The HIV-1 envelope protein gp120 impairs B cell proliferation by inducing TGF-ß1 production and FcRL4 expression.

The humoral immune response after acute infection with HIV-1 is delayed and ineffective. The HIV-1 envelope protein gp120 binds to and signals through integrin α4ß7 on T cells. We found that gp120 also bound to and signaled through α4ß7 on naive B cells, which resulted in an abortive proliferative response. In primary B cells, signaling by gp120 through α4ß7 resulted in increased expression of the immunosuppressive cytokine TGF-ß1 and FcRL4, an inhibitory receptor expressed on B cells. Coculture of B cells with HIV-1-infected autologous CD4(+) T cells also increased the expression of FcRL4 by B cells. Our findings indicated that in addition to mediating chronic activation of the immune system, viral proteins contributed directly to HIV-1-associated B cell dysfunction. Our studies identify a mechanism whereby the virus may subvert the early HIV-1-specific humoral immune response.

Multiomics Characterization of a Less Invasive Microfluidic-Based Cell Sorting Technique.

Fluorescence-activated cell sorting (FACS) is a specialized technique to isolate specific cell subpopulations with a high level of recovery and accuracy. However, the cell sorting procedure can impact the viability and metabolic state of cells. Here, we performed a comparative study and evaluated the impact of traditional high-pressure charged droplet-based and microfluidic chip-based sorting on the metabolic and phosphoproteomic profile of different cell types. While microfluidic chip-based sorted cells more closely resembled the unsorted control group for most cell types tested, the droplet-based sorted cells showed significant metabolic and phosphoproteomic alterations. In particular, greater changes in redox and energy status were present in cells sorted with the droplet-based cell sorter along with larger shifts in proteostasis. 13C-isotope tracing analysis on cells recovering postsorting revealed that the sorter-induced suppression of mitochondrial TCA cycle activity recovered faster in the microfluidic chip-based sorted group. Apart from this, amino acid and lipid biosynthesis pathways were suppressed in sorted cells, with minimum impact and faster recovery in the microfluidic chip-based sorted group. These results indicate microfluidic chip-based sorting has a minimum impact on metabolism and is less disruptive compared to droplet-based sorting.

Development of a Parallel Reaction Monitoring Mass Spectrometry Assay for the Detection of SARS-CoV-2 Spike Glycoprotein and Nucleoprotein.

There is an urgent need for robust and high-throughput methods for SARS-CoV-2 detection in suspected patient samples to facilitate disease management, surveillance, and control. Although nucleic acid detection methods such as reverse transcription polymerase chain reaction (RT-PCR) are the gold standard, during the current pandemic, the deployment of RT-PCR tests has been extremely slow, and key reagents such as PCR primers and RNA extraction kits are at critical shortages. Rapid point-of-care viral antigen detection methods have been previously employed for the diagnosis of respiratory viruses such as influenza and respiratory syncytial viruses. Therefore, the direct detection of SARS-CoV-2 viral antigens in patient samples could also be used for diagnosis of active infection, and alternative methodologies for specific and sensitive viral protein detection should be explored. Targeted mass spectrometry techniques have enabled the identification and quantitation of a defined subset of proteins/peptides at single amino acid resolution with attomole level sensitivity and high reproducibility. Herein, we report a targeted mass spectrometry assay for the detection of SARS-CoV-2 spike protein and nucleoprotein in a relevant biological matrix. Recombinant full-length spike protein and nucleoprotein were digested and proteotypic peptides were selected for parallel reaction monitoring (PRM) quantitation using a high-resolution Orbitrap instrument. A spectral library, which contained seven proteotypic peptides (four from spike protein and three from nucleoprotein) and the top three to four transitions, was generated and evaluated. From the original spectral library, we selected two best performing peptides for the final PRM assay. The assay was evaluated using mock test samples containing inactivated SARS-CoV-2 virions, added to in vitro derived mucus. The PRM assay provided a limit of detection of ∼200 attomoles and a limit of quantitation of ∼ 390 attomoles. Extrapolating from the test samples, the projected titer of virus particles necessary for the detection of SARS-CoV-2 spike and nucleoprotein detection was approximately 2 × 105 viral particles/mL, making it an attractive alternative to RT-PCR assays. Potentially, mass spectrometry-based methods for viral antigen detection may deliver higher throughput and could serve as a complementary diagnostic tool to RT-PCR. Furthermore, this assay could be used to evaluate the presence of SARS-CoV-2 in archived or recently collected biological fluids, in vitro-derived research materials, and wastewater samples.

Glycosylation, hypogammaglobulinemia, and resistance to viral infections.

Genetic defects in MOGS, the gene encoding mannosyl-oligosaccharide glucosidase (the first enzyme in the processing pathway of N-linked oligosaccharide), cause the rare congenital disorder of glycosylation type IIb (CDG-IIb), also known as MOGS-CDG. MOGS is expressed in the endoplasmic reticulum and is involved in the trimming of N-glycans. We evaluated two siblings with CDG-IIb who presented with multiple neurologic complications and a paradoxical immunologic phenotype characterized by severe hypogammaglobulinemia but limited clinical evidence of an infectious diathesis. A shortened immunoglobulin half-life was determined to be the mechanism underlying the hypogammaglobulinemia. Impaired viral replication and cellular entry may explain a decreased susceptibility to infections.

Neurons Internalize Functionalized Micron-Sized Silicon Dioxide Microspheres.

Microparticles have potential as neuron-specific delivery platforms and devices with many applications in neuroscience, pharmacology, and biomedicine. To date, most literature suggests that neurons are not phagocytic cells capable of internalizing microparticles larger than 0.5 µm. We report that neurons transport fluorescently labeled silica microspheres with diameters of 1-2 µm into neurons in vitro and in rat brain without having overt effects on cell viability. Using flow cytometry, fluorescence-activated cell sorting, and confocal and electron microscopy, we first found that SH-SY5Y human neuroblastoma cells internalized 1-µm silicon microspheres with surface charges of -70 mV (hydroxyl and carboxyl), -30 mV (amino), and +40 mV (ammonio). Uptake was rapid, within 2-4 h, and did not affect cell viability 48 h later. Flow cytometry assays indicate that SH-SY5Y cells internalize 1- and 1.5-µm microspheres at the same rate over a 24-h incubation period. Electron microscopy confirms that SH-SY5Y cells internalize 1-, 1.5-, and 2-µm microspheres. Confocal microscopy demonstrated that primary cortical neurons also internalized 1-, 1.5-, and 2-µm amino microspheres within 4 h. Finally, we injected 1-µm amino microspheres into rat striatum and found microspheres inside neurons. Overall, neurons can internalize microspheres up to 2 µm in diameter with a range of surface chemical groups and charges. These findings allow a host of neuroscience and neuroengineering applications including intracellular microdevices within neurons.

Tyrosine sulfation in the second variable loop (V2) of HIV-1 gp120 stabilizes V2-V3 interaction and modulates neutralization sensitivity.

Elicitation of broadly neutralizing antibodies is essential for the development of a protective vaccine against HIV-1. However, the native HIV-1 envelope adopts a protected conformation that conceals highly conserved sites of vulnerability from antibody recognition. Although high-definition structures of the monomeric core of the envelope glycoprotein subunit gp120 and, more recently, of a stabilized soluble gp140 trimer have been solved, fundamental aspects related to the conformation and function of the native envelope remain unresolved. Here, we show that the conserved central region of the second variable loop (V2) of gp120 contains sulfated tyrosines (Tys173 and Tys177) that in the CD4-unbound prefusion state mediate intramolecular interaction between V2 and the conserved base of the third variable loop (V3), functionally mimicking sulfated tyrosines in CCR5 and anti-coreceptor-binding-site antibodies such as 412d. Recombinant gp120 expressed in continuous cell lines displays low constitutive levels of V2 tyrosine sulfation, which can be enhanced markedly by overexpression of the tyrosyl sulfotransferase TPST2. In contrast, virion-associated gp120 produced by primary CD4(+) T cells is inherently highly sulfated. Consistent with a functional role of the V2 sulfotyrosines, enhancement of tyrosine sulfation decreased binding and neutralization of HIV-1 BaL by monomeric soluble CD4, 412d, and anti-V3 antibodies and increased recognition by the trimer-preferring antibodies PG9, PG16, CH01, and PGT145. Conversely, inhibition of tyrosine sulfation increased sensitivity to soluble CD4, 412d, and anti-V3 antibodies and diminished recognition by trimer-preferring antibodies. These results identify the sulfotyrosine-mediated V2-V3 interaction as a critical constraint that stabilizes the native HIV-1 envelope trimer and modulates its sensitivity to neutralization.

Incubation of methamphetamine craving is associated with selective increases in expression of Bdnf and trkb, glutamate receptors, and epigenetic enzymes in cue-activated fos-expressing dorsal striatal neurons.

Cue-induced methamphetamine seeking progressively increases after withdrawal (incubation of methamphetamine craving), but the underlying mechanisms are largely unknown. We determined whether this incubation is associated with alterations in candidate genes in dorsal striatum (DS), a brain area implicated in cue- and context-induced drug relapse. We first measured mRNA expression of 24 candidate genes in whole DS extracts after short (2 d) or prolonged (1 month) withdrawal in rats following extended-access methamphetamine or saline (control condition) self-administration (9 h/d, 10 d). We found minimal changes. Next, using fluorescence-activated cell sorting, we compared gene expression in Fos-positive dorsal striatal neurons, which were activated during "incubated" cue-induced drug-seeking tests after prolonged withdrawal, with nonactivated Fos-negative neurons. We found significant increases in mRNA expression of immediate early genes (Arc, Egr1), Bdnf and its receptor (Trkb), glutamate receptor subunits (Gria1, Gria3, Grm1), and epigenetic enzymes (Hdac3, Hdac4, Hdac5, GLP, Dnmt3a, Kdm1a) in the Fos-positive neurons only. Using RNAscope to determine striatal subregion and cell-type specificity of the activated neurons, we measured colabeling of Fos with Drd1 and Drd2 in three DS subregions. Fos expression was neither subregion nor cell-type specific (52.5 and 39.2% of Fos expression colabeled with Drd1 and Drd2, respectively). Finally, we found that DS injections of SCH23390 (C17H18ClNO), a D1-family receptor antagonist known to block cue-induced Fos induction, decreased incubated cue-induced methamphetamine seeking after prolonged withdrawal. Results demonstrate a critical role of DS in incubation of methamphetamine craving and that this incubation is associated with selective gene-expression alterations in cue-activated D1- and D2-expressing DS neurons.

Context-induced reinstatement of methamphetamine seeking is associated with unique molecular alterations in Fos-expressing dorsolateral striatum neurons.

Context-induced reinstatement of drug seeking is a well established animal model for assessing the neural mechanisms underlying context-induced drug relapse, a major factor in human drug addiction. Neural activity in striatum has previously been shown to contribute to context-induced reinstatement of heroin, cocaine, and alcohol seeking, but not yet for methamphetamine seeking. In this study, we found that context-induced reinstatement of methamphetamine seeking increased expression of the neural activity marker Fos in dorsal but not ventral striatum. Reversible inactivation of neural activity in dorsolateral but not dorsomedial striatum using the GABA agonists muscimol and baclofen decreased context-induced reinstatement. Based on our previous findings that Fos-expressing neurons play a critical role in conditioned drug effects, we assessed whether context-induced reinstatement was associated with molecular alterations selectively induced within context-activated Fos-expressing neurons. We used fluorescence-activated cell sorting to isolate reinstatement-activated Fos-positive neurons from Fos-negative neurons in dorsal striatum and used quantitative PCR to assess gene expression within these two populations of neurons. Context-induced reinstatement was associated with increased expression of the immediate early genes Fos and FosB and the NMDA receptor subunit gene Grin2a in only Fos-positive neurons. RNAscope in situ hybridization confirmed that Grin2a, as well as Grin2b, expression were increased in only Fos-positive neurons from dorsolateral, but not dorsomedial, striatum. Our results demonstrate an important role of dorsolateral striatum in context-induced reinstatement of methamphetamine seeking and that this reinstatement is associated with unique gene alterations in Fos-expressing neurons.

A methodology for exploring biomarker--phenotype associations: application to flow cytometry data and systemic sclerosis clinical manifestations.

BACKGROUND: This work seeks to develop a methodology for identifying reliable biomarkers of disease activity, progression and outcome through the identification of significant associations between high-throughput flow cytometry (FC) data and interstitial lung disease (ILD) - a systemic sclerosis (SSc, or scleroderma) clinical phenotype which is the leading cause of morbidity and mortality in SSc. A specific aim of the work involves developing a clinically useful screening tool that could yield accurate assessments of disease state such as the risk or presence of SSc-ILD, the activity of lung involvement and the likelihood to respond to therapeutic intervention. Ultimately this instrument could facilitate a refined stratification of SSc patients into clinically relevant subsets at the time of diagnosis and subsequently during the course of the disease and thus help in preventing bad outcomes from disease progression or unnecessary treatment side effects. The methods utilized in the work involve: (1) clinical and peripheral blood flow cytometry data (Immune Response In Scleroderma, IRIS) from consented patients followed at the Johns Hopkins Scleroderma Center. (2) machine learning (Conditional Random Forests - CRF) coupled with Gene Set Enrichment Analysis (GSEA) to identify subsets of FC variables that are highly effective in classifying ILD patients; and (3) stochastic simulation to design, train and validate ILD risk screening tools. RESULTS: Our hybrid analysis approach (CRF-GSEA) proved successful in predicting SSc patient ILD status with a high degree of success (>82% correct classification in validation; 79 patients in the training data set, 40 patients in the validation data set). CONCLUSIONS: IRIS flow cytometry data provides useful information in assessing the ILD status of SSc patients. Our new approach combining Conditional Random Forests and Gene Set Enrichment Analysis was successful in identifying a subset of flow cytometry variables to create a screening tool that proved effective in correctly identifying ILD patients in the training and validation data sets. From a somewhat broader perspective, the identification of subsets of flow cytometry variables that exhibit coordinated movement (i.e., multi-variable up or down regulation) may lead to insights into possible effector pathways and thereby improve the state of knowledge of systemic sclerosis pathogenesis.

The CD8-derived chemokine XCL1/lymphotactin is a conformation-dependent, broad-spectrum inhibitor of HIV-1.

CD8+ T cells play a key role in the in vivo control of HIV-1 replication via their cytolytic activity as well as their ability to secrete non-lytic soluble suppressive factors. Although the chemokines that naturally bind CCR5 (CCL3/MIP-1α, CCL4/MIP- 1ß, CCL5/RANTES) are major components of the CD8-derived anti-HIV activity, evidence indicates the existence of additional, still undefined, CD8-derived HIV-suppressive factors. Here, we report the characterization of a novel anti-HIV chemokine, XCL1/lymphotactin, a member of the C-chemokine family that is produced primarily by activated CD8+ T cells and behaves as a metamorphic protein, interconverting between two structurally distinct conformations (classic and alternative). We found that XCL1 inhibits a broad spectrum of HIV-1 isolates, irrespective of their coreceptor-usage phenotype. Experiments with stabilized variants of XCL1 demonstrated that HIV-1 inhibition requires access to the alternative, all-ß conformation, which interacts with proteoglycans but does not bind/activate the specific XCR1 receptor, while the classic XCL1 conformation is inactive. HIV-1 inhibition by XCL1 was shown to occur at an early stage of infection, via blockade of viral attachment and entry into host cells. Analogous to the recently described anti-HIV effect of the CXC chemokine CXCL4/PF4, XCL1-mediated inhibition is associated with direct interaction of the chemokine with the HIV-1 envelope. These results may open new perspectives for understanding the mechanisms of HIV-1 control and reveal new molecular targets for the design of effective therapeutic and preventive strategies against HIV-1.

Identification of the platelet-derived chemokine CXCL4/PF-4 as a broad-spectrum HIV-1 inhibitor.

The natural history of HIV-1 infection is highly variable in different individuals, spanning from a rapidly progressive course to a long-term asymptomatic infection. A major determinant of the pace of disease progression is the in vivo level of HIV-1 replication, which is regulated by a complex network of cytokines and chemokines expressed by immune and inflammatory cells. The chemokine system is critically involved in the control of HIV-1 replication by virtue of the role played by specific chemokine receptors, most notably CCR5 and CXCR4, as cell-surface coreceptors for HIV-1 entry; hence, the chemokines that naturally bind such coreceptors act as endogenous inhibitors of HIV-1. Here, we show that the CXC chemokine CXCL4 (PF-4), the most abundant protein contained within the α-granules of platelets, is a broad-spectrum inhibitor of HIV-1 infection. Unlike other known HIV-suppressive chemokines, CXCL4 inhibits infection by the majority of primary HIV-1 isolates regardless of their coreceptor-usage phenotype or genetic subtype. Consistent with the lack of viral phenotype specificity, blockade of HIV-1 infection occurs at the level of virus attachment and entry via a unique mechanism that involves direct interaction of CXCL4 with the major viral envelope glycoprotein, gp120. The binding site for CXCL4 was mapped to a region of the gp120 outer domain proximal to the CD4-binding site. The identification of a platelet-derived chemokine as an endogenous antiviral factor may have relevance for the pathogenesis and treatment of HIV-1 infection.

Treatment with IL-7 prevents the decline of circulating CD4+ T cells during the acute phase of SIV infection in rhesus macaques.

Although treatment with interleukin-7 (IL-7) was shown to transiently expand the naïve and memory T-cell pools in patients with chronic HIV-1 infection receiving antiretroviral therapy (ART), it is uncertain whether a full immunologic reconstitution can be achieved. Moreover, the effects of IL-7 have never been evaluated during acute HIV-1 (or SIV) infection, a critical phase of the disease in which the most dramatic depletion of CD4(+) T cells is believed to occur. In the present study, recombinant, fully glycosylated simian IL-7 (50 µg/kg, s.c., once weekly for 7 weeks) was administered to 6 rhesus macaques throughout the acute phase of infection with a pathogenic SIV strain (mac251); 6 animals were infected at the same time and served as untreated controls. Treatment with IL-7 did not cause clinically detectable side effects and, despite the absence of concomitant ART, did not induce significant increases in the levels of SIV replication except at the earliest time point tested (day 4 post-infection). Strikingly, animals treated with IL-7 were protected from the dramatic decline of circulating naïve and memory CD4(+) T cells that occurred in untreated animals. Treatment with IL-7 induced only transient T-cell proliferation, but it was associated with sustained increase in the expression of the anti-apoptotic protein Bcl-2 on both CD4(+) and CD8(+) T cells, persistent expansion of all circulating CD8(+) T-cell subsets, and development of earlier and stronger SIV Tat-specific T-cell responses. However, the beneficial effects of IL-7 were not sustained after treatment interruption. These data demonstrate that IL-7 administration is effective in protecting the CD4(+) T-cell pool during the acute phase of SIV infection in macaques, providing a rationale for the clinical evaluation of this cytokine in patients with acute HIV-1 infection.

IL-7 induces expression and activation of integrin α4ß7 promoting naive T-cell homing to the intestinal mucosa.

Interleukin-7 (IL-7) is a nonredundant cytokine that plays a critical role in T-cell homeostasis and promotes immunologic reconstitution in lymphopenic hosts. Here, we show that IL-7, at doses that reflect suprahomeostatic concentrations achieved in lymphopenic hosts, is a potent and selective inducer of the gut-homing integrin α4ß7 in human T cells, as documented both ex vivo and in vivo in patients enrolled in a clinical trial of IL-7 treatment. Induction of α4ß7 by IL-7 occurs primarily in naive T cells and is associated with functional activation of the integrin, as indicated by increased binding activity for the specific α4ß7 ligand, MAdCAM-1. The physiologic relevance of these findings was validated by the preferential homing of IL-7-treated naive human T cells to the intestinal compartment in humanized NOD/SCID/IL-2 receptor-γ(null) (NSG) mice. We also show that IL-7 triggers a peculiar activation program in naive T cells, characterized by the acquisition of memory-like phenotypic features and proliferation uncoupled from expression of classic T-cell activation markers. These findings provide a mechanism for the transient in vivo depletion of circulating T cells after IL-7 administration and suggest that intestinal homing and memory-like conversion of naive T cells are critical steps in the IL-7-driven immunologic reconstitution of lymphopenic hosts.

Intraprotomer masking of third variable loop (V3) epitopes by the first and second variable loops (V1V2) within the native HIV-1 envelope glycoprotein trimer.

Within the trimeric HIV-1 envelope (Env) spike, the first and second variable loops (V1V2 region) and the third variable loop (V3) of the gp120 subunit play dual roles in antibody recognition, because they contain neutralization epitopes and also participate in epitope masking. The spatial relationships between V1V2 and V3 and the associated mechanisms of epitope masking remain unclear. Here we investigated interactions between these domains using two monoclonal antibodies recognizing distinct conserved linear epitopes that are subject to masking in the functional trimer, which limits their neutralizing activities. Using Env pseudotype virus infection assays, we found that deleting the V1V2 region greatly enhanced neutralization by both antibodies, leading us to consider two alternative models: V1V2 on one gp120 protomer masks V3 on the same protomer (intraprotomer or cis masking) versus on an adjacent protomer (interprotomer or trans masking). Our experimental approach exploited a previously described complementation system wherein two variant Envs harboring different inactivating mutations (one in gp120, the other in gp41) are coexpressed in the same cell; functional Env results only from cooperative interactions within mixed trimers, thereby enabling selective examination of mixed trimer activity. We introduced additional mutations that either promoted (V1V2 deletion, i.e., unmasking) or prevented (GPGR to GPGQ mutation, i.e., epitope destruction) interaction with the antibodies. The observed neutralization sensitivities of mixed trimers produced from various combinations of constructs support the intraprotomer (cis) model of V1V2 masking of V3 epitopes.

Targeted protein degradation using chimeric human E2 ubiquitin-conjugating enzymes.

Proteins can be targeted for degradation by engineering biomolecules that direct them to the eukaryotic ubiquitination machinery. For instance, the fusion of an E3 ubiquitin ligase to a suitable target binding domain creates a 'biological Proteolysis-Targeting Chimera' (bioPROTAC). Here we employ an analogous approach where the target protein is recruited directly to a human E2 ubiquitin-conjugating enzyme via an attached target binding domain. Through rational design and screening we develop E2 bioPROTACs that induce the degradation of the human intracellular proteins SHP2 and KRAS. Using global proteomics, we characterise the target-specific and wider effects of E2 vs. VHL-based fusions. Taking SHP2 as a model target, we also employ a route to bioPROTAC discovery based on protein display libraries, yielding a degrader with comparatively weak affinity capable of suppressing SHP2-mediated signalling.

Sialic acid-binding Ig-like lectin-7 interacts with HIV-1 gp120 and facilitates infection of CD4pos T cells and macrophages.

BACKGROUND: Sialic acid-binding Ig-like lectin-7 (Siglec-7) expression is strongly reduced on natural killer (NK) cells from HIV-1 infected viremic patients. To investigate the mechanism(s) underlying this phenomenon, we hypothesized that Siglec-7 could contribute to the infection of CD4pos target cells following its interaction with HIV-1 envelope (Env) glycoprotein 120 (gp120). RESULTS: The ability of Siglec-7 to bind gp120 Env in a sialic acid-dependent manner facilitates the infection of both T cells and monocyte-derived macrophages (MDMs). Indeed, pre-incubation of HIV-1 with soluble Siglec-7 (sSiglec-7) increases the infection rate of CD4pos T cells, which do not constitutively express Siglec-7. Conversely, selective blockade of Siglec-7 markedly reduces the degree of HIV-1 infection in Siglec-7pos MDMs. Finally, the sSiglec-7 amount is increased in the serum of AIDS patients with high levels of HIV-1 viremia and inversely correlates with CD4pos T cell counts. CONCLUSIONS: Our results show that Siglec-7 binds HIV-1 and contributes to enhance the susceptibility to infection of CD4pos T cells and MDMs. This phenomenon plays a role in HIV-1 pathogenesis and in disease progression, as suggested by the inverse correlation between high serum level of sSiglec-7 and the low CD4pos T cell count observed in AIDS patients in the presence of chronic viral replication.

Ageing-Induced Decline in Primary Myeloid Cell Phagocytosis Is Unaffected by Optineurin Insufficiency.

Optineurin is a ubiquitin-binding adaptor protein involved in multiple cellular processes, including innate inflammatory signalling. Mutations in optineurin were found in amyotrophic lateral sclerosis, an adult-onset fatal neurodegenerative disease that targets motor neurons. Neurodegeneration results in generation of neuronal debris, which is primarily cleared by myeloid cells. To assess the role of optineurin in phagocytosis, we performed a flow cytometry-based phagocytic assay of apoptotic neuronal debris and E. coli bioparticles in bone marrow-derived macrophages (BMDMs), and primary neonatal microglia from wild-type (WT) and optineurin-insufficient (Optn470T) mice. We found no difference in phagocytosis efficiency and the accompanying cytokine secretion in WT and Optn470T BMDMs and microglia. This was true at both steady state and upon proinflammatory polarization with lipopolysaccharide. When we analysed the effect of ageing as a major risk factor for neurodegeneration, we found a substantial decrease in the percentage of phagocytic cells and proinflammatory cytokine secretion in BMDMs from 2-year-old mice. However, this ageing-induced phagocytic decline was unaffected by optineurin insufficiency. All together, these results indicate that ageing is the factor that perturbs normal phagocytosis and proinflammatory cytokine secretion, but that optineurin is dispensable for these processes.

Neuroimmune characterization of optineurin insufficiency mouse model during ageing.

Optineurin is a multifunctional polyubiquitin-binding protein implicated in inflammatory signalling. Optineurin mutations are associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), neurodegenerative diseases characterised by neuronal loss, neuroinflammation, and peripheral immune disbalance. However, the pathogenic role of optineurin mutations is unclear. We previously observed no phenotype in the unmanipulated young optineurin insufficiency mice (Optn470T), designed to mimic ALS/FTD-linked truncations deficient in polyubiquitin binding. The purpose of this study was to investigate whether ageing would trigger neurodegeneration. We performed a neurological, neuropathological, and immunological characterization of ageing wild-type (WT) and Optn470T mice. No motor or cognitive differences were detected between the genotypes. Neuropathological analyses demonstrated signs of ageing including lipofuscin accumulation and microglial activation in WT mice. However, this was not worsened in Optn470T mice, and they did not exhibit TAR DNA-binding protein 43 (TDP-43) aggregation or neuronal loss. Spleen immunophenotyping uncovered T cell immunosenescence at two years but without notable differences between the WT and Optn470T mice. Conventional dendritic cells (cDC) and macrophages exhibited increased expression of activation markers in two-year-old Optn470T males but not females, although the numbers of innate immune cells were similar between genotypes. Altogether, a combination of optineurin insufficiency and ageing did not induce ALS/FTD-like immune imbalance and neuropathology in mice.

Cellular and molecular dynamics in the lungs of neonatal and juvenile mice in response to E. coli.

Bacterial pneumonia in neonates can cause significant morbidity and mortality when compared to other childhood age groups. To understand the immune mechanisms that underlie these age-related differences, we employed a mouse model of Escherichia coli pneumonia to determine the dynamic cellular and molecular differences in immune responsiveness between neonates (PND 3-5) and juveniles (PND 12-18), at 24, 48, and 72 hr. Cytokine gene expression from whole lung extracts was also quantified at these time points, using quantitative RT-PCR. E. coli challenge resulted in rapid and significant increases in neutrophils, monocytes, and γδT cells, along with significant decreases in dendritic cells and alveolar macrophages in the lungs of both neonates and juveniles. E. coli-challenged juvenile lung had significant increases in interstitial macrophages and recruited monocytes that were not observed in neonatal lungs. Expression of IFNγ-responsive genes was positively correlated with the levels and dynamics of MHCII-expressing innate cells in neonatal and juvenile lungs. Several facets of immune responsiveness in the wild-type neonates were recapitulated in juvenile MHCII-/- juveniles. Employing a pre-clinical model of E. coli pneumonia, we identified significant differences in the early cellular and molecular dynamics in the lungs that likely contribute to the elevated susceptibility of neonates to bacterial pneumonia and could represent targets for intervention to improve respiratory outcomes and survivability of neonates.

The XIST lncRNA is a sex-specific reservoir of TLR7 ligands in SLE.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with a dramatic sex bias, affecting 9 times more women than men. Activation of Toll-like receptor 7 (TLR7) by self-RNA is a central pathogenic process leading to aberrant production of type I interferon (IFN) in SLE, but the specific RNA molecules that serve as TLR7 ligands have not been defined. By leveraging gene expression data and the known sequence specificity of TLR7, we identified the female-specific X-inactive specific transcript (XIST) long noncoding RNA as a uniquely rich source of TLR7 ligands in SLE. XIST RNA stimulated IFN-α production by plasmacytoid DCs in a TLR7-dependent manner, and deletion of XIST diminished the ability of whole cellular RNA to activate TLR7. XIST levels were elevated in blood leukocytes from women with SLE compared with controls, correlated positively with disease activity and the IFN signature, and were enriched in extracellular vesicles released from dying cells in vitro. Importantly, XIST was not IFN inducible, suggesting that XIST is a driver, rather than a consequence, of IFN in SLE. Overall, our work elucidated a role for XIST RNA as a female sex-specific danger signal underlying the sex bias in SLE.