Immunopathogenesis in pulmonary tuberculosis : impact of immunomodulation and diabetes co-morbidity

Even in the 21st century, tuberculosis (TB) remains a major global health threat, primarily due to the emergence of antibiotic resistance. Presence of co-morbidities such as diabetes mellitus (DM) has worsened the current situation and made it more difficult to treat this deadly disease, especially in resource-poor settings. It is well-known that Mtb (Mycobacterium tuberculosis) bacilli can manipulate both innate and adaptive arms of the human immune system, but how Mtb evade host antimicrobial mechanism is not fully understood. Therefore, a deeper understanding of the immunomodulation caused by Mtb, with and without co-existing illnesses, is essential to develop more effective treatment strategies. The work in this thesis was intended to uncover Mtb-mediated immune alterations, particularly in TB-DM disease, and to examine the feasibility of novel host-directed therapy (HDT). In Paper I, we set out to study the efficacy of HDT using phenylbutyrate (PBA) and vitamin D (vitD) to strengthen host immune defenses upon administration to pulmonary TB patients. In a randomized controlled trial conducted in Bangladesh, we previously reported positive effects on clinical as well as microbiological TB outcomes upon daily PBA and vitD treatment together with standard chemotherapy for 8 weeks. Stored samples obtained from the clinical trial subjects were now used to assess secondary outcomes including cytokine/chemokine secretion by peripheral blood mononuclear cell (PBMC) cultures (Luminex assay), endoplasmic reticulum (ER) stress markers expressed in monocyte-derived-macrophages (MDMs) (quantitative real-time PCR), and activation of LC3-dependent autophagy in Mtb-infected MDMs (confocal microscopy). We observed a marked reduction in the concentration of inflammatory mediators including tumor necrosis factor (TNF)-α, CC motif chemokine ligand (CCL)-11 and CCL5 after 8 weeks of PBA treatment compared to the placebo group. Similarly, vitD treatment effectively reduced CCL11, C-X-C motif chemokine ligand (CXCL)-10 and PDGF concentrations after 8 weeks of treatment. Both PBA- and vitD-treatment contributed to reduced mRNA levels of the ER stress marker, x-box binding protein1spliced (XBP1sp)-l. Autophagy was enhanced in MDMs obtained from all intervention groups after 8 weeks of treatment as compared to placebo. These findings suggested that the improvement of primary outcomes observed in the clinical trial, were associated with reduced inflammation and ER stress and instead enhanced autophagy in Mtb-infected patient cells. In paper II, we aimed to explore DM-associated immune alterations of clinical, radiological, and immunological outcomes in TB disease using TB and TB-DM study cohorts collected in Bangladesh. Clinical samples from peripheral blood and sputum from patients and controls were analyzed (blood chemistry, Luminex, quantitative real-time PCR) along with clinical data (composite clinical TB score and demographics) and chest radiography (chest X-ray score) before and after 1, 2 and 6 months of standard anti-TB treatment. TB-DM patients were significantly older, had higher body mass index (BMI), were less anemic and from a better socio-economic background compared to TB patients. Intriguingly, clinical TB symptoms and time to bacterial clearance in sputum were similar comparing TB and TB-DM patients. Even so, TB-DM patients had poorly managed glycemic control throughout the study period and glycemic status was positively associated with BMI. Importantly, the TB-DM cohort showed reduced resolution of inflammation in the middle and lower lung zones compared with TB patients, which was correlated to plasma leptin concentrations at all time points. These changes were associated with upregulated mRNA expression of inflammatory TNF-α and IL-1β in PBMCs as well as higher CD8 mRNA levels but downregulated CD4 and IL-10 transcripts in sputum cells after standard treatment in TB-DM compared to TB patients. Additionally, glycemic status in TB-DM patients was inversely correlated to sputum IL-10 transcript levels observed after start of anti-TB treatment. These results indicate that TB-DM disease is characterized by low-grade inflammation that persist even after completion of successful anti-TB chemotherapy. In Paper III, we developed a protocol for assessment of M1/M2 polarization of human myeloid-derived cells using 10-color flow cytometry of adherent macrophages infected with green fluorescent protein (GFP)-expressing Mtb. The experimental protocol involved in vitro polarization of MDMs into classically activated (M1) or alternatively activated (M2) macrophages and assessment of phenotype and function before, and 4 to 24 hours after Mtb infection. M1 or M2 cells were successfully differentiated with granulocyte monocyte colony stimulating factor (GM-CSF) or monocyte colony stimulating factor (M-CSF), followed by polarization with interferon (IFN)-γ and lipopolysaccharide (LPS), or interleukin (IL)-4, respectively. This protocol allowed us to polarize and define M1 cells by elevated levels of CD64 and CD86 co-expression, while M2 cells were characterized by a high CD163 and CD200R co-expression. The level of Mtb infection was generally higher in M2 as compared to M1 cells, although the relative increase in infected cells from 4 to 24 hours was higher in M1- compared with M2-polarized cells. Manual gating as well as unsupervised analysis using dimensionality reduction with Uniform Manifold Approximation and Projection (UMAP) and phenograph clustering, showed that Mtb infection altered the expression of M1 and M2 markers after 24 hours and generated clearly separated cell clusters of different sizes. This M1/M2 flow cytometry protocol could be used as a backbone in Mtb-macrophage research and be adopted for special needs including assessment of cells cultured in vitro or obtained ex vivo from clinical patient samples

Efficacy of sodium butyrate adjunct therapy in shigellosis: a randomized, double-blind, placebo-controlled clinical trial

BACKGROUND: Treatment of shigellosis in rabbits with butyrate reduces clinical severity and counteracts the downregulation of cathelicidin (CAP-18) in the large intestinal epithelia. Here, we aimed to evaluate whether butyrate can be used as an adjunct to antibiotics in the treatment of shigellosis in patients. METHODS: A randomized, double-blind, placebo-controlled, parallel-group designed clinical trial was conducted. Eighty adult patients with shigellosis were randomized to either the Intervention group (butyrate, n = 40) or the Placebo group (normal saline, n = 40). The Intervention group was given an enema containing sodium butyrate (80 mM), twice daily for 3 days, while the Placebo group received the same dose of normal saline. The primary endpoint of the trial was to assess the efficacy of butyrate in improving clinical, endoscopic and histological features of shigellosis. The secondary endpoint was to study the effect of butyrate on the induction of antimicrobial peptides in the rectum. Clinical outcomes were assessed and concentrations of antimicrobial peptides (LL-37, human beta defensin1 [HBD-1] and human beta defensin 3 [HBD-3]) and pro-inflammatory cytokines (interleukin-1β [IL-1β] and interleukin-8 [IL-8]) were measured in the stool. Sigmoidoscopic and histopathological analyses, and immunostaining of LL-37 in the rectal mucosa were performed in a subgroup of patients. RESULTS: Compared with placebo, butyrate therapy led to the early reduction of macrophages, pus cells, IL-8 and IL-1β in the stool and improvement in rectal histopathology. Butyrate treatment induced LL-37 expression in the rectal epithelia. Stool concentration of LL-37 remained significantly higher in the Intervention group on days 4 and 7. CONCLUSION: Adjunct therapy with butyrate during shigellosis led to early reduction of inflammation and enhanced LL-37 expression in the rectal epithelia with prolonged release of LL-37 in the stool. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00800930

Immune responses in the treatment of drug-sensitive pulmonary tuberculosis with phenylbutyrate and vitamin D3 as host directed therapy

Background We have previously shown that 8 weeks’ treatment with phenylbutyrate (PBA) (500mgx2/day) with or without vitamin D3 (vitD3) (5000 IU/day) as host-directed therapy (HDT) accelerated clinical recovery, sputum culture conversion and increased expression of cathelicidin LL-37 by immune cells in a randomized, placebo-controlled trial in adults with pulmonary tuberculosis (TB). In this study we further aimed to examine whether HDT with PBA and vitD3 promoted clinically beneficial immunomodulation to improve treatment outcomes in TB patients. Methods Cytokine concentration was measured in supernatants of peripheral blood mononuclear cells (PBMC) from patients (n = 31/group). Endoplasmic reticulum stress-related genes (GADD34 and XBP1spl) and human beta-defensin-1 (HBD1) gene expression were studied in monocyte-derived-macrophages (MDM) (n = 18/group) from PBMC of patients. Autophagy in MDM (n = 6/group) was evaluated using LC3 expression by confocal microscopy. Results A significant decline in the concentration of cytokines/chemokines was noted from week 0 to 8 in the PBA-group [TNF-α (β = − 0.34, 95% CI = − 0.68, − 0.003; p = 0.04), CCL11 (β = − 0.19, 95% CI = − 0.36, − 0.03; p = 0.02) and CCL5 (β = − 0.08, 95% CI = − 0.16, 0.002; p = 0.05)] and vitD3-group [(CCL11 (β = − 0.17, 95% CI = − 0.34, − 0.001; p = 0.04), CXCL10 (β = − 0.38, 95% CI = − 0.77, 0.003; p = 0.05) and PDGF-β (β = − 0.16, 95% CI = − 0.31, 0.002; p = 0.05)] compared to placebo. Both PBA- and vitD3-groups showed a decline in XBP1spl mRNA on week 8 (p < 0.03). All treatment groups demonstrated increased LC3 expression in MDM compared to placebo over time (p < 0.037). Conclusion The use of PBA and vitD3 as adjunct therapy to standard TB treatment promoted favorable immunomodulation to improve treatment outcomes.This study was supported by the International Centre for Diarrheal Disease Research, Bangladesh (icddr,b), Sida (Sida-icddrb Agreement support; Grant 384, SWE-2008-065) and Swedish Strategic Foundation (SSF, Grant No. RBd08–0014), the Swedish Heart-Lung Foundation (Grant No. 2013–0366) and Swedish Research Council (Grant No. 2016–01496). No funding bodies had any role in the design of the study, collection, analysis, and interpretation of data and in writing the manuscript.Peer Reviewe

Memory profiles distinguish cross-reactive and virus-specific T cell immunity to mpox

Mpox represents a persistent health concern with varying disease severity. Reinfections with mpox virus (MPXV) are rare, possibly indicating effective memory responses to MPXV or related poxviruses, notably vaccinia virus (VACV) from smallpox vaccination. We assessed cross-reactive and virus-specific CD4+ and CD8+ T cells in healthy individuals and mpox convalescent donors. Cross-reactive T cells were most frequently observed in healthy donors over 45 years. Notably, long-lived memory CD8+ T cells targeting conserved VACV/MPXV epitopes were identified in older individuals more than four decades after VACV exposure and exhibited stem-like characteristics, defined by T cell factor-1 (TCF-1) expression. In mpox convalescent donors, MPXV-reactive CD4+ and CD8+ T cells were more prevalent compared to controls, demonstrating enhanced functionality and skewing towards effector phenotypes, which correlated with milder disease. Collectively, we report robust effector memory MPXV-specific T cell responses in mild mpox and long-lived TCF-1+ VACV/MPXV-specific CD8+ T cells decades after smallpox vaccination

Ciprofloxacin Affects Host Cells by Suppressing Expression of the Endogenous Antimicrobial Peptides Cathelicidins and Beta-Defensin-3 in Colon Epithelia

Antibiotics exert several effects on host cells including regulation of immune components. Antimicrobial peptides (AMPs), e.g., cathelicidins and defensins display multiple functions in innate immunity. In colonic mucosa, cathelicidins are induced by butyrate, a bacterial fermentation product. Here, we investigated the effect of antibiotics on butyrate-induced expression of cathelicidins and beta-defensins in colon epithelial cells. Real-time PCR analysis revealed that ciprofloxacin and clindamycin reduce butyrate-induced transcription of the human cathelicidin LL-37 in the colonic epithelial cell line HT-29. Suppression of LL-37 peptide/protein by ciprofloxacin was confirmed by Western blot analysis. Immunohistochemical analysis demonstrated that ciprofloxacin suppresses the rabbit cathelicidin CAP-18 in rectal epithelia of healthy and butyrate-treated Shigella-infected rabbits. Ciprofloxacin also down-regulated butyrate-induced transcription of the human beta-defensin-3 in HT-29 cells. Microarray analysis of HT-29 cells revealed upregulation by butyrate with subsequent down-regulation by ciprofloxacin of additional genes encoding immune factors. Dephosphorylation of histone H3, an epigenetic event provided a possible mechanism of the suppressive effect of ciprofloxacin. Furthermore, LL-37 peptide inhibited Clostridium difficile growth in vitro. In conclusion, ciprofloxacin and clindamycin exert immunomodulatory function by down-regulating AMPs and other immune components in colonic epithelial cells. Suppression of AMPs may contribute to the overgrowth of C. difficile, causing antibiotic-associated diarrhea

Significant Effects of Oral Phenylbutyrate and Vitamin D3 Adjunctive Therapy in Pulmonary Tuberculosis: A Randomized Controlled Trial.

Development of new tuberculosis (TB) drugs and alternative treatment strategies are urgently required to control the global spread of TB. Previous results have shown that vitamin D3 (vitD3) and 4-phenyl butyrate (PBA) are potent inducers of the host defense peptide LL-37 that possess anti-mycobacterial effects.To examine if oral adjunctive therapy with 5,000IU vitD3 or 2x500 mg PBA or PBA+vitD3 to standard chemotherapy would lead to enhanced recovery in sputum smear-positive pulmonary TB patients.Adult TB patients (n = 288) were enrolled in a randomized, double-blind, placebo-controlled trial conducted in Bangladesh. Primary endpoints included proportions of patients with a negative sputum culture at week 4 and reduction in clinical symptoms at week 8. Clinical assessments and sputum smear microscopy were performed weekly up to week 4, fortnightly up to week 12 and at week 24; TB culture was performed at week 0, 4 and 8; concentrations of LL-37 in cells, 25-hydroxyvitamin D3 (25(OH)D3) in plasma and ex vivo bactericidal function of monocyte-derived macrophages (MDM) were determined at week 0, 4, 8, 12 and additionally at week 24 for plasma 25(OH)D3.At week 4, 71% (46/65) of the patients in the PBA+vitD3-group (p = 0.001) and 61.3% (38/62) in the vitD3-group (p = 0.032) were culture negative compared to 42.2% (27/64) in the placebo-group. The odds of sputum culture being negative at week 4 was 3.42 times higher in the PBA+vitD3-group (p = 0.001) and 2.2 times higher in vitD3-group (p = 0.032) compared to placebo. The concentration of LL-37 in MDM was significantly higher in the PBA-group compared to placebo at week 12 (p = 0.034). Decline in intracellular Mtb growth in MDM was earlier in the PBA-group compared to placebo (log rank 11.38, p = 0.01).Adjunct therapy with PBA+vitD3 or vitD3 or PBA to standard short-course therapy demonstrated beneficial effects towards clinical recovery and holds potential for host-directed-therapy in the treatment of TB.clinicaltrials.gov NCT01580007

Additional file 1: of Immune responses in the treatment of drug-sensitive pulmonary tuberculosis with phenylbutyrate and vitamin D3 as host directed therapy

Table S1. Descriptive statistics of the current studied patients and the patients who were not studied. Table S2. Longitudinal change (week 0, 4, 8 and 12) in TB score in the intervention groups compared to placebo. Table S3. Concentrations of inflammatory cytokines and chemokines in treatment groups at different time intervals. (DOCX 24 kb

Mean TB score in TB patients in the four intervention arms during the study period.

<p>Standard deviation is shown as vertical bar. Comparisons of intervention arms are made with the placebo arm with statistically significant differences being shown in asterisks. The PBA-group demonstrated significantly lower TB scores than the placebo group at week 2, 4, 8, 10 and 12. At week 10 all three intervention groups showed lower scores than the placebo group. Multivariate regression analysis was utilized for comparison of mean effect of clinical scores in the different intervention groups.</p

Baseline characteristics of patients with freshly diagnosed pulmonary tuberculosis in the four treatment groups.

<p>Data is presented as mean ± standard deviation or number with percentage in parentheses. BCG, <i>Bacillus Calmette–Guérin</i>.; ESR, erythrocyte sedimentation rate; Hb, hemoglobin; WBC, white blood cells. History of contacts, BCG given and Tuberculin skin test done are dichotomous variable and age, weight, duration of illness, ESR, Hb, WBC and vitamin D status are continuous variable.</p><p>Baseline characteristics of patients with freshly diagnosed pulmonary tuberculosis in the four treatment groups.</p