Understanding how drugs affect the immune system has consequences for treating disease and minimizing unwanted side effects. Here we present an integrative computational approach for predicting interactions between drugs and immune cells in a system-wide manner. The approach matches gene sets between transcriptional signatures to determine their similarity. We apply the method to model the interactions between 1,309 drugs and 221 immune cell types and predict 69,995 interactions. The resulting immune-cell pharmacology map is used to predict how five drugs influence four immune cell types in humans and mice. To validate the predictions, we analyzed patient records and examined cell population changes from in vivo experiments. Our method offers a tool for screening thousands of interactions to identify relationships between drugs and the immune system.
At a glance
Pharmaceutical drugs of all types and classes influence the immune system1, 2, 3, 4 but the mechanisms of these perturbations are often poorly understood. Some drugs target immune cells specifically to treat immunological diseases, such as B-cell lymphomas (e.g., rituximab (Rituxan)5), whereas others have broad immunosuppressive or anti-inflammatory effects (e.g., thalidomide (Thalomid)6, leflunomide (Arava)7 or sirolimus (Rapamune)8). However, many drugs that were not developed to be immunomodulatory are nevertheless associated with mild to severe immune reactions. For example, several anti-infectives, anti-convulsants and anti-diabetic drugs are believed to induce the skin hypersensitivity reaction urticaria9, 10, 11, and psychoanaleptics, such as respiridone (Risperdal), memantine (Namenda) and citalopram (Celexa), have the rare, but life-threatening side effect of an immune-complex hypersensitivity called Stevens-Johnson syndrome1. Our lack of understanding of the global interactions between pharmaceuticals and the immune system confounds drug development, conceals potentially serious side effects of marketed compounds12, 13, 14 and limits discovery of drugs that could be repurposed for immune diseases.
Published studies on the effects of drugs on immune cells have mainly examined the consequences of administering one drug to a single cell type15, 16. Even when high-throughput screens have been performed, they usually have focused on a specific target or readout (e.g., changes in select cell surface markers)17, 18, 19 and ignored other perturbations to the system. In the present report, we build on previous systems-level approaches that compare and integrate differential expression profiles of disease with drug perturbation profiles to discover potential new drug indications20, 21, 22. Recent large-scale collaborative efforts have produced compendia of molecular profiles for both pharmaceutical drugs23 and immune cells24. To our knowledge, systematic integration and analysis of chemogenomic and immunogenomic data have not been performed.
We integrated drug perturbation data obtained with human cancer cells and gene expression data obtained from mouse immune cells. Our analysis quantifies the likelihood that a drug affects an immune cell state change in the form of an 'immunemod score'. In total, we generated 304 immune cell state transitions from 221 immune cell types. We studied all combinations of 1,309 drugs and 304 immune cell state transitions, and found 69,995 significant interactions (of 397,936 possible interactions). From these interactions, we constructed an immune-cell pharmacology (IP) map of predicted drug–immune cell connections, which includes both known interactions as well as ones that have—to our knowledge—not been reported previously. To address concerns about integrating data across species, we examined predictions in both mouse and humans. We performed in vivo experimental validation of three predictions and obtained 100% agreement. In addition, we found evidence in patient data that supported our predicted interactions between drugs and immune cells in two independent sets of electronic medical records. Our results suggest that integrative computational analysis can improve understanding of the effects of drugs on the immune system and provide a framework for rational manipulation of these effects.
Generating molecular signatures of immune cells
The Immunological Genome Project (ImmGen) is the largest publicly available compendium of genome-wide transcriptional expression profiles for more than 250 distinct immunological cell states in mice25, 26, 27. The data comprise 14 categories of immune cell types collected from 25 tissue locations (Supplementary Fig. 1). These states reflect diverse stages of lineage differentiation, collected from various tissues, using an assortment of genetic variants, in response to stimulations with chemicals, bacteria or viruses, and at separate effector stages. One challenge with using ImmGen data for probing immune perturbations is that gene expression profiles were captured at a single state, which provides limited information on cellular response to external stimuli. Thus we created a data set that reflects immune cell responses to perturbations by generating differentially expressed gene signatures between two immunological states that differ by a single parameter (e.g., cell types with identical surface markers isolated from separate tissues or two cell types that differ by one surface marker such as naive vs. memory CD4+ T cells).
We compiled a set of 304 immune cell state change signatures from 221 unique cell types in the ImmGen compendium to explore how drug perturbations alter the immune system (Fig. 1a,c and Supplementary Table 1). The full ranked lists are provided in Supplementary Table 2. These signatures group by similar cell types when clustered by the Jaccard distance between sets of the extreme fold-change genes (Supplementary Fig. 2). The average Jaccard distances between related cell types exceeded the overall average background distance (Supplementary Fig. 3) and showed significant differences between immune cell subsets (P = 6 × 10−15, ANOVA).
Generating drug chemogenomic profiles
The Connectivity Map (CMap) is a data repository of genome-wide transcriptional expression profiles collected from 6,100 experimental conditions of 1,309 unique compounds applied to human cell lines23. Each perturbation is represented by a list of differentially expressed genes that we ranked based on fold change. To capture the consensus profile of a compound across conditions, we merged multiple experiments (i.e., different drug concentrations or cell lines) for the same compound into a single prototype ranked list (PRL), using a hierarchical majority-voting scheme28, 29 (Fig. 1a). The collection of PRLs created a comprehensive resource for developing a systematic screening tool to look for connections between drug perturbations and immunological states (Supplementary Table 3).
IP map construction
We created a system-wide interaction map between drugs and immune cells by matching the 1,309 drug perturbation profiles in CMap to the 304 immune cell state changes we curated from the ImmGen compendia. Our matching algorithm evaluates the similarity between two transcriptional expression patterns by comparing the top- and bottom-ranked genes from both profiles20, 21 (Fig. 1b, Supplementary Fig. 4 and Online Methods). Specifically, we tested the similarity between the immunological state–change profiles (state B vs. state A) to each of the drug perturbation profiles (treated vs. untreated) by computing an immunemod score based on the overlap of the top- and bottom-ranked genes in each profile. A positive immunemod score indicates that the specific drug treatment profile is similar to immune cell state B and suggests the drug biases the immune cell toward state B, whereas a negative score signals the drug treatment shifts the cell toward state A (Fig. 1b,d).
To evaluate the significance of our predicted drug-cell interactions, we generated random drug perturbation profiles for each compound and repeated the analysis 1,000 times for each immune cell state change (Supplementary Fig. 4a). A complete computational integration of the CMap and ImmGen data sets produced 397,936 potential connections between drugs and immune cell state changes. To assess whether a predicted connection was robust, we varied the set size of the top- and bottom-ranked genes used for the matching algorithm and recalculated all ~400,000 immunemod score P-value pairs. The proportion each drug-cell interaction was significant among all gene set sizes provided a relative weight for each predicted interaction (Supplementary Fig. 4b,c). Larger weights indicate that a given drug-cell interaction depends less on the set size chosen to calculate the immunemod score and signifies a robust connection. This selection process enabled discovery of previously unknown interactions while minimizing spurious connections (Supplementary Fig. 5).
Using the significant and robust interactions, we made connections between drugs and immune cell state changes to generate a comprehensive IP map. The IP map contains 69,995 connections (Supplementary Table 4) that are significant at a false-discovery rate (FDR) < 5% and that appear in > 85% of gene set sizes. Although every drug showed a significant association with at least one of the 304 immunological state changes, the most frequent number of state transitions is 26, and 144 drugs influence 100 or more state changes (Supplementary Fig. 6). Drugs predicted to influence the largest number of immune cell state transitions include potent immunomodulators, many of which induce significant immunosuppression (Table 1). Drugs with immunomodulatory activity (e.g., anti-inflammatory agents, anti-histamines and immunosuppresants) show a significant enrichment for immune cell interactions (E = 1.5, P = 0.002, E = 1.4, P = 0.04, E = 2.1, P = 0.02, respectively, and Supplementary Table 5).
Global properties of the IP map
To examine the global landscape of the IP map, we used the immunemod score as a similarity metric and organized the complete set of drug and immune cell interactions through unsupervised hierarchical clustering (Fig. 2a). We found that drugs with similar therapeutic classes cluster together. For example, anti-psychotics (clozapine (Clozaril), loxapine (Adasuve), haloperidol (Haldol) and fluphenazine) formed a cluster, as did purine analogs (mercaptopurine (Purixan) and tioguanine), and calcium channel blockers (dexverapamil, bepridil and perhexiline). These three clusters are predicted to interact with the largest number of immune cell subset transitions. Drug clusters also showed enrichment for the same molecular target. For example, the anti-diabetic drugs troglitazone (Rezulin) and rosiglitazone (Avandia) both target PPARG and ACSL4 as part of their mechanism of action for reducing blood glucose. Based on their immunemod scores, these drugs are predicted to influence T cell and natural killer (NK) cell differentiation, providing a potential explanation for the therapeutic benefits observed in patients with autoimmune disease30, 31.
We identified 28 drugs associated with later stages of lineage development across multiple cell types (e.g., stem/progenitor and pre-B cells; Fig. 2a and Supplementary Table 6). These drugs include compounds used to treat diseases of metabolism and the nervous, musculo-skeletal and respiratory systems, or are anti-infectives. Moreover, these 28 drugs are enriched for (i) agents with immunosuppressant, anti-psoriatic and dopaminergic activity, and (ii) compounds that target the chromatin-associated enzyme PARP1, which is a key regulatory molecule for differentiation and proliferation32, 33. By contrast, 17 compounds influence immature hematopoietic cell subsets (Fig. 2a and Supplementary Table 7). These compounds aren't associated with a single therapeutic class, yet their molecular targets are enriched for processes associated with oxidoreductase activity and alkylation repair, both of which are important for differentiation and maintaining stem cell integrity34, 35.
To further characterize the drug-immune cell interactome, we performed unsupervised hierarchical clustering with multiscale bootstrap resampling36. Out of the 143 cell type changes, 119 fit into one of 25 stable cell clusters (P < 0.05, multiscale bootstrap analysis) (Supplementary Table 8). Almost half (13/25) of the stable cell clusters exhibited a significant over-enrichment (E > 2, P < 0.05) for one or more cell types (Supplementary Table 9). By comparison, 1,089 drugs out of the 1,309 in total fell into one of 409 stable drug clusters (P < 0.05, multiscale bootstrap analysis) (Supplementary Table 10). Almost 87% (356/409) of the stable drug clusters showed a significant over-enrichment (E > 4, P < 0.05) for one or more therapeutic classes (anatomical therapeutic chemical (ATC) classification levels 1–3) or a molecular target (Supplementary Table 11). For example, 88 drug clusters showed significant enrichment for at least one ATC classification system level 1 description. The enrichments are driven by an abundance of anti-thrombotic agents or vitamin K and other hemostatics (B), contrast agents or diagnostic radiopharmaceuticals (V), and alkylating agents, cytotoxic antibiotics, hormone antagonists or immunosuppressants (L) (Supplementary Fig. 7).
To examine the features of stable clusters in greater detail, we identified 53 drug clusters enriched for a therapeutic class and molecular target, and intersected these clusters with the 13 cell clusters enriched for one or more cell type (Fig. 2b). The intersecting clusters revealed that specific immune cell subsets (e.g., pre-lymphocytes, monocytes, NKT cells, and gamma-delta T cells) overlap with multiple drug categories, whereas other subsets (e.g., B cells and macrophages) intersect with a couple categories. A few drug clusters (e.g., cl195, cl335) influenced multiple cell types, whereas other clusters (e.g., cl2, cl48, cl49, cl97) influenced a single cell type. The drug cluster with the greatest overlap across immune cells (cl195) was enriched for anti-neoplastic drugs that are cytotoxic antibiotics (e.g., doxorubicin (Doxil) and mitoxantrone).
We discovered a strong positive association between the number of molecular targets for a given drug and the number of interactions predicted to influence immunological state transitions (P = 1.7 × 10−5, linear regression). When we examined adverse drug interactions using the side effect resource (version 2)1, we found no relationship between the reported number of side effects for a drug and the number of immune cell interactions (P = 0.8, linear regression). However, as side effect data have a broad frequency distribution and are difficult to measure accurately2, this lack of correlation may reflect the variation inherent in the bias of capturing and reporting side effects.
Connections and substructures in the IP map
To examine possible immunological outcomes that might result from connections in the IP map, we focused on immune cell state changes between cell subsets or tissues. Drugs and cell state transitions were organized by therapeutic class and category of immune cell state transition (e.g., within a subset or between tissue) to provide an overview of all the predicted connections that are statistically significant (Fig. 3a and Supplementary Fig. 8). We identified promiscuous drugs by their interactions with a large number of subset transitions (Table 2). Given the uneven cell type distribution within the subset state changes (Supplementary Table 1), we defined drug hubs based on interactions with the greatest number of cell types, which we hypothesize could have the largest influence on the immune system. By contrast, drug islands were defined on the basis of interacting with the fewest number of cells (Fig. 3b).
Hubs were enriched for anti-neoplastic drugs (E = 13.8, P < 1 × 10−5; Supplementary Fig. 9), which could be expected given the influence these compounds have on immune cells1, 2. Hubs were also enriched for nervous system compounds, such as the selective serotonin reuptake inhibitor zimeldine (E = 1.3, P < 0.04), which was pulled off the market due to a rare, but severe adverse reaction leading to the autoimmune condition known as Guillain-Barré syndrome37, and the anti-seizure drug topiramate (Topamax), which was shown to be an effective treatment for inflammatory bowel disease in a preclinical model21 (Supplementary Fig. 10a). By contrast, drug islands were enriched for metabolic drugs that included the anti-diabetic compounds gliquidone and metformin (Fortamet, Glumetza; Supplementary Fig. 10b). This metabolic island in the IP map mirrors the low connectivity found in metabolic diseases in the human disease network38.
To explore drugs predicted to influence a portion of the adaptive immune system, we identified a subnetwork based on the largest magnitude immunemod scores for T-cell subsets and tissues (Supplementary Fig. 11). This subnetwork includes more than 1,000 compounds predicted to influence CD4+ or CD8+ subsets, with 113 and 202 compounds unique to each group, respectively. The top immunemod score for this subnetwork is between CD4+FoxP3+ T cells and guanfacine (Tenex, Intuniv; Supplementary Figs. 12 and 13a). Guanfacine is an α2A receptor (ADRA2A) agonist used for lowering blood pressure and treating attention deficit hyperactivity disorder (ADHD)39.
To verify that the immunemod score identifies a drug's influence on a specific cell subset, we administered the anti-hypertensive drug guanfacine to mice and measured the percentages of regulatory T-cell subsets isolated from spleens. Based on the immunemod score direction, we reasoned the frequency of regulatory T cells should increase following treatment with guanfacine. In comparison to untreated mice, the treated mice showed a 5% increase in the average frequency of CD62L+ cells within the CD4+FoxP3+ T-cell compartment (42.0% vs. 37.0%: n = 15 treated vs. n = 14 untreated, P = 0.01; Supplementary Fig. 13b).
When we examined the CD8+ subsets, the top immunemod score was to the anti-Parkinsonian drug trihexyphenidyl (Artane). One molecular target for trihexyphenidyl is the muscarinic acetylcholine receptor M1 (CHRM1). When the gene that encodes for this molecular target is knocked out in a mouse model, CD8+ cells from these mice exhibit defective cytotoxic capability40.
Validation of drug–immune cell interactions in humans
Immune cell data were collected from mice, and drug perturbation data were gathered from human cancer cell lines. One concern with integrating these data is whether the findings from mice translate to humans. We tested whether interactions predicted by the IP map could be observed in humans by examining immune cell counts of patients administered drugs versus untreated patients. To compare patient populations, we examined complete blood counts for more than 2.3 million electronic medical records in the Mount Sinai Hospital System and selected individuals who were treated and had blood cell counts collected within 1 month of receiving a drug.
Given the constraints of routine clinical laboratory tests found in electronic medical records, we restricted IP map predictions to two common drugs predicted to influence monocytes and neutrophils. The IP map predicted the general anesthetic propofol (Diprivan) and the anti-hypertensive spironolactone (Aldactone) would increase neutrophils and monocytes, respectively (Fig. 3c). Propofol increased neutrophil counts by 2,500 cells/mm3, and spironolactone increased monocyte frequencies 1.6% (Fig. 3d). Although the cell population changes were small, both shifts were significant (P < 1 × 10−100, Wilcoxon rank sum). Furthermore, the neutrophil increase shifted most patients beyond the upper normal range. To validate these observations, we examined the same drug-immune cell pairings in the electronic medical records of Columbia University Medical Center. This independent data source showed the same direction and significance for both drugs and their predicted influence on immune cells (Supplementary Table 12).
Validation of clioquinol influencing neutrophil migration
To assess the accuracy and specificity of a predicted interaction in the IP map, we experimentally validated the influence of the drug clioquinol on neutrophil migration from the bone marrow to the blood. This hypothesis emerged from a prediction with a top immunemod score (Fig. 4a), as well as the desire to identify a drug that could modify immune system dynamics between tissues and would be straightforward to evaluate with an abundant cell type in vivo. Moreover, neutrophil regulation plays a critical role in health and disease so new drugs that modify their kinetics might have therapeutic potential41, 42, 43.
We selected two drugs for the experiment on the basis of their immunemod scores and P-values. These statistical metrics identified the highest immunemod score, and the corresponding lowest P-value, for the predicted pairing between neutrophils and clioquinol (Fig. 4a). Clioquinol is an anti-fungal and anti-protozoal drug without a known mechanism of action, but the compound has been tested in a preclinical model for Alzheimer's disease44, 45. As a control, we selected the anti-viral and anti-Parkinsonian drug amantadine (Symmetrel) as a control drug because our algorithm predicted an immunemod score of zero for the amantadine-neutrophil interaction (Fig. 4b). Based on the immunemod scores, we reasoned that clioquinol would influence neutrophil migration from the bone marrow to the blood, whereas amantadine would have no influence on neutrophil migration (Fig. 4b).
To evaluate the predicted influence of clioquinol on neutrophils in vivo, we injected C57BL/6 mice with clioquinol or vehicle control (n = 8 treated and n = 8 untreated). Following the treatment with clioquinol, but not the control, neutrophils were recruited to the peritoneal cavity (Fig. 4c,d and Supplementary Fig. 14). In mice treated with clioquinol, more than 70% of the hematopoietic cells in the cavity were neutrophils, whereas in mice treated with vehicle alone, less than 5% were neutrophils, similar to untreated mice. This recruitment coincided with neutrophil mobilization from bone marrow to blood (Fig. 4c,d and Supplementary Fig. 14). Moreover, qPCR analysis of collected tissue samples revealed increased abundance of transcripts of neutrophil-specific chemokines such as Cxcl1, Cxcl2 and Cxcl5 (Fig. 4e). We also detected Cxcr2 transcript, which suggests infiltration of neutrophils in the analyzed samples.
As predicted, in mice treated with amantadine there was no significant change in the frequencies of neutrophils in blood, bone marrow or peritoneal cavity (Fig. 4d). To survey a broader array of immune cell changes following treatment, we performed mass cytometry (CyTOF) using a panel of 19 markers to evaluate cell differences in the spleens of mice treated with clioquinol or vehicle control. Consistent with what we observed by flow cytometry, neutrophil numbers increased (Ly6G+CD11b+ cells) after treatment with clioquinol (Fig. 4f). Furthermore, when we examined the complete set of markers using SPADE trees46, we found an increase in naive CD8+ cells (CD45+CD3+CD4−CD8+Thy1.2+TCRb+CD62LhiCD44lo) (Supplementary Fig. 15), which was also predicted by our algorithm albeit with a lower immunemod score (Fig. 4a).
We describe an integrative computational approach to map the effects of drugs on immune function. We compared chemogenomic and immunogenomic profiles and created an immunemod score to quantify the likely influence of a drug perturbation on an immune cell based on the overlap of their transcriptional profiles. Although the complete set of interactions between drugs and immune cells is larger than what we have modeled here, our systematic examination of almost 400,000 potential interactions is a step toward mapping this massive space. In vivo experiments to confirm one prediction—that the selective alpha-2A adrenergic receptor agonist guanfacine increases the proportion of regulatory T cells—suggested that a drug used to treat hypertension and anxiety might be repurposed to promote peripheral tolerance. To examine the utility of using the IP map to identify drugs with the highest immunemod scores for a given immunological state transition, we predicted that the change in neutrophil proportions between the blood and bone marrow would be most influenced by the drug clioquinol. This inference is supported by the proportions of neutrophils collected from blood and bone marrow and the pattern of neutrophil-specific genes expressed in various tissues. A previous study that used compounds to manipulate Cxcr2 and Cxcr4 expression levels showed neutrophil mobilization patterns similar to what we observed47. Although clioquinol has neurotoxic effects, these findings imply its potential use as a short-term neutrophil booster in certain contexts. Additionally, this finding suggests that our approach could enable the discovery of compounds that control neutrophil kinetics to resolve inflammatory responses48.
A limitation of the IP map is that it combines data sets from mouse and human, and therefore the predicted connections might not translate to human immune cells. Several recent studies have shown both similarities and differences between the transcriptional profiles of immune cells in mice and humans49, 50, 51, 52. However, both of our predictions were confirmed in electronic medical records at Mount Sinai and in an independent data set from Columbia University. Taken together, our computational analyses and experimental results suggest that the IP map captures immune responses in both humans and mice.
Another limitation is that the CMap drug profiles were generated on a limited set of cancer cell lines using whole-genome transcriptional profiling. Although the efforts of the LINCS project (http://www.lincsproject.org/) will greatly expand the number of compounds and include more cell lines, it is likely worthwhile to generate immune-specific CMap signatures on at least a subset of immune cell types to further evaluate and improve the immunemod scoring method. Efforts such as the extensive transcriptional profiling of human cell lineage differentiation53 must be extended for a more comprehensive picture of human immunity, which will help us better understand how the ImmGen data will translate across species. We did not use chemical structure information, and we acknowledge that incorporating structural information and data other than transcriptional profiles54, 55 would provide a more complete picture of the complexity of drug effects on the immune system.
The statistical bioinformatics method we used for systematically exploring drug-immune cell interactions follows a Kolmogorov-Smirnov (KS) approach similar to that used by numerous other studies20, 23, 56. This method has been useful at identifying numerous biological connections that have been subsequently validated by experimentation. However, a limitation of the traditional KS approach using transcriptional data is an assumption of statistical independence among transcripts. Others have recently proposed potential solutions for this limitation57, 58, 59, 60. When we incorporated a principal component analysis (PCA)-based approach57 into our methods, the P-values did rise as expected. Under this alternative null model, the number of significant interactions decreases by about a factor of 3 from the independent shuffling method (Supplementary Fig. 5). However, it appears that PCA-based correction may be overly pessimistic at low FDR thresholds57, 58, 59, 60. This observation seems to be reflected in our own analysis where we find that all of the experimentally tested and validated interactions fall above the significance threshold subsequent to PCA-adjusted permutation. To our knowledge, there has not yet been a systematic analysis of the various proposed enrichment analysis methods on chemogenomic data based on uncorrelated gene sets. Systematic evaluation of permutation and expression de-correlation approaches for large-scale chemogenomic connectivity mapping is a fruitful area for future studies, especially as the chemogenomics community embraces reduced probe set arrays using the L1000 platform.
The apparently unknown interactions identified in the IP map may include many that warrant experimental follow-up. Other possible applications of our data include studying the contribution of immune cells to adverse drug reactions, the role of immune cell subsets in cancer and other diseases, and combination drug therapies. Moreover, global trends extracted from our data could provide guidelines and specific predictions on how to manipulate immune cells, uncover drug mechanisms of action, and select alternative compounds from the same therapeutic category with fewer immune cell side effects.
Drug and immune cell gene expression data.
Drug-induced transcriptional profile changes determined from human cancer cell lines were obtained from the Connectivity Map (CMap) database23. We processed and analyzed version 2, which included 6,100 experiments using 1,309 compounds. Preprocessing and normalization steps were performed, as described previously23. To make cross-platform comparisons compatible, we standardized gene identifiers from microarray-specific probe identifiers to NCBI GeneID identifiers, selecting the maximum across individual probe expression values. To create a single rank-ordered expression profile for each of the 1,309 compounds, we merged multiple experiments for the same compound into a single Prototype Ranked List (PRL) following the processing described previously28, 29. The final data set included 13,071 differential gene expression values for each of the 1,309 compounds.
Immune cell gene expression data collected from steady-state profiling of 249 distinct cell types were obtained from the ImmGen24. Preprocessing and normalization were performed as described previously61. As cell profiles were collected at steady state, we selected 221 unique cell types and created 304 differential state signatures from the difference between two steady-state profiles (Supplementary Table 13). To make cross-species and cross-platform comparisons reasonable, we standardized gene identifiers from microarray-specific probe identifiers to NCBI GeneID identifiers, mapped mouse GeneID identifiers to their human ortholog, and selected the maximum across individual probe expression values. Finally, we converted differential state profiles to ranked lists ordered by differential expression values, creating a data set with 11,153 differential gene expression values for each of the 304 immunological state changes.
IP map construction.
We constructed a matrix of predicted interactions between each of the 1,309 drugs and 304 immunological state changes using a rank-based, pattern-matching strategy described previously20. (All Input Data, and Supplementary Source Code have been deposited at Synapse hosted by Sage Bionetworks. https://www.synapse.org/#!Synapse:syn4877787/files/) Briefly, for each trio of drug, cell and gene set size (d, c, s), we calculated an immunemod score (ImS) based on the degree of overlap between drug and immune cell gene sets at the extremes of the two ranked signatures. To obtain a measure of significance for the immunemod score, we shuffled the genes in the drug rank signature and calculated a permuted immunemod score (ImS*) for each drug, cell and gene set triplet (ImS*(di, cj, sk)]. We calculated the P-value for each ImS by counting the number of randomized scores ImS*(di, cj, sk) that were greater than or equal to the absolute value of the actual scores ImS(di, cj, sk) and dividing by the number of permutations (nperms = 1,000). This permutation strategy sets the lower bound for P-values at 0.001, which yields a biased estimate for the number of false positives given the number of hypotheses under consideration. To provide accurate P-values at the lower range while containing the computational cost, we used the generalized Pareto distribution to model the P-value distribution and calculated improved estimates for low P-values (counts < 1/100) based on the distribution of permuted immunemod scores62. We adjusted the P-values63 and selected an FDR of 5% as the cutoff for significance. To control for spurious interactions based on the size of the gene set used for matching, we varied the size of the matching set between 100 and 250 genes for each of the top and bottom extremes and recalculated all immunemod score, P-value pairs for every drug-cell interaction. The proportion of times each drug-cell interaction was significant among all sizes of gene sets provides a relative strength for any given interaction. A predicted interaction was considered to be strong and stable if it was significant for 85% or more of the set sizes.
To assess the similarity between expression profiles of immune cell subsets, we calculated the Jaccard distance among all pairs of extreme fold-change genes, and used an ANOVA to evaluate the differences between immune cell subsets. We investigated a series of diagnostic plots and did not find significant deviations that would violate the assumptions of normality or homoscedasticity.
To organize the drugs and immune cells in an unbiased manner, we applied hierarchical clustering to the full interaction matrix using the computed Pearson correlation coefficient as a distance metric between immunemod scores and complete linkage clustering to agglomerate drugs or cells. We used the pvclust R package36 to compute a bootstrap analysis of the clusters and identified a significant cluster if the approximately unbiased probability was >95%.
To determine the enrichment of an anatomical therapeutic class category, we calculated the fold-change and P-value. Fold-change enrichment (E) was calculated as a ratio of ratios E = (a/b)/(c/d), where a is the number of drugs with a particular category (e.g., “L”) in the cluster of interest, b is the number of drugs with that category in the overall data set, c is the total number of drugs in cluster and d is the total number of drugs overall. We used the hypergeometric distribution to calculate the P-value and assess the significance of each enrichment calculation.
To examine the association between chemical features (e.g., molecular targets and drug side effects) and number of immune cell interactions, we implemented a simple linear regression model. Chemical features followed a skewed distribution so we log-transformed the data, which adjusted the values so they followed a normal distribution. Based on diagnostic plots of the transformed data, we did not find deviations that violated the assumptions of normality and homoscedasticity that are central to regression models.
To test whether drug treatment with clioquinol or amantadine produced any difference in neutrophil cell frequencies in various tissue compartments, we used an ANOVA model to compare the treatment groups. Multiple group testing and P-values were evaluated using Tukey's honest significant difference. For all ANOVA tests, we generated a series of diagnostic plots to examine: (i) the residual errors for outliers, (ii) the QQ plots for normality, and (iii) the square root of the standardized residuals for heteroscedasticity. In all cases, we did not find significant deviations that would violate the assumptions used in the ANOVA model. For comparison, we evaluated the treatment and control groups directly via the Wilcoxon rank sum test and found the median differences between treatment (clioquinol, amantadine) and controls (PEG400, PBS) to follow the exact same pattern obtained using the ANOVA, with a similar maximum P-value for significant differences (P ≤ 0.01). To test whether guanfacine influence regulatory T-cell frequencies, we used a meta-analysis strategy64 to combine experimental conditions and groups, which allowed us to ascertain whether the overall differences from each independent experiment were robust and significant.
Electronic medical records.
We pulled all patient entries from the Mount Sinai Electronic Medical Records that contained complete blood count information on neutrophils and monocytes (more than 2.3 million entries in total). To determine if either propofol or spironolactone were associated with a change in cell counts, we identified patient entries that had laboratory values measured within 30 d of drug administration versus patient entries that never received drug. We tested for group-level differences using the nonparametric Wilcoxon rank sum test. The findings were validated using the Electronic Medical Records of Columbia University Medical Center, where we employed the same criteria for patient selection and performed a Wilcoxon rank sum test for group differences.
Circos plots created using the circlize R package (version 0.0.7 https://github.com/jokergoo/circlize). Network diagrams produced using Cytoscape65 and SPADE trees generated with CytoSPADE66. All other plots created using the R statistical package.
Mice and drug treatment.
6- to 12-week-old female C57Bl/c mice were obtained from Jackson Laboratories. Mice received intraperitoneal injections three times every 12 h with clioquinol, amantadine (both at 30 mg/kg per dose, Sigma Aldrich) or appropriate controls. Dosing level and frequency were chosen based on previous experiments using clioquinol in mice45, 67 and the drug half-life (11–14 h). Clioquinol was dissolved in 8% PEG400/PBS heated to 37 °C; amantadine was dissolved in PBS. Before injection, the solutions were shaken several times. Mice were euthanized for tissue collection between 2.5 and 3 h after the last treatment. Blood collection was obtained from the tail. For guanfacine treatment, mice received initial injection of 5 mg/kg of drug or vehicle control (PBS) on day 1, followed by two (experiment 1) or six (experiments 2–5) intraperitoneal injections at 2 mg/kg every 12 h starting on day 2. Mice were euthanized for tissue collection 12 h after the last treatment. All animal procedures were done according to protocols approved by the Mount Sinai School of Medicine Institutional Animal Care and Use Committee.
Peritoneal cavity cells were collected by washing with cold PBS containing 4% FBS. Single-cell suspensions of bone marrow were obtained by flushing femurs, followed by filtration through a 100-μm cell strainer (BD Biosciences). Red blood cells were lysed for 2 min at room temperature with RBC lysis buffer (eBioscience). Samples were stained with the following antibodies (all from eBioscience): allophycocyanin-eFluor780-conjugated CD45 (30-F11), peridinin chlorophyll protein–cyanine 5.5-conjugated CD11b (M1/70), phycoerythrin-conjugated Ly6G (RE6-8C5), phycoerythrin-conjugated CD3 (145-2C11), peridinin chlorophyll protein–cyanine 5.5-conjugated CD25 (PC61.5), fluorescein isothiocyanate-conjugated CD62L (MEL-14), allophycocyanin-conjugated FoxP3 (FJK-16s), eFluor450-conjugated CD4 (GK1.5), allophycocyanin-eFluor780-conjugated CD8a (53-6.7), and allophycocyanin-conjugated CD44 (clone IM7, BD Pharmingen). DAPI was used to label dead cells. LSR Fortessa was used for sample acquisition and FlowJo software for data analysis.
RNA isolation and quantitative PCR.
Total RNA was extracted from pieces of lung, liver, spleen and bone marrow cells using QIAzol Lysis Reagent (Qiagen) and glycogen blue (Ambion, Life Technologies) according to the manufacturer's instruction. For cDNA synthesis, 2 μg total RNA was reverse-transcribed for 1 h at 37 °C with an RNA-to-cDNA kit (Applied Biosystems). For quantitative PCR, SYBR green qPCR master mix 2× (Fermentas, Thermo Scientific) and the following primers were used: mouse Actb forward, 5′-CTAAGGCCAACCGTGAAAAG-3′, and reverse, 5′-ACCAGAGGCATACAGGGACA-3′; mouse Cxcl1 forward, 5′-GTGTTGCCCTCAGGGCC-3′, and reverse, 5′-GCCTCGCGACCATTCTTG-3′; mouse Cxcl2 forward, 5′-ACGCCCCCAGGACCC-3′, and reverse, 5′-CTTTTTGACCGCCCTTGAGA-3′; mouse Cxcl5 forward, 5′-CTCGCCATTCATGCGGAT-3′, and reverse, 5′-CTTCAGCTAGATGCTGCGGC-3′; mouse Cxcr2 forward, 5′-CTTTGCCCTGACCTTGCCT-3′, and reverse, 5′-GCACAGGGTTGAGCCAAAA-3′; mouse Cxcr4 forward, 5′-TGGCCTTCATCAGCCTGG-3′, and reverse, 5′-TTGGCCTTTGACTGTTGGTG-3′.
Mass cytometry (CyTOF) analysis of mouse spleen.
To obtain single-cell suspensions, spleens were digested for 20 min at 37 °C in HBSS containing 8% FBS and 0.2 mg/ml collagenase IV (Sigma Aldrich). After filtration through a 100-μm cell strainer, red blood cells were lysed for 2 min at RT with RBC lysis buffer. Cells (5 × 106 per sample) were stained for the following surface markers: 141Pr-Ly6G, 153Eu-PDCA1, 162Dy-Ly6C, 166Er-CD138 (all prepared in-house) and 142Nd-CD11c, 147Sm-CD45, 148Nd-CD11b, 149Sm-CD19, 151Eu-CD25, 152Sm-CD3e, 156Gd-Thy1.2, 160Gd-CD62L, 168Er-CD8, 169Tm-TCRb, 170Er-NK1.1, 171Yb-CD44, 172Yb-CD4, 174Yb-MHCII and 176Yb-B220 (all from DVS Sciences). Cisplatin was added for the final 5 min to label dead cells and samples were fixed using Fix and Perm buffer (DVS Sciences). Immediately before injection, EQ Four Element Calibration Beads were added and samples were run on CyTOF 2 mass cytometer (DVS Sciences) in three 10-min acquisitions rounds. Data were normalized to EQ Beads and files concatenated using DVS software.
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We thank G. Hoffman and B. Readhead for useful conversations about the computational methods and suggestions on the manuscript, L. Li for assistance with the electronic medical records from Mount Sinai, K. Oguntuyo for assistance with the qPCR reactions, and A. Rahman for sample preparation and acquisition on the CyTOF. This work was supported by the US National Institutes of Health grants R01AI104848, R33CA182377 and R01AI113221 (to B.D.B.), U54CA189201 and R01DK098242, and Jonathan E. Gray TCI Young Scientist Cancer Research Award (to J.T.D.).
- Supplementary Figure 1: Cell type and tissue location counts in the Immunological Genome data set. (76 KB)
Barplots indicate the sums of the table rows and columns. Abbreviated cell type labels represent: Neut = neutrophil, Mono = monocyte, NKT = natural killer T cell, NK = natural killer cell, Mac = macrophage, T4 = CD4+ T cell, T8 = CD8+ T cell, Tgd = gamma-delta T cell, DC = dendritic cell. Abbreviated tissue labels represent: BM = Bone marrow, scLN = Subcutaneous lymph nodes, msLN = mesenteric lymph nodes, sdLN = skin-draining lymph nodes, SI = Small intestine, FLN = Follicular lymph nodes, IE = Intraepithelial, mdLN = mediastinal lymph nodes, PP = Peyer’s patch, AA = abdominal adipose, LN = Lymph nodes, MsLN = Medullary sinus lymph nodes, PLN = Pancreatic lymph nodes, SF = synovial fluid, ssLN = Subcapsular lymph nodes.
- Supplementary Figure 2: Clustered heatmap of cell state change similarity. (163 KB)
Hierarchical clustering applied to the matrix of Jaccard distances calculated between gene sets from the extremes—top/bottom 5% fold-change—of each cell state change expression profile. The full heatmap shows complete linkage clustering for all pairwise combinations of the 304 cell state changes. Colored bars on the rows and columns represent the categories of state changes and the cell types respectively.
- Supplementary Figure 3: Jaccard distance between immune cell state change gene sets. (61 KB)
Sets based on the extremes—top/bottom 5% fold-change—of each cell state change expression profile. Each bar height reflects the average distance within a cell type and the error bars are the s.e.m. Gray horizontal line shows the average distance between all gene sets. Group differences assessed by ANOVA.
- Supplementary Figure 4: Schematic depiction of our method to test for significant interactions and identify robust connections. (98 KB)
(a) Diagram of ranked list comparison to calculate the immunemod score for each pair of drug (j) and cell state change (k) at a particular gene set size (l). (b) Gene set size variation procedure to evaluate the robustness of each predicted interaction. (c) Schematic of the permutation testing to assess the significance of an immunemod score for a given drug (j), cell state change (k), and gene set size (l).
- Supplementary Figure 5: Quantile-Quantile plot for gene expression models. (54 KB)
P-value distribution of independent (naïve) and correlated permutation models for the IP map.
- Supplementary Figure 6: Number of predicted interactions between drugs and immune cells across all categories of immunological cell state changes. (98 KB)
(a) Overall, (b) Subset, (c) Tissue, (d) Genetic, (e) Time, and (f) Perturbation.
- Supplementary Figure 7: Drug class enrichments among the stable drug clusters. (42 KB)
Enrichment value for each of the 13 level 1 ATC categories: H = Systemic hormonal preparations, excl. sex hormones and insulins, V = Various, B = Blood and blood forming organs, P = Anti-parasitic products, M = Musculo-skeletal system, L = Anti-neoplastic and immunomodulating agents, G = Genito urinary system and sex hormones, R = Respiratory system, A = Alimentary tract and metabolism, D = Dermatologicals, J = Anti-infectives for systemic use, S = Sensory organs, N = Nervous system, and C = Cardiovascular system. Asterisks indicate significant over- or under-enrichment (P < 0.01 and E > 1 or E < 1 respectively). Significance assessed by hypergeometric distribution.
- Supplementary Figure 8: Circular layout of the predicted connections between drugs and cell subsets found in different tissues. (262 KB)
All connections statistically significant P < 0.05 with FDR < 5%, see methods. Line widths correspond to the number of interactions. A drug-tissue connection represents a drug that is predicted to influence a particular immune cell in the specific tissue as compared to the same cell in a contrasting tissue. The diagram has been organized by sorting the tissues counter-clockwise (drugs clockwise) in order of decreasing number of connections. Each single letter for of the drug half corresponds to a level 1 label of the ATC system. H = Systemic hormonal preparations, excl. sex hormones and insulins, V = Various, B = Blood and blood forming organs, P = Anti-parasitic products, M = Musculo-skeletal system, L = Anti-neoplastic and immunomodulating agents, G = Genito urinary system and sex hormones, R = Respiratory system, A = Alimentary tract and metabolism, D = Dermatologicals, J = Anti-infectives for systemic use, S = Sensory organs, N = Nervous system, and C = Cardiovascular system. Codes for each tissue label are as follows: PC = peritoneal cavity, br = brain, AA = abdominal adipose, pan = pancreas, SI = small intestine, SF = synovial fluid, LN = lymph nodes, PLN = pancreatic lymph nodes, mdLN = mediastinal lymph nodes, FLN = follicular lymph nodes, thy = thymus, sdLN = skin-draining lymph nodes, PP = Peyer’s patch, scLN = subcutaneous lymph nodes, msLN = mesenteric lymph nodes, and BM = bone marrow.
- Supplementary Figure 9: Distribution and enrichment of drug hubs and islands. (94 KB)
(a) Histogram of drugs binned according to the number of unique cell types they are predicted to interact with (top right shows drug hubs with connections to 11 or more cell types). ATC level 1 categories enriched in drug (b) islands and (c) hubs. Significance assessed by fold-enrichment analysis and hypergeometric distribution.
- Supplementary Figure 10: Drug hubs and islands. (89 KB)
Examples of drug (a) hubs or (b) islands associated with immune cell types.
- Supplementary Figure 11: Two-dimensional colored heatmap of the T cell subnetwork pulled from the IP map. (209 KB)
Data organized by drug (rows) and T cell categories (columns). Predicted interactions between drug categories (ATC level 1 categories: H = Systemic hormonal preparations, excl. sex hormones and insulins, B = Blood and blood forming organs, V = Various, L = Anti-neoplastic and immunomodulating agents, P = Anti-parasitic products, M = Musculo-skeletal system, G = Genito urinary system and sex hormones, R = Respiratory system, S = Sensory organs, D = Dermatologicals, A = Alimentary tract and metabolism, N = Nervous system, J = Anti-infectives for systemic use, and C = Cardiovascular system) and (left) T cell subsets “Cell Types” or (right) identical T cell subset found in different tissues. All connections are statistically significant (P < 0.05 and FDR < 5%, see methods). Each colored cell in the heatmap represents the fraction of drugs from a particular ATC level 1 category (0–0.7, max = 1.0) predicted to influence the specific Cell Type or Tissue location. Colored circles represent the number of drugs in each category where the size is proportional to the number of unique drugs (16–144).
- Supplementary Figure 12: Drugs predicted to influence T cell subset changes. (175 KB)
T4 = CD4+ cells, T8 = CD8+ cells, FP3 = FoxP3, mem = memory subset. Drugs ranked in descending order by immunemod score.
- Supplementary Figure 13: Influence of guanfacine on regulatory T cells. (57 KB)
(a) Graph showing connections for a single drug, Guanfacine, predicted to influence regulatory T cells based on the immunemod score (shown as numbers next to each node). (b) Percentage of regulatory T cells (CD4+FoxP3+CD62L+) isolated from mouse spleens following treatment with the anti-hypertensive drug guanfacine. Each circle is a separate mouse from five separate experiments—grouped by color—for a total of 14 or 15 mice in the Ctrl and Guanfacine groups respectively. Dashed lines connect the mean frequency values between groups in each experiment. Significance assessed by meta analysis (see below).
Morgan, A.A., Khatri, P., Jones, R.H., Sarwal, M.M. & Butte, A.J. Comparison of multiplex meta analysis techniques for understanding the acute rejection of solid organ transplants. BMC Bioinformatics 11 Suppl 9, S6 (2010).
- Supplementary Figure 14: Flow cytometry plots. (174 KB)
Representative biaxial flow cytometry plots showing the gating strategy for identifying Ly6G+CD11b+ cells from blood, bone marrow and peritoneal cavity of treated and untreated mice. Data are representative of three to four experiments with 8 mice in each condition.
- Supplementary Figure 15: SPADE trees of CyTOF data collected during a single experiment. (199 KB)
Cells isolated from spleens of mice treated with drug (clioquinol) and vehicle control (PEG400). Trees show the lymphocyte lineage of CD45+ cells. Key for trees shown to orient toward CD4+ and CD8+ cells. Arrows highlight CD45+CD3+CD4–CD8+Thy1.2+TCRb+CD62LhiCD44lo population increase after clioquinol treatment. Horizontal color scales represent median intensity (blue = low, red = high) for marker indicated.
- Supplementary Text and Figures (15,726 KB)
Supplementary Figures 1–15