA metabarcoding analysis revealed the presence of seven phyla: Ascomycota, Basidiomycota, Mortierellomycota, Mucoromycota, Glomeromycota, Chytridiomycota, and Monoblepharomycota. A total of 440 identified species were recorded. They mainly belonged to Ascomycota (263) and Basidiomycota (127). In Mortierellomycota, 12 species were recorded, among which Podila verticillata is extremely frequent and represents the dominant species in the entire mycobiota of Aguarongo. The present research provides the first account of the entire soil mycobiota in the Aguarongo forest, where many fungal species exist that have strong application potential in agriculture, bioremediation, chemical, and the food industry. The Aguarongo forest hides a huge number of unknown fungal species that could be assessed, and its protection is of the utmost importance.

Metabolic Footprints of Burkholderia Sensu Lato Rhizosphere Bacteria Active against Maize Fusarium Pathogens.

Consistent with their reported abundance in soils, several Burkholderia sensu lato strains were isolated from the rhizosphere of maize plants cultivated at different sites in central México. Comparative analysis of their 16S rRNA gene sequences permitted their separation into three distinctive clades, which were further subdivided into six other clusters by their close resemblance to (1) Trinickia dinghuensis; (2) Paraburkholderia kirstenboschensis, P. graminis, P. dilworthii and P. rhynchosiae; (3) B. gladioli; (4) B. arboris; (5) B. contaminans, or (6) B. metallica representative species. Direct confrontation assays revealed that these strains inhibited the growth of pathogenic Fusarium oxysporum f. sp. radicis-lycopersici, and F. verticillioides within a roughly 3-55% inhibition range. The use of a DIESI-based non-targeted mass spectroscopy experimental strategy further indicated that this method is an option for rapid determination of the pathogen inhibitory capacity of Burkholderia sensu lato strains based solely on the analysis of their exometabolome. Furthermore, it showed that the highest anti-fungal activity observed in B. contaminans and B. arboris was associated with a distinctive abundance of certain m/z ions, some of which were identified as components of the ornbactin and pyochelin siderophores. These results highlight the chemical diversity of Burkholderia sensu lato bacteria and suggest that their capacity to inhibit the Fusarium-related infection of maize in suppressive soils is associated with siderophore synthesis.

Inflammation drives alternative first exon usage to regulate immune genes including a novel iron-regulated isoform of Aim2.

Determining the layers of gene regulation within the innate immune response is critical to our understanding of the cellular responses to infection and dysregulation in disease. We identified a conserved mechanism of gene regulation in human and mouse via changes in alternative first exon (AFE) usage following inflammation, resulting in changes to the isoforms produced. Of these AFE events, we identified 95 unannotated transcription start sites in mice using a de novo transcriptome generated by long-read native RNA-sequencing, one of which is in the cytosolic receptor for dsDNA and known inflammatory inducible gene, Aim2. We show that this unannotated AFE isoform of Aim2 is the predominant isoform expressed during inflammation and contains an iron-responsive element in its 5'UTR enabling mRNA translation to be regulated by iron levels. This work highlights the importance of examining alternative isoform changes and translational regulation in the innate immune response and uncovers novel regulatory mechanisms of Aim2.

Generation and utilization of a HEK-293T murine GM-CSF expressing cell line.

Macrophages and dendritic cells (DCs) are innate immune cells that play a key role in defense against pathogens. In vitro cultures of bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) are well-established and valuable methods for immunological studies. Typically, commercially available recombinant GM-CSF is utilized to generate BMDCs and is also used to culture alveolar macrophages. We have generated a new HEK-293T cell line expressing murine GM-CSF that secretes high levels of GM-CSF (~180 ng/ml) into complete media as an alternative to commercial GM-CSF. Differentiation of dendritic cells and expression of various markers were kinetically assessed using the GM-CSF HEK293T cell line, termed supGM-CSF and compared directly to purified commercial GMCSF. After 7-9 days of cell culture the supGM-CSF yielded twice as many viable cells compared to the commercial purified GM-CSF. In addition to differentiating BMDCs, the supGM-CSF can be utilized to culture functionally active alveolar macrophages. Collectively, our results show that supernatant from our GM-CSF HEK293T cell line supports the differentiation of mouse BMDCs or alveolar macrophage culturing, providing an economical alternative to purified GM-CSF.

A conserved long noncoding RNA, GAPLINC, modulates the immune response during endotoxic shock.

Recent studies have identified thousands of long noncoding RNAs (lncRNAs) in mammalian genomes that regulate gene expression in different biological processes. Although lncRNAs have been identified in a variety of immune cells and implicated in immune response, the biological function and mechanism of the majority remain unexplored, especially in sepsis. Here, we identify a role for a lncRNA-gastric adenocarcinoma predictive long intergenic noncoding RNA (GAPLINC)-previously characterized for its role in cancer, now in the context of innate immunity, macrophages, and LPS-induced endotoxic shock. Transcriptome analysis of macrophages from humans and mice reveals that GAPLINC is a conserved lncRNA that is highly expressed following macrophage differentiation. Upon inflammatory activation, GAPLINC is rapidly down-regulated. Macrophages depleted of GAPLINC display enhanced expression of inflammatory genes at baseline, while overexpression of GAPLINC suppresses this response. Consistent with GAPLINC-depleted cells, Gaplinc knockout mice display enhanced basal levels of inflammatory genes and show resistance to LPS-induced endotoxic shock. Mechanistically, survival is linked to increased levels of nuclear NF-κB in Gaplinc knockout mice that drives basal expression of target genes typically only activated following inflammatory stimulation. We show that this activation of immune response genes prior to LPS challenge leads to decreased blood clot formation, which protects Gaplinc knockout mice from multiorgan failure and death. Together, our results identify a previously unknown function for GAPLINC as a negative regulator of inflammation and uncover a key role for this lncRNA in modulating endotoxic shock.

Submicroscopic 11p13 deletion including the elongator acetyltransferase complex subunit 4 gene in a girl with language failure, intellectual disability and congenital malformations: A case report.

BACKGROUND: We described the main features of an infant diagnosed with facial dysmorphic, language failure, intellectual disability and congenital malformations to strengthen our understanding of the disease. Currently, treatment is only rehabilitation and surgery for cleft lip and palate. CASE SUMMARY: The proband was a 2-years-8-months-old girl. Familial history was negative for congenital malformations or intellectual disability. The patient had microcephaly, upward-slanting palpebral fissures, depressed nasal bridge, bulbous nose and bilateral cleft lip and palate. Brain magnetic resonance imaging showed cortical atrophy and band heterotopia. Her motor and intellectual development is delayed. A submicroscopic deletion in 11p13 involving the elongator acetyltransferase complex subunit 4 gene (ELP4) and a loss of heterozygosity in Xq25-q26.3 were detected. CONCLUSION: There is no treatment for the ELP4 deletion caused by a submicroscopic 11p3 deletion. We describe a second case of deletion of the ELP4 gene without aniridia, which confirms the association between ELP4 gene with several defects and absence of this ocular defect. Additional clinical data in the deletion of the ELP4 gene as cleft palate, facial dysmorphism, and changes at level brain could be associated to this gene or be part of the effect of the recessives genes involved in the loss of heterozygosity region of Xq25-26.3.

High-Throughput CRISPR Screening Identifies Genes Involved in Macrophage Viability and Inflammatory Pathways.

Macrophages are critical effector cells of the immune system, and understanding genes involved in their viability and function is essential for gaining insights into immune system dysregulation during disease. We use a high-throughput, pooled-based CRISPR-Cas screening approach to identify essential genes required for macrophage viability. In addition, we target 3' UTRs to gain insights into previously unidentified cis-regulatory regions that control these essential genes. Next, using our recently generated nuclear factor κB (NF-κB) reporter line, we perform a fluorescence-activated cell sorting (FACS)-based high-throughput genetic screen and discover a number of previously unidentified positive and negative regulators of the NF-κB pathway. We unravel complexities of the TNF signaling cascade, showing that it can function in an autocrine manner in macrophages to negatively regulate the pathway. Utilizing a single complex library design, we are capable of interrogating various aspects of macrophage biology, thus generating a resource for future studies.

Macrophage Exosomes Resolve Atherosclerosis by Regulating Hematopoiesis and Inflammation via MicroRNA Cargo.

Developing strategies that promote the resolution of vascular inflammation and atherosclerosis remains a major therapeutic challenge. Here, we show that exosomes produced by naive bone marrow-derived macrophages (BMDM-exo) contain anti-inflammatory microRNA-99a/146b/378a that are further increased in exosomes produced by BMDM polarized with IL-4 (BMDM-IL-4-exo). These exosomal microRNAs suppress inflammation by targeting NF-κB and TNF-α signaling and foster M2 polarization in recipient macrophages. Repeated infusions of BMDM-IL-4-exo into Apoe-/- mice fed a Western diet reduce excessive hematopoiesis in the bone marrow and thereby the number of myeloid cells in the circulation and macrophages in aortic root lesions. This also leads to a reduction in necrotic lesion areas that collectively stabilize atheroma. Thus, BMDM-IL-4-exo may represent a useful therapeutic approach for atherosclerosis and other inflammatory disorders by targeting NF-κB and TNF-α via microRNA cargo delivery.

Expression of metalloproteinases MMP-2 and MMP-9 is associated to the presence of androgen receptor in epithelial ovarian tumors.

BACKGROUND: The current study evaluated the metalloproteinases MMP-2 and MMP-9 expression in epithelial cells and the surrounding stroma in ovarian tumors and the association of MMPs with the histological subtypes, the clinical stage and the presence of steroid hormone receptors. Tumor samples were obtained from 88 patients undergoing surgical cytoreduction of primary ovarian tumors in Instituto Nacional de Cancerología, from México City. The formalin fixed and paraffin embedded samples were processed in order to demonstrate the presence of androgen receptor,estrogen receptor alpha, progesterone receptor, MMP-2,MMP-9 and collagen IV by immunohistochemistry and/or immunofluorescence. RESULTS: MMP-2 and MMP-9 were differentially expressed in the epithelium and the stroma of ovarian tumors associated to histological subtype, clinical stage and sexual steroid hormone receptor expression. Based on Cox proportional hazard regression model we demonstrated that MMP-2 located in the epithelium and the stroma are independent prognostic biomarkers for overall survival in epithelial ovarian tumors. Kaplan Meir analysis of the combination of AR (+) with MMP-2 (+) in epithelium and AR (+) with MMP-2 (-) in stroma displayed a significant reduction of survival. CONCLUSIONS: The presence of MMP-2 in the stroma of the tumor was a protective factor while the presence of MMP-2 in the epithelium indicated an adverse prognosis. The presence of AR associated with MMP-2 in the tumor cells was a risk factor for overall survival in epithelial ovarian cancer.

The how and why of lncRNA function: An innate immune perspective.

Next-generation sequencing has provided a more complete picture of the composition of the human transcriptome indicating that much of the "blueprint" is a vastness of poorly understood non-protein-coding transcripts. This includes a newly identified class of genes called long noncoding RNAs (lncRNAs). The lack of sequence conservation for lncRNAs across species meant that their biological importance was initially met with some skepticism. LncRNAs mediate their functions through interactions with proteins, RNA, DNA, or a combination of these. Their functions can often be dictated by their localization, sequence, and/or secondary structure. Here we provide a review of the approaches typically adopted to study the complexity of these genes with an emphasis on recent discoveries within the innate immune field. Finally, we discuss the challenges, as well as the emergence of new technologies that will continue to move this field forward and provide greater insight into the biological importance of this class of genes. This article is part of a Special Issue entitled: ncRNA in control of gene expression edited by Kotb Abdelmohsen.

Gender-specific differences in clinical and metabolic variables associated with NAFLD in a Mexican pediatric population.

INTRODUCTION AND OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in children and it is more prevalent in Hispanic males. The gender differences can be explained by body fat distribution, lifestyle, or sex hormone metabolism. We evaluated anthropometric and metabolic differences by gender in children with and without NAFLD. METHODS: We included 194 participants (eutrophic, overweight, and individuals with obesity). The presence of NAFLD was determined using ultrasonography, and we evaluated the association between this disease with metabolic and anthropometric variables by gender. RESULTS: The mean age was 10.64±2.54 years. The frequency of NAFLD in boys was 24.51% and in girls was 11.96% (OR=2.39; 95%CI=1.10-5.19; p=0.025). For girls, NAFLD was significantly associated with triglycerides (p=0.012), homeostatic model assessment of insulin resistance (HOMA-IR) (p=0.048), and the visceral adiposity index (VAI) (p=0.024). The variables related to NAFLD in a gender-specific manner were body mass index (BMI) (p=0.001), waist circumference (WC) (p<0.001), HDL cholesterol (p=0.021), alanine aminotransferase (ALT) (p<0.001), and aspartate aminotransferase (AST) (p=0.002). CONCLUSIONS: In our study NAFLD is more frequent in boys, only ALT, and no other clinical or metabolic variables, were associated with NAFLD in these patients. HOMA-IR, VAI, triglyceride levels, and ALT were associated with NAFLD only in girls. The ALT cut-off points for the development of NAFLD in our study were 28.5U/L in females and 27.5U/L in males. Our findings showed that NAFLD should be intentionally screened in patients with obesity, particularly in boys.

Tracing cellular heterogeneity in pooled genetic screens via multi-level barcoding.

BACKGROUND: While pooled loss- and gain-of-function CRISPR screening approaches have become increasingly popular to systematically investigate mammalian gene function, the large majority of them have thus far not investigated the influence of cellular heterogeneity on screen results. Instead most screens are analyzed by averaging the abundance of perturbed cells from a bulk population of cells. RESULTS: Here we developed multi-level barcoded sgRNA libraries to trace multiple clonal Cas9 cell lines exposed to the same environment. The first level of barcoding allows monitoring growth kinetics and treatment responses of multiplexed clonal cell lines under identical conditions while the second level enables in-sample replication and tracing of sub-clonal lineages of cells expressing the same sgRNA. CONCLUSION: Using our approach, we illustrate how heterogeneity in growth kinetics and treatment response of clonal cell lines impairs the results of pooled genetic screens.

Malonylation of GAPDH is an inflammatory signal in macrophages.

Macrophages undergo metabolic changes during activation that are coupled to functional responses. The gram negative bacterial product lipopolysaccharide (LPS) is especially potent at driving metabolic reprogramming, enhancing glycolysis and altering the Krebs cycle. Here we describe a role for the citrate-derived metabolite malonyl-CoA in the effect of LPS in macrophages. Malonylation of a wide variety of proteins occurs in response to LPS. We focused on one of these, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In resting macrophages, GAPDH binds to and suppresses translation of several inflammatory mRNAs, including that encoding TNFα. Upon LPS stimulation, GAPDH undergoes malonylation on lysine 213, leading to its dissociation from TNFα mRNA, promoting translation. We therefore identify for the first time malonylation as a signal, regulating GAPDH mRNA binding to promote inflammation.

Whole sequence of the mitochondrial DNA genome of Kearns Sayre Syndrome patients: Identification of deletions and variants.

Mitochondria both produce the energy of the cell as ATP via respiration and regulate cellular metabolism. Accordingly, any deletion or mutation in the mitochondrial DNA (mtDNA) may result in a disease. One of these diseases is Kearns Sayre syndrome (KSS), described for the first time in 1958, where different large-scale deletions of different sizes and at different positions have been reported in the mitochondrial genome of patients with similar clinical symptoms. In this study, sequences of the mitochondrial genome of three patients with clinic features of KSS were analyzed. Our results revealed the position, heteroplasmy percentage, size of deletions, and their haplogroups. Two patients contained deletions reported previously and one patient showed a new deletion not reported previously. These results display for the first time a systematic analysis of mtDNA variants in the whole mtDNA genome of patients with KSS to help to understand their association with the disease.

Genetic Models Reveal cis and trans Immune-Regulatory Activities for lincRNA-Cox2.

An inducible gene expression program is a hallmark of the host inflammatory response. Recently, long intergenic non-coding RNAs (lincRNAs) have been shown to regulate the magnitude, duration, and resolution of these responses. Among these is lincRNA-Cox2, a dynamically regulated gene that broadly controls immune gene expression. To evaluate the in vivo functions of this lincRNA, we characterized multiple models of lincRNA-Cox2-deficient mice. LincRNA-Cox2-deficient macrophages and murine tissues had altered expression of inflammatory genes. Transcriptomic studies from various tissues revealed that deletion of the lincRNA-Cox2 locus also strongly impaired the basal and inducible expression of the neighboring gene prostaglandin-endoperoxide synthase (Ptgs2), encoding cyclooxygenase-2, a key enzyme in the prostaglandin biosynthesis pathway. By utilizing different genetic manipulations in vitro and in vivo, we found that lincRNA-Cox2 functions through an enhancer RNA mechanism to regulate Ptgs2. More importantly, lincRNA-Cox2 also functions in trans, independently of Ptgs2, to regulate critical innate immune genes in vivo.