Inflammation-mediated tissue damage in pulmonary tuberculosis and host-directed therapeutic strategies.

Treatment of tuberculosis (TB) involves the administration of anti-mycobacterial drugs for several months. The emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb, the causative agent) together with increased disease severity in people with co-morbidities such as diabetes mellitus and HIV have hampered efforts to reduce case fatality. In severe disease, TB pathology is largely attributable to over-exuberant host immune responses targeted at controlling bacterial replication. Non-resolving inflammation driven by host pro-inflammatory mediators in response to high bacterial load leads to pulmonary pathology including cavitation and fibrosis. The need to improve clinical outcomes and reduce treatment times has led to a two-pronged approach involving the development of novel antimicrobials as well as host-directed therapies (HDT) that favourably modulate immune responses to Mtb. HDT strategies incorporate aspects of immune modulation aimed at downregulating non-productive inflammatory responses and augmenting antimicrobial effector mechanisms to minimise pulmonary pathology and accelerate symptom resolution. HDT in combination with existing antimycobacterial agents offers a potentially promising strategy to improve the long-term outcome for TB patients. In this review, we describe components of the host immune response that contribute to inflammation and tissue damage in pulmonary TB, including cytokines, matrix metalloproteinases, lipid mediators, and neutrophil extracellular traps. We then proceed to review HDT directed at these pathways.

Host-directed therapy to combat mycobacterial infections.

Upon infection, mycobacteria, such as Mycobacterium tuberculosis (Mtb) and nontuberculous mycobacteria (NTM), are recognized by host innate immune cells, triggering a series of intracellular processes that promote mycobacterial killing. Mycobacteria, however, have developed multiple counter-strategies to persist and survive inside host cells. By manipulating host effector mechanisms, including phagosome maturation, vacuolar escape, autophagy, antigen presentation, and metabolic pathways, pathogenic mycobacteria are able to establish long-lasting infection. Counteracting these mycobacteria-induced host modifying mechanisms can be accomplished by host-directed therapeutic (HDT) strategies. HDTs offer several major advantages compared to conventional antibiotics: (a) HDTs can be effective against both drug-resistant and drug-susceptible bacteria, as well as potentially dormant mycobacteria; (b) HDTs are less likely to induce bacterial drug resistance; and (c) HDTs could synergize with, or shorten antibiotic treatment by targeting different pathways. In this review, we will explore host-pathogen interactions that have been identified for Mtb for which potential HDTs impacting both innate and adaptive immunity are available, and outline those worthy of future research. We will also discuss possibilities to target NTM infection by HDT, although current knowledge regarding host-pathogen interactions for NTM is limited compared to Mtb. Finally, we speculate that combinatorial HDT strategies can potentially synergize to achieve optimal mycobacterial host immune control.

Adjunctive pascolizumab in rifampicin-susceptible pulmonary tuberculosis: proof-of-concept, partially-randomised, double-blind, placebo-controlled, dose-escalation trial.

BACKGROUND: Interleukin-4 (IL-4), increased in tuberculosis infection, may impair bacterial killing. Blocking IL-4 confers benefit in animal models. We evaluated safety and efficacy of pascolizumab (humanised anti-IL-4 monoclonal antibody) as adjunctive tuberculosis treatment. METHODS: Participants with rifampicin-susceptible pulmonary tuberculosis received a single intravenous infusion of pascolizumab or placebo; and standard 6-month tuberculosis treatment. Pascolizumab dose increased in successive cohorts: [1] non-randomised 0.05 mg/kg (n = 4); [2] non-randomised 0.5 mg/kg (n = 4); [3] randomised 2.5 mg/kg (n = 9) or placebo (n = 3); [4] randomised 10 mg/kg (n = 9) or placebo (n = 3). Co-primary safety outcome was study-drug-related grade 4 or serious adverse event (G4/SAE); in all cohorts (1-4). Co-primary efficacy outcome was week-8 sputum culture time-to-positivity (TTP); in randomised cohorts (3-4) combined. RESULTS: Pascolizumab levels exceeded IL-4 50% neutralising dose for 8 weeks in 78-100% of participants in cohorts 3-4. There were no study-drug-related G4/SAEs. Median week-8 TTP was 42 days in pascolizumab and placebo groups (p = 0.185). Rate of TTP increase was greater with pascolizumab (difference from placebo 0.011 [95% Bayesian credible interval 0.006 to 0.015] log10TTP/day. CONCLUSIONS: There was no evidence to suggest blocking IL-4 was unsafe. Preliminary efficacy findings are consistent with animal models. This supports further investigation of adjunctive anti-IL-4 interventions for tuberculosis in larger phase 2 trials.

The Cyclooxygenase 2 Inhibitor Etoricoxib as Adjunctive Therapy in Tuberculosis Impairs Macrophage Control of Mycobacterial Growth.

BACKGROUND: Current tuberculosis treatment regimens could be improved by adjunct host-directed therapies (HDT) targeting host responses. We investigated the antimycobacterial capacity of macrophages from patients with tuberculosis in a phase 1/2 randomized clinical trial (TBCOX2) of the cyclooxygenase-2 inhibitor etoricoxib. METHODS: Peripheral blood mononuclear cells from 15 patients with tuberculosis treated with adjunctive COX-2i and 18 controls (standard therapy) were collected on day 56 after treatment initiation. The ex vivo capacity of macrophages to control mycobacterial infection was assessed by challenge with Mycobacterium avium, using an in vitro culture model. Macrophage inflammatory responses were analyzed by gene expression signatures, and concentrations of cytokines were analyzed in supernatants by multiplex. RESULTS: Macrophages from patients receiving adjunctive COX-2i treatment had higher M. avium loads than controls after 6 days, suggesting an impaired capacity to control mycobacterial infection compared to macrophages from the control group. Macrophages from the COX-2i group had lower gene expression of TNF, IL-1B, CCL4, CXCL9, and CXCL10 and lowered production of cytokines IFN-ß and S100A8/A9 than controls. CONCLUSIONS: Our data suggest potential unfavorable effects with impaired macrophage capacity to control mycobacterial growth in patients with tuberculosis receiving COX-2i treatment. Larger clinical trials are required to analyze the safety of COX-2i as HDT in patients with tuberculosis. CLINICAL TRIALS REGISTRATION: NCT02503839.

Mimicry of Short Linear Motifs by Bacterial Pathogens: A Drugging Opportunity.

Bacterial pathogens have developed complex strategies to successfully survive and proliferate within their hosts. Throughout the infection cycle, direct interaction with host cells occurs. Many bacteria have been found to secrete proteins, such as effectors and toxins, directly into the host cell with the potential to interfere with cell regulatory processes, either enzymatically or through protein-protein interactions (PPIs). Short linear motifs (SLiMs) are abundant peptide modules in cell signaling proteins. Here, we cover the reported examples of eukaryotic-like SLiM mimicry being used by pathogenic bacteria to hijack host cell machinery and discuss how drugs targeting SLiM-regulated cell signaling networks are being evaluated for interference with bacterial infections. This emerging anti-infective opportunity may become an essential contributor to antibiotic replacement strategies.

Inhibition of leukotriene-B4 signalling-mediated host response to tuberculosis is a potential mode of adjunctive host-directed therapy.

Treatment of tuberculosis (TB) is faced with several challenges including the long treatment duration, drug toxicity and tissue pathology. Host-directed therapy provides promising avenues to find compounds for adjunctively assisting antimycobacterials in the TB treatment regimen, by promoting pathogen eradication or limiting tissue destruction. Eicosanoids are a class of lipid molecules that are potent mediators of inflammation and have been implicated in aspects of the host response against TB. Here, we have explored the blood transcriptome of pulmonary TB patients to understand the activity of leukotriene B4, a pro-inflammatory eicosanoid. Our study shows a significant upregulation in the leukotriene B4 signalling pathway in active TB patients, which is reversed with TB treatment. We have further utilized our in-house network analysis algorithm, ResponseNet, to identify potential downstream signal effectors of leukotriene B4 in TB patients including STAT1/2 and NADPH oxidase at a systemic as well as local level, followed by experimental validation of the same. Finally, we show the potential of inhibiting leukotriene B4 signalling as a mode of adjunctive host-directed therapy against TB. This study provides a new mode of TB treatment along with mechanistic insights which can be further explored in pre-clinical trials.

Advances in the targeted theragnostics of osteomyelitis caused by Staphylococcus aureus.

Bone infections caused by Staphylococcus aureus may lead to an inflammatory condition called osteomyelitis, which results in progressive bone loss. Biofilm formation, intracellular survival, and the ability of S. aureus to evade the immune response result in recurrent and persistent infections that present significant challenges in treating osteomyelitis. Moreover, people with diabetes are prone to osteomyelitis due to their compromised immune system, and in life-threatening cases, this may lead to amputation of the affected limbs. In most cases, bone infections are localized; thus, early detection and targeted therapy may prove fruitful in treating S. aureus-related bone infections and preventing the spread of the infection. Specific S. aureus components or overexpressed tissue biomarkers in bone infections could be targeted to deliver active therapeutics, thereby reducing drug dosage and systemic toxicity. Compounds like peptides and antibodies can specifically bind to S. aureus or overexpressed disease markers and combining these with therapeutics or imaging agents can facilitate targeted delivery to the site of infection. The effectiveness of photodynamic therapy and hyperthermia therapy can be increased by the addition of targeting molecules to these therapies enabling site-specific therapy delivery. Strategies like host-directed therapy focus on modulating the host immune mechanisms or signaling pathways utilized by S. aureus for therapeutic efficacy. Targeted therapeutic strategies in conjunction with standard surgical care could be potential treatment strategies for S. aureus-associated osteomyelitis to overcome antibiotic resistance and disease recurrence. This review paper presents information about the targeting strategies and agents for the therapy and diagnostic imaging of S. aureus bone infections.

Cell death induced by NLRP3-palmitate axis impairs pulmonary damage tolerance and aggravates immunopathology during obesity-tuberculosis comorbidity.

A low-grade and persistent inflammation, which is the hallmark of obesity, requires the participation of NLRP3 and cell death. During Mycobacterium tuberculosis infection, NLRP3 signaling is important for bacterial killing by macrophages in vitro but was shown to be dispensable for host protection in vivo. We hypothesized that during obesity-tuberculosis (TB) comorbidity, NLRP3 signaling might play a detrimental role by inducing excessive inflammation. We employed a model of high-fat-diet-induced obesity, followed by M. tuberculosis infection in C57BL/6 mice. Obese mice presented increased susceptibility to infection and pulmonary immunopathology compared to lean mice. Using treatment with NLRP3 antagonist and Nlrp3-/- mice, we showed that NLRP3 signaling promoted cell death, with no effect in bacterial loads. The levels of palmitate were higher in the lungs of obese infected mice compared to lean counterparts, and we observed that this lipid increased M. tuberculosis-induced macrophage death in vitro, which was dependent on NLRP3 and caspase-1. At the chronic phase, although lungs of obese Nlrp3-/- mice showed an indication of granuloma formation compared to obese wild-type mice, there was no difference in the bacterial load. Our findings indicate that NLRP3 may be a potential target for host-directed therapy to reduce initial and severe inflammation-mediated disease and to treat comorbidity-associated TB. © 2022 The Pathological Society of Great Britain and Ireland.

The first-in-class pyrazole-based dual InhA-VEGFR inhibitors towards integrated antitubercular host-directed therapy.

Several facets of the host response to tuberculosis have been tapped for clinical investigation, especially targeting angiogenesis mediated by VEGF signaling from infected macrophages. Herein, we rationalized combining the antiangiogenic effects of VEGFR-2 blockade with direct antitubercular InhA inhibition in single hybrid dual inhibitors as advantageous alternatives to the multidrug regimens. Inspired by expanded triclosans, the ether ligation of triclosan was replaced by rationalized linkers to assemble the VEGFR-2 inhibitors thematic scaffold. Accordingly, new series of 3-(p-chlorophenyl)-1-phenylpyrazole derivatives tethered to substituted ureas and their isosteres were synthesized, evaluated against Mycobacterium tuberculosis virulent cell line H37Rv, and assessed for their InhA inhibitory activities. The urea derivatives 8d and 8g exhibited the most promising antitubercular activity (MIC = 6.25 µg/mL) surpassing triclosan (MIC = 20 µg/mL) with potential InhA inhibition, thus identified as the study hits. Interestingly, both compounds inhibited VEGFR-2 at nanomolar IC50 (15.27 and 24.12 nM, respectively). Docking and molecular dynamics simulations presumed that 8d and 8g could bind to their molecular targets InhA and VEGFR-2 posing essential stable interactions shared by the reference inhibitors triclosan and sorafenib. Finally, practical LogP, Lipinski's parameters and in silico ADMET calculations highlighted their drug-likeness as novel leads in the arsenal against TB.

Harnessing endogenous peptide compounds as potential therapeutics for severe influenza.

BACKGROUND: Excessive pulmonary inflammation and damage are characteristic features of severe influenza virus infections. LAT8881 is a synthetic, 16 amino acid cyclic peptide form of a naturally occurring C-terminal fragment of human growth hormone with therapeutic efficacy against influenza. Shorter, linear peptides are typically easier to manufacture and formulate for delivery than larger cyclic peptides. A 6 amino acid linear peptide fragment of LAT8881, LAT9997, was investigated as a potential influenza therapy. METHODS: LAT9997 was evaluated for its potential to limit disease in a preclinical mouse model of severe influenza infection. RESULTS: Intranasal treatment of mice with either LAT8881 or LAT9997 from day 1 following influenza infection significantly improved survival outcomes. Initiating LAT9997 treatment at the onset of severe disease, also significantly improved disease severity. Greater disease resistance in LAT9997-treated mice correlated with reduced lung immunopathology, damage markers, vascular leak, and epithelial cell death. Treatment reduced viral loads, cytokines, and neutrophil infiltration in the airways, yet maintained protective alveolar macrophages in a dose-dependent manner. Sequential trimming of N- and C-terminal amino acids from LAT9997 revealed a structure-activity relationship. CONCLUSIONS: These findings provide preclinical evidence that therapeutic LAT9997 treatment limits viral burden and characteristic features of severe influenza, including hyperinflammation and lung damage.

TFEB - at the crossroads of host-pathogen interactions.

The existence of constantly evolving dynamic interactions between the host and the pathogen determines their fate in this continuous arms race. Hence, identifying the molecular basis of processes that reinforce host defensive strategies to eliminate intracellular pathogens is of utmost significance. Pathogenic intrusion activates autophagy and phagocytic pathways that culminate in the lysosome, a vital organelle responsible for pathogen clearance. The transcription factor TFEB plays a pivotal role in autophagy-lysosomal function. Although TFEB is an emerging transcription factor in the field of immune signaling pathways, its role in infectious diseases remains contentious. Recent evidence suggests that infection with certain bacterial and viral pathogens causes TFEB, which is normally located in the cytoplasm, to translocate to the nucleus. There, it activates the transcription of genes that trigger the autophagy-lysosomal and inflammatory pathways to target intracellular pathogens. It is known that some pathogens modulate TFEB to establish themselves inside the host; in some cases, pathogens restrict TFEB to the cytoplasm, whereas in others, functional TFEB fuels pathogen survival and replication. However, the key regulators and molecular mechanisms that decide the outcome of TFEB function during intracellular infection are not clear. In this Review, we attempt to dissect the complex functions of TFEB in host-pathogen interactions and explore the suitability of TFEB as a therapeutic target of clinical relevance.

Mesenchymal Stromal Cells: an Antimicrobial and Host-Directed Therapy for Complex Infectious Diseases.

There is an urgent need for new antimicrobial strategies for treating complex infections and emerging pathogens. Human mesenchymal stromal cells (MSCs) are adult multipotent cells with antimicrobial properties, mediated through direct bactericidal activity and modulation of host innate and adaptive immune cells. More than 30 in vivo studies have reported on the use of human MSCs for the treatment of infectious diseases, with many more studies of animal MSCs in same-species models of infection. MSCs demonstrate potent antimicrobial effects against the major classes of human pathogens (bacteria, viruses, fungi, and parasites) across a wide range of infection models. Mechanistic studies have yielded important insight into their immunomodulatory and bactericidal activity, which can be enhanced through various forms of preconditioning. MSCs are being investigated in over 80 clinical trials for difficult-to-treat infectious diseases, including sepsis and pulmonary, intra-abdominal, cutaneous, and viral infections. Completed trials consistently report MSCs to be safe and well tolerated, with signals of efficacy against some infectious diseases. Although significant obstacles must be overcome to produce a standardized, affordable, clinical-grade cell therapy, these studies suggest that MSCs may have particular potential as an adjunct therapy in complex or resistant infections.

Phosphatidylserine Liposomes Reduce Inflammatory Response, Mycobacterial Viability, and HIV Replication in Coinfected Human Macrophages.

Chronic immune activation is the key pathogenetic event of Mycobacterium tuberculosis-human immunodeficiency virus (HIV) coinfection. We assessed the therapeutic value of phosphatidylserine-liposome (PS-L) in an in vitro model of M. tuberculosis-HIV coinfection. PS-L reduced nuclear factor-κB activation and the downstream production of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6 in bacille Calmette-Guérin-infected macrophages and of TNF-α and IL-1ß in M. tuberculosis-infected and M. tuberculosis-HIV-coinfected macrophages. Importantly, a significant reduction of intracellular M. tuberculosis viability and HIV replication were also observed. These results support the further exploitation of PS-L as host-directed therapy for M. tuberculosis-HIV coinfection.

Different modalities of host cell death and their impact on Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis (Mtb) is the pathogen that causes tuberculosis (TB), a leading infectious disease of humans worldwide. One of the main histopathological hallmarks of TB is the formation of granulomas comprised of elaborately organized aggregates of immune cells containing the pathogen. Dissemination of Mtb from infected cells in the granulomas due to host and mycobacterial factors induces multiple cell death modalities in infected cells. Based on molecular mechanism, morphological characteristics, and signal dependency, there are two main categories of cell death: programmed and nonprogrammed. Programmed cell death (PCD), such as apoptosis and autophagy, is associated with a protective response to Mtb by keeping the bacteria encased within dead macrophages that can be readily phagocytosed by arriving in uninfected or neighboring cells. In contrast, non-PCD necrotic cell death favors the pathogen, resulting in bacterial release into the extracellular environment. Multiple types of cell death in the PCD category, including pyroptosis, necroptosis, ferroptosis, ETosis, parthanatos, and PANoptosis, may be involved in Mtb infection. Since PCD pathways are essential for host immunity to Mtb, therapeutic compounds targeting cell death signaling pathways have been experimentally tested for TB treatment. This review summarizes different modalities of Mtb-mediated host cell deaths, the molecular mechanisms underpinning host cell death during Mtb infection, and its potential implications for host immunity. In addition, targeting host cell death pathways as potential therapeutic and preventive approaches against Mtb infection is also discussed.

Surveying the Epigenetic Landscape of Tuberculosis in Alveolar Macrophages.

Tuberculosis (TB) remains the leading cause of bacterial disease-related death and is among the top 10 overall causes of death worldwide. The complex nature of this infectious lung disease has proven difficult to treat, and significant research efforts are now evaluating the feasibility of host-directed, adjunctive therapies. An attractive approach in host-directed therapy targets host epigenetics, or gene regulation, to redirect the immune response in a host-beneficial manner. Substantial evidence exists demonstrating that host epigenetics are dysregulated during TB and that epigenetic-based therapies may be highly effective to treat TB. However, the caveat is that much of the knowledge that exists on the modulation of the host epigenome during TB has been gained using in vitro, small-animal, or blood-derived cell models, which do not accurately reflect the pulmonary nature of the disease. In humans, the first and major target cells of Mycobacterium tuberculosis are alveolar macrophages (AM). As such, their response to infection and treatment is clinically relevant and ultimately drives the outcome of disease. In this review, we compare the fundamental differences between AM and circulating monocyte-derived macrophages in the context of TB and summarize the recent advances in elucidating the epigenomes of these cells, including changes to the transcriptome, DNA methylome, and chromatin architecture. We will also discuss trained immunity in AM as a new and emerging field in TB research and provide some perspectives for the translational potential of targeting host epigenetics as an alternative TB therapy.

Randomized Trial of Metformin With Anti-Tuberculosis Drugs for Early Sputum Conversion in Adults With Pulmonary Tuberculosis.

BACKGROUND: Metformin, by reducing intracellular Mycobacterium tuberculosis growth, can be considered an adjunctive therapy to anti-tuberculosis treatment (ATT). We determined whether metformin with standard ATT reduces time to sputum culture conversion and tissue inflammation in adults with pulmonary tuberculosis (PTB). METHODS: In a randomized, 8-week, clinical trial, newly diagnosed, culture-positive PTB patients were randomized to standard ATT (HREZ = control arm) or standard ATT plus daily 1000 mg metformin (MET-HREZ = Metformin with Rifampicin [METRIF] arm) for 8 weeks during 2018-2020 at 5 sites in India. The primary end point was time to sputum culture conversion by liquid culture during 8 weeks of ATT. Plasma inflammatory markers were estimated in a subset. A Cox proportional hazard model was used to estimate time and predictors of culture conversion. RESULTS: Of the 322 patients randomized, 239 (74%) were male, and 212 (66%) had bilateral disease on chest radiograph with 54 (18%) showing cavitation. The median time to sputum culture conversion by liquid culture was 42 days in the METRIF arm and 41 days in the control arm (hazard ratio, 0.8; 95% confidence interval [CI], .624-1.019). After 8 weeks of ATT, cavitary lesions on X-ray (7, 5.3% vs 18, 12.9%; relative risk, 0.42; 95% CI, .18-.96; P = .041) and inflammatory markers were significantly lower in the METRIF arm. Higher body mass index and lower sputum smear grading were associated with faster sputum culture conversion. CONCLUSIONS: The addition of metformin to standard ATT did not hasten sputum culture conversion but diminished excess inflammation, thus reducing lung tissue damage as seen by faster clearance on X-ray and reduced inflammatory markers. CLINICAL TRIALS REGISTRATION: Clinical Trial Registry of India (CTRI/2018/01/011176).

Quantitative Systems Pharmacology Modeling Framework of Autophagy in Tuberculosis: Application to Adjunctive Metformin Host-Directed Therapy.

Quantitative systems pharmacology (QSP) modeling of the host immune response against Mycobacterium tuberculosis can inform the rational design of host-directed therapies (HDTs). We aimed to develop a QSP framework to evaluate the effects of metformin-associated autophagy induction in combination with antibiotics. A QSP framework for autophagy was developed by extending a model for host immune response to include adenosine monophosphate-activated protein kinase (AMPK)-mTOR-autophagy signaling. This model was combined with pharmacokinetic-pharmacodynamic models for metformin and antibiotics against M. tuberculosis. We compared the model predictions to mice infection experiments and derived predictions for the pathogen- and host-associated dynamics in humans treated with metformin in combination with antibiotics. The model adequately captured the observed bacterial load dynamics in mice M. tuberculosis infection models treated with metformin. Simulations for adjunctive metformin therapy in newly diagnosed patients suggested a limited yet dose-dependent effect of metformin on reduction of the intracellular bacterial load when the overall bacterial load is low, late during antibiotic treatment. We present the first QSP framework for HDTs against M. tuberculosis, linking cellular-level autophagy effects to disease progression and adjunctive HDT treatment response. This framework may be extended to guide the design of HDTs against M. tuberculosis.

Targeting immunometabolism in host-directed therapies to fungal disease.

Fungal infections affect over a billion people and are responsible for more than 1.5 million deaths each year. Despite progress in diagnostic and therapeutic approaches, the management of severe fungal infections remains a challenge. Recently, the reprogramming of cellular metabolism has emerged as a central mechanism through which the effector functions of immune cells are supported to promote antifungal activity. An improved understanding of the immunometabolic signatures that orchestrate antifungal immunity, together with the dissection of the mechanisms that underlie heterogeneity in individual immune responses, may therefore unveil new targets amenable to adjunctive host-directed therapies. In this review, we highlight recent advances in the metabolic regulation of host-fungus interactions and antifungal immune responses, and outline targetable pathways and mechanisms with promising therapeutic potential.

Preparation and Evaluation of Low-Dose Calcitriol Dry Powder Inhalation as Host-Directed Adjunct Therapy for Tuberculosis.

BACKGROUND: It is unclear whether Vitamin D is efficacious as a host-directed therapy (HDT) for patients of tuberculosis (TB). We investigated pulmonary delivery of the active metabolite of Vitamin D3, i.e., 1, 25-dihydroxy vitamin D3 (calcitriol) in a mouse model of infection with Mycobacterium tuberculosis (Mtb). METHODS: We optimized a spray drying process to prepare a dry powder inhalation (DPI) of calcitriol using a Quality by Design (QbD) approach. We then compared outcomes when Mtb-infected mice were treated with inhaled calcitriol at 5 ng/kg as a stand-alone intervention versus DPI as adjunct to standard oral anti-tuberculosis therapy (ATT). RESULTS: The DPI with or without concomitant ATT markedly improved the morphology of the lungs and mitigated histopathology in both the lungs and the spleens. The number of nodular lesions on the lung surface decreased from 43.7 ± 3.1 to 22.5 ± 3.9 with the DPI alone and to 9.8 ± 2.5 with DPI + ATT. However, no statistically significant induction of host antimicrobial peptide cathelicidin or reduction in bacterial burden was seen with the DPI alone. DPI + ATT did not significantly reduce the bacterial burden in the lungs compared to ATT alone. CONCLUSIONS: We concluded that HDT using the low dose calcitriol DPI contributed markedly to mitigation of pathology, but higher dose may be required to evoke significant induction of bactericidal host response and bactericidal activity in the lung.

n-3 long-chain PUFA promote antibacterial and inflammation-resolving effects in Mycobacterium tuberculosis-infected C3HeB/FeJ mice, dependent on fatty acid status.

Non-resolving inflammation is characteristic of tuberculosis (TB). Given their inflammation-resolving properties, n-3 long-chain PUFA (n-3 LCPUFA) may support TB treatment. This research aimed to investigate the effects of n-3 LCPUFA on clinical and inflammatory outcomes of Mycobacterium tuberculosis-infected C3HeB/FeJ mice with either normal or low n-3 PUFA status before infection. Using a two-by-two design, uninfected mice were conditioned on either an n-3 PUFA-sufficient (n-3FAS) or -deficient (n-3FAD) diet for 6 weeks. One week post-infection, mice were randomised to either n-3 LCPUFA supplemented (n-3FAS/n-3+ and n-3FAD/n-3+) or continued on n-3FAS or n-3FAD diets for 3 weeks. Mice were euthanised and fatty acid status, lung bacterial load and pathology, cytokine, lipid mediator and immune cell phenotype analysed. n-3 LCPUFA supplementation in n-3FAS mice lowered lung bacterial loads (P = 0·003), T cells (P = 0·019), CD4+ T cells (P = 0·014) and interferon (IFN)-γ (P < 0·001) and promoted a pro-resolving lung lipid mediator profile. Compared with n-3FAS mice, the n-3FAD group had lower bacterial loads (P = 0·037), significantly higher immune cell recruitment and a more pro-inflammatory lipid mediator profile, however, significantly lower lung IFN-γ, IL-1α, IL-1ß and IL-17, and supplementation in the n-3FAD group provided no beneficial effect on lung bacterial load or inflammation. Our study provides the first evidence that n-3 LCPUFA supplementation has antibacterial and inflammation-resolving benefits in TB when provided 1 week after infection in the context of a sufficient n-3 PUFA status, whilst a low n-3 PUFA status may promote better bacterial control and lower lung inflammation not benefiting from n-3 LCPUFA supplementation.