Multiple Cytokines Are Released When Blood from Patients with Tuberculosis Is Stimulated with Mycobacterium tuberculosis Antigens

Mycobacterium tuberculosis (Mtb) infection may cause overt disease or remain latent. Interferon gamma release assays (IGRAs) detect Mtb infection, both latent infection and infection manifesting as overt disease, by measuring whole-blood interferon gamma (IFN-γ) responses to Mtb antigens such as early secreted antigenic target-6 (ESAT-6), culture filtrate protein 10 (CFP-10), and TB7.7. Due to a lack of adequate diagnostic standards for confirming latent Mtb infection, IGRA sensitivity for detecting Mtb infection has been estimated using patients with culture-confirmed tuberculosis (CCTB) for whom recovery of Mtb confirms the infection. In this study, cytokines in addition to IFN-γ were assessed for potential to provide robust measures of Mtb infection.Cytokine responses to ESAT-6, CFP-10, TB7.7, or combinations of these Mtb antigens, for patients with CCTB were compared with responses for subjects at low risk for Mtb infection (controls). Three different multiplexed immunoassays were used to measure concentrations of 9 to 20 different cytokines. Responses were calculated by subtracting background cytokine concentrations from cytokine concentrations in plasma from blood stimulated with Mtb antigens.Two assays demonstrated that ESAT-6, CFP-10, ESAT-6+CFP-10, and ESAT-6+CFP-10+TB7.7 stimulated the release of significantly greater amounts of IFN-γ, IL-2, IL-8, MCP-1 and MIP-1β for CCTB patients than for controls. Responses to combination antigens were, or tended to be, greater than responses to individual antigens. A third assay, using whole blood stimulation with ESAT-6+CFP-10+TB7.7, revealed significantly greater IFN-γ, IL-2, IL-6, IL-8, IP-10, MCP-1, MIP-1β, and TNF-α responses among patients compared with controls. One CCTB patient with a falsely negative IFN-γ response had elevated responses with other cytokines.Multiple cytokines are released when whole blood from patients with CCTB is stimulated with Mtb antigens. Measurement of multiple cytokine responses may improve diagnostic sensitivity for Mtb infection compared with assessment of IFN-γ alone

Comparisons of cytokine responses to different antigens measured by in-house MMIA among patients with culture-confirmed tuberculosis.

<p>Median cytokine responses (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026545#pone-0026545-t004" target="_blank">Table 4</a>) to early secreted antigenic target-6 (ESAT-6), culture filtrate protein 10 (CFP-10), TB7.7, and combinations of these <i>M. tuberculosis</i> (<i>Mtb</i>) antigens for patients with culture-confirmed tuberculosis (patients) are listed with p values in parenthesis calculated by Mann-Whitney U Rank Sum test. Significant differences (<i>p</i><0.05) are indicated in <b>bold type</b>.</p

Cytokine concentrations and responses measured with a commercial ELISA.

<p>Median background IFN-γ concentrations (Nil, pg/mL) and IFN-γ responses (pg/mL) to phytohemagglutinin (Mitogen Response), early secreted antigenic target-6 (ESAT-6 Response), culture filtrate protein 10 (CFP-10 Response), TB7.7 (TB7.7 Response), and combinations of these <i>M. tuberculosis</i> (<i>Mtb</i>) antigens (ESAT-6+CFP-10 Response, and ESAT-6+CFP-10+TB7.7 Response) are listed with ranges in parentheses. Cytokine concentrations were measured with a commercial enzyme-linked immunosorbent assay (ELISA) for patients with culture-confirmed tuberculosis (patients) and subjects at low-risk of <i>Mtb</i> exposure (controls). Responses were calculated by subtracting the background cytokine concentration in plasma from blood incubated with saline (Nil) from the cytokine concentration in plasma from blood incubated with mitogen or <i>Mtb</i> antigens. N = 21 for patients and N = 24 for controls unless indicated (*n = 20, **n = 23). <i>P</i> values were calculated by Mann-Whitney U Rank Sum test comparing cytokine concentrations or responses for patients with controls. Significant differences (<i>p</i><0.05) are indicated in <b>bold type</b>.</p

Comparisons of IFN-γ responses to different antigens measured by commercial ELISA among patients with culture-confirmed tuberculosis.

<p>Median IFN-γ responses (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026545#pone-0026545-t002" target="_blank">Table 2</a>) to early secreted antigenic target-6 (ESAT-6), culture filtrate protein 10 (CFP-10), TB7.7, and combinations of these antigens for patients with culture-confirmed tuberculosis (patients) are listed with p values in parenthesis calculated by Mann-Whitney U Rank Sum test. Significant differences (<i>p</i><0.05) are indicated in <b>bold type</b>.</p

Cytokine concentrations and responses measured with an in-house MMIA.

<p>Median background cytokine concentrations (Nil, pg/mL) and cytokine responses (pg/mL) to phytohemagglutinin (Mitogen Response), early secreted antigenic target-6 (ESAT-6 Response), culture filtrate protein 10 (CFP-10 Response), TB7.7 (TB7.7 Response), and combinations of these <i>M. tuberculosis</i> (<i>Mtb</i>) antigens (ESAT-6+CFP-10 Response and ESAT-6+CFP-10+TB7.7 Response) are listed with ranges in parentheses. Cytokine concentrations were measured with an in-house multiplexed microsphere-based immunoassay (in-house MMIA) for patients with culture-confirmed tuberculosis (patients) and subjects at low-risk of <i>Mtb</i> exposure (controls). For concentrations that were designated “out of range” by Bio-Plex Manager software, low values are reported as 0 and high values are reported as the upper limit of the standard curve multiplied by the dilution factor (25,000 pg/mL). Responses were calculated by subtracting the background cytokine concentration in plasma from blood incubated with saline (Nil) from the cytokine concentration in plasma from blood incubated with mitogen or <i>Mtb</i> antigens. N = 6 for patients and N = 10 for controls unless indicated (*n = 6). <i>P</i> values were calculated by Mann-Whitney U Rank Sum test comparing cytokine concentrations or responses for patients with controls. Significant differences (<i>p</i><0.05) are indicated in <b>bold type</b>.</p

Cytokine concentrations and responses measured by a commercial MMIA.

<p>Median background cytokine concentrations (Nil, pg/mL) and cytokine responses (pg/mL) to phytohemagglutinin (Mitogen Response) and to a combination of early secreted antigenic target-6, culture filtrate protein 10, and TB7.7 (ESAT-6+CFP-10+TB7.7 Response) are listed with ranges in parentheses. Cytokine concentrations were measured with a custom commercial multiplexed microsphere-based immunoassay (commercial MMIA) for patients with culture-confirmed tuberculosis (patients) and subjects at low risk for <i>M. tuberculosis</i> (<i>Mtb</i>) infection (controls). For values that were designated “out of range” by Bio-Plex Manager software, low values are reported as 0 and high values are reported as the observed value of the highest standard concentration multiplied by the dilution factor. Responses were calculated by subtracting the background cytokine concentration in plasma from blood incubated with saline (Nil) from the cytokine concentration in plasma from blood incubated with mitogen or <i>Mtb</i> antigen. N = 12 for patients and N = 12 for controls. <i>P</i> values were calculated by Mann-Whitney U Rank Sum test comparing cytokine concentrations or responses for patients versus controls. Significant differences (<i>p</i><0.05) are indicated in <b>bold type</b>.</p

Cytokine concentrations and responses measured with a microarray.

<p>Median background cytokine concentrations (Nil, pg/mL) and cytokine responses (pg/mL) to phytohemagglutinin (Mitogen Response), early secreted antigenic target-6 (ESAT-6 Response), culture filtrate protein 10 (CFP-10 Response), TB7.7 (TB7.7 Response), and combinations of these <i>M. tuberculosis</i> (<i>Mtb</i>) antigens (ESAT-6+CFP-10 Response and ESAT-6+CFP-10+TB7.7 Response) are listed with ranges in parentheses. Cytokine concentrations were measured by a commercial quantitative immuno-microaray (microarray) for patients with culture-confirmed tuberculosis (patients) and subjects at low risk for <i>Mtb</i> exposure (controls). Responses were calculated by subtracting the background cytokine concentration in plasma from blood incubated with saline (Nil) from the cytokine concentration in plasma from blood incubated with mitogen or <i>Mtb</i> antigens. Cytokine responses for some patients and controls were not determined due to poor performance of the respective standard curves. N = 5 or 6 for patients subjects and N = 6 or 7 for controls unless indicated (*n = 4, **n = 5). <i>P</i> values were calculated by Mann-Whitney U Rank Sum test comparing cytokine concentrations or responses for patients with controls. Significant differences (<i>p</i><0.05) are indicated in <b>bold type</b>.</p

(A–B): Patterns of cytokine responses.

<p>Patterns of cytokine response to a peptide cocktail representing early secreted antigenic target-6, culture filtrate protein 10, and TB7.7 (ESAT-6+CFP-10+TB7.7) are depicted for 12 patients with culture-confirmed tuberculosis (patients) in <b>Panel A,</b> and for 12 subjects at low risk for <i>Mtb</i> infection (controls) in <b>Panel B</b> that were summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026545#pone-0026545-t009" target="_blank">Table 9</a>. Cytokine concentrations were measured with a custom commercial multiplexed microsphere-based immunoassay (commercial MMIA). Responses were calculated by subtracting the background cytokine concentration in plasma from blood incubated with saline (Nil) from the cytokine concentration in plasma from blood incubated with <i>Mtb</i> antigens.</p

Cytokine concentrations and responses measured with a microarray.

<p>Median background cytokine concentrations (Nil, pg/mL) and cytokine responses (pg/mL) to phytohemagglutinin (Mitogen Response), early secreted antigenic target-6 (ESAT-6 Response), culture filtrate protein 10 (CFP-10 Response), TB7.7 (TB7.7 Response), and combinations of these <i>M. tuberculosis</i> (<i>Mtb</i>) antigens (ESAT-6+CFP-10 Response and ESAT-6+CFP-10+TB7.7 Response) are listed with ranges in parentheses. Cytokine concentrations were measured by a commercial quantitative immuno-microaray (microarray) for patients with culture-confirmed tuberculosis (patients) and subjects at low risk for <i>Mtb</i> exposure (controls). Responses were calculated by subtracting the background cytokine concentration in plasma from blood incubated with saline (Nil) from the cytokine concentration in plasma from blood incubated with mitogen or <i>Mtb</i> antigens. Cytokine responses for some patients and controls were not determined due to poor performance of the respective standard curves. N = 5 or 6 for patients subjects and N = 6 or 7 for controls unless indicated (*n = 4, **n = 5). <i>P</i> values were calculated by Mann-Whitney U Rank Sum test comparing cytokine concentrations or responses for patients with controls. Significant differences (<i>p</i><0.05) are indicated in <b>bold type</b>.</p

Comparisons of cytokine responses to different antigens measured by Microarray among patients with culture-confirmed tuberculosis.

<p>Median cytokine responses (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026545#pone-0026545-t006" target="_blank">Tables 6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026545#pone-0026545-t007" target="_blank">7</a>) to early secreted antigenic target-6 (ESAT-6), culture filtrate protein 10 (CFP-10), and combinations of these <i>M. tuberculosis</i> (<i>Mtb</i>) antigens (with or without TB7.7) for patients with culture-confirmed tuberculosis (patients) are listed with p values in parenthesis calculated by Mann-Whitney U Rank Sum test. Significant differences (<i>p</i><0.05) are indicated in <b>bold type</b>.</p