Gas6/TAM Signalling Negatively Regulates Inflammatory Induction of GM-CSF in Mouse Brain Microglia

Microglia and astrocytes are the main CNS glial cells responsible for the neuroinflammatory response, where they release a plethora of cytokines into the CNS inflammatory milieu. The TAM (Tyro3, Axl, Mer) receptors and their main ligand Gas6 are regulators of this response, however, the underlying mechanisms remain to be determined. We investigated the ability of Gas6 to modulate the CNS glial inflammatory response to lipopolysaccharide (LPS), a strong pro-inflammatory agent, through a qPCR array that explored Toll-like receptor signalling pathway-associated genes in primary cultured mouse microglia. We identified the Csf2 gene, encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), as a major Gas6 target gene whose induction by LPS was markedly blunted by Gas6. Both the Csf2 gene induction and the suppressive effect of Gas6 on this were emulated through measurement of GM-CSF protein release by cells. We found distinct profiles of GM-CSF induction in different glial cell types, with microglia being most responsive during inflammation. Also, Gas6 markedly inhibited the LPS-stimulated nuclear translocation of NF-κB p65 protein in microglia. These results illustrate microglia as a major resident CNS cellular source of GM-CSF as part of the neuroinflammatory response, and that Gas6/TAM signalling inhibits this response through suppression of NF-κB signalling.

Generation of Mesenchymal Cell Lines Derived from Aged Donors

Background: Mesenchymal stromal cells (MSCs) have the capacity for self-renewal and multi-differentiation, and for this reason they are considered a potential cellular source in regenerative medicine of cartilage and bone. However, research on this field is impaired by the predisposition of primary MSCs to senescence during culture expansion. Therefore, the aim of this study was to generate and characterize immortalized MSC (iMSC) lines from aged donors. Methods: Primary MSCs were immortalized by transduction of simian virus 40 large T antigen (SV40LT) and human telomerase reverse transcriptase (hTERT). Proliferation, senescence, phenotype and multi-differentiation potential of the resulting iMSC lines were analyzed. Results: MSCs proliferate faster than primary MSCs, overcome senescence and are phenotypically similar to primary MSCs. Nevertheless, their multi-differentiation potential is unbalanced towards the osteogenic lineage. There are no clear differences between osteoarthritis (OA) and non-OA iMSCs in terms of proliferation, senescence, phenotype or differentiation potential. Conclusions: Primary MSCs obtained from elderly patients can be immortalized by transduction of SV40LT and hTERT. The high osteogenic potential of iMSCs converts them into an excellent cellular source to take part in in vitro models to study bone tissue engineering.

A Cellular Reference Resource for the Mouse Urinary Bladder

The urinary bladder functions as a reservoir to store and extrude liquid bodily waste. Significant debate exists as to this tissue's cellular composition and genes associated with their functions. We use a repertoire of cell profiling tools to comprehensively define and spatial resolve cell types. We characterize spatially validated, basal-to-luminal gene expression dynamics within the urothelium, the cellular source of most bladder cancers. We define three distinct populations of fibroblasts that spatially organize from the sub-urothelial layer through to the detrusor muscle, clarifying knowledge around these controversial interstitial cells, and associate increased fibroblasts with aging. We overcome challenges of profiling the detrusor muscle, absence from earlier single cell studies, to report on its transcriptome with many novel and neuronal-like features presumably associated with neuromuscular junctions. Our approach provides a blueprint for tissue atlas construction and the data provides the foundation for future studies of bladder function in health and disease.

T cell derived HIV-1 is present in the CSF in the face of suppressive antiretroviral therapy

HIV cerebrospinal fluid (CSF) escape, where HIV is suppressed in blood but detectable in CSF, occurs when HIV persists in the CNS despite antiretroviral therapy (ART). To determine the virus producing cell type and whether lowered CSF ART levels are responsible for CSF escape, we collected blood and CSF from 156 neurosymptomatic participants from Durban, South Africa. We observed that 28% of participants with an undetectable HIV blood viral load showed CSF escape. We detected host cell surface markers on the HIV envelope to determine the cellular source of HIV in participants on the first line regimen of efavirenz, emtricitabine, and tenofovir. We confirmed CD26 as a marker which could differentiate between T cells and macrophages and microglia, and quantified CD26 levels on the virion surface, comparing the result to virus from in vitro infected T cells or macrophages. The measured CD26 level was consistent with the presence of T cell produced virus. We found no significant differences in ART concentrations between CSF escape and fully suppressed individuals in CSF or blood, and did not observe a clear association with drug resistance mutations in CSF virus which would allow HIV to replicate. Hence, CSF HIV in the face of ART may at least partly originate in CD4+ T cell populations.

Astrocytic and Neuronal Apolipoprotein E Isoforms Differentially Affect Neuronal Excitability

Synaptic changes and neuronal network dysfunction are among the earliest changes in Alzheimer’s disease (AD). Apolipoprotein E4 (ApoE4), the major genetic risk factor in AD, has been shown to be present at synapses and to induce hyperexcitability in mouse knock-in brain regions vulnerable to AD. ApoE in the brain is mainly generated by astrocytes, however, neurons can also produce ApoE under stress conditions such as aging. The potential synaptic function(s) of ApoE and whether the cellular source of ApoE might affect neuronal excitability remain poorly understood. Therefore, the aim of this study was to elucidate the synaptic localization and effects on neuronal activity of the two main human ApoE isoforms from different cellular sources in control and AD-like in vitro cultured neuron models. In this study ApoE is seen to localize at or near to synaptic terminals. Additionally, we detected a cellular source-specific effect of ApoE isoforms on neuronal activity measured by live cell Ca2+ imaging. Neuronal activity increases after acute but not long-term administration of ApoE4 astrocyte medium. In contrast, ApoE expressed by neurons appears to induce the highest neuronal firing rate in the presence of ApoE3, rather than ApoE4. Moreover, increased neuronal activity in APP/PS1 AD transgenic compared to wild-type neurons is seen in the absence of astrocytic ApoE and the presence of astrocytic ApoE4, but not ApoE3. In summary, ApoE can target synapses and differentially induce changes in neuronal activity depending on whether ApoE is produced by astrocytes or neurons. Astrocytic ApoE induces the strongest neuronal firing with ApoE4, while the most active and efficient neuronal activity induced by neuronal ApoE is caused by ApoE3. ApoE isoforms also differentially affect neuronal activity in AD transgenic compared to wild-type neurons.

Interleukin-17A Drives IL-19 and IL-24 Expression in Skin Stromal Cells Regulating Keratinocyte Proliferation

IL-17A has been shown to be up-regulated in psoriasis lesions and is central to psoriasis pathogenesis. IL-19, along with other IL-20 subfamily cytokines such as IL-20 and IL-24, is induced by IL-17A and contributes especially to epidermal hyperplasia in psoriasis. However, the regulation, cellular sources of IL-19 and whether targeting of IL-17A by biologics influence IL-19 expression is not completely understood. To investigate the regulation of IL-19 by IL-17A in psoriasis, the imiquimod-induced psoriasis mouse (IMQ) model was used. Enhanced expression of IL-17A in the IMQ model was achieved by anti-IL-10 antibody treatment. Assessments of skin inflammation macroscopically, by histology and flow cytometry, all confirmed increased psoriatic symptoms. Interestingly, depletion of IL-10 markedly upregulated IL-23/IL-17 pathway related cytokines followed by a significant increase in IL-19 and IL-24. The up-regulation of IL-19 and IL-24, but not IL-17A, coincided with increased keratinocyte proliferation. To investigate the cellular source and effects of biologics on IL-19, human skin fibroblasts from healthy controls and psoriasis patients were cultured alone or co-cultured with activated memory CD4+ T cells. Besides IL-1β, IL-17A induced direct expression of IL-19 and IL-24 in skin fibroblasts and keratinocytes. Importantly, intrinsic higher expression of IL-19 in psoriatic skin fibroblasts was observed in comparison to healthy skin fibroblasts. Neutralization of IL-17A in the human skin fibroblast-T cell co-culture system significantly suppressed IL-19 and IL-24 expression. Together, our data show that IL-17A-induced IL-19 and IL-24 expression in skin stromal cells contribute to keratinocyte proliferation.

Anti-Carbamylated LL37 Antibodies Promote Pathogenic Bone Resorption in Rheumatoid Arthritis

ObjectiveAntibodies against carbamylated proteins (anti-CarP) are associated with poor prognosis and the development of bone erosions in rheumatoid arthritis (RA). RA neutrophils externalize modified autoantigens through the formation of neutrophil extracellular traps (NETs). Increased levels of the cathelicidin LL37 have been documented in the synovium of RA patients, but the cellular source remains unclear. We sought to determine if post-translational modifications of LL37, specifically carbamylation, occur during NET formation, enhance this protein’s autoantigenicity, and contribute to drive bone erosion in the synovial joint.MethodsELISA and Western blot analyses were used to identify carbamylated LL37 (carLL37) in biological samples. Anti-carLL37 antibodies were measured in the serum of HLA-DRB1*04:01 transgenic mice and in human RA synovial fluid.ResultsElevated levels of carLL37 were found in plasma and synovial fluid from RA patients, compared to healthy controls. RA NETs release carLL37 and fibroblast-like synoviocytes (FLS) internalized NET-bound carLL37 and loaded it into their MHCII compartment. HLA-DRB1*04:01 transgenic mice immunized with FLS containing NETs developed autoantibodies against carLL37. Anti-carLL37 antibodies were present in RA sera and synovial fluid and they correlated with radiologic bone erosion scores of the hands and feet in RA patients. CarLL37-IgG immune complexes enhanced the ability of monocytes to differentiate into osteoclasts and potentiated osteoclast-mediated extracellular matrix resorption.ConclusionsNETs are a source of carLL37 leading to induction of anti-carbamylated autoantibody responses. Furthermore, carLL37-IgG immune complexes may be implicated in the bone damage characteristic of RA. These results support that dysregulated NET formation has pathogenic roles in RA.

Single-cell metabolite detection and genomics reveals uncultivated talented producer

The production of bioactive metabolites is increasingly recognized as an important function of host-associated bacteria. An example is defensive symbiosis that might account for much of the chemical richness of marine invertebrates including sponges (Porifera), one of the oldest metazoans. However, as most complex microbiomes remain largely uncultivated and lack reference genomes, unequivocally linking metabolic functions to a cellular source is a challenge. Here we report an analysis pipeline of microfluidic encapsulation, Raman microscopy, and integrated digital genomics (MERMAID) for an efficient identification of uncultivated producers. We applied this method to the chemically rich bacteriosponge Theonella swinhoei, previously shown to contain ′Entotheonella′ symbionts providing most of its bioactive substances except for the antifungal aurantosides that lacked biosynthetic gene candidates in the metagenome. Raman-guided single-bacterial analysis and sequencing revealed a cryptic, distinct multiproducer, ′Candidatus Poriflexus aureus′ from a new Chloroflexi lineage. Its exceptionally large genome contains numerous biosynthetic loci and suggested an even higher chemical richness of this sponge than previously appreciated. This study highlights the importance of complementary technologies to uncover microbiome functions, reveals remarkable parallels between distantly related symbionts of the same host, and adds functional support for diverse chemically prolific lineages being present in microbial dark matter.

Reactive Oxygen Species: Not Omnipresent but Important in Many Locations

Reactive oxygen species (ROS), such as the superoxide anion or hydrogen peroxide, have been established over decades of research as, on the one hand, important and versatile molecules involved in a plethora of homeostatic processes and, on the other hand, as inducers of damage, pathologies and diseases. Which effects ROS induce, strongly depends on the cell type and the source, amount, duration and location of ROS production. Similar to cellular pH and calcium levels, which are both strictly regulated and only altered by the cell when necessary, the redox balance of the cell is also tightly regulated, not only on the level of the whole cell but in every cellular compartment. However, a still widespread view present in the scientific community is that the location of ROS production is of no major importance and that ROS randomly diffuse from their cellular source of production throughout the whole cell and hit their redox-sensitive targets when passing by. Yet, evidence is growing that cells regulate ROS production and therefore their redox balance by strictly controlling ROS source activation as well as localization, amount and duration of ROS production. Hopefully, future studies in the field of redox biology will consider these factors and analyze cellular ROS more specifically in order to revise the view of ROS as freely flowing through the cell.

Immune cell and tumor cell-derived CXCL10 is indicative of immunotherapy response in metastatic melanoma

A T cell-inflamed tumor microenvironment is characterized by the accumulation and local activation of CD8+ T cells and Bat3-lineage dendritic cells, which together are associated with clinical response to anti-programmed cell death protein 1 (anti-PD-1)-based immunotherapy. Preclinical models have demonstrated a crucial role for the chemokine CXCL10 in the recruitment of effector CD8+ T cells into the tumor site, and a chemokine gene signature is also seen in T cell-inflamed tumors from patients. However, the cellular source of CXCL10 in human solid tumors is not known. To identify the cellular source of CXCL10 we analyzed 22 pretreatment biopsy samples of melanoma metastases from patients who subsequently underwent checkpoint blockade immunotherapy. We stained for CD45+ and Sox10+ cells with multiparameter immunofluorescence staining, and RNA in situ hybridization technology was used in concert to identify CXCL10 transcripts. The results were correlated with the expression levels of CXCL10 transcripts from bulk RNA sequencing and the best overall response to immune checkpoint inhibition (anti-PD-1 alone or with anti-CTLA-4) in the same patients. We identified CD45+ cells as the major cellular source for CXCL10 in human melanoma metastases, with additional CXCL10 production seen by Sox10+ cells. Up to 90% of CD45+ cells and up to 69% of Sox10+ cells produced CXCL10 transcripts. The CXCL10 staining result was consistent with the level of CXCL10 expression determined by bulk RNA sequencing. The percentages of CD45+ CXCL10+ cells and Sox10+ CXCL10+ cells independently predicted response (p<0.001). The average number of transcripts per cell correlated with the CD45+ cell infiltrate (R=0.37). Immune cells and melanoma cells produce CXCL10 in human melanoma metastases. Intratumoral CXCL10 is a positive prognostic factor for response to immunotherapy, and the RNAscope technique is achievable using paraffin tissue. Strategies that support effector T cell recruitment via induction of CXCL10 should be considered as a mechanism-based intervention to expand immunotherapy efficacy.