Neurovascular injury with complement activation and inflammation in COVID-19, 2022, Lee ... Nath et al

[SNT Gatchaman]

Neurovascular injury with complement activation and inflammation in COVID-19
Myoung-Hwa Lee, Daniel P. Perl, Joseph Steiner, Nicholas Pasternack, Wenxue Li, Dragan Maric, Farinaz Safavi, Iren Horkayne-Szakaly, Robert Jones, Michelle N. Stram, Joel T. Moncur, Marco Hefti, Rebecca D. Folkerth, Avindra Nath

The underlying mechanisms by which severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to acute and long-term neurological manifestations remains obscure. We aimed to characterize the neuropathological changes in patients with coronavirus disease 2019 and determine the underlying pathophysiological mechanisms.

In this autopsy study of the brain, we characterized the vascular pathology, the neuroinflammatory changes and cellular and humoral immune responses by immunohistochemistry.

All patients died during the first wave of the pandemic from March to July 2020. All patients were adults who died after a short duration of the infection, some had died suddenly with minimal respiratory involvement. Infection with SARS-CoV-2 was confirmed on ante-mortem or post-mortem testing. Descriptive analysis of the pathological changes and quantitative analyses of the infiltrates and vascular changes were performed.

All patients had multifocal vascular damage as determined by leakage of serum proteins into the brain parenchyma. This was accompanied by widespread endothelial cell activation. Platelet aggregates and microthrombi were found adherent to the endothelial cells along vascular lumina. Immune complexes with activation of the classical complement pathway were found on the endothelial cells and platelets. Perivascular infiltrates consisted of predominantly macrophages and some CD8+ T cells. Only rare CD4+ T cells and CD20+ B cells were present. Astrogliosis was also prominent in the perivascular regions. Microglial nodules were predominant in the hindbrain, which were associated with focal neuronal loss and neuronophagia.

Antibody-mediated cytotoxicity directed against the endothelial cells is the most likely initiating event that leads to vascular leakage, platelet aggregation, neuroinflammation and neuronal injury. Therapeutic modalities directed against immune complexes should be considered.

Link to DOI | PDF

 

[SNT Gatchaman]

9 patients (7 males, 2 females, 24–73 years) died during the first wave. Co-morbidities included diabetes, hypertension and substance use disorder. 5 patients had died suddenly, 4 at home. The remaining patients died within days to weeks after onset of symptoms.

The control group consisted of 9 males and 1 female, 43–74 years. They were diagnosed with pulmonary infections, systemic infections, hypertension, bipolar disorder and substance use disorder. Doesn't look like diabetes in control group.

 

[SNT Gatchaman]

Some quotes (slightly edited) —

Fibrinogen is a large abundant protein in blood but is unable to cross the intact blood–brain barrier. Immunostaining for this protein showed areas of multifocal staining throughout the brain. Strong diffuse immunostaining was present in the perivascular regions with a gradient of weaker staining at increasing distance from the blood vessels.

Fibrinogen leakage was evident in all COVID-19 cases. The non-COVID cases showed none or only minimal weak immunostaining (P = 0.0011).

Fibrinogen deposition was positively correlated with the amount of CD68+ cells (P < 0.0001), TMEM119+ cells (P = 0.0034), CD3+ cells (P < 0.0001) and CD8+ cells (P = 0.0031).

All techniques failed to detect any virus in the brain, including regions where there were obvious signs of inflammation.

Using post-mortem high-resolution MRI, we previously found that most patients had widespread multifocal microvascular disease which corelated with vascular leakage and injury.

The loss of vascular integrity was evident by the presence of several large proteins in the perivascular regions that normally do not cross the blood–brain barrier. These included fibrinogen, C1q, IgG and IgM. Fibrinogen was present in high concentrations around the blood vessels with a gradual decrease in concentrations at greater distances from the vasculature. This suggests a leaky blood–brain barrier. All markers of vascular injury were more common in the hindbrain. Similar deposition of fibrinogen in the lumen of the blood vessels has been described in active inflammatory lesions of multiple sclerosis. But even in these patients there was no perivascular leakage of fibrinogen as described in our patients.

the mechanism of increased PECAM-1 levels in the endothelial cells remains unclear. However, this molecule can serve as an adhesion molecule for platelets and platelet aggregates that were adhered to the endothelial cells was a prominent observation in this study. The platelets were activated and, in some instances, caused occlusion of the small blood vessels.

Deposition of complement cascade and immunoglobulins suggests an immune-mediated injury to the endothelial cells. The antigen against which this immune response is targeted remains unknown. Possibly, the antibodies are directed against an antigen on the endothelial cells, e.g. anti-idiotypic antibodies against the spike protein would bind to the ACE-2 receptor on endothelial cells. Alternatively, immune complexes formed by the antibodies and spike protein that may bind to the ACE-2 receptor on the endothelial cells. The spike protein has been shown to compromise the blood–brain barrier in vitro.

Thus, while the damage to endothelial cells may not be unique to the CNS, the consequences of the breakdown of the blood–brain barrier are unique to the CNS.

Since several of the patients in our series died suddenly with very minor lung involvement, we believe that had these patients survived they would probably have progressed to develop long-COVID. Hence the pathological findings here are relevant to this population as well.

 

[SNT Gatchaman]

Conclusions

Injury to the microvasculature by immune complexes with complement activation is the key central event that results in breakdown of the blood–brain barrier, microthromboses, perivascular inflammation and neuronal injury. We postulate that these events are central to the development of the neurological manifestations seen in acute COVID-19 and possibly in long-COVID. Importantly, these studies suggest that therapeutic approaches targeted against the development of immune complexes should be considered.

 

[Hutan]

Thanks for posting this @SNTGatchaman

I acknowledge the important gift the people who were the subject of this study and their families made to science. I think we need more brain autopsy studies like this, especially of people with Long Covid and ME/CFS, (and of women with these conditions - there were only 2 in this study). I hope patient charities will work towards making this possible.

For those like me who missed it on the first pass, this is a NIH/NINDS study, with the senior author being Nath. It gives us more of an idea of what Nath is thinking about Long Covid, and reinforces his recent comments about the need to investigate

Therapeutic modalities directed against immune complexes

 

[LarsSG]

So, naively, if they think something (antibodies or antibodies + spike) is binding to ACE-2 on endothelial cells, that seems like something you could test in vitro with acute Covid or Long Covid patient plasma.

 

[Hutan]

Additionally, many patients complain of persistent symptoms of cognitive difficulties, extreme fatigue, sleep and autonomic dysfunc- tion lasting several months after recovery from the acute infection suggesting a post-viral CNS syndrome that has been termed long-COVID or post-acute sequelae of SARS-CoV-2 infection, which resembles myalgic encephalomyelitis/chronic fatigue syndrome.

There's acknowledgement of the similarities of long-covid and ME/CFS.

Patients in this series represent a subset of patients who showed micro- vascular abnormalities on post-mortem MRI.

So, these patients were selected specifically because they showed micro-vascular abnormalities - which amplifies the concern that @SNT Gatchaman expressed (that the co-morbidities of these people might explain some or all of the brain pathology).

Fibrinogen leakage was evident in all COVID-19 cases

Fibrinogen definitely shouldn't be getting into the brain - if it is, the Blood Brain Barrier is compromised. It was getting in, in all of the cases; the controls had virtually none.




It looks as though fibrinogen in the brain is a bit like a bull in a china shop:
Fibrinogen in neurological diseases: mechanisms, imaging and therapeutics 2018

The blood coagulation protein fibrinogen is deposited in the brain in a wide range of neurological diseases and traumatic injuries with blood–brain barrier (BBB) disruption. Recent research has uncovered pleiotropic roles for fibrinogen in the activation of CNS inflammation, induction of scar formation in the brain, promotion of cognitive decline and inhibition of repair. Such diverse roles are possible in part because of the unique structure of fibrinogen, which contains multiple binding sites for cellular receptors and proteins expressed in the nervous system. The cellular and molecular mechanisms underlying the actions of fibrinogen are beginning to be elucidated, providing insight into its involvement in neurological diseases, such as multiple sclerosis, Alzheimer disease and traumatic CNS injury. Selective drug targeting to suppress the damaging functions of fibrinogen in the nervous system without affecting its beneficial effects in haemostasis opens a new fibrinogen therapeutics pipeline for neurological disease.

It's not just fibrinogen that was found to be leaking in -

plasma proteins from blood vessels into the brain parenchyma was also confirmed by IgM staining (Supplementary Fig. 6). The pattern is similar to that of fibrinogen leakage, although the fibrinogen leak- age was much more prominent, probably because its molecular mass is smaller and hence can diffuse further into the parenchyma.

 

[Hutan]

I'm jumping the gun here, because we don't yet know if this fibrinogen leakage is happening in Long Covid (or ME/CFS generally). But if fibrinogen is found in the brains of people with Long Covid, then this comment from the 2018 paper I quoted above is interesting:

Fibrinogen is not expressed in brain and must cross a disrupted BBB from the circulation to be found in the nervous system. Fibrinogen is highly polymorphic with over 300 single-nucleotide polymorphisms. Metagenome-wide association studies are needed to test for a genetic link with disease.

Perhaps any sort of fibrinogen can breach the BBB given the right circumstances. But perhaps some versions of fibrinogen can get into the brain easier than others. Maybe this fibrinogen leakage issue is something for the DecodeME team to watch @Andy, @Simon M.

 

[Hoopoe]

When endothelial cells are activated, they express proteins called adhesion molecules that cause platelets to stick together.

Adhesion molecules are cell surface proteins that mediate the interaction between cells, or between cells and the extracellular matrix (ECM). There are four families of adhesion molecules: immunoglobulin-like adhesion molecules, integrins, cadherins and selectins.

ME/CFS was associated with increased expression of cadherins in a proteomics study by Hanson. Would cadherins fit with the process that the paper is suggestng is occurring?

 

[Andy]

Maybe this fibrinogen leakage issue is something for the DecodeME team to watch @Andy, @Simon M.

Watch in what way? The idea behind a GWAS is that it is hypothesis free and reports on what has been found by the analysis, rather than going searching for results in an attempt to support a particular idea.

 

[Hutan]

Watch in what way? The idea behind a GWAS is that it is hypothesis free and reports on what has been found by the analysis, rather than going searching for results in an attempt to support a particular idea.

At one point Simon was asking for ideas about particular questions that the DecodeME data might be applied to. With so much variation around the composition of fibrinogen, it just seemed like an interesting question to look at, if it turns out that fibrinogen breaching the blood brain barrier is characteristic of Long Covid/ME/CFS. The interaction of genes is complex e.g.

single nucleotide polymorphisms (SNPs) and haplotypes of the fibrinogen gene-cluster (fibrinogen chains alpha [FGA], beta [FGB], and gamma [FGG])

so, maybe it's useful to have some ideas of things to dig into, as well as seeing what obvious things pop out? I don't know, clearly I'm not an expert, feel free to ignore the suggestion.

 

[Andy]

At one point Simon was asking for ideas about particular questions that the DecodeME data might be applied to.

Unless I've forgotten something I think Simon was asking for particular questions to put into the optional additional DecodeME questionnaire that will be offered to participants to complete.

 

[Hutan]

Unless I've forgotten something I think Simon was asking for particular questions to put into the optional additional DecodeME questionnaire that will be offered to participants to complete.

Yes, my bad.

 

[SNT Gatchaman]

The review paper @Hutan referenced above probably warrants its own thread. There's a lot in it — fibrinogen has unusual characteristics that are important. The relationship between fibrinogen and the innate immune system and neuroinflammation looks potentially very relevant. (There is also reference to rheumatoid arthritis and other peripheral inflammatory diseases.)

The inflammatory functions of fibrinogen mediated primarily via binding to the CD11b/CD18 integrin receptor (also known as αMβ2 or complement receptor 3) in microglia and macrophages contribute to neurological deficits in neuroinflammatory disease. At the same time, fibrinogen binding to proteins such as latent transforming growth factor-β (TGFβ) or Aβ contributes to brain trauma and AD, respectively. Thus, fibrinogen is at the nexus of crosstalk between neurons and glia, the vasculature and immune cells and is a key molecular integrator of neurological, cerebrovascular and immune mechanisms of CNS injury and disease

This thread's paper is discussing evidence for antibody-mediated BBB disruption. In part, the 2018 review article discusses the (substantial) links between fibrinogen and amyloid-β. I would also wonder whether the process could involve amyloid-form fibrinogen directly. Perhaps it might be more inflammogenic and could promote an endotheliitis, thereby disrupting the blood brain-barrier, which then allows (amyloid-) fibrinogen etc into the perivascular parenchyma. Normal fibrinogen promotes Aβ but could amyloid form fibrinogen accelerate this?

Interestingly, fibrin can physically interact with Aβ to promote Aβ fibril formation. In turn, Aβ can activate the coagulation factor XII (FXII)-mediated contact pathway to further drive the conversion of fibrinogen into fibrin. Fibrin–Aβ interactions also alter fibrin clot structure and block plasminogen binding to fibrin, which results in degradation-resistant clots and may further enhance fibrin-induced AD pathology.

 

[Midnattsol]

Would cadherins fit with the process that the paper is suggestng is occurring?

Cadherins are found in the blood brain barrier and likely play a role in its integrity, they may also modulate other proteins found in the tight junctions complexes that hold cells together such as claudins:

VE-cadherin and β-catenin as modulators of TJs
In addition to TJs, junctional complexes between endothelial cells include adherens junctions (AJs), constituted by transmembrane proteins VE-cadherin linked to the actin cytoskeleton through catenins (eg: p120-catenin, α- and β-catenin) [107–109]. Interestingly, AJ and TJ complexes functionally interact in brain endothelial cells: indeed, VE-cadherin engagement induces claudin-5 transcription through inhibition of FoxO1 activity (a transcription repressor of claudin-5 gene) and β-catenin sequestration (a stabilizer of FoxO1 activity) in AJ complexes [97], in line with the above-mentioned capacity of β-catenin, downstream of Wnt receptor activation, to control claudin gene expression [96]. These findings clearly place VE-cadherin upstream of claudin-5 in the establishment, maturation and maintenance of endothelial cell-cell junctions.[\QUOTE]
From the open access article "Tight junctions at the blood brain barrier: physiological architecture and disease-associated dysregulation".

 

[Midnattsol]

Not that I think it would have mattered much, but I would have wanted to know the patient's vitamin D status. There are multiple compounds that influence tight junction complexes and thus barrier integrity, in cell culture studies vitamin D has shown protective effects against many of these by stopping the signal cascade that cause the tight junctions to fall apart.

 

[Hoopoe]

Hanson et al did not report which type of cadherin was elevated in ME/CFS, if any. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7931008/

If one type of cadherin stood out from the others, it would hint which type of tissue was involved. VE-cadherin would suggest a vascular problem and whatever other tissues also happen to express the VE type. N-cadherin would suggest a brain problem. There are many types of cadherins. This approach might be a way to find out where in the body things are going wrong.

 

[Midnattsol]

Hanson et al did not report which type of cadherin was elevated in ME/CFS, if any. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7931008/

If one type of cadherin stood out from the others, it would hint which type of tissue was involved. VE-cadherin would suggest a vascular problem and whatever other tissues also happen to express the VE type. N-cadherin would suggest a brain problem. There are many types of cadherins. This approach might be a way to find out where in the body things are going wrong.

The problem with these is that their localisation, not just the amount of them, can influence the integrity of the barrier are part of. So we might have the same amount as healthy controls but if the localisation is changed by something the barrier might still be impaired.

 

[dreampop]

I remember this from when they started because of how atypical the cohort was, and therefore a stretch to connect it to me/cfs. This is@Forbin 's quoting Nath during an NIH call.

Dr. Avi Nath: Thank you Dr. Koroshetz. So I'm going to tell you three things. One is I'll tell you where we stand currently with the ME/CFS study that we were doing. Second thing I want to tell you is what we've been doing with COVID and the third thing I'm going to tell you is the overlap between COVID and ME/CFS and what we're doing about that.

So first of all with the current ME/CFS intramural study that we were conducting, it came to a grinding halt when the COVID outbreak occurred but we took that opportunity to look at the data that we have collected on these patients. And we have a huge amount of data and so what we did was, we formed working groups, several different working groups, one for immunology, one for the virology, one for the physiology that we've done, and so on and so forth. All those people have started looking at the data, analyzing it and when you analyze data it leads sometimes to more questions than answers, which is the way science works, but I think that's perfectly fine. So now we're doing some follow-up experiments to answer those things, trying to make correlations between each of these working groups. So anyhow that's where we stand with that. I think we have some really interesting findings that are coming out of this stuff and so our hope is to get this out as soon as we can.

The second thing is when COVID hit, I mean my area of expertise is infections of the nervous system, so as you can imagine we got very heavily involved and with Dr. Koroshetz' help we were able to send messages around to all over the country to collect brain samples from patients who were dying of COVID and we were fortunate to be able to get some samples because these autopsies had to be done in BSL3 labs. We didn't have a BSL3 functioning lab here in the intramural NIH at that time that we could take our brains from and there were very few that were available around the country and even some places who had it they didn't have PPE to actually do these things. So multiple challenges but we overcame all of them.

And what we did was we accessed the brains and we studied the pathology. We found that actually there is a fair bit of inflammation in the brain, there was damage to the blood vessels in the brain, and these were very unique individuals because they were individuals we got from the New York medical examiner's office. Some of them had died in bed, or in a subway, and so they did not have much respiratory symptoms. They were not critically ill individuals but they still had pathology in their brain, so my suspicion is that had these individuals survived they would have had these long-haul COVID symptoms for sure. That gives us an opportunity to understand the brain pathology of long-haul COVID patients.

And I think that brings me to the relevance for ME/CFS because we suspect that there's overlap between the two syndromes and so what we learned from these patients is applicable to ME/CFS. What we've done now is, we are very eager to bring in patients with so-called long-haul COVID who look exactly like ME/CFS, they meet all the criteria for ME/CFS. We bring them to NIH, study them exactly the same way as we've done for the ME/CFS patients and try to see if we can determine what the similarities and differences might be. For that there's no intramural funding currently available, so we wrote a grant like everybody else and we've submitted it and if the grant gets funded we plan to bring in about 50 individuals with ME/CFS-like symptoms and another 50 who had COVID and got better completely, so we can compare the two. So that's where we stand. We're waiting to see if the funds arrive, we'll initiate the studies. So I'll stop over here.

https://www.nih.gov/mecfs/nih-me-cfs-advocacy-call-march-30-2021