Tbc1d10c is a selective, constitutive suppressor of the CD8 T-cell anti-tumor response.

Cancer immunotherapy approaches target signaling pathways that are highly synonymous between CD4 and CD8 T-cell subsets and, therefore, often stimulate nonspecific lymphocyte activation, resulting in cytotoxicity to otherwise healthy tissue. The goal of our study was to identify intrinsic modulators of basic T lymphocyte activation pathways that could discriminately bolster CD8 anti-tumor effector responses. Using a Tbc1d10c null mouse, we observed marked resistance to a range of tumor types conferred by Tbc1d10c deficiency. Moreover, tumor-bearing Tbc1d10c null mice receiving PD-1 or CTLA-4 monotherapy exhibited a 33% or 90% cure rate, respectively. While Tbc1d10c was not expressed in solid tumor cells, Tbc1d10c disruption selectively augmented CD8 T-cell activation and cytotoxic effector responses and adoptive transfer of CD8 T cells alone was sufficient to recapitulate Tbc1d10c null tumor resistance. Mechanistically, Tbc1d10c suppressed CD8 T-cell activation and anti-tumor function by intersecting canonical NF-κB pathway activation via regulation of Map3k3-mediated IKKß phosphorylation. Strikingly, none of these cellular or molecular perturbations in the NF-κB pathway were featured in Tbc1d10c null CD4 T cells. Our findings identify a Tbc1d10c-Map3k3-NF-κB signaling axis as a viable therapeutic target to promote CD8 T-cell anti-tumor immunity while circumventing CD4 T cell-associated cytotoxicity and NF-κB activation in tumor cells.

Mechanosensor Piezo1 mediates bimodal patterns of intracellular calcium and FAK signaling.

Piezo1 belongs to mechano-activatable cation channels serving as biological force sensors. However, the molecular events downstream of Piezo1 activation remain unclear. In this study, we used biosensors based on fluorescence resonance energy transfer (FRET) to investigate the dynamic modes of Piezo1-mediated signaling and revealed a bimodal pattern of Piezo1-induced intracellular calcium signaling. Laser-induced shockwaves (LIS) and its associated shear stress can mechanically activate Piezo1 to induce transient intracellular calcium (Ca[i] ) elevation, accompanied by an increase in FAK activity. Interestingly, multiple pulses of shockwave stimulation caused a more sustained calcium increase and a decrease in FAK activity. Similarly, tuning the degree of Piezo1 activation by titrating either the dosage of Piezo1 ligand Yoda1 or the expression level of Piezo1 produced a similar bimodal pattern of FAK responses. Further investigations revealed that SHP2 serves as an intermediate regulator mediating this bimodal pattern in Piezo1 sensing and signaling. These results suggest that the degrees of Piezo1 activation induced by both mechanical LIS and chemical ligand stimulation may determine downstream signaling characteristics.

Spatiotemporal dynamics of PIEZO1 localization controls keratinocyte migration during wound healing.

Keratinocytes, the predominant cell type of the epidermis, migrate to reinstate the epithelial barrier during wound healing. Mechanical cues are known to regulate keratinocyte re-epithelialization and wound healing; however, the underlying molecular transducers and biophysical mechanisms remain elusive. Here, we show through molecular, cellular, and organismal studies that the mechanically activated ion channel PIEZO1 regulates keratinocyte migration and wound healing. Epidermal-specific Piezo1 knockout mice exhibited faster wound closure while gain-of-function mice displayed slower wound closure compared to littermate controls. By imaging the spatiotemporal localization dynamics of endogenous PIEZO1 channels, we find that channel enrichment at some regions of the wound edge induces a localized cellular retraction that slows keratinocyte collective migration. In migrating single keratinocytes, PIEZO1 is enriched at the rear of the cell, where maximal retraction occurs, and we find that chemical activation of PIEZO1 enhances retraction during single as well as collective migration. Our findings uncover novel molecular mechanisms underlying single and collective keratinocyte migration that may suggest a potential pharmacological target for wound treatment. More broadly, we show that nanoscale spatiotemporal dynamics of Piezo1 channels can control tissue-scale events, a finding with implications beyond wound healing to processes as diverse as development, homeostasis, disease, and repair.

The skin is the largest organ of the body. It enables touch sensation and protects against external insults. Wounding of the skin exposes the body to an increased risk of infection, disease and scar formation. During wound healing, the cells in the topmost layer of the skin, called keratinocytes, move in from the edges of the wound to close the gap. This helps to restore the skin barrier. Previous research has shown that the mechanical forces experienced by keratinocytes play a role in wound closure. Several proteins, called mechanosensors, perceive these forces and instruct the cells what to do. Until now, it was unclear what kind of mechanosensors control wound healing. To find out more, Holt et al. studied a recently discovered mechanosensor (for which co-author Ardem Pataputian received the Nobel Prize in 2021), called Piezo1, using genetically engineered mice. The experiments revealed that skin wounds in mice without Piezo1 in their keratinocytes healed faster than mice with normal levels of Piezo1. In contrast, skin wounds of mice with increased levels of Piezo1 in their keratinocytes healed slower than mice with normal levels of Piezo1. The same pattern held true for keratinocytes grown in the laboratory that had been treated with chemicals to increase the activity of Piezo1. To better understand how Piezo1 slows wound healing, Holt et al. tracked its location inside the keratinocytes. This revealed that the position of Piezo1 changes over time. It builds up near the edge of the wound in some places, and at those regions makes the cells move backwards rather than forwards. In extreme cases, an increased activity of Piezo1 can cause an opening of the wound instead of closing it. These findings have the potential to guide research into new wound treatments. But first, scientists must confirm that blocking Piezo1 would not cause side effects, like reducing the sensation of touch. Moreover, it would be interesting to see if Piezo1 also plays a role in other important processes, such as development or certain diseases.

Air pollution from wildfires and human health vulnerability in Alaskan communities under climate change.

Alaskan wildfires are becoming more frequent and severe, but very little is known regarding exposure to wildfire smoke, a risk factor for respiratory and cardiovascular illnesses. We estimated long-term, present-day and future exposure to wildfire-related fine particulate matter (PM2.5) across Alaska for the general population and subpopulations to assess vulnerability using observed data for the present day (1997-2010), modelled estimates for the present day (1997-2001), and modelled estimates for the future (2047-2051). First, we assessed wildfire-PM2.5 exposure by estimating monthly-average wildfire-specific PM2.5 levels across 1997-2010 for 158 Alaskan census tracts, using atmospheric transport modelling based on observed area-burned data. Second, we estimated changes in future (2047-2051) wildfire-PM2.5 exposure compared to the present-day (1997-2001) by estimating the monthly-average wildfire-specific PM2.5 levels for 29 boroughs/census areas (county-equivalent areas), under the Intergovernmental Panel on Climate Change (IPCC) A1B scenario from an ensemble of 13 climate models. Subpopulation risks for present and future exposure levels were estimated by summing area-weighted exposure levels utilizing the 2000 Census and State of Alaska's population projections. We assessed vulnerability by several subpopulation characteristics (e.g. race/ethnicity, urbanicity). Wildfire-PM2.5 exposure levels during 1997-2010 were highest in interior Alaska during July. Among subpopulations, average summer (June-August) exposure levels for urban dwellers and African-American/Blacks were highest at 9.1 µg m-3 and 10 µg m-3, respectively. Estimated wildfire-PM2.5 varied by Native American tribe, ranging from average summer levels of 2.4 µg m-3 to 13 µg m-3 for Tlingit-Haida and Alaskan Athabascan tribes, respectively. Estimates indicate that by the mid-21st century, under climate change, almost all of Alaska could be exposed to increases of 100% or more in levels of wildfire-specific PM2.5 levels. Exposure to wildfire-PM2.5 likely presents a substantial public health burden in the present day for Alaska communities, with different impacts by subpopulation. Under climate change, wildfire smoke could pose an even greater public health risks for most Alaskans.

Merkel Cells Activate Sensory Neural Pathways through Adrenergic Synapses.

Epithelial-neuronal signaling is essential for sensory encoding in touch, itch, and nociception; however, little is known about the release mechanisms and neurotransmitter receptors through which skin cells govern neuronal excitability. Merkel cells are mechanosensory epidermal cells that have long been proposed to activate neuronal afferents through chemical synaptic transmission. We employed a set of classical criteria for chemical neurotransmission as a framework to test this hypothesis. RNA sequencing of adult mouse Merkel cells demonstrated that they express presynaptic molecules and biosynthetic machinery for adrenergic transmission. Moreover, live-cell imaging directly demonstrated that Merkel cells mediate activity- and VMAT-dependent release of fluorescent catecholamine neurotransmitter analogs. Touch-evoked firing in Merkel-cell afferents was inhibited either by pre-synaptic silencing of SNARE-mediated vesicle release from Merkel cells or by neuronal deletion of ß2-adrenergic receptors. Together, these results identify both pre- and postsynaptic mechanisms through which Merkel cells excite mechanosensory afferents to encode gentle touch. VIDEO ABSTRACT.

Piezo2 senses airway stretch and mediates lung inflation-induced apnoea.

Respiratory dysfunction is a notorious cause of perinatal mortality in infants and sleep apnoea in adults, but the mechanisms of respiratory control are not clearly understood. Mechanical signals transduced by airway-innervating sensory neurons control respiration; however, the physiological significance and molecular mechanisms of these signals remain obscured. Here we show that global and sensory neuron-specific ablation of the mechanically activated ion channel Piezo2 causes respiratory distress and death in newborn mice. Optogenetic activation of Piezo2+ vagal sensory neurons causes apnoea in adult mice. Moreover, induced ablation of Piezo2 in sensory neurons of adult mice causes decreased neuronal responses to lung inflation, an impaired Hering-Breuer mechanoreflex, and increased tidal volume under normal conditions. These phenotypes are reproduced in mice lacking Piezo2 in the nodose ganglion. Our data suggest that Piezo2 is an airway stretch sensor and that Piezo2-mediated mechanotransduction within various airway-innervating sensory neurons is critical for establishing efficient respiration at birth and maintaining normal breathing in adults.

Piezo2 is the principal mechanotransduction channel for proprioception.

Proprioception, the perception of body and limb position, is mediated by proprioceptors, specialized mechanosensory neurons that convey information about the stretch and tension experienced by muscles, tendons, skin and joints. In mammals, the molecular identity of the stretch-sensitive channel that mediates proprioception is unknown. We found that the mechanically activated nonselective cation channel Piezo2 was expressed in sensory endings of proprioceptors innervating muscle spindles and Golgi tendon organs in mice. Two independent mouse lines that lack Piezo2 in proprioceptive neurons showed severely uncoordinated body movements and abnormal limb positions. Moreover, the mechanosensitivity of parvalbumin-expressing neurons that predominantly mark proprioceptors was dependent on Piezo2 expression in vitro, and the stretch-induced firing of proprioceptors in muscle-nerve recordings was markedly reduced in Piezo2-deficient mice. Together, our results indicate that Piezo2 is the major mechanotransducer of mammalian proprioceptors.

The effect on emotions and brain activity by the direct/indirect lighting in the residential environment.

This study was performed to explore how direct/indirect lighting affects emotions and brain oscillations compared to the direct lighting when brightness and color temperature are controlled. Twenty-eight subjects (12 females; mean age 22.5) participated. The experimental conditions consisted of two lighting environments: direct/indirect lighting (400 lx downlight, 300 lx uplight) and direct lighting (700 lx downlight). On each trial, a luminance environment was presented for 4 min, followed by participants rated their emotional feelings of the lighting environment. EEG data were recorded during the experiment. Spectral analysis was performed for the range of delta, theta, alpha, beta, and gamma ranges. The participants felt cooler and more pleasant and theta oscillations on the F4, F8, T4, and TP7 electrodes were more enhanced in the direct/indirect lighting environment compared to the direct lighting environment. There was significant correlation between the "cool" rating and the theta power of the F8 electrode. The participants felt more pleasant in the direct/indirect lighting environment, indicating that space with direct/indirect lighting modulated subjective perception. Additionally, our results suggest that theta oscillatory activity can be used as a biological marker that reflects emotional status in different lighting environments.

Merkel cells and neurons keep in touch.

The Merkel cell-neurite complex is a unique vertebrate touch receptor comprising two distinct cell types in the skin. Its presence in touch-sensitive skin areas was recognized more than a century ago, but the functions of each cell type in sensory transduction have been unclear. Three recent studies demonstrate that Merkel cells are mechanosensitive cells that function in touch transduction via Piezo2. One study concludes that Merkel cells, rather than sensory neurons, are principal sites of mechanotransduction, whereas two other studies report that both Merkel cells and neurons encode mechanical inputs. Together, these studies settle a long-standing debate on whether or not Merkel cells are mechanosensory cells, and enable future investigations of how these skin cells communicate with neurons.

Piezo2 is the major transducer of mechanical forces for touch sensation in mice.

The sense of touch provides critical information about our physical environment by transforming mechanical energy into electrical signals. It is postulated that mechanically activated cation channels initiate touch sensation, but the identity of these molecules in mammals has been elusive. Piezo2 is a rapidly adapting, mechanically activated ion channel expressed in a subset of sensory neurons of the dorsal root ganglion and in cutaneous mechanoreceptors known as Merkel-cell-neurite complexes. It has been demonstrated that Merkel cells have a role in vertebrate mechanosensation using Piezo2, particularly in shaping the type of current sent by the innervating sensory neuron; however, major aspects of touch sensation remain intact without Merkel cell activity. Here we show that mice lacking Piezo2 in both adult sensory neurons and Merkel cells exhibit a profound loss of touch sensation. We precisely localize Piezo2 to the peripheral endings of a broad range of low-threshold mechanoreceptors that innervate both hairy and glabrous skin. Most rapidly adapting, mechanically activated currents in dorsal root ganglion neuronal cultures are absent in Piezo2 conditional knockout mice, and ex vivo skin nerve preparation studies show that the mechanosensitivity of low-threshold mechanoreceptors strongly depends on Piezo2. This cellular phenotype correlates with an unprecedented behavioural phenotype: an almost complete deficit in light-touch sensation in multiple behavioural assays, without affecting other somatosensory functions. Our results highlight that a single ion channel that displays rapidly adapting, mechanically activated currents in vitro is responsible for the mechanosensitivity of most low-threshold mechanoreceptor subtypes involved in innocuous touch sensation. Notably, we find that touch and pain sensation are separable, suggesting that as-yet-unknown mechanically activated ion channel(s) must account for noxious (painful) mechanosensation.

Piezo1, a mechanically activated ion channel, is required for vascular development in mice.

Mechanosensation is perhaps the last sensory modality not understood at the molecular level. Ion channels that sense mechanical force are postulated to play critical roles in a variety of biological processes including sensing touch/pain (somatosensation), sound (hearing), and shear stress (cardiovascular physiology); however, the identity of these ion channels has remained elusive. We previously identified Piezo1 and Piezo2 as mechanically activated cation channels that are expressed in many mechanosensitive cell types. Here, we show that Piezo1 is expressed in endothelial cells of developing blood vessels in mice. Piezo1-deficient embryos die at midgestation with defects in vascular remodeling, a process critically influenced by blood flow. We demonstrate that Piezo1 is activated by shear stress, the major type of mechanical force experienced by endothelial cells in response to blood flow. Furthermore, loss of Piezo1 in endothelial cells leads to deficits in stress fiber and cellular orientation in response to shear stress, linking Piezo1 mechanotransduction to regulation of cell morphology. These findings highlight an essential role of mammalian Piezo1 in vascular development during embryonic development.

Piezo2 is required for Merkel-cell mechanotransduction.

How we sense touch remains fundamentally unknown. The Merkel cell-neurite complex is a gentle touch receptor in the skin that mediates slowly adapting responses of Aß sensory fibres to encode fine details of objects. This mechanoreceptor complex was recognized to have an essential role in sensing gentle touch nearly 50 years ago. However, whether Merkel cells or afferent fibres themselves sense mechanical force is still debated, and the molecular mechanism of mechanotransduction is unknown. Synapse-like junctions are observed between Merkel cells and associated afferents, and yet it is unclear whether Merkel cells are inherently mechanosensitive or whether they can rapidly transmit such information to the neighbouring nerve. Here we show that Merkel cells produce touch-sensitive currents in vitro. Piezo2, a mechanically activated cation channel, is expressed in Merkel cells. We engineered mice deficient in Piezo2 in the skin, but not in sensory neurons, and show that Merkel-cell mechanosensitivity completely depends on Piezo2. In these mice, slowly adapting responses in vivo mediated by the Merkel cell-neurite complex show reduced static firing rates, and moreover, the mice display moderately decreased behavioural responses to gentle touch. Our results indicate that Piezo2 is the Merkel-cell mechanotransduction channel and provide the first line of evidence that Piezo channels have a physiological role in mechanosensation in mammals. Furthermore, our data present evidence for a two-receptor-site model, in which both Merkel cells and innervating afferents act together as mechanosensors. The two-receptor system could provide this mechanoreceptor complex with a tuning mechanism to achieve highly sophisticated responses to a given mechanical stimulus.

Epidermal Merkel cells are mechanosensory cells that tune mammalian touch receptors.

Touch submodalities, such as flutter and pressure, are mediated by somatosensory afferents whose terminal specializations extract tactile features and encode them as action potential trains with unique activity patterns. Whether non-neuronal cells tune touch receptors through active or passive mechanisms is debated. Terminal specializations are thought to function as passive mechanical filters analogous to the cochlea's basilar membrane, which deconstructs complex sounds into tones that are transduced by mechanosensory hair cells. The model that cutaneous specializations are merely passive has been recently challenged because epidermal cells express sensory ion channels and neurotransmitters; however, direct evidence that epidermal cells excite tactile afferents is lacking. Epidermal Merkel cells display features of sensory receptor cells and make 'synapse-like' contacts with slowly adapting type I (SAI) afferents. These complexes, which encode spatial features such as edges and texture, localize to skin regions with high tactile acuity, including whisker follicles, fingertips and touch domes. Here we show that Merkel cells actively participate in touch reception in mice. Merkel cells display fast, touch-evoked mechanotransduction currents. Optogenetic approaches in intact skin show that Merkel cells are both necessary and sufficient for sustained action-potential firing in tactile afferents. Recordings from touch-dome afferents lacking Merkel cells demonstrate that Merkel cells confer high-frequency responses to dynamic stimuli and enable sustained firing. These data are the first, to our knowledge, to directly demonstrate a functional, excitatory connection between epidermal cells and sensory neurons. Together, these findings indicate that Merkel cells actively tune mechanosensory responses to facilitate high spatio-temporal acuity. Moreover, our results indicate a division of labour in the Merkel cell-neurite complex: Merkel cells signal static stimuli, such as pressure, whereas sensory afferents transduce dynamic stimuli, such as moving gratings. Thus, the Merkel cell-neurite complex is an unique sensory structure composed of two different receptor cell types specialized for distinct elements of discriminative touch.

The touch dome defines an epidermal niche specialized for mechanosensory signaling.

In mammalian skin, Merkel cells are mechanoreceptor cells that are required for the perception of gentle touch. Recent evidence indicates that mature Merkel cells descend from the proliferative layer of skin epidermis; however, the stem cell niche for Merkel cell homeostasis has not been reported. Here, we provide genetic evidence for maintenance of mature Merkel cells during homeostasis by Krt17+ stem cells located in epidermal touch domes of hairy skin and in the tips of the rete ridges of glabrous skin. Lineage tracing analysis indicated that the entire pool of mature Merkel cells is turned over every 7-8 weeks in the adult epidermis and that Krt17+ stem cells also maintain squamous differentiation in the touch dome and in glabrous skin. Finally, selective genetic ablation of Krt17+ touch-dome keratinocytes indicates that these cells, and not mature Merkel cells, are primarily responsible for maintaining innervation of the Merkel cell-neurite complex.

Excitatory glutamate is essential for development and maintenance of the piloneural mechanoreceptor.

The piloneural collar in mammalian hairy skin comprises an intricate pattern of circumferential and longitudinal sensory afferents that innervate primary and secondary pelage hairs. The longitudinal afferents tightly associate with terminal Schwann cell processes to form encapsulated lanceolate nerve endings of rapidly adapting mechanoreceptors. The molecular basis for piloneural development, maintenance and function is poorly understood. Here, we show that Nefh-expressing glutamatergic neurons represent a major population of longitudinal and circumferential sensory afferents innervating the piloneural collar. Our findings using a VGLUT2 conditional-null mouse model indicate that glutamate is essential for innervation, patterning and differentiation of NMDAR(+) terminal Schwann cells during piloneural collar development. Similarly, treatment of adult mice with a selective NMDAR antagonist severely perturbed piloneural collar structure and reduced excitability of these mechanosensory neurons. Collectively, these results show that DRG-derived glutamate is essential for the proper development, maintenance and sensory function of the piloneural mechanoreceptor.

Identification of epidermal progenitors for the Merkel cell lineage.

Epithelial stem cells in adult mammalian skin are known to maintain epidermal, follicular and sebaceous lineages during homeostasis. Recently, Merkel cell mechanoreceptors were identified as a fourth lineage derived from the proliferative layer of murine skin epithelium; however, the location of the stem or progenitor population for Merkel cells remains unknown. Here, we have identified a previously undescribed population of epidermal progenitors that reside in the touch domes of hairy skin, termed touch dome progenitor cells (TDPCs). TDPCs are epithelial keratinocytes and are distinguished by their unique co-expression of α6 integrin, Sca1 and CD200 surface proteins. TDPCs exhibit bipotent progenitor behavior as they give rise to both squamous and neuroendocrine epidermal lineages, whereas the remainder of the α6(+) Sca1(+) CD200(-) epidermis does not give rise to Merkel cells. Finally, TDPCs possess a unique transcript profile that appears to be enforced by the juxtaposition of TDPCs with Merkel cells within the touch dome niche.

A distinct population of clonogenic and multipotent murine follicular keratinocytes residing in the upper isthmus.

The bulge region of adult murine hair follicles harbors epidermal stem cells with multipotent capacity; however, the restricted contributions of these cells under homeostatic conditions indicates that additional stem or progenitor cell populations may be required to maintain squamous and sebaceous lineages. We have identified a distinct population of murine hair follicle keratinocytes residing in the upper isthmus (UI) between the infundibulum and bulge regions that are distinguished by low alpha6 integrin levels and are negative for CD34 and Sca-1. Purified UI cells give rise to long-term, stable epidermal, follicular and sebaceous lineages and can self-renew in vivo. These cells are non-quiescent and possess a unique transcript profile compared with bulge stem cells and may represent a distinct reservoir of epidermal stem or progenitor cells.

Separate cavity margin sampling at the time of initial breast lumpectomy significantly reduces the need for reexcisions.

In breast conservation therapy, the margin status of the specimen predicts local recurrence and determines the need for reexcision. Many surgeons now take, at the time of lumpectomy, multiple separate "cavity margins" (CM) (the entire wall of the residual cavity) as final margins that supersede the oriented lumpectomy margins (LMs). We studied the efficacy of this method in 126 patients (23 with ductal carcinoma in situ [DCIS] only and 103 with invasive carcinoma with or without DCIS) who had an oriented lumpectomy specimen and also had four to six additional CMs. The tumors were evaluated for the following: size, grade, LM status (distance of tumor from margin and, if involved, extent of involvement), vascular invasion, lymph node status, and presence or absence of extensive intraductal component. The additional CM specimens were evaluated for residual carcinoma (if any) and its distance from the inked true margins, and the results were correlated with the corresponding LMs. Only approximately 50% of patients (52 of 103) with histologically positive LMs (defined as carcinoma within 2 mm of the inked surface) had residual carcinoma in their CMs. Additional CM sampling rendered the overall final margin status histologically negative in 61 of 103 (59%) cases with histologically positive LMs, therefore significantly reducing the need for reexcision. Younger patient age, higher number of positive LMs, high tumor grade, and the presence of extensive intraductal component were predictive of residual carcinoma in CM specimens, whereas the distance of carcinoma from the inked surface and the extent of tumor involvement of histologically positive LMs were not. Because CM specimens taken from patients with histologically positive LMs usually lack tumor, we suspect that many positive LMs are likely false positives. Possible factors accounting for false-positive LMs include seepage of ink into crevices of the specimen promoted by excessive inking, tumor friability promoting displacement of tumor into ink, manipulation of specimens for radiographs, and retraction artifact.