Materials and Methods s18

Supplement Data

MATERIALS AND METHODS

Cytokine measurements

For tuberculosis (TB) patients, plasma levels of interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-alpha, and interferon-gamma were measured using commercialized enzyme-linked immunosorbent assay (ELISA) kits (all Beckton Dickinson Pharmingen, San Diego, CA, USA) before and after two months of anti-TB therapy. The detection limit was 2.2 pg/mL for IL-6, 2.0 pg/mL for IL-10, 1.0 pg/mL for interferon-gamma, and 2.0 pg/mL for TNF-alpha.

Antibodies used in flow cytometry

The flow cytometry study used the following monoclonal antibodies (IgG1, kappa): phycoerythrin [PE]-conjugated mouse anti-human CD14 antibody as monocyte surface marker, isotype control mouse IgG1, fluorescein isothiocyanate [FITC]-conjugated mouse anti-human TLR2, and isotype control IgG1 (all Beckton Dickinson Pharmingen, San Diego, CA, USA).

Expression of TLR2 protein by flow cytometry

Whole blood was drawn into EDTA tubes and 50 μl was transferred into 12 x 75 mm polystyrene tubes (Falcon No. 2052) mixed with monoclonal antibodies (20 μl PE-conjugated anti-CD14, 10 μl FITC-conjugated anti-TLR2, or isotype controls) and incubated on ice in the dark for 30 minutes as described previously [1]. For lysis of erythrocytes, 1.5 ml of lysis buffer was added to the mixture at room temperature in the dark for 15 min.

The samples were then centrifuged at 300 x g for 5 min at 4°C. The supernatant was discarded, leaving approximately 50 μl of fluid, including the cell pellet in the tube. The cell suspension was washed with phosphate-buffered saline and re-suspended in 1% para-formaldehyde, acquired in a fluorescence-activated cell sorter (FACS) (Becton Dickinson), and analyzed using CellQuest software (Becton Dickinson). Different leukocyte populations (i.e., granulocytes, lymphocytes and monocytes) were identified by their characteristic forward and side scattering properties. All monocytes were enclosed in an electronic gate (Figure S1A). From this gated sub-population of monocytes, at least 10,000 CD14+ cells were acquired for each sample (Figure S1B) and analyzed for TLR2 expression (Figure S1C).

Choosing the cut-off value to define high or low TLR2 expression

Locally weighted scatter-plot smoothing (LOESS) was selected as the technique for the smooth function of TLR2 expression level in TB patients and in healthy subjects. From the partial prediction curve of the smooth function, the cut-off value of TLR2 expression associated with TB was determined.

Anti-tuberculosis treatment

All TB patients received directly observed standard anti-TB treatment, including daily isoniazid, rifampin, ethambutol, and pyrazinamide in the first two months, and daily isoniazid and rifampin for the next four months [2]. The regimen was modified if necessary by the primary care physician in case of concomitant hepatic/renal diseases or adverse events.

Chest radiography score

All patients underwent chest radiography before starting anti-TB therapy. The radiographic score was recorded according to a previous study to define the extent of the pulmonary disease [3]. Briefly, each lung was divided into three areas, with each area rated on a 4-point scale of 0 to 3 for the extent of infiltration, for a maximum radiographic score of 18. Greater the extent of infiltration was associated with higher score. After two months of treatment, all patients had a follow-up chest radiograph, and thus, a second radiographic score. Each patient also submitted three sputum samples for mycobacterial culture.

Definition of disseminated tuberculosis, severe chronic kidney disease, and alcoholism

Disseminated TB was defined if the patient had (1) M. tuberculosis isolated from blood, bone marrow, or liver biopsy specimen, or from more than two non-contiguous organs; (2) M. tuberculosis isolated from one organ with histologic demonstration of caseating granulomatous inflammation from a non-contiguous organ; and (3) M. tuberculosis isolated from one organ and radiographic findings of miliary lung lesions [4]. The estimated glomerular filtration rate (eGFR) was obtained using the Modification of Diet in Renal Disease (MDRD) study equation [5]. Severe chronic kidney disease (CKD) was defined as eGFR ≤30 ml/min/1.73 m2.

Statistical analysis

Proportions or means were used to describe the demographic, clinical, and radiographic characteristics of each group upon enrollment and during follow-up.

While conducting multivariate logistic, linear, and Cox proportional hazard regression analyses, basic model-fitting techniques, including variable selection, goodness-of-fit, area under the receiver operating characteristic curve (AUC), adjusted generalized R2, and regression diagnostics (e.g., residual analysis, detection of influential cases, and check for multi-collinearity) were applied to ensure the quality of multivariate analysis. The possibility of interactions between selected variables was also checked. In a step-wise variable selection procedure, all potential predictors were included and significance levels for entry and stay were set at 0.15.

RESULTS

Underlying co-morbidities in tuberculosis patients

Thirty-five (40%) patients had underlying co-morbidities, including malignancy in 15 (seven lung cancer, two breast cancer, and one each with chronic myeloid leukemia, Hodgkin’s lymphoma, nasopharyngeal carcinoma, hypopharyngeal carcinoma, cholangeal carcinoma, and prostate cancer), diabetes mellitus in 10, alcoholism in six, severe chronic kidney disease in four, autoimmune disease in three (none received anti-tumor necrosis factor therapy), and hepatitis B virus-related liver cirrhosis in one.

Of the 7 (8%; male, n=3) TB patients who died within six months after the start of anti-TB treatment, six had underlying co-morbidities, including malignancy in three (i.e., hypopharyngeal cancer, breast cancer, and chronic myeloid leukemia), diabetes mellitus in two, and severe chronic kidney disease, liver cirrhosis, and rheumatoid arthritis in one each. Three (male, n=2) died of bilateral severe pulmonary tuberculosis with acute respiratory distress syndrome. One man died of nosocomial pneumonia due to multi-drug-resistant Acinetobacter baumannii. Causes of death for the other three women are Escherichia coli urosepsis, end-stage breast cancer with multiple metastases, and acute myocardial infarction.

Cut-off value defining high or low TLR2 expression

The smoothing curve of TLR2 expression in the 87 TB patients and 94 healthy controls showed that rMFI ≥9 was associated with TB (Figure S2). Thus, in the following analysis, TLR2 expression was considered high if rMFI ≥9.

Predicting sputum culture conversion

Among the cytokines assayed in this study (i.e., IL-6, IL-10, TNF-alpha, and interferon-gamma) and TLR2 expression, TLR2 expression was the best predictor for sputum culture conversion after two months of anti-TB treatment. The area under the receiver operating characteristic (ROC) curve of TLR2 was 0.657. Combinations of several cytokines measured failed to improve the prediction for sputum culture conversion.

REFERENCES

1. Droemann D, Goldmann T, Tiedje T, Zabel P, Dalhoff K, Schaaf B (2005) Toll-like receptor 2 expression is decreased on alveolar macrophages in cigarette smokers and COPD patients. Respir Res 6:68

2. American Thoracic Society, CDC, and Infectious Diseases Society of America (2003) Treatment of tuberculosis. MMWR Recomm Rep 52 (RR-11):1-77

3. Snider GL, Doctor L, Demas TA, Shaw AR (1971) Obstructive airway disease in patients with treated pulmonary tuberculosis. Am Rev Respir Dis 103 (5):625-640

4. Wang JY, Hsueh PR, Wang SK, Jan IS, Lee LN, Liaw YS, Yang PC, Luh KT (2007) Disseminated tuberculosis: a 10-year experience in a medical center. Medicine 86 (1):39-46

5. Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D (1999) A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med 130 (6):461-470

Table S1. Factors associated with plasma levels of TLR2-downstream cytokines

Abbreviations: rMFI, relative mean fluorescent intensity; SE, standard error; TLR, toll-like receptor

Table S2. Initial and follow-up plasma levels of toll-like receptor 2-downstream cytokines in different sub-groups

Abbreviations: IFN, interferon; IL, interleukin; RS, radiographic score; TB, tuberculosis; TNF, tumor necrosis factor

Numbers were mean values (standard deviations)

Figure S1.

Figure S2.

LEGEND

Figure S1. Toll-like receptor (TLR) 2 expression on CD14+ peripheral blood monocytes. (A) Peripheral blood cells from a representative patient with pulmonary tuberculosis (TB) were incubated with PE-conjugated anti-CD14 and either FITC-conjugated anti-TLR2 or IgG isotype control, and analyzed by flow cytometry. Cell populations were separated and identified by their characteristic forward and side scattering property. Monocytes were enclosed in an electronic gate. (B) From the gated sub-population of monocytes, at least 10,000 CD14+ cells were acquired for TLR2 / isotype expression analysis. (C) Representative histogram of isotype control-stained cells and TLR2-stained cells from a healthy subject, and TLR2-stained cells from a TB patient.

Figure S2. Partial prediction curve describing the Toll-like receptor (TLR) 2 expression level on CD14+ peripheral blood monocytes affected the probability of tuberculosis (TB). The shaded areas denoted the 95% confidence intervals. The probability of TB increased when the TLR2 expression was ≥9 rMFI.