Primary Innate Immune Cells Research Articles

PGRMC2 and HLA-G regulate immune homeostasis in a microphysiological model of human maternal-fetal membrane interface

Chorion trophoblasts (CTCs) and immune cell-enriched decidua (DECs) comprise the maternal-fetal membrane interface called the chorio-decidual interface (CDi) which constantly gets exposed to maternal stressors without leading to labor activation. This study explored how CTCs act as a barrier at CDi. The roles of human leukocyte antigen (HLA)-G and progesterone receptor membrane component 2 (PGRMC2) in mediating immune homeostasis were also investigated. The CDi was recreated in a two-chamber microfluidic device (CDi-on-chip) with an outer chamber of primary DECs and immune cell line-derived innate immune cells and an inner chamber of wild-type or PGRMC2 or HLA-G knockout immortalized CTCs. To mimic maternal insults, DECs were treated with lipopolysaccharide, poly(I:C), or oxidative stress inducer cigarette smoke extract. Expression levels of inflammation and immunity genes via targeted RNA sequencing, production of soluble mediators, and immune cell migration into CTCs were determined. In CDi-on-chip, decidua and immune cells became inflammatory in response to insults while CTCs were refractory, highlighting their barrier function. HLA-G and PGRMC2 are found to be vital to immune homeostasis at the CDi, with PGRMC2 serving as an upstream regulator of inflammation, HLA-G expression, and mesenchymal-epithelial transition, and HLA-G serving as a frontline immunomodulatory molecule, thus preventing fetal membrane compromise.

The transcriptome of MHV-infected RAW264.7 cells offers an alternative model for macrophage innate immunity research.

Macrophages are the primary innate immune cells encountered by the invading coronaviruses, and their abilities to initiate inflammatory reactions, to maintain the immunity homeostasis by differential polarization, to train the innate immune system by epigenic modification have been reported in laboratory animal research. In the current in vitro research, murine macrophage RAW 264.7 cell were infected by mouse hepatitis virus, a coronavirus existed in mouse. At 3-, 6-, 12-, 24-, and 48-h post infection (hpi.), the attached cells were washed with PBS and harvested in Trizol reagent. Then The harvest is subjected to transcriptome sequencing. The transcriptome analysis showed the immediate (3 hpi.) up regulation of DEGs related to inflammation, like Il1b and Il6. DEGs related to M2 differential polarization, like Irf4 showed up regulation at 24 hpi., the late term after viral infection. In addition, DEGs related to metabolism and histone modification, like Ezh2 were detected, which might correlate with the trained immunity of macrophages. The current in vitro viral infection study showed the key innated immunity character of macrophages, which suggested the replacement value of viral infection cells model, to reduce the animal usage in preclinical research.

Prenatal Immune Challenge Differentiates the Effect of Aripiprazole and Risperidone on CD200-CD200R and CX3CL1-CX3CR1 Dyads and Microglial Polarization: A Study in Organotypic Cortical Cultures.

Microglia are the primary innate immune cells of the central nervous system and extensively contribute to brain homeostasis. Dysfunctional or excessive activity of microglia may be associated with several neuropsychiatric disorders, including schizophrenia. Therefore, we examined whether aripiprazole and risperidone could influence the expression of the Cd200-Cd200r and Cx3cl1-Cx3cr1 axes, which are crucial for the regulation of microglial activity and interactions of these cells with neurons. Additionally, we evaluated the impact of these drugs on microglial pro- and anti-inflammatory markers (Cd40, Il-1β, Il-6, Cebpb, Cd206, Arg1, Il-10 and Tgf-β) and cytokine release (IL-6, IL-10). The research was executed in organotypic cortical cultures (OCCs) prepared from the offspring of control rats (control OCCs) or those exposed to maternal immune activation (MIA OCCs), which allows for the exploration of schizophrenia-like disturbances in animals. All experiments were performed under basal conditions and after additional stimulation with lipopolysaccharide (LPS), following the "two-hit" hypothesis of schizophrenia. We found that MIA diminished the mRNA level of Cd200r and affected the OCCs' response to additional LPS exposure in terms of this parameter. LPS downregulated the Cx3cr1 expression and profoundly changed the mRNA levels of pro- and anti-inflammatory microglial markers in both types of OCCs. Risperidone increased Cd200 expression in MIA OCCs, while aripiprazole treatment elevated the gene levels of the Cx3cl1-Cx3cr1 dyad in control OCCs. The antipsychotics limited the LPS-generated increase in the expression of proinflammatory factors (Il-1β and Il-6) and enhanced the mRNA levels of anti-inflammatory components (Cd206 and Tgf-β) of microglial polarization, mostly in the absence of the MIA procedure. Finally, we observed a more pronounced modulating impact of aripiprazole on the expression of pro- and anti-inflammatory cytokines when compared to risperidone in MIA OCCs. In conclusion, our data suggest that MIA might influence microglial activation and crosstalk of microglial cells with neurons, whereas aripiprazole and risperidone could beneficially affect these changes in OCCs.

Microglia-neuron interactions in schizophrenia.

Multiple lines of evidence implicate increased neuroinflammation mediated by glial cells to play a key role in neurodevelopmental disorders such as schizophrenia. Microglia, which are the primary innate immune cells of the brain, are crucial for the refinement of the synaptic circuitry during early brain development by synaptic pruning and the regulation of synaptic plasticity during adulthood. Schizophrenia risk factors as genetics or environmental influences may further be linked to increased activation of microglia, an increase of pro-inflammatory cytokine levels and activation of the inflammasome resulting in an overall elevated neuroinflammatory state in patients. Synaptic loss, one of the central pathological hallmarks of schizophrenia, is believed to be due to excess removal of synapses by activated microglia, primarily affecting glutamatergic neurons. Therefore, it is crucial to investigate microglia-neuron interactions, which has been done by multiple studies focusing on post-mortem brain tissues, brain imaging, animal models and patient iPSC-derived 2D culture systems. In this review, we summarize the major findings in patients and in vivo and in vitro models in the context of neuron-microglia interactions in schizophrenia and secondly discuss the potential of anti-inflammatory treatments for the alleviation of positive, negative, and cognitive symptoms.

DOP67 MB310: From Clinical FMT to a Phase 1 Study with a Defined, Orally Administered Live Bacterial Therapeutic for Mild Moderate Ulcerative Colitis

Abstract Background The composition of the gut microbiome is a key determinant in inflammatory bowel disease. Consequently, modifying the composition of the gut microbiota has the potential to transform the treatment of ulcerative colitis (UC). The clinical use of faecal microbiota transplantation (FMT) has become widespread, especially for the treatment of C. difficile associated diseases, as a method of changing the composition of the gut microbiome from a diseased to a healthy state. FMT is, by its nature, an undefined and variable therapy due to differences between donors. Microbiotica has taken a "patient-first" discovery approach to identify a defined consortium, which is being developed as a live bacterial therapeutic for treatment of mild-to-moderate UC patients in a Phase 1b clinical trial, the COMPOSER-1 study, in 2024. Methods A clinical trial in UC patients (Costello et al., 2019), in which patients were treated with FMT from healthy donors, showed a 32% clinical remission rate in the active treatment group, as compared to 9% in the placebo arm. Donor, pre-treatment recipient, and post-FMT recipient samples were analysed using shotgun metagenomic sequencing. All samples were analysed using Microbiotica’s precision microbiome analysis platform to identify bacteria associated with patient response. These bacteria were assessed in cellular assays using relevant human cell/cell lines including: Caco2, dendritic cells, M1 macrophages, and CD4+ T-cells. Results We defined a signature of therapeutic response based on bacteria that engrafted from the donor into UC patients and were associated with clinical benefit. A sub-species level analysis identified 8 bacteria, which have been developed into a defined live bacterial therapeutic, MB310. The consortium bacteria enhanced the barrier integrity of an epithelial cell monolayer, and protected the barrier from inflammatory challenge by LPS. In vitro, the bacteria also have an anti-inflammatory effect when incubated with different primary innate immune cells, dendritic cells and M1 macrophages. Specific MB310 bacteria, either directly or via metabolites, are able to regulate T-cell responses. Conclusion In summary, we have identified a consortium of bacteria using patient-first discovery in a successful FMT study that is being developed as a treatment for UC. The consortium of 8 bacteria is able to impact multiple disease-relevant mechanisms including epithelial barrier integrity and immunomodulation. MB310 is being advanced to a first-in-human study in 2024 to explore safety and initial signs of efficacy. Costello, et al. (2019) Jama 321.2: 156-164.

TIAM2S-positive microglia enhance inflammation and neurotoxicity through soluble ICAM-1-mediated immune priming.

TIAM Rac1-associated GEF 2 short form (TIAM2S) as an oncoprotein alters the immunity of peripheral immune cells to construct an inflammatory tumor microenvironment. However, its role in the activation of microglia, the primary innate immune cells of the brain, and neuroinflammation remains unknown. This study investigated the mechanism underlying TIAM2S shapes immune properties of microglia to facilitate neuron damage. Human microglial clone 3 cell line (HMC3) and human brain samples were applied to determine the presence of TIAM2S in microglia by western blots and double immunostaining. Furthermore, TIAM2S transgenic mice combined with multiple reconstituted primary neuron-glial culture systems and a cytokine array were performed to explore how TIAM2S shaped immune priming of microglia and participated in lipopolysaccharide (LPS)-induced neuron damage. TIAM2S protein was detectable in HMC3 cells and presented in a small portion (~11.1%) of microglia in human brains referred to as TIAM2S-positive microglia. With the property of secreted soluble factor-mediated immune priming, TIAM2S-positive microglia enhanced LPS-induced neuroinflammation and neural damage in vivo and in vitro. The gain- and loss-of-function experiments showed soluble intercellular adhesion molecule-1 (sICAM-1) participated in neurotoxic immune priming of TIAM2S+ microglia. Together, this study demonstrated a novel TIAM2S-positive microglia subpopulation enhances inflammation and neurotoxicity through sICAM-1-mediated immune priming.

Histone deacetylase 3 regulates microglial function through histone deacetylation

ABSTRACT As the primary innate immune cells of the brain, microglia respond to damage and disease through pro-inflammatory release of cytokines and neuroinflammatory molecules. Histone acetylation is an activating transcriptional mark that regulates inflammatory gene expression. Inhibition of histone deacetylase 3 (Hdac3) has been utilized in pre-clinical models of depression, stroke, and spinal cord injury to improve recovery following injury, but the molecular mechanisms underlying Hdac3’s regulation of inflammatory gene expression in microglia is not well understood. To address this lack of knowledge, we examined how pharmacological inhibition of Hdac3 in an immortalized microglial cell line (BV2) impacted histone acetylation and gene expression of pro- and anti-inflammatory genes in response to immune challenge with lipopolysaccharide (LPS). Flow cytometry and cleavage under tags & release using nuclease (CUT & RUN) revealed that Hdac3 inhibition increases global and promoter-specific histone acetylation, resulting in the release of gene repression at baseline and enhanced responses to LPS. Hdac3 inhibition enhanced neuroprotective functions of microglia in response to LPS through reduced nitric oxide release and increased phagocytosis. The findings suggest Hdac3 serves as a regulator of microglial inflammation, and that inhibition of Hdac3 facilitates the microglial response to inflammation and its subsequent clearing of debris or damaged cells. Together, this work provides new mechanistic insights into therapeutic applications of Hdac3 inhibition which mediate reduced neuroinflammatory insults through microglial response.

Divergent functional outcomes of NLRP3 blockade downstream of multi-inflammasome activation: therapeutic implications for ALS.

NOD-Like Receptor Family Pyrin Domain Containing 3 (NLRP3) inflammasome modulation has emerged as a potential therapeutic approach targeting inflammation amplified by pyroptotic innate immune cell death. In diseases characterized by non-cell autonomous neurodegeneration including amyotrophic lateral sclerosis (ALS), the activation of several inflammasomes has been reported. Since functional redundancy can exist among inflammasome pathways, here we investigate the effects of NLRP3 inhibition on NLRP3, NLR family CARD Domain Containing 4 (NLRC4) and non-canonical pathways to understand whether NLRP3 blockade alone can mitigate pro-inflammatory cytokine release and pyroptotic cell death in contexts where single or multiple inflammasome pathways independent of NLRP3 are activated. In this study we do not limit our insights into inflammasome biology by solely relying on the THP-1 monocytic line under the LPS/nigericin-mediated NLRP3 pathway activation paradigm. We assess therapeutic potential and limitations of NLRP3 inhibition in multi-inflammasome activation contexts utilizing various human cellular systems including cell lines expressing gain of function (GoF) mutations for several inflammasomes, primary human monocytes, macrophages, healthy and Amyotrophic Lateral Sclerosis (ALS) patient induced pluripotent stem cells (iPSC)-derived microglia (iMGL) stimulated for canonical and non-canonical inflammasome pathways. We demonstrate that NLRP3 inhibition can modulate the NLRC4 and non-canonical inflammasome pathways; however, these effects differ between immortalized, human primary innate immune cells, and iMGL. We extend our investigation in more complex systems characterized by activation of multiple inflammasomes such as the SOD1G93A mouse model. Through deep immune phenotyping by single-cell mass cytometry we demonstrate that acute NLRP3 inhibition does not ameliorate spinal cord inflammation in this model. Taken together, our data suggests that NLRP3 inhibition alone may not be sufficient to address dynamic and complex neuroinflammatory pathobiological mechanisms including dysregulation of multiple inflammasome pathways in neurodegenerative disease such as ALS.

Mixtures of per- and poly-fluoroalkyl substances (PFAS) reduce the in vitro activation of human T cells and basophils

In the last decades, per- and poly-fluoroalkyl substances (PFAS), widely used industrial chemicals, have been in the center of attention because of their omnipotent presence in water and soils worldwide. Although efforts have been made to substitute long-chain PFAS towards safer alternatives, their persistence in humans still leads to exposure to these compounds. PFAS immunotoxicity is poorly understood as no comprehensive analyses on certain immune cell subtypes exist. Furthermore, mainly single entities and not PFAS mixtures have been assessed. In the present study we aimed to investigate the effect of PFAS (short-chain, long-chain and a mixture of both) on the in vitro activation of primary human immune cells.Our results show the ability of PFAS to reduce T cells activation. In particular, exposure to PFAS affected T helper cells, cytotoxic T cells, Natural Killer T cells, and Mucosal associated invariant T (MAIT) cells, as assessed by multi-parameter flow cytometry. Furthermore, the exposure to PFAS reduced the expression of several genes involved in MAIT cells activation, including chemokine receptors, and typical proteins of MAIT cells, such as GZMB, IFNG and TNFSF15 and transcription factors. These changes were mainly induced by the mixture of both short- and long-chain PFAS. In addition, PFAS were able to reduce basophil activation induced by anti-FcεR1α, as assessed by the decreased expression of CD63.Our data clearly show that the exposure of immune cells to a mixture of PFAS at concentrations mimicking real-life human exposure resulted in reduced cell activation and functional changes of primary innate and adaptive human immune cells.

Ovariectomy in mice primes hippocampal microglia to exacerbate behavioral sickness responses

Estrogens are a group of steroid hormones that promote the development and maintenance of the female reproductive system and secondary sex characteristics. Estrogens also modulate immune responses; estrogen loss at menopause increases the risk of inflammatory disorders. Elevated inflammatory responses in the brain can lead to affective behavioral changes, which are characteristic of menopause. Thus, here we examined whether loss of estrogens sensitizes microglia, the primary innate immune cell of the brain, leading to changes in affective behaviors. To test this question, adult C57BL/6 mice underwent an ovariectomy to remove endogenous estrogens and then received estradiol hormone replacement or vehicle. After a one-month recovery, mice received an immune challenge with lipopolysaccharide (LPS) or vehicle control treatment and underwent behavioral testing. Ovariectomized, saline-treated mice exhibited reduced social investigation compared to sham-operated mice. Furthermore, ovariectomized mice that received LPS exhibited an exacerbated decrease in sucrose preference, which was ameliorated by estradiol replacement. These results indicate that ovariectomy modulates affective behaviors at baseline and in response to an inflammatory challenge. Ovariectomy-related behavioral changes were associated with downregulation of Cx3cr1, a microglial receptor that limits activation, suggesting that estrogen loss can disinhibit microglia to immune stimuli. Indeed, estradiol treatment reduced ovariectomy-induced increases in Il1b and Il6 expression after an immune challenge. Changes in microglial reactivity following ovariectomy are likely subtle, as overt changes in microglial morphology (e.g., soma size and branching) were limited. Collectively, these results suggest that a lack of estrogens may allow microglia to confer exaggerated neuroimmune responses, thereby raising vulnerability to adverse affective- and sickness-related behavioral changes.

Origin and Emergence of Microglia in the CNS-An Interesting (Hi)story of an Eccentric Cell.

Microglia belong to tissue-resident macrophages of the central nervous system (CNS), representing the primary innate immune cells. This cell type constitutes ~7% of non-neuronal cells in the mammalian brain and has a variety of biological roles integral to homeostasis and pathophysiology from the late embryonic to adult brain. Its unique identity that distinguishes its "glial" features from tissue-resident macrophages resides in the fact that once entering the CNS, it is perennially exposed to a unique environment following the formation of the blood-brain barrier. Additionally, tissue-resident macrophage progenies derive from various peripheral sites that exhibit hematopoietic potential, and this has resulted in interpretation issues surrounding their origin. Intensive research endeavors have intended to track microglial progenitors during development and disease. The current review provides a corpus of recent evidence in an attempt to disentangle the birthplace of microglia from the progenitor state and underlies the molecular elements that drive microgliogenesis. Furthermore, it caters towards tracking the lineage spatiotemporally during embryonic development and outlining microglial repopulation in the mature CNS. This collection of data can potentially shed light on the therapeutic potential of microglia for CNS perturbations across various levels of severity.

Inhibition of caspase-1-dependent apoptosis suppresses peste des petits ruminants virus replication.

Peste des petits ruminants (PPR), caused by the PPR virus (PPRV), is an acute and fatal contagious disease that mainly infects goats, sheep, and other artiodactyls. Peripheral blood mononuclear cells (PBMCs) are considered the primary innate immune cells. PBMCs derived from goats were infected with PPRV and analyzed to detect the relationship between PPRV replication and apoptosis or the inflammatory response. Quantitative real-time polymerase chain reaction was used to identify PPRV replication and cytokines expression. Flow cytometry was conducted to detect apoptosis and the differentiation of CD4+ and CD8+ T cells after PPRV infection. PPRV stimulated the differentiation of CD4+ and CD8+ T cells. In addition, PPRV induced apoptosis in goat PBMCs. Furthermore, apoptosis and the inflammatory response induced by PPRV could be suppressed by Z-VAD-FMK and Z-YVAD-FMK, respectively. Moreover, the virus titer of PPRV was attenuated by inhibiting caspase-1-dependent apoptosis and inflammation. This study showed that apoptosis and the inflammatory response play an essential role in PPR viral replication in vitro, providing a new mechanism related to the cell host response.

Vagus nerve stimulation is a potential treatment for ischemic stroke.

Microglia are the brain's primary innate immune cells, and they are activated and affect pro-inflammatory phenotype or regulatory phenotype after ischemic stroke. Vagus nerve stimulation was shown to activate microglial phenotypic changes and exhibit neuroprotective effects in ischemia/reperfusion injury. In this study, we established rat models of ischemic stroke by occlusion of the middle cerebral artery and performed vagus nerve stimulation 30 minutes after modeling. We found that vagus nerve stimulation caused a shift from a pro-inflammatory phenotype to a regulatory phenotype in microglia in the ischemic penumbra. Vagus nerve stimulation decreased the levels of pro-inflammatory phenotype markers inducible nitric oxide synthase and tumor necrosis factor α and increased the expression of regulatory phenotype markers arginase 1 and transforming growth factor β through activating α7 nicotinic acetylcholine receptor expression. Additionally, α7 nicotinic acetylcholine receptor blockade reduced the inhibition of Toll-like receptor 4/nuclear factor kappa-B pathway-associated proteins, including Toll-like receptor 4, myeloid differentiation factor 88, I kappa B alpha, and phosphorylated-I kappa B alpha, and also weakened the neuroprotective effects of vagus nerve stimulation in ischemic stroke. Vagus nerve stimulation inhibited Toll-like receptor 4/nuclear factor kappa-B expression through activating α7 nicotinic acetylcholine receptor and regulated microglial polarization after ischemic stroke, thereby playing a role in the treatment of ischemic stroke. Findings from this study confirm the mechanism underlying vagus nerve stimulation against ischemic stroke.

Co-culturing Human Intestinal Enteroid Monolayers with Innate Immune Cells.

The coordinated interaction between the intestinal epithelium and immune cells is required to maintain proper barrier function and mucosal host defenses to the harsh external environment of the gut lumen. Complementary to in vivo models, there is a need for practical and reproducible in vitro models that employ primary human cells to confirm and advance our understanding of mucosal immune responses under physiologic and pathophysiologic conditions. Here we describe the methods to co-culture human intestinal stem cell-derived enteroids grown as confluent monolayers on permeable supports with primary human innate immune cells (e.g., monocyte-derived macrophages and polymorphonuclear neutrophils). This co-culture model reconstructs the cellular framework of the human intestinal epithelial-immune niche with distinct apical and basolateral compartments to recreate host responses to luminal and submucosal challenges, respectively. Enteroid-immune co-cultures enable multiple outcome measures to interrogate important biological processes such as epithelial barrier integrity, stem cell biology, cellular plasticity, epithelial-immune cellscrosstalk, immune cell effector functions, changes in gene expression (i.e., transcriptomic, proteomic, epigenetic), and host-microbiome interactions.

The Role of Mononuclear Phagocytes in the Testes and Epididymis

The mononuclear phagocytic system (MPS) is the primary innate immune cell group in male reproductive tissues, maintaining the balance of pro-inflammatory and immune tolerance. This article aims to outline the role of mononuclear macrophages in the immune balance of the testes and epididymis, and to understand the inner immune regulation mechanism. A review of pertinent publications was performed using the PubMed and Google Scholar databases on all articles published prior to January 2021. Search terms were based on the following keywords: 'MPS', 'mononuclear phagocytes', 'testes', 'epididymis', 'macrophage', 'Mφ', 'dendritic cell', 'DC', 'TLR', 'immune', 'inflammation', and 'polarization'. Additionally, reference lists of primary and review articles were reviewed for other publications of relevance. This review concluded that MPS exhibits a precise balance in the male reproductive system. In the testes, MPS cells are mainly suppressed subtypes (M2 and cDC2) under physiological conditions, which maintain the local immune tolerance. Under pathological conditions, MPS cells will transform into M1 and cDC1, producing various cytokines, and will activate T cell specific immunity as defense to foreign pathogens or self-antigens. In the epididymis, MPS cells vary in the different segments, which express immune tolerance in the caput and pro-inflammatory condition in the cauda. Collectively, MPS is the control point for maintaining the immune tolerance of the testes and epididymis as well as for eliminating pathogens.

Human Neutrophils Present Mild Activation by Zika Virus But Reduce the Infection of Susceptible Cells.

The emergence of the Zika virus (ZIKV) has highlighted the need for a deeper understanding of virus-host interactions in order to pave the way for the development of antiviral therapies. The present work aimed to address the response of neutrophils during ZIKV infection. Neutrophils are important effector cells in innate immunity implicated in the host’s response to neurotropic arboviruses. Our results indicate that human neutrophils were not permissive to Asian or African ZIKV strain replication. In fact, after stimulation with ZIKV, neutrophils were mild primed against the virus as evaluated through CD11b and CD62L modulation, secretion of inflammatory cytokines and granule content, production of reactive oxygen species, and neutrophil extracellular traps formation. Overall, neutrophils did not affect ZIKV infectivity. Moreover, in vitro ZIKV infection of primary innate immune cells did not trigger neutrophil migration. However, neutrophils co-cultured with ZIKV susceptible cell lineages resulted in lower cell infection frequencies, possibly due to cell-to-cell contact. In vivo, neutrophil depletion in immunocompetent mice did not affect ZIKV spreading to the draining lymph nodes. The data suggest that human neutrophils do not play an antiviral role against ZIKV per se, but these cells might participate in an infected environment shaping the ZIKV infection in other target cells.

Functional genome-wide short hairpin RNA library screening identifies key molecules for extracellular vesicle secretion from microglia

SUMMARYActivated microglia release extracellular vesicles (EVs) as modulators of brain homeostasis and innate immunity. However, the molecules critical for regulating EV production from microglia are poorly understood. Here we establish a murine microglial cell model to monitor EV secretion by measuring the fluorescence signal of tdTomato, which is linked to tetraspanin CD63. Stimulation of tdTomato+ cells with ATP induces rapid secretion of EVs and a reduction in cellular tdTomato intensity, reflecting EV secretion. We generate a GFP+ tdTomato+ cell library expressing TurboGFP and barcoded short hairpin RNAs for genome-wide screening using next-generation sequencing. We identify Mcfd2, Sepp1, and Sdc1 as critical regulators of ATP-induced EV secretion from murine microglia. Small interfering RNA (siRNA-based) silencing of each of these genes suppresses lipopolysaccharide- and ATP-induced inflammasome activation, as determined by interleukin-1β release from primary cultured murine microglia. These molecules are critical for microglial EV secretion and are potential therapeutic targets for neuroinflammatory disorders.

Small extracellular vesicles induced gene expression changes in microglia

BackgroundSmall extracellular vesicles (sEVs) are 30‐150 nm membranous particles released by a variety of cells and serve as a mediator in intercellular communication between adjacent and distal cells. sEVs carry biomolecular cargo including miRNAs, mRNAs, proteins, and lipids, which are selectively sorted and packaged and mirror the physiological state of the donor cells. Disease states can alter sEV composition affecting the message carried and thereby, its functional impact. Microglia, as the tissue‐resident macrophages and primary innate immune cells of the central nervous system, play an important role in neuropathic pain. Here, we investigated alterations in the composition of serum sEVs from a mouse model of neuropathic pain and assessed the functional consequences of sEV uptake by primary cortical microglia.MethodssEVs were purified from the serum of a spared nerve injury (SNI) mouse model of neuropathic pain and sham‐operated mice four weeks after surgery. sEV uptake by primary cortical microglia was confirmed by incubating microglia with 1 µg of PKH26‐labeled sEVs for 24 h and visualized using confocal microscopy. Transcriptome and cytokine alterations induced by sEV uptake in microglia (1 µg for 24 h) were determined using RNA‐seq and cytokine array.ResultsWe identified a differential expression of 22 miRNAs in serum sEVs derived from SNI and sham control mice. Confocal microscopy confirmed the presence of sEVs in microglia following 24 h sEV incubation. We observed a significant difference in gene expression resulting from the uptake of distinct serum sEVs – alterations were more prominent in microglia treated with sham sEVs. Gene Ontology analysis indicated gene expression changes induced by the uptake of sEVs from both SNI and sham surgery were both implicated in immune responses, but genes upregulated by sham sEVs were also enriched in inflammatory responses. Secretions of pro‐inflammatory cytokines and chemokines including CXCL10, CXCL2, and TNF‐α were higher in microglia incubated with sEVs from mice that had sham surgery compared to PBS control or SNI sEVs.ConclusionsOur data demonstrate alterations in sEV composition from both SNI and sham surgeries, and the molecular changes captured in donor sEVs could impact gene expression in the recipient cells. The differences observed in microglial phenotypes and gene expression suggest sEVs from sham surgery are directed at wound healing and thus differ from nerve injury‐induced sEVs.

Ehrlichia SLiM Ligand Mimetic Activates Notch Signaling in Human Monocytes.

ABSTRACTEhrlichia chaffeensis evades innate host defenses by reprogramming the mononuclear phagocyte through mechanisms that involve the exploitation of multiple evolutionarily conserved cellular signaling pathways, including Notch. This immune evasion strategy is directed in part by tandem repeat protein (TRP) effectors. Specifically, the TRP120 effector activates and regulates Notch signaling through interactions with the Notch receptor and the negative regulator, F-Box and WD repeat domain-containing 7 (FBW7). However, the specific molecular interactions and motifs required for E. chaffeensis TRP120-Notch receptor interaction and activation have not been defined. To investigate the molecular basis of TRP120 Notch activation, we compared TRP120 with endogenous canonical/noncanonical Notch ligands and identified a short region of sequence homology within the tandem repeat (TR) domain. TRP120 was predicted to share biological function with Notch ligands, and a function-associated sequence in the TR domain was identified. To investigate TRP120-Notch receptor interactions, colocalization between TRP120 and endogenous Notch-1 was observed. Moreover, direct interactions between full-length TRP120, the TRP120 TR domain containing the putative Notch ligand sequence, and the Notch receptor LBR were demonstrated. To molecularly define the TRP120 Notch activation motif, peptide mapping was used to identify an 11-amino acid short linear motif (SLiM) located within the TRP120 TR that activated Notch signaling and downstream gene expression. Peptide mutants of the Notch SLiM or anti-Notch SLiM antibody reduced or eliminated Notch activation and NICD nuclear translocation. This investigation reveals a novel molecularly defined pathogen encoded Notch SLiM mimetic that activates Notch signaling consistent with endogenous ligands.

Transcriptional response of murine microglia in Alzheimer\u2019s disease and inflammation

BackgroundAlzheimer’s disease (AD) is a neurodegenerative disorder and is the most common cause of late-onset dementia. Microglia, the primary innate immune cells of the central nervous system (CNS), have a complex role in AD neuropathology. In the initial stages of AD, microglia play a role in limiting pathology by removing amyloid-β (Aβ) by phagocytosis. In contrast, microglia also release pro-inflammatory cytokines and chemokines to promote neuroinflammation and exacerbate AD neuropathology. Therefore, investigating microglial gene networks could identify new targets for therapeutic strategies for AD.ResultsWe identified 465 differentially expressed genes (DEG) in 5XFAD versus wild-type mice by microarray, 354 DEG in lipopolysaccharide (LPS)-stimulated N9 microglia versus unstimulated control cells using RNA-sequencing (RNA-seq), with 32 DEG common between both datasets. Analyses of the 32 common DEG uncovered numerous molecular functions and pathways involved in Aβ phagocytosis and neuroinflammation associated with AD. Furthermore, multiplex ELISA confirmed the induction of several cytokines and chemokines in LPS-stimulated microglia.ConclusionsIn summary, AD triggered multiple signaling pathways that regulate numerous genes in microglia, contributing to Aβ phagocytosis and neuroinflammation. Overall, these data identified several regulatory factors and biomarkers in microglia that could be useful in further understanding AD neuropathology.