Abstract Unavailable

Abstract Unavailable

Knowledge of immune activation in the brain during acute HIV infection is crucial for the prevention and treatment of HIV-associated neurological disorders. We determined regional brain (basal ganglia, thalamus, and frontal cortex) immune and virological profiles at 7 and 14 days post infection (dpi) with SIVmac239 in rhesus macaques. The basal ganglia and thalamus had detectable viruses earlier (7 dpi) than the frontal cortex (14 dpi) and contained higher quantities of viruses than the latter. Increased immune activation of astrocytes and significant infiltration of macrophages in the thalamus at 14 dpi coincided with elevated plasma viral load, and SIV colocalized only within macrophages. RNA signatures of proinflammatory responses, including IL-6, were detected at 7 dpi in microglia and interestingly, preceded reliable detection of virus in tissues and were maintained in the chronically infected macaques. Countering the proinflammatory response, the antiinflammatory response was not detected until increased TGF-β expression was found in perivascular macrophages at 14 dpi. But this response was not detected in chronic infection. Our data provide evidence that the interplay of acute proinflammatory and antiinflammatory responses in the brain likely contributed to the overt neuroinflammation, where the immune activation preceded reliable viral detection.

Mosquito immune cells, known as hemocytes, are integral to cellular and humoral responses that limit pathogen survival and mediate immune priming. However, without reliable cell markers and genetic tools, studies of mosquito immune cells have been limited to morphological observations, leaving several aspects of their biology uncharacterized. Here, we use single-cell RNA sequencing (scRNA-seq) to characterize mosquito immune cells, demonstrating an increased complexity to previously defined prohemocyte, oenocytoid, and granulocyte subtypes. Through functional assays relying on phagocytosis, phagocyte depletion, and RNA-FISH experiments, we define markers to accurately distinguish immune cell subtypes and provide evidence for immune cell maturation and differentiation. In addition, gene-silencing experiments demonstrate the importance of lozenge in defining the mosquito oenocytoid cell fate. Together, our scRNA-seq analysis provides an important foundation for future studies of mosquito immune cell biology and a valuable resource for comparative invertebrate immunology.

Macrophages sense and respond to pathogens by induction of antimicrobial and inflammatory programs to alert other immune cells and eliminate the infectious threat. We have previously identified the transcription factor IRF1 to be consistently activated in macrophages during Mycobacterium avium infection, but its precise role during infection is not clear. Here, we show that tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) autocrine/paracrine signaling contributes to controlling the intracellular growth of M. avium in human primary macrophages through activation of IRF1 nuclear translocation and expression of IRG1, a mitochondrial enzyme that produces the antimicrobial metabolite itaconate. Small interfering RNA (siRNA)-mediated knockdown of IRF1 or IRG1 increased the mycobacterial load, whereas exogenously provided itaconate was bacteriostatic at high concentrations. While the overall level of endogenous itaconate was low in M. avium-infected macrophages, the repositioning of mitochondria to M. avium phagosomes suggests a mechanism by which itaconate can be delivered directly to M. avium phagosomes in sufficient quantities to inhibit growth. Using mRNA hybridization, we further show that uninfected bystander cells actively contribute to the resolution of infection by producing IL-6 and TNF-α, which, via paracrine signaling, activate IRF1/IRG1 and strengthen the antimicrobial activity of infected macrophages. This mechanism contributes to the understanding of why patients on anti-inflammatory treatment, e.g., with tocilizumab or infliximab, can be more susceptible to mycobacterial disease. IMPORTANCE The prevalence of lung diseases caused by nontuberculous mycobacteria, such as Mycobacterium avium, is increasing in countries where tuberculosis is not endemic, most likely because of an aging population that is immunocompromised from underlying disease or immunosuppressive therapy. Our study contributes to the understanding of mycobacterial survival and killing in human macrophages and, more broadly, to the impact of immunometabolism during infection. We show evidence of an antimicrobial program in human primary macrophages where activation of the transcription factor IRF1 and expression of the mitochondrial enzyme IRG1 restrict the intracellular growth of M. avium, possibly by directed delivery of itaconate to M. avium phagosomes. The study also sheds light on why patients on immunosuppressive therapy are more susceptible to mycobacterial infections, since TNF-α and IL-6 contribute to driving the described antimycobacterial program.

Tianeptine is an atypical antidepressant used in Europe to treat patients who respond poorly to selective serotonin reuptake inhibitors (SSRIs). The recent discovery that tianeptine is a mu opioid receptor (MOR) agonist has provided a potential avenue for expanding our understanding of antidepressant treatment beyond the monoamine hypothesis. Thus, our studies aim to understand the neural circuits underlying tianeptine's antidepressant effects. We show that tianeptine induces rapid antidepressant-like effects in mice after as little as one week of treatment. Critically, we also demonstrate that tianeptine's mechanism of action is distinct from fluoxetine in two important aspects: (1) tianeptine requires MORs for its chronic antidepressant-like effect, while fluoxetine does not, and (2) unlike fluoxetine, tianeptine does not promote hippocampal neurogenesis. Using cell-type specific MOR knockouts we further show that MOR expression on GABAergic cells-specifically somatostatin-positive neurons-is necessary for the acute and chronic antidepressant-like responses to tianeptine. Using central infusion of tianeptine, we also implicate the ventral hippocampus as a potential site of antidepressant action. Moreover, we show a dissociation between the antidepressant-like phenotype and other opioid-like phenotypes resulting from acute tianeptine administration such as analgesia, conditioned place preference, and hyperlocomotion. Taken together, these results suggest a novel entry point for understanding what circuit dysregulations may occur in depression, as well as possible targets for the development of new classes of antidepressant drugs.

Stress-induced release of dynorphins (Dyn) activates kappa opioid receptors (KOR) in serotonergic neurons to produce dysphoria and potentiate drug reward; however, the circuit mechanisms responsible for this effect are not known. In male mice, we found that conditional deletion of KOR from Slc6a4 (SERT)-expressing neurons blocked stress-induced potentiation of cocaine conditioned place preference (CPP). Within the dorsal raphe nucleus (DRN), two overlapping populations of KOR-expressing neurons: Slc17a8 (VGluT3) and SERT, were distinguished functionally and anatomically. Optogenetic inhibition of these SERT+ neurons potentiated subsequent cocaine CPP, whereas optical inhibition of the VGluT3+ neurons blocked subsequent cocaine CPP. SERT+/VGluT3- expressing neurons were concentrated in the lateral aspect of the DRN. SERT projections from the DRN were observed in the medial nucleus accumbens (mNAc), but VGluT3 projections were not. Optical inhibition of SERT+ neurons produced place aversion, whereas optical stimulation of SERT+ terminals in the mNAc attenuated stress-induced increases in forced swim immobility and subsequent cocaine CPP. KOR neurons projecting to mNAc were confined to the lateral aspect of the DRN, and the principal source of dynorphinergic (Pdyn) afferents in the mNAc was from local neurons. Excision of Pdyn from the mNAc blocked stress-potentiation of cocaine CPP. Prior studies suggested that stress-induced dynorphin release within the mNAc activates KOR to potentiate cocaine preference by a reduction in 5-HT tone. Consistent with this hypothesis, a transient pharmacological blockade of mNAc 5-HT1B receptors potentiated subsequent cocaine CPP. 5-HT1B is known to be expressed on 5-HT terminals in NAc, and 5-HT1B transcript was also detected in Pdyn+, Adora2a+ and ChAT+ (markers for direct pathway, indirect pathway, and cholinergic interneurons, respectively). Following stress exposure, 5-HT1B transcript was selectively elevated in Pdyn+ cells of the mNAc. These findings suggest that Dyn/KOR regulates serotonin activation of 5HT1B receptors within the mNAc and dynamically controls stress response, affect, and drug reward.

Castleman disease (CD) represents a group of rare, heterogeneous and poorly understood disorders that share characteristic histopathological features. Unicentric CD (UCD) typically involves a single enlarged lymph node whereas multicentric CD (MCD) involves multiple lymph node stations. To understand the cellular basis of CD, we undertook a multi-platform analysis using targeted RNA sequencing, RNA in-situ hybridization (ISH), and adaptive immune receptor rearrangements (AIRR) profiling of archived tissue from 26 UCD, 14 MCD, and 31 non-CD reactive controls. UCD showed differential expression and upregulation of follicular dendritic cell markers (CXCL13, clusterin), angiogenesis factors (LPL, DLL4), extracellular matrix remodeling factors (TGFβ, SKIL, LOXL1, IL-1β, ADAM33, CLEC4A), complement components (C3, CR2) and germinal center activation markers (ZDHHC2 and BLK) compared to controls. MCD showed upregulation of IL-6 (IL-6ST, OSMR and LIFR), IL-2, plasma cell differentiation (XBP1), FDC marker (CXCL13, clusterin), fibroblastic reticular cell cytokine (CCL21), angiogenesis factor (VEGF), and mTORC1 pathway genes compared to UCD and controls. ISH studies demonstrated that VEGF was increased in the follicular dendritic cell-predominant atretic follicles and the interfollicular macrophages of MCD compared to UCD and controls. IL-6 expression was higher along interfollicular vasculature-associated cells of MCD. Immune repertoire analysis revealed oligoclonal expansions of T-cell populations in MCD cases (2/6) and UCD cases (1/9) that are consistent with antigen-driven T cell activation. The findings highlight the unique genes, pathways and cell types involved in UCD and MCD. We identify potential novel targets in CD that may be harnessed for therapeutics.

KLF5 plays a crucial role in stem cells of colorectum in cooperation with Lgr5 gene. In this study, we aimed to explicate a regulatory mechanism of the KLF5 gene product from a view of three-dimensional genome structure in colorectal cancer (CRC).In vitro engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP)-seq method was used to identify the regions that bind to the KLF5 promoter.We revealed that the KLF5 promoter region interacted with the KLF5 enhancer region as well as the transcription start site (TSS) region of the Colon Cancer Associated Transcript 1 (CCAT1) gene. Notably, the heterodeletion mutants of KLF5 enhancer impaired the cancer stem-like properties of CRC cells. The KLF5 protein participated in the core-regulatory circuitry together with co-factors (BRD4, MED1, and RAD21), which constructs the three-dimensional genome structures consisting of KLF5 promoter, enhancer and CCAT1 TSS region. In vitro analysis indicated that KLF5 regulated CCAT1 expression and we found that CCAT1 expression was highly correlated with KLF5 expression in CRC clinical samples.Our data propose the mechanistic insight that the KLF5 protein constructs the core-regulatory circuitry with co-factors in the three-dimensional genome structure and coordinately regulates KLF5 and CCAT1 expression in CRC.

The absence of the putative DNA/RNA helicase Schlafen11 (SLFN11) is thought to cause resistance to DNA-damaging agents (DDAs) and PARP inhibitors.We developed and validated a clinically applicable SLFN11 immunohistochemistry assay and retrospectively correlated SLFN11 tumour levels to patient outcome to the standard of care therapies and olaparib maintenance.High SLFN11 associated with improved prognosis to the first-line treatment with DDAs platinum-plus-etoposide in SCLC patients, but was not strongly linked to paclitaxel-platinum response in ovarian cancer patients. Multivariate analysis of patients with relapsed platinum-sensitive ovarian cancer from the randomised, placebo-controlled Phase II olaparib maintenance Study19 showed SLFN11 tumour levels associated with sensitivity to olaparib. Study19 patients with high SLFN11 had a lower progression-free survival (PFS) hazard ratio compared to patients with low SLFN11, although both groups had the benefit of olaparib over placebo. Whilst caveated by small sample size, this trend was maintained for PFS, but not overall survival, when adjusting for BRCA status across the olaparib and placebo treatment groups, a key driver of PARP inhibitor sensitivity.We provide clinical evidence supporting the role of SLFN11 as a DDA therapy selection biomarker in SCLC and highlight the need for further clinical investigation into SLFN11 as a PARP inhibitor predictive biomarker.

Previous studies suggest that microglial-expressed Apolipoprotein E (ApoE) is necessary to shift microglia into a neurodegenerative transcriptional state in Alzheimer's disease (AD) mouse models. On the other hand, elimination of microglia shifts amyloid beta (Aβ) accumulation from parenchymal plaques to cerebral amyloid angiopathy (CAA), mimicking the effects of global APOE*4 knock-in. Here, we specifically knock-out microglial-expressed ApoE while keeping astrocytic-expressed ApoE intact. When microglial-specific ApoE is knocked-out of a 5xFAD mouse model of AD, we found a ~35% increase in average Aβ plaque size, but no changes in plaque load, microglial number, microglial clustering around Aβ plaques, nor the formation of CAA. Immunostaining revealed ApoE protein present in plaque-associated microglia in 5xFAD mice with microglial-specific ApoE knockout, suggesting that microglia can take up ApoE from other cellular sources. Mice with Apoe knocked-out of microglia had lower synaptic protein levels than control mice, indicating that microglial-expressed ApoE may have a role in synapse maintenance. Surprisingly, microglial-specific ApoE knock-out resulted in few differentially expressed genes in both 5xFAD and control mice; however, some rescue of 5xFAD associated neuronal networks may occur with microglial-specific ApoE knock-out as shown by weighted gene co-expression analysis. Altogether, our data indicates that microglial-expressed ApoE may not be necessary for plaque formation or for the microglial transcriptional shift into a Disease Associated Microglia state that is associated with reactivity to plaques but may be necessary for plaque homeostasis in disease and synaptic maintenance under normal conditions.

Kallikrein related peptidase 6 (Klk6) is a secreted serine protease highly expressed in oligodendrocytes and implicated in demyelinating conditions. To gain insights into the significance of Klk6 to oligodendrocyte biology, we investigated the impact of global Klk6 gene knockout on CNS developmental myelination using the spinal cord of male and female mice as a model. Results demonstrate that constitutive loss of Klk6 expression accelerates oligodendrocyte differentiation developmentally, including increases in the expression of myelin proteins such as MBP, PLP and CNPase, in the number of CC-1+ mature oligodendrocytes, and myelin thickness by the end of the first postnatal week. Co-ordinate elevations in the pro-myelinating signaling pathways ERK and AKT, expression of fatty acid 2-hydroxylase, and myelin regulatory transcription factor were also observed in the spinal cord of 7d Klk6 knockouts. LC/MS/MS quantification of spinal cord lipids showed sphingosine and sphingomyelins to be elevated in Klk6 knockouts at the peak of myelination. Oligodendrocyte progenitor cells (OPCs)-derived from Klk6 knockouts, or wild type OPCs-treated with a Klk6 inhibitor (DFKZ-251), also showed increased MBP and PLP. Moreover, inhibition of Klk6 in OPC cultures enhanced brain derived neurotrophic factor-driven differentiation. Altogether, these findings suggest that oligodendrocyte-derived Klk6 may operate as an autocrine or paracrine rheostat, or brake, on pro-myelinating signaling serving to regulate myelin homeostasis developmentally and in the adult. These findings document for the first time that inhibition of Klk6 globally, or specifically in oligodendrocyte progenitors, is a strategy to increase early stages of oligodendrocyte differentiation and myelin production in the CNS.