See the published version of this preprint at BMC Medical Genomics.
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Research article
Irina St. Louis
University of Minnesota Twin Cities
Corresponding Author
ORCiD: https://orcid.org/0000-0003-0795-4723
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Background: Cryptococcal meningitis (CM) is a major cause of death in HIV-infected patients in sub-Saharan Africa. Many CM patients experience cryptococcosis-associated immune reconstitution inflammatory syndrome (C-IRIS), which is often fatal. We sought to identify transcriptomic biomarker pathways in peripheral blood that are associated with, or, predict the development of death or fatal C-IRIS among patients with CM who were enrolled in the Cryptococcal Optimal ART Timing (COAT) Trial.
Methods: We assessed peripheral blood gene expression using next-generation RNA sequencing in 4 groups of patients with CM: 1) no C-IRIS or Death; 2) C-IRIS survivors; 3) fatal C-IRIS; 4) Death without C-IRIS. Gene expression was assessed at the time of ART initiation, at 1, 4, and 8 weeks on ART, and at the time of C-IRIS events.
Results: We identified 12 inflammatory and stress response pathways, including interferon type 1 signaling, that were upregulated at the time of ART initiation in patients with future fatal C-IRIS, as compared with survivors. The upregulation of transcripts involved in innate immunity (inflammasome, Toll-like receptor signaling), was observed at the time of fatal or nonfatal C-IRIS events. At the time of fatal C-IRIS events, numerous transcripts within fMLP, Rho family GTPases, HMGB1, and other acute phase response signaling pathways were upregulated, which reflect the severity of inflammation and systemic oxidative stress. Those who died without recognized C-IRIS also had increased expression of pathways associated with oxidative stress and tissue damage. The Partial Least Squares model estimated above mentioned pathways as predictors of fatal outcome.
Conclusions: Our results showed that overactivated innate immunity, involving Toll-like receptor/inflammasome, and inflammation-induced oxidative stress, are associated with fatal outcomes. The results of this study provide insight into the molecular drivers of death and fatal C-IRIS to inform future diagnostic test development or guide targeted treatments.
Keywords
fatal immune reconstitution inflammatory syndrome, transcriptomic biomarkers, AIDS/HIV, cryptococcal meningitis IRIS
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Figure 4
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementalTable1ANOVA.xlsx
-File name: Supplemental Table 1 ANOVA. -Title of data: Supplemental Table 1. ANOVA multivariate analysis was performed asa described in the methods. -Description of data: in the methods.
- SupplementalTable2DiedNoIRISweek0BMCG.xlsx
-File name: Supplemental Table 2. Supplemental Table 2 Died-No IRIS week0 BMCG. -Title of data: Supplementary table 2. Fold change in expression for genes within phagocytes- and T cell pathways. -Description of data: The comparison performed between Death without C-IRIS groups and No C-IRIS or death groups (from early and deferred ART arms combined). Benjamini-Hochberg-corrected Z scores are <1 for these pathways identification (=not significant for all). The expression fold changes and p-values are from ANOVA.
- SupplementalTable3PLSbiomarkers.xlsx
-File name: Supplemental Table 3 PLS biomarkers. -Title of data: Supplemental Table 3. Partial Least Squares (PLS) regression model coefficients ( R ) for centered and scaled Various Importance in Projection (VIP) data. -Description of data: This data was analyzed by Groups: earlier and deferred ART arms combined (see the methods section and text).
- SupplementalFigure1.pptx
-File name: Supplemental Figure 1. -Title of data: Oneway Wilcoxon and ANOVA -Description of data: Assessment of differences in age, CD4+ T cell count, pre-ART HIV viral load, and LFA (lateral flow immunoassay) between groups. Wilcoxon rank sum paired test and one way ANOVA (analyses of variances) test for nonparametric data were used for the comparison between the groups (JMP14.0pro SAS Institute Inc.). The cut-off significance level for all P values was 0.05. Blue line, Mean and Standard deviations (Wilcoxon). Green, Mean and Standard deviation by ANOVA. See tables. The pre-ART serum cryptococcal antigen titers, as measured by LFA assay, were significantly higher in C-IRIS survivors group, as compared to patients who died in earlier study arm with or without C-IRIS.
- SupplementalFigure2.docx
-File name: Supplemental Figure 2. -Title of data: The fold change expression values for the Interferon-STAT and antiviral defense pathways in C-IRIS Survivor Group, as compared to No C-IRIS or Death Group. -Description of data: The low antiviral gene expressions at the time of antiretroviral therapy (ART) initiation in cryptococcosis-associated immune reconstitution inflammatory syndrome (C-IRIS) group. C-IRIS survivors group exhibited significantly lower expression of genes associated with innate immune responses: specifically, those that involve activation of antiviral defense (for example, interferon-inducible genes (IFI), and other, connected with orange lines), or genes encoding enzymes, that directly restrict or inhibit viral replication (for example, oligoadenylate synthase [OAS] or helicases [DHX]). The transcription of interferon-induced protein with tetratricopeptide repeats (IFIT) family genes is predominantly activated by type I and type III interferons and transmitted by the pattern recognition and the JAK-STAT signaling pathways. The OAS genes encode a synthase family that is induced by interferons and catalyze the 2’,5’-oligomers of adenosine to bind and activate RNase L. The IFIT and OAS families of genes are one of many interferon-stimulated genes that play a significant role in the inhibition of cellular protein synthesis in infected cells and is particularly important in cellular resistance to viral infections. Transcripts (nodes), colored in blue, were downregulated (>1.5-fold). Fold change for these transcripts’ expression is shown under the nodes. The orange line represents activation effect of the encoded protein. The blue line represents inactivation effect of the encoded protein. Transcripts were identified through functional analysis using Ingenuity Pathway Assistant (IPA) software.
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View the published article at BMC Medical Genomics.
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See the published version of this preprint at BMC Medical Genomics.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research article
Irina St. Louis
University of Minnesota Twin Cities
Corresponding Author
ORCiD: https://orcid.org/0000-0003-0795-4723
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at BMC Medical Genomics.
Version 1
Posted 21 Sep, 2020
No community comments so far
Editorial decision: Minor revision
On 11 Nov, 2020
Review #1 received
Received 10 Nov, 2020
Review #2 received
Received 10 Nov, 2020
Reviewer #2 agreed
On 03 Nov, 2020
Reviewer #1 agreed
On 30 Oct, 2020
Editor assigned
On 19 Sep, 2020
Reviewers invited
Invitations sent on 19 Sep, 2020
Submission checks complete
On 18 Sep, 2020
Editor invited
On 18 Sep, 2020
Version (no preprint)
Posted 29 Jul, 2020
This version was not preprinted
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Epigenetics & Genomics
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at BMC Medical Genomics.
Background: Cryptococcal meningitis (CM) is a major cause of death in HIV-infected patients in sub-Saharan Africa. Many CM patients experience cryptococcosis-associated immune reconstitution inflammatory syndrome (C-IRIS), which is often fatal. We sought to identify transcriptomic biomarker pathways in peripheral blood that are associated with, or, predict the development of death or fatal C-IRIS among patients with CM who were enrolled in the Cryptococcal Optimal ART Timing (COAT) Trial.
Methods: We assessed peripheral blood gene expression using next-generation RNA sequencing in 4 groups of patients with CM: 1) no C-IRIS or Death; 2) C-IRIS survivors; 3) fatal C-IRIS; 4) Death without C-IRIS. Gene expression was assessed at the time of ART initiation, at 1, 4, and 8 weeks on ART, and at the time of C-IRIS events.
Results: We identified 12 inflammatory and stress response pathways, including interferon type 1 signaling, that were upregulated at the time of ART initiation in patients with future fatal C-IRIS, as compared with survivors. The upregulation of transcripts involved in innate immunity (inflammasome, Toll-like receptor signaling), was observed at the time of fatal or nonfatal C-IRIS events. At the time of fatal C-IRIS events, numerous transcripts within fMLP, Rho family GTPases, HMGB1, and other acute phase response signaling pathways were upregulated, which reflect the severity of inflammation and systemic oxidative stress. Those who died without recognized C-IRIS also had increased expression of pathways associated with oxidative stress and tissue damage. The Partial Least Squares model estimated above mentioned pathways as predictors of fatal outcome.
Conclusions: Our results showed that overactivated innate immunity, involving Toll-like receptor/inflammasome, and inflammation-induced oxidative stress, are associated with fatal outcomes. The results of this study provide insight into the molecular drivers of death and fatal C-IRIS to inform future diagnostic test development or guide targeted treatments.
Figure 1
Figure 2
Figure 3
Figure 4
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementalTable1ANOVA.xlsx
-File name: Supplemental Table 1 ANOVA. -Title of data: Supplemental Table 1. ANOVA multivariate analysis was performed asa described in the methods. -Description of data: in the methods.
- SupplementalTable2DiedNoIRISweek0BMCG.xlsx
-File name: Supplemental Table 2. Supplemental Table 2 Died-No IRIS week0 BMCG. -Title of data: Supplementary table 2. Fold change in expression for genes within phagocytes- and T cell pathways. -Description of data: The comparison performed between Death without C-IRIS groups and No C-IRIS or death groups (from early and deferred ART arms combined). Benjamini-Hochberg-corrected Z scores are <1 for these pathways identification (=not significant for all). The expression fold changes and p-values are from ANOVA.
- SupplementalTable3PLSbiomarkers.xlsx
-File name: Supplemental Table 3 PLS biomarkers. -Title of data: Supplemental Table 3. Partial Least Squares (PLS) regression model coefficients ( R ) for centered and scaled Various Importance in Projection (VIP) data. -Description of data: This data was analyzed by Groups: earlier and deferred ART arms combined (see the methods section and text).
- SupplementalFigure1.pptx
-File name: Supplemental Figure 1. -Title of data: Oneway Wilcoxon and ANOVA -Description of data: Assessment of differences in age, CD4+ T cell count, pre-ART HIV viral load, and LFA (lateral flow immunoassay) between groups. Wilcoxon rank sum paired test and one way ANOVA (analyses of variances) test for nonparametric data were used for the comparison between the groups (JMP14.0pro SAS Institute Inc.). The cut-off significance level for all P values was 0.05. Blue line, Mean and Standard deviations (Wilcoxon). Green, Mean and Standard deviation by ANOVA. See tables. The pre-ART serum cryptococcal antigen titers, as measured by LFA assay, were significantly higher in C-IRIS survivors group, as compared to patients who died in earlier study arm with or without C-IRIS.
- SupplementalFigure2.docx
-File name: Supplemental Figure 2. -Title of data: The fold change expression values for the Interferon-STAT and antiviral defense pathways in C-IRIS Survivor Group, as compared to No C-IRIS or Death Group. -Description of data: The low antiviral gene expressions at the time of antiretroviral therapy (ART) initiation in cryptococcosis-associated immune reconstitution inflammatory syndrome (C-IRIS) group. C-IRIS survivors group exhibited significantly lower expression of genes associated with innate immune responses: specifically, those that involve activation of antiviral defense (for example, interferon-inducible genes (IFI), and other, connected with orange lines), or genes encoding enzymes, that directly restrict or inhibit viral replication (for example, oligoadenylate synthase [OAS] or helicases [DHX]). The transcription of interferon-induced protein with tetratricopeptide repeats (IFIT) family genes is predominantly activated by type I and type III interferons and transmitted by the pattern recognition and the JAK-STAT signaling pathways. The OAS genes encode a synthase family that is induced by interferons and catalyze the 2’,5’-oligomers of adenosine to bind and activate RNase L. The IFIT and OAS families of genes are one of many interferon-stimulated genes that play a significant role in the inhibition of cellular protein synthesis in infected cells and is particularly important in cellular resistance to viral infections. Transcripts (nodes), colored in blue, were downregulated (>1.5-fold). Fold change for these transcripts’ expression is shown under the nodes. The orange line represents activation effect of the encoded protein. The blue line represents inactivation effect of the encoded protein. Transcripts were identified through functional analysis using Ingenuity Pathway Assistant (IPA) software.
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