Background
Upon
M. tuberculosis infection, a host can develop a wide range of disease manifestations ranging from asymptomatic infection to severe progressive disease [
1]. Importantly, only 10 % of individuals exposed to
M. tuberculosis develop active disease, which highlights the importance of understanding the key determinants of susceptibility to infection. The determinants of the TB clinical presentation are described to involve a complex relationship between the mycobacterium and the host immune responses [
2,
3]. Successful host response against
M. tuberculosis requires the production of pro-inflammatory cytokines including IFN-γ and TNF-α [
4,
5]. Indeed, individuals genetically deficient in molecules from the IFN pathway, as well as those under treatment of chronic conditions with TNF-α blockers, are highly susceptible to severe TB [
6].
Existing blood-based tests, such as IFN gamma release assays (IGRA) are inadequate for monitoring treatment response [
7]. Therefore, host biomarkers for monitoring treatment response have been considered as important priorities for TB research [
8].
It has been recently described in distinct TB patient cohorts from Brazil and India that TB disease severity is associated elevated circulating levels of CRP and other pro-inflammatory cytokines and lipid mediators [
9,
10]. Although there have been indications from experimental models that
M. tuberculosis loads in the lungs are associated with the inflammatory profile and lung disease severity [
11,
12] this relationship has not been yet fully investigated prospectively.
The present study addresses this question in a cohort of PTB patients from a highly endemic area in Brazil. The findings argue that there is a strong relationship between pre-treatment mycobacterial loads and systemic inflammation, which may set the stage for persistence of sputum culture positivity and radiographic improvement after 2 months of antibiotic chemotherapy.
Discussion
Several exploratory studies have evaluated the diagnostic potential of cytokine biomarkers other than IFN-γ for monitoring anti-tuberculous therapy [
2,
3], but no prospective study have measured simultaneously the direct associations between mycobacterial loads, Th1/Th2 cytokines, acute phase parameters and pulmonary lesions upon treatment initiation, as we performed in our longitudinal study. A recent systematic review by Clifford et al. [
26] identified only 12 longitudinal studies that measured ex vivo serum cytokine concentrations without a stimulation step during anti-TB treatment. The largest studies were those by Azzuri et al. [
27] and Wang et al. [
28], with 220 and 87 TB cases, respectively. Thus, additional prospective studies assessing effects of ATT on the host inflammatory profile are greatly needed, especially in endemic areas such as Brazil.
In the present study, we prospectively depict the immune profile, microbial clearance and evolution of radiographic lesions in 73 pulmonary TB patients before and 60 days after anti-tuberculous treatment initiation. Logistic regression analysis adjusted for age, gender and hemoglobin levels confirmed that an AFB smear grade above 1+ was indeed associated with increased risk for
M. tuberculosis culture positivity at day 60 of ATT. These results confirm that monitoring sputum and culture conversion may be a good indicators of treatment outcome for drug-sensitive TB but may present low sensitivity and modest specificity for predicting failure and relapse [
29]. In our study, we found that pre-ATT mycobacterial loads in sputum and systemic inflammation synergistically associated with the status of radiographic lesions during treatment. This approach could potentially improve the predictive value of non-favorable outcomes associated with ATT in future clinical trials.
Within the inflammatory parameters evaluated, values of CRP, IL-2, IL-4, TNF-α and ESR significantly decreased upon treatment initiation similarly to other studies [
28,
30‐
36]. On the converse, IL-10 levels substantially increased at day 60 of ATT, as also described elsewhere [
5,
27,
28,
30,
33‐
40]. Nevertheless, the reports on the changes in IL-10 levels are conflicting, as other studies have described decreases of the systemic levels of this cytokine during ATT [
5,
27,
28,
35]. It is possible that differences in study populations as well as distinct epidemiological/genetic background could explain these conflicting findings.
In our study, concentrations of IL-6 and IFN-γ remained unchanged during the anti-TB treatment in disagreement with different investigations where IL-6 and IFN-γ tended to decrease during ATT [
28,
30,
31,
33,
36‐
38] and with other reports which showed increases in IFN-γ levels [
39,
41‐
43]. Additional studies systematically investigating patients from different ethnicity/genetic backgrounds should be performed to narrow down the mechanisms underlying these discrepancies. Importantly, at day 60 of treatment, the cytokine network of associations based on Spearman ranks revealed that the cytokine exhibiting the higher number of significant correlations with the remaining biomarkers was shifted from TNF-α to IFN-γ. It is possible that these two cytokines may be critical for driving activation of specific immune pathways evoked by ATT; however, this hypothesis requires additional mechanistic studies to be tested.
Multidimensional analyses revealed that ESR, IL-2, IL-4 and CRP were the parameters with the highest power to discriminate individuals before and after treatment initiation. As highlighted previously [
44], our findings indicate that monitoring acute phase parameters (ESR, CRP), Th1 cytokines (IL-2) and anti-inflammatory (IL-4) may help understanding the multidimensionality of immunopathology in TB and point to novel potential therapeutic targets for pulmonary TB. Recently, Mihret et al. [
39], while evaluating HIV negative and positive TB cases, have demonstrated that the ratios of IFN-γ/IL-10 and IFN-γ/IL-4 display significant increases after treatment in HIV negative TB cases but not in HIV positive TB cases.
Remarkably, we observed that individuals who were AFB+ at pre-ATT remained with heightened levels of IL-6, IL-10 and IFN-γ upon treatment initiation compared to those with negative smears, similarly to that described by Djoba et al. [
33] and Chowdhury et al. [
37] where serum levels of IFN-γ, TNF-α, IL-6 and IL-10 were shown to be directly associated with the bacterial load. As expected, the values of the pro-inflammatory markers were markedly decreased at 60 days of antimicrobial treatment. Our results also revealed that there is a strong association between pre-treatment mycobacterial loads and risk for culture positivity at day 60 of ATT initiation similarly to results described by Djoba et al. [
33].
The apparent association between increased levels of only IL-10, among all the other markers, and presence of
M. tuberculosis culture positivity at day 60 of ATT is intriguing and may underline a potential inability of the immune system to clear the bacteria from the lung. Indeed, IL-10 induction is described to be a potential scape mechanism used by
M. tuberculosis to survive in macrophages [
45].
M. tuberculosis can be recognized by TLR-2 and induce production of IL-10, that promotes differentiation into a M2-like macrophage phenotype and possibly Th2 adaptive responses [
46]. Moreover, recent studies demonstrated a direct association between TLR2 polymorphisms and enhanced bacterial loads in pulmonary tuberculosis patients [
45]. Our group is currently assessing the influence of immune related polymorphisms in cytokine levels in TB patients undergoing ATT.
More recently, some studies have reported the association between sputum conversion at 60 day of TB treatment with decreases in serum levels of CRP, CXCL10, IL-17, MMP-8, as well as with cytokine responses after
M. tuberculosis-specific in vitro stimulation of peripheral blood. Moreover, sputum conversion has also been associated with decreases in IFN-γ, TNF-α and IL-2 levels as well as in frequency of IFN-γ
+CD4
+ T-cells that express immune activation markers CD38 and HLA-DR and the proliferation marker Ki-67 in peripheral blood [
32,
34,
46‐
49]. Whether our findings reflect changes in T-cell compartment or in other immune cells deserves further investigation.
In the present study, we detected an increased frequency of AFB+ smears at pre-ATT in patients presenting with cavitary lesions. This finding is consistent with the idea that cavitary lesions increase risk for TB transmission [
22,
24]. Interestingly, bilateral cavity formation is thought to be the most significant predictor of treatment failure for extensively drug-resistant TB [
23]. Our current understanding of the mechanisms implicated in cavity formation is still elusive and derive mostly from experimental models and observational studies of human specimens [
50,
51]. We observed that the circulating levels of all the parameters assessed could not distinguish patients with non-cavitary disease from those presenting with cavitary lung lesions at pre-treatment. In addition, the magnitude of changes in levels of IL-2, IL-4, IL-6 and IFN-γ between day 0 and 60 of ATT was similar between the groups of patients stratified according to cavitary disease status. Although CRP concentrations consistently decreased at day 60 of ATT in both subgroups of patients, individuals with cavitary disease exhibited heightened values compared to patients with non-cavitary TB. Furthermore, serum levels of IL-10 significantly increased while TNF-and ESR values substantially decreased from day 0 to day 60 of ATT in patients with cavitary TB but not in those with non-cavitary lung disease. These differential changes detected may indicate that individuals with lung cavities exhibit a slightly different inflammatory response to ATT. However, these disparities in biomarker levels may not be biologically relevant as they did not reflect differences in early treatment responses, as the frequencies of patients presenting with culture positive or improvement of lung lesions by radiographic evaluation on day 60 of ATT were similar between the subgroups with or without cavitary TB. It is possible that de directly observed therapy employed here, together with the close monitoring of patients who remained in the hospital for the first 60 days of treatment, resulted in good treatment response regardless of cavitary disease.
Individuals presenting with pre-ATT AFB smear < 2+ and CRP levels < 4.7 mg/L were more than 12 times more likely to exhibit radiographic improvement of lung disease. Few studies have evaluated the interaction within bacterial load, inflammation response and radiographic changes [
33]. Nevertheless, other studies have reported associations between a number of cytokines, such as TNF-α, IL-1β, IL-6, IL17 and TGF-β1, and radiographic TB lung disease presentation [
30,
33,
36,
48,
52]. CRP is acute inflammatory protein whose production by hepatic cells is stimulated by TNF-α, IL-1 and IL-6. This protein has important functions, which include induction of inflammatory responses and participation in immune activation following infection. Many authors suggest that a fine control of inflammatory responses is crucial to promote clinical and microbiological improvement of TB patients [
8,
10,
53]. Exhaustive inflammatory responses lead to immunopathology-driven pulmonary injury, which directly impacts disease severity in pulmonary tuberculosis [
12,
54]. Lower CRP concentrations found in a subset of TB patients prior to ATT may be indicative of a mild/moderate systemic inflammatory response reflected by increase odds for improvement in chest-X-ray 60 days after treatment initiation.
The strengths of our study include: a) cohort of hospital-based pulmonary TB patients that assessed simultaneously the direct associations between mycobacterial loads, Th1/Th2 cytokines, acute phase parameters and pulmonary lesions upon treatment initiation, b) our study population was followed up very closely to assure treatment adherence: during the study period, all pulmonary TB patients remained hospitalized and received anti-TB drugs under directly observed manner, c) the personnel performing the laboratory and chest radiographs analysis were unaware of the treatment outcomes, d) innovative statistical analyses employing multidimensional approaches were used to define a biosignature that better relates to the clinical outcomes assessed.
Our study has some limitations. We did not evaluate other diseases, which could result in similar disease presentation than TB. Identification of a biosignature distinguishing TB from other clinical disease groups has been published by us and others [
9,
10,
55‐
57] and it was not an aim of the present study. Our study has a very narrowed goal of testing associations between bacillary loads, systemic inflammation and radiological changes in individuals with active TB undergoing treatment. Moreover, we have not screened our patients for a number of co-infections, such as viral hepatitis and helminth infections, as well as for comorbidities such as steatohepatitis, which could affect immune responses in TB patients [
58,
59]. Additional studies with larger numbers of patients will be required to investigate whether the results described here in terms of prospective changes in inflammatory biomarkers in the context of ATT are considerably affected by these co-morbidities. We have assessed serum levels of inflammatory markers and have not studied specific cellular immune responses against
M. tuberculosis infection. The use of serum samples may result in lower specificity than measuring cytokine responses induced by stimulations with TB antigen in cell cultures. Few HIV positive TB cases were included in the analysis, due to the lower prevalence of TB-HIV co-infection in our study setting. Finally, we did not evaluate different patterns of the host immune response according to different
M. tuberculosis lineages [
60]. Nevertheless, our results were able to demonstrate unique associations between host inflammatory markers in serum and microbiological and radiological outcomes after the onset of anti-TB treatment. Due to the relatively small number of patients studied in our exploratory study, it would be important to validate the results reported here in additional cohorts with larger patient populations as well as in different epidemiological settings.