Respiratory viral infection promotes the awakening and outgrowth of dormant metastatic breast cancer cells in lungs.

Breast cancer is the second most common cancer globally. Most deaths from breast cancer are due to metastatic disease which often follows long periods of clinical dormancy1. Understanding the mechanisms that disrupt the quiescence of dormant disseminated cancer cells (DCC) is crucial for addressing metastatic progression. Infection with respiratory viruses (e.g. influenza or SARS-CoV-2) is common and triggers an inflammatory response locally and systemically2,3. Here we show that influenza virus infection leads to loss of the pro-dormancy mesenchymal phenotype in breast DCC in the lung, causing DCC proliferation within days of infection, and a greater than 100-fold expansion of carcinoma cells into metastatic lesions within two weeks. Such DCC phenotypic change and expansion is interleukin-6 (IL-6)-dependent. We further show that CD4 T cells are required for the maintenance of pulmonary metastatic burden post-influenza virus infection, in part through attenuation of CD8 cell responses in the lungs. Single-cell RNA-seq analyses reveal DCC-dependent impairment of T-cell activation in the lungs of infected mice. SARS-CoV-2 infected mice also showed increased breast DCC expansion in lungs post-infection. Expanding our findings to human observational data, we observed that cancer survivors contracting a SARS-CoV-2 infection have substantially increased risks of lung metastatic progression and cancer-related death compared to cancer survivors who did not. These discoveries underscore the significant impact of respiratory viral infections on the resurgence of metastatic cancer, offering novel insights into the interconnection between infectious diseases and cancer metastasis.

Restoring connexin-36 function in diabetogenic environments precludes mouse and human islet dysfunction.

The secretion of insulin from ß-cells in the islet of Langerhans is governed by a series of metabolic and electrical events, which can fail during the progression of type 2 diabetes (T2D). ß-cells are electrically coupled via connexin-36 (Cx36) gap junction channels, which coordinates the pulsatile dynamics of [Ca2+ ] and insulin release across the islet. Factors such as pro-inflammatory cytokines and free fatty acids disrupt gap junction coupling under in vitro conditions. Here we test whether gap junction coupling and coordinated [Ca2+ ] dynamics are disrupted in T2D, and whether recovery of gap junction coupling can recover islet function. We examine islets from donors with T2D, from db/db mice, and islets treated with pro-inflammatory cytokines (TNF-α, IL-1ß, IFN-É£) or free fatty acids (palmitate). We modulate gap junction coupling using Cx36 over-expression or pharmacological activation via modafinil. We also develop a peptide mimetic (S293) of the c-terminal regulatory site of Cx36 designed to compete against its phosphorylation. Cx36 gap junction permeability and [Ca2+ ] dynamics were disrupted in islets from both human donors with T2D and db/db mice, and in islets treated with pro-inflammatory cytokines or palmitate. Cx36 over-expression, modafinil treatment and S293 peptide all enhanced Cx36 gap junction coupling and protected against declines in coordinated [Ca2+ ] dynamics. Cx36 over-expression and S293 peptide also reduced apoptosis induced by pro-inflammatory cytokines. Critically, S293 peptide rescued gap junction coupling and [Ca2+ ] dynamics in islets from both db/db mice and a sub-set of T2D donors. Thus, recovering or enhancing Cx36 gap junction coupling can improve islet function in diabetes. KEY POINTS: Connexin-36 (Cx36) gap junction permeability and associated coordination of [Ca2+ ] dynamics is diminished in human type 2 diabetes (T2D) and mouse models of T2D. Enhancing Cx36 gap junction permeability protects against disruptions to the coordination of [Ca2+ ] dynamics. A novel peptide mimetic of the Cx36 c-terminal regulatory region protects against declines in Cx36 gap junction permeability. Pharmacological elevation in Cx36 or Cx36 peptide mimetic recovers [Ca2+ ] dynamics and glucose-stimulated insulin secretion in human T2D and mouse models of T2D.

Reduced PU.1 Expression Collaborates with Tet2 Loss to Trigger Myeloid Leukemogenesis.

The leukemic transformation of hematopoietic stem and progenitor cells in the setting of Tet2 deficiency is driven by PU.1 gene network loss through complementary reduction in PU.1 expression and hypermethylation of ETS loci at the enhancers of PU.1 target genes. See related article by Aivalioti et al., p. 444 (6).

Functional architecture of pancreatic islets identifies a population of first responder cells that drive the first-phase calcium response.

Insulin-secreting ß-cells are functionally heterogeneous. Whether there exist cells driving the first-phase calcium response in individual islets, has not been examined. We examine "first responder" cells, defined by the earliest [Ca2+] response during first-phase [Ca2+] elevation, distinct from previously identified "hub" and "leader" cells. We used islets isolated from Mip-CreER; Rosa-Stop-Lox-Stop-GCamP6s mice (ß-GCamP6s) that show ß-cell-specific GCamP6s expression following tamoxifen-induced CreER-mediated recombination. First responder cells showed characteristics of high membrane excitability and lower electrical coupling to their neighbors. The first-phase response time of ß-cells in the islet was spatially organized, dependent on the cell's distance to the first responder cell, and consistent over time up to approximately 24 h. When first responder cells were laser ablated, the first-phase [Ca2+] was slowed down, diminished, and discoordinated compared to random cell ablation. Cells that were next earliest to respond often took over the role of the first responder upon ablation. In summary, we discover and characterize a distinct first responder ß-cell state, critical for the islet first-phase response to glucose.

Modulation of Gap Junction Coupling Within the Islet of Langerhans During the Development of Type 1 Diabetes.

In type 1 diabetes (T1D), islet dysfunction occurs prior to diabetes onset. Pro-inflammatory cytokines can disrupt insulin secretion and Ca2+ homeostasis. Connexin36 (Cx36) gap junctions electrically couple ß-cells to coordinate glucose-stimulated Ca2+ and insulin secretion. Cx36 gap junction coupling can also protect against cytokine-induced apoptosis. Our goal was to determine how islet gap junction coupling and Ca2+ dynamics are altered in mouse models of T1D prior to diabetes. Glucose tolerance was assessed in NOD and immunodeficient NOD-RAG1KO mice at 6-12 weeks age. Glucose-stimulated insulin secretion, Ca2+ dynamics, and gap junction coupling were measured in islets isolated at each age. Gap junction coupling was also measured in islets from mice that underwent transfer of diabetogenic splenocytes and from chromograninA knockout NOD mice. Cell death was measured in islets isolated from wild-type, Cx36 knockout or Cx36 over-expression mice, each treated with a cocktail of pro-inflammatory cytokines and KATP or SERCA activators/inhibitors. NOD mice over-expressing Cx36 were also monitored for diabetes development, and islets assessed for insulitis and apoptosis. NOD and NOD-RAG1KO controls showed similar glucose tolerance at all ages. Ca2+ dynamics and gap junction coupling were disrupted in islets of NOD mice at 9 weeks, compared to controls. Transfer of diabetogenic splenocytes also decreased gap junction coupling. Islets from chromograninA knockout mice displayed normal coupling. Overexpression of Cx36 protected islets from cytokine-induced apoptosis. A knockout of Cx36 amplified cytokine-induced apoptosis, which was reversed by KATP activation or SERCA activation. Cx36 overexpression in NOD mice delayed diabetes development compared to NOD controls. However, apoptosis and insulitis were not improved. Decreases in islet gap junction coupling occur prior to T1D onset. Such decreases alter islet susceptibility to apoptosis due to altered Ca2+. Future studies will determine if increasing Cx36 gap junction coupling in combination with restoring Ca2+ homeostasis protects against islet decline in T1D.

Dynamic changes in ß-cell [Ca2+] regulate NFAT activation, gene transcription, and islet gap junction communication.

OBJECTIVE: Diabetes occurs because of insufficient insulin secretion due to ß-cell dysfunction within the islet of Langerhans. Elevated glucose levels trigger ß-cell membrane depolarization, action potential generation, and slow sustained free-Ca2+ ([Ca2+]) oscillations, which trigger insulin release. Nuclear factor of activated T-cell (NFAT) is a transcription factor, which is regulated by the increases in [Ca2+] and calceineurin (CaN) activation. NFAT regulation links cell activity with gene transcription in many systems and regulates proliferation and insulin granule biogenesis within the ß-cell. However, the link between the regulation of ß-cell electrical activity and oscillatory [Ca2+] dynamics with NFAT activation and downstream transcription is poorly understood. Here, we tested whether dynamic changes to ß-cell electrical activity and [Ca2+] regulate NFAT activation and downstream transcription. METHODS: In cell lines, mouse islets, and human islets, including those from donors with type 2 diabetes, we applied both agonists/antagonists of ion channels together with optogenetics to modulate ß-cell electrical activity. We measured the dynamics of [Ca2+] and NFAT activation as well as performed whole transcriptome and functional analyses. RESULTS: Both glucose-induced membrane depolarization and optogenetic stimulation triggered NFAT activation as well as increased the transcription of NFAT targets and intermediate early genes (IEGs). Importantly, slow, sustained [Ca2+] oscillation conditions led to NFAT activation and downstream transcription. In contrast, in human islets from donors with type2 diabetes, NFAT activation by glucose was diminished, but rescued upon pharmacological stimulation of electrical activity. NFAT activation regulated GJD2 expression and increased Cx36 gap junction permeability upon elevated oscillatory [Ca2+] dynamics. However, it is unclear if NFAT directly binds the GJD2 gene to regulate expression. CONCLUSIONS: This study provides an insight into the specific patterns of electrical activity that regulate NFAT activation, gene transcription, and islet function. In addition, it provides information on how these factors are disrupted in diabetes.

Optogenetic stimulation of cholinergic fibers for the modulation of insulin and glycemia.

Previous studies have demonstrated stimulation of endocrine pancreas function by vagal nerve electrical stimulation. While this increases insulin secretion, expected concomitant reductions in circulating glucose do not occur. A complicating factor is the non-specific nature of electrical nerve stimulation. Optogenetic tools, however, provide the potential for cell-type specific neural stimulation using genetic targeting and/or spatially shaped excitation light. Here, we demonstrate light-activated stimulation of the endocrine pancreas by targeting parasympathetic (cholinergic) axons. In a mouse model expressing ChannelRhodopsin2 (ChR2) in cholinergic cells, serum insulin and glucose were measured in response to (1) ultrasound image-guided optical stimulation of axon terminals in the pancreas or (2) optical stimulation of axons of the cervical vagus nerve. Measurements were made in basal-glucose and glucose-stimulated conditions. Significant increases in plasma insulin occurred relative to controls under both pancreas and cervical vagal stimulation, while a rapid reduction in glycemic levels were observed under pancreatic stimulation. Additionally, ultrasound-based measurements of blood flow in the pancreas were increased under pancreatic stimulation. Together, these results demonstrate the utility of in-vivo optogenetics for studying the neural regulation of endocrine pancreas function and suggest its therapeutic potential for the control of insulin secretion and glucose homeostasis.

Caloric restriction recovers impaired ß-cell-ß-cell gap junction coupling, calcium oscillation coordination, and insulin secretion in prediabetic mice.

Caloric restriction can decrease the incidence of metabolic diseases, such as obesity and Type 2 diabetes mellitus. The mechanisms underlying the benefits of caloric restriction involved in insulin secretion and glucose homeostasis are not fully understood. Intercellular communication within the islets of Langerhans, mediated by Connexin36 (Cx36) gap junctions, regulates insulin secretion dynamics and glucose homeostasis. The goal of this study was to determine whether caloric restriction can protect against decreases in Cx36 gap junction coupling and altered islet function induced in models of obesity and prediabetes. C57BL6 mice were fed with a high-fat diet (HFD), showing indications of prediabetes after 2 mo, including weight gain, insulin resistance, and elevated fasting glucose and insulin levels. Subsequently, mice were submitted to 1 mo of 40% caloric restriction (2 g/day of HFD). Mice under 40% caloric restriction showed reversal in weight gain and recovered insulin sensitivity, fasting glucose, and insulin levels. In islets of mice fed the HFD, caloric restriction protected against obesity-induced decreases in gap junction coupling and preserved glucose-stimulated calcium signaling, including Ca2+ oscillation coordination and oscillation amplitude. Caloric restriction also promoted a slight increase in glucose metabolism, as measured by increased NAD(P)H autofluorescence, as well as recovering glucose-stimulated insulin secretion. We conclude that declines in Cx36 gap junction coupling that occur in obesity can be completely recovered by caloric restriction and obesity reversal, improving Ca2+ dynamics and insulin secretion regulation. This suggests a critical role for caloric restriction in the context of obesity to prevent islet dysfunction.

How Heterogeneity in Glucokinase and Gap-Junction Coupling Determines the Islet [Ca2+] Response.

Understanding how cell subpopulations in a tissue impact overall system function is challenging. There is extensive heterogeneity among insulin-secreting ß-cells within islets of Langerhans, including their insulin secretory response and gene expression profile, and this heterogeneity can be altered in diabetes. Several studies have identified variations in nutrient sensing between ß-cells, including glucokinase (GK) levels, mitochondrial function, or expression of genes important for glucose metabolism. Subpopulations of ß-cells with defined electrical properties can disproportionately influence islet-wide free-calcium activity ([Ca2+]) and insulin secretion via gap-junction electrical coupling. However, it is poorly understood how subpopulations of ß-cells with altered glucose metabolism may impact islet function. To address this, we utilized a multicellular computational model of the islet in which a population of cells deficient in GK activity and glucose metabolism was imposed on the islet or in which ß-cells were heterogeneous in glucose metabolism and GK kinetics were altered. This included simulating GK gene (GCK) mutations that cause monogenic diabetes. We combined these approaches with experimental models in which gck was genetically deleted in a population of cells or GK was pharmacologically inhibited. In each case, we modulated gap-junction electrical coupling. Both the simulated islet and the experimental system required 30-50% of the cells to have near-normal glucose metabolism, fewer than cells with normal KATP conductance. Below this number, the islet lacked any glucose-stimulated [Ca2+] elevations. In the absence of electrical coupling, the change in [Ca2+] was more gradual. As such, electrical coupling allows a large minority of cells with normal glucose metabolism to promote glucose-stimulated [Ca2+]. If insufficient numbers of cells are present, which we predict can be caused by a subset of GCK mutations that cause monogenic diabetes, electrical coupling exacerbates [Ca2+] suppression. This demonstrates precisely how metabolically heterogeneous ß-cell populations interact to impact islet function.

Exendin-4 overcomes cytokine-induced decreases in gap junction coupling via protein kinase A and Epac2 in mouse and human islets.

KEY POINTS: The pancreatic islets of Langerhans maintain glucose homeostasis through insulin secretion, where insulin secretion dynamics are regulated by intracellular Ca2+ signalling and electrical coupling of the insulin producing ß-cells in the islet. We have previously shown that cytokines decrease ß-cell coupling and that compounds which increase cAMP can increase coupling. In both mouse and human islets exendin-4, which increases cAMP, protected against cytokine-induced decreases in coupling and in mouse islets preserved glucose-stimulated calcium signalling by increasing connexin36 gap junction levels on the plasma membrane. Our data indicate that protein kinase A regulates ß-cell coupling through a fast mechanism, such as channel gating or membrane organization, while Epac2 regulates slower mechanisms of regulation, such as gap junction turnover. Increases in ß-cell coupling with exendin-4 may protect against cytokine-mediated ß-cell death as well as preserve insulin secretion dynamics during the development of diabetes. ABSTRACT: The pancreatic islets of Langerhans maintain glucose homeostasis. Insulin secretion from islet ß-cells is driven by glucose metabolism, depolarization of the cell membrane and an influx of calcium, which initiates the release of insulin. Gap junctions composed of connexin36 (Cx36) electrically couple ß-cells, regulating calcium signalling and insulin secretion dynamics. Cx36 coupling is decreased in pre-diabetic mice, suggesting a role for altered coupling in diabetes. Our previous work has shown that pro-inflammatory cytokines decrease Cx36 coupling and that compounds which increase cAMP can increase Cx36 coupling. The goal of this study was to determine if exendin-4, which increases cAMP, can protect against cytokine-induced decreases in Cx36 coupling and altered islet function. In both mouse and human islets, exendin-4 protected against cytokine-induced decreases in coupling and preserved glucose-stimulated calcium signalling. Exendin-4 also protected against protein kinase Cδ-mediated decreases in Cx36 coupling. Exendin-4 preserved coupling in mouse islets by preserving Cx36 levels on the plasma membrane. Exendin-4 regulated Cx36 coupling via both protein kinase A (PKA)- and Epac2-mediated mechanisms in cytokine-treated islets. In mouse islets, modulating Epac2 had a greater impact in mediating Cx36 coupling, while in human islets modulating PKA had a greater impact on Cx36 coupling. Our data indicate that PKA regulates Cx36 coupling through a fast mechanism, such as channel gating, while Epac2 regulates slower mechanisms of regulation, such as Cx36 turnover in the membrane. Increases in Cx36 coupling with exendin-4 may protect against cytokine-mediated ß-cell dysfunction to insulin secretion dynamics during the development of diabetes.

Spinal Cord Injury in Rats Dysregulates Diurnal Rhythms of Fecal Output and Liver Metabolic Indicators.

Spinal cord injury (SCI) dysregulates metabolic homeostasis. Metabolic homeostasis is optimized across the day by the circadian system. Despite the prevalence of metabolic pathologies after SCI, post-SCI circadian regulation of metabolism remains understudied. Here, we hypothesized that SCI in rats would disrupt circadian regulation of key metabolic organs, leading to metabolic dysregulation. Female and male Sprague-Dawley rats received moderate thoracic (T)-9 contusion SCI (or sham surgery). First, SCI disrupted diurnal rhythms in two metabolic behaviors: fecal production and food intake rhythms were ablated acutely. SCI also expedited whole-gut transit time. In parallel, acute SCI increased plasma glucose. Diurnal glucose storage-release cycles regulated by the liver were disrupted by SCI, which also increased liver glucose metabolism messenger RNAs (mRNAs). Further, SCI disrupted liver clock gene expression and suppressed inflammatory gene rhythms. Together, our novel data suggest that SCI disrupts typical metabolic and circadian function. Improving post-SCI metabolic function could enhance recovery of homeostasis.

Journal Club: MRI reveals acute inflammation in cortical lesions during early MS.

Early multiple sclerosis is characterized by immune-associated demyelination of CNS axons. In a recent Neurology® article, Maranzano et al. evaluated MRI scans of patients with early multiple sclerosis to study the evolution of leukocortical lesions. Their novel data suggest that acute inflammation after blood-brain barrier leakage may contribute to gray matter cortical lesions in early multiple sclerosis.

Spinal Cord Injury in Rats Disrupts the Circadian System.

Spinal cord injury (SCI) perturbs many physiological systems. The circadian system helps maintain homeostasis throughout the body by synchronizing physiological and behavioral functions to predictable daily events. Whether disruption of these coordinated daily rhythms contributes to SCI-associated pathology remains understudied. Here, we hypothesized that SCI in rats would dysregulate several prominent circadian outputs including glucocorticoids, core temperature, activity, neuroinflammation, and circadian gene networks. Female and male Sprague Dawley rats were subjected to clinically relevant thoracic 9 moderate contusion SCI (or laminectomy sham surgery). Diurnal measures-including rhythms of plasma corticosterone (CORT), body temperature, and activity (using small implanted transmitters), and intraspinal circadian and inflammatory gene expression-were studied prior to and after surgery. SCI caused overall increases and disrupted rhythms of the major rodent glucocorticoid, CORT. Presurgery and sham rats displayed expected rhythms in body temperature and activity, whereas rats with SCI had blunted daily rhythms in body temperature and activity. In parallel, SCI disrupted intraspinal rhythms of circadian clock gene expression. Circadian clock genes can act as transcriptional regulators of inflammatory pathways. Indeed, SCI rats also showed dysregulated rhythms in inflammatory gene expression in both the epicenter and distal spinal cord. Our data show that moderate SCI in rats causes wide-ranging diurnal rhythm dysfunction, which is severe at acute time points and gradually recovers over time. Normalizing post-SCI diurnal rhythms could enhance the recovery of homeostasis and quality of life.

Exploring acute-to-chronic neuropathic pain in rats after contusion spinal cord injury.

Spinal cord injury (SCI) causes chronic pain in 65% of individuals. Unfortunately, current pain management is inadequate for many SCI patients. Rodent models could help identify how SCI pain develops, explore new treatment strategies, and reveal whether acute post-SCI morphine worsens chronic pain. However, few studies explore or compare SCI-elicited neuropathic pain in rats. Here, we sought to determine how different clinically relevant contusion SCIs in male and female rats affect neuropathic pain, and whether acute morphine worsens later chronic SCI pain. First, female rats received sham surgery, or 150kDyn or 200kDyn midline T9 contusion SCI. These rats displayed modest mechanical allodynia and long-lasting thermal hyperalgesia. Next, a 150kDyn (1s dwell) midline contusion SCI was performed in male and female rats. Interestingly, males, but not females showed SCI-elicited mechanical allodynia; rats of both sexes had thermal hyperalgesia. In this model, acute morphine treatment had no significant effect on chronic neuropathic pain symptoms. Unilateral SCIs can also elicit neuropathic pain that could be exacerbated by morphine, so male rats received unilateral T13 contusion SCI (100kDyn). These rats exhibited significant, transient mechanical allodynia, but not thermal hyperalgesia. Acute morphine did not exacerbate chronic pain. Our data show that specific rat contusion SCI models cause neuropathic pain. Further, chronic neuropathic pain elicited by these contusion SCIs was not amplified by our course of early post-trauma morphine. Using clinically relevant rat models of SCI could help identify novel pain management strategies.