Airway derived emphysema-specific alveolar type II cells exhibit impaired regenerative potential in COPD.

Emphysema, the progressive destruction of gas exchange surfaces in the lungs, is a hallmark of chronic obstructive pulmonary disease (COPD) that is presently incurable. This therapeutic gap is largely due to a poor understanding of potential drivers of impaired tissue regeneration, such as abnormal lung epithelial progenitor cells, including alveolar type II (ATII) and airway club cells. We discovered an emphysema-specific sub-population of ATII cells located in enlarged distal alveolar sacs, termed asATII cells. Single cell RNA-seq and in situ localisation revealed that asATII cells co-express the alveolar marker surfactant protein C (SPC) and the club cell marker secretaglobin-3A2 (SCGB3A2). A similar ATII sub-population derived from club cells was also identified in mouse COPD models using lineage labeling. Human and mouse ATII sub-populations formed 80-90% fewer alveolar organoids than healthy controls, indicating reduced progenitor function. Targeting asATII cells or their progenitor club cells could reveal novel COPD treatment strategies.

Idiosyncratic nature of lactation reveals link to breast cancer risk.

Breastfeeding protects against breast cancer in some women but not others, however the mechanism remains elusive. Lactation requires intense secretory activity of the endoplasmic reticulum (ER) for the production of milk proteins and ER-mitochondria contacts for lipid synthesis. We show that in female mice that share the same nuclear genome (BL/6) but differ in mitochondrial genomes (C57 or NZB), the biological processes engaged during lactation are entirely different at the sub-cellular organization and transcriptional levels resulting in anti-tumorigenic lactation in BL/6C57 females and pro-tumorigenic lactation in BL/6NZB females. Single cell sequencing identified a sub-population of cells, uniquely amplified during lactation in BL/6NZB females, which shares the genetic signature that characterizes post-partum breast cancer (PPBC) in humans relative to matched breast cancers in never pregnant women. Our data indicate that differences in ER and mitochondrial-stress responses during lactation between genotypes inadvertently leads to loss of p53 tumor suppressor function in BL/6NZB females allowing the expansion of the PPBC-like sub-population of cells. Overall, our data reveals the unexpected idiosyncratic nature of lactation and its impacts on the risk of the development of PPBC.

Deep brain stimulation induces white matter remodeling and functional changes to brain-wide networks.

Deep brain stimulation (DBS) is an emerging therapeutic option for treatment resistant neurological and psychiatric disorders, most notably depression. Despite this, little is known about the anatomical and functional mechanisms that underlie this therapy. Here we targeted stimulation to the white matter adjacent to the subcallosal anterior cingulate cortex (SCC-DBS) in macaques, modeling the location in the brain proven effective for depression. We demonstrate that SCC-DBS has a selective effect on white matter macro- and micro-structure in the cingulum bundle distant to where stimulation was delivered. SCC-DBS also decreased functional connectivity between subcallosal and posterior cingulate cortex, two areas linked by the cingulum bundle and implicated in depression. Our data reveal that white matter remodeling as well as functional effects contribute to DBS's therapeutic efficacy.

Circulating myeloid-derived MMP8 in stress susceptibility and depression.

Psychosocial stress has profound effects on the body, including the immune system and the brain1,2. Although a large number of pre-clinical and clinical studies have linked peripheral immune system alterations to stress-related disorders such as major depressive disorder (MDD)3, the underlying mechanisms are not well understood. Here we show that expression of a circulating myeloid cell-specific proteinase, matrix metalloproteinase 8 (MMP8), is increased in the serum of humans with MDD as well as in stress-susceptible mice following chronic social defeat stress (CSDS). In mice, we show that this increase leads to alterations in extracellular space and neurophysiological changes in the nucleus accumbens (NAc), as well as altered social behaviour. Using a combination of mass cytometry and single-cell RNA sequencing, we performed high-dimensional phenotyping of immune cells in circulation and in the brain and demonstrate that peripheral monocytes are strongly affected by stress. In stress-susceptible mice, both circulating monocytes and monocytes that traffic to the brain showed increased Mmp8 expression following chronic social defeat stress. We further demonstrate that circulating MMP8 directly infiltrates the NAc parenchyma and controls the ultrastructure of the extracellular space. Depleting MMP8 prevented stress-induced social avoidance behaviour and alterations in NAc neurophysiology and extracellular space. Collectively, these data establish a mechanism by which peripheral immune factors can affect central nervous system function and behaviour in the context of stress. Targeting specific peripheral immune cell-derived matrix metalloproteinases could constitute novel therapeutic targets for stress-related neuropsychiatric disorders.

The Neurovascular Unit as a Locus of Injury in Low-Level Blast-Induced Neurotrauma.

Blast-induced neurotrauma has received much attention over the past decade. Vascular injury occurs early following blast exposure. Indeed, in animal models that approximate human mild traumatic brain injury or subclinical blast exposure, vascular pathology can occur in the presence of a normal neuropil, suggesting that the vasculature is particularly vulnerable. Brain endothelial cells and their supporting glial and neuronal elements constitute a neurovascular unit (NVU). Blast injury disrupts gliovascular and neurovascular connections in addition to damaging endothelial cells, basal laminae, smooth muscle cells, and pericytes as well as causing extracellular matrix reorganization. Perivascular pathology becomes associated with phospho-tau accumulation and chronic perivascular inflammation. Disruption of the NVU should impact activity-dependent regulation of cerebral blood flow, blood-brain barrier permeability, and glymphatic flow. Here, we review work in an animal model of low-level blast injury that we have been studying for over a decade. We review work supporting the NVU as a locus of low-level blast injury. We integrate our findings with those from other laboratories studying similar models that collectively suggest that damage to astrocytes and other perivascular cells as well as chronic immune activation play a role in the persistent neurobehavioral changes that follow blast injury.

Interstitial Macrophages Mediate Efferocytosis of Alveolar Epithelium during Influenza Infection.

Efferocytosis is a process whereby apoptotic cells are cleared to maintain tissue homeostasis. In the lungs, efferocytosis has been implicated in several acute and chronic inflammatory diseases. A long-standing method to study efferocytosis in vivo is to instill apoptotic cells into the lungs to evaluate macrophage uptake. However, this approach provides nonphysiologic levels of cells to the airspaces, where there is preferential access to the alveolar macrophages. To circumvent this limitation, we developed a new method to study efferocytosis of damaged alveolar type 2 (AT2) epithelial cells in vivo. A reporter mouse that expresses TdTomato in AT2 epithelial cells was injured with influenza (strain PR8) to induce apoptosis of AT2 cells. We were able to identify macrophages that acquire red fluorescence after influenza injury, indicating efferocytosis of AT2 cells. Furthermore, evaluation of macrophage populations led to the surprising finding that lung interstitial macrophages were the primary efferocyte in vivo. In summary, we present a novel finding that the interstitial macrophage, not the alveolar macrophage, primarily mediates clearance of AT2 cells in the lungs after influenza infection. Our method of studying efferocytosis provides a more physiologic approach in evaluating the spatiotemporal dynamics of apoptotic cell clearance in vivo and opens new avenues to study the mechanisms by which efferocytosis regulates inflammation.

Single basolateral amygdala neurons in macaques exhibit distinct connectional motifs with frontal cortex.

Basolateral amygdala (BLA) projects widely across the macaque frontal cortex, and amygdalo-frontal projections are critical for appropriate emotional responding and decision making. While it is appreciated that single BLA neurons branch and project to multiple areas in frontal cortex, the organization and frequency of this branching has yet to be fully characterized. Here, we determined the projection patterns of more than 3,000 macaque BLA neurons. We found that one-third of BLA neurons had two or more distinct projection targets in frontal cortex and subcortical structures. The patterns of single BLA neuron projections to multiple areas were organized into repeating motifs that targeted distinct sets of areas in medial and ventral frontal cortex, indicative of separable BLA networks. Our findings begin to reveal the rich structure of single-neuron connections in the non-human primate brain, providing a neuroanatomical basis for the role of BLA in coordinating brain-wide responses to valent stimuli.

Activation and pro-inflammatory cytokine production by unswitched memory B cells during SARS-CoV-2 infection.

Memory B cells are comprised of unswitched (CD27+IgD+) and switched (CD27+IgD-) subsets. The origin and function of unswitched human memory B cells are debated in the literature, whereas switched memory B cells are primed to respond to recurrent infection. Unswitched memory B cells have been described to be reduced in frequency with severe SARS-CoV2 infection and here we characterize their activation status, BCR functionality, and contribution to virally-induced cytokine production. Analyses of whole blood from healthy individuals, people immunized against SARS-CoV2, and those who have had mild and severe SARS-CoV2 infection, confirm a reduction in the frequency of unswitched memory B cells during severe SARS-CoV2 infection and demonstrate this reduction is associated with increased levels of systemic TNFα. We further document how severe viral infection is associated with an increased frequency of 'IgD+' only memory B cells that correlate with increased IgG autoantibody levels. Unswitched and switched memory B cells from severe SARS-CoV2 infection displayed evidence of heightened activation with a concomitant reduction in the expression of the inhibitory receptor CD72. Functionally, both populations of memory B cells from severe SARS-COV2 infection harbored a signaling-competent BCR that displayed enhanced BCR signaling activity in the unswitched population. Finally, we demonstrate that B cells from mild SARS-CoV2 infection are poised to secrete pro-inflammatory cytokines IL-6 and TNFα. Importantly, unswitched memory B cells were a major producer of IL-6 and switched memory B cells were a major producer of TNFα in response to viral TLR ligands. Together these data indicate that B cells contribute to the inflammatory milieu during viral infection.

Myelodysplastic neoplasm-associated U2AF1 mutations induce host defense defects by compromising neutrophil chemotaxis.

Myelodysplastic neoplasm (MDS) is a hematopoietic stem cell disorder that may evolve into acute myeloid leukemia. Fatal infection is among the most common cause of death in MDS patients, likely due to myeloid cell cytopenia and dysfunction in these patients. Mutations in genes that encode components of the spliceosome represent the most common class of somatically acquired mutations in MDS patients. To determine the molecular underpinnings of the host defense defects in MDS patients, we investigated the MDS-associated spliceosome mutation U2AF1-S34F using a transgenic mouse model that expresses this mutant gene. We found that U2AF1-S34F causes a profound host defense defect in these mice, likely by inducing a significant neutrophil chemotaxis defect. Studies in human neutrophils suggest that this effect of U2AF1-S34F likely extends to MDS patients as well. RNA-seq analysis suggests that the expression of multiple genes that mediate cell migration are affected by this spliceosome mutation and therefore are likely drivers of this neutrophil dysfunction.

Dectin-1-Independent Macrophage Phagocytosis of Mycobacterium abscessus.

Mycobacterium abscessus, a species of nontuberculous mycobacteria (NTM), is an opportunistic pathogen that is readily cleared by healthy lungs but can cause pulmonary infections in people with chronic airway diseases. Although knowledge pertaining to molecular mechanisms of host defense against NTM is increasing, macrophage receptors that recognize M. abscessus remain poorly defined. Dectin-1, a C-type lectin receptor identified as a fungal receptor, has been shown to be a pathogen recognition receptor (PRR) for both M. tuberculosis and NTM. To better understand the role of Dectin-1 in host defense against M. abscessus, we tested whether blocking Dectin-1 impaired the uptake of M. abscessus by human macrophages, and we compared M. abscessus pulmonary infection in Dectin-1-deficient and wild-type mice. Blocking antibody for Dectin-1 did not reduce macrophage phagocytosis of M. abscessus, but did reduce the ingestion of the fungal antigen zymosan. Laminarin, a glucan that blocks Dectin-1 and other PRRs, caused decreased phagocytosis of both M. abscessus and zymosan. Dectin-1-/- mice exhibited no defects in the control of M. abscessus infection, and no differences were detected in immune cell populations between wild type and Dectin-1-/- mice. These data demonstrate that murine defense against M. abscessus pulmonary infection, as well as ingestion of M. abscessus by human macrophages, can occur independent of Dectin-1. Thus, additional PRR(s) recognized by laminarin participate in macrophage phagocytosis of M. abscessus.

Polymorphic KIR3DL3 expression modulates tissue-resident and innate-like T cells.

Most human killer cell immunoglobulin-like receptors (KIR) are expressed by natural killer (NK) cells and recognize HLA class I molecules as ligands. KIR3DL3 is a conserved but polymorphic inhibitory KIR recognizing a B7 family ligand, HHLA2, and is implicated for immune checkpoint targeting. The expression profile and biological function of KIR3DL3 have been somewhat elusive, so we searched extensively for KIR3DL3 transcripts, revealing highly enriched expression in γδ and CD8+ T cells rather than NK cells. These KIR3DL3-expressing cells are rare in the blood and thymus but more common in the lungs and digestive tract. High-resolution flow cytometry and single-cell transcriptomics showed that peripheral blood KIR3DL3+ T cells have an activated transitional memory phenotype and are hypofunctional. The T cell receptor (TCR) usage is biased toward genes from early rearranged TCR-α variable segments or Vδ1 chains. In addition, we show that TCR-mediated stimulation can be inhibited through KIR3DL3 ligation. Whereas we detected no impact of KIR3DL3 polymorphism on ligand binding, variants in the proximal promoter and at residue 86 can reduce expression. Together, we demonstrate that KIR3DL3 is up-regulated alongside unconventional T cell stimulation and that individuals may vary in their ability to express KIR3DL3. These results have implications for the personalized targeting of KIR3DL3/HHLA2 checkpoint inhibition.

Thiol-Michael Addition Microparticles: Their Synthesis, Characterization, and Uptake by Macrophages.

Polymeric microparticles are promising biomaterial platforms for targeting macrophages in the treatment of disease. This study investigates microparticles formed by a thiol-Michael addition step-growth polymerization reaction with tunable physiochemical properties and their uptake by macrophages. The hexafunctional thiol monomer dipentaerythritol hexa-3-mercaptopropionate (DPHMP) and tetrafunctional acrylate monomer di(trimethylolpropane) tetraacrylate (DTPTA) were reacted in a stepwise dispersion polymerization, achieving tunable monodisperse particles over a size range (1-10 µm) relevant for targeting macrophages. An off-stoichiometry thiol-acrylate reaction afforded facile secondary chemical functionalization to create particles with different chemical moieties. Uptake of the microparticles by RAW 264.7 macrophages was highly dependent on treatment time, particle size, and particle chemistry with amide, carboxyl, and thiol terminal chemistries. The amide-terminated particles were non-inflammatory, while the carboxyl- and thiol-terminated particles induced pro-inflammatory cytokine production in conjunction with particle phagocytosis. Finally, a lung-specific application was explored through time-dependent uptake of amide-terminated particles by human alveolar macrophages in vitro and mouse lungs in vivo without inducing inflammation. The findings demonstrate a promising microparticulate delivery vehicle that is cyto-compatible, is non-inflammatory, and exhibits high rates of uptake by macrophages.

Late chronic local inflammation, synaptic alterations, vascular remodeling and arteriovenous malformations in the brains of male rats exposed to repetitive low-level blast overpressures.

In the course of military operations in modern war theaters, blast exposures are associated with the development of a variety of mental health disorders associated with a post-traumatic stress disorder-related features, including anxiety, impulsivity, insomnia, suicidality, depression, and cognitive decline. Several lines of evidence indicate that acute and chronic cerebral vascular alterations are involved in the development of these blast-induced neuropsychiatric changes. In the present study, we investigated late occurring neuropathological events associated with cerebrovascular alterations in a rat model of repetitive low-level blast-exposures (3 × 74.5 kPa). The observed events included hippocampal hypoperfusion associated with late-onset inflammation, vascular extracellular matrix degeneration, synaptic structural changes and neuronal loss. We also demonstrate that arteriovenous malformations in exposed animals are a direct consequence of blast-induced tissue tears. Overall, our results further identify the cerebral vasculature as a main target for blast-induced damage and support the urgent need to develop early therapeutic approaches for the prevention of blast-induced late-onset neurovascular degenerative processes.

Marked elevations in lung and plasma ceramide in COVID-19 linked to microvascular injury.

The pathogenesis of the marked pulmonary microvasculature injury, a distinguishing feature of COVID-19 acute respiratory distress syndrome (COVID-ARDS), remains unclear. Implicated in the pathophysiology of diverse diseases characterized by endothelial damage, including ARDS and ischemic cardiovascular disease, ceramide and in particular palmitoyl ceramide (C16:0-ceramide) may be involved in the microvascular injury in COVID-19. Using deidentified plasma and lung samples from COVID-19 patients, ceramide profiling by mass spectrometry was performed. Compared with healthy individuals, a specific 3-fold C16:0-ceramide elevation in COVID-19 patient plasma was identified. Compared with age-matched controls, autopsied lungs of individuals succumbing to COVID-ARDS displayed a massive 9-fold C16:0-ceramide elevation and exhibited a previously unrecognized microvascular ceramide-staining pattern and markedly enhanced apoptosis. In COVID-19 plasma and lungs, the C16-ceramide/C24-ceramide ratios were increased and reversed, respectively, consistent with increased risk of vascular injury. Indeed, exposure of primary human lung microvascular endothelial cell monolayers to C16:0-ceramide-rich plasma lipid extracts from COVID-19, but not healthy, individuals led to a significant decrease in endothelial barrier function. This effect was phenocopied by spiking healthy plasma lipid extracts with synthetic C16:0-ceramide and was inhibited by treatment with ceramide-neutralizing monoclonal antibody or single-chain variable fragment. These results indicate that C16:0-ceramide may be implicated in the vascular injury associated with COVID-19.

Peripheral immune-derived matrix metalloproteinase promotes stress susceptibility.

Psychosocial stress has profound effects on the body, including the peripheral immune system and the brain1,2. Although a large number of pre-clinical and clinical studies have linked peripheral immune system alterations to stress-related disorders such as major depressive disorder (MDD)3,4,5, the underlying mechanisms are not well understood. Here we show that a peripheral myeloid cell-specific proteinase, matrix metalloproteinase 8 (MMP8), is elevated in serum of subjects with MDD as well as in stress-susceptible (SUS) mice following chronic social defeat stress (CSDS). In mice, we show that this increase leads to alterations in extracellular space and neurophysiological changes in the nucleus accumbens (NAc), thereby altering social behaviour. Using a combination of mass cytometry and single-cell RNA-sequencing, we performed high-dimensional phenotyping of immune cells in circulation and brain and demonstrate that peripheral monocytes are strongly affected by stress. Both peripheral and brain-infiltrating monocytes of SUS mice showed increased Mmp8 expression following CSDS. We further demonstrate that peripheral MMP8 directly infiltrates the NAc parenchyma to control the ultrastructure of the extracellular space. Depleting MMP8 prevented stress-induced social avoidance behaviour and alterations in NAc neurophysiology and extracellular space. Collectively, these data establish a novel mechanism by which peripheral immune factors can affect central nervous system function and behaviour in the context of stress. Targeting specific peripheral immune cell-derived matrix metalloproteinases could constitute novel therapeutic targets for stress-related neuropsychiatric disorders.

Single-cell RNA sequencing reveals unique monocyte-derived interstitial macrophage subsets during lipopolysaccharide-induced acute lung inflammation.

Interstitial macrophages (IMs) reside in the lung tissue surrounding key structures including airways, vessels, and alveoli. Recent work has described IM heterogeneity during homeostasis, however, there are limited data on IMs during inflammation. We sought to characterize IM origin, subsets, and transcriptomic profiles during homeostasis and lipopolysaccharide (LPS) induced acute lung inflammation. During homeostasis, we used three complementary methods, spectral flow cytometry, single-cell RNA-sequencing, and gene regulatory network enrichment, to demonstrate that IMs can be divided into two core subsets distinguished by surface and transcriptional expression of folate receptor ß (Folr2/FRß). These subsets inhabited distinct niches within the lung interstitium. Within FRß+ IMs we identified a subpopulation marked by coexpression of LYVE1. During acute LPS-induced inflammation, lung IM numbers expand. Lineage tracing revealed IM expansion was due to recruitment of monocyte-derived IMs. At the peak of inflammation, recruited IMs were comprised two unique subsets defined by expression of genes associated with interferon signaling and glycolytic pathways. As recruited IMs matured, they adopted the overall transcriptional state of FRß- resident IMs but retained expression in several origin-specific genes, such as IL-1ß. FRß+ IMs were of near-pure resident origin. Taken together our data show that during LPS-induced inflammation, there are distinct populations of IMs that likely have unique functions. FRΒ+ IMs comprise a stable, resident population, whereas FRß- ΙΜs represent a mixed population of resident and recruited IMs.

Neuronal vulnerability to brain aging and neurodegeneration in cognitively impaired marmoset monkeys (Callithrix jacchus).

The investigation of neurobiological and neuropathological changes that affect synaptic integrity and function with aging is key to understanding why the aging brain is vulnerable to Alzheimer's disease. We investigated the cellular characteristics in the cerebral cortex of behaviorally characterized marmosets, based on their trajectories of cognitive learning as they transitioned to old age. We found increased astrogliosis, increased phagocytic activity of microglial cells and differences in resting and reactive microglial cell phenotypes in cognitively impaired compared to nonimpaired marmosets. Differences in amyloid beta deposition were not related to cognitive trajectory. However, we found age-related changes in density and morphology of dendritic spines in pyramidal neurons of layer 3 in the dorsolateral prefrontal cortex and the CA1 field of the hippocampus between cohorts. Overall, our data suggest that an accelerated aging process, accompanied by neurodegeneration, that takes place in cognitively impaired aged marmosets and affects the plasticity of dendritic spines in cortical areas involved in cognition and points to mechanisms of neuronal vulnerability to aging.

High-throughput sequencing of macaque basolateral amygdala projections reveals dissociable connectional motifs with frontal cortex.

The basolateral amygdala (BLA) projects widely across the macaque frontal cortex1-4, and amygdalo-frontal projections are critical for optimal emotional responding5 and decision-making6. Yet, little is known about the single-neuron architecture of these projections: namely, whether single BLA neurons project to multiple parts of the frontal cortex. Here, we use MAPseq7 to determine the projection patterns of over 3000 macaque BLA neurons. We found that one-third of BLA neurons have two or more distinct targets in parts of frontal cortex and of subcortical structures. Further, we reveal non-random structure within these branching patterns such that neurons with four targets are more frequently observed than those with two or three, indicative of widespread networks. Consequently, these multi-target single neurons form distinct networks within medial and ventral frontal cortex consistent with their known functions in regulating mood and decision-making. Additionally, we show that branching patterns of single neurons shape functional networks in the brain as assessed by fMRI-based functional connectivity. These results provide a neuroanatomical basis for the role of the BLA in coordinating brain-wide responses to valent stimuli8 and highlight the importance of high-resolution neuroanatomical data for understanding functional networks in the brain.