Immune profiling in Puerto Rican injection drug users with and without HIV-1 infection

Antiretroviral therapy has been effective in suppressing HIV viral load and enabling people living with HIV to experience longer, more conventional lives. However, as people living with HIV are living longer, they are developing aging-related diseases prematurely and are more susceptible to comorbidities that have been linked to chronic inflammation. Coincident with HIV infection and aging, drug abuse has also been independently associated with gut dysbiosis, microbial translocation, and inflammation. Here, we hypothesized that injection drug use would exacerbate HIV-induced immune activation and inflammation, thereby intensifying immune dysfunction. We recruited 50 individuals not using injection drugs (36/50 HIV+) and 47 people who inject drugs (PWID, 12/47 HIV+). All but 3 of the HIV+ subjects were on antiretroviral therapy. Plasma immune profiles were characterized by immunoproteomics, and cellular immunophenotypes were assessed using mass cytometry. The immune profiles of HIV+/PWID−, HIV−/PWID+, and HIV+/PWID+ were each significantly different from controls; however, few differences between these groups were detected, and only 3 inflammatory mediators and 2 immune cell populations demonstrated a combinatorial effect of injection drug use and HIV infection. In conclusion, a comprehensive analysis of inflammatory mediators and cell immunophenotypes revealed remarkably similar patterns of immune dysfunction in HIV-infected individuals and in people who inject drugs with and without HIV-1 infection

Single-cell profiling of human dura and meningioma reveals cellular meningeal landscape and insights into meningioma immune response

BACKGROUND: Recent investigations of the meninges have highlighted the importance of the dura layer in central nervous system immune surveillance beyond a purely structural role. However, our understanding of the meninges largely stems from the use of pre-clinical models rather than human samples. METHODS: Single-cell RNA sequencing of seven non-tumor-associated human dura samples and six primary meningioma tumor samples (4 matched and 2 non-matched) was performed. Cell type identities, gene expression profiles, and T cell receptor expression were analyzed. Copy number variant (CNV) analysis was performed to identify putative tumor cells and analyze intratumoral CNV heterogeneity. Immunohistochemistry and imaging mass cytometry was performed on selected samples to validate protein expression and reveal spatial localization of select protein markers. RESULTS: In this study, we use single-cell RNA sequencing to perform the first characterization of both non-tumor-associated human dura and primary meningioma samples. First, we reveal a complex immune microenvironment in human dura that is transcriptionally distinct from that of meningioma. In addition, we characterize a functionally diverse and heterogenous landscape of non-immune cells including endothelial cells and fibroblasts. Through imaging mass cytometry, we highlight the spatial relationship among immune cell types and vasculature in non-tumor-associated dura. Utilizing T cell receptor sequencing, we show significant TCR overlap between matched dura and meningioma samples. Finally, we report copy number variant heterogeneity within our meningioma samples. CONCLUSIONS: Our comprehensive investigation of both the immune and non-immune cellular landscapes of human dura and meningioma at single-cell resolution builds upon previously published data in murine models and provides new insight into previously uncharacterized roles of human dura

Imaging mass cytometry reveals tissue-specific cellular immune phenotypes in the mouse knee following ACL injury

Objective: To develop an imaging mass cytometry method for identifying complex cell phenotypes, inter-cellular interactions, and population changes in the synovium and infrapatellar fat pad (IFP) of the mouse knee following a non-invasive compression injury. Design: Fifteen male C57BL/6 mice were fed a high-fat diet for 8 weeks prior to random assignment to sham, 0.88 ​mm, or 1.7 ​mm knee compression displacement at 24 weeks of age. 2-weeks after loading, limbs were prepared for histologic and imaging mass cytometry analysis, focusing on myeloid immune cell populations in the synovium and IFP. Results: 1.7 ​mm compression caused anterior cruciate ligament (ACL) rupture, development of post-traumatic osteoarthritis, and a 2- to 3-fold increase in cellularity of synovium and IFP tissues compared to sham or 0.88 ​mm compression. Imaging mass cytometry identified 11 myeloid cell subpopulations in synovium and 7 in IFP, of which approximately half were elevated 2 weeks after ACL injury in association with the vasculature. Notably, two monocyte/macrophage subpopulations and an MHC IIhi population were elevated 2-weeks post-injury in the synovium but not IFP. Vascular and immune cell interactions were particularly diverse in the synovium, incorporating 8 unique combinations of 5 myeloid cell populations, including a monocyte/macrophage population, an MHC IIhi population, and 3 different undefined F4/80+ myeloid populations. Conclusions: Developing an imaging mass cytometry method for the mouse enabled us to identify a diverse array of synovial and IFP vascular-associated myeloid cell subpopulations. These subpopulations were differentially elevated in synovial and IFP tissues 2-weeks post injury, providing new details on tissue-specific immune regulation

Insulin-like growth factor receptor signaling regulates working memory, mitochondrial metabolism, and amyloid-β uptake in astrocytes

Objective: A decline in mitochondrial function and biogenesis as well as increased reactive oxygen species (ROS) are important determinants of aging. With advancing age, there is a concomitant reduction in circulating levels of insulin-like growth factor-1 (IGF-1) that is closely associated with neuronal aging and neurodegeneration. In this study, we investigated the effect of the decline in IGF-1 signaling with age on astrocyte mitochondrial metabolism and astrocyte function and its association with learning and memory. Methods: Learning and memory was assessed using the radial arm water maze in young and old mice as well as tamoxifen-inducible astrocyte-specific knockout of IGFR (GFAP-CreTAM/igfrf/f). The impact of IGF-1 signaling on mitochondrial function was evaluated using primary astrocyte cultures from igfrf/f mice using AAV-Cre mediated knockdown using Oroboros respirometry and Seahorse assays. Results: Our results indicate that a reduction in IGF-1 receptor (IGFR) expression with age is associated with decline in hippocampal-dependent learning and increased gliosis. Astrocyte-specific knockout of IGFR also induced impairments in working memory. Using primary astrocyte cultures, we show that reducing IGF-1 signaling via a 30–50% reduction IGFR expression, comparable to the physiological changes in IGF-1 that occur with age, significantly impaired ATP synthesis. IGFR deficient astrocytes also displayed altered mitochondrial structure and function and increased mitochondrial ROS production associated with the induction of an antioxidant response. However, IGFR deficient astrocytes were more sensitive to H2O2-induced cytotoxicity. Moreover, IGFR deficient astrocytes also showed significantly impaired glucose and Aβ uptake, both critical functions of astrocytes in the brain. Conclusions: Regulation of astrocytic mitochondrial function and redox status by IGF-1 is essential to maintain astrocytic function and coordinate hippocampal-dependent spatial learning. Age-related astrocytic dysfunction caused by diminished IGF-1 signaling may contribute to the pathogenesis of Alzheimer's disease and other age-associated cognitive pathologies. Keywords: Primary astrocytes, IGF-1, Amyloid, Mitochondria, ROS, Alzheimer's diseas

Sjögren’s Syndrome Minor Salivary Gland CD4+ Memory T Cells Associate with Glandular Disease Features and Have a Germinal Center T Follicular Helper Transcriptional Profile

To assess the types of salivary gland (SG) T cells contributing to Sjögren’s syndrome (SS), we evaluated SG T cell subtypes for association with disease features and compared the SG CD4+ memory T cell transcriptomes of subjects with either primary SS (pSS) or non-SS sicca (nSS). SG biopsies were evaluated for proportions and absolute numbers of CD4+ and CD8+ T cells. SG memory CD4+ T cells were evaluated for gene expression by microarray. Differentially-expressed genes were identified, and gene set enrichment and pathways analyses were performed. CD4+CD45RA− T cells were increased in pSS compared to nSS subjects (33.2% vs. 22.2%, p < 0.0001), while CD8+CD45RA− T cells were decreased (38.5% vs. 46.0%, p = 0.0014). SG fibrosis positively correlated with numbers of memory T cells. Proportions of SG CD4+CD45RA− T cells correlated with focus score (r = 0.43, p < 0.0001), corneal damage (r = 0.43, p < 0.0001), and serum Ro antibodies (r = 0.40, p < 0.0001). Differentially-expressed genes in CD4+CD45RA− cells indicated a T follicular helper (Tfh) profile, increased homing and increased cellular interactions. Predicted upstream drivers of the Tfh signature included TCR, TNF, TGF-β1, IL-4, and IL-21. In conclusion, the proportions and numbers of SG memory CD4+ T cells associate with key SS features, consistent with a central role in disease pathogenesis