Here, we highlight the potential translational benefits of delivering FLASH radiotherapy using ultra-high dose rates (>100 Gy·s−1). Compared with conventional dose-rate (CONV; 0.07–0.1 Gy·s−1) ...modalities, we showed that FLASH did not cause radiation-induced deficits in learning and memory in mice. Moreover, 6 months after exposure, CONV caused permanent alterations in neurocognitive end points, whereas FLASH did not induce behaviors characteristic of anxiety and depression and did not impair extinction memory. Mechanistic investigations showed that increasing the oxygen tension in the brain through carbogen breathing reversed the neuroprotective effects of FLASH, while radiochemical studies confirmed that FLASH produced lower levels of the toxic reactive oxygen species hydrogen peroxide. In addition, FLASH did not induce neuroinflammation, a process described as oxidative stress-dependent, and was also associated with a marked preservation of neuronal morphology and dendritic spine density. The remarkable normal tissue sparing afforded by FLASH may someday provide heretofore unrealized opportunities for dose escalation to the tumor bed, capabilities that promise to hasten the translation of this groundbreaking irradiation modality into clinical practice.
Durable CD4+ T cell immunity: cherchez la stem Hughes, Erik P.; Syage, Amber R.; Tantin, Dean
Trends in immunology,
March 2024, 2024-03-00, 20240301, Volume:
45, Issue:
3
Journal Article
Peer reviewed
Different CD4+ T cell subsets have been described to have ‘stem-like’ features, frequently without a clear indication of what that means functionally or devoid of a systematic set of criteria for how ...to apply the terminology.Bona fide stem cells are themselves varied and their definitions have been evolving recently.CD4+ T cell types with clear stem-like features are naive cells and subsets of memory cells central memory (Tcm) and stem cell memory (Tscm). These also display stability over time, specific niche homing, high replicative potential, and the capacity to differentiate into many other T cell types. Other cells fall along a continuum of more versus less stem-like features.In some cases, more functional characterization of these cells is required before appending a ‘stem-like’ moniker is justified. For others, substituting ‘progenitor’ or ‘precursor’ terminology along the lines of the hematopoietic system is likely to be more accurate.
T cells with stem-like features mediate durable immunity to pathogens and tumors but can also promote tissue damage. The complexity of the literature labeling T cells as ‘stem-like’ suggests that both the criteria for ascribing stem cell character to T cells, and the nomenclature used to relate them to one another, need refinement. This would help systemize the field, allowing better inference of function from name.
Mammalian stem cells govern development, tissue homeostasis, and regeneration. Following years of study, their functions have been delineated with increasing precision. The past decade has witnessed heightened widespread use of stem cell terminology in association with durable T cell responses to infection, antitumor immunity, and autoimmunity. Interpreting this literature is complicated by the fact that descriptions are diverse and criteria for labeling ‘stem-like’ T cells are evolving. Working under the hypothesis that conceptual frameworks developed for actual stem cells can be used to better evaluate and organize T cells described to have stem-like features, we outline widely accepted properties of stem cells and compare these to different ‘stem-like’ CD4+ T cell populations.
Mammalian stem cells govern development, tissue homeostasis, and regeneration. Following years of study, their functions have been delineated with increasing precision. The past decade has witnessed heightened widespread use of stem cell terminology in association with durable T cell responses to infection, antitumor immunity, and autoimmunity. Interpreting this literature is complicated by the fact that descriptions are diverse and criteria for labeling ‘stem-like’ T cells are evolving. Working under the hypothesis that conceptual frameworks developed for actual stem cells can be used to better evaluate and organize T cells described to have stem-like features, we outline widely accepted properties of stem cells and compare these to different ‘stem-like’ CD4+ T cell populations.
Cosmic radiation exposures have been found to elicit cognitive impairments involving a wide-range of underlying neuropathology including elevated oxidative stress, neural stem cell loss, and ...compromised neuronal architecture. Cognitive impairments have also been associated with sustained microglia activation following low dose exposure to helium ions. Space-relevant charged particles elicit neuroinflammation that persists long-term post-irradiation. Here, we investigated the potential neurocognitive benefits of microglia depletion following low dose whole body exposure to helium ions.
Adult mice were administered a dietary inhibitor (PLX5622) of colony stimulating factor-1 receptor (CSF1R) to deplete microglia 2 weeks after whole body helium irradiation (
He, 30 cGy, 400 MeV/n). Cohorts of mice maintained on a normal and PLX5622 diet were tested for cognitive function using seven independent behavioral tasks, microglial activation, hippocampal neuronal morphology, spine density, and electrophysiology properties 4-6 weeks later.
PLX5622 treatment caused a rapid and near complete elimination of microglia in the brain within 3 days of treatment. Irradiated animals on normal diet exhibited a range of behavioral deficits involving the medial pre-frontal cortex and hippocampus and increased microglial activation. Animals on PLX5622 diet exhibited no radiation-induced cognitive deficits, and expression of resting and activated microglia were almost completely abolished, without any effects on the oligodendrocyte progenitors, throughout the brain. While PLX5622 treatment was found to attenuate radiation-induced increases in post-synaptic density protein 95 (PSD-95) puncta and to preserve mushroom type spine densities, other morphologic features of neurons and electrophysiologic measures of intrinsic excitability were relatively unaffected.
Our data suggest that microglia play a critical role in cosmic radiation-induced cognitive deficits in mice and, that approaches targeting microglial function are poised to provide considerable benefit to the brain exposed to charged particles.
The present study examines functional contributions of microglia in host defense, demyelination, and remyelination following infection of susceptible mice with a neurotropic coronavirus. Treatment ...with PLX5622, an inhibitor of colony stimulating factor 1 receptor (CSF1R) that efficiently depletes microglia, prior to infection of the central nervous system (CNS) with the neurotropic JHM strain of mouse hepatitis virus (JHMV) resulted in increased mortality compared with control mice that correlated with impaired control of viral replication. Single cell RNA sequencing (scRNASeq) of CD45+ cells isolated from the CNS revealed that PLX5622 treatment resulted in muted CD4+ T cell activation profile that was associated with decreased expression of transcripts encoding MHC class II and CD86 in macrophages but not dendritic cells. Evaluation of spinal cord demyelination revealed a marked increase in white matter damage in PLX5622‐treated mice that corresponded with elevated expression of transcripts encoding disease‐associated proteins Osteopontin (Spp1), Apolipoprotein E (Apoe), and Triggering receptor expressed on myeloid cells 2 (Trem2) that were enriched within macrophages. In addition, PLX5622 treatment dampened expression of Cystatin F (Cst7), Insulin growth factor 1 (Igf1), and lipoprotein lipase (Lpl) within macrophage populations which have been implicated in promoting repair of damaged nerve tissue and this was associated with impaired remyelination. Collectively, these findings argue that microglia tailor the CNS microenvironment to enhance control of coronavirus replication as well as dampen the severity of demyelination and influence repair.
Main points
Microglia augment host defense in response to CNS infection by the neurotropic coronavirus JHMV.
A role for microglia in restricting demyelination and promoting remyelination in JHMV infected mice also is demonstrated.
Abstract Background Cystatin F is a secreted lysosomal cysteine protease inhibitor that has been implicated in affecting the severity of demyelination and enhancing remyelination in pre-clinical ...models of immune-mediated demyelination. How cystatin F impacts neurologic disease severity following viral infection of the central nervous system (CNS) has not been well characterized and was the focus of this study. We used cystatin F null-mutant mice ( Cst7-/- ) with a well-established model of murine coronavirus-induced neurologic disease to evaluate the contributions of cystatin F in host defense, demyelination and remyelination. Methods Wildtype controls and Cst7-/- mice were intracranially (i.c.) infected with a sublethal dose of the neurotropic JHM strain of mouse hepatitis virus (JHMV), with disease progression and survival monitored daily. Viral plaque assays and qPCR were used to assess viral levels in CNS. Immune cell infiltration into the CNS and immune cell activation were determined by flow cytometry and 10X genomics chromium 3’ single cell RNA sequencing (scRNA-seq). Spinal cord demyelination was determined by luxol fast blue (LFB) and Hematoxylin/Eosin (H&E) staining and axonal damage assessed by immunohistochemical staining for SMI-32. Remyelination was evaluated by electron microscopy (EM) and calculation of g -ratios. Results JHMV-infected Cst7-/- mice were able to control viral replication within the CNS, indicating that cystatin F is not essential for an effective Th1 anti-viral immune response. Infiltration of T cells into the spinal cords of JHMV-infected Cst7-/- mice was increased compared to infected controls, and this correlated with increased axonal damage and demyelination associated with impaired remyelination. Single-cell RNA-seq of CD45 + cells enriched from spinal cords of infected Cst7-/- and control mice revealed enhanced expression of transcripts encoding T cell chemoattractants, Cxcl9 and Cxcl10 , combined with elevated expression of interferon-g ( Ifng) and perforin ( Prf1 ) transcripts in CD8 + T cells from Cst7-/- mice compared to controls. Conclusions Cystatin F is not required for immune-mediated control of JHMV replication within the CNS. However, JHMV-infected Cst7-/- mice exhibited more severe clinical disease associated with increased demyelination and impaired remyelination. The increase in disease severity was associated with elevated expression of T cell chemoattractant chemokines, concurrent with increased neuroinflammation. These findings support the idea that cystatin F influences expression of proinflammatory gene expression impacting neuroinflammation, T cell activation and/or glia cell responses ultimately impacting neuroinflammation and neurologic disease.
Intracranial (i.c.) infection of susceptible C57BL/6 mice with the neurotropic JHM strain of mouse hepatitis virus (JHMV) (a member of the
family) results in acute encephalomyelitis and viral ...persistence associated with an immune-mediated demyelinating disease. The present study was undertaken to better understand the molecular pathways evoked during innate and adaptive immune responses as well as the chronic demyelinating stage of disease in response to JHMV infection of the central nervous system (CNS). Using single-cell RNA sequencing analysis (scRNAseq) on flow-sorted CD45-positive (CD45
) cells enriched from brains and spinal cords of experimental mice, we demonstrate the heterogeneity of the immune response as determined by the presence of unique molecular signatures and pathways involved in effective antiviral host defense. Furthermore, we identify potential genes involved in contributing to demyelination as well as remyelination being expressed by both microglia and macrophages. Collectively, these findings emphasize the diversity of the immune responses and molecular networks at defined stages following viral infection of the CNS.
Understanding the immunological mechanisms contributing to both host defense and disease following viral infection of the CNS is of critical importance given the increasing number of viruses that are capable of infecting and replicating within the nervous system. With this in mind, the present study was undertaken to evaluate the molecular signatures of immune cells within the CNS at defined times following infection with a neuroadapted murine coronavirus using scRNAseq. This approach has revealed that the immunological landscape is diverse, with numerous immune cell subsets expressing distinct mRNA expression profiles that are, in part, dictated by the stage of infection. In addition, these findings reveal new insight into cellular pathways contributing to control of viral replication as well as to neurologic disease.
Intracranial inoculation of the neuroadapted JHM strain of mouse hepatitis virus (JHMV) into susceptible strains of mice results in acute encephalomyelitis followed by a cimmune-mediated ...demyelination similar to the human demyelinating disease multiple sclerosis (MS). JHMV infection of transgenic mice in which expression of the neutrophil chemoattractant chemokine CXCL1 is under the control of a tetracycline-inducible promoter active within GFAP-positive cells results in sustained neutrophil infiltration in the central nervous system (CNS) that correlates with an increase in spinal cord demyelination. We used single cell RNA sequencing (scRNAseq) and flow cytometry to characterize molecular and cellular changes within the CNS associated with increased demyelination in transgenic mice compared to control animals. These approaches revealed the presence of activated neutrophils as determined by expression of mRNA transcripts associated with neutrophil effector functions, including
,
,
, and
, as well as altered neutrophil morphology and protein expression. Collectively, these findings reveal insight into changes in the profile of neutrophils associated with increased white matter damage in mice persistently infected with a neurotropic coronavirus.
The molecular mechanisms leading to the establishment of immunological memory are inadequately understood, limiting the development of effective vaccines and durable antitumor immune therapies. Here, ...we show that ectopic OCA-B expression is sufficient to improve antiviral memory recall responses, while having minimal effects on primary effector responses. At peak viral response, short-lived effector T cell populations are expanded but show increased
and
expression, while memory precursor effector cells show increased expression of
,
and
on a per-cell basis. Using an OCA-B mCherry reporter mouse line, we observe high OCA-B expression in CD4
central memory T cells. We show that early in viral infection, endogenously elevated OCA-B expression prospectively identifies memory precursor cells with increased survival capability and memory recall potential. Cumulatively, the results demonstrate that OCA-B is both necessary and sufficient to promote CD4 T cell memory in vivo and can be used to prospectively identify memory precursor cells.
Microglia are the primary resident myeloid cells of the brain responsible for maintaining homeostasis and protecting the central nervous system (CNS) from damage and infection. Monocytes and ...monocyte‐derived macrophages arising from the periphery have also been implicated in CNS pathologies, however, distinguishing between different myeloid cell populations in the CNS has been difficult. Here, we set out to develop a reliable histological marker that can assess distinct myeloid cell heterogeneity and functional contributions, particularly in the context of disease and/or neuroinflammation. scRNAseq from brains of mice infected with the neurotropic JHM strain of the mouse hepatitis virus (JHMV), a mouse coronavirus, revealed that Lgals3 is highly upregulated in monocyte and macrophage populations, but not in microglia. Subsequent immunostaining for galectin‐3 (encoded by Lgals3), also referred to as MAC2, highlighted the high expression levels of MAC2 protein in infiltrating myeloid cells in JHMV‐infected and bone marrow (BM) chimeric mice, in stark contrast to microglia, which expressed little to no staining in these models. Expression of MAC2 was found even 6–10 months following BM‐derived cell infiltration into the CNS. We also demonstrate that MAC2 is not a specific label for plaque‐associated microglia in the 5xFAD mouse model, but only appears in a distinct subset of these cells in the presence of JHMV infection or during aging. Our data suggest that MAC2 can serve as a reliable and long‐lasting histological marker for monocyte/macrophages in the brain, identifying an accessible approach to distinguishing resident microglia from infiltrating cells in the CNS under certain conditions.
Main Points
Lgals3 is upregulated in monocyte/macrophage populations.
Galectin‐3/MAC2 labels bone marrow‐derived monocytes 6–10 months following cell infiltration into the CNS, but not microglia.
MAC2 is not a specific marker for plaque‐associated microglia.
Numerous clinical studies have established the debilitating neurocognitive side effects of chemotherapy in the treatment of breast cancer, often referred as chemobrain. We hypothesize that cognitive ...impairments are associated with elevated microglial inflammation in the brain. Thus, either elimination of microglia or restoration of microglial function could ameliorate cognitive dysfunction. Using a rodent model of chronic Adriamycin (ADR) treatment, a commonly used breast cancer chemotherapy, we evaluated two strategies to ameliorate chemobrain: 1) microglia depletion using the colony stimulating factor-1 receptor (CSF1R) inhibitor PLX5622 and 2) human induced pluripotent stem cell-derived microglia (iMG)-derived extracellular vesicle (EV) treatment. In strategy 1 mice received ADR once weekly for 4 weeks and were then administered CSF1R inhibitor (PLX5622) starting 72 h post-ADR treatment. ADR-treated animals given a normal diet exhibited significant behavioral deficits and increased microglial activation 4-6 weeks later. PLX5622-treated mice exhibited no ADR-related cognitive deficits and near complete depletion of IBA-1 and CD68
microglia in the brain. Cytokine and RNA sequencing analysis for inflammation pathways validated these findings. In strategy 2, 1 week after the last ADR treatment, mice received retro-orbital vein injections of iMG-EV (once weekly for 4 weeks) and 1 week later, mice underwent behavior testing. ADR-treated mice receiving EV showed nearly complete restoration of cognitive function and significant reductions in microglial activation as compared to untreated ADR mice. Our data demonstrate that ADR treatment elevates CNS inflammation that is linked to cognitive dysfunction and that attenuation of neuroinflammation reverses the adverse neurocognitive effects of chemotherapy.
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