Single-cell transcriptomics of human traumatic brain injury reveals activation of endogenous retroviruses in oligodendroglia, 2023, Garza et al.

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Single-cell transcriptomics of human traumatic brain injury reveals activation of endogenous retroviruses in oligodendroglia
Raquel Garza; Yogita Sharma; Diahann A.M. Atacho; Arun Thiruvalluvan; Sami Abu Hamdeh; Marie E. Jönsson; Vivien Horvath; Anita Adami; Martin Ingelsson; Patric Jern; Molly Gale Hammell; Elisabet Englund; Agnete Kirkeby; Johan Jakobsson; Niklas Marklund

Traumatic brain injury (TBI) is a leading cause of chronic brain impairment and results in a robust, but poorly understood, neuroinflammatory response that contributes to the long-term pathology. We used single-nuclei RNA sequencing (snRNA-seq) to study transcriptomic changes in different cell populations in human brain tissue obtained acutely after severe, life-threatening TBI.

This revealed a unique transcriptional response in oligodendrocyte precursors and mature oligodendrocytes, including the activation of a robust innate immune response, indicating an important role for oligodendroglia in the initiation of neuroinflammation. The activation of an innate immune response correlated with transcriptional upregulation of endogenous retroviruses in oligodendroglia. This observation was causally linked in vitro using human glial progenitors, implicating these ancient viral sequences in human neuroinflammation.

In summary, this work provides insight into the initiating events of the neuroinflammatory response in TBI, which has therapeutic implications.


Link | PDF (Cell Reports)

 

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To investigate cell-type-specific transcriptional responses after severe, acute TBI, we performed snRNA-seq from fresh-frozen human brain tissue. We recruited 12 patients with severe TBI, defined as having a post-resuscitation Glasgow Coma Scale score ≤8. [...] The age (mean ± standard deviation) of the patients (10 males, 2 females) was 49.5 ± 18.2 years. In these patients, decompressive surgery was a life-saving measure to remove injured and swollen space-occupying brain tissue causing marked mass effect or increased intracranial pressure (ICP) refractory to conservative, medical neurointensive care treatment. The injured and contused brain regions (typically the injured part of a temporal or frontal lobe) were surgically removed between 4 h and 8 days after injury. As control tissue, we used five fresh-frozen post-mortem samples from the frontal and temporal lobes obtained from three non-neurological deaths of patients aged 69, 75, and 87 years.

There is no perfect control material to obtain for the study of human TBI tissue. We chose to use post-mortem tissue obtained from acute, non-neurological deaths from our clinic. These samples have been handled in a similar way to the TBI samples, with the exception that they are coming from deceased individuals. Aware of these limitations, we decided to look only at transcriptional alterations with robust differences in magnitude. For example, many of the genes related to an IFN response are completely silent in oligodendrocytes and OPCs (such as STAT1 and STAT2) and are then robustly transcriptionally activated in TBI tissue. By using strict thresholds, we limit the possibilities to draw wrong conclusions due to differences in tissue origin or tissue quality.

 

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Particularly in mature oligodendrocytes, we also found the activation of terms related to a response to interferon-gamma and cytokine stimulus showing clear upregulation of interferon regulatory factor IRF1

When we performed a similar analysis on microglia and astrocytes, we found very limited evidence of an innate or interferon response or the upregulation of MHC molecules. The induction of immune genes in oligodendroglia was robust between individuals, with a trend for stronger induction of immune genes in samples collected a short time after injury (up to 4 h)

Taken together, these results suggest that oligodendroglia undergo a unique transcriptional response following TBI, which activates an interferon response and turns on MHC-related genes—thereby transforming to an immune-like cell state.

The transcriptional response in OPCs and mature oligodendrocytes after TBI is reminiscent to that which occurs after viral infection. In this respect, the transcriptional activation of ERVs has been linked to an interferon response. The aberrant expression of ERVs results in the formation of double-stranded RNAs, reverse-transcribed DNA molecules, and ERV-derived peptides that induce a ‘‘viral mimicry,’’ where cells respond as though infected and this triggers or boosts inflammation.

we found that several ERV (long terminal repeat [LTR]) subfamilies were transcriptionally activated in oligodendrocytes and OPCs. This response was especially noticeable in ERV subfamilies such as HERV-K [...] HERV-W[...], and HERV-H [...], which are evolutionary young ERVs found specifically in primates. We found no transcriptional activation of other families of TEs such as LINE-1s and no evidence for the activation of ERVs or other TEs in other cell types, except for microglia, which also displayed some evidence of ERV activation

Notably, the transcriptional activation of some of these ERVs resulted in a readthrough transcript extending into the nearby genome

 

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The induction of an interferon response has been associated with the transcriptional activation of ERVs, which is thought to play a part in boosting the interferon response.

When investigating changes in gene expression profile upon IFNg stimulation of hGPCs, we found a distinct and robust transcriptional response. Genes linked to IFN signaling and innate immune activation were highly upregulated in the IFNg-treated hGPCs. We found a clear correlation between the upregulated genes in IFNg-treated hGPCs and those observed to be activated in the TBI samples

hGPC is human glial progenitor cell

 

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Neuroinflammation is a hallmark of acute and chronic neurodegenerative states, including TBI, and is therefore a promising route for urgently needed disease-modifying therapies. However, information on how the neuroinflammatory response starts and then transforms to a chronic state is scarce, and the molecular events needed to initiate and maintain this process, and in which cell types, have not been established.

In this study, we used snRNA-seq to perform a detailed analysis of human tissue samples obtained in conjunction with acute surgery for TBI. Our results provide two main insights into the start of a human neuroinflammatory response. First, we found that OPCs and oligodendrocytes may play an unanticipated key role in this process by adopting an immune-like cell state, including evidence for an IFN response. Secondly, we found that the activation of an IFN response in OPCs and oligodendrocytes is mechanistically linked to the transcriptional activation of ERVs.

Neuronal cell death is common in acute, severe TBI due to hemorrhages, increased ICP, and energy metabolic failure. In addition, there is a distinct inflammatory response, associated with white matter abnormalities, that persists from the acute post-injury phase for many years post-injury. Prior studies have established that there are numerous contributors to the neuroinflammatory response after TBI, including microglial activation, infiltration of systemic immune cells, and the subsequent release of proinflammatory cytokines.

Our observations confirm that microglia are likely important players in this process. We observe an increased number of microglia after TBI that are coupled to cell proliferation. However, our results indicate that oligodendroglia also seem to play an important role in the initiation of this process. Oligodendroglia are vulnerable to the TBI induced neuroinflammation as well as to excitotoxicity and reactive oxygen species formation

Our results suggest that, following TBI, oligodendroglia adopt a different cell state characterized by the expression of immune genes. These results are reminiscent of what has been observed in multiple sclerosis, where OPCs and oligodendrocytes initiate a similar transcriptional program.

Almost 10% of the human genome is made up of ERVs, a consequence of their colonization of our germline throughout evolution. In adult tissues, including the brain, ERVs are normally transcriptionally silenced via epigenetic mechanisms, including DNA and histone methylation. However, there is emerging evidence, both from animal models and the analysis of human material, indicating that ERVs can be transcriptionally activated in the diseased brain and that this correlates with neuroinflammation. ERV expression has been found to be elevated in the cerebrospinal fluid and in post-mortem brain biopsies from patients with multiple sclerosis, amyotrophic lateral sclerosis, and Alzheimer’s or Parkinson’s disease.

Our results now provide direct evidence that ERV proviruses are transcriptionally activated at the start of human neuroinflammation.

 

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One of the references is Regulatory evolution of innate immunity through co-option of endogenous retroviruses (2016, Science) which concluded —

Together, our findings reveal IFN-inducible enhancers introduced and amplified by ERVs in many mammalian genomes. On occasion, these elements have been co-opted to regulate host genes encoding immunity factors. Although we have shown that ERVs play a functional role regulating innate immune pathways in human HeLa cells, further studies will be necessary to extend our findings to primary hematopoietic cells and other species such as mouse.

We speculate that the prevalence of IFN-inducible enhancers in the LTRs of these ancient retroviruses is not coincidental, but may reflect former viral adaptations to exploit immune signaling pathways promoting viral transcription and replication. Indeed, several extant viruses, including HIV, possess IFN-inducible cis-regulatory elements.

It would be ironic if viral molecular adaptations had been evolutionarily recycled to fuel innovation and turnover of the host immune repertoire. Regardless of how these sequences originated, our study illuminates how selfish genetic elements have contributed raw material that has been repurposed for cellular innovation.