bioRxiv : the preprint server for biology
Dorman, LC;Nguyen, PT;Escoubas, CC;Vainchtein, ID;Xiao, Y;Lidsky, PV;Nakajo, H;Silva, NJ;Lagares-Linares, C;Wang, EY;Taloma, SE;Cuevas, B;Nakao-Inoue, H;Rivera, BM;Schwer, B;Condello, C;Andino, R;Nowakowski, TJ;Molofsky, AV;
PMID: 35233577 | DOI: 10.1101/2021.04.29.441889
Microglia, the innate immune cells of the brain, are exquisitely sensitive to dynamic changes in the neural environment. Using single cell RNA sequencing of the postnatal somatosensory cortex during topographic remapping, we identified a type I interferon (IFN-I) responsive microglia population that expanded with this developmental stressor. Using the marker gene IFITM3 we found that IFN-I responsive microglia were engulfing whole neurons. Loss of IFN-I signaling ( Ifnar1 -/- ) resulted in dysmorphic 'bubble' microglia with enlarged phagolysosomal compartments. We also observed a reduction in dead cells and an accumulation of neurons with double strand DNA breaks, a marker of cell stress. Conversely, IFN-I gain of function in zebrafish was sufficient to drive microglial engulfment of whole neurons. We identified IFITM3+ microglia in two murine disease models: SARS-CoV-2 infection and the 5xFAD model of Alzheimer's disease. These data reveal a novel role for IFN-I signaling in regulating efficient neuronal clearance by microglia.
Brain Struct Funct. 2018 Oct 20.
Gasparini S, Resch JM, Narayan SV, Peltekian L, Iverson GN, Karthik S, Geerling JC.
PMID: 30343334 | DOI: 10.1007/s00429-018-1778-y
Sodium deficiency elevates aldosterone, which in addition to epithelial tissues acts on the brain to promote dysphoric symptoms and salt intake. Aldosterone boosts the activity of neurons that express 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), a hallmark of aldosterone-sensitive cells. To better characterize these neurons, we combine immunolabeling and in situ hybridization with fate mapping and Cre-conditional axon tracing in mice. Many cells throughout the brain have a developmental history of Hsd11b2 expression, but in the adult brain one small brainstem region with a leaky blood-brain barrier contains HSD2 neurons. These neurons express Hsd11b2, Nr3c2 (mineralocorticoid receptor), Agtr1a (angiotensin receptor), Slc17a6 (vesicular glutamate transporter 2), Phox2b, and Nxph4; many also express Cartpt or Lmx1b. No HSD2 neurons express cholinergic, monoaminergic, or several other neuropeptidergic markers. Their axons project to the parabrachial complex (PB), where they intermingle with AgRP-immunoreactive axons to form dense terminal fields overlapping FoxP2 neurons in the central lateral subnucleus (PBcL) and pre-locus coeruleus (pLC). Their axons also extend to the forebrain, intermingling with AgRP- and CGRP-immunoreactive axons to form dense terminals surrounding GABAergic neurons in the ventrolateral bed nucleus of the stria terminalis (BSTvL). Sparse axons target the periaqueductal gray, ventral tegmental area, lateral hypothalamic area, paraventricular hypothalamic nucleus, and central nucleus of the amygdala. Dual retrograde tracing revealed that largely separate HSD2 neurons project to pLC/PB or BSTvL. This projection pattern raises the possibility that a subset of HSD2 neurons promotes the dysphoric, anorexic, and anhedonic symptoms of hyperaldosteronism via AgRP-inhibited relay neurons in PB.
March-Riera, S;Wilson, AA;Bhatia, SN;Muhlberger, E;
| DOI: 10.1016/j.stemcr.2022.08.003
Liver damage and an exacerbated inflammatory response are hallmarks of Ebola virus (EBOV) infection. Little is known about the intrinsic response to infection in human hepatocytes and their contribution to inflammation. Here, we present an induced pluripotent stem cell (iPSC)-derived hepatocyte-like cell (HLC) platform to define the hepato-intrinsic response to EBOV infection. We used this platform to show robust EBOV infection, with characteristic ultrastructural changes and evidence for viral replication. Transcriptomics analysis revealed a delayed response with minimal early transcriptomic changes, followed by a general downregulation of hepatic function and upregulation of interferon signaling, providing a potential mechanism by which hepatocytes participate in disease severity and liver damage. Using RNA-fluorescence in situ hybridization (FISH), we showed that IFNB1 and CXCL10 were mainly expressed in non-infected bystander cells. We did not observe an inflammatory signature during infection. In conclusion, iPSC-HLCs are an immune competent platform to study responses to EBOV infection.
J Comp Pathol. 2015 Jul 16.
Palmer MV, Thacker TC, Waters WR.
PMID: 26189773 | DOI: 10.1016/j.jcpa.2015.06.004.
Mycobacterium bovis is the cause of tuberculosis in most animal species including cattle and is a serious zoonotic pathogen. In man, M. bovis infection can result in disease clinically indistinguishable from that caused by Mycobacterium tuberculosis, the cause of most human tuberculosis. Regardless of host, the typical lesion induced by M. bovis or M. tuberculosis is the tuberculoid granuloma. Tuberculoid granulomas are dynamic structures reflecting the interface between host and pathogen and, therefore, pass through various morphological stages (I to IV). Using a novel in-situ hybridization assay, transcription of various cytokine and chemokine genes was examined qualitatively and quantitatively using image analysis. In experimentally infected cattle, pulmonary granulomas of all stages were examined 150 days after aerosol exposure to M. bovis. Expression of mRNA encoding tumour necrosis factor (TNF)-α, transforming growth factor-β, interferon (IFN)-γ, interleukin (IL)-17A, IL-16, IL-10, CXCL9 and CXCL10 did not differ significantly between granulomas of different stages. However, relative expression of the various cytokines was characteristic of a Th1 response, with high TNF-α and IFN-γ expression and low IL-10 expression. Expression of IL-16 and the chemokines CXCL9 and CXCL10 was high, suggestive of granulomas actively involved in T-cell chemotaxis.
Schulz D, Zanotelli VRT, Fischer JR, Schapiro D, Engler S, Lun XK, Jackson HW, Bodenmiller B.
PMID: 29289569 | DOI: 10.1016/j.cels.2017.12.001
To build comprehensive models of cellular states and interactions in normal and diseased tissue, genetic and proteomic information must be extracted with single-cell and spatial resolution. Here, we extended imaging mass cytometry to enable multiplexed detection of mRNA and proteins in tissues. Three mRNA target species were detected by RNAscope-based metal in situ hybridization with simultaneous antibody detection of 16 proteins. Analysis of 70 breast cancer samples showed that HER2 and CK19 mRNA and protein levels are moderately correlated on the single-cell level, but that only HER2, and not CK19, has strong mRNA-to-protein correlation on the cell population level. The chemoattractant CXCL10 was expressed in stromal cell clusters, and the frequency of CXCL10-expressing cells correlated with T cell presence. Our flexible and expandable method will allow an increase in the information content retrieved from patient samples for biomedical purposes, enable detailed studies of tumor biology, and serve as a tool to bridge comprehensive genomic and proteomic tissue analysis.
Linker KE, Elabd MG, Tawadrous P, Cano M, Green KN, Wood MA, Leslie FM
PMID: 31949158 | DOI: 10.1038/s41467-019-14173-3
With the rise of e-cigarette use, teen nicotine exposure is becoming more widespread. Findings from clinical and preclinical studies show that the adolescent brain is particularly sensitive to nicotine. Animal studies have demonstrated that adolescent nicotine exposure increases reinforcement for cocaine and other drugs. However, the mechanisms that underlie these behaviors are poorly understood. Here, we report reactive microglia are critical regulators of nicotine-induced increases in adolescent cocaine self-administration. Nicotine has dichotomous, age-dependent effects on microglial morphology and immune transcript profiles. A multistep signaling mechanism involving D2 receptors and CX3CL1 mediates nicotine-induced increases in cocaine self-administration and microglial activation. Moreover, nicotine depletes presynaptic markers in a manner that is microglia-, D2- and CX3CL1-dependent. Taken together, we demonstrate that adolescent microglia are uniquely susceptible to perturbations by nicotine, necessary for nicotine-induced increases in cocaine-seeking, and that D2 receptors and CX3CL1 play a mechanistic role in these phenomena
Liu, X;Wang, Y;Zeng, Y;Wang, D;Wen, Y;Fan, L;He, Y;Zhang, J;Sun, W;Liu, Y;Tao, A;
PMID: 36876522 | DOI: 10.1111/all.15699
Spinal astrocytes contribute to chronic itch via sensitization of itch-specific neurons expressing gastrin-releasing peptide receptor (GRPR). However, whether microglia-neuron interactions contribute to itch remains unclear. In this study, we aimed to explore how microglia interact with GRPR+ neurons and promote chronic itch.RNA sequencing, quantitative real-time PCR, western blot, immunohistochemistry, RNAscope ISH, pharmacologic and genetic approaches were performed to examine the roles of spinal NLRP3 (The NOD-like receptor family, pyrin-containing domain 3) inflammasome activation and IL-1β-IL1R1 signaling in chronic itch. Grpr-eGFP and Grpr KO mice were used to investigate microglia-GRPR+ neuron interactions.We observed NLRP3 inflammasome activation and IL-1β production in spinal microglia under chronic itch conditions. Blockade of microglial activation and the NLRP3/caspase-1/IL-1β axis attenuated chronic itch and neuronal activation. Type 1 IL-1 receptor (IL-1R1) was expressed in GRPR+ neurons, which are essential for the development of chronic itch. Our studies also find that IL-1β+ microglia are localized in close proximity to GRPR+ neurons. Consistently, intrathecal injection of IL1R1 antagonist or exogenous IL-1β indicate that the IL-1β-IL-1R1 signaling pathway enhanced the activation of GRPR+ neurons. Furthermore, our results demonstrate that the microglial NLRP3/caspase-1/IL-1β axis contributes to several different chronic itches triggered by small molecules and protein allergens from the environment and drugs.Our findings reveal a previously unknown mechanism in which microglia enhances the activation of GRPR+ neurons through the NLRP3/caspase-1/IL-1β/IL1R1 axis. These results will provide new insights into the pathophysiology of pruritus and novel therapeutic strategies for patients with chronic itch.
Golden, JW;Li, R;Cline, CR;Zeng, X;Mucker, EM;Fuentes-Lao, AJ;Spik, KW;Williams, JA;Twenhafel, N;Davis, N;Moore, JL;Stevens, S;Blue, E;Garrison, AR;Larson, DD;Stewart, R;Kunzler, M;Liu, Y;Wang, Z;Hooper, JW;
PMID: 35073750 | DOI: 10.1128/mbio.02906-21
The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created a global health emergency. While most human disease is mild to moderate, some infections lead to a severe disease characterized by acute respiratory distress, hypoxia, anosmia, ageusia, and, in some instances, neurological involvement. Small-animal models reproducing severe disease, including neurological sequela, are needed to characterize the pathophysiological mechanism(s) of disease and to identify medical countermeasures. Transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE2) viral receptor under the control of the K18 promoter develop severe and lethal respiratory disease subsequent to SARS-CoV-2 intranasal challenge when high viral doses are used. Here, we report on SARS-CoV-2 infection of hamsters engineered to express the hACE2 receptor under the control of the K18 promoter. K18-hACE2 hamsters infected with a relatively low dose of 100 or 1,000 PFU of SARS-CoV-2 developed a severe and lethal disease, with most animals succumbing by day 5 postinfection. Hamsters developed severe lesions and inflammation within the upper and lower respiratory system, including infection of the nasal cavities causing marked destruction of the olfactory epithelium as well as severe bronchopneumonia that extended deep into the alveoli. Additionally, SARS-CoV-2 infection spread to the central nervous system (CNS), including the brain stem and spinal cord. Wild-type (WT) hamsters naturally support SARS-CoV-2 infection, with the primary lesions present in the respiratory tract and nasal cavity. Overall, infection in the K18-hACE2 hamsters is more extensive than that in WT hamsters, with more CNS involvement and a lethal outcome. These findings demonstrate the K18-hACE2 hamster model will be valuable for studying SARS-CoV-2. IMPORTANCE The rapid emergence of SARS-CoV-2 has created a global health emergency. While most human SARS-CoV-2 disease is mild, some people develop severe, life-threatening disease. Small-animal models mimicking the severe aspects of human disease are needed to more clearly understand the pathophysiological processes driving this progression. Here, we studied SARS-CoV-2 infection in hamsters engineered to express the human angiotensin-converting enzyme 2 viral receptor under the control of the K18 promoter. SARS-CoV-2 produces a severe and lethal infection in transgenic hamsters that mirrors the most severe aspects of COVID-19 in humans, including respiratory and neurological injury. In contrast to other animal systems, hamsters manifest disease with levels of input virus more consistent with natural human infection. This system will be useful for the study of SARS-CoV-2 disease and the development of drugs targeting this virus.
Stempel AV, Stumpf A, Zhang HY, Özdoğan T, Pannasch U, Theis AK, Otte DM, Wojtalla A, Rácz I, Ponomarenko A, Xi ZX, Zimmer A, Schmitz D.
PMID: 27133464 | DOI: 10.1016/j.neuron.2016.03.034
Endocannabinoids (eCBs) exert major control over neuronal activity by activating cannabinoid receptors (CBRs). The functionality of the eCB system is primarily ascribed to the well-documented retrograde activation of presynaptic CB1Rs. We find that action potential-driven eCB release leads to a long-lasting membrane potential hyperpolarization in hippocampal principal cells that is independent of CB1Rs. The hyperpolarization, which is specific to CA3 and CA2 pyramidal cells (PCs), depends on the activation of neuronal CB2Rs, as shown by a combined pharmacogenetic and immunohistochemical approach. Upon activation, they modulate the activity of the sodium-bicarbonate co-transporter, leading to a hyperpolarization of the neuron. CB2R activation occurred in a purely self-regulatory manner, robustly altered the input/output function of CA3 PCs, and modulated gamma oscillations in vivo. To conclude, we describe a cell type-specific plasticity mechanism in the hippocampus that provides evidence for the neuronal expression of CB2Rs and emphasizes their importance in basic neuronal transmission.
Gibson EM, Nagaraja S, Ocampo A, Tam LT, Wood LS, Pallegar PN, Greene JJ, Geraghty AC, Goldstein AK, Ni L, Woo PJ, Barres BA, Liddelow S, Vogel H, Monje M.
| DOI: 10.1016/j.cell.2018.10.049
Chemotherapy results in a frequent yet poorly understood syndrome of long-term neurological deficits. Neural precursor cell dysfunction and white matter dysfunction are thought to contribute to this debilitating syndrome. Here, we demonstrate persistent depletion of oligodendrocyte lineage cells in humans who received chemotherapy. Developing a mouse model of methotrexate chemotherapy-induced neurological dysfunction, we find a similar depletion of white matter OPCs, increased but incomplete OPC differentiation, and a persistent deficit in myelination. OPCs from chemotherapy-naive mice similarly exhibit increased differentiation when transplanted into the microenvironment of previously methotrexate-exposed brains, indicating an underlying microenvironmental perturbation. Methotrexate results in persistent activation of microglia and subsequent astrocyte activation that is dependent on inflammatory microglia. Microglial depletion normalizes oligodendroglial lineage dynamics, myelin microstructure, and cognitive behavior after methotrexate chemotherapy. These findings indicate that methotrexate chemotherapy exposure is associated with persistent tri-glial dysregulation and identify inflammatory microglia as a therapeutic target to abrogate chemotherapy-related cognitive impairment.
Mishra D, Richard JE, Maric I, Porteiro B, Häring M, Kooijman S, Musovic S, Eerola K, López-Ferreras L, Peris E, Grycel K, Shevchouk OT, Micallef P, Olofsson CS, Wernstedt Asterholm I, Grill HJ, Nogueiras R, Skibicka KP.
PMID: 30865890 | DOI: 10.1016/j.celrep.2019.02.044
Chronic low-grade inflammation and increased serum levels of the cytokine IL-6 accompany obesity. For brain-produced IL-6, the mechanisms by which it controls energy balance and its role in obesity remain unclear. Here, we show that brain-produced IL-6 is decreased in obese mice and rats in a neuroanatomically and sex-specific manner. Reduced IL-6 mRNA localized to lateral parabrachial nucleus (lPBN) astrocytes, microglia, and neurons, including paraventricular hypothalamus-innervating lPBN neurons. IL-6 microinjection into lPBN reduced food intake and increased brown adipose tissue (BAT) thermogenesis in male lean and obese rats by increasing thyroid and sympathetic outflow to BAT. Parabrachial IL-6 interacted with leptin to reduce feeding. siRNA-mediated reduction of lPBN IL-6 leads to increased weight gain and adiposity, reduced BAT thermogenesis, and increased food intake. Ambient cold exposure partly normalizes the obesity-induced suppression of lPBN IL-6. These results indicate that lPBN-produced IL-6 regulates feeding and metabolism and pinpoints (patho)physiological contexts interacting with lPBN IL-6.
Geraghty AC, Gibson EM, Ghanem RA, Greene JJ, Ocampo A, Goldstein AK, Ni L, Yang T, Marton RM, Paşca SP, Greenberg ME, Longo FM, Monje M.
PMID: 31122677 | DOI: 10.1016/j.neuron.2019.04.032
Activity-dependent myelination is thought to contribute to adaptive neurological function. However, the mechanisms by which activity regulates myelination and the extent to which myelin plasticity contributes to non-motor cognitive functions remain incompletely understood. Using a mouse model of chemotherapy-related cognitive impairment (CRCI), we recently demonstrated that methotrexate (MTX) chemotherapy induces complex glial dysfunction for which microglial activation is central. Here, we demonstrate that remote MTX exposure blocks activity-regulated myelination. MTX decreases cortical Bdnf expression, which is restored by microglial depletion. Bdnf-TrkB signaling is a required component of activity-dependent myelination. Oligodendrocyte precursor cell (OPC)-specific TrkB deletion in chemotherapy-naive mice results in impaired cognitive behavioral performance. A small-molecule TrkB agonist rescues both myelination and cognitive impairment after MTX chemotherapy. This rescue after MTX depends on intact TrkB expression in OPCs. Taken together, these findings demonstrate a molecular mechanism required for adaptive myelination that is aberrant in CRCI due to microglial activation
