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Identification of promising plasma immune biomarkers to

differentiate active pulmonary tuberculosis
Fabiana A. Zambuzi a, Priscilla M. Cardoso-Silva a, Milena S. Espindola a, Luana S. Soares
a
, Leonardo J. Galvão-Lima a, Verônica S. Brauer a, Matheus S. Gomes b, Laurence R.
Amaral b,
Matthew Schaller c, Valdes R. Bollela d, Fabiani G. Frantz a,⇑
a
Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, SP, Brazil
b
Laboratorio de Bioinformatica e Analises Moleculares – INGEB/FACOM, Universidade Federal de Uberlandia, Patos de Minas, MG, Brazil
c
Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, USA
d
Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, SP, Brazil

articleinfo
abstract
Article history:
Received 6 June 2016 Although much research has been done related to biomarker discovery for tuberculosis infection, a
Received in revised form 23 August 2016 set of biomarkers that can discriminate between active and latent TB diseases remains elusive. In
Accepted 26 August 2016 the current study we correlate clinical aspects of TB disease with changes in the immune response
as determined by biomarkers detected in plasma. Our study measured 18 molecules in human
plasma in 17 patients with active disease (APTB), 14 individuals with latent tuberculosis infection
Keywords: (LTBI) and 16 uninfected controls (CTRL). We found that active tuberculosis patients have increased
Pulmonary tuberculosis plasma levels of IL-6, IP-10, TNF-a, sCD163 and sCD14. Statistical analysis of these biomarkers
Immune response indicated that simultaneous measure- ment of sCD14 and IL-6 was able to diagnose active
Biosignature tuberculosis infection with 83% accuracy. We also demonstrated that TNF-a and sCD163 were
Diagnostic correlated with tuberculosis severity. We showed that the simultaneous detection of both plasma
Prognostic
sCD14 and IL-6 is a promising diagnostic approach to identify APTB, and further, measurement of
TNF-a and sCD163 can identify the most severe cases of tuberculosis.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction ⇑ Corresponding author at: Faculdade de Ciencias Farmaceuticas de Ribeirao Preto

- FCFRP, Universidade de Sao Paulo, ZIP Code: 14041-903, Brazil.
E-mail addresses: fabianazambuzi@usp.br (F.A. Zambuzi), priscilla.mariane@
Tuberculosis (TB) represents an important public health hotmail.com (P.M. Cardoso-Silva), msespindola@usp.br (M.S. Espindola), luanasi-
prob- lem worldwide. In 2014, it was estimated that 9.6 soares@usp.br (L.S. Soares), galvaolima@usp.br (L.J. Galvão-Lima), veronica.
million people developed TB and 1.5 million died from M. brauer@hotmail.com (V.S. Brauer), matheus@ingeb.ufu.br (M.S. Gomes), laurence
@ufu.br (L.R. Amaral), mschalle@umich.edu (M. Schaller), bollela@gmail.com (V.R.
tuberculosis (Mtb) infec- tion [1]. Accordingly to the World
Bollela), frantz@usp.br (F.G. Frantz).
Health Organization, in its Glo- bal Tuberculosis report, the
male to female ratio of prevalence is around 1.5–2. This
prevalence is true whether for Africa countries and for those
considered industrialized [1]. Factors contributing to the
spread of TB include the ineffectiveness of the BCG vaccine
[2,3], the absence of rapid and sensitive techniques for
diagnosis
[4] and a lengthy and complex treatment regimen to reduce
bacte- rial burden in the host [5,6]. The incidence and the
course of TB are
related with alcohol abuse [7] tobacco, diabetes and low body
mass index, that are risk factors and worsen the scenario,
when are com- bined could triple or quadruple the risk of
active TB development [8].
Improved diagnosis of Mtb infection may lead to
increases in effective treatment of infected individuals and
decreased spread of the disease. To this end there has been
a focus on the discovery of biomarkers that can distinguish
between latent and active tuberculosis, with the goal of a
rapid and simple diagnosis of active infection [9]. A
biomarker is a molecule that can be objectively measured
and evaluated as a disease indicator and thus used as a
http://dx.doi.org/10.1016/j.cyto.2016.08.030
1043-4666/© 2016 Elsevier Ltd. All rights reserved.
diagnostic tool. Ideally, TB biomarkers would be used to
determine the stages of TB infection and also as tools for
monitoring the success of clinical intervention [10]. Although
a single biomarker has not proved sufficient for the
identification of TB infection, it is possible that a combination
of independent biomarkers (disease biosignature [11]) will
provide information needed to accurately diagnose this
disease.
Biomarkers and biosignatures are described in many infectious
diseases. Sepsis is an example of this, where measuring
levels of polymorphonuclear (PMN) CD64, procalcitonin (PCT)
and soluble
 1
triggering receptor expressed on myeloid cells-1 (TREM-1) in
serum currently function as a diagnostic tool in critically ill
sepsis patients [12]. For TB diagnosis, the accepted approach
for biomar- ker discovery has been the use of gene expression
profiles, as demonstrated by Berry et al. [13], in which
peripheral blood leuko- cytes from TB patients were found to
have elevated expression of upstream and downstream
elements of the type I and II interferon signaling pathway.
Another study has demonstrated that expres- sion of CD64,
LTF, and Rab33A constitute a biosignature, which allows
discrimination between TB patients and healthy donors [14].
Furthermore, serum levels of IL-6 may be a marker to deter-
mine the effectiveness of antibiotic treatment in patients with
active TB infection [15]. Other investigators have identified
immune mediators [reviewed by [16]], metabolic markers
[17] and a proteomics fingerprint [18] that form possible
biosignatures indicative of TB. If widely used these
biosignatures may aid in diagnosis and monitoring of
treatment effectiveness in pulmonary TB patients.
In the current study we correlated clinical aspects of
specific
stages of TB infection with changes in the immune response,
as determined by measurement of cytokines detected in
plasma. We demonstrated that the plasma levels of cytokines
in patients with active pulmonary TB were elevated when
compared to latently infected individuals or uninfected
controls. Specifically we found increased levels of TNF-a, IL-6,
IP-10 and monocyte acti- vation markers, sCD163 and sCD14.
Of these, we found that IL-6 and sCD14 were sufficient to
discriminate active TB disease from both latently infected and
uninfected individuals. Analyzed together, levels of these
molecules may discriminate between latent and active
infection and thus provide an effective TB biosig- nature.
Additionally we demonstrate that sCD163 and TNF-a may be
used to indicate the severity of Mtb infection.
2. Materials and methods
2.1. Ethical aspects
This research has approval of the Ethics Committee at the
Hospital das Clínicas de Ribeirão Preto and FMRP-USP
(Protocol #6481/2013). All patients and controls provided a
written informed consent form for participation in the study.
diameter of cavitation did not exceed 4 cm in patients
classified at this stage. Patients with advanced disease (stage
3), had lesions that were of greater extent than defined in
moderate disease. Table 1 presents the demographic and
clinical characteristics of active pulmonary tuberculosis
patients (APTB), latent infected subjects (LTBI) and non-
infected controls (CTRL).
2.3. Immune biomarkers quantification
Blood was collected using Heparin blood collection tubes
(BD Biosciences, San Diego, CA), and plasma separation was
performed to quantify cytokine, chemokine and
monocyte/macrophage biomarkers. To evaluate IL-1b, IL-4, IL-
5, IL-6, IL-10, IL-12p70,
IFN-a2, TNF-a, IFN-ɣ, IP-10, RANTES, MCP-1, GM-CSF, IL-17, MIP-
1a and MIP-1b, we used a customized multiplex kit (16-plex,
EMD Millipore Corporation, Billerica, Massachusetts, USA) and
fol- lowed the manufacturer’s instructions. Following
incubation with fluorescent magnetic microspheres coated with
capture antibodies specific for the above listed cytokines,
samples were evaluated in a fluorescent bead-based instrument
(Luminex® MAGPIX® System; Luminex Corporation, Austin, Texas,
USA). Luminex bead-based data were analyzed using Milliplex
Analyst software v3.5 (Millipore; VigeneTech Inc., Boston,
Massachusetts, USA) and a three-parameter logistic curve fit.
The concentration of soluble monocytes/macrophages plasma
biomarkers CD14 and CD163 were measured using a DuoSet
ELISA kit (R&D Systems, Minneapolis, Minn), according to the
manufacturer’s instructions.
2.4. Statistical analysis
Conventional statistical analyses were performed using
GraphPad Prism 5.0 software (Graph-Pad Software, San
Diego, CA, USA). Differences between groups were first
evaluated to test their normality. Considering the
nonparametric nature of all data sets, statistical analyses
between the groups were performed by the Mann-Whitney
test. The correlation between immune biomarkers and stages
of pulmonary lesions visualized by radiog- raphy were
calculated using Spearman rank correlation. Values of P <
0.05 were considered significant.

2.2. Study group Table 1

Baseline characteristics of the cohort.
Active TB patients with treatment-time less than one
Demographic and clinical characteristics CTRL LTBI APTB
month (n = 17), latent infected individuals (n = 14) and non-
Age (yr) 27± 31,4± 39,6±
infected con- trols (n = 16) from both genders, aged between
Men:Women 3:13 3:11 14:3
18 and 65 years and without HIV coinfection, were recruited Smoking (%) 0 0 29,4
from Hospital das Clínicas de Ribeirão Preto e Centro de Alcoholism (%) 0 0 23,5
Saúde Escola, FMRP-USP. Active disease was diagnosed Drug addiction (%) 0 0 23,5
through clinical or radiological signs and confirmed by Sign and symptoms
microbiological results. Healthy individuals were classified Productive or unproductive cough (n) – – 15
Weight loss (n) – – 14
based on the Tuberculin Skin Test (TST) status into latent (TST
Fever (n) – – 11
reactor) or non-infected (TST-) groups [19]. Active TB patients
Smear (Number of individual)
were also classified regarding the severity of disease
+ NA NA 9
according to pulmonary radiographic images using a double ++ NA NA 2
blind test and classified as minimal, moderate and advanced +++ NA NA 3
disease, as described by Abakay et al. [20]. Thus, the lesions Negative NA NA 3
were considered minimal (stage 1), when there was no Status of chest radiograph (Number of individual)
evidence of cavitation, the tissue density was classified as Minimal NA NA 8
Moderate NA NA 5
mild or moderate, and location was above the second Advanced NA NA 4
chondrosternal junction. Moreover, these stage 1
lesions involved only a single segment of one or both lungs and
the extent of combined lesions did not exceed the volume of a sin- gle lung. In moderate disease (stage 2), the lesions were
densely confluent, but the area occupied by these lesions
could not occupy more than a third of the volume of a lung.
In addition, the overall
General characteristics of the groups involved in the study showing the number of 1
subjects per group (n), the mean age (yr: years old), gender, addictions, sign/
symptoms; n: number; and status of chest radiograph. CTRL: Non-infected
Control; LTBI: Latent Tuberculosis Infection; APTB: Active Pulmonary
Tuberculosis, NA: not applied.
1
To perform biomarker networks analysis, we used the
Spearman correlation test. Additionally, the cytokine profile of
each individual was assessed to determine individual immune
biosignatures [21] and heatmap analysis was performed.
Values of P < 0.05 were con- sidered significant. The significant
correlations found amongst these
markers were compiled using Cytoscape software (version 3.2)
[22]. The networks were constructed for each study group, and
the corre- lation coefficient (r) was used to categorize the
interactions as negative (r < 0), weak (r 6 0.35), moderate
(0.36 6 r 6 0.67) and strong (r P 0.68), as proposed
previously [23].

Fig. 1. Quantification of cytokines and chemokines from plasma. Plasma was separated from whole blood drawn from pulmonary tuberculosis patients (APTB), latent
infected subjects (LTBI) and non-infected controls (CTRL), by centrifugation and chemokines/cytokines were measured using a customized kit (MILLIPLEX ® MAP Human
Cytokine/ Chemokine; Millipore). Each point represents an individual sample. The results are expressed in pg/mL and were analyzed by the non-parametric Mann-Whitney
test, with significance P < 0.01667 (corrected by multiple comparisons).

Additionally, the cytokine profile of each individual was
assessed to determine individual immune biosignatures. Initially,
all data were used to calculate the global median value of
each marker, which was used as a cut-off point to classify
each individ- ual as ‘‘low” or ‘‘high” producers of a given
immune marker. Data were organized in gray-scale diagrams
to calculate the frequency of high producers for each clinical
group. Relevant data (P50%) were highlighted in both bold
and underline format. Radar charts was performed in
Microsoft Excel (Microsoft Office 2013, Las Vegas, USA) to
characterize the overall frequency of individuals with high
levels of each marker for each study group, showing potential
immune biomarker signature.
Finally, to understand how these markers could stratify our
groups, we performed a heatmaps using the heatmap.2
function in the R (Project for Statistical Computing Version
3.3.0) and gplots package. A decision tree was generated
using machine-learning algorithms and built using the WEKA
software (Waikato Environ- ment for Knowledge Analysis,
version 3.6.11, University of Waikato, New Zealand). The
algorithm C4.5 was used to build the decision tree using the
parameter J48. This method analyzed all phenotypic
characteristics to select a minimum set of markers that could
efficiently separate study groups.
3. Results
3.1. Identifying the biosignature of patients infected with Mtb
During the progression of infectious disease, it is the
activation of the immune system and the resulting release of
cytokines and chemokines that causes both bacterial
clearance and disease- associated pathology. In TB infection,
cytokines and chemokines
Fig. 2. Network patterns among plasma cytokines. Networks were organized to determine the interactions among cytokines detected in the plasma from pulmonary
tuberculosis patients (APTB), latent infected subjects (LTBI) and non-infected controls (CTRL). The significant correlations with p < 0.05 are represented by lines according
1
play an important role in the immune response against bacilli,
in one case, acting to contain the spread of the
microorganism through the Th1 INF-c/TNF-a axis that promotes
macrophage acti- vation and increases the intracellular killing
of Mtb, while the Th2 associated cytokines IL-4, IL-5 and IL-
13 have an inhibitory effect on macrophage mediated killing
of microbes [24]. In this current study, we evaluated levels of
these inflammatory mediators in plasma at different stages of
TB infection (Fig. 1). Therefore, we found an increased level
of IL-6, IP-10 and TNF-a in APTB patients compared to both
other groups (Fig. 1C–F).
To determine if any of the cytokine levels correlated with
each other, we analyzed this data set using the Spearman
rank correla- tion test using Cytoscape software (Fig. 2). We
found significant interactions between cytokines and
chemokines in plasma from non-infected controls (CTRL),
latent tuberculosis infection (LTBI) subjects and active
pulmonary tuberculosis (APTB) patients. These interactions were
further classified according to the value of the correlation
coefficient (r) into strong, moderate, weak and negative
correlations. Overall, patients with active tuberculosis had an
ele- vated number of correlations amongst most of plasma
cytokines analyzed when compared to the other groups, and
these correla- tions existed between cytokines that are known
to play an impor- tant role during Mtb infection. For example,
APTB patients had increased number of correlations amongst
TNF-a, IFN-c, IP-10, IL-17 and IL-6 when compared with
LTBI subjects. In contrast to our findings in APTB patients,
increased IL-10 levels were corre- lated with latent infection.
This correlation was reduced during active disease,
demonstrating that an inflammatory profile charac- terized the
progression of Mtb infection to the active form. Other studies
have reported a protective role of IL-10, and found that IL-10
and IFN-c levels were elevated and expressed simultaneously
 1
to the intensity of the association, classified as weak, moderate, strong and negative according to correlation coefficient value (r).
 1
and were related to the disease resolution in clinically cured (corrected by multiple comparitions).

patients (revised by [25]).

We hypothesized that there may be other markers of
immune activation that extend beyond the cytokine and
chemokine levels we measured in plasma. For example,
monocytes are known to produce soluble forms of CD14 and
CD163 when activated by inflammatory stimulus including
LPS, oxidative stress and Toll-like receptor (TLR) activation
[26–28]. We analyzed plasma from our cohort of patients for
the presence of these markers and found that APTB patients
exhibited higher levels of soluble CD163 and CD14, indicating
an increased inflammatory response and an increased degree
of monocyte activation compared to con- trol and LTBI
individuals (Fig. 3).
To fully characterize the immune profile of patients at
different stages of TB, we analyzed the biosignature of each
patient, includ- ing plasma levels of cytokines, chemokines and
soluble mediators. We compared cytokine production by
individual donors to the global median for the entire cohort of
each marker (Fig. 4). High producers, with production over
50% of the global median, are depicted with a black square
and those with a production lower than 50% of the median
are depicted as a white square. The control group presented
frequencies lower than 50% for almost all of the analyzed
markers, results that were similar to frequencies for the LTBI
subjects. However, all markers from APTB patients had fre-
quencies higher than 50%. GM-CSF, IFN-a2, IL-12, IL-4, IL- 5,
IL-6, IP-10, MIP-1a, RANTES, TNF-a, sCD163 and sCD14 were at
respec- tive frequencies of 73%, 59%, 59%, 59%, 65%, 77%,
77%, 59%, 53%,
65%, 82% and 88% for APTB patients.

Fig. 3. Quantification of plasma levels of sCD163 and sCD14. The levels of soluble
CD163 (a) and sCD14 (b) in plasma from pulmonary tuberculosis patients (APTB),
latent infected subjects (LTBI) and non-infected controls (CTRL) were measured
by ELISA. Each point is representative of an individual. The results, expressed in
ng/mL, were analyzed by the non-parametric Mann Whitney test, p < 0.01667
1 Using all markers that were significantly different among
the groups in our analysis showed in Figs. 1 and 3 (i.e., IL- Mtb infection of macrophages initiates a cascade of
10, TNF-a, IL-6, sCD163 and sCD14) we calculated the Z cytokine production including IL-12p40, IFN-c, TNF-a, IL-1b, IL-
score for all individu- als included in our study groups and 6 [30,31].
generated a heatmap, as shown in Fig. 5. This analysis
indicated that sCD14 was consis- tently highly expressed in
APTB individuals compared to the other two groups. This
analysis also highlighted differences in IL-6, TNF- a and
sCD163. These data indicate that these mediators could dif-
ferentiate active TB infection from both latent and uninfected
individuals.
Finally, using these results, we constructed a decision
tree to demonstrate how these markers can segregate APTB
individuals from other groups. Using this tree, we divided all
individuals into two groups, those with active pulmonary
tuberculosis and those without disease. This latter group
was comprised of individuals with latent infection and the
no-infection controls. From the 47 individuals included in the
construction of the decision tree, we obtained an accuracy
median of 97.87%, classifying 46 out of 47 individuals, as
shown in Fig. 6. To test if our model could accurately
diagnose TB we performed an analysis of leave-one-out
cross vali- dation (LOOCV) [29]. This analysis is commonly
used to validate statistical tests performed on data that are
difficult to confirm because of the difficulty of obtaining
replicates. Applying this vali- dation approach to our data,
we obtained an accuracy median of 82.97%, demonstrating
that the decision tree we built and thus tested can be used
to segregate patients with active disease from those with
latent or no disease. These data demonstrate that the most
important markers used in determination of active disease
status were sCD14, followed by IL-6. Importantly, sCD14 by
itself could classify 15 out of 17 APTB patients, highlighting the
effective- ness of this marker. When we analyze the AUC
(area under the ROC curve), i.e., true positive versus false
positives, good results were obtained. The classifier obtained
true positive results in 89.2%, for the both classes, APTB and
Non-disease (data not shown).

3.2. Severity of disease correlated with biomarkers

We have now shown that APTB patients recruited into

this study had high levels of cytokines in their plasma as
well as increased levels of the monocyte activation marker
sCD14 com- pared to the other groups. Furthermore we
have shown that sCD14 and IL-6 can be used to determine
active disease status when compared to latently infected
and uninfected individuals. We next wanted to determine if
the increased level of cytokines present in the plasma of
APTB patients correlated with the degree of lung injury in
these same patients. To this end, chest radiographs of patients
diagnosed with pulmonary tuberculosis were analyzed in a
double-blind test and the lesions classified into three cate-
gories based on the extent of lung injury. The degree of
injury for each patient was correlated with those markers
that we found to have statistically significant elevation in
APTB patients compared to those without disease. We
observed that only sCD163 (Fig. 7A) and TNF-a (Fig. 7B)
positively correlated with the degree of lung injury found in
our group of APTB patients, and that high levels of these
markers correlated with clinical severity. Thus, the increase
of sCD163 and TNF-a in plasma of patients with active
disease may be indicative of disease progression. Further
charac- terization of these findings may prove that these
molecules are a prognostic tool for determining the severity
of tuberculosis infection.

4. Discussion
104 F.A. Zambuzi et al. / Cytokine 88 (2016) 99–107
1

Fig. 4. Representation of a possible global signature of pulmonary tuberculosis. (A) This diagram was designed using the value of the global median of each soluble
mediator as the cutoff point to mark each individual as ‘‘low” or ‘‘high” regarding their expression levels of a given analyte. Those marked in black correspond to values
higher than the overall median of the respective analyte, and blanks are those with less than the median values. Below each table are shown the frequency (%) of
individuals with high levels of mediators while the relevant data (P50%) are in bold and underlined format. (B) Radar charts summarize the signature of the mediators
analyzed in pulmonary tuberculosis, relative to control and latent subjects, wherein each axis shows the proportion of subjects with high levels of a given mediator. The
relevant data (P50%) are indicated by arrows (?). APTB: pulmonary tuberculosis patients, LTBI: latent infected subjects, CTRL: non-infected controls.

The present study screened 16 cytokines and chemokines,

The variability of cytokine measurements across several
includ- ing those mentioned above, in plasma from APTB
studies reflects that changes in the balance of cytokine levels
patients and LTBI individuals in order to characterize the
in plasma is primarily related to the progression of Mtb
overall immune profile of TB in these cohorts and determine if
infection and only secondarily related to the level of other
measurement of multiple cytokines can distinguish between
cytokine levels in the host [24]. In this study, we measured
latent and active infection. At baseline, APTB patients had
multiple cytokines within the same host to determine if these
elevated levels of TNF-a, IP-10 and IL-6 (Fig. 1), each of
measurements, assessed individu- ally or in concert, yielded an
which constitute an important mediator of the immune
accurate assessment of infection sta- tus for a specific cohort
response against Mtb as previously described [15,32,33].
of patients. Analyzing the correlation between cytokines
detected on plasma samples, we found
 1
IL-4 and

Fig. 5. The expression pattern of immunological markers determines a

segregation profile for active TB patients. The values of the significantly
different markers (IL-6, TNF-a, IP-10, IL-5, sCD163 and sCD14) were used to
calculate the Z score and then this data was used to generate a heatmap. The
Z score scale ranged from —4 to +4 and the intensity of production was
illustrated with green (less production), black and red (high production) colors.
(For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)

Fig. 6. Decision tree to segregate APTB patients from individuals without active
infection. The values of IL-6, TNF- a, IP-10, sCD163 and sCD14 were used to
construct the data set. The decision tree was generated based on the clustering of
all individuals into two groups, those with active tuberculosis disease (APTB) and
without disease (CTRL and LTBI individuals). The numbers beside the name of the
groups correspond to the values of correct/incorrect register.

differences in the number and intensity of them. Uninfected

control individuals demonstrated a characteristic profile of the
immune response in a basal way, represented in Fig. 2.
However, it is clear to note that Mtb infection altered the
degree of cellular activation, since new correlations were
established in LTBI individ- uals and in APTB patients. Mainly,
it was also possible to observe that new correlations were
formed, especially in individuals with active disease.
Generally, new correlations were formed by cytokines that
plays an important effect in the immune response against
bacilli, highlighting positive relationship between pro-
inflammatory cytokines such as TNF-a, IP-10, IL-12, IL-6,
IFN-c, IL- 17 and correlations between Th2 cytokines such as
IL-10, which were not seen in non-infected individuals. Most obtained from our heat map and decision tree analysis
of these correlations formed in APTB patients were not indicate that these markers could act as a promising
observed in individuals with latent infection and controls, biosignature for tuberculosis and from those parameters, with
and therefore could be related to specific correlations of sCD14 and IL-6 acting as decisive markers to diagnose APTB
disease progression. Thus, we conclude that together, these patients. Overall, our analysis indicates that measuring
cytokines can comprise a biosigna- ture indicative of APTB. multiple markers elevated
We also measured the levels of sCD163 and sCD14,
mediators that indicate activation of the innate immune
system. These two mediators, produced by activated
monocytes/macrophages were elevated in plasma from patients
with APTB. Our research indicates that these markers, in
concert with cytokine and chemokine levels, may be used as
part of the biosignature of the immune response to Mtb
infection. CD163 is a scavenger receptor of haptoglobin-
hemoglobin complexes [34,35] that is cleaved from the cell
surface by inflammatory stimuli including TLR activation
[27], oxidative stress [26] and Fcc cross-linking [36]. This
cleaved formed is soluble and can be detected in biological
fluids [37]. CD14 is an associated membrane receptor that
recognizes high affinity LPS from Gram-negative bacteria
[28] and other pathogen associated molecules including
mycobacterial derived Lipoarabinmannan [38]. Membrane
bound CD14 can be cleaved to form soluble CD14 (sCD14)
as a result of monocyte activation [39]. This soluble form
can be detected in plasma and biological fluids as a marker
for inflammatory conditions. Soluble CD14 has also been
detected in the acute phase response to sepsis, burns and
trauma [40].
In this current study, we have demonstrated that APTB
patients presented with increased levels of these two soluble
mediators, indicating a systemic activation state of
monocytes during disease development. It reinforces other
studies showing that patients with active TB have increased
levels of sCD14 in both serum [41] and bronchoalveolar
lavage fluid [42] and elevated levels of sCD163 [43,44]. Our
study has demonstrated that these two mole- cules, when
measured simultaneously, are key components of an
important biosignature that can distinguish APTB patients
from CTRL and LTBI cohorts.
To fully characterize the TB biosignature in our patient
cohort, we analyzed the frequency of individuals
characterized as high producers as related to the global
median of each marker. Our cohort of APTB patients
presented an increased frequency of high expression scores
for most of the parameters tested compared with the other
two groups, a finding in line with this group’s activated
immune state. Thus, we have identified a possible
biosignature of pulmonary TB, characterized by an intense
active immunological profile and marked by increased
frequency of individuals that are high producers of GM-CSF,
IFN-a2, IL-4, IL-5 IL-6, IP-10, MIP-1a, RANTES, TNF-a,
sCD163 and sCD14.
Further, a heat map constructed based on these TB
biosignature
markers, also showed that LTBI and CTRL individuals had
similar expression of all analyzed parameters and again, these
were differ- ent from the expression patterns of APTB
patients. Our work con- firmed the results of Feruglio and
colleagues showing that sCD14 could strongly segregate
patients with disease and suggested that this marker could
be used to discriminate between the active and latent forms
of TB [45].
Our studies also demonstrated that sCD14 and IL-6 were
useful markers for discriminating amongst the different
stages of TB. Previous studies have identified that both
sCD14 and IL-6 [15,45] are elevated in patients with Mtb
infection, and both can be used as diagnostic and treatment
monitoring tools. Our current study demonstrates that solely
measuring the level of sCD14 in plasma can identify 15 of
17 APTB patients in our cohort. Together, the results
106 F.A. Zambuzi et al. / Cytokine 88 (2016) 99–107
1

Fig. 7. Correlation between plasma levels of TNF- a and sCD163 and lung injury in APTB patients. Lung injury of APTB patients was classified in a double-blind test and then
divided into three grades. Images are representative of each grade – minimal (1), moderate (2) and advanced (3) disease. Levels of sCD163 (A), TNF-a (B), IL-6 (C),
sCD14 (D), IP-10 (E) and IL-5 (F) were correlated with the degree of lung injury in patients with active disease through Spearman’s rank correlation test. The values of
p and r2 are
specified in each chart.

during Mtb infection is a better predictor of active disease The authors declare that they have no commercial or other
than any one marker alone. association that might pose a conflict of interest on the
Our study also demonstrated that plasma levels of sCD163 manuscript
and TNF-a correlated with disease severity in APTB patients.
Other studies have demonstrated that increased sCD163 in
plasma corre- late with mortality and advanced TB disease.
Our study confirms these results and demonstrates that sCD163
is a critical component in the TB biosignature. Although TNF-a
is associated with immune protection in patients with Mtb
infection, there is also evidence that high levels of TNF-a are
related to immunopathological responses and contribute
significantly to lung damage [46]. Other studies have
demonstrated a positive association between TNF-a serum
levels and the severity of TB [47]. Here we suggest that TNF-
a, along with sCD163, can be used to stage the progression
of pulmonary TB.
In conclusion, we have demonstrated that quantification of a
set of well characterized biomarkers in plasma can be used in
a novel way to classify different stages of disease progression
in patients infected with Mtb. Specifically, we have shown
that the simultane- ous detection of plasma sCD14 and IL-6
can identify APTB, and measurement of sCD163 and TNF-a in
APTB patients can further classify the degree of disease
progression. Measurement of these molecules in a larger
cohort of patients is needed to confirm the accuracy and
utility of these markers as an accurate diagnostic tool for Mtb
infection.

Funding

This work was supported by: Fundação de Amparo à

Pesquisa do Estado de São Paulo – Brazil [Grant No.
2011/12199-0]; Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior – Brazil (CAPES) and Conselho Nacional de
Pesquisa – Brazil (CNPq).

Conflict of interest
Acknowledgements

The authors are grateful to Caroline Fontanari, Thaísa

da Silva Araújo, Margarida Maria Passeri Nascimento and
Alyne Fávero Galvão for their technical support and Dr.
Judith Connett for her critical reading of the manuscript.

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