Patent Application
20230075138
The COVID-19 pandemic is unquestionably one of the most urgent global health crises of the modern era. Its onset and dissemination around the world has sparked clinical and basic research in efforts to discover interventions which can inhibit or prevent infection or mitigate the clinical course of disease progression. Intensive Care Unit (ICU) admissions and mortality in severe COVID-19 patients are driven by “cytokine storms” and acute respiratory distress syndrome (ARDS). Interim clinical trial results suggest that the corticosteroid dexamethasone displays superior 28-day survival in severe COVID-19 patients requiring ventilation or oxygen. Longitudinal analysis of lab test results pre-and post-corticosteroid administration from 10 hospitalized COVID-19 patients shows ICU duration positively correlates with a change in plasma IL-6 levels. Among a larger cohort of 20 patients with only post-corticosteroid IL-6 measurement, a consistent trend of higher IL-6 levels indicating longer ICU stays is observed.
Infection with SARS-CoV-2 is largely asymptomatic or presents with mild-to-moderate symptoms1, but can result in progressive respiratory illness leading to acute respiratory distress syndrome (ARDS) in a subset of patients.2, 3 Multiple clinical trials are underway to investigate therapies ranging from antivirals (e.g. Lopinavir-Remdesivir) to corticosteroids and targeted immunosuppressive agents (e.g. Dexamethasone, Tocilizumab)4. While antiviral drugs, antibiotics, and antimalarials have shown little to no clinical benefit in COVID-19 patients modulation of host inflammatory responses with drugs that inhibit IL-6 signaling (tocilizumab) or stimulate glucocorticoid receptor activity (dexamethasone) have been shown to improve clinical outcomes in critically ill patients.6 In line with previous reports of IL-6 as a biomarker of severe disease5, some studies examining small patient cohorts have suggested treatment with tocilizumab (anti-IL6 receptor) may improve outcomes in severely ill patients.6, 7 The use of IL-6 as a biomarker for disease severity has gained traction during the ongoing COVID-19 pandemic. The high sensitivity plasma IL-6 tests conducted at the Mayo Clinic from 2010 and 2019 show only 274 out of 1463 tests (18%) were above 10 pg/mL (normal range between 0.31-5 pg/mL), compared to 377 out of 537 tests (70%) in the first half of 2020, including both COVID-19 and unrelated IL-6 testing. These estimates suggest that physicians have begun using plasma IL-6 levels to assess COVID-19 disease severity, and further that higher plasma IL-6 levels are likely to continue being reported during the ongoing pandemic relative to the pre-COVID era. In some cases, serial measurements of plasma IL-6 levels may have been used clinically to determine disease progression and treatment efficacy, motivating a thorough examination of the available longitudinal real-world evidence from the Mayo Clinic platform.
A recent interim update from the RECOVERY (Randomised Evaluation of COVid-19 thERapY) trial that is examining larger cohorts of severe COVID-19 patient outcomes revealed the maximal reduction in mortality among patients treated with dexamethasone over other therapies.8 In this trial, 2,104 randomized patients received 6 mg of dexamethasone once per day for ten days, compared with 4,321 patients that received standard care alone. Dexamethasone reduced mortality by one-third in ventilated patients (rate ratio 0.65 [95% confidence interval 0.48 to 0.88]; p=0.0003) and by one-fifth in other patients receiving oxygen only (0.80 [0.67 to 0.96]; p=0.0021).8
Although corticosteroids have long been utilized clinically for their immunosuppressive and anti-inflammatory capacities, 9, 10, 11, 12 the precise mechanisms by which dexamethasone mediates clinical improvement in severe COVID-19 patients are not well understood. Indeed, the success of dexamethasone in treating COVID-19 was somewhat unexpected in light of an early report during this pandemic urging clinicians to not prescribe corticosteroids for lung injury in COVID-19 patients, based on the lack of efficacy as well as elevated risk of adverse events associated with steroid use in the previous SARS and MERS epidemics.13 Such ostensibly contradictory evidence and guidance underline the need for an improved mechanistic stratification of corticosteroid efficacy in different subsets of severely ill COVID-19 patients.
The present disclosure is based, at least in part, on the longitudinal analysis of real-world data (RWD) of COVID-19 patients and a comprehensive profiling of NR3C1 and triangulate expression-derived insights with human genetic and pharmacologic datasets to nominate putative mechanisms by which dexamethasone results in reduced mortality. Analysis of the single cell RNA-sequence data of bronchoalveolar lavage fluid from severe COVID-19 patients and nearly 2 million human cells from a pan-tissue scan shows alveolar macrophages, smooth muscle cells, and endothelial cells co-express the glucocorticoid receptor NR3C1 and IL-6. In some aspects of the invention disclosed herein, corticosteroids that are NR3C1 agonists may reduce pulmonary and multi-organ inflammation in COVID-19 patients with respiratory failure, by antagonizing IL-6 production in lung macrophages and vasculature. Thus, in some aspects, the invention provides methods of treating a coronavirus infection in a subject in need thereof, comprising a) determining whether the subject is a responder or a non-responder to an immunomodulating therapy by determining the expression level of at least one mRNA of a corticosteroid receptor in a sample obtained from said subject. In preferred embodiments, said expression level is compared with a reference value, wherein the expression level of the corticosteroid receptor relative to the reference value indicates whether the subject will respond to an immunomodulating therapy; and the immunomodulating therapy is administered to the subject whose expression level is indicative of responding to said immunomodulating therapy.
In certain aspects, provided herein are methods of determining whether an immunomodulating therapy is effective for treating a subject having a coronavirus infection. In some embodiments, the method comprises determining the expression level of at least one mRNA of a corticosteroid receptor in a sample obtained from said subject; and comparing the expression level of the corticosteroid receptor with a reference value, wherein a the expression level of the corticosteroid receptor relative to the reference value indicates whether the immunomodulating therapy is effective for treating said subject.
In some aspects, disclosed herein are methods for distinguishing a human subject suffering from coronavirus disease 2019 (CoVID-19) responsive to an immunomodulating therapy from non-responsive subjects, comprising a) obtaining a sample from said subject; b) obtaining a gene expression profile from the sample, wherein the expression profile comprises expression levels for one or more genes; wherein said one or more genes comprise at least one corticosteroid receptor; and c) comparing the gene expression profile of the sample with at least one reference gene expression profile.
In certain aspects of the invention, disclosed herein are methods for monitoring a human subject suffering from CoVID-19 for potential treatment with an immunomodulating therapy. In some embodiments, such methods comprise obtaining a sample from the subject at predetermined intervals. Preferably, a gene expression profile is obtained from the sample(s), wherein the expression profile comprises expression levels for one or more genes; wherein said one or more genes comprises at least one corticosteroid receptor; and the gene expression profile of each sample is compared chronologically, wherein a decrease in corticosteroid receptor expression over time identifies the subject as a responder to an immunomodulating therapy. In some such embodiments, the method further comprising administering an immunomodulating therapy to the subject if the expression of corticosteroid receptor decreases over time. In other embodiments, the method further comprises withholding immunomodulating therapy from the subject if the expression of corticosteroid receptor does not decrease over time.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the presently disclosed methods and compositions. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
Rapid advances in genomic and transcriptomic technologies over the past decade hold great potential to characterize drug targets at unprecedented levels. The nƒerX® platform Single Cell application was released as a resource to help researchers analyze publicly deposited single cell RNA-sequencing datasets and readily contextualize these expression-derived insights using quantified literature associations14. this wealth of gene expression data at single cell resolution has been harnessed to profile human tissues and cells based on their expression of ACE2, the putative entry receptor for SARS-CoV-2. 14,15,16,17 Notably, although the primary glucocorticoid receptor (NR3C1) has been previously reported to be ubiquitously expressed,18,19,20 the global expression profile of this important drug target has not been systematically evaluated across the hundreds of thousands of bulk RNA-seq samples and millions of single cell RNA-sequencing data points which are available.
The present disclosure provides molecular support for the immunomodulatory effects of corticosteroids and the immune cell types which are most likely to be affected by systemic and pulmonary exposure to dexamethasone. Notably, multiple populations of alveolar macrophages in coronavirus-infected patients (e.g., COVID-19 patients) co-express NR3C1 and IL-6 genes, including one population which tends to express lower levels of NR3C1 and higher levels of IL-6 in severely ill patients compared to those with mild disease. Moreover, non-immune cells including endothelial cells and smooth muscle cells are among the cell types which most frequently co-express these genes. Taken together, said disclosure as provided herein, presents molecular evidence that the clinical improvement observed with dexamethasone treatment may be due to NR3C1-mediated antagonism of IL-6 production both systemically and locally in the lungs. Triangulating real world evidence with multi-omics inference over a privacy-preserving ‘precision COVID platform’ thus offered a promising methodology for dissecting the complex immunosuppressive mechanisms underlying corticosteroid therapy.
In certain aspects of the invention, provided herein are methods of treating a coronavirus infection in a subject in need thereof, comprising a) determining whether the subject is a responder or a non-responder to an immunomodulating therapy by determining the expression level of at least one mRNA of a corticosteroid receptor in a sample obtained from said subject. In preferred embodiments, said expression level is compared with a reference value, wherein the expression level of the corticosteroid receptor relative to the reference value indicates whether the subject will respond to an immunomodulating therapy; and the immunomodulating therapy is administered to the subject whose expression level is indicative of responding to said immunomodulating therapy.
In some aspects, provided herein are methods of determining whether an immunomodulating therapy is effective for treating a subject having a coronavirus infection. In some embodiments, the method comprises determining the expression level of at least one mRNA of a corticosteroid receptor in a sample obtained from said subject; and comparing the expression level of the corticosteroid receptor with a reference value, wherein a the expression level of the corticosteroid receptor relative to the reference value indicates whether the immunomodulating therapy is effective for treating said subject.
In some embodiments, the method further comprises determining the expression level of mRNA of IL-6 in the sample obtained from said subject. Preferably the relative expression levels are compared, wherein lower mRNA expression level of the corticosteroid receptor relative to the reference value and concomitant higher IL-6 mRNA expression level relative to an IL-6 reference value indicates that the subject will respond to an immunomodulating therapy. In some such embodiments, the corticosteroid receptor reference value and/or IL-6 reference value indicative of responsiveness to the immunomodulating therapy is representative of one or more subjects who are responsive to the therapy. In other embodiments, a corticosteroid receptor reference value and/or a IL-6 reference value indicative of non-responsiveness to the immunomodulating therapy is representative of one or more subjects who are non-responsive to the therapy. In preferred embodiments, the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV2).
In some aspects, disclosed herein are methods for distinguishing a human subject suffering from coronavirus disease 2019 (CoVID-19) responsive to an immunomodulating therapy from non-responsive subjects, comprising a) obtaining a sample from said subject; b) obtaining a gene expression profile from the sample, wherein the expression profile comprises expression levels for one or more genes; wherein said one or more genes comprise at least one corticosteroid receptor; and c) comparing the gene expression profile of the sample with at least one reference gene expression profile. In preferred embodiments, the gene expression profile of the sample is compared with a reference gene expression profile indicative of responsiveness to the therapy obtained from one or more subjects who are responsive to the therapy and/or a reference gene expression profile indicative of non-responsiveness to the therapy obtained from one or more subjects who are non-responsive to the therapy, wherein similarity in expression profiles between the sample and reference profiles indicates sensitivity to the therapy in the subject from whom the sample was obtained, thereby identifying the subject as a responder or non-responder to the therapy.
In certain aspects of the invention, disclosed herein are methods for monitoring a human subject suffering from CoVID-19 for potential treatment with an immunomodulating therapy. In some embodiments, such methods comprise obtaining a sample from the subject at predetermined intervals. Preferably, a gene expression profile is obtained from the sample(s), wherein the expression profile comprises expression levels for one or more genes; wherein said one or more genes comprises at least one corticosteroid receptor; and gene expression profile of each sample is compared chronologically, wherein a decrease in corticosteroid receptor expression over time identifies the subject as a responder to an immunomodulating therapy. In some such embodiments, the method further comprises administering an immunomodulating therapy to the subject if the expression of corticosteroid receptor decreases over time. In other embodiments, the method further comprises withholding immunomodulating therapy from the subject if the expression of corticosteroid receptor does not decrease over time.
In some embodiments, the gene expression profile comprises the expression level of IL-6. In preferred embodiments, a decrease in expression level of the corticosteroid receptor and concomitant increase in IL-6 expression level indicates that the subject will be a responder to an immunomodulating therapy.
In some embodiments the sample comprises, or is derived from, a biological sample from the subject that comprises the cells of the subject, such as a tissue sample or a bodily fluid sample. Such samples include, but are not limited to an organ sample (e.g., lung) or a sample of any fluid present in the body (for example and without limitation, blood, plasma, serum, saliva, synovial fluid, lymph, urine, or cerebrospinal fluid). In preferred embodiments, the sample comprises bronchoalveolar lavage fluid.
In some such embodiments, the sample comprises fibroblasts, epithelial cells, endothelial cells, and smooth muscle cells. In preferred embodiments, the sample comprises peripheral blood mononuclear cells (PBMCs). More preferably, the sample comprises immune cells, such as T cells, B cells, monocytes, natural killer cells, dendritic cells, and macrophages. Most preferably, the sample comprises macrophages, such as alveolar macrophages.
Expression levels and/or gene expression profiles may be obtained from the samples by methods known in the art, e.g., by using RNA-sequencing methods. In some such embodiments, expression levels are determined by bulk RNA-sequencing and/or single cell RNA-sequencing. In preferred embodiments, the expression level is determined by single cell RNA-sequencing, e.g., in immune cells of the sample. In some such embodiments, the immune cells are macrophages and/or T cells.
The immunomodulating therapies contemplated herein may comprise administering a corticosteroid, a corticosteroid receptor agonist, a therapeutic antibody, or any combination thereof. In some embodiments the corticosteroid is a glucocorticoid or a mineralocorticoid. Corticosteroids and agonists contemplated herein include, but are not limited to dexamethasone, prednisone, triamcinolone, methylprednisolone, prednisolone, betamethasone, or hydrocortisone. Preferably, the corticosteroid is dexamethasone. Therapeutic antibodies of the invention (e.g., immunotherapeutic chimeric, humanized, or human monoclonal antibodies, and the like) include anti-cytokine and/or anti-cytokine receptor antibodies. In some such embodiments the therapeutic antibody is an anti-granulocyte macrophage colony-stimulating factor receptor (GM-CSF-R) antibody, such as mavrilimumab. In preferred embodiments, the therapeutic antibody is an anti-IL6 receptor antibody, such as tocilizumab.
The corticosteroid receptors contemplated herein include glucocorticoid receptors and mineralocorticoid receptors. Preferably, the corticosteroid receptor of the invention is a glucocorticoid receptor, also known as NR3C1 (nuclear receptor subfamily 3, group C, member 1).
Cohort Definition for Patients Receiving IL-6 Measurements and Corticosteroids
All patients who tested positive for SARS-CoV-2 (COVIDpos), as determined by at least one positive PCR test, within the Mayo Clinic Health System and were hospitalized, were selected as candidates for further analysis. These COVIDpos patients were then filtered by requiring an administration of a systemic corticosteroid at some point during their hospital stay. Corticosteroids included dexamethasone, prednisone, triamcinolone, methylprednisolone, prednisolone, betamethasone, and hydrocortisone. Tocilizumab was also included as a control given it's known effect on IL-6 signaling. In patients who received a systemic corticosteroid, it was required that they underwent plasma IL-6 testing at least once both before and after the first administration of any of the corticosteroid agents listed above. Only 13 patients met these criteria, with 7 patients receiving IL-6 testing before and after methylprednisolone administration, 4 patients receiving IL-6 testing before and after prednisone administration, and 4 patients receiving IL-6 testing before and after hydrocortisone administration. Two patients overlapped drug categories, with one receiving first administrations of both methylprednisolone and hydrocortisone between IL-6 tests and the other receiving first administrations of both prednisone and hydrocortisone between IL-6 tests. In total, six patients received IL-6 testing both before and after any first corticosteroid administration within the timeframe considered (−25 days to +64 days). In addition to IL-6 levels and corticosteroid/tocilizumab administration data, outcomes including death, admission to an ICU, and length of time in ICU, as well as demographic information such as age, sex, and race were extracted.
Data Accession and Processing:
Datasets were downloaded in raw fastq format and uniformly processed using salmon.
Global Expression Analysis:
To identify highly expressing cells and tissues for a given gene, the following steps were followed:
1. The distribution of gene expression was plotted (in units of transcripts per million, or TPM) across all samples from all studies.
2. The distribution was divided into “High Expression Group” (e.g., cells in top 5% of expressing samples for query gene) and “Low Expression Group” (e.g. cells in bottom 25% of expressing samples for query gene).
3. The number of individual samples from each annotated cell or tissue type falling in the High and Low Expression Groups was counted. Cells and tissues were extracted from sample-level metadata available through the Gene Expression Omnibus (GEO) and other databases including the Genotype-Tissue Expression project (GTEx), The Cancer Genome Atlas (TCGA), and the Cancer Cell Line Encyclopedia (CCLE).
4. Fisher's Exact Test p-value was computed to measure the enrichment of cell type C (or tissue T) among the High Expression Group. Enrichment Scores displayed correspond to-log10(adjusted p-value), where p-values are adjusted using the Benjamini-Hochberg (BH) correction.
Data Accession and Processing:
Datasets were downloaded and processed as previously described14, and processed datasets were made available for investigation upon on-line registration in the nferX Single Cell platform.
Global Expression Analysis:
To identify highly expressing cells and tissues for a given gene, the following steps were followed:
1. The distribution of gene expression was plotted (in units of counts per 10,000, or CP10K) across all single cells from all studies.
2. The distribution was divided into “High Expression Group” (e.g., cells in top 10% of expressing samples for query gene) and “Low Expression Group” (e.g. cells in bottom 90% of expressing samples for query gene).
3. The number of individual cells from each annotated cell population (or tissue) falling in the High and Low Expression Groups was counted.
4. Fisher's Exact Test p-value was computed to measure the enrichment of cell population C (or tissue T) among the High Expression Group. Enrichment Scores displayed correspond to-log10(adjusted p-value), where p-values are adjusted using the Benjamini-Hochberg (BH) correction.
Coexpression Analysis:
For a given set of genes, a single coexpression vector was computed as the geometric mean of CP10K values of all genes in each cell. The geometric mean was used as a coexpression metric as it will only yield a positive value in cells which express all genes in the defined set (i.e. one or more zero values in an individual cell will result in a coexpression value of zero for that cell). As such, all cells with a coexpression value (CP10Kgm) greater than zero were considered as “coexpressing cells”, whereas all cells with a CP10Kgm values equal to zero were considered as “non-coexpressing cells.” After this coexpression vector was computed, it was treated identically to a gene expression vector for a single gene in the context of the Global Expression or single study-level analyses described above.
Comparison of Patient IL6/ICU Pattern Between Drug Classes:
Four categories of patient IL-6 level/ICU stay length were defined (i.e. the 4 quadrants described in Table 2). For each drug class with at least 10 patients, and for each quadrant with nonzero number of patients, the proportion of patients in the quadrant out of those taking a drug from the class were compared to the proportion of patients in the quadrant out of those not taking a drug from the class. Fisher exact test was performed to compute p-values on these proportion comparisons. A Benjamini-Hochberg adjustment was also applied to these p-values for multiple hypotheses.
Kaplan-Meier curves were generated for COVID-19 positive patients that were hospitalized at the Mayo Clinic (see
To further understand the specific effects of the different corticosteroids given to COVID positive patients, survival rates of hospitalized and ICU patients given dexamethasone, hydrocortisone, prednisone and methylprednisolone were also generated. For hospitalized patients, dexamethasone (n=30) and prednisone (n=39) performed similarly (90% and 92% 60-day survival, respectively) and have higher 60-day survival post-diagnosis compared to hydrocortisone (n=34) and methylprednisolone (n=22) (79% and 77% 60-day survival, respectively). While a decrease in survival is observed in the more severe subset of each of these cohorts who are admitted to the ICU, dexamethasone (n=11) shows the most significant decrease in 60-day survival, dropping from 90% to 72%.
Given the early clinical data showing that both dexamethasone and tocilizumab independently improve outcomes in severely ill COVID-19 patients, the beneficial effect of some anti-inflammatory agents may be in part due to the suppression of IL-6 production. This mechanism would be consistent with the known role of glucocorticoids in reducing IL-6 transcription in macrophages21. Plasma IL-6 measurements were taken for 63 hospitalized COVID-19 patients who also received corticosteroids during the course of their care at the Mayo Clinic. The timing of SARS-CoV-2 PCR testing, corticosteroid administration, tocilizumab administration, and IL-6 measurements amongst these patients are highlighted in
The IL-6 levels remained stable or decreased after steroid administration in 7 of 10 patients, while the remaining 3 patients showed striking increases in IL-6 (see
Longitudinal (i.e. pre-and post-corticosteroid) IL-6 testing in similar COVID positive cohorts, with severe symptoms requiring ICU administration, could assist in identifying patients who respond to corticosteroids alone versus those who may require additional anti-inflammatory therapy (e.g. tocilizumab) in combination.
Along these lines, only one of the 10 patients received tocilizumab along with corticosteroid therapy. In this patient, a single dose of tocilizumab was administered on the same day as the first corticosteroid (oral prednisone) administration; the patient subsequently received methylprednisolone. Notably, mean IL-6 levels increased more than 6-fold in this patient following tocilizumab and corticosteroid administration, and the patient had an extended ICU stay (30 days).
Because only 10 patients met the aforementioned criteria, 10 additional COVID positive, ICU patients who only had IL-6 measured post-corticosteroid administration were also examined. Their IL-6 levels were plotted against the duration of ICU stay (see
While not statistically significant due to small cohort sizes (see Table 3), together the results may indicate that patients with high IL-6 levels post-corticosteroids are more likely to have longer ICU durations compared to the other drugs disclosed herein.
Again, this points to the necessity for IL-6 measurement post-corticosteroid administration to monitor if further intervention is needed.
To better understand the mechanisms of potential corticosteroid-induced effects on IL-6 levels, the gene expression of NR3C1—the highest affinity target of dexamethasone, methylprednisolone, and prednisone—systematically profiled across over 450,000 human bulk RNA-sequencing (bulk RNA-seq) samples from more than 10,000 studies (see
Various immune cells, including T cells, B cells, monocytes, natural killer cells, dendritic cells, and macrophages, were found to be enriched among the samples with the highest (top 5%) of NR3C1 expression (see
NR3C1 expression was further characterized by single cell RNA-sequencing (single cell RNA-seq) across 1.9 million human cells using the nƒerX® Single Cell platform14. Consistent with the findings from bulk RNA-seq, various hematopoietic lineages were enriched among the cells with highest expression of NR3C1 (see
To connect the expression profile to the clinical observation of reduced plasma IL-6 in a subset of patients following corticosteroid administration, co-expression of NR3C1 and IL-6 was specifically evaluated across these same 1.9 million human single cells. Two populations of alveolar macrophages from a study of healthy controls (n=3) and COVID-19 patients (n=9) were among the most strongly enriched cell types for this co-expression (see
Given that dexamethasone appears to reduce mortality among only severe cases of COVID-19, how NR3C1 and IL-6 co-expression varies between mild and severe disease was investigated. NR3C1 and IL-6 expression levels were assessed in each recovered BALF cell population between cases of mild COVID-19 (n=3) and severe COVID-19 (n=6), along with healthy controls (n=3). Notably, expression of IL-6 and NR3C1 was positively correlated in the strongest co-expressing population (“Macrophages 4” identified above), and further that NR3C1 expression in this cell population was significantly lower in patients with severe disease compared to mild disease (see
For a subset of 700k patients at the Mayo clinic with rich longitudinal data (i.e. at least 4 encounters, with each being 2-6 weeks apart), it was observed that
Further, ICD (International Classification of Diseases) codes enriched among patients with high IL-6 levels in 2019 included “Nonspecific Abnormal Finding of the Lung” whereas those enriched in 2020 include “Acute Respiratory Failure with Hypoxia” and “COVID-19 Infection.” This captured the strong clinical association between high IL-6 levels and severe COVID-19 infection, and suggests the clinical benefit of corticosteroid therapy in critically ill COVID-19 patients is related to an ability to reduce the transcriptional expression of IL-6 and other inflammatory mediators in various cell types.
The integrated analysis of publicly available molecular data and curated electronic health record (EHR) data was prepared to study mechanisms of dexamethasone activity in COVID-19. Despite the long history of corticosteroid use in the clinic, expression of the glucocorticoid receptor (NR3C1) has never been systematically profiled in the modern genomic era to understand the tissues and cell types which are most likely to be directly targeted by systemic steroid therapies. The analysis of gene expression by bulk and single cell RNA-sequencing provided herein suggests that hematopoietic cells are the cells most prominently impacted by dexamethasone. In particular, T cells are the most strongly enriched human cell type for high NR3C1 expression, but other adaptive and innate immune cells are also notably strong expressers.
To analyze the cellular context around the intersection of glucocorticoids and IL-6, the first directed co-expression analysis of NR3C1 and IL-6 across human single cell RNA-sequencing datasets was conducted, totaling almost 2 million cells. The analysis highlighted that while NR3C1 alone is highly expressed in T cells throughout the human body, co-expression with IL-6 is prominently observed in alveolar macrophages of the lung along with various non-immune cells across multiple tissues including endothelial, smooth muscle, epithelial, and stromal cells. This suggests that engagement of the glucocorticoid receptor by dexamethasone in these co-expressing cell types reduces local and systemic IL-6 production, which in turn restores immune homeostasis and mitigates the progression of the COVID-19 associated acute respiratory distress syndrome. The analysis of EHR data from a large academic medical center shows that plasma IL-6 levels are reduced after steroid administration in a subset of critically ill COVID-19 patients. Patients with this steroid-associated IL-6 decline tended to have shorter ICU admissions than patients whose IL-6 levels increased even after steroid administration.
Other inflammatory pathways were also notably activated, and excessively so, in COVID-19 patients. Blockade of these pathways may provide clinical benefit similar to that seen with tocilizumab. For example, early results indicate that blockade of the granulocyte-macrophage colony-stimulating factor (GM-CSF) pathway with the monoclonal antibody mavrilimumab may improve outcomes in severely' several ill patients. GM-CSF is known to orchestrate the activity of various innate immune cells including dendritic cells and macrophages, and the single cell RNA-seq data confirms that NR3C1 is co-expressed with both the alpha and beta subunits of the GM-CSF receptor (CSF2RA and CSF2Rb) in several macrophage populations from the BALF of COVID-19 patients (see
Without being bound by any particular theory, monitoring expression levels of N3CR1 and/or IL-6, or of plasma IL-6 levels after initiation of steroids, may be warranted in clinical practice to determine whether a patient is likely to respond to corticosteroid therapy alone or if they should be considered as candidates for alternative intervention such as tocilizumab.
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All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.
This application claims the benefit of U.S. Provisional Application No. 63/221,667, filed on Jul. 14, 2021, the entire contents of which are incorporated herein in its entirety by this reference.
| Number | Date | Country | |
|---|---|---|---|
| 63221667 | Jul 2021 | US |
