FEZF2 and AIRE1: An Evolutionary Trade-off in the Elimination of Auto-reactive T Cells in the Thymus.

Autoimmune Regulator 1 (AIRE1) and Forebrain Embryonic Zinc Finger-Like Protein 2 (FEZF2) play pivotal roles in orchestrating the expression of tissue-restricted antigens (TRA) to facilitate the elimination of autoreactive T cells. AIRE1's presence in the gonads of various vertebrates has raised questions about its potential involvement in gene expression control for germline cell selection. Nevertheless, the evolutionary history of these genes has remained enigmatic, as has the rationale behind their apparent redundancy in vertebrates. Furthermore, the origin of the elimination process itself has remained elusive. To shed light on these mysteries, we conducted a comprehensive evolutionary analysis employing a range of tools, including multiple sequence alignment, phylogenetic tree construction, ancestral sequence reconstruction, and positive selection assessment. Our investigations revealed intriguing insights. AIRE1 homologs emerged during the divergence of T cells in higher vertebrates, signifying its role in this context. Conversely, FEZF2 exhibited multiple homologs spanning invertebrates, lampreys, and higher vertebrates. Ancestral sequence reconstruction demonstrated distinct origins for AIRE1 and FEZF2, underscoring that their roles in regulating TRA have evolved through disparate pathways. Furthermore, it became evident that both FEZF2 and AIRE1 govern a diverse repertoire of genes, encompassing ancient and more recently diverged targets. Notably, FEZF2 demonstrates expression in both vertebrate and invertebrate embryos and germlines, accentuating its widespread role. Intriguingly, FEZF2 harbors motifs associated with autophagy, such as DKFPHP, SYSELWKSSL, and SYSEL, a process integral to cell selection in invertebrates. Our findings suggest that FEZF2 initially emerged to regulate self-elimination in the gonads of invertebrates. As organisms evolved toward greater complexity, AIRE1 likely emerged to complement FEZF2's role, participating in the regulation of cell selection for elimination in both gonads and the thymus. This dynamic interplay between AIRE1 and FEZF2 underscores their multifaceted contributions to TRA expression regulation across diverse evolutionary contexts.

Investigation of Mutated in Colorectal Cancer (MCC) Gene Family Evolution History Indicates a Putative Role in Th17/Treg Differentiation.

The MCC family of genes plays a role in colorectal cancer development through various immunological pathways, including the Th17/Treg axis. We have previously shown that MCC1 but not MCC2 plays a role in Treg differentiation. Our understanding of the genetic divergence patterns and evolutionary history of the MCC family in relation to its function, in general, and the Th17/Treg axis, in particular, remains incomplete. In this investigation, we explored 12 species' genomes to study the phylogenetic origin, structure, and functional specificity of this family. In vertebrates, both MCC1 and MCC2 homologs have been discovered, while invertebrates have a single MCC homolog. We found MCC homologs as early as Cnidarians and Trichoplax, suggesting that the MCC family first appeared 741 million years ago (Ma), whereas MCC divergence into the MCC1 and MCC2 families occurred at 540 Ma. In general, we did not detect significant positive selection regulating MCC evolution. Our investigation, based on MCC1 structural similarity, suggests that they may play a role in the evolutionary changes in Tregs' emergence towards complexity, including the ability to utilize calcium for differentiation through the use of the EFH calcium-binding domain. We also found that the motif NPSTGE was highly conserved in MCC1, but not in MCC2. The NPSTGE motif binds KEAP1 with high affinity, suggesting an Nrf2-mediated function for MCC1. In the case of MCC2, we found that the "modifier of rudimentary" motif is highly conserved. This motif contributes to the regulation of alternative splicing. Overall, our study sheds light on how the evolution of the MCC family is connected to its function in regulating the Th17/Treg axis.

Factors regulating the differences in frequency of infiltration of Th17 and Treg of the blood-brain barrier.

Controlling CD4+ immune cell infiltration of the brain is a leading aim in designing therapeutic strategies for a range of neuropathological disorders such as multiple sclerosis, Alzheimer's disease, and depression. CD4+ T cells are a highly heterogeneous and reprogrammable family, which includes various distinctive cell types such as Th17, Th1, and Treg cells. Interestingly Th17 and Treg cells share a related transcriptomic profile, where the TGFß-SMADS pathway plays a fundamental role in regulating the differentiation of both of these cell types. However, Th17 could be highly pathogenic and was shown to promote inflammation in various neuropathological disorders. Conversely, Treg is anti-inflammatory and is known to inhibit Th17. It could be noticed that Th17 frequencies of infiltration of the blood-brain barrier in various neurological disorders are significantly upregulated. However, Treg infiltration numbers are significantly low. The reasons behind these contradicting observations are still unknown. In this perspective, we propose that the difference in the T-cell receptor repertoire diversity, diapedesis pathways, chemokine expression, and mechanical properties of these two cell types could be contributing to answering this intriguing question.

Investigation of the Molecular Evolution of Treg Suppression Mechanisms Indicates a Convergent Origin.

Regulatory T cell (Treg) suppression of conventional T cells is a central mechanism that ensures immune system homeostasis. The exact time point of Treg emergence is still disputed. Furthermore, the time of Treg-mediated suppression mechanisms' emergence has not been identified. It is not yet known whether Treg suppression mechanisms diverged from a single pathway or converged from several sources. We investigated the evolutionary history of Treg suppression pathways using various phylogenetic analysis tools. To ensure the conservation of function for investigated proteins, we augmented our study using nonhomology-based methods to predict protein functions among various investigated species and mined the literature for experimental evidence of functional convergence. Our results indicate that a minority of Treg suppressor mechanisms could be homologs of ancient conserved pathways. For example, CD73, an enzymatic pathway known to play an essential role in invertebrates, is highly conserved between invertebrates and vertebrates, with no evidence of positive selection (w = 0.48, p-value < 0.00001). Our findings indicate that Tregs utilize homologs of proteins that diverged in early vertebrates. However, our findings do not exclude the possibility of a more evolutionary pattern following the duplication degeneration−complementation (DDC) model. Ancestral sequence reconstruction showed that Treg suppression mechanism proteins do not belong to one family; rather, their emergence seems to follow a convergent evolutionary pattern.

Trends of Anger and Physical Aggression in Russian Women During COVID-19 Lockdown.

The effect of social lockdown during the COVID-19 outbreak on female aggressiveness is not well known. The strict measures of lockdown have resulted in millions of people, worldwide, confined to their homes during the pandemic. However, the consequence of lockdown strategies on females' psychological status including aggressiveness has not yet been investigated. We conducted a cross-sectional study on 31 Russian females' homemakers who are participants in an online fitness platform to investigate the immediate anxiety, depression, and aggression experienced under strict lockdown measures. The participants were surveyed using the hospital anxiety depression scale (HADS) and the Buss-Perry Aggression Questionnaire. We used descriptive and statistical methods to investigate the prevalence of these emotions among two age groups (20-35 and 36-65 years). We found that moderate anxiety prevalence was 77.4% in the entire group while mild depression was 54.8%. Interestingly, the whole sample showed a high level of angriness (p = 0.0002) and physical aggression (p = 0.019). These two emotions seem to be more prevalent than other negative emotions such as hostility, verbal aggression. This relationship was not dependent on age. Overall, there is a significant worsening in female aggression that could lead to higher chances of female victimization and being subjected to partner violence. Future policies designing lockdown strategies should consider this effect on active female homemakers. Due to the small size of our cohort, our results are only indicative of data trends. Larger studies are still needed to confirm the current findings.

Investigation of Evolutionary History and Origin of the Tre1 Family Suggests a Role in Regulating Hemocytes Cells Infiltration of the Blood-Brain Barrier.

Understanding the evolutionary relationship between immune cells and the blood-brain barrier (BBB) is important to devise therapeutic strategies. In vertebrates, immune cells follow either a paracellular or a transcellular pathway to infiltrate the BBB. In Drosophila, glial cells form the BBB that regulates the access of hemocytes to the brain. However, it is still not known which diapedesis route hemocytes cells follow. In vertebrates, paracellular migration is dependent on PECAM1, while transcellular migration is dependent on the expression of CAV1. Interestingly Drosophila genome lacks both genes. Tre1 family (Tre1, moody, and Dmel_CG4313) play a diverse role in regulating transepithelial migration in Drosophila. However, its evolutionary history and origin are not yet known. We performed phylogenetic analysis, together with HH search, positive selection, and ancestral reconstruction to investigate the Tre1 family. We found that Tre1 exists in Mollusca, Arthropoda, Ambulacraria, and Scalidophora. moody is shown to be a more ancient protein and it has existed since Cnidaria emergence and has a homolog (e.g., GPCR84) in mammals. The third family member (Dmel_CG4313) seems to only exist in insects. The origin of the family seems to be related to the rhodopsin-like family and in particular family α. We found that opsin is the nearest receptor to have a common ancestor with the Tre1 family that has diverged in sponges. We investigated the positive selection of the Tre1 family using PAML. Tre1 seems to have evolved under negative selection, whereas moody has evolved during positive selection. The sites that we found under positive selection are likely to play a role in the speciation of function in the case of moody. We have identified an SH3 motif, in Tre1 and, moody and Dmel_CG4313. SH3 is known to play a fundamental role in regulating actin movement in a Rho-dependent manner in PECAM1. Our results suggest that the Tre1 family could be playing an important role in paracellular diapedesis in Drosophila.

Interleukins and Interleukin Receptors Evolutionary History and Origin in Relation to CD4+ T Cell Evolution.

Understanding the evolution of interleukins and interleukin receptors is essential to control the function of CD4+ T cells in various pathologies. Numerous aspects of CD4+ T cells' presence are controlled by interleukins including differentiation, proliferation, and plasticity. CD4+ T cells have emerged during the divergence of jawed vertebrates. However, little is known about the evolution of interleukins and their origin. We traced the evolution of interleukins and their receptors from Placozoa to primates. We performed phylogenetic analysis, ancestral reconstruction, HH search, and positive selection analysis. Our results indicated that various interleukins' emergence predated CD4+ T cells divergence. IL14 was the most ancient interleukin with homologs in fungi. Invertebrates also expressed various interleukins such as IL41 and IL16. Several interleukin receptors also appeared before CD4+ T cells divergence. Interestingly IL17RA and IL17RD, which are known to play a fundamental role in Th17 CD4+ T cells first appeared in mollusks. Furthermore, our investigations showed that there is not any single gene family that could be the parent group of interleukins. We postulate that several groups have diverged from older existing cytokines such as IL4 from TGFß, IL10 from IFN, and IL28 from BCAM. Interleukin receptors were less divergent than interleukins. We found that IL1R, IL7R might have diverged from a common invertebrate protein that contained TIR domains, conversely, IL2R, IL4R and IL6R might have emerged from a common invertebrate ancestor that possessed a fibronectin domain. IL8R seems to be a GPCR that belongs to the rhodopsin-like family and it has diverged from the Somatostatin group. Interestingly, several interleukins that are known to perform a critical function for CD4+ T cells such as IL6, IL17, and IL1B have gained new functions and evolved under positive selection. Overall evolution of interleukin receptors was not under significant positive selection. Interestingly, eight interleukin families appeared in lampreys, however, only two of them (IL17B, IL17E) evolved under positive selection. This observation indicates that although lampreys have a unique adaptive immune system that lacks CD4+ T cells, they could be utilizing interleukins in homologous mode to that of the vertebrates' immune system. Overall our study highlights the evolutionary heterogeneity within the interleukins and their receptor superfamilies and thus does not support the theory that interleukins evolved solely in jawed vertebrates to support T cell function. Conversely, some of the members are likely to play conserved functions in the innate immune system.

Paracellular and Transcellular Leukocytes Diapedesis Are Divergent but Interconnected Evolutionary Events.

Infiltration of the endothelial layer of the blood-brain barrier by leukocytes plays a critical role in health and disease. When passing through the endothelial layer during the diapedesis process lymphocytes can either follow a paracellular route or a transcellular one. There is a debate whether these two processes constitute one mechanism, or they form two evolutionary distinct migration pathways. We used artificial intelligence, phylogenetic analysis, HH search, ancestor sequence reconstruction to investigate further this intriguing question. We found that the two systems share several ancient components, such as RhoA protein that plays a critical role in controlling actin movement in both mechanisms. However, some of the key components differ between these two transmigration processes. CAV1 genes emerged during Trichoplax adhaerens, and it was only reported in transcellular process. Paracellular process is dependent on PECAM1. PECAM1 emerged from FASL5 during Zebrafish divergence. Lastly, both systems employ late divergent genes such as ICAM1 and VECAM1. Taken together, our results suggest that these two systems constitute two different mechanical sensing mechanisms of immune cell infiltrations of the brain, yet these two systems are connected. We postulate that the mechanical properties of the cellular polarity is the main driving force determining the migration pathway. Our analysis indicates that both systems coevolved with immune cells, evolving to a higher level of complexity in association with the evolution of the immune system.

What has single-cell RNA sequencing revealed about microglial neuroimmunology?

The use of single-cell RNA sequencing (scRNA-seq) in microglial research is increasing rapidly. The basic workflow of this approach consists of isolating single cells, followed by sequencing. scRNA-seq is capable of examining microglial heterogeneity on a cellular level. However, the results gained from applying this technique suffer from discrepancies due to differences between applied methods characteristics such as the number of cells sequenced and the depth of sequencing. This review aims to shed more light on the recent developments that happened in this field and how they are related to the methods used. To do that, we track the progress and limitations of various scRNA-seq methods currently available. The review then summarizes the current knowledge gained using scRNA-seq in the field of microglia, including novel subpopulations associated with function and development under homeostasis as well during several pathological conditions such as Alzheimer, lipopolysaccharide response, and HIV in relation to the methods employed. Our review points out that despite major developments found using this technique, current scRNA-seq methods suffer from high cost, low yields, and nonstandardization of generated data. Additional development of scRNA-seq methods will raise our awareness of microglia's heterogeneity and plasticity under healthy and pathological conditions.

Drugs Modulating CD4+ T Cells Blood-Brain Barrier Interaction in Alzheimer's Disease.

The effect of Alzheimer's disease (AD) medications on CD4+ T cells homing has not been thoroughly investigated. CD4+ T cells could both exacerbate and reduce AD symptoms based on their infiltrating subpopulations. Proinflammatory subpopulations such as Th1 and Th17 constitute a major source of proinflammatory cytokines that reduce endothelial integrity and stimulate astrocytes, resulting in the production of amyloid ß. Anti-inflammatory subpopulations such as Th2 and Tregs reduce inflammation and regulate the function of Th1 and Th17. Recently, pathogenic Th17 has been shown to have a superior infiltrating capacity compared to other major CD4+ T cell subpopulations. Alzheimer's drugs such as donepezil (Aricept), rivastigmine (Exelon), galantamine (Razadyne), and memantine (Namenda) are known to play an important part in regulating the mechanisms of the neurotransmitters. However, little is known about the effect of these drugs on CD4+ T cell subpopulations' infiltration of the brain during AD. In this review, we focus on understanding the influence of AD drugs on CD4+ T cell subpopulation interactions with the BBB in AD. While current AD therapies improve endothelial integrity and reduce astrocytes activations, they vary according to their influence on various CD4+ T cell subpopulations. Donepezil reduces the numbers of Th1 but not Th2, Rivastigmine inhibits Th1 and Th17 but not Th2, and memantine reduces Th1 but not Treg. However, none of the current AD drugs is specifically designed to target the dysregulated balance in the Th17/Treg axis. Future drug design approaches should specifically consider inhibiting CD4+ Th17 to improve AD prognosis.

Repurposing Zileuton as a Depression Drug Using an AI and In Vitro Approach.

Repurposing drugs to target M1 macrophages inflammatory response in depression constitutes a bright alternative for commonly used antidepressants. Depression is a significant type of mood disorder, where patients suffer from pathological disturbances associated with a proinflammatory M1 macrophage phenotype. Presently, the most commonly used antidepressants such as Zoloft and Citalopram can reduce inflammation, but suffer from dangerous side effects without offering specificity toward macrophages. We employed a new strategy for drug repurposing based on the integration of RNA-seq analysis and text mining using deep neural networks. Our system employs a Google semantic AI universal encoder to compute sentences embedding. Sentences similarity is calculated using a sorting function to identify drug compounds. Then sentence relevance is computed using a custom-built convolution differential network. Our system highlighted the NRF2 pathway as a critical drug target to reprogram M1 macrophage response toward an anti-inflammatory profile (M2). Using our approach, we were also able to predict that lipoxygenase inhibitor drug zileuton could modulate NRF2 pathway in vitro. Taken together, our results indicate that reorienting zileuton usage to modulate M1 macrophages could be a novel and safer therapeutic option for treating depression.

Molecular Evolution and Functional Divergence of the IgLON Family.

IgLON family is a subgroup of cell adhesion molecules which is known to have diverse roles in neuronal development. IgLONs are characterized by possessing 3 Ig-like C2 domains, which play a part in mediating various cellular interactions. Recently, IgLONs have been shown to be expressed at the blood-brain barrier (BBB). However, our understanding of the genetic divergence patterns and evolutionary rates of these proteins in relation to their functions, in general, and at the BBB, in particular, remains inadequate. In this study, 12 species were explored to shed more light on the phylogenetic origins, structure, functional specificity, and divergence of this family. A total of 40 IgLON genes were identified from vertebrates and invertebrates. The absence of IgLON family genes in Hydra vulgaris and Nematostella vectensis but not in Drosophila melanogaster suggests that this family appeared during the time of divergence of Arthropoda 455 Mya. In general, IgLON genes have been subject to strong positive selection in vertebrates. Our study, based on IgLONs' structural similarity, suggests that they may play a role in the evolutionary changes in the brain anatomy towards complexity including regulating neural growth and BBB permeability. IgLONs' functions seem to be performed through complex interactions on the level of motifs as well as single residues. We identified several IgLON motifs that could be influencing cellular migration and proliferation as well as BBB integrity through interactions with SH3 or integrin. Our motif analysis also revealed that NEGR1 might be involved in MAPK pathway as a form of a signal transmitting receptor through its motif (KKVRVVVNF). We found several residues that were both positively selected and with highly functional specificity. We also located functional divergent residues that could act as drug targets to regulate BBB permeability. Furthermore, we identified several putative metalloproteinase cleavage sites that support the ectodomain shedding hypothesis of the IgLONs. In conclusion, our results present a bridge between IgLONs' molecular evolution and their functions.