A three-stage intrathymic development pathway for the mucosal-associated invariant T cell lineage.

Mucosal-associated invariant T cells (MAIT cells) detect microbial vitamin B2 derivatives presented by the antigen-presenting molecule MR1. Here we defined three developmental stages and checkpoints for the MAIT cell lineage in humans and mice. Stage 1 and stage 2 MAIT cells predominated in thymus, while stage 3 cells progressively increased in abundance extrathymically. Transition through each checkpoint was regulated by MR1, whereas the final checkpoint that generated mature functional MAIT cells was controlled by multiple factors, including the transcription factor PLZF and microbial colonization. Furthermore, stage 3 MAIT cell populations were expanded in mice deficient in the antigen-presenting molecule CD1d, suggestive of a niche shared by MAIT cells and natural killer T cells (NKT cells). Accordingly, this study maps the developmental pathway and checkpoints that control the generation of functional MAIT cells.

Drosha controls dendritic cell development by cleaving messenger RNAs encoding inhibitors of myelopoiesis.

To investigate if the microRNA (miRNA) pathway is required for dendritic cell (DC) development, we assessed the effect of ablating Drosha and Dicer, the two enzymes central to miRNA biogenesis. We found that while Dicer deficiency had some effect, Drosha deficiency completely halted DC development and halted myelopoiesis more generally. This indicated that while the miRNA pathway did have a role, it was a non-miRNA function of Drosha that was particularly critical. Drosha repressed the expression of two mRNAs encoding inhibitors of myelopoiesis in early hematopoietic progenitors. We found that Drosha directly cleaved stem-loop structure within these mRNAs and that this mRNA degradation was necessary for myelopoiesis. We have therefore identified a mechanism that regulates the development of DCs and other myeloid cells.

Highlight of 2023: big impacts of microRNAs in T cells.

In 2023, several significant discoveries on the function of microRNAs in the immune system were reported. Here we discuss several notable papers that revealed important functions in T cells.

Mapping the two distinct proliferative bursts early in T-cell development.

T-cell development occurs in the thymus and is tightly regulated to produce a diverse enough repertoire of mature T cells that can recognize any potential pathogen. The development of T cells is dependent on small numbers of uncommitted precursors that continually seed the thymus from the bone marrow. As they progress along the developmental pathway, there is a massive expansion in cell number to generate the necessary diversity in T-cell receptor chain usage. It is recognized that there are two proliferative bursts that occur early in T-cell development, one prior to ß-selection and one after, and these are responsible for the expansion. While the proliferation following ß-selection is well-characterized, the earlier proliferative burst has yet to be precisely defined. In this study, we employ single-cell RNA sequencing coupled to trajectory inference methods to pinpoint when in T-cell development thymocytes are induced into cell cycle. We show that the first proliferative burst is initiated in the double-negative (DN) 2a stage before T lineage commitment occurs, with cell cycling downregulated by the DN3a stage. A second burst is then initiated at the DN3b stage, immediately after ß-selection. We subsequently employ fluorescence-activated cell sorting-based analysis for DNA content to confirm these two proliferative bursts.

Single-Cell RNA Sequencing Approaches for Tracing T Cell Development.

T cell development occurs in the thymus, where uncommitted progenitors are directed into a range of sublineages with distinct functions. The goal is to generate a TCR repertoire diverse enough to recognize potential pathogens while remaining tolerant of self. Decades of intensive research have characterized the transcriptional programs controlling critical differentiation checkpoints at the population level. However, greater precision regarding how and when these programs orchestrate differentiation at the single-cell level is required. Single-cell RNA sequencing approaches are now being brought to bear on this question, to track the identity of cells and analyze their gene expression programs at a resolution not previously possible. In this review, we discuss recent advances in the application of these technologies that have the potential to yield unprecedented insight to T cell development.

Higher well-being individuals are more receptive to cultivated meat: An investigation of their reasoning for consuming cultivated meat.

It is evident that over-consumption of meat can contribute to the emission of hazardous greenhouse gases. One viable way to address such climate impact is to make people become more aware of more sustainable diet options, such as cultivated meat. However, it is challenging to instigate change in people's meat-eating habit, and empirical works have been examining the psychological factors that are related to consumers' willingness to consume cultivated meat. Research has suggested that psychological well-being can play a role in the meaning-making of food consumption, with higher well-being individuals showing more recognition of other sociocultural benefits of consuming food beyond just fulfilling their sustenance needs. As existing works have yet to understand the link between well-being and consumption of novel foods, the current research set out to fill this gap by examining the relationship between people's psychological well-being and their willingness to consume cultivated meat via different reasons (mediators) for consuming cultivated meat. We recruited a representative sample of 948 adults in Singapore to complete an online survey. The study offered the first evidence that there is a positive relationship between people's psychological well-being and their willingness to consume cultivated meat. Further, results revealed that their higher willingness can be motivated by the perception that cultivated meat is as healthy and nutritious, as safe as, and has the same sensory quality as real meat, and is beneficial to the society. This investigation adds to the growing literature on consumer acceptance of cultivated meat by showing the novel finding that well-being and receptivity to cultivated meat is positively linked, and such a positive link can be explained by people's better recognition of the prospective benefits offered by this alternative food.

Expression of the miR-17~92a cluster of microRNAs by regulatory T cells controls blood glucose homeostasis.

Regulatory T cells (Tregs) are a specialized immune cell type that play important roles in regulating immune responses. However, those found in adipose tissue, particularly visceral adipose tissue (VAT), have also been shown to exert metabolic regulatory functions. This study investigated the requirement of the miR-17~92a cluster of microRNAs in VAT Tregs and the impact on blood glucose. This cluster of microRNAs is one that we previously showed to be important for the fitness of Tregs found in secondary lymphoid organs. It was found that male mice with Treg-specific miR-17~92a deficiency are resistant to impaired glucose tolerance induced by a high-fat diet. However, high-fat feeding still impaired glucose tolerance in female mice with Treg-specific miR-17~92a deficiency. There was an increase in KLRG1- naïve Tregs and a loss of KLRG1+ terminally differentiated Tregs in the VAT of Treg-specific miR-17~92a-deficient male mice but not in female mice. The protection of male mice from high-fat feeding was also associated with increased interleukin-10 and reduced interferonγ expression by conventional CD4+ T cells and reduced interleukin-2 expression by both CD4+ and CD8+ T cells in the VAT. Together this suggests that expression of miR-17~92a by VAT Tregs regulates the effector phenotype of conventional T cells and in turn the metabolic function of adipose tissue and blood glucose homeostasis.

A cross-country investigation of social image motivation and acceptance of lab-grown meat in Singapore and the United States.

This research has three goals. First, it sets out to compare consumer acceptance of lab-grown meat in the U.S. and in Singapore. Second, it seeks to explain the difference in Americans' and Singaporeans' acceptance of lab-grown meat by examining their eating motivations. Specifically, we focused on social image motivations - the motivations to present oneself positively in social contexts. Third, this study also aims to assess if exposure to information about lab-grown meat communicated by celebrity versus expert social media influencers (SMIs) can impact people's acceptance of lab-grown meat products. Our analysis showed that Singaporean participants had greater acceptance of lab-grown meat compared to their American counterparts, and this cultural difference was explained by Singaporeans' stronger social image eating motivations. In other words, cross-country differences in motivation to eat for a favorable social image can explain differences in consumer acceptance of lab-grown meat. The Singaporean cultural trait of kiasuism, which is exemplified by the fear of losing out or being left behind, may explain Singaporeans' motivation to project an image of being 'trailblazers' (vis-a-vis other nationalities) by expressing a higher acceptance of novel foods such as lab-grown meat. Results also revealed that the information about lab-grown meat being communicated by a celebrity or an expert SMI did not make a difference in participants' acceptance of lab-grown meat in both countries. Together, this research suggests an interesting implication that novel food industries and marketers can promote product branding by boosting media coverage (including online social media) of their lab-grown products' 'firsts' (e.g., the first production line in the world, the first technological breakthrough), especially in markets with high social image concerns.

Runx-CBFbeta complexes control expression of the transcription factor Foxp3 in regulatory T cells.

The transcription factor Foxp3 has an indispensable role in establishing stable transcriptional and functional programs of regulatory T cells (T(reg) cells). Loss of Foxp3 expression in mature T(reg) cells results in a failure of suppressor function, yet the molecular mechanisms that ensure steady, heritable Foxp3 expression in the T(reg) cell lineage remain unknown. Using T(reg) cell-specific gene targeting, we found that complexes of the transcription factors Runx and CBFbeta were required for maintenance of Foxp3 mRNA and protein expression in T(reg) cells. Consequently, mice lacking CBFbetab exclusively in the T(reg) cell lineage had a moderate lymphoproliferative syndrome. Thus, Runx-CBFbeta complexes maintain stable high expression of Foxp3 and serve as an essential determinant of T(reg) cell lineage stability.

Do Patients With Diabetes Have Poorer Improvements in Patient-Reported Outcomes After Total Knee Arthroplasty?

BACKGROUND: Diabetes is one of the most common comorbidities in patients undergoing total knee arthroplasty (TKA) for osteoarthritis. However, the evidence remains unclear on how it affects patient-reported outcome measures after TKA. METHODS: We reviewed prospectively collected data of 2840 patients who underwent primary unilateral TKA between 2008 and 2018, of which 716 (25.2%) had diabetes. All patients had their HbA1c measured within 1 month before surgery, and only well-controlled diabetics (HbA1c <8.0%) were allowed to proceed with surgery. Patient demographics and comorbidities were recorded, and multiple regression was performed to evaluate the impact of diabetes on improvements in patient-reported outcome measures (Short Form 36 (SF-36), Western Ontario and McMaster University Osteoarthritis Index (WOMAC), Knee Society Score (KSS)) and knee range of motion (ROM). RESULTS: Compared with nondiabetics, patients with diabetes were more likely to possess a higher body mass index (P-value <.001), more comorbidities (P-value <.001), and poorer preoperative SF-36 Physical Component Summary (PCS) (P-value <.001), WOMAC (P-value = .002), KSS-function (P-value <.001), and knee ROM (P-value <.001). Multiple regression showed that diabetic patients experienced marginally poorer improvements in KSS-knee (-1.22 points, P-value = .025) and knee ROM (-1.67°, P-value = .013) than nondiabetics. However, there were no significant differences in improvements for SF-36 PCS (P-value = .163), Mental Component Summary (P-value = .954), WOMAC (P-value = .815), and KSS-function (P-value = .866). CONCLUSION: Patients with well-controlled diabetes (HbA1c <8.0%) can expect similar improvements in general health and osteoarthritis outcomes (SF-36 PCS and Mental Component Summary, WOMAC, and KSS-function) compared with nondiabetics after TKA. Despite having marginally poorer improvements in knee-specific outcomes (KSS-knee and knee ROM), these differences are unlikely to be clinically significant.

RUNX transcription factor-mediated association of Cd4 and Cd8 enables coordinate gene regulation.

T cell fate is associated with mutually exclusive expression of CD4 or CD8 in helper and cytotoxic T cells, respectively. How expression of one locus is temporally coordinated with repression of the other has been a long-standing enigma, though we know RUNX transcription factors activate the Cd8 locus, silence the Cd4 locus, and repress the Zbtb7b locus (encoding the transcription factor ThPOK), which is required for CD4 expression. Here we found that nuclear organization was altered by interplay among members of this transcription factor circuitry: RUNX binding mediated association of Cd4 and Cd8 whereas ThPOK binding kept the loci apart. Moreover, targeted deletions within Cd4 modulated CD8 expression and pericentromeric repositioning of Cd8. Communication between Cd4 and Cd8 thus appears to enable long-range epigenetic regulation to ensure that expression of one excludes the other in mature CD4 or CD8 single-positive (SP) cells.

Dicer1-mediated miRNA processing shapes the mRNA profile and function of murine platelets.

Human platelets contain microRNAs (miRNAs) and miRNA processing machinery, but their contribution to platelet function remains incompletely understood. Here, we show that murine megakaryocyte (MK)-specific knockdown of Dicer1, the ribonuclease that cleaves miRNA precursors into mature miRNAs, reduces the level of the majority of miRNAs in platelets. This leads to altered platelet messenger RNA (mRNA) expression profiles and mild thrombocytopenia. Fibrinogen receptor subunits Itga2b (αIIb) and Itgb3 (ß3) mRNAs were among the differentially expressed transcripts that are increased in platelets lacking Dicer1. Argonaute 2 (Ago2), a member of the miRNA silencing complex, co-immunoprecipitated with αIIband ß3mRNAs in wild-type platelets. Furthermore, co-immunoprecipitation experiments suggested reduced αIIb/ß3/Ago2 complexes in miRNA-deficient platelets. These results suggested that miRNAs regulate both integrin subunits. Subsequent 3' untranslated region luciferase reporter assays confirmed that the translation of both αIIband ß3mRNAs can be regulated by miRNAs miR-326, miR-128, miR-331, and miR-500. Consistent with these molecular changes, the deletion ofDicer1resulted in increased surface expression of integrins αIIband ß3, and enhanced platelet binding to fibrinogen in vivo and in vitro. Heightened platelet reactivity, shortened tail-bleeding time, and reduced survival following collagen/epinephrine-induced pulmonary embolism were also observed in Dicer1-deficient animals. CombinedPf4-cre-mediated deletion of Drosha and Dicer1 did not significantly exacerbate phenotypes observed in single Dicer1 knockout mice. In summary, these findings indicate that Dicer1-dependent generation of mature miRNAs in late-stage MKs and platelets modulates the expression of target mRNAs important for the hemostatic and thrombotic function of platelets.

Plasticity of CD4+ T cell lineage differentiation.

The differentiation of naive CD4(+) T cells into lineages with distinct effector functions has been considered to be an irreversible event. T helper type 1 (Th1) cells stably express IFN-gamma, whereas Th2 cells express IL-4. The discovery and investigation of two other CD4(+) T cell subsets, induced regulatory T (iTreg) cells and Th17 cells, has led to a rethinking of the notion that helper T cell subsets represent irreversibly differentiated endpoints. Accumulating evidence suggests that CD4(+) T cells, particularly iTreg and Th17 cells, are more plastic than previously appreciated. It appears that expression of Foxp3 by iTreg cells or IL-17 by Th17 cells may not be stable and that there is a great degree of flexibility in their differentiation options. Here, we will discuss recent findings that demonstrate the plasticity of CD4(+) T cell differentiation and the biological implications of this flexibility.

Diverse endonucleolytic cleavage sites in the mammalian transcriptome depend upon microRNAs, Drosha, and additional nucleases.

The life span of a mammalian mRNA is determined, in part, by the binding of regulatory proteins and small RNA-guided complexes. The conserved endonuclease activity of Argonaute2 requires extensive complementarity between a small RNA and its target and is not used by animal microRNAs, which pair with their targets imperfectly. Here we investigate the endonucleolytic function of Ago2 and other nucleases by transcriptome-wide profiling of mRNA cleavage products retaining 5' phosphate groups in mouse embryonic stem cells (mESCs). We detect a prominent signature of Ago2-dependent cleavage events and validate several such targets. Unexpectedly, a broader class of Ago2-independent cleavage sites is also observed, indicating participation of additional nucleases in site-specific mRNA cleavage. Within this class, we identify a cohort of Drosha-dependent mRNA cleavage events that functionally regulate mRNA levels in mESCs, including one in the Dgcr8 mRNA. Together, these results highlight the underappreciated role of endonucleolytic cleavage in controlling mRNA fates in mammals.

The Social Amplification of Haze-Related Risks on the Internet.

This study explores the implications of the digital network society for public health communication and management through an empirical study on communication related to the transboundary haze crisis in Singapore. Using the Social Amplification of Risk Framework (SARF), the authors applied sentiment and thematic analysis on haze-related posts on an online discussion forum (HardwareZone) and a social networking site (Facebook), and to haze-related articles in The Straits Times (a newspaper). The study shows that the medium matters in social amplification of risk: Facebook had an effect on the amplification of emotions, while HardwareZone and The Straits Times did not. In addition, the results show that spikes in online risk amplification were strongly influenced by unprecedented events. They also suggest that anger expressed online may be linked to a sense of futility. Implications for practice and recommendations for future research are provided in the conclusion.

Canonical and alternate functions of the microRNA biogenesis machinery.

The canonical microRNA (miRNA) biogenesis pathway requires two RNaseIII enzymes: Drosha and Dicer. To understand their functions in mammals in vivo, we engineered mice with germline or tissue-specific inactivation of the genes encoding these two proteins. Changes in proteomic and transcriptional profiles that were shared in Dicer- and Drosha-deficient mice confirmed the requirement for both enzymes in canonical miRNA biogenesis. However, deficiency in Drosha or Dicer did not always result in identical phenotypes, suggesting additional functions. We found that, in early-stage thymocytes, Drosha recognizes and directly cleaves many protein-coding messenger RNAs (mRNAs) with secondary stem-loop structures. In addition, we identified a subset of miRNAs generated by a Dicer-dependent but Drosha-independent mechanism. These were distinct from previously described mirtrons. Thus, in mammalian cells, Dicer is required for the biogenesis of multiple classes of miRNAs. Together, these findings extend the range of function of RNaseIII enzymes beyond canonical miRNA biogenesis, and help explain the nonoverlapping phenotypes caused by Drosha and Dicer deficiency.

Epigenetic propagation of CD4 expression is established by the Cd4 proximal enhancer in helper T cells.

The stability of a lineage program (cellular memory) is dependent on mechanisms that epigenetically maintain active or repressed states of gene expression (transcriptional memory). Although epigenetic silencing of genes has been clearly demonstrated from yeast to mammals, heritable maintenance of active transcription has been less clearly defined. To investigate the potential role of active transcriptional memory during lineage diversification, we employed targeted mutation of a positive-acting cis element in the Cd4 locus to determine the impact on CD4 expression and the differentiation of CD4(+) helper T cells in mice. We show that the proximal enhancer (E4(P)) of Cd4 is essential for CD4 expression in immature CD4(+)8(+) thymocytes. Furthermore, its loss resulted in reduced and unstable expression of CD4 in mature T cells. However, if the enhancer was deleted after cells had already committed to the helper T-cell lineage, CD4 expression remained high and was stable upon cell division. "Active" histone modifications, once initiated by E4(P), were also propagated independently of the enhancer. Thus, E4(P) is responsible for establishing an epigenetically inherited active Cd4 locus in the helper T-cell lineage. To our knowledge, this is the first genetic demonstration of active transcriptional memory in mammalian cells.

DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration.

Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.

In-vivo evaluation of subcutaneously implanted cell-loaded apatite microcarriers for osteogenic potency.

Cell-loaded apatite microcarriers present as potential scaffolds for direct in-vivo delivery of cells post-expansion to promote bone regeneration. The objective of this study was to evaluate the osteogenic potency of human foetal mesenchymal stem cells (hfMSC)-loaded apatite microcarriers when implanted subcutaneously in a mouse model. This was done by examining for ectopic bone formation at 2 weeks, 1 month and 2 months, which were intended to coincide with the inflammation, healing and remodelling phases, respectively. Three histological examinations including haematoxylin and eosin staining to examine general tissue morphology, Masson's trichrome staining to identify tissue type, and Von Kossa staining to examine extent of tissue mineralisation were performed. In addition, immunohistochemistry assay of osteopontin was conducted to confirm active bone formation by the seeded hfMSCs. Results showed a high level of tissue organisation and new bone formation, with active bone remodelling being observed at the end of 2 months, and an increase in tissue density, organisation, and mineralisation could also be observed for hfMSC-loaded apatite microcarriers. Various cell morphology resembling that of osteoblasts and osteoclasts could be seen on the surfaces of the hfMSC-loaded apatite microcarriers, with presence of woven bone tissue formation being observed at the intergranular space. These observations were consistent with evidence of ectopic bone formation, which were absent in group containing apatite microcarriers only. Overall, results suggested that hfMSC-loaded apatite microcarriers retained their osteogenic potency after implantation, and provided an effective platform for bone tissue regeneration.