Small-molecule CBP/p300 histone acetyltransferase inhibition mobilizes leukocytes from the bone marrow via the endocrine stress response.

Mutations of the CBP/p300 histone acetyltransferase (HAT) domain can be linked to leukemic transformation in humans, suggestive of a checkpoint of leukocyte compartment sizes. Here, we examined the impact of reversible inhibition of this domain by the small-molecule A485. We found that A485 triggered acute and transient mobilization of leukocytes from the bone marrow into the blood. Leukocyte mobilization by A485 was equally potent as, but mechanistically distinct from, granulocyte colony-stimulating factor (G-CSF), which allowed for additive neutrophil mobilization when both compounds were combined. These effects were maintained in models of leukopenia and conferred augmented host defenses. Mechanistically, activation of the hypothalamus-pituitary-adrenal gland (HPA) axis by A485 relayed shifts in leukocyte distribution through corticotropin-releasing hormone receptor 1 (CRHR1) and adrenocorticotropic hormone (ACTH), but independently of glucocorticoids. Our findings identify a strategy for rapid expansion of the blood leukocyte compartment via a neuroendocrine loop, with implications for the treatment of human pathologies.

Central regulation of stress-evoked peripheral immune responses.

Stress-linked psychiatric disorders, including anxiety and major depressive disorder, are associated with systemic inflammation. Recent studies have reported stress-induced alterations in haematopoiesis that result in monocytosis, neutrophilia, lymphocytopenia and, consequently, in the upregulation of pro-inflammatory processes in immunologically relevant peripheral tissues. There is now evidence that this peripheral inflammation contributes to the development of psychiatric symptoms as well as to common co-morbidities of psychiatric disorders such as metabolic syndrome and immunosuppression. Here, we review the specific brain and spinal regions, and the neuronal populations within them, that respond to stress and transmit signals to peripheral tissues via the autonomic nervous system or neuroendocrine pathways to influence immunological function. We comprehensively summarize studies that have employed retrograde tracing to define neurocircuits linking the brain to the bone marrow, spleen, gut, adipose tissue and liver. Moreover, we highlight studies that have used chemogenetic or optogenetic manipulation or intracerebroventricular administration of peptide hormones to control somatic immune responses. Collectively, this growing body of literature illustrates potential mechanisms through which stress signals are conveyed from the CNS to immune cells to regulate stress-relevant behaviours and comorbid pathophysiology.

Interleukin-3 coordinates glial-peripheral immune crosstalk to incite multiple sclerosis.

Glial cells and central nervous system (CNS)-infiltrating leukocytes contribute to multiple sclerosis (MS). However, the networks that govern crosstalk among these ontologically distinct populations remain unclear. Here, we show that, in mice and humans, CNS-resident astrocytes and infiltrating CD44hiCD4+ T cells generated interleukin-3 (IL-3), while microglia and recruited myeloid cells expressed interleukin-3 receptor-ɑ (IL-3Rɑ). Astrocytic and T cell IL-3 elicited an immune migratory and chemotactic program by IL-3Rɑ+ myeloid cells that enhanced CNS immune cell infiltration, exacerbating MS and its preclinical model. Multiregional snRNA-seq of human CNS tissue revealed the appearance of IL3RA-expressing myeloid cells with chemotactic programming in MS plaques. IL3RA expression by plaque myeloid cells and IL-3 amount in the cerebrospinal fluid predicted myeloid and T cell abundance in the CNS and correlated with MS severity. Our findings establish IL-3:IL-3RA as a glial-peripheral immune network that prompts immune cell recruitment to the CNS and worsens MS.

ZBTB7A regulates MDD-specific chromatin signatures and astrocyte-mediated stress vulnerability in orbitofrontal cortex.

Hyperexcitability in the orbitofrontal cortex (OFC) is a key clinical feature of anhedonic domains of Major Depressive Disorder (MDD). However, the cellular and molecular substrates underlying this dysfunction remain unknown. Here, cell-population-specific chromatin accessibility profiling in human OFC unexpectedly mapped genetic risk for MDD exclusively to non-neuronal cells, and transcriptomic analyses revealed significant glial dysregulation in this region. Characterization of MDD-specific cis-regulatory elements identified ZBTB7A - a transcriptional regulator of astrocyte reactivity - as an important mediator of MDD-specific chromatin accessibility and gene expression. Genetic manipulations in mouse OFC demonstrated that astrocytic Zbtb7a is both necessary and sufficient to promote behavioral deficits, cell-type-specific transcriptional and chromatin profiles, and OFC neuronal hyperexcitability induced by chronic stress - a major risk factor for MDD. These data thus highlight a critical role for OFC astrocytes in stress vulnerability and pinpoint ZBTB7A as a key dysregulated factor in MDD that mediates maladaptive astrocytic functions driving OFC hyperexcitability.

Monocytes re-enter the bone marrow during fasting and alter the host response to infection.

Diet profoundly influences physiology. Whereas over-nutrition elevates risk for disease via its influence on immunity and metabolism, caloric restriction and fasting appear to be salutogenic. Despite multiple correlations observed between diet and health, the underlying biology remains unclear. Here, we identified a fasting-induced switch in leukocyte migration that prolongs monocyte lifespan and alters susceptibility to disease in mice. We show that fasting during the active phase induced the rapid return of monocytes from the blood to the bone marrow. Monocyte re-entry was orchestrated by hypothalamic-pituitary-adrenal (HPA) axis-dependent release of corticosterone, which augmented the CXCR4 chemokine receptor. Although the marrow is a safe haven for monocytes during nutrient scarcity, re-feeding prompted mobilization culminating in monocytosis of chronologically older and transcriptionally distinct monocytes. These shifts altered response to infection. Our study shows that diet-in particular, a diet's temporal dynamic balance-modulates monocyte lifespan with consequences for adaptation to external stressors.

Innate Immunity in Cardiovascular Diseases-Identification of Novel Molecular Players and Targets.

During the past few years, unexpected developments have driven studies in the field of clinical immunology. One driver of immense impact was the outbreak of a pandemic caused by the novel virus SARS-CoV-2. Excellent recent reviews address diverse aspects of immunological re-search into cardiovascular diseases. Here, we specifically focus on selected studies taking advantage of advanced state-of-the-art molecular genetic methods ranging from genome-wide epi/transcriptome mapping and variant scanning to optogenetics and chemogenetics. First, we discuss the emerging clinical relevance of advanced diagnostics for cardiovascular diseases, including those associated with COVID-19-with a focus on the role of inflammation in cardiomyopathies and arrhythmias. Second, we consider newly identified immunological interactions at organ and system levels which affect cardiovascular pathogenesis. Thus, studies into immune influences arising from the intestinal system are moving towards therapeutic exploitation. Further, powerful new research tools have enabled novel insight into brain-immune system interactions at unprecedented resolution. This latter line of investigation emphasizes the strength of influence of emotional stress-acting through defined brain regions-upon viral and cardiovascular disorders. Several challenges need to be overcome before the full impact of these far-reaching new findings will hit the clinical arena.

tRNA-like Transcripts from the NEAT1-MALAT1 Genomic Region Critically Influence Human Innate Immunity and Macrophage Functions.

The evolutionary conserved NEAT1-MALAT1 gene cluster generates large noncoding transcripts remaining nuclear, while tRNA-like transcripts (mascRNA, menRNA) enzymatically generated from these precursors translocate to the cytosol. Whereas functions have been assigned to the nuclear transcripts, data on biological functions of the small cytosolic transcripts are sparse. We previously found NEAT1-/- and MALAT1-/- mice to display massive atherosclerosis and vascular inflammation. Here, employing selective targeted disruption of menRNA or mascRNA, we investigate the tRNA-like molecules as critical components of innate immunity. CRISPR-generated human ΔmascRNA and ΔmenRNA monocytes/macrophages display defective innate immune sensing, loss of cytokine control, imbalance of growth/angiogenic factor expression impacting upon angiogenesis, and altered cell-cell interaction systems. Antiviral response, foam cell formation/oxLDL uptake, and M1/M2 polarization are defective in ΔmascRNA/ΔmenRNA macrophages, defining first biological functions of menRNA and describing new functions of mascRNA. menRNA and mascRNA represent novel components of innate immunity arising from the noncoding genome. They appear as prototypes of a new class of noncoding RNAs distinct from others (miRNAs, siRNAs) by biosynthetic pathway and intracellular kinetics. Their NEAT1-MALAT1 region of origin appears as archetype of a functionally highly integrated RNA processing system.

Sleep exerts lasting effects on hematopoietic stem cell function and diversity.

A sleepless night may feel awful in its aftermath, but sleep's revitalizing powers are substantial, perpetuating the idea that convalescent sleep is a consequence-free physiological reset. Although recent studies have shown that catch-up sleep insufficiently neutralizes the negative effects of sleep debt, the mechanisms that control prolonged effects of sleep disruption are not understood. Here, we show that sleep interruption restructures the epigenome of hematopoietic stem and progenitor cells (HSPCs) and increases their proliferation, thus reducing hematopoietic clonal diversity through accelerated genetic drift. Sleep fragmentation exerts a lasting influence on the HSPC epigenome, skewing commitment toward a myeloid fate and priming cells for exaggerated inflammatory bursts. Combining hematopoietic clonal tracking with mathematical modeling, we infer that sleep preserves clonal diversity by limiting neutral drift. In humans, sleep restriction alters the HSPC epigenome and activates hematopoiesis. These findings show that sleep slows decay of the hematopoietic system by calibrating the hematopoietic epigenome, constraining inflammatory output, and maintaining clonal diversity.

Brain motor and fear circuits regulate leukocytes during acute stress.

The nervous and immune systems are intricately linked1. Although psychological stress is known to modulate immune function, mechanistic pathways linking stress networks in the brain to peripheral leukocytes remain poorly understood2. Here we show that distinct brain regions shape leukocyte distribution and function throughout the body during acute stress in mice. Using optogenetics and chemogenetics, we demonstrate that motor circuits induce rapid neutrophil mobilization from the bone marrow to peripheral tissues through skeletal-muscle-derived neutrophil-attracting chemokines. Conversely, the paraventricular hypothalamus controls monocyte and lymphocyte egress from secondary lymphoid organs and blood to the bone marrow through direct, cell-intrinsic glucocorticoid signalling. These stress-induced, counter-directional, population-wide leukocyte shifts are associated with altered disease susceptibility. On the one hand, acute stress changes innate immunity by reprogramming neutrophils and directing their recruitment to sites of injury. On the other hand, corticotropin-releasing hormone neuron-mediated leukocyte shifts protect against the acquisition of autoimmunity, but impair immunity to SARS-CoV-2 and influenza infection. Collectively, these data show that distinct brain regions differentially and rapidly tailor the leukocyte landscape during psychological stress, therefore calibrating the ability of the immune system to respond to physical threats.

Straightforward Analysis of Sulfated Glycosaminoglycans by MALDI-TOF Mass Spectrometry from Biological Samples.

Glycosaminoglycans (GAGs) are considered to be the most difficult type of glycoconjugates to analyze as they are constituted of linear long polysaccharidic chains having molecular weights reaching up to several million daltons. Bottom-up analysis of glycosaminoglycans from biological samples is a long and work-extensive procedure due to the many preparation steps involved. In addition, so far, only few research articles have been dedicated to the analysis of GAGs by means of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) because their intact ionization can be problematic due to the presence of labile sulfate groups. In this work, we had the aim of exploring the sulfation pattern of monosulfated chondroitin/dermatan sulfate (CS/DS) disaccharides in human tissue samples because they represent the most abundant form of sulfation in disaccharides. We present here an optimized strategy to analyze on-target derivatized CS/DS disaccharides via MALDI-TOF-MS using a fast workflow that does not require any purification after enzymatic cleavage. For the first time, we show that MALDI-TOF/TOF experiments allow for discrimination between monosulfated CS disaccharide isomers via specific fragments corresponding to glycosidic linkages and to cross-ring cleavages. This proof of concept is illustrated via the analysis of CS/DS disaccharides of atherosclerotic lesions of different histological origins, in which we were able to identify their monosulfation patterns.

Astrocytic interleukin-3 programs microglia and limits Alzheimer's disease.

Communication within the glial cell ecosystem is essential for neuronal and brain health1-3. The influence of glial cells on the accumulation and clearance of ß-amyloid (Aß) and neurofibrillary tau in the brains of individuals with Alzheimer's disease (AD) is poorly understood, despite growing awareness that these are therapeutically important interactions4,5. Here we show, in humans and mice, that astrocyte-sourced interleukin-3 (IL-3) programs microglia to ameliorate the pathology of AD. Upon recognition of Aß deposits, microglia increase their expression of IL-3Rα-the specific receptor for IL-3 (also known as CD123)-making them responsive to IL-3. Astrocytes constitutively produce IL-3, which elicits transcriptional, morphological, and functional programming of microglia to endow them with an acute immune response program, enhanced motility, and the capacity to cluster and clear aggregates of Aß and tau. These changes restrict AD pathology and cognitive decline. Our findings identify IL-3 as a key mediator of astrocyte-microglia cross-talk and a node for therapeutic intervention in AD.

Liver X receptors are required for thymic resilience and T cell output.

The thymus is a primary lymphoid organ necessary for optimal T cell development. Here, we show that liver X receptors (LXRs)-a class of nuclear receptors and transcription factors with diverse functions in metabolism and immunity-critically contribute to thymic integrity and function. LXRαß-deficient mice develop a fatty, rapidly involuting thymus and acquire a shrunken and prematurely immunoinhibitory peripheral T cell repertoire. LXRαß's functions are cell specific, and the resulting phenotypes are mutually independent. Although thymic macrophages require LXRαß for cholesterol efflux, thymic epithelial cells (TECs) use LXRαß for self-renewal and thymocytes for negative selection. Consequently, TEC-derived LXRαß protects against homeostatic premature involution and orchestrates thymic regeneration following stress, while thymocyte-derived LXRαß limits cell disposal during negative selection and confers heightened sensitivity to experimental autoimmune encephalomyelitis. These results identify three distinct but complementary mechanisms by which LXRαß governs T lymphocyte education and illuminate LXRαß's indispensable roles in adaptive immunity.

Hematopoiesis and Cardiovascular Disease.

A central feature of atherosclerosis, the most prevalent chronic vascular disease and root cause of myocardial infarction and stroke, is leukocyte accumulation in the arterial wall. These crucial immune cells are produced in specialized niches in the bone marrow, where a complex cell network orchestrates their production and release. A growing body of clinical studies has documented a correlation between leukocyte numbers and cardiovascular disease risk. Understanding how leukocytes are produced and how they contribute to atherosclerosis and its complications is, therefore, critical to understanding and treating the disease. In this review, we focus on the key cells and products that regulate hematopoiesis under homeostatic conditions, during atherosclerosis and after myocardial infarction.

Initial interaction of citrate-coated iron oxide nanoparticles with the glycocalyx of THP-1 monocytes assessed by real-time magnetic particle spectroscopy and electron microscopy.

Interaction with biological material can alter physicochemical parameters of magnetic nanoparticles and might thereby change their magnetic behavior with potentially important implications for various nanoparticle applications. Little is known about changes of the magnetic behavior that occur during the initial phase of cell binding and uptake. We investigate the magnetic behavior of very small superparamagnetic iron-oxide nanoparticles (VSOP) during initial contact with THP-1 monocytes. We combine real-time magnetic particle spectroscopy (MPS), a fast and sensitive method for specific detection of magnetic nanoparticles in biological specimen with high-pressure-freezing/freeze-substitution transmission electron microscopy (HPF/FS-TEM), enabling us to generate snapshots of the interaction of VSOP with the cellular glycocalyx. MPS reveals significant changes of the dynamic magnetic behavior within seconds after VSOP injection into monocyte suspensions that correlate with the formation of nanoparticle clusters in the glycocalyx. The combination of real-time MPS and HPF/FS-TEM provides an ideal platform to analyze magnetic behaviors of nanoparticles upon interaction with cells and tissues.

Gut intraepithelial T cells calibrate metabolism and accelerate cardiovascular disease.

The biochemical response to food intake must be precisely regulated. Because ingested sugars and fats can feed into many anabolic and catabolic pathways1, how our bodies handle nutrients depends on strategically positioned metabolic sensors that link the intrinsic nutritional value of a meal with intermediary metabolism. Here we describe a subset of immune cells-integrin ß7+ natural gut intraepithelial T lymphocytes (natural IELs)-that is dispersed throughout the enterocyte layer of the small intestine and that modulates systemic metabolism. Integrin ß7- mice that lack natural IELs are metabolically hyperactive and, when fed a high-fat and high-sugar diet, are resistant to obesity, hypercholesterolaemia, hypertension, diabetes and atherosclerosis. Furthermore, we show that protection from cardiovascular disease in the absence of natural IELs depends on the enteroendocrine-derived incretin GLP-12, which is normally controlled by IELs through expression of the GLP-1 receptor. In this metabolic control system, IELs modulate enteroendocrine activity by acting as gatekeepers that limit the bioavailability of GLP-1. Although the function of IELs may prove advantageous when food is scarce, present-day overabundance of diets high in fat and sugar renders this metabolic checkpoint detrimental to health.

Sleep modulates haematopoiesis and protects against atherosclerosis.

Sleep is integral to life1. Although insufficient or disrupted sleep increases the risk of multiple pathological conditions, including cardiovascular disease2, we know little about the cellular and molecular mechanisms by which sleep maintains cardiovascular health. Here we report that sleep regulates haematopoiesis and protects against atherosclerosis in mice. We show that mice subjected to sleep fragmentation produce more Ly-6Chigh monocytes, develop larger atherosclerotic lesions and produce less hypocretin-a stimulatory and wake-promoting neuropeptide-in the lateral hypothalamus. Hypocretin controls myelopoiesis by restricting the production of CSF1 by hypocretin-receptor-expressing pre-neutrophils in the bone marrow. Whereas hypocretin-null and haematopoietic hypocretin-receptor-null mice develop monocytosis and accelerated atherosclerosis, sleep-fragmented mice with either haematopoietic CSF1 deficiency or hypocretin supplementation have reduced numbers of circulating monocytes and smaller atherosclerotic lesions. Together, these results identify a neuro-immune axis that links sleep to haematopoiesis and atherosclerosis.

Macrophage uptake switches on OCT contrast of superparamagnetic nanoparticles for imaging of atherosclerotic plaques.

Background: Optical coherence tomography (OCT) is an intravascular, high-resolution imaging technique that is used to characterize atherosclerotic plaques. However, the identification of macrophages as important markers of inflammation and plaque vulnerability remains difficult. Here, we investigate whether the uptake of very small iron oxide particles (VSOP) in macrophages, that cluster in phagolysosomes and allow high-quality magnetic resonance imaging (MRI) of atherosclerotic plaques, and uptake of ferumoxytol nanoparticles enhance detection of macrophages by OCT. Materials and methods: RAW 264.7 macrophage cells were incubated with VSOP (1 and 2 mM Fe) that have been clinically tested and ferumoxytol (8.9 mM Fe) that is approved for iron deficiency treatment and currently investigated as an MRI contrast agent. The light scattering of control macrophages, nanoparticle-labeled macrophages (2,000,000 in 500 µL) and nanoparticle suspensions was measured in synchronous wavelength scan mode using a fluorescence spectrophotometer. For OCT analyses, pellets of 8,000,000 non-labeled, VSOP-labeled and ferumoxytol-labeled RAW 264.7 macrophages were imaged and analyzed on an OPTIS™ OCT imaging system. Results: Incubation with 1 and 2 mM VSOP resulted in uptake of 7.1±1.5 and 12±1.5 pg Fe per cell, which increased the backscattering of the macrophages in spectrophotometry 2.5- and 3.6-fold, whereas incubation with 8.9 mM Fe ferumoxytol resulted in uptake of 6.6±2 pg Fe per cell, which increased the backscattering 1.5-fold at 700 nm. In contrast, backscattering of non-clustered nanoparticles in suspension was negligible. Accordingly, OCT imaging could visualize significantly increased backscattering and signal attenuation of nanoparticle-labeled macrophages in comparison with controls. Conclusion: We conclude that VSOP and, to a lesser extent, ferumoxytol increase light scattering and attenuation when taken up by macrophages and can serve as a multimodal imaging probe for MRI and OCT to improve macrophage detection in atherosclerotic plaques by OCT in the future.

Very small superparamagnetic iron oxide nanoparticles: Long-term fate and metabolic processing in atherosclerotic mice.

We investigated the biotransformation of very small superparamagnetic iron oxide nanoparticles (VSOP) in atherosclerotic LDLR-/- mice. Transmission electron microscopy revealed an uptake of VSOP not only by macrophages but also by endothelial cells in liver, spleen, and atherosclerotic lesions and their accumulation in the lysosomal compartment. Using magnetic particle spectroscopy (MPS), we show that the majority of VSOP's superparamagnetic iron was degraded within 28 days. MPS spectrum shape indicated changes in the magnetic properties of VSOP during the biodegradation process. Experiments with primary murine bone marrow derived macrophages, primary murine liver sinusoidal endothelial cells, and primary human aortic endothelial cells demonstrated that loading with VSOP induced a differential response of cellular iron homeostasis mechanisms with increased levels of ferritin and iron transport proteins in macrophages and increased levels of ferritin in endothelial cells.

Lipoprotein apheresis in patients with peripheral artery disease and lipoprotein(a)-hyperlipoproteinemia: 2-year follow-up of a prospective single center study.

OBJECTIVE: Elevated plasma levels of lipoprotein(a) [Lp(a)], referred to as lipoprotein(a)-hyperlipoproteinemia [Lp(a)-HLP], are an independent risk factor for atherosclerosis. Lipoprotein apheresis (LA) enables an effective reduction of Lp(a) plasma levels. The present study investigates the effects of LA in patients with Lp(a)-HLP and peripheral artery disease (PAD). METHODS: Ten patients with isolated Lp(a)-HLP and severe PAD and who had recently undergone a revascularization (index procedure) were prospectively included in this observational single center study. All patients received weekly LA. Ankle-brachial-index (ABI), transcutaneous partial oxygen pressure (tcpO2), pain level, and walking distance were assessed at baseline and at the follow ups scheduled 1, 3, 6, 12, and 24 months after initiation of LA. The number of revascularizations within 12 months prior and within 24 months after the index procedure was determined. RESULTS: As early as 1 month after initiation of LA, all investigated parameters had improved significantly compared to baseline. This improvement was further substantiated under LA throughout the entire follow-up period. Comparing baseline results with the 24-month follow-up, the average ABI increased from 0.53 ± 0.15 to 0.97 ± 0.08 (P < 0.001). The mean tcpO2 also increased from 42.9 ± 2.3 mmHg to 61 ± 4.6 mmHg (P < 0.001). The improved perfusion led to a reduction of the mean pain level from 7.0 ± 1.5 to 1.1 ± 0.4 (P < 0.001) on a visual analogue scale (VAS) and an extension of the mean walking distance from 87 ± 60 m to 402 ± 119 m (P < 0.001). All patients suffered from severe PAD with a high number of revascularizations in the 12 months prior to the index procedure (35 procedures in 120 patient-months). Since initiation of LA, the number of revascularizations dropped significantly and remained very low during the entire follow-up period (2 procedures in 229 patient-months, P < 0.001). CONCLUSION: In patients with Lp(a)-HLP and severe PAD, LA results in sustained improvement of circulation, pain level and walking distance. The number of repeat revascularizations is strongly reduced under LA treatment.

Anterior and posterior parts of the rat ventral tegmental area and the rostromedial tegmental nucleus receive topographically distinct afferents from the lateral habenular complex.

That activation of the reward system involves increased activity of dopaminergic (DA) neurons in the ventral tegmental area (VTA) is widely accepted. In contrast, the lateral habenular complex (LHb), which is known as the center of the anti-reward system, directly and indirectly inhibits DA neurons in the VTA. The VTA, however, is not a homogenous entity. Instead, it displays major functional differences between its anterior (aVTA) and posterior (pVTA) regions. It is not precisely known, whether habenular input to the aVTA, pVTA, and the newly recognized rostromedial tegmental nucleus (RMTg) are similarly or differently organized. Consequently, the present investigation addressed the connections between LHb and aVTA, pVTA, and RMTg using retrograde and anterograde tracing techniques in the rat. Our experiments disclosed strictly reciprocal and conspicuously focal interconnections between LHbM (LHbMPc/LHbMC) and PN, as well as between RLi and LHbLO. In addition, we found that LHb inputs to the aVTA are dorsoventrally ordered. Dorsal parts of the aVTA receive afferents from LHbL and LHbM, whereas ventral parts of the aVTA are preferentially targeted by the LHbM. LHb afferents to the pVTA are distinct from those to the RMTg, given that the RMTg is primarily innervated from the LHbL, whereas pVTA receives afferents from LHbM and LHbL. These data indicate the existence of two separate pathways from the LHb to the VTA, a direct and an indirect one, which may subserve distinct biological functions.