Defining the inflammatory pathway and prognostic neutrophil-derived biomarkers associated with pulmonary morbidity and long-term outcome of patients following TB therapy

Exacerbated neutrophil activity is generally linked to Tuberculosis (TB) disease severity. How-ever, TB-induced lung severity is dynamic with equal proportions of patients showing mild vs severe forms of lung damage after successful treatment. With recent studies revealing previously unseen neutrophil heterogeneity, it is likely that differential neutrophil profiles in patients contrib-ute to the extent of disease severity and lung recovery. Hence, monitoring neutrophil responses could allow identification of target molecules for host-directed therapies which can be coupled to antibiotic TB treatment (ATT) to limit lung damage and promote good lung recovery. The under-lying hypothesis for this thesis was that neutrophil heterogeneity is associated with differences in severity of lung pathology pre- and post-TB treatment in ATB patients. The main aim was to as-sess neutrophil phenotypes and function, and their soluble mediators in patients with differential lung function before and after TB therapy in The Gambia. First, focusing on ATB-related lung pathology, TB patients were grouped based on two clinically relevant parameters of ATB severity: chest x-ray scores (based on the well-defined Ralph Score incorporating extent of lung infiltrate and presence or absence of cavities) and GeneXpert Ultra bacterial load (Cycle threshold (Ct) value). For patients with severe lung damage at baseline, Ralph scores were also used to determine if they had good or poor recovery of lung pathology post treatment. We are currently analysing lung function recovery post treatment using spirometry readouts to complement the present findings. Analysis of neutrophils using stimulation of fresh whole blood was performed for the first time in The Gambia to monitor neutrophil function and determine differences between groups at baseline and following treatment. Considering that neu-trophils react to pathogen- and damage-associated molecular patterns (PAMPs and DAMPs, re-spectively) which trigger proinflammatory and antimicrobial responses in innate immune cells, I used Mtb H37Rv whole cell lysate (WCL) and ESAT-6/CFP-10 fusion protein which contain Mtb-specific molecular structures and nucleic acids (PAMPs) to elicit neutrophilic Mtb-specific re-sponse. Unstimulated blood samples were used to account for basal inflammatory levels and phorbol, 12-myristate, 13-acetate (PMA) was used as a positive control. Neutrophil subsets have been identified based on their CD16 and CD62L expression levels and shown to have varying functional attributes. Activated, they secrete differing levels of pro- and anti-inflammatory cytokines and regulate the inflammatory response to Mtb. In this study, we re-ported the relationship between functionality of the subsets following in-vitro activation and TB disease severity. At baseline, flow cytometry revealed WCL-specific activation of multiple neutro-phil subsets, increased ROS generation capacity, increased levels of banded neutrophils (CD16dimCD62Lbr) and higher levels of IL10-expressing CD16dimCD62Llo neutrophils in patients with mild lung pathology compared to severe pathology. However, TB-specific stimulation did not result in any differences in frequencies of these phenotypes with treatment time. Meanwhile, there was heterogeneity in frequencies of CD16|CD62L-defined neutrophil subsets in unstimulated samples with treatment compared to baseline. Having observed that neutrophil subsets play a role in ATB-related lung damage severity at base-line, we measured the concentrations of major neutrophil-derived mediators in antigen-stimulated WBA supernatants, together with ex vivo plasma and sputum samples from the same patients. This downstream assay involved analysis of a comprehensive neutrophil-focused panel from priming/activation markers like GM-CSF, IL8/CXCL8, TNF and IFNγ, through to damage-associ-ated molecular pattern (DAMP), Calprotectin (S100A8/9), to IL12/23(p40) and IL10 which pos-sess regulatory roles and finally matrix metalloproteinases (MMP1, MMP3, MMP9, MMP8) and myeloperoxidase (MPO) which regulate tissue damage. Overall, patients with severe lung pathol-ogy at baseline also had higher levels of S100A8/9, MMP8 and TNF. High S100A8/9 levels in patients with severe lung damage is in accordance with the increased neutrophil levels and neu-trophil activation seen by flow cytometry. S100A8/A9 has been previously shown to regulate CD11b expression and neutrophil recruitment. Moreover, our data showed that MMP8 association with TB disease severity is on par with that of S100A8/9, which to our knowledge is novel and underscores the importance of targeting this protein in further mechanistical studies. Interestingly, MPO was the only neutrophil-derived marker to be associated with all three disease severity criteria and the only one (together with IL10) to be associated with a protective effect against lung damage. Lower levels of MPO in sputum were associated with more severe lung damage, higher Mtb burden and poorer lung recovery after treatment. Lower levels of MPO were also found in females: who generally develop less severe inflammatory responses compared to males. MPO is also closely linked to ROS metabolism as it catalyses peroxide degradation. Whilst both MPO and ROS are usually linked to increased acute inflammation, our observations reveal their unexpected protective role—in limiting pulmonary pathology and resolving inflammation—during ATB. This supports investigating their potential for host-directed therapies. Finally, unre-solved lung damage after treatment was associated with persistently high sputum MMP9 and S100A9 levels at month 6 despite microbiological clearance. MMP9 and S100A8/9 are neutrophil-derived molecules reported to exacerbate tissue damage in chronic TB, which suggests that un-resolved lung damage even after treatment is also influenced by persistent neutrophil activity. In conclusion, our study contributes first-hand knowledge to the field by demonstrating that unlike other major neutrophil-derived inflammatory mediators (S100A8/9 and MMP8) and neutrophil subtypes, MPO, ROS (against Mtb burden), IL10 and banded (CD16dimCD62Lbr) neutrophils, play a protective role against TB-related lung pathology. These observations support the hypothesis that neutrophils harbour both pro-inflammatory and immunosuppressive characteristics in chronic TB pathogenesis. It also supports targeting neutrophils in further studies aimed at developing host-directed therapies against severe pathology in chronic ATB patients.Eine erhöhte Neutrophilenaktivität wird häufig mit der Schwere der Tuberkulose (TB) in Verbindung gebracht. Die Schwere der TB Lungenerkrankung ist jedoch dynamisch, wobei der Anteil der Patienten, die nach erfolgreicher Behandlung leichte und schwere Formen der Lung-enschädigung aufweisen, in etwa gleich groß ist. Studien, welche eine hohe Heterogenität der Neutrophilen aufzeigen, lassen vermuten, dass unterschiedliche Neutrophilenpopulationen bei den Patienten das Ausmaß der Krankheitsschwere und der Lungenheilung beeinflussen. Daher könnte das Monitoren verschiedener Neutrophilenpopulationen und deren Effektormoleküle die Identifizierung von Zielmolekülen für wirtsspezifische Therapien ermöglichen. Solche wirtsspezifischen Therapien könnten dann mit einer antibiotischen Tuberkulosebehandlung (ATT) kombiniert werden, um wiederum Lungenschäden zu begrenzen und eine gute Lungenheilung zu fördern. Die dieser Arbeit zugrunde liegende Hypothese war, dass Unterschiede in neutrophilen Zellpopulationen und Effektormolekülen mit Unterschieden in der Lungenpathologie vor und nach der Tuberkulosebehandlung einhergehen. Das Hauptziel bestand also darin, Phänotyp und die Funktion der Neutrophilen Zellpopulationen sowie ihre löslichen Mediatoren bei Patienten mit un-terschiedlicher Lungenfunktion vor und nach einer TB-Therapie in Gambia zu untersuchen. Zunächst wurden die TB-Patienten auf der Grundlage von zwei klinisch relevanten Parametern für den Schweregrad der ATB in Gruppen eingeteilt: Thoraxröntgen-Scores (basierend auf dem gut definierten Ralph-Score, der das Ausmaß des Lungeninfiltrats und das Vorhandensein oder Fehlen von Hohlräumen (engl: „Cavities“) und die GeneXpert-Ultra-Bakterienlast (PCR Cycle-Schwellenwert (Ct)). Bei Patienten mit schweren Lungenschäden zu Beginn der Studie wurden die Ralph-Scores auch verwendet, um zu untersuchen ob sich die Lungenpathologie nach der Behandlung verbesserte. Derzeit analysieren wir die Erholung der Lungenfunktion nach Behand-lung anhand von Spirometrie-Messungen, um die vorliegenden Ergebnisse weiter zu ergänzen. Die Analyse der Neutrophilen durch Stimulation von frischem Vollblut wurde durchgeführt, um die Neutrophilenfunktion zu studieren und Unterschiede zwischen den Gruppen zu Beginn und nach der Behandlung festzustellen. In Anbetracht der Tatsache, dass Neutrophile auf pathogen- und schadensassoziierte molekulare Muster (PAMPs bzw. DAMPs) reagieren, die proinflammato-rischen und antimikrobiellen Reaktionen in angeborenen Immunzellen auslösen, wurden Mtb H37Rv-Vollzelllysat (WCL) und ESAT-6/CFP-10-Fusionsprotein verwendet, die Mtb-spezifische molekulare Strukturen und Nukleinsäuren (PAMPs) enthalten. Unstimulierte Blutproben wurden verwendet, um basale Entzündungswerte zu berücksichtigen, und Phorbol-12-Myristat-13-Acetat (PMA) diente als Positivkontrolle. Unterschiedliche neutrophile Zellpopulationen wurden auf der Grundlage ihrer CD16- und CD62L-Expressionsmuster identifiziert, welche unterschiedliche funktionelle Eigenschaften aufweisen. Wenn sie aktiviert sind, scheiden sie unterschiedliche Mengen an pro- und anti-in-flammatorischen Zytokinen aus und regulieren die Entzündungsreaktion auf Mtb auf unterschie-dliche Art und Weise. In dieser Studie untersuchten wir den Zusammenhang zwischen der Funk-tionalität der unterschiedlichen neutrophilen Zellpopulationen nach in-vitro-Aktivierung und dem Schweregrad der TB-Erkrankung. Zu Beginn der Studie zeigten die Durchflusszytome-trieergebnisse eine WCL-spezifische Aktivierung mehrerer neutrophiler Subpopulationen, eine erhöhte ROS-Erzeugungskapazität, erhöhte Mengen an gebänderten Neutrophilen (CD16dimCD62Lbr) und höhere Mengen an IL10-exprimierenden CD16dimCD62Llo-Neutro-philen bei Patienten mit leichter Lungenpathologie im Vergleich zu solchen mit schwerer Pathol-ogie. Die TB-spezifische Stimulation führte jedoch nicht zu Unterschieden in der Häufigkeit dieser Phänotypen mit der Behandlungsdauer. Die Häufigkeit der CD16|CD62L-definierten neutrophilen Untergruppen in den nicht stimulierten Proben war im Vergleich zum Ausgangswert vor der Behandlung heterogen. Nachdem wir festgestellt hatten, dass unterschiedliche neutrophile Zellpopulationen bei der Schwere der ATB-bedingten Lungenschädigung vor Behandlungsbeginn eine Rolle spielen, ha-ben wir die Konzentrationen der wichtigsten neutrophilen Effektormediatoren in antigenstimu-lierten Vollblut-Überständen zusammen mit Plasma- und Sputumproben derselben Patienten ge-messen. Unser Assay beinhaltete die Analyse von Neutrophilen-assoziierten Zytokinen und weiteren Effektormolekülen wie GM-CSF, IL8/CXCL8, TNF und IFNγ, Calprotectin (S100A8/9), IL12/23(p40) und IL10, und schließlich Matrix-Metalloproteinasen (MMP1, MMP3, MMP9, MMP8) und Myeloperoxidase (MPO), die Gewebeschäden regulieren. Insgesamt wiesen Patienten mit schwerer Lungenpathologie bei Studienbeginn höhere Werte von S100A8/9, MMP8 und TNF auf. Die hohen S100A8/9-Werte bei Patienten mit schweren Lungenschäden stehen im Einklang mit den erhöhten Neutrophilenspiegeln und deren Aktivierung. Es wurde bereits gezeigt, dass S100A8/A9 die CD11b-Expression sowie die Rekrutierung von Neutrophilen reguliert. Darüber hinaus zeigten unsere Daten, dass die MMP8 Konzentration mit dem Schweregrad der TB-Erkrankung und der von S100A8/9 korreliert, was neu ist und in weiteren mechanistischen Studien untersucht werden sollte. Interessanterweise war MPO der einzige von Neutrophilen stammende Marker, der mit allen drei Kriterien für den Schweregrad der Erkrankung assoziiert war, und zusammen mit IL10 der einzige Marker, der mit geringen Lungenschäden assoziiert war. Niedrigere MPO-Werte im Sputum wurden mit schwereren Lungenschäden, einer höheren Mtb-Belastung und einer schlechteren Erholung der Lunge nach der Behandlung in Verbindung gebracht. Niedrigere MPO-Werte wurden auch bei Frauen festgestellt, die im Allgemeinen eher weniger schwere Entzündungs-reaktionen entwickeln als Männer. MPO ist auch eng mit dem ROS-Stoffwechsel verbunden, da es den Peroxidabbau katalysiert. Während MPO und ROS in der Regel mit einer verstärkten akuten Entzündung in Verbindung gebracht werden, zeigen unsere Beobachtungen eine unerwartete schützende Rolle - die Begrenzung der TB Lungenpathologie und das Abklingen der Entzündung. Dies spricht dafür, das Potenzial von MPO auch für wirtsspezifische Therapien zu untersuchen. Schließlich wurden Lungenschädigungen, welche nach der erfolgreichen 6-mona-tigen Behandlung und mikrobiologischer Clearance immer noch evident waren, mit hohen Spu-tum MMP9 and S100A9 Werten assoziiert. MMP9 und S100A8/9 sind von Neutrophilen stam-mende Moleküle, welche die Gewebeschäden bei chronischer Tuberkulose verschlimmern. Dies deutet darauf hin, dass diese persistierenden Lungenschäden auch nach der Behandlung durch die anhaltende Aktivität der Neutrophilen beeinflusst werden. Zusammenfassend lässt sich sagen, dass unsere Studie neue Erkenntnisse aus erster Hand liefert. Sie weisen darauf hin, dass im Gegensatz zu anderen wichtigen neutrophilen Entzün-dungsmediatoren (S100A8/9 und MMP8) und Neutrophilen-Subtypen, MPO, ROS (gegen die Mtb-Belastung), IL10 und stabkernige (CD16dimCD62Lbr) Neutrophile eine schützende Rolle gegen TB-bedingte Lungenpathologie spielen. Diese Beobachtungen stützen die Hypothese, dass Neutrophile bei der chronischen TB-Pathogenese sowohl entzündungsfördernde als auch immunosuppressive Eigenschaften aufweisen können. Sie sprechen auch dafür, neutrophile Zell-populationen und Effektormoleküle in weiteren Studien zur Entwicklung wirtsspezifischer Tuberk-ulosetherapien zu berücksichtigen

Neutrophils in Tuberculosis-Associated Inflammation and Lung Pathology.

Protective immunity to Mycobacterium tuberculosis (Mtb)-the causative agent of tuberculosis (TB)-is not fully understood but involves immune responses within the pulmonary airways which can lead to exacerbated inflammation and immune pathology. In humans, this inflammation results in lung damage; the extent of which depends on specific host pro-inflammatory processes. Neutrophils, though increasingly linked to the development of inflammatory disorders, have been less well studied in relation to TB-induced lung pathology. Neutrophils mode of action and their specialized functions can be directly linked to TB-specific lung tissue damage observed on patient chest X-rays at diagnosis and contribute to long-term pulmonary sequelae. This review discusses aspects of neutrophil activity associated with active TB, including the resulting inflammation and pulmonary impairment. It highlights the significance of neutrophil function on TB disease outcome and underlines the necessity of monitoring neutrophil function for better assessment of the immune response and severity of lung pathology associated with TB. Finally, we propose that some MMPs, ROS, MPO, S100A8/A9 and Glutathione are neutrophil-related inflammatory mediators with promising potential as targets for developing host-directed therapies for TB

Neutrophils Contribute to Severity of Tuberculosis Pathology and Recovery From Lung Damage Pre- and Posttreatment.

BACKGROUND: Despite microbiological cure, about 50% of tuberculosis (TB) patients have poor lung recovery. Neutrophils are associated with lung pathology; however, CD16/CD62L-defined subsets have not been studied in TB. Using flow cytometry, we monitored frequencies, phenotype, and function of neutrophils following stimulation with Mycobacterium tuberculosis (Mtb) whole cell lysate (WCL) and ESAT-6/CFP-10 fusion protein (EC) in relation to lung pathology. METHODS: Fresh blood from 42 adult, human immunodeficiency virus (HIV)-negative TB patients were analyzed pre- and post-therapy, with disease severity determined using chest radiography and bacterial load. Flow cytometry was used to monitor frequencies, phenotype, and function (generation of reactive oxygen species [ROS], together with CD11b, tumor necrosis factor, and interleukin 10 [IL-10] expression) of neutrophils following 2-hour stimulation with Mtb-specific antigens. RESULTS: Total neutrophils decreased by post-treatment compared to baseline (P = .0059); however, CD16brCD62Lbr (segmented) neutrophils increased (P = .0031) and CD16dimCD62Lbr (banded) neutrophils decreased (P = .038). Banded neutrophils were lower in patients with severe lung damage at baseline (P = .035). Following WCL stimulation, ROS from segmented neutrophils was higher in patients with low Mtb loads even after adjusting for sex (P = .038), whereas IL-10-expressing CD16dimCD62Llo cells were higher in patients with mild damage (P = .0397) at baseline. CONCLUSIONS: High ROS generation, low levels of banded neutrophils, and high levels of IL-10-expressing CD16dimCD62Llo neutrophils are associated with reduced lung pathology at diagnosis. Hence, neutrophils are potential early indicators of TB severity and promising targets for TB host-directed therapy

Major Neutrophil-Derived Soluble Mediators Associate With Baseline Lung Pathology and Post-Treatment Recovery in Tuberculosis Patients

Background: The inflammatory response to Mycobacterium tuberculosis results in variable degrees of lung pathology during active TB (ATB) with central involvement of neutrophils. Little is known about neutrophil-derived mediators and their role in disease severity at baseline and recovery upon TB treatment initiation. Methods: 107 adults with confirmed pulmonary TB were categorised based on lung pathology at baseline and following successful therapy using chest X-ray scores (Ralph scores) and GeneXpert bacterial load (Ct values). Plasma, sputum, and antigen-stimulated levels of MMP1, MMP3, MMP8, MMP9, MPO, S100A8/9, IL8, IL10, IL12/23(p40), GM-CSF, IFNγ, and TNF were analysed using multiplex cytokine arrays. Results: At baseline, neutrophil counts correlated with plasma levels of MMP8 (rho = 0.45, p = 2.80E-06), S100A8 (rho = 0.52, p = 3.00E-08) and GM-CSF (rho = 0.43, p = 7.90E-06). Levels of MMP8 (p = 3.00E-03), MMP1 (p = 1.40E-02), S100A8 (p = 1.80E-02) and IL12/23(p40) (p = 1.00E-02) were associated with severe lung damage, while sputum MPO levels were directly linked to lung damage (p = 1.80E-03), Mtb load (p = 2.10E-02) and lung recovery (p = 2.40E-02). Six months of TB therapy significantly decreased levels of major neutrophil-derived pro-inflammatory mediators: MMP1 (p = 4.90E-12 and p = 2.20E-07), MMP8 (p = 3.40E-14 and p = 1.30E-05) and MMP9 (p = 1.60E-04 and p = 1.50E-03) in plasma and sputum, respectively. Interestingly, following H37Rv whole cell lysate stimulation, S100A8 (p = 2.80E-02), MMP9 (p = 3.60E-02) and MPO (p = 9.10E-03) levels at month 6 were significantly higher compared to baseline. Sputum MMP1 (p = 1.50E-03), MMP3 (p = 7.58E-04), MMP9 (p = 2.60E-02) and TNF (p = 3.80E-02) levels were lower at month 6 compared to baseline in patients with good lung recovery. Conclusion: In this study, patients with severe lung pathology at baseline and persistent lung damage after treatment were associated with higher plasma and sputum levels of major pro-inflammatory neutrophil-derived mediators. Interestingly, low sputum MPO levels were associated with severe lung damage, higher Mtb burden and low recovery. Our data suggest that therapeutic agents which target these mediators should be considered for future studies on biomarkers and host-directed therapeutic approaches against TB-related lung pathology and/or lung recovery

Pathogenesis of Post-Tuberculosis Lung Disease: Defining Knowledge Gaps and Research Priorities at the 2 International Post-Tuberculosis Symposium.

Post-tuberculosis (TB) lung disease (PTLD) is increasingly recognized as a major contributor to the global burden of chronic lung disease, with recent estimates indicating that over half of TB survivors have impaired lung function after successful completion of TB treatment. However, the pathologic mechanisms that contribute to PTLD are not well understood, thus limiting the development of therapeutic interventions to improve long-term outcomes after TB. This report summarizes the work of the "Pathogenesis and Risk Factors Committee" for the Second International Post-Tuberculosis Symposium, which took place in Stellenbosch, South Africa in April 2023. The committee first identified six areas with high translational potential: (1) tissue matrix destruction, including the role of matrix metalloproteinase dysregulation and neutrophil activity, (2) fibroblasts and profibrotic activity, (3) granuloma fate and cell death pathways, (4) mycobacterial factors including pathogen burden, (5) animal models, and (6) the impact of key clinical risk factors including HIV, diabetes, smoking, malnutrition, and alcohol. We share here the key findings from a literature review of those areas, highlighting knowledge gaps and areas where further research is needed

Pathogenesis of post-tuberculosis lung disease: defining knowledge gaps and research priorities at the 2nd International Post-Tuberculosis Symposium

Post-tuberculosis (TB) lung disease (PTLD) is increasingly recognized as a major contributor to the global burden of chronic lung disease, with recent estimates indicating that over half of TB survivors have impaired lung function after successful completion of TB treatment. However, the pathologic mechanisms that contribute to PTLD are not well understood, thus limiting the development of therapeutic interventions to improve long-term outcomes after TB. This report summarizes the work of the "Pathogenesis and Risk Factors Committee" for the Second International Post-Tuberculosis Symposium, which took place in Stellenbosch, South Africa in April 2023. The committee first identified six areas with high translational potential: (1) tissue matrix destruction, including the role of matrix metalloproteinase dysregulation and neutrophil activity, (2) fibroblasts and profibrotic activity, (3) granuloma fate and cell death pathways, (4) mycobacterial factors including pathogen burden, (5) animal models, and (6) the impact of key clinical risk factors including HIV, diabetes, smoking, malnutrition, and alcohol. We share here the key findings from a literature review of those areas, highlighting knowledge gaps and areas where further research is needed.</p