Complement Component C1q as Serum Biomarker to Detect Active Tuberculosis

Abstract

Background: Tuberculosis (TB) remains a major threat to global health. Currently, diagnosis of active TB is hampered by the lack of specific biomarkers that discriminate active TB disease from other (lung) diseases or latent TB infection (LTBI). Integrated human gene expression results have shown that genes encoding complement components, in particular different C1q chains, were expressed at higher levels in active TB compared to LTBI. Methods: C1q protein levels were determined using ELISA in sera from patients, from geographically distinct populations, with active TB, LTBI as well as disease controls. Results: Serum levels of C1q were increased in active TB compared to LTBI in four independent cohorts with an AUC of 0.77 [0.70; 0.83]. After 6 months of TB treatment, levels of C1q were similar to those of endemic controls, indicating an association with disease rather than individual genetic predisposition. Importantly, C1q levels in sera of TB patients were significantly higher as compared to patients with sarcoidosis or pneumonia, clinically important differential diagnoses. Moreover, exposure to other mycobacteria, such as Mycobacterium leprae (leprosy patients) or BCG (vaccinees) did not result in elevated levels of serum C1q. In agreement with the human data, in non-human primates challenged with Mycobacterium tuberculosis, increased serum C1q levels were detected in animals that developed progressive disease, not in those that controlled the infection. Conclusions: In summary, C1q levels are elevated in patients with active TB compared to LTBI in four independent cohorts. Furthermore, C1q levels from patients with TB were also elevated compared to patients with sarcoidosis, leprosy and pneumonia. Additionally, also in NHP we observed increased C1q levels in animals with active progressive TB, both in serum and in broncho-alveolar lavage. Therefore, we propose that the addition of C1q to current biomarker panels may provide added value in the diagnosis of active TB.

Keywords: C1q; blood; complement; infection; innate immunity; mycobacterium; tuberculosis.

Figure 1

Differentially expressed complement genes in whole blood tuberculosis transcript signatures. Tuberculosis specific transcript signatures, from various populations, were investigated for the presence of differentially expressed complement genes using a tuberculosis RNA biomarker database. Publically available transcriptome data was retrieved from Gene Expression Omnibus (–, –37) and analyzed using GEO2R. Data were available for nine populations comparing active TB with LTBI and for 8 populations comparing active TB with other diseases. For each population we determined if the complement family genes were differentially expressed between TB and LTBI or other diseases. Differential expression was defined as an adjusted p-value <0.05 and more than 2-fold change. Differential expression of a gene between TB and LTBI or other diseases was expressed as percentage of the total number of populations investigated. The differential expression of complement genes was scored (A,B) as well as the mean factorial change for the C1q genes, C1QA, C1QB, and C1QC (C,D) for the comparisons TB vs. LTBI (A,C) and TB vs. other diseases (B,D). As a reference two other highly upregulated potential diagnostic TB markers, FCGR1A, and GBP5, were included in the analyses.

Figure 2

C1q serum levels are increased in patients with pulmonary Tuberculosis. C1q levels (μg/ml) were measured with ELISA in sera from TB patients (active disease) and controls from different cohorts. First the results from the independent and geographically different cohorts are depicted. TB patients from Italy were compared to Latent TB infected (LTBI), to Past TB (patients that were successfully treated for TB) and to endemic controls (A). TB patients from The Gambia were compared to TB contacts (B). TB patients from Korea were compared to endemic controls (C). From South Africa the TB patients were compared to patients suspected for TB but confirmed non-TB (D). Subsequently, data from the cohorts were pooled: control (CTRL) comprises Dutch healthy controls, combined with the CTRL from Italy and Korea; moreover, healthy individuals prior to vaccination with BCG were included (n = 117). Latent TB infected (LTBI) comprises LTBI from Italy, TB suspects (confirmed non-TB) from South Africa and TB contacts from the Gambia (n = 100). Active tuberculosis (TB) from Italy, the Gambia, Korea and South Africa (n = 99); Past TB are patients that were successfully treated for TB and combined the past TB from Italy and the Gambia samples after 6 months of treatment (n = 71) (E). From the Gambia the TB patients were followed over time during treatment, TB contacts are shown on the right (n = 50) (F). Results were analyzed using the Mann-Whitney U test, >2 groups Kruskall-Wallis and Dunn's multiple comparisons test. The treatment months were compared to TB diagnosis with Friedman Test and Dunn's multiple comparisons test.

Figure 3

BCG vaccination does not induce a similar C1q upregulation. Healthy individuals who were vaccinated with BCG (n = 13), were followed over time and C1q serum levels were measured. C1q levels were first calculated to μg/ml and for each individual set to 1 using the measurement of the C1q level before vaccination. The dotted lines indicate the variation that is present in C1q levels at the time of screening and prior to vaccination.

Figure 4

Increased C1q serum levels are associated with TB. C1q was measured in various cohorts by ELISA. The pooled data from Figure 2E control population (CTRL) and TB (active disease) is now compared to other diseases: leprosy (n = 86), sarcoidosis (n = 50), and community acquired pneumonia (n = 68) (A). Differences between groups were analyzed using Kruskall-Wallis and Dunn's multiple comparisons test. Both the leprosy and the pneumonia cohort comprise two patient groups. The data for these individual groups is visualized in Supplementary Figure E1. ROC analysis of the ability of C1q to distinguish TB from CTRL, LTBI, sarcoidosis, and pneumonia are plotted together (B) and for all comparisons the Area Under the Curve (AUC) was calculated and summarized in the table (C).

Figure 5

C1q accumulates in lung tissue of patients with fatal pulmonary TB. Lung tissue, obtained at autopsy from a non-pulmonary disease control (n = 1), fatal active TB patients (n = 3), or patients with lethal pneumonia (n = 3) were stained for the presence of C1q. The left column shows the presence of C1q in a section of the control, two active TB patients and a pneumonia patient, scale bar at 200 μm. The middle column shows the same samples stained for C1q, while the right column shows consecutive tissue slides stained with a matched control antibody, scale bars at 50 μm. Necrotic areas that stain positive for C1q are highlighted with an asterisk (*) and individual cells that stain positive for C1q are highlighted with arrows.

Figure 6

Rhesus macaques infected with Mycobacterium tuberculosis display increased levels of C1q in serum and broncho-alveolar lavage fluid. C1q levels were measured in serum samples from rhesus macaques (n = 23) which were infected with Mycobacterium tuberculosis and followed over time. C1q levels were first calculated as Arbitrary Units (AU) per ml after which the C1q level of each animal at baseline was set as 1. Animals which controlled the infection are termed non-progressors and animals reaching a premature humane endpoint due to active disease progression are termed acute progressors (A). Separately, C1q was measured in broncho-alveolar lavage (BAL) fluid obtained from animals before and 6 or 12 weeks after infection with M.tb (n = 6) and calculated as AU/ml (B).