HMGB1/RAGE Signaling and Pro-Inflammatory Cytokine Responses in Non-HIV Adults with Active Pulmonary Tuberculosis

<div><p>Background</p><p>We aimed to study the pathogenic roles of High-Mobility Group Box 1 (HMGB1) / Receptor-for-Advanced-Glycation-End-products (RAGE) signaling and pro-inflammatory cytokines in patients with active pulmonary tuberculosis (PTB).</p><p>Methods</p><p>A prospective study was conducted among non-HIV adults newly-diagnosed with active PTB at two acute-care hospitals (n = 80); age-and-sex matched asymptomatic individuals (tested for latent TB) were used for comparison (n = 45). Plasma concentrations of 8 cytokines/chemokines, HMGB1, soluble-RAGE, and transmembrane-RAGE expressed on monocytes/dendritic cells, were measured. Gene expression (mRNA) of HMGB1, RAGE, and inflammasome-NALP3 was quantified. Patients’ PBMCs were stimulated with recombinant-HMGB1 and MTB-antigen (lipoarabinomannan) for cytokine induction e<i>x vivo</i>.</p><p>Results</p><p>In active PTB, plasma IL-8/CXCL8 [median(IQR), 6.0(3.6–15.1) <i>vs</i> 3.6(3.6–3.6) pg/ml, P<0.001] and IL-6 were elevated, which significantly correlated with mycobacterial load, extent of lung consolidation (<b><i>r</i></b>_{<b><i>s</i></b>} +0.509, P<0.001), severity-score (<b><i>r</i></b>_{<b><i>s</i></b>} +0.317, P = 0.004), and fever and hospitalization durations (<b><i>r</i></b>_{<b><i>s</i></b>} +0.407, P<0.001). IL-18 and sTNFR1 also increased. Plasma IL-8/CXCL8 (adjusted OR 1.12, 95%CI 1.02–1.23 per unit increase, P = 0.021) and HMGB1 (adjusted OR 1.42 per unit increase, 95%CI 1.08–1.87, P = 0.012) concentrations were independent predictors for respiratory failure, as well as for ICU admission/death. Gene expression of HMGB1, RAGE, and inflammasome-NALP3 were upregulated (1.2−2.8 fold). Transmembrane-RAGE was increased, whereas the decoy soluble-RAGE was significantly depleted. RAGE and HMGB1 gene expressions positively correlated with cytokine levels (IL-8/CXCL8, IL-6, sTNFR1) and clinico-/radiographical severity (e.g. extent of consolidation <i>r</i>_s +0.240, P = 0.034). <i>Ex vivo</i>, recombinant-HMGB1 potentiated cytokine release (e.g. TNF-α) when combined with lipoarabinomannan.</p><p>Conclusion</p><p>In patients with active PTB, HMGB1/RAGE signaling and pro-inflammatory cytokines may play important roles in pathogenesis and disease manifestations. Our clinico-immunological data can provide basis for the development of new strategies for disease monitoring, management and control.</p></div

Expressions of RAGE, HMGB1 and inflammasome in active PTB patients, compared with IGRA-positive and IGRA-negative asymptomatic individuals.

<p>Expressions of RAGE, HMGB1 and inflammasome in active PTB patients, compared with IGRA-positive and IGRA-negative asymptomatic individuals.</p

Correlations between plasma cytokine levels, and clinical and bacteriological variables in patients with active PTB (n = 80).

<p>Correlations between plasma cytokine levels, and clinical and bacteriological variables in patients with active PTB (n = 80).</p

High plasma IL-6 and IL-8/CXCL8 concentrations were significantly associated with development of respiratory failure (hypoxemia, upper panels), and the adverse outcomes of ICU admission/death (lower panels) in patients with confirmed active PTB (n = 80).

<p><b>Note</b>. Other cytokines, hypoxemia [yes <i>vs</i> no]: IL-18 [median (IQR), 1655.0 (1266.6–2185.8) <i>vs</i> 1516.3 (1285.8–1882.3) pg/mL]; sTNFR1 [median (IQR), 1460.2 (1054.1–2008.7) <i>vs</i> 1664.6 (1089.2–2571.4) pg/mL; HMGB1 [median(IQR), 3.5(2.1–5.2) <i>vs</i> 3.2 (1.7–4.5)], outliers excluded; all P >0.05.</p

<i>Ex vivo</i> stimulation of PBMC with recombinant-HMGB1 alone, LAM alone and their combinations.

<p>TNF-α release was substantially higher with HMGB1 and LAM co-stimulation; the response in active PTB patients was about 2 times greater than in the uninfected (PTB <i>vs</i> IGRA-negative subjects, median(IQR) fold-change, 14.4(4.7–39.9) <i>vs</i> 6.6(2.6–11.1), P = 0.005). <b>Footnotes</b>: ATB, patients with active PTB; Control, IGRA-negative individuals. Fold change: TNF-α release with/without ligand stimulation. Box and whisker represent 50^th and 75^th percentiles respectively; Mann Whitney U test. Median fold-change of TNF-α with HMGB1+LAM stimulation in ATB cases <i>vs</i> all IGRA-positive/negative individuals: 14.4 (4.7–39.9) vs 6.1 (2.8–11.0), P = 0.001.</p

Independent variables associated with development of respiratory failure in active PTB, as shown in final multivariable logistic regression models.

<p>Independent variables associated with development of respiratory failure in active PTB, as shown in final multivariable logistic regression models.</p

Plasma concentrations of pro-inflammatory cytokines/chemokines, HMGB1, and RAGE in patients with active PTB, compared with IGRA-positive and IGRA-negative asymptomatic individuals.

<p>Plasma concentrations of pro-inflammatory cytokines/chemokines, HMGB1, and RAGE in patients with active PTB, compared with IGRA-positive and IGRA-negative asymptomatic individuals.</p

Correlations between initial cytokine/chemokine concentrations and clinical parameters at presentation (temperature, respiratory rate, oxygen saturation) and the clinical outcomes (hospital length-of-stay, ICU admission).

<p>For correlations with temperature, respiratory rate, oxygen saturation, and length-of-stay, the Spearman's rank coefficients (<i>rho</i>) were shown. For risk of ICU admission, the adjusted odds ratio and the 95% confidence interval (CI) per log₁₀ unit increase in cytokine concentration were shown (adjusted for age, comorbidity and time from onset). Data on respiratory rate was incomplete in seasonal influenza cases, and there were too few ICU admissions to allow meaningful analysis.</p><p>*p<0.05,</p><p>**p<0.01.</p

PBMC activation and <i>ex vivo</i> cytokine/chemokine expression during seasonal influenza infection.

<p>PBMC actively expressed IL-6, (CXCL8/IL-8), CCL2/MCP-1, CXCL10/IP-10, and CXCL9/MIG during acute influenza infection; upon illness recovery, cytokine production decreased, and there was a corresponding increase in cellular responsiveness to stimuli. Cytokine response pre-/post-stimulation and the trend changes in cytokine expression across time points (with PHA/LPS stimulation – red bars; without stimulation – blue bars; folds increase in expression or the ‘responsiveness’ – gray bars) were compared using the <i>Mann-Whitney U</i> test (asterisks, underlined), and the <i>Jonckheere-Terpstra</i> test (blue/gray triangles and asterisks), respectively. IL-17A did not appear to be activated via the PHA/LPS stimulation pathway.</p

Serial changes in plasma cytokine/chemokine concentrations during the course of hospitalization.

<p>There was sustained elevation of the proinflammatory cytokines (IL-6, CXCL8/IL-8, CCL2/MCP-1, sTNFR-1) in severe pH1N1 pneumonia; the adaptive-immunity related cytokines (CXCL10/IP-10, CXCL9/MIG, IL-17A) were markedly suppressed compared with seasonal influenza. All patients with pH1N1 influenza (severe pneumonia, n = 34; milder illness, n = 29) received antiviral treatment soon after hospitalization/recruitment; none had received high-dose corticosteroids or other immunosuppressants for ‘viral pneumonitis’ or ‘ARDS’<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026050#pone.0026050-Lee2" target="_blank">[8]</a>. Among seasonal influenza patients (most had complicated illnesses, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026050#pone-0026050-t001" target="_blank">Table 1</a> footnotes), 30(57%) received antiviral treatment. Median concentrations at each time point are shown for each group; the interquartile ranges (presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026050#pone-0026050-t001" target="_blank">Table 1</a>) are omitted here for clarity. Fewer mild pH1N1 and untreated seasonal influenza patients remained hospitalized at day 6–7 for study (Day 1, n = 116; Day 3–4, n = 62; Day 6–7, n = 30).</p