scRNA-seq reveals persistent aberrant differentiation of nasal epithelium driven by TNFα and TGFβ in post-COVID syndrome, 2025, Reddy et al.

[SNT Gatchaman]

scRNA-seq reveals persistent aberrant differentiation of nasal epithelium driven by TNFα and TGFβ in post-COVID syndrome

Spoiler: Authors

Reddy, K D; Maluje, Y; Ott, F; Saurabh, R; Schaaf, A; Bohnhorst, A; Biedermann, S B; Pierstorf, J; Winkelmann, S; Voß, B; Laudien, M; Bahmer, T; Heyckendorf, J; Brinkmann, F; Schreiber, S; Lieb, W; Jakwerth, C A; Schmidt-Weber, C B; Hansen, G; von Mutius, E; Rabe, K F; Dittrich, A M; Maison, N; Schaub, B; Kopp, M V; Busch, H; Weckmann, M; Fähnrich, A

Post-COVID syndrome (PCS) affects approximately 3-17% of individuals following acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and poses a potential global health burden. While improved assessment strategies are emerging, mechanistic insights and treatment options remain limited.

This study investigates molecular mechanisms underlying PCS using single-cell RNA (scRNA) transcriptomics combined with in vitro validation. scRNA analysis is performed on nasal biopsies from 25 patients with moderate or severe PCS to investigate differential cell types, signalling pathways, and cell-cell communication. Air-liquid interface cultures are used to validate findings, focusing on the TNFα-TGFβ axis.

Severe PCS shows reduced numbers of ciliated cells, increased immune cell infiltration, and heightened inflammatory signaling that drives TGFβ and TNFα upregulation, in the absence of a detectable viral load. These changes trigger epithelial-mesenchymal transition, basal cell expansion and a mis-stratified nasal epithelium. In vitro experiments confirm TGFβ and TNFα as causal cytokines promoting ciliated cell loss and increased basal cell abundance.

These findings indicate a sustained severe PCS is not driven by ongoing viral load but by immune cell activity and chronic cytokine production. Targeting the TNFα-TGFβ axis may mitigate immune-mediated nasal tissue damage and support epithelium restoration, offering a potential therapeutic strategy for PCS.

Web | PDF | Nature Communications | Open Access

 

[V.R.T.]

scRNA-seq reveals persistent aberrant differentiation of nasal epithelium driven by TNFα and TGFβ in post-COVID syndrome

Spoiler: Authors

Reddy, K D; Maluje, Y; Ott, F; Saurabh, R; Schaaf, A; Bohnhorst, A; Biedermann, S B; Pierstorf, J; Winkelmann, S; Voß, B; Laudien, M; Bahmer, T; Heyckendorf, J; Brinkmann, F; Schreiber, S; Lieb, W; Jakwerth, C A; Schmidt-Weber, C B; Hansen, G; von Mutius, E; Rabe, K F; Dittrich, A M; Maison, N; Schaub, B; Kopp, M V; Busch, H; Weckmann, M; Fähnrich, A

Post-COVID syndrome (PCS) affects approximately 3-17% of individuals following acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and poses a potential global health burden. While improved assessment strategies are emerging, mechanistic insights and treatment options remain limited.

This study investigates molecular mechanisms underlying PCS using single-cell RNA (scRNA) transcriptomics combined with in vitro validation. scRNA analysis is performed on nasal biopsies from 25 patients with moderate or severe PCS to investigate differential cell types, signalling pathways, and cell-cell communication. Air-liquid interface cultures are used to validate findings, focusing on the TNFα-TGFβ axis.

Severe PCS shows reduced numbers of ciliated cells, increased immune cell infiltration, and heightened inflammatory signaling that drives TGFβ and TNFα upregulation, in the absence of a detectable viral load. These changes trigger epithelial-mesenchymal transition, basal cell expansion and a mis-stratified nasal epithelium. In vitro experiments confirm TGFβ and TNFα as causal cytokines promoting ciliated cell loss and increased basal cell abundance.

These findings indicate a sustained severe PCS is not driven by ongoing viral load but by immune cell activity and chronic cytokine production. Targeting the TNFα-TGFβ axis may mitigate immune-mediated nasal tissue damage and support epithelium restoration, offering a potential therapeutic strategy for PCS.

Web | PDF | Nature Communications | Open Access

Two papers mentioning TGF beta today!

We haven't talked about the idea that abberant cell signalling could be occurring in the nose. Perhaps these findings support the case for more tissue biopsy studies generally?

Personal note- Ever since I got covid at the tail end of my ME/CFS deterioration five years ago, my nose has been almost permanently blocked from passages swelling halfway shut.

 

[jaded]

Just to add to that only since Covid my neuropathic pain has spread to my nose and all trigeminal nerves and it’s absolutely excruciating. Prior to this it was “only” in all body areas below my shoulders. I’m a firm believer there’s neuroimmune dysfunction of some flavour all over which can worsen over time/spread with each significant immune challenge.

 

[SNT Gatchaman]

The nasal epithelium supports respiratory functions involving air filtration, humidification, and pathogen protection. Basal cells differentiate into various cell types that produce mucus to trap foreign particles and pathogens. SARS-CoV-2 primarily infects ciliated epithelial cells and replicates more efficiently in the nose compared to the lower respiratory tract. […] we hypothesize a residual disease state in the nasal epithelia in post-COVID patients.

Respiratory epithelium (Wikipedia).

Here, we show that a severe post-COVID syndrome (PCS) phenotype is characterized by immune-mediated damage to the nasal epithelium, occurring independently of viral persistence.

we observed significant changes in cell-type composition, signaling pathways, and communication between cells. Severe PCS is associated with a decrease in proximal ciliated cells, an increase in immune cell infiltration, and heightened inflammatory signaling, particularly involving TNFα and TGFβ. This inflammatory response drives epithelial-mesenchymal transition and the expansion of basal cells.

We selected 33 patients (whose entire metadata was available) recruited from the NAPKON study cohort (n = 1270). Using the PCS questionnaire developed previously, these 33 participants were classified into mild (n = 4), moderate (n = 11), and severe (n = 18) PCS based on long-term symptom complexes.

The group of patients with mild PCS (n = 4), consisting solely of males, was statistically smaller and significantly imbalanced. […] excluded to ensure statistically robust and meaningful comparisons.

Using a curette, the nasal epithelium was collected from the anterior and medial heads of the middle turbinate

 

[SNT Gatchaman]

analysis of IL6 and CXCL8 expression corroborated a lack of active viral infection, as IL6 and CXCL8 are upregulated during acute infection. This indicates viral clearance and the absence of an active inflammatory response to infection. Nonetheless, a significant change in the relative airway cell composition occurs with PCS severity. Proximal ciliated cells, their precursors, and deuterosomal epithelial cells decreased in relative abundance with increasing PCS scores.

Deuterosome (Wikipedia) relates to ciliated cells.

Conversely, T-cell (1.5 log2FC), basal cell (1.3 log2 FC), and myeloid-dendritic cell (0.6 log2 FC) abundance increased in severe PCS. Hereby, we report that proximal ciliated cells are depleted, whilst basal epithelium, T cells, and myeloid dendritic cells are increased in severe PCS compared to moderate PCS.

we examined the pathways enriched upstream of the differentially regulated genes. Severe PCS reported inflammation-related enrichment (TNFα, TGF β , NF-κB). This, combined with increased myeloid and T-cell abundance, indicated an activated immune response in severe PCS. The PI3K pathway, enriched in moderate PCS, implicates reduced cell proliferation. Conversely, in severe PCS, TGFβ signaling is enriched, whilst cellular migration pathways are enriched in moderate PCS, as indicated by PI3K enrichment signaling.

we deduced paracrine cell-cell communication strength and direction via differential expression of known receptor ligand gene pairs in severe vs. moderate PCS. We observe that basal proliferating cells and myeloid cells form a communication nexus in severe PCS. Comparatively, in moderate PCS, mucous and basal cells send signals, whilst T cells receive cellular signals in moderate PCS.

cell-cell communication strength in severe PCS was dominated by cell-cell adhesion (CD46, CD99, JAM), immune signaling (GALECTIN), and cell growth and survival (MK, PROS1). The macrophage migration inhibitory factor (MIF) was enriched in severe PCS ligand-receptor interactions. MIF is an inflammatory cytokine associated with TNFα and TGF β production. MIF signaling via CD74, CXCR4, and CD44 receptors activates downstream NF-κB, MAPK, and AKT pathways, regulating immune responses, cell proliferation, and cell survival. MIF and CD74 were highly expressed across all cell types irrespective of PCS severity. We observed increasing CXCR4 (myeloid) and CD44 (myeloid and basal cells) expression with PCS severity.

 

[SNT Gatchaman]

Analysis of PROGENy pathway enrichment per cell type revealed an enrichment of EGFR and NF-κB in both myeloid-dendritic cells and T cells, confirming the downstream effects of CD74 activation.

TNF expression was observed primarily in the immune cell compartments, likely driven by the NF-κB pathway. TGFβ1 expression was increased in immune and basal epithelial cells. In the former, TGFβ1 expression is putatively caused by upstream TGFβ signaling, whilst the latter is driven by WNT or MAPK pathway activation.

To check whether TGFβ signaling leads to EMT [Epidermal-Mesenchymal Transition] (important for the nasal epithelium remodeling during inflammation) […] GSEA confirmed TNFα, NF-κB, and EMT as the most upregulated pathways in the nasal mucosa of severe PCS patients.

Taken together, our analysis shows that basal and dendritic myeloid cells are integral communicators across worsening PCS. However, in severe PCS, basal proliferating cells feature a TGFβ-driven response, with myeloid cells maintaining an inflammatory profile. This inflammation is triggered through MIF and propagated by TNFα signaling, leading to the activation of TGFβ and EMT in severe PCS nasal epithelium.

Checking in vitro —

we exposed basal nasal epithelial cells (NECs) to an air-liquid interface (ALI) model with or without TNFα and TGF β stimulation either alone or in combination […] Six distinct clusters were identified

TGFβ exposure promoted increased abundance of differentiating basal cells, with reduced ciliated cell abundance compared to control (PBS) conditions. Comparatively, TNFα exposure resulted in greater basal cell differentiation towards ciliated cells and the secretory type cells). When combined, TGFβ and TNFα caused an amelioration of multiple cell fates, with basal cells apparently starting to differentiate but never committing to a terminal cell state.

Upstream pathway enrichment analysis across treatment conditions highlights activation of VEGF, hypoxia, p53, JAK-STAT, and EGFR pathways, and most prominently combined TGFβ + TNFα stimulation.

Stimulation with only TGFβ during differentiation partially replicated the epithelial phenotype seen in severe PCS patients, with this effect amplified by combined TGFβ/TNFα stimulation.

Our model demonstrates that TGFβ causes a loss of basal epithelial differentiation towards ciliated cells, which is exaggerated by co-exposure with TNFα, as illustrated in Fig. 6. This corroborates what we observe on a transcriptional level in our PCS cohort.

 

[SNT Gatchaman]

Discussion —

In this study, we observed an apparent malformation of pseudostratified nasal mucosa in severe PCS patients compared to moderate PCS patients. This was characterized by reduced ciliated epithelium and increased immune cell presence in severe PCS. The prolonged presence of myeloid and T cells in severe PCS was associated with a persistent inflammatory response via MIF-CD74 signaling and increased expression of TNFα and TGFβ. The proposed effects of this perpetuating inflammatory response and EMT of basal epithelial cells were reinforced in vitro by TGFβ combined with TNFα exposure, impairing basal cell differentiation into ciliated cells using an air-liquid interface model.

scRNA-seq revealed a persistence of myeloid and T cells and latent activation of the MIF-CD74 axis in severe PCS. […] A recent study has linked post-COVID exercise intolerance to resident immune cell induced artery remodeling in skeletal muscle despite viral clearance, suggesting long-lasting tissue remodeling is a PCS feature. Immune cell clearance is impeded in PCS and potentially prolonged inflammation-promoting paracrine cell-cell communication via MIFCD74 and subsequent expression of TNFα and TGF β .

CD74 signals through the ERK and Akt pathway activation. The critical function of CD74 is to stabilize the presentation of antigens for T cells. When combined with MIF, it may drive the persistent activation of pro-inflammatory pathways in severe PCS. In addition, latent production of TGFβ promotes continued activation of the MIF-CD74 axis via the ERK pathway, generating a self-perpetuating cycle in severe PCS patients.

So a reinforcing loop of —

MIF-CD74 ➝ ↑TNF⍺ & TGFβ ➝ ↑MIF-CD74

Severity might be aggravated by the appearance of a new immune cell subset linked to two MIF co-receptors, CXCR4 and CD44.

Our data also indicates fibronectin induced by TGFβ stimulation is enriched in severe PCS. Fibronectin is well described to induce epithelial proliferation and differentiation. Conversely, laminin B1 is produced more in moderate PCS […] These ECM changes, combined with a hypothesized increased deposition of ECM-bound TGFβ, could cause ‘crowding’ of basal epithelial cells followed by anoikis (luminal cell extrusion) as evident by the reduced activation of the PI3K pathway in severe PCS.

 

[SNT Gatchaman]

Our results highlight a potential mechanism for virus-free PCS progression. […] Notably, these cellular and molecular responses occur in the absence of detectable SARS-CoV-2 viral RNA. This contradicts the prevailing hypothesis that PCS is driven by the persistence of a residual viral load.

 

[ScoutB]

I wonder if there have been any proper studies on Epipharyngeal Abrasive Therapy, that thing where they scrape people’s noses out (it sounds brutal)

 

[SNT Gatchaman]

I think I'd rather wait for a bit more validation and some more precisely targeted drugs. Also this process could be involving out of reach respiratory epithelium in the lower respiratory tract, rather than confined to the upper airway.

It would though be quite something if it could modify the situation in some people and sometimes favour reconstitution of the normal pattern of respiratory epithelium (maybe you have to hold your tongue just right ) — rather than for the usual reasons given in the Japanese literature. Eg from Autonomic Nervous System Regulation Effects of Epipharyngeal Abrasive Therapy for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Associated With Chronic Epipharyngitis (2023)

The direct autonomic nerve stimulating effect of EAT may be, first, to stimulate the parasympathetic function and restore the vagus nerve reflex. Second, by stimulating both sympathetic and parasympathetic nerves simultaneously to induce the pharyngeal reflex, it may work to normalize the onset of the pharyngeal reflex. However, the autonomic reflex activity by EAT may induce pathological vagal reflexes or Reilly's phenomenon because the response differs depending on the stimulus intensity, stimulus duration, stimulus site, and sensitivity of the individual.

or Epipharyngeal Abrasive Therapy EAT Reduces the mRNA Expression of Major Proinflammatory Cytokine IL-6 in Chronic Epipharyngitis (2022)

Epipharyngitis is involved not only in otolaryngological disorders, but also in the development of systemic diseases such as IgA nephropathy, palmoplantar pustulosis, rheumatic arthritis, psoriasis, myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), and Long COVID. We have previously shown endoscopically that patients with Long COVID have residual epipharyngeal inflammation. Epipharyngeal abrasive therapy (EAT), which is a treatment for chronic epipharyngitis in Japan, has been reported to be effective in improving these pathological conditions. It has been pointed out that the anti-inflammatory effect of ZnCl2 applied to cotton swabs used in EAT with a sufficient abrasive procedure inhibit the epipharyngeal-activated lymphocytes, but the underlying mechanism has not been elucidated.

As an aside, I'm not sure how the term "epipharynx" is defined. It may be an anachronism, but I think I'm with most of the world with use of nasopharynx, oropharynx and hypopharynx. I think it's used to indicate the back wall of the nasopharynx.

 

[V.R.T.]

Targeting the TNFα-TGFβ axis may mitigate immune-mediated nasal tissue damage and support epithelium restoration, offering a potential therapeutic strategy for PCS.

Are there drugs on the market that target this pathway?

Is this model positing that the abberant signalling is occuring exclusively in nasal tissues or elsewhere too?

And do we think this type of issue could cause the symptoms of ME/CFS?

 

[SNT Gatchaman]

Are there drugs on the market that target this pathway?

The paper discusses the potential roles of infliximab, perfinidone and metformin.

They go on to say —

Dysregulation of inflammation is shown to alter basal cell differentiation. Although reduced, in our model, TNFα-stimulation still reports differentiation of basal cells to ciliated epithelium, indicating a protective effect of the anti-inflammatory regents present in the ALI media. This differentiation is lost with combined exposure to TNFα and TGFβ. These results support why clinical observations that broad-spectrum anti-inflammatory interventions, rather than pathway-specific therapeutics, appear more effective at mitigating PCS progression [56–58]. As such, this model presents as a tool to elucidate the therapeutic potential of current interventions. However, more complex experimental designs are required to appropriately explore this notion.

Quoting from the other paper this week Blood diagnostic biomarkers for neurologic manifestations of long COVID (2025) —

Pirfenidone is an anti-fibrotic drug that can prevent lung injury during SARS-CoV-2 infection by blocking the maturation process of TGF-β1. The TGF-β1 signalling pathway, has been proposed as a potential therapeutic target to treat the Neuro-PASC symptoms in the chronic phase based on its immune suppressing effect. TGF-β1 has the potential to dampen inflammation and immune suppression can reduce the ability of the body to eradicate the virus consistent with recent reports of the virus presence in Long Covid. TGF-β inhibitors have not been successfully used to treat hospitalized patients with severe COVID-19 infections where TGF-β1 is increased though Pirfenidone works mostly in the lung. The use of Pirfenidone to treat Neuro-PASC is thus unclear.

Is this model positing that the abberant signalling is occuring exclusively in nasal tissues or elsewhere too?

I would start with the assumption it's not confined to the nasopharynx. They made a related comment about viral persistence —

Notably, these cellular and molecular responses occur in the absence of detectable SARS-CoV-2 viral RNA. This contradicts the prevailing hypothesis that PCS is driven by the persistence of a residual viral load. However, this conclusion may be influenced by the sampling location with potential viral load persistence in the lower respiratory tract.

And do we think this type of issue could cause the symptoms of ME/CFS?

One for @Jonathan Edwards, but maybe it could relate to the "writing on the wall" idea?