Proximal immune-epithelial progenitor interactions drive chronic tissue sequelae post COVID-19, 2023, Harish Narasimhan et al

[Mij]

Abstract
The long-term health effects of SARS-CoV-2, termed Post-Acute Sequelae of COVID-19 (PASC), are quickly evolving into a major public health concern, but the underlying cellular and molecular etiology remain poorly defined. There is growing evidence that PASC is linked to abnormal immune responses and/or poor organ recovery post-infection. However, the exact processes linking non-resolving inflammation, impaired tissue repair, and PASC are still unclear.

In this report, we utilized a cohort of respiratory PASC patients with viral infection-mediated pulmonary fibrosis and a clinically relevant mouse model of post-viral lung sequelae to investigate the pathophysiology of respiratory PASC. Using a combination of imaging and spatial transcriptomics, we identified dysregulated proximal interactions between immune cells and epithelial progenitors unique to respiratory PASC but not acute COVID-19 or idiopathic pulmonary fibrosis (IPF). Specifically, we found a central role for lung-resident CD8+ T cell-macrophage interactions in maintaining Krt8hi transitional and ectopic Krt5+ basal cell progenitors, and the development of fibrotic sequelae after acute viral pneumonia.

Mechanistically, CD8+ T cell derived IFN-γ and TNF stimulated lung macrophages to chronically release IL-1β, resulting in the abnormal accumulation of dysplastic epithelial progenitors in fibrotic areas. Notably, therapeutic neutralization of IFN-γ and TNF, or IL-1β after the resolution of acute infection resulted in markedly improved alveolar regeneration and restoration of pulmonary function.

Together, our findings implicate a dysregulated immune-epithelial progenitor niche in driving respiratory PASC and identify potential therapeutic targets to dampen chronic pulmonary sequelae post respiratory viral infections including SARS-CoV-2.

https://www.biorxiv.org/content/10.1101/2023.09.13.557622v1

 

[Hutan]

Mostly University of Virginia work

Given the extensive damage to the respiratory tract during primary infection, the lungs are particularly susceptible to sustained impairments including dyspnea, compromised lung function, and radiological abnormalities which persist up to 2 years post infection in contrast to the majority of extrapulmonary sequelae (1, 2, 4). Some individuals also develop a non-resolving fibroproliferative response – PASC pulmonary fibrosis (PASC-PF) and typically require persistent oxygen supplementation and eventual lung transplantation (4-8).

This paper is about 'respiratory PASC'. They appear to be suggesting that the compromise to lung function can last a long time whereas PASC impacts elsewhere do not.

Despite the paper being focused on lungs, maybe it can tell us something about Long covid more generally, maybe it can't.

It has long been established that, type 2 alveolar epithelial (AT2) cells are the facultative stem cell of the lungs that undergo self-renewal and differentiate into type 1 alveolar (AT1) cells to replenish the denuded epithelial niche after alveolar injury (17-19). Recent work has further delineated the AT2 to AT1 trans-differentiation process and identified a transitional state characterized by high expression of cytokeratin 8 (Krt8hi) (18-22). Severe alveolar damage also induces the recruitment of Krt5+ basal cell progenitors to the distal lung, which persist in an undifferentiated state or differentiate into upper airway cell fates, causing alveolar “bronchiolization” (23-27). The accumulation of undifferentiated Krt8hi transitional cells and/or the emergence of ectopic Krt5+ pods are hallmarks of lung injury, and their persistence have pathologically been associated with chronic diseases such as lung fibrosis (5, 28-33).

So, Type 2 epithelial cells in the furthest reaches of the lungs differentiate into new epithelial cells when there is injury. Differentiating cells express high levels of cytokeratin 8 (Krt8hi).

If there is severe damage of the alveoli (distal lung is just the part of the lung at the end of the branching tubes, where the alveoli are), then Krt5+ basal cells are recruited to the distal lung. Some of these just stay there and some differentiate into the airway tubes (bronchiolisation). Essentially, the repaired tissue gets messed up and doesn't work properly. They talk about these 'ectopic' pods of the Krt5+ cells - I'm imagining it's a bit like endometriosis, where cells of one type migrate and settle in places where they shouldn't be. This 'cells in the wrong place' seems to result in lung fibrosis.

In this study, they did a lot of things, including looking at the lungs that had been removed from people who needed a lung transplant as a result of a Covid-19 infection.

Lung histology was notable for extensive immune cell infiltration and collagen deposition in the alveolar epithelium (Fig. 1a). Consistent with the observed fibrotic sequelae, we found reduced levels of AT1 and AT2 cells in PASC-PF lungs compared to controls, suggesting a persistent defect in alveolar regeneration (Fig. 1b,c) (39). We also observed chronic persistence of ectopic Krt5+ basal cells and Krt8hi transitional cells in PASC-PF lungs compared to controls, which is concordant with recent reports

PASC-PF lungs also harbored widespread expression of alpha smooth muscle actin (αSMA), indicative of myofibroblast activity in addition to pockets of Krt5- Krt17+ aberrant basaloid cells previously found in IPF lungs

Therefore, PASC-PF is characterized by the sustained loss of functional alveolar epithelial cells, and the persistence of dysplastic Krt5+ pods and Krt8hi transitional cells, which is histologically akin to other fibrotic lung diseases such as idiopathic pulmonary fibrosis (IPF)

They seem to be suggesting that the normal cell replacement process in the alveoli wasn't happening so well, with more cells in the stage between AT1 and AT2 cells which don't function so well and also more of those upper airway cells down further in the lungs where they shouldn't be. I don't yet know what they mean by the 'expression of alpha smooth muscle actin, indicative of myofibrobalst activity' that they found in the lungs, along with pockets of aberrant basaloid cells.

 

[Hutan]

Notably, this correlation between CD8+ T cells, and Krt8hi and Krt5+ dysplastic areas was unique to PASC-PF lungs but not seen in lungs from control, acute COVID-19 or IPF conditions (Fig. 1k-m, Extended data Fig. 1d-g,i,j). Collectively, these data indicate that the spatiotemporal colocalization of CD8+ T cells and areas of dysplastic repair is a unique feature of post-viral pulmonary fibrosis and supports immune-epithelial progenitor interactions potentially contributing to the observed defects in alveolar regeneration and chronic pulmonary sequelae.

They seem to be suggesting that increased levels of CD8+ T cells and areas where the tissue repair wasn't happening properly was a feature of post-viral pulmonary fibrosis.

The researchers tried to recreate this situation in mice by exposing them to a SARS-CoV-2 but they couldn't. However, when they infected mice with a flu virus, they could. There was immune cell infiltration and collagen deposition and persistent lung dysfunction.

Moreover, lungs from aged mice harbored significantly larger Krt5+ and Krt8hi areas of dysplastic repair as well as higher levels of CD8+ T cells post influenza viral pneumonia, similar to human PASC-PF lungs

"Exuberant tissue CD8+ T cell responses impair alveolar regeneration and promote dysplastic lung repair following viral pneumonia"
(I'm wondering if there is similar damage to the micro-vascular system in muscles and that some dysfunctional healing might result in in the cardiovascular dysfunction that has been reported by people like Systrom, where there is inadequate oxygen extraction. Of course, there is the issue of how people who seem very mildly affected by an infection would sustain such damage. Anyway, onwards.)

Furthermore, the association between CD8+ T cells and Krt8hi areas was seen only at post-acute timepoints and strengthened over time, recapitulating features of human lungs after severe SARS-CoV-2 infection and suggesting these immune-epithelial progenitor interactions are primarily a feature of chronic sequelae of viral infections

Importantly, Krt5+ and Krt8hi areas were significantly reduced by depletion of CD8+ T cells (Fig. 3i,j), suggesting that CD8+ T cells are essential for the maintenance of dysplastic repair areas after recovery from acute disease. Interestingly, AT1 and AT2 cells were markedly increased, and the alveolar architecture was restored after CD8+ T- cell depletion

Depleting the CD8+ T cells (after they had done their job in the acute disease) seemed to stop the faulty tissue repair, but primarily in aged mice as opposed to the young mice.

We found that the resolution of areas of dysplastic repair only occurs upon depletion of pulmonary CD8+ T cells but not circulating CD8+ T cells (Extended data Fig. 3j,k) (34), suggesting that lung-resident CD8+ T cells are required for the maintenance of dysplastic lung repair.

Taken together, our results strongly implicate the persistent tissue CD8+ T cell responses in the development of chronic pulmonary sequelae post viral pneumonia in aged animals.

They say they achieved the removal of circulating T cells with a low dose of an antiCD8 treatment, and the removal of lung-resident T cells with a high dose of the antiCD8 treatment. What I take away from that, is that you could apply a treatment that removes T cells from the blood, but that they could still be hanging out in the tissues, potentially causing problems. So, if you are testing an anti-T cell treatment and you think the T cells are causing a problem in tissues, then you need to test for the removal of the T cells from the tissue, not just the blood.

 

[Hutan]

We further performed an agnostic evaluation of signaling pathways differentially regulated within the healthy alveolar epithelium compared to Krt5+ and Krt8hi-rich areas of dysplastic repair using Gene Set Enrichment Analysis (GSEA) (Fig. 4c). Several pathways associated with inflammatory responses were highly active within areas of dysplastic repair, whereas growth factor responses and restoration of the vasculature were prominently observed in the healthy alveolar epithelium.

Analysis of gene expression seemed to point to highly active inflammatory responses in the areas of dysfunctional repair. There were a whole range of immune cells active in the areas of dysfunctional repair, including macrophages.

To further characterize the immune-epithelial progenitor niche within areas of dysplastic repair, we performed a correlation analysis and found that CD8+ T cells and monocyte-derived macrophages were physically clustered around Krt5+ and Krt8hi areas of dysplastic repair, and excluded from areas enriched with alveolar macrophages and mature alveolar epithelial cells

Together, our data revealed a conserved finding in both mouse and human post-viral lungs, where CD8+ T cells are present in fibrotic regions and in proximity to fibroproliferative mediators such as monocyte-derived macrophages, and Krt5+ and Krt8hi dysplastic epithelial progenitors, representing a pathological niche after acute respiratory viral infections.

A CD8+ T cell-macrophage axis induces IL-1β release to arrest AT2 trans-differentiation in the transitional cell state

We postulated the interactions between CD8+ T cells, macrophages, and epithelial progenitors within this pathological niche generated molecular cues to create a profibrotic microenvironment.

IL-1β has been shown to promote the expansion of transitional Krt8+ cells upon bleomycin injury (21, 50). Therefore, we investigated if IL-1β mediated the development of post-viral pulmonary fibrosis. First, we found that CD64+ macrophages were major producers of pro-IL-1β compared to CD64- cells in influenza-infected aged lungs (Fig. 5b and Extended data Fig. 5b). Since mature IL-1β release from cells requires caspase-1 mediated pro-IL-1β cleavage (51), we examined caspase-1 activity in lung macrophages after CD8+ T cell depletion. Strikingly, CD8+ T cell depletion significantly reduced caspase-1 activity in lung macrophages (Fig. 5c). Moreover, inflammasome gene signatures were attenuated with αCD8 treatment (Extended data Fig. 5c), supporting the notion that CD8+ T cells persisting in the lungs post infection promote chronic inflammasome activation and IL-1β release by macrophages.

They are proposing that the CD8+ T cells persisting in tissue makes macrophages release IL-1B, which promotes the expansion of the cells that cause fibrosis.

They did a number of studies showing that the CD8+ T cells are having these effects by releasing IFN-y and TNF. Knocking out the T cells or the IFN-y and TNF stopped the pathology.

The synergistic activity between macrophages and CD8+ T cells to produce IL-1β in vitro was not observed when isolated from naïve mice, suggesting that prior infection is necessary to prime the cells (Extended data Fig. 5f). Moreover, IL-1β released into the supernatant was significantly reduced upon treatment with IFN-γ and TNF neutralizing Ab in the coculture system, confirming the role of IFN-γ and TNF in promoting IL-1β release by macrophages

They found that the relationship between the macrophages and the CD8+ T cells required an infection to prime the cells.

 

[Hutan]

I haven't looked at the figures, but my impression is that these researchers did a lot of work, checking their assumptions in several ways. It seems like good work that should move the understanding of post-infectious lung fibrosis forward. I think it might possibly have some relevance to ME/CFS too.

Since depletion of CD8+ T cells is not a clinically feasible treatment strategy, we explored the therapeutic efficacy of neutralizing the cytokines that are effectors of the profibrotic CD8+ T-cell. As expected, blocking IFN-γ and TNF activity in aged influenza-infected mice ameliorated fibrotic sequelae when compared to isotype controls (Fig. 6a,b). Moreover, IFN-γ and TNF neutralization attenuated Krt5+ and Krt8hi areas of dysplastic repair (Fig. 6c,d),
enhanced alveolar regeneration as evidenced by the increased numbers of AT1 and AT2 cells (Fig. 6c,e). The observed cellular changes also reflected in physiological benefit, with improved lung function following treatment (Fig. 6f).

The researchers say that depletion of CD8+ T cells isn't clinically feasible, so they blocked IFN-y and TNF, and that worked. They say that neutralising the IL-1B worked too.

Thus, these data suggest that neutralization of IFN-γ and TNF, or IL-1β in the post-acute stage of viral infection may serve as viable therapeutic options to augment alveolar regeneration and dampen fibrotic sequelae observed following respiratory viral infections (Extended data Fig. 7). Consistent with the observed improvement in outcomes and a recent study identifying increased BAL IL-1β levels in respiratory PASC (7), we found that circulating IL-1β levels were elevated in individuals exhibiting persistent abnormal pulmonary function compared to those that had fully recovered (Fig. 6n), suggesting that chronic IL-1β activity may impede the restoration of normal lung function after acute SARS-CoV-2 infection.

They found higher levels of IL-1B in people with persistent abnormal pulmonary function. I did check that out - here it is:


I think we've seen variable reports on blood IL-1B in people with ME/CFS. I think if levels were consistently higher, we would know by now. But, that said, the chart does show overlap in the levels with healthy controls

 

[Hutan]

What I like about this idea in terms of it being useful for thinking about ME/CFS is that it is the process of healing following infection-induced injury that causes the harm.
From the discussion:

Excessive infiltration and accumulation of profibrotic monocyte derived macrophages has been reported in the context of severe acute COVID-19, IPF, and PASC (12, 53, 57, 58).
Here, we elucidate a previously unknown role for pulmonary CD8+ T cells in impaired recovery and fibrotic remodeling in PASC-PF but not acute COVID-19 or IPF lungs. This distinction is likely the product of a dysregulated and protracted antiviral response, originally aimed towards the clearance of the virus and virus-infected cells. Following viral infection-mediated alveolar injury, lung resident CD8+ T cells are recruited and maintained at sites of severe damage in order to protect these vulnerable sites in case of reinfection – previously termed as repair associated memory depots (59). These pulmonary CD8+ T cells typically gradually contract with successful alveolar regeneration in individuals that successfully recover from acute COVID-19 (9). However, long-term persistence of CD8+ T cells in human PASC, and aged influenza-infected mice impairs lung recovery post infection and drives the development of fibrotic disease.

The authors mention two drugs that they think could stop the pathological process they have set out:

Given the observed benefits in adults and pediatric patients hospitalized with acute COVID-19, United States Food and Drug Administration has already granted emergency use authorization to the IL-1 receptor antagonist, Anakinra and the JAK- inhibitor, Baricitinib, in acute COVID-19. Our data strongly suggest that these drugs may also serve as promising candidates to treat ongoing respiratory PASC in the clinic.

(Alternatively, they could just tell the people to join a choir and work on their disordered breathing...)

 

[Hutan]

Those references that are given to support the claim that 'excessive infiltration and accumulation of profibrotic monocyte derived macrophages have been reported in ..PASC'?
12. Phetsouphanh C, Darley DR, Wilson DB, Howe A, Munier CML, Patel SK, et al. Immunological dysfunction persists for 8 months following initial mild-to-moderate SARS-CoV-2 infection. Nature Immunology. 2022;23(2):210-6.

53. Joseph IB, Connor HP, Karolina JS, Nikolay SM, Estefani D, Emmy J, et al. Expansion of profibrotic monocyte-derived alveolar macrophages in patients with persistent respiratory symptoms and radiographic abnormalities after COVID-19. bioRxiv. 2023:2023.07.30.551145.

57. Wendisch D, Dietrich O, Mari T, von Stillfried S, Ibarra IL, Mittermaier M, et al. SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis. Cell. 2021;184(26):6243-61.e27.

58. Misharin AV, Morales-Nebreda L, Reyfman PA, Cuda CM, Walter JM, McQuattie-Pimentel AC, et al. Monocyte-derived alveolar macrophages drive lung fibrosis and persist in the lung over the life span. Journal of Experimental Medicine. 2017;214(8):2387-404.

So, mostly very lung focused. I think we have seen similar reports for other parts of the body, both in Long covid and PASC.

 

[Hutan]

On baricitinib for Long Covid:

Wes Ely has applied to study Baricitinib in LC. https://www.youtube.com/watch?v=GYi9NJelm2A

 

[SNT Gatchaman]

On baricitinib for Long Covid

See The JAK/STAT signaling pathway: from bench to clinic (2021, Nature Signal Transduction and Targeted Therapy) for a detailed review paper on the JAK/STAT pathway, which includes mechanism of baricitinib, other JAK inhibitors and STAT inhibitors.