Stem-like T cells are associated with the pathogenesis of ulcerative colitis in humans.

To understand the role of T cells in the pathogenesis of ulcerative colitis (UC), we analyzed colonic T cells isolated from patients with UC and controls. Here we identified colonic CD4+ and CD8+ T lymphocyte subsets with gene expression profiles resembling stem-like progenitors, previously reported in several mouse models of autoimmune disease. Stem-like T cells were increased in inflamed areas compared to non-inflamed regions from the same patients. Furthermore, TCR sequence analysis indicated stem-like T cells were clonally related to proinflammatory T cells, suggesting their involvement in sustaining effectors that drive inflammation. Using an adoptive transfer colitis model in mice, we demonstrated that CD4+ T cells deficient in either BCL-6 or TCF1, transcription factors that promote T cell stemness, had decreased colon T cells and diminished pathogenicity. Our results establish a strong association between stem-like T cell populations and UC pathogenesis, highlighting the potential of targeting this population to improve clinical outcomes.

Intratumoral follicular regulatory T cells curtail anti-PD-1 treatment efficacy.

Immune-checkpoint blockade (ICB) has shown remarkable clinical success in boosting antitumor immunity. However, the breadth of its cellular targets and specific mode of action remain elusive. We find that tumor-infiltrating follicular regulatory T (TFR) cells are prevalent in tumor tissues of several cancer types. They are primarily located within tertiary lymphoid structures and exhibit superior suppressive capacity and in vivo persistence as compared with regulatory T cells, with which they share a clonal and developmental relationship. In syngeneic tumor models, anti-PD-1 treatment increases the number of tumor-infiltrating TFR cells. Both TFR cell deficiency and the depletion of TFR cells with anti-CTLA-4 before anti-PD-1 treatment improve tumor control in mice. Notably, in a cohort of 271 patients with melanoma, treatment with anti-CTLA-4 followed by anti-PD-1 at progression was associated with better a survival outcome than monotherapy with anti-PD-1 or anti-CTLA-4, anti-PD-1 followed by anti-CTLA-4 at progression or concomitant combination therapy.

Tissue-resident memory features are linked to the magnitude of cytotoxic T cell responses in human lung cancer.

Therapies that boost the anti-tumor responses of cytotoxic T lymphocytes (CTLs) have shown promise; however, clinical responses to the immunotherapeutic agents currently available vary considerably, and the molecular basis of this is unclear. We performed transcriptomic profiling of tumor-infiltrating CTLs from treatment-naive patients with lung cancer to define the molecular features associated with the robustness of anti-tumor immune responses. We observed considerable heterogeneity in the expression of molecules associated with activation of the T cell antigen receptor (TCR) and of immunological-checkpoint molecules such as 4-1BB, PD-1 and TIM-3. Tumors with a high density of CTLs showed enrichment for transcripts linked to tissue-resident memory cells (TRM cells), such as CD103, and CTLs from CD103hi tumors displayed features of enhanced cytotoxicity. A greater density of TRM cells in tumors was predictive of a better survival outcome in lung cancer, and this effect was independent of that conferred by CTL density. Here we define the 'molecular fingerprint' of tumor-infiltrating CTLs and identify potentially new targets for immunotherapy.

Genome-Wide Posttranscriptional Dysregulation by MicroRNAs in Human Asthma as Revealed by Frac-seq.

MicroRNAs are small noncoding RNAs that inhibit gene expression posttranscriptionally, implicated in virtually all biological processes. Although the effect of individual microRNAs is generally studied, the genome-wide role of multiple microRNAs is less investigated. We assessed paired genome-wide expression of microRNAs with total (cytoplasmic) and translational (polyribosome-bound) mRNA levels employing subcellular fractionation and RNA sequencing (Frac-seq) in human primary bronchoepithelium from healthy controls and severe asthmatics. Severe asthma is a chronic inflammatory disease of the airways characterized by poor response to therapy. We found genes (i.e., isoforms of a gene) and mRNA isoforms differentially expressed in asthma, with novel inflammatory and structural pathophysiological mechanisms related to bronchoepithelium disclosed solely by polyribosome-bound mRNAs (e.g., IL1A and LTB genes or ITGA6 and ITGA2 alternatively spliced isoforms). Gene expression (i.e., isoforms of a gene) and mRNA expression analysis revealed different molecular candidates and biological pathways, with differentially expressed polyribosome-bound and total mRNAs also showing little overlap. We reveal a hub of six dysregulated microRNAs accounting for ∼90% of all microRNA targeting, displaying preference for polyribosome-bound mRNAs. Transfection of this hub in bronchial epithelial cells from healthy donors mimicked asthma characteristics. Our work demonstrates extensive posttranscriptional gene dysregulation in human asthma, in which microRNAs play a central role, illustrating the feasibility and importance of assessing posttranscriptional gene expression when investigating human disease.

Mycobacterium tuberculosis subverts negative regulatory pathways in human macrophages to drive immunopathology.

Tuberculosis remains a global pandemic and drives lung matrix destruction to transmit. Whilst pathways driving inflammatory responses in macrophages have been relatively well described, negative regulatory pathways are less well defined. We hypothesised that Mycobacterium tuberculosis (Mtb) specifically targets negative regulatory pathways to augment immunopathology. Inhibition of signalling through the PI3K/AKT/mTORC1 pathway increased matrix metalloproteinase-1 (MMP-1) gene expression and secretion, a collagenase central to TB pathogenesis, and multiple pro-inflammatory cytokines. In patients with confirmed pulmonary TB, PI3Kδ expression was absent within granulomas. Furthermore, Mtb infection suppressed PI3Kδ gene expression in macrophages. Interestingly, inhibition of the MNK pathway, downstream of pro-inflammatory p38 and ERK MAPKs, also increased MMP-1 secretion, whilst suppressing secretion of TH1 cytokines. Cross-talk between the PI3K and MNK pathways was demonstrated at the level of eIF4E phosphorylation. Mtb globally suppressed the MMP-inhibitory pathways in macrophages, reducing levels of mRNAs encoding PI3Kδ, mTORC-1 and MNK-1 via upregulation of miRNAs. Therefore, Mtb disrupts negative regulatory pathways at multiple levels in macrophages to drive a tissue-destructive phenotype that facilitates transmission.

Immunological biomarkers as indicators for outcome after discontinuation of nucleos(t)ide analogue therapy in patients with HBeAg-negative chronic hepatitis B.

The optimal duration of treatment with nucleos(t)ide analogues (NAs) for patients with HBeAg-negative chronic hepatitis B (CHB) is unknown. The aim of this study was to identify an immune signature associated with off-treatment remission to NA therapy. We performed microarray analysis of peripheral blood mononuclear cell (PBMCs) from six patients with chronic hepatitis B who stopped NA therapy (three with off-treatment remission, three with relapse) and five patients with chronic HBV infection (previously termed 'inactive carriers') served as controls. Results were validated using qRT-PCR on a second group of 21 individuals (17 patients who stopped treatment and four controls). PBMCs from 38 patients on long-term NA treatment were analysed for potential to stop treatment. Microarray analysis indicated that patients with off-treatment remission segregated as a distinct out-group. Twenty-one genes were selected for subsequent validation. Ten of these were expressed at significantly lower levels in the patients with off-treatment remission compared to the patients with relapse and predicted remission with AUC of 0.78-0.92. IFNγ, IL-8, FASLG and CCL4 were the most significant by logistic regression. Twelve (31.6%) of 38 patients on long-term NA therapy had expression levels of all these four genes below cut-off values and hence were candidates for stopping treatment. Our data suggest that patients with HBeAg-negative CHB who remain in off-treatment remission 3 years after NA cessation have a distinct immune signature and that PBMC RNA levels of IFNγ, IL-8, FASLG and CCL4 may serve as potential biomarkers for stopping NA therapy.

Modulation of nonsense mediated decay by rapamycin.

Rapamycin is a naturally occurring macrolide whose target is at the core of nutrient and stress regulation in a wide range of species. Despite well-established roles as an inhibitor of cap-dependent mRNA translation, relatively little is known about its effects on other modes of RNA processing. Here, we characterize the landscape of rapamycin-induced post-transcriptional gene regulation. Transcriptome analysis of rapamycin-treated cells reveals genome-wide changes in alternative mRNA splicing and pronounced changes in NMD-sensitive isoforms. We demonstrate that despite well-documented attenuation of cap-dependent mRNA translation, rapamycin can augment NMD of certain transcripts. Rapamycin-treatment significantly reduces the levels of both endogenous and exogenous Premature Termination Codon (PTC)-containing mRNA isoforms and its effects are dose-, UPF1- and 4EBP-dependent. The PTC-containing SRSF6 transcript exhibits a shorter half-life upon rapamycin-treatment as compared to the non-PTC isoform. Rapamycin-treatment also causes depletion of PTC-containing mRNA isoforms from polyribosomes, underscoring the functional relationship between translation and NMD. Enhanced NMD activity also correlates with an enrichment of the nuclear Cap Binding Complex (CBC) in rapamycin-treated cells. Our data demonstrate that rapamycin modulates global RNA homeostasis by NMD.

IFN-γ Influences Epithelial Antiviral Responses via Histone Methylation of the RIG-I Promoter.

The asthmatic lung is prone to respiratory viral infections that exacerbate the symptoms of the underlying disease. Recent work has suggested that a deficient T-helper cell type 1 response in early life may lead to these aberrant antiviral responses. To study the development of long-term dysregulation of innate responses, which is a hallmark of asthma, we investigated whether the inflammatory environment of the airway epithelium can modulate antiviral gene expression via epigenetic mechanisms. We primed AALEB cells, a human bronchial epithelial cell line, with IFN-γ and IL-13, and subsequently infected the cells with respiratory syncytial virus (RSV). We then analyzed the expression of innate antiviral genes and their epigenetic markers. Priming epithelial cells with IFN-γ reduced the RSV viral load. Microarray analysis identified that IFN-γ priming enhanced retinoic acid-inducible gene (RIG)-I mRNA expression, and this expression correlated with epigenetic changes at the RIG-I promoter that influenced its transcription. Using chromatin immunoprecipitation, we observed a reduction of trimethylated histone 3 lysine 9 at the RIG-I promoter. Addition of inhibitor BIX-01294 to this model indicated an involvement of lysine methyltransferase G9a in RIG-I epigenetic regulation. These data suggest that prior exposure to IFN-γ may leave an epigenetic mark on the chromatin that enhances airway cells' ability to resist infection, possibly via epigenetic upregulation of RIG-I. These observations provide further evidence for a crucial role of IFN-γ in the development of mature antiviral responses within a model of respiratory infection. Further clinical validation is required to determine whether this effect in early life leads to changes in antiviral responses associated with asthma.

Toll-like Receptor 7 Is Reduced in Severe Asthma and Linked to an Altered MicroRNA Profile.

RATIONALE: Asthma is one of the most common chronic diseases worldwide, and individuals with severe asthma experience recurrent exacerbations. Exacerbations are predominantly viral associated and have been linked to defective airway IFN responses. Ascertaining the molecular mechanisms underlying this deficiency is a major research goal to identify new therapeutic targets. OBJECTIVES: We investigated the hypothesis that reduced Toll-like receptor 7 (TLR7)-derived signaling drove the impaired IFN responses to rhinovirus by asthmatic alveolar macrophages (AMs); the molecular mechanisms underlying this deficiency were explored. METHODS: AMs were recovered from bronchoalveolar lavage from healthy subjects and patients with severe asthma. Expression of pattern-recognition receptors and microRNAs was evaluated by quantitative polymerase chain reaction and Western blotting. A TLR7-luciferase reporter construct was created to evaluate binding of microRNAs to the 3' untranslated region of TLR7. IFN production was measured by quantitative polymerase chain reaction and ELISA. MEASUREMENTS AND MAIN RESULTS: The expression of TLR7 was significantly reduced in severe asthma AMs and was associated with reduced rhinovirus and imiquimod-induced IFN responses by these cells compared with healthy AMs. Severe asthma AMs also expressed increased levels of three microRNAs, which we showed were able to directly reduce TLR7 expression. Ex vivo knockdown of these microRNAs restored TLR7 expression with concomitant augmentation of virus-induced IFN production. CONCLUSIONS: In severe asthma, TLR7 deficiency drives impaired innate immune responses to virus by AMs. Blocking a group of microRNAs that are up-regulated in these cells can restore antiviral innate responses, providing a novel approach for therapy in asthma.

Genetic factors in susceptibility to contact sensitivity.

There are clear differences in individual susceptibility to the development of contact allergies; some individuals readily become allergic to many chemicals, and others remain clinically tolerant of everything that they come into contact with. A great number of molecules and pathways can contribute to the perturbation by xenobiotics and the subsequent possible immune response. It is necessary to consider susceptibility in two ways: as allergen-specific and as non-allergen-specific. It is likely that different receptor pathways and processes will be involved in the different forms of susceptibility. As investigations of the genetic control of such susceptibility have failed to identify major genetic control, it is likely that small contributions will be made by many components. Whereas genome-wide associations and transcriptome analyses may reveal genetic clues in the future, explanation of how/why the expression of multiple molecular components can be controlled in a coordinated fashion may follow from investigation of microRNAs. It is becoming clear that microRNAs can regulate the expression of multiple genes and even multiple components of biochemical pathways.

MicroRNAs and respiratory diseases.

MicroRNAs (miRNAs) are a family of endogenous, small, noncoding RNA molecules that modulate physiological and pathological processes by post-transcriptional inhibition of gene expression. They were first recognised as regulators of development in worms and fruitflies. In recent years extensive research has explored their pivotal role in the pathogenesis of human diseases. Over 1,000 human miRNAs have been discovered to date; however, the biological function and protein targets for the majority remain to be uncovered. Within the respiratory system, miRNAs are important in normal pulmonary development and maintaining lung homeostasis. Recent studies have also begun to reveal that altered miRNA expression profiles may be associated with pathological processes within the lung and lead to the development of various pulmonary diseases, ranging from inflammatory diseases to lung cancers. Advancing our understanding of the role of miRNAs in the respiratory system will help provide new perspectives on disease mechanisms and reveal intriguing therapeutic targets and diagnostic markers for respiratory disorders.

The interleukin 13 (IL-13) pathway in human macrophages is modulated by microRNA-155 via direct targeting of interleukin 13 receptor alpha1 (IL13Ralpha1).

Macrophages play a central role in the balance and efficiency of the immune response and are at the interface between innate and adaptive immunity. Their phenotype is a delicate equilibrium between the M1 (classical, pro-Th(1)) and M2 (alternative, pro-Th(2)) profiles. This balance is regulated by cytokines such as interleukin 13 (IL-13), a typical pro-M2-Th(2) cytokine that has been related to allergic disease and asthma. IL-13 binds to IL-13 receptor α1 (IL13Rα1), a component of the Type II IL-4 receptor, and exerts its effects by activating the transcription factor signal transducer and activator of transcription 6 (STAT6) through phosphorylation. MicroRNAs are short (∼22 nucleotide) inhibitory non-coding RNAs that block the translation or promote the degradation of their specific mRNA targets. By bioinformatics analysis, we found that microRNA-155 (miR-155) is predicted to target IL13Rα1. This suggested that miR-155 might be involved in the regulation of the M1/M2 balance in macrophages by modulating IL-13 effects. miR-155 has been implicated in the development of a healthy immune system and function as well as in the inflammatory pro-Th(1)/M1 immune profile. Here we have shown that in human macrophages, miR-155 directly targets IL13Rα1 and reduces the levels of IL13Rα1 protein, leading to diminished activation of STAT6. Finally we also demonstrate that miR-155 affects the IL-13-dependent regulation of several genes (SOCS1, DC-SIGN, CCL18, CD23, and SERPINE) involved in the establishment of an M2/pro-Th(2) phenotype in macrophages. Our work shows a central role for miR-155 in determining the M2 phenotype in human macrophages.

Interactions of human endothelial cells with gold nanoparticles of different morphologies.

The interactions between noncancerous, primary endothelial cells and gold nanoparticles with different morphologies but the same ligand capping are investigated. The endothelial cells are incubated with gold nanospheres, nanorods, hollow gold spheres, and core/shell silica/gold nanocrystals, which are coated with monocarboxy (1-mercaptoundec-11-yl) hexaethylene glycol (OEG). Cell viability studies show that all types of gold particles are noncytotoxic. The number of particles taken up by the cells is estimated using inductively coupled plasma (ICP), and are found to differ depending on particle morphology. The above results are discussed with respect to heating efficiency. Using experimental data reported earlier and theoretical model calculations which take into account the physical properties and distribution of particles in the cellular microenvironment, it is found that collective heating effects of several cells loaded with nanoparticles must be included to explain the observed viability of the endothelial cells.

Laser-induced damage and recovery of plasmonically targeted human endothelial cells.

Laser-induced techniques that employ the surface plasmon resonances of nanoparticles have recently been introduced as an effective therapeutic tool for destroying tumor cells. Here, we adopt a low-intensity laser-induced technique to manipulate the damage and repair of a vital category of noncancerous cells, human endothelial cells. Endothelial cells construct the interior of blood vessels and play a pivotal role in angiogenesis. The degree of damage and repair of the cells is shown to be influenced by laser illumination in the presence of gold nanoparticles of different morphologies, which either target the cellular membrane or are endocytosed. A pronounced influence of the plasmonic nanoparticle laser treatment on the expression of critical angiogenic genes is shown. Our results show that plasmon-mediated mild laser treatment, combined with specific targeting of cellular membranes, enables new routes for controlling cell permeability and gene regulation in endothelial cells.

MicroRNAs-A Promising Tool for Asthma Diagnosis and Severity Assessment: A Systematic Review.

Micro RNAs (miRNAs) are short, non-coding RNAs (Ribonucleic acids) with regulatory functions that could prove useful as biomarkers for asthma diagnosis and asthma severity-risk stratification. The objective of this systematic review is to identify panels of miRNAs that can be used to support asthma diagnosis and severity-risk assessment. Three databases (Medline, Embase, and SCOPUS) were searched up to 15 September 2020 to identify studies reporting differential expression of specific miRNAs in the tissues of adults and children with asthma. Studies reporting miRNAs associations in animal models that were also studied in humans were included in this review. We identified 75 studies that met our search criteria. Of these, 66 studies reported more than 200 miRNAs that are differentially expressed in asthma patients when compared to non-asthmatic controls. In addition, 16 studies reported 17 miRNAs that are differentially expressed with differences in asthma severity. We were able to construct two panels of miRNAs that are expressed in blood and can serve as core panels to further investigate the practicality and efficiency of using miRNAs as non-invasive biomarkers for asthma diagnosis and severity-risk assessment, respectively.

Targeting the tumor mutanome for personalized vaccination in a TMB low non-small cell lung cancer.

BACKGROUND: Cancer is characterized by an accumulation of somatic mutations, of which a significant subset can generate cancer-specific neoepitopes that are recognized by autologous T cells. Such neoepitopes are emerging as important targets for cancer immunotherapy, including personalized cancer vaccination strategies. METHODS: We used whole-exome and RNA sequencing analysis to identify potential neoantigens for a patient with non-small cell lung cancer. Thereafter, we assessed the autologous T-cell reactivity to the candidate neoantigens using a long peptide approach in a cultured interferon gamma ELISpot and tracked the neoantigen-specific T-cells in the tumor by T-cell receptor (TCR) sequencing. In parallel, identified gene variants were incorporated into a Modified Vaccinia Ankara-based vaccine, which was evaluated in the human leucocyte antigen A*0201 transgenic mouse model (HHD). RESULTS: Sequencing revealed a tumor with a low mutational burden: 2219 sequence variants were identified from the primary tumor, of which 23 were expressed in the transcriptome, involving 18 gene products. We could demonstrate spontaneous T-cell responses to 5/18 (28%) mutated gene variants, and further analysis of the TCR repertoire of neoantigen-specific CD4+ and CD8+ T cells revealed TCR clonotypes that were expanded in both blood and tumor tissue. Following vaccination of HHD mice, de novo T-cell responses were generated to 4/18 (22%) mutated gene variants; T cells reactive against two variants were also evident in the autologous setting. Subsequently, we determined the major histocompatibility complex restriction of the T-cell responses and used in silico prediction tools to determine the likely neoepitopes. CONCLUSIONS: Our study demonstrates the feasibility of efficiently identifying tumor-specific neoantigens that can be targeted by vaccination in tumors with a low mutational burden, promising successful clinical exploitation, with trials currently underway.

MicroRNA-155 targets SMAD2 and modulates the response of macrophages to transforming growth factor-{beta}.

Transforming growth factor-beta (TGF-ß) is a pleiotropic cytokine with important effects on processes such as fibrosis, angiogenesis, and immunosupression. Using bioinformatics, we identified SMAD2, one of the mediators of TGF-ß signaling, as a predicted target for a microRNA, microRNA-155 (miR-155). MicroRNAs are a class of small non-coding RNAs that have emerged as an important class of gene expression regulators. miR-155 has been found to be involved in the regulation of the immune response in myeloid cells. Here, we provide direct evidence of binding of miR-155 to a predicted binding site and the ability of miR-155 to repress SMAD2 protein expression. We employed a lentivirally transduced monocyte cell line (THP1-155) containing an inducible miR-155 transgene to show that endogenous levels of SMAD2 protein were decreased after sustained overexpression of miR-155. This decrease in SMAD2 led to a reduction in both TGF-ß-induced SMAD-2 phosphorylation and SMAD-2-dependent activation of the expression of the CAGA(12)LUC reporter plasmid. Overexpression of miR-155 altered the cellular responses to TGF-ß by changing the expression of a set of genes that is involved in inflammation, fibrosis, and angiogenesis. Our study provides firm evidence of a role for miR-155 in directly repressing SMAD2 expression, and our results demonstrate the relevance of one of the two predicted target sites in SMAD2 3'-UTR. Altogether, our data uncover an important role for miR-155 in modulating the cellular response to TGF-ß with possible implications in several human diseases where homeostasis of TGF-ß might be altered.

Receptor-mediated interactions between colloidal gold nanoparticles and human umbilical vein endothelial cells.

A new strategy to manipulate cell operations is demonstrated, based on membrane-receptor-specific interactions between colloidal peptide-capped gold nanoparticles and human umbilical vein endothelial cells. It is shown that colloidal gold nanoparticles of similar charge and size but capped with different peptide sequences can deliberately trigger specific cell functions related to the important biological process of blood vessel growth known as angiogenesis. Specific binding of the peptide-capped particles to two endothelial-expressed receptors (VEGFR-1, NRP-1), which control angiogenesis, is achieved. The cellular fate of the functional nanoparticles is imaged and the influence of the different peptide-coated nanoparticles on the gene expression profile of hypoxia-related and angiogenic genes is monitored. The findings open up new avenues towards the deliberate biological control of cellular functions using strategically designed nanoparticles.

Human Intestinal Macrophages Are Involved in the Pathology of Both Ulcerative Colitis and Crohn Disease.

BACKGROUND: Intestinal macrophages are key immune cells in the maintenance of intestinal immune homeostasis and have a role in the pathogenesis of inflammatory bowel disease (IBD). However, the mechanisms by which macrophages exert a pathological influence in both ulcerative colitis (UC) and Crohn disease (CD) are not yet well understood. METHODS: We purified intestinal macrophages from gastrointestinal mucosal biopsies (patients with UC, patients with CD, and healthy donors) and analyzed their transcriptome by RNA sequencing and bioinformatics, confirming results with quantitative polymerase chain reaction and immunohistochemistry. RESULTS: Compared with those of healthy donors, intestinal macrophages in patients with UC and with CD showed cellular reprograming of 1287 and 840 dysregulated genes, respectively (false discovery rate ≤ 0.1). The UC and CD intestinal macrophages showed an activated M1 inflammatory phenotype and the downregulation of genes engaged in drug/xenobiotic metabolism. Only macrophages from CD showed, concomitant to an M1 phenotype, a significant enrichment in the expression of M2 and fibrotic and granuloma-related genes. For the first time, we showed (and validated by quantitative polymerase chain reaction and immunohistochemistry) that intestinal macrophages in patients with IBD present both M1 and M2 features, as recently described for tumor-associated macrophages, that affect key pathways for IBD pathology, represented by key markers such as MMP12 (fibrosis), CXCL9 (T-cell attraction), and CD40 (T-cell activation). CONCLUSIONS: Our data support the therapeutic targeting of macrophages to maintain remission in IBD but also indicate that a shift toward an M2 program-as proposed by some reports-may not limit the recruitment and activation of T cells because M2 features do not preclude M1 activation in patients with UC or CD and could exacerbate M2-related CD-specific features such as fibrosis and the formation of granulomas.

Circulating miRNAs-A potential tool to identify severe asthma risk?

BACKGROUND: Identifying patients at risk of severe asthma is vitally important given the disproportionate burden of disease imposed by that state. However, biomarkers to support such needs remain elusive. METHODS: In this letter, we assessed whether specific panels of circulating miRNAs (microRNAs) can differentiate between mild and severe asthma patients as well as between healthy subjects and severe asthma patients. RESULTS: To our knowledge, the miRNAs identified in our work such as miR-28-3p, miR-16-2-3p, and miR-210-3p have not been previously reported as differentially expressed in the serum of severe asthma patients. CONCLUSION: Our findings suggest that miRNA expression profiles may have the capability as potential biomarkers that signal the risk of having severe asthma. As such, these findings have significant novelty and merit wider dissemination to facilitate further work in this field.