Early in the pandemic, a team of immunologists from the Max Planck Institute of Immunobiology and Epigenetics in Freiburg and physicians from the University of Freiburg Medical Center joined forces to learn more about immunity in people recovering from COVID infections.
The study revealed a yet unknown involvement of Interleukin 33, an important alarm-signal, when immune cells get exposed to SARS-CoV-2 for a second time.
Since the beginning of the coronavirus pandemic, scientists and physicians worldwide undertook enormous efforts to understand the disease caused by the virus. In their latest collaborative study, researchers from the Max Planck Institute of Immunobiology and Epigenetics in Freiburg and physicians from the University of Freiburg Medical Center unveil a novel feature of COVID-19 immunity, which could have implications for future therapies.
The study points to the involvement of Interleukin 33, an important danger signal, when immune cells encounter SARS-CoV-2 for a second time.
“We started the study at a very early stage of the pandemic in 2020 when not much was known about the immune response post-infection,” says Erika Pearce, group leader at the Max Planck Institute of Immunobiology and Epigenetics.
“Our aim was to examine the development of immunity in people recovering from COVID-19.”
Antibodies stick around
An infection with SARS-CoV-2 triggers a complex immune response necessary for the development of immunity to the virus. In simple terms, two linked branches of our immune system need to remember the virus to prevent reinfection, namely antibody-producing B cells and memory T cells.
Understanding how this happens in SARS-CoV-2 infection is key for controlling the COVID-19 pandemic and critical for the success of the vaccination efforts.
For the study, the team examined blood samples of 155 individuals who mostly had mild disease. They measured the amount of antibodies against the SARS-CoV-2 spike protein and found that patients maintain high levels of antibodies more than two months after infection, indicating that they will likely be protected from re-infection.
“We thought this was very encouraging, but we also wanted to understand better how the immune system would react to a second encounter with the virus,” says Petya Apostolova, physician and researcher in the lab of Erika Pearce.
When the virus hits the second time
Effective immunity to a virus is reached when sufficient antibodies and memory T cells are present in the blood of a person who has recovered from the disease or has been vaccinated. To test how this happens after COVID-19, the team exposed blood cells from participants who had antibodies against SARS-CoV-2 to a portion of the virus.
They observed that memory T cells had developed and quickly responded to viral proteins. “We measured a broad panel of molecules that our immune cells use to communicate with each other. It was most fascinating to us that of all these measurements, the amount of Interleukin 33 was the closest match to the amount of antibodies people had, and to the activation of their memory T cells,” explains Apostolova.
Interleukin 33 (IL-33) is released by cells that sense danger in their environment and has been previously linked to chronic lung disease. IL-33 can have beneficial effects by activating T cells and inducing antibody production, but it can also promote inflammation of the lung. For the first time, this study has linked IL-33 production to immunity to SARS-CoV-2.
“We believe that Interleukin 33, which is normally produced as an alarm-signal, could be an important link between protection and disease severity,” says Cornelius Waller from the University of Freiburg Medical Center. Indeed, by analyzing public data of lung cells taken from patients during SARS-CoV-2 infection, the researchers were able to show that Interleukin 33 was produced in their lungs. However, identifying the implications of these findings also in the context of lung tissue damage after severe COVID-19 infections will require more investigation.
The group of researchers hopes this collaboration will continue. Waller said, “We were able to discover this much so quickly through this fantastic synergy between clinicians experienced in the care for COVID-19 patients and experts in the immunology field.” The researchers hope that this study might pave the way to better understanding immunity to SARS-CoV-2 and other viral infections.
Intensive efforts are underway to unravel the immunopathology of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and to control the pandemic. Given the public health emergency, scarcity of effective antiviral therapies, and rapid evolution of lung disease associated with COVID-19, patients who are critically ill with COVID-19 and have exuberant inflammation, life-threatening acute respiratory distress syndrome, and coagulopathy, are basically treated as if they had secondary haemophagocytic lymphohistiocytosis or virus-associated macrophage activation syndrome (MAS).
These treatments are focused on therapies that neutralise key cytokines driving classical MAS, such as interleukin-6 ([IL]-6; eg, tocilizumab) or interferon gamma (IFNγ; eg, emapalumab).1, 2 In fact, some fatal cases of COVID-19 are accompanied not only by severe respiratory disease, but also by increased systemic inflammation as shown by higher ferritin concentrations.2 However, in many aspects, COVID-19 does not resemble typical MAS. We propose that the cytokine storm syndrome seen in COVID-19 is dissimilar to that seen in canonical MAS and should be regarded as a distinct entity and approached in a novel way reflecting its unique qualities.
Cellular immunity and T-cell polarisation in COVID-19
Whereas virus-induced MAS shows the classic hallmarks of a T-helper (Th)-1 profile, with high production of IFNγ,1, 3 COVID-19 is instead characterised by circulating T cells that show an activated Th17 membrane phenotype (CD38+HLA-DR+CD4+CCR6+)4 and express granulocyte–macrophage colony-stimulating factor (GM-CSF) in part along with IFNγ.5 Concentrations of both IL-17 and IFNγ are increased in serum from patients with COVID-19 in proportion with viral load and lung injury.6
Similarly, Middle East respiratory syndrome has been associated with a combined Th1–Th17 inflammatory response.7 Notably, the cytokine storm composition induced by SARS-CoV-2 differs from that induced by severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus, with lower production of type 1 cytokines (eg, IL-12p70, IL-15), and high concentrations of type 2 cytokines (eg, IL-4, IL-9, IL-10, transforming growth factor β [TGFβ], IL-13).6, 7, 8, 9, 10, 11 These findings might provide important clues to the specific immunopathology of COVID-19.
Transcriptomic analyses of bronchoalveolar lavage fluid from patients with COVID-19 have revealed a strong upregulation of IL-33.11 IL-33 is a cytokine of the IL-1 family that is expressed in barrier tissues and exerts pleiotropic functions. In the lungs, IL-33 is promptly released, mainly by injured epithelial alveolar cells, following infection and cellular damage.12 Among its functions, IL-33 enhances TGFβ-mediated differentiation of Foxp3+ regulatory T (Treg) cells13 and stimulates CD11c+ myeloid dendritic cells to secrete IL-2, which drives Treg cell expansion, thus ultimately promoting resolution of inflammation.14
Individuals infected with SARS-CoV-2 who develop milder symptoms tend to have large numbers of Treg cells10 and alveolar macrophages showing a scavenger resolving (FABP4+) phenotype.15 In the presence of an adequate immune response and virus clearance, IL-33 might drive rapid Treg cell-dependent restoration of respiratory tissue homoeostasis, which probably accounts for the mild or asymptomatic forms of COVID-19 seen in most individuals.
IL-33 and cellular drivers of mild–moderate COVID-19
In susceptible individuals who develop symptomatic SARS-CoV-2 infection and COVID-19 pneumonia (eg, in the presence of individual cytokine or receptor polymorphisms), IL-33 might abnormally upregulate expression of its own receptor ST2 (also known as IL-1RL1) on Treg cells, resulting in increased expression of the canonical Th2 transcription factor GATA-binding factor 3 (GATA3), which impairs the suppressive function of Treg cells.
The dysregulation of GATA3+ Foxp3+ Treg cells might result in impaired immunological tolerance and increased secretion of type 2 cytokines, thus promoting autoinflammatory lung disease.16 TGFβ2, which is also increased in the bronchoalveolar lavage fluid of patients with COVID-19,11 might further enhance ST2 expression in innate lymphoid cells, and IL-33 is the key cytokine that drives these cells to differentiate into type 2 innate lymphoid cells (ILC2).17 ILC2 subsequently elicit lung inflammation by releasing large amounts of IL-9, which promotes their own survival and expands γδ T cells.18, 19 IL-9 is known to stimulate proliferation and expansion of Vγ9Vδ2+ T cells that have a predominantly effector memory phenotype and a combined Th1–Th17 cytokine response profile.19 When exposed to TGFβ, γδ T cells can also become an important source of IL-9.20 By acting in both autocrine and paracrine manners, IL-33-induced IL-9 might sustain a proinflammatory ILC2–γδT cell axis in the lungs of patients with COVID-19, thus initiating mild–moderate forms of pneumonia.
Both ILC2 and γδ T cells are centrally involved in lung homoeostasis and are rapidly activated in response to pathogens including viruses;19, 21 in COVID-19, IL-4 is upregulated at early stages and in milder forms of the disease,10 whereas IL-9 and activated γδ T cells are observed more frequently in mild-to-moderate disease,9, 22 and IFNγ and IL-17 progressively increase with disease severity.6 Vγ9Vδ2+ T cells from patients with COVID-19 have been found to express an effector memory phenotype three times more frequently than do conventional αβ T cells,23 thus suggesting that this T cell subset is selectively stimulated in COVID-19. Because of significantly higher expression of the chemokine receptor CXCR3 compared with their αβ counterparts,24 γδ T cells might be rapidly recruited into inflamed lungs of patients with COVID-19 in response to the observed strong upregulation of the CXCR3 ligands CXCL9 and CXCL10 (figure 1 ).6, 9, 11, 15, 25, 26, 27, 28
T-cell polarisation in COVID-19
IL-33 released from virus-damaged cells might induce dysregulated GATA3+Foxp3+ Tregs and promote IL-2 production by dendritic cells, resulting in further expansion of Tregs. IL-33 might also elicit differentiation of ILC2, with TGFβ enhancing ST2 expression on these cells and facilitating production of IL-9. IL-9 in turn stimulates expansion of effector memory Vγ9Vδ2+ T cells with mixed Th1 and Th17 profiles that express CXCR3 and are recruited to the lungs by CXCL9 and CXCL10. IL-9 possibly induces its own transcription factor PU.1 and thus act in an autocrine and paracrine manner (along with TGFβ) to drive proliferation and survival of ILC2 and γδ T cells. Additional positive loops might be fed by IFNγ, which triggers production of CXCL9 and CXCL10 by macrophages. In severe forms of COVID-19, IL-33, along with IL-2 and IL-7 released by dendritic cells, might further stimulate T-cell expansion through STAT5 and induce production of large amounts of GM-CSF by γδ and T helper cells. At advanced stages of disease, aberrant activation of the MyD88-related NF-κB pathway and activation of the NLRP3 inflammasome might induce virus-exposed cells and infiltrating monocytes–macrophages to overproduce IL-1β, IL-23, and IL-6. IL-1β, IL-23, IL-6, and IL-7 act on STAT3 and RORC, thus promoting differentiation of CCR2+ T cells that are recruited to the lungs by CCL2 and CCL8 into γδT17 and Th17 cells producing IL-17 and GM-CSF. In turn, GM-CSF might further recruit and activate proinflammatory monocytes–macrophages. CCR=C-C motif chemokine receptor. CCL=C-C motif chemokine ligand. CXCL=C-X-C motif chemokine ligand. CXCR=C-X-C chemokine receptor. Foxp=forkhead box protein. GATA=GATA-binding factor. GM-CSF=granulocyte-macrophage colony-stimulating factor. IL=interleukin. ILC2=type 2 innate lymphoid cell. MyD88=myeloid differentiation primary response protein. NF-κB=nuclear factor-kappa B. NLRP=NACHT, LRR, and PYD domains-containing protein. PU.1=transcription factor PU.1. RORC=nuclear receptor ROR-gamma. ST2=ST2 receptor. STAT=signal transducer and transcription activator. TGF=transforming growth factor. Th=T-helper. TLR=toll-like receptors. Treg=regulatory T cell.
IL-33 induction of GM-CSF-expressing T cells in severe COVID-19
The cellular composition of lung infiltrates in patients with COVID-19 pneumonia changes with the progression of disease. Infiltrates in patients with moderate pneumonia include mainly lymphoid and dendritic cells; whereas, severe forms of disease are characterised by massive infiltration of macrophages and neutrophils.15
In patients with COVID-19, expression of T-cell chemoattractants (eg, CXCL9, CXCL10) and their receptors (eg, CXCR3) precedes expression of monocyte and neutrophil chemoattractants (eg, CCL2, CCL3, CCL4, CCL7, CXCL8) and their corresponding receptors (eg, CCR1, CXCR2).15 The composition and phenotypes of lung macrophages also change with disease severity. Resident alveolar (A-FABP4+) macrophages, which show scavenger and lipid metabolic functions typical of anti-inflammatory or resolving M2-like cells (eg, macrophage receptor MARCO, PPAR-γ, Apo-CI), predominate in mild and moderate forms, whereas CD14+ monocyte-derived macrophages (FCN1high) and chemoattractant (FCN1lowSPP-1+) macrophages, which show highly inflammatory M1-like profiles (eg, nuclear factor-kappa B [NF-κB], CCL2, CCL3), dominate tissue specimens from patients with severe forms of COVID-19 and who are critically ill.15
In the circulation of patients with COVID-19, amounts of proinflammatory CD14+CD16+ intermediate monocytes increase with disease severity, and upregulation of GM-CSF in CD4+ and CD8+ T cells might account for tissue recruitment and activation of neutrophils and monocyte-derived macrophages in most severe forms of the disease.5
Although described as Th1 cells, at least half of the GM-CSF-producing T cells observed in the circulation of patients with severe COVID-19 do not coexpress the canonical Th1 cytokine IFNγ.5 Lymphocytes from patients with COVID-19 appear to be functionally exhausted, producing lower amounts of IFNγ, IL-2, and tumour necrosis factor (TNF), and having decreased cytotoxic function.29
Many factors could possibly explain this lymphocyte dysfunction, in particular the upregulation of multiple coinhibitory receptors such as CD94, CD152 (cytotoxic T-lymphocyte-associated antigen 4), programmed cell death protein 1 (PD-1), and T-cell immunoglobulin mucin receptor 3 (TIM-3).29 However, suboptimal production of IFNγ, poor cytotoxic capabilities, a shorter lymphocyte lifespan, and lymphopenia might also be attributable to a scarcity in type I and III interferons (IFNα, IFNβ, and IFNλ), in the blood as well as in the lungs of patients with COVID-19.27 Interferons are more highly suppressed by SARS-CoV-2 than by SARS-CoV infection,27, 28 and this most likely accounts for the impaired antiviral responses and spontaneous apoptosis of dysfunctional lymphocytes.11, 30
Lymphocyte impairment in COVID-19 resembles the cytotoxic dysfunction of CD8+ cytotoxic T lymphocytes and natural killer cells observed in familial haemophagocytic lymphohistiocytosis, in which T cell dysfunction is the result of heterozygous mutations in genes affecting the expression of perforin or other proteins involved in the trafficking and docking of cytolytic granules,1 and in patients who are predisposed to MAS, in whom IL-6 overexpression can reduce perforin and granzyme B concentration inside granules.31
The inability to kill infected or activated antigen-presenting cells in patients with either MAS or COVID-19 could result in persistent interactions between T cells and antigen presenting cells, culminating in hyperproduction of cytokines as a result of overstimulation of both cell types.1 However, by contrast with COVID-19, IFNγ is not impaired in MAS, and is a major driver of disease. In MAS, IFNγ-producing CD8+ T-cell populations are elevated in primary and secondary lymphoid organs, leading to IFNγ-driven macrophage hyperactivation and haemophagocytosis.1, 3
The effects of IFNγ deficiency have been investigated in an experimental model of haemophagocytic lymphohistiocytosis, which develops when perforin deficient (Prf1−/−) mice are infected with the lymphocytic choriomeningitis virus. Surprisingly, mice lacking both IFNγ and perforin (IFNγ−/−Prf1−/−) still develop a severe MAS-like disease that requires the IL-33–ST2 axis and is downstream mediated by GM-CSF-producing CD8+ T cells. The inflammatory burden in infected IFNγ−/−Prf1−/− mice is even higher than in Prf1−/− mice, being characterised by a 10–15 times increase in neutrophils and stronger upregulation of IL-1β and IL-6.32 The same interplay between IL-33 and GM-CSF might occur in patients with COVID-19, which would initiate the cytokine storm syndrome. Thus, severe forms of COVID-19 might represent atypical MAS or MAS-like reactions with incorporated interferon deficiencies.
Cellular expansion in severe–critical COVID-19
Failure of lymphocytes to adequately respond to viral antigens and proapoptotic signals might induce dendritic cells to produce large amounts of the lymphocyte growth factors IL-7 and IL-2, thereby stimulating T-cell survival and expansion. High concentrations of IL-2 and IL-7 in serum are characteristic of severe COVID-19 cases.6, 9 However, IL-2 and IL-7 might amplify ILC2 survival and differentiation induced by IL-33,33 expand γδ T cells, which produce IL-17 through the signal transducer and activator of transcription (STAT)3,34 and enhance IL-33-induced pathologic expansion of T cells expressing GM-CSF through STAT5.35, 36 Patients infected with SARS-CoV-2 show an increase in circulating CD4+ γδ T cells that overexpress the IL-2 receptor CD25 but not PD-1, suggesting that these cells are not exhausted, but are specifically activated in response to IL-2.22
IL-7 enables γδ T cells to fully differentiate into γδT17 cells34 that coproduce IL-17F along with IL-17A, and rapidly migrate into inflamed tissues in response to CCR2 and CCR5 ligands such as CCL2 and CCL8.37, 38 As shown for murine γδT17 cells, human Vδ2+ T cells that co-express CCR2 and CCR5 also express the IL-7 receptor and show a Th17-like phenotype (CCR6+CD161+IL-23R+).39 Transcriptional analyses of respiratory cell populations in response to SARS-CoV-2 infection reveal strong upregulation of CCL8, CCL2, CXCL9, CXCL10 and their respective receptors,11, 15, 27 and global upregulation of IL-17 and IL-17F-related pathways,26 including the CCR6 ligand CCL20 and IL-23.27
IL-23 and IL-1β are required for GM-CSF production by γδT17 cells and conventional αβ Th17 cells.40, 41 In models of autoimmunity in which GM-CSF is a key pathogenic molecule, such as experimental autoimmune encephalomyelitis, γδ T cells have been identified as the major source of GM-CSF.42 Whereas conventional αβ Th17 cells evolve to produce IFNγ during the development of the disease, γδT17 cells are less likely to produce IFNγ and will more likely evolve to produce GM-CSF.42 As for γδT17, recruitment of IL-23-driven, GM-CSF-producing Th17 cells requires CCR2.43
By promptly releasing multiple cytokines such as IL-9, IL-17, IL-17F, TNF, IFNγ, and GM-CSF, γδT17 might be instrumental in recruiting neutrophils and proinflammatory monocytes into the capillaries and alveoli of patients with COVID-19. Moreover, activation of γδ T cells might be important in the cytokine-driven induction of procoagulant tissue factor in endothelial cells,44 thus also having a potential role in vascular manifestations and pulmonary thromboses associated with COVID-19 pneumonia.2, 29
Cytotoxicity against virus-infected alveolar epithelial cells by γδ T cells has been shown for influenza virus45 and might involve atypical pathways alternative to granzyme B and perforin, which are more commonly used by CD8+ T cells and natural killer cells and could be impaired in COVID-19 and MAS.1, 29, 31 Specifically, γδ T cells might exert cytotoxic effects through the TNF-related apoptosis-inducing ligand, Fas ligand, and granzyme K,39, 45 which are all overexpressed in the lungs of patients with COVID-19,11, 15 and might therefore explain how γδ T cells cause diffuse damage to the alveolar epithelium (figure 1).
Suppression of antiviral responses and hyperinflammation
Advanced stages of COVID-19 are characterised by high circulating and pulmonary concentrations of IL-1α, IL-1β, and IL-1 receptor antagonist (IL-1RA).6, 15, 25 The increased production of these molecules probably relates to high viral loads resulting in increased viroporins and subsequent activation of the NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome. The strong expression of IL-1α, IL-1β, and IL-1RA is also due to monocyte activation and intense lung infiltration of monocyte-derived macrophages at later stages, as suggested by an abundance of CD14+IL-1β+ monocytes in the circulation of patients with COVID-19 in the early stages of recovery.46 Active IL-1β is produced following NLRP3 assembly and consequent caspase-1 activation. By modulating ion fluxes across host cell membranes, viroporins (in particular the ORF3a protein) have been shown to activate NLRP3 during SARS-CoV infection, and a similar mechanism might be at play during SARS-CoV-2 infection.47
An imbalance in signalling from toll-like receptor (TLR) pathways, with the myeloid differentiation primary response protein (MyD88) pathway predominating over the TIR domain-containing adapter molecule 1 (TICAM-1, also known as TRIF) pathways, might further increase NLRP3 activation.48 Signalling downstream of IL-1 family receptors, including the IL-33 receptor ST2, and downstream of membrane TLRs, can activate MyD88 and elicit inflammation; whereas TRIF-mediated pathways downstream of endosomal TLRs would be expected to mount antiviral interferon responses and protect against coronaviruses.48, 49 Although coronaviruses are single-stranded RNA viruses that are predicted to bind directly to endosomal TLR7 and TLR8, and indirectly to TLR3 (using double-stranded RNA replication intermediates), aberrant inflammation induced by coronaviruses might instead involve membrane-expressed TLR2, as suggested by virus spike protein interactions with heparan sulphate-enriched regions of TLR2 in studies of the mouse hepatitis coronavirus.50 A predominance of MyD88 signalling over TRIF signalling would lead virus-exposed cells to produce high amounts of IL-1β, and NF-κB-induced cytokines and chemokines (eg, TNF, IL-8, IL-6, IL-12p40, IL-23, CCL2) rather than interferons, IL-12p35 and IL-12p70.30, 49, 50
High concentrations of MyD88-related cytokines and reduced expression of TRIF-related cytokines characterise the cytokine milieu observed in the lungs of patients with severe and life-threatening COVID-19.15, 27, 28 Such an altered cytokine environment would polarise the immune response towards detrimental (Th17-sustained and GM-CSF-induced) hyperinflammation40, 41 caused by monocyte-derived macrophages and neutrophils, in place of protective (Th1-sustained and IFN-induced) antiviral responses exerted by cytotoxic T lymphocytes, natural killer cells, and B cells.29, 30 Altogether, coronaviruses seem to deceive and escape the immune system by eliciting a response that is generally more appropriate for extracellular rather than intracellular pathogens.
IL-33 and pathway synergisms in critical systemic COVID-19
In addition to NLRP3 stimulation and IL-1 release,47 substantial amounts of viroporins in patients with life-threatening COVID-19 might also account for extensive injury of alveolar epithelial cells and overproduction of IL-33.51 IL-33, IL-1α, and GM-CSF also stimulate each other’s release by alveolar type 2 pneumocytes.52, 53 Accordingly, diffuse alveolar damage with alveolar denudation and reactive type 2 pneumocyte hyperplasia are histological hallmarks of COVID-19 with acute respiratory distress syndrome.4
Feedforward loops might also engage mast cells, macrophages, endothelial cells, T cells, and neutrophils.40, 54 Although whether mast cells and macrophages produce IL-33 is still up for debate,51 it is well established that mast cells, infiltrating neutrophils, and cytotoxic T lymphocytes secrete serine proteases (eg, tryptase, cathepsin G, elastase, granzymes) that cleave IL-33 released from damaged epithelial and endothelial barriers into a mature form of IL-33 that is 10–30 times more active.51 IL-33 amplifies lung inflammation by inducing various proinflammatory cytokines (eg, GM-CSF, IL-1β, IL-6, TNF, granulocyte colony-stimulating factor [G-CSF]), chemokines (eg, CXCL1, CXCL2, CXCL6, CXCL8, CCL2, CCL20), and adhesion molecules (eg, E-selectin, ICAM1, VCAM1) in several target cells.32, 54, 55, 56, 57 Conversely, by inhibiting type 1 interferons and IL-12p35, IL-33 might contribute to impaired antiviral cytotoxic responses.58 In models of MAS-like disease, IL-33 is a crucial contributor to the weight loss and hyperferritinaemia related to systemic hyperinflammation, and to the expansion of GM-CSF-producing CD8+ T cells, upregulation of IL-1β and IL-6, and tissue neutrophilia.32 These features are the same as key characteristics seen in patients with critical COVID-19.5, 15, 26
IL-33 has also been implicated in the formation of neutrophil extracellular traps during virus-induced asthma exacerbation.58 Similarly, neutrophil priming with GM-CSF might promote the production of neutrophil extracellular traps.59 By releasing neutrophil elastase and other proteinases, neutrophil extracellular traps could in turn cleave and further activate IL-33. These pathways might be relevant in patients with critical COVID-19, since neutrophilia and the neutrophil-to-lymphocyte ratio are associated with poor prognosis, and high concentrations of neutrophil extracellular traps have been detected in patients with COVID-19 admitted to hospital and receiving mechanical ventilation.60
Neutrophil extracellular traps might propagate inflammation and microvascular thrombosis in patients with COVID-19 and severe acute respiratory distress syndrome.60 Along with IL-33, IL-1, TNF, and other cytokines, neutrophil extracellular traps might increase endothelial permeability and induce a procoagulant phenotype in endothelial tissues by inducing expression of tissue factor,61, 62, 63 thus representing a possible link between hyperinflammation and hypercoagulability that could account for D-dimer elevation, pulmonary thrombosis, and microvascular manifestations affecting the heart, kidneys, and small bowel seen in patients with critical COVID-19.64, 65 Endothelialitis and endothelial dysfunction would also account for predominant exudative-phase diffuse alveolar damage characterised by hyaline membranes and fibrin deposits typically observed in patients with COVID-19 and severe acute respiratory distress syndrome.4
IL-33 has also been shown to stimulate expression of IL-1β, IL-6, CCL2, CXCL2, and G-CSF by adipocytes.57 Elevated circulating concentrations of soluble ST2 (measured more often than IL-33 because of its higher concentration and stability) are associated with obesity, diabetes, hypertension, and acute cardiovascular diseases. High soluble ST2 concentrations also predict worse outcomes and are associated with extension of heart damage, heart failure, increased cardiovascular death, and all-cause mortality.54 Notably, diabetes, hypertension, and cardiovascular diseases are common comorbidities in patients with COVID-19, and obesity has been independently associated with increased severity and mortality among younger patients with COVID-19.66 Circulating concentrations of soluble ST2 correlate with the extent of tissue damage, and might represent an indicator in plasma of IL-33 release and bioactivity in tissues. Production of soluble ST2 might be reduced by anti-ST2 treatment, and such reduction would modulate T-cell polarisation by decreasing pathogenic Th1 and Th17 cells, and increasing IL-10-producing Treg cells.67 Future research should focus on whether soluble ST2 concentrations in plasma have prognostic value in patients with COVID-19 (figure 2 ).
IL-33 might orchestrate all pathogenic phases of COVID-19
IL-33 might induce numerous cytokines and chemokines as well as its own receptor, ST2, in various cell types. In asymptomatic or paucisymptomatic patients, IL-33 might expand anti-inflammatory Foxp3+ Treg cells or induce IL-4 production by GATA3+Foxp3+ Tregs and ILC2, thus stimulating mast cells, which might account for minor, allergy-like symptoms. In individuals with mild-to-moderate disease, IL-33 (along with TGFβ) might induce ILC2 to release large amounts of IL-9, driving local expansion of effector memory Vγ9Vδ2+ T cells in the lungs. In moderate–to-severe pneumonia, IL-33 combined with IL-2 and IL-7 from dendritic cells might further expand ILC2, γδT cells, and GM-CSF-producing T cells. In severe–critical COVID-19, IL-33, GM-CSF, and IL-1 might stimulate each other’s release by acting on multiple cell types. IL-33 induction of cytokines, chemokines, adhesion molecules, tissue factor, and neutrophil extracellular traps might contribute to endothelialitis, thrombosis, and extrapulmonary involvement in patients with MAS-like disease. Neutrophil extracellular traps and mast cell degranulation could provoke protease-mediated cleavage of IL-33 into a 10–30 times more potent form, and IL-33-induced release of its soluble receptor ST2 might further polarise T cells and contribute to cardiovascular manifestations. In patients who survive, IL-33 might drive the post-acute fibrotic phase thorugh induction of IL-13 and TGFβ in M2-differentiated macrophages and ILC2, thereby stimulating myofibroblasts and eliciting the epithelial–to–mesenchymal transition of type 2 pneumocytes. Molecules inside brackets are part of self-amplifying proinflammatory loops fed by IL-33 and outside brackets indicate different factors possibly induced by IL-33. Question mark indicates the uncertainty of whether mast cells produce IL-33. bFGF=fibroblast growth factor. CCL=C-C motif chemokine ligand. CTGF=connective tissue growth factor. CXCL=C-X-C motif chemokine ligand. DIC=(systemic vascular thromboses mimicking) diffuse intravascular coagulation. EMT=epithelial-mesenchymal transition. Foxp=forkhead box protein. GATA=GATA-binding factor. G-CSF=granulocyte colony-stimulating factor. GM-CSF=granulocyte-macrophage colony-stimulating factor. ICU=intensive care unit. IFN=interferon. IL=interleukin. ILC2=type 2 innate lymphoid cell. MAS=macrophage activation syndrome. MOF=multiple organ failure. NET=neutrophil extracellular trap. PDGF=platelet-derived growth factor. P/F ratio=arterial oxygen partial pressure to fractional inspired oxygen ratio. sST2=soluble ST2. ST2=ST2 receptor. TGF=transforming growth factor. TF-1=tissue factor-1. TNF=tumour necrosis factor. TRAIL=TNF-related apoptosis-inducing ligand. Treg=regulatory T cell.
Similarities between COVID-19, Kawasaki disease, and Behçet’s disease
Parallels between COVID-19 and rheumatic disorders can be made by referring to discrete autoinflammatory syndromes that share symptoms with COVID-19 such as fever, frequent conjunctivitis, and—most remarkably—vasculitic manifestations with neutrophilia, thrombosis, and aneurysmal dilations, involving coronary vessels (eg, Kawasaki disease in infants) or pulmonary vessels (eg, Behçet’s disease in adults).
Case series of children infected with SARS-CoV-2 who develop Kawasaki-like disease with MAS features have been described.68 GM-CSF produced by cardiac fibroblasts is key in disease progression in mouse models of Kawasaki disease, and significantly increased soluble ST2, E-selectin, CXCL10, IL-17F, and in some cases IL-9, have been reported in the circulation of patients with acute Kawasaki disease compared with other children who are febrile.69, 70, 71 Similarly, Behçet’s disease has been associated with high concentrations of both soluble ST2 and IL-33, as well as increased CXCL10 and CCL2, Vγ9Vδ2 T-cell expansion, IL-17F gene polymorphisms, and intense recruitment of T cells producing IL-9 and IL-17 to the lungs.72, 73, 74, 75, 76 Some patients with either Behçet’s disease77 or COVID-1978 also show positivity for antiphospholipid antibodies, which might further contribute to the coagulopathy seen in both conditions.
IL-33 induction of pulmonary fibrosis in chronic COVID-19
Lung alveolar inflammation in COVID-19 is accompanied by loose interstitial fibrosis and can result in widespread fibrotic changes.79 IL-33 could also be important at these later stages of the disease. In a bleomycin-induced pulmonary fibrosis mouse model, the IL-33–ST2 axis is required to induce alternatively activated M2 macrophages and ILC2 to release key profibrotic cytokines.80 IL-33-activated mast cells might also play a role in organ fibrosis.81 Most remarkably, IL-33 has been shown to induce epithelial-to-mesechymal transition of type 2 pneumocytes through TGFβ signalling.82
IL-33 concentrations are elevated in patients with systemic sclerosis and correlate with the severity of pulmonary fibrosis, and patients with idiopathic pulmonary fibrosis show increased serum concentrations of soluble ST2 when the disease is exacerbated.82 IL-33 can induce cytokines (eg, TGFβ, IL-13) and chemokines (eg, CCL2, CXCL6) involved in pulmonary fibrosis, which are also increased in patients infected with SARS-CoV-2,6, 9, 11, 15, 26 thus suggesting additional roles for IL-33 in driving the post-acute fibrotic phase of COVID-19. Growth factors such as vascular endothelial growth factor, platelet-derived growth factor, and fibroblast growth factor are all involved in fibrotic processes and are overexpressed in patients with COVID-19,9 and γδ T cells exposed to TGFβ might produce connective tissue growth factor (figure 2).83
reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546716/
More information: Michal A. Stanczak et al. IL-33 expression in response to SARS-CoV-2 correlates with seropositivity in COVID-19 convalescent individuals, Nature Communications (2021). DOI: 10.1038/s41467-021-22449-w
