Probes for INS

Tissue-based T cells are important effectors in the prevention and control of mucosal viral infections; less is known about tissue-based B cells. We demonstrate that B cells and antibody-secreting cells (ASCs) are present in inflammatory infiltrates in skin biopsy specimens from study participants during symptomatic herpes simplex virus 2 (HSV-2) reactivation and early healing. Both CD20+ B cells, most of which are antigen inexperienced based on their coexpression of IgD, and ASCs - characterized by dense IgG RNA expression in combination with CD138, IRF4, and Blimp-1 RNA - were found to colocalize with T cells. ASCs clustered with CD4+ T cells, suggesting the potential for crosstalk. HSV-2-specific antibodies to virus surface antigens were also present in tissue and increased in concentration during HSV-2 reactivation and healing, unlike in serum, where concentrations remained static over time. B cells, ASCs, and HSV-specific antibody were rarely detected in biopsies of unaffected skin. Evaluation of samples from serial biopsies demonstrated that B cells and ASCs followed a more migratory than resident pattern of infiltration in HSV-affected genital skin, in contrast to T cells. Together, these observations suggest the presence of distinct phenotypes of B cells in HSV-affected tissue; dissecting their role in reactivation may reveal new therapeutic avenues to control these infections.

Diffuse Large B-Cell Lymphoma (DLBCL) presents a high clinical and biological heterogeneity, and the tumor microenvironment chracteristics are important in its  progression. The aim of this study was to evaluate tumor T, B cells, macrophages and mast cells distribution in GBC and ABC DLBCL subgroups through a set of morphometric parameters allowing to provide a quantitative evaluation of the morphological features of the spatial patterns generated by these inflammatory cells.   Histological ABC and GCB samples were immunostained for CD4, CD8, CD68, CD 163, and tryptase in order to determine both percentage and position of positive cells in the tissue characterizing their spatial distribution. The results evidenced that cell patterns generated by CD4-, CD8-, CD68-, CD163- and tryptase-positive cell profiles exhibited a significantly higher uniformity index in ABC than in GCB subgroup. The positive-cell distributions appeared clustered in tissues from GCB, while in tissues from ABC such a feature was lower or absent. The combinations of spatial statistics-derived parameters can lead to better predictions of tumor cell infiltration than any classical morphometric method providing a more accurate description of the functional status of the tumor, useful for patient prognosis.

Microglial-derived inflammation has been linked to a broad range of neurodegenerative and neuropsychiatric conditions, including amyotrophic lateral sclerosis (ALS). Using single-cell RNA sequencing, a class of Disease-Associated Microglia (DAMs) have been characterized in neurodegeneration. However, the DAM phenotype alone is insufficient to explain the functional complexity of microglia, particularly with regard to regulating inflammation that is a hallmark of many neurodegenerative diseases. Here, we identify a subclass of microglia in mouse models of ALS which we term RIPK1-Regulated Inflammatory Microglia (RRIMs). RRIMs show significant up-regulation of classical proinflammatory pathways, including increased levels of Tnf and Il1b RNA and protein. We find that RRIMs are highly regulated by TNFα signaling and that the prevalence of these microglia can be suppressed by inhibiting receptor-interacting protein kinase 1 (RIPK1) activity downstream of the TNF receptor 1. These findings help to elucidate a mechanism by which RIPK1 kinase inhibition has been shown to provide therapeutic benefit in mouse models of ALS and may provide an additional biomarker for analysis in ongoing phase 2 clinical trials of RIPK1 inhibitors in ALS.

Nonconvulsive epileptiform activity and microglial alterations have been detected in people with Alzheimer’s disease (AD) and related mouse models. However, the relationship between these abnormalities remains to be elucidated. We suppressed epileptiform activity by treatment with the antiepileptic drug levetiracetam or by genetic ablation of tau and found that these interventions reversed or prevented aberrant microglial gene expression in brain tissues of aged human amyloid precursor protein transgenic mice, which simulate several key aspects of AD. The most robustly modulated genes included multiple factors previously implicated in AD pathogenesis, including TREM2, the hypofunction of which increases disease risk. Genetic reduction of TREM2 exacerbated epileptiform activity after mice were injected with kainate. We conclude that AD-related epileptiform activity markedly changes the molecular profile of microglia, inducing both maladaptive and adaptive alterations in their activities. Increased expression of TREM2 seems to support microglial activities that counteract this type of network dysfunction.

Activation of the complement system propagates neuroinflammation and brain damage early and chronically after traumatic brain injury (TBI). The complement system is complex and comprises more than 50 components, many of which remain to be characterized in the normal and injured brain. Moreover, complement therapeutic studies have focused on a limited number of histopathological outcomes, which while informative, do not assess the effect of complement inhibition on neuroprotection and inflammation in a comprehensive manner. Using high throughput gene expression technology (NanoString), we simultaneously analyzed complement gene expression profiles with other neuroinflammatory pathway genes at different time points after TBI. We additionally assessed the effects of complement inhibition on neuropathological processes. Analyses of neuroinflammatory genes were performed at days 3, 7, and 28 post injury in male C57BL/6 mice following a controlled cortical impact injury. We also characterized the expression of 59 complement genes at similar time points, and also at 1- and 2-years post injury. Overall, TBI upregulated the expression of markers of astrogliosis, immune cell activation, and cellular stress, and downregulated the expression of neuronal and synaptic markers from day 3 through 28 post injury. Moreover, TBI upregulated gene expression across most complement activation and effector pathways, with an early emphasis on classical pathway genes and with continued upregulation of C2, C3 and C4 expression 2 years post injury. Treatment using the targeted complement inhibitor, CR2-Crry, significantly ameliorated TBI-induced transcriptomic changes at all time points. Nevertheless, some immune and synaptic genes remained dysregulated with CR2-Crry treatment, suggesting adjuvant anti-inflammatory and neurotropic therapy may confer additional neuroprotection. In addition to characterizing complement gene expression in the normal and aging brain, our results demonstrate broad and chronic dysregulation of the complement system after TBI, and strengthen the view that the complement system is an attractive target for TBI therapy.

METHODS : Retinal, optic nerve head (ONH) and distal optic nerve (ON) tissues from 8 juvenile 10-12 week-old cats (4 males and 4 females) with feline congenital glaucoma (FCG) and 5 age-matched normal control cats (3 males and 2 females) were used. Data for weekly intraocular pressure (IOP) and optic nerve axon counts were available for all subjects. Protein and gene expression in tissue cryosections were examined by immunofluorescence labeling (IF) and RNAscope in situ hybridization (ISH), respectively. Retinal tissue was IF labeled for myeloid cell marker, IBA-1 and flat-mounted. ISH for markers of infiltrating monocytes/macrophages (_CCR2_) and proinflammatory cytokines (_IL1A_, _C1QA_, _TNF_) was performed. Microglia were identified by IF of homeostatic microglial marker, P2RY12. Microscopy images wereanalyzed using Image J, QuPath and Imaris. Two-tailed unpaired t-test or Mann-Whitney test or ANOVA were used for between-group comparisons (p