Studies of effector functions in mycobacterium tuberculosis-infected macrophages with implications for host-directed therapies

Tuberculosis infection remains a global health problem and the emergence of multidrugresistant TB (MDR-TB) adds further challenges in the battle to stop the spread of infection. Macrophages are the primary host cells to be infected with Mycobacterium tuberculosis (Mtb), and are therefore key cells in the initial control of TB infection. However, to survive and persist in activated macrophages, Mtb has developed strategies to counteract antimicrobial immunity. This thesis work aimed to explore macrophage responses in Mtbinfected cells, the nature of innate effector mechanisms and how these can be enhanced. For this purpose, we used an in vitro macrophage infection model as well as an organotypic lung tissue model system. The thesis work is based on four papers, Study I and III, involved studies on the effects of the immunomodulatory compounds, vitamin D3 (VitD3) and phenylbutyrate (PBA), on the induction of antimicrobial peptides, primarily human cathelicidin LL-37, and the ability to kill intracellular Mtb. In Study I, we observed that Mtb can down-regulate the expression of LL-37 in infected macrophages, but this effect was effectively counteracted by treatment with VitD3 and/or PBA. PBA and/or VitD3 prevented intracellular Mtb growth via induction of LL-37 as well as activation of autophagy in Mtb-infected macrophages. In Study III, we further demonstrated that PBA+VitD3 enhanced intracellular Mtb killing of both MDR-TB and drug-susceptible strains and this effect was dependent on LL-37. In Study IV, we explored the role of VitD3 in polarization of macrophages in comparison to activation with conventional stimuli such as GM-CSF (polarization of inflammatory M1 macrophages) or MCSF (polarization of anti-inflammatory M2 macrophages). The findings from Study IV, suggested that polarization with VitD3 enhanced the ability of Mtb-infected macrophages to control intracellular Mtb growth in comparison to M1 and M2 subsets. Enhanced Mtb growth control was associated with elevated levels of pro-inflammatory cytokines and LL-37, but reduced Mtb-induced expression of the immunosuppressive enzyme IDO (indoleamine 2,3-dioxygenase). Study II, aimed to investigate the expression and function of matrix metalloproteinases (MMPs) in early TB granuloma formation using the organotypic lung tissue model that enabled studies on macrophage-Mtb interactions in a more physiological environment in tissue. In this study, we found that pre-treatment of macrophages with the global MMP inhibitor, marimastat, resulted in effective inhibition of TB granuloma formation, which was associated with reduced Mtb growth detected in the lung tissue model. Thus, reduced degradation of extracellular matrix proteins, could prevent Mtb multiplication and spread of the infection. The main conclusion from this thesis work is that immunomodulatory compounds with the ability to boost or block innate effector mechanisms in Mtb-infected macrophages may be used as adjunct host-directed therapies that could support standard anti-TB drugs to enhance clinical recovery from TB

Polarization of Human Monocyte-Derived Cells With Vitamin D Promotes Control of Mycobacterium tuberculosis Infection

Background: Understanding macrophage behavior is key to decipher Mycobacterium tuberculosis (Mtb) pathogenesis. We studied the phenotype and ability of human monocyte-derived cells polarized with active vitamin D [1,25(OH)(2)D-3] to control intracellular Mtb infection compared with polarization of conventional subsets, classical M1 or alternative M2. Methods: Human blood-derived monocytes were treated with active vitamin D or different cytokines to obtain 1,25(OH)(2)D-3-polarized as well as M1- and M2-like cells or fully polarized M1 and M2 subsets. We used an in vitro macrophage Mtb infection model to assess both phenotype and functional markers i.e., inhibitory and scavenger receptors, costimulatory molecules, cytokines, chemokines, and effector molecules using flow cytometry and quantitative mRNA analysis. Intracellular uptake of bacilli and Mtb growth was monitored using flow cytometry and colony forming units. Results: Uninfected M1 subsets typically expressed higher levels of CCR7, TLR2, and CD86, while M2 subsets expressed higher CD163, CD200R, and CD206. Most of the investigated markers were up-regulated in all subsets after Mtb infection, generating a mixed M1/M2 phenotype, while the expression of CD206, HLADR, and CD80 was specifically up-regulated (P amp;lt; 0.05) on 1,25(OH)(2)D-3-polarized macrophages. Consistent with the pro-inflammatory features of M1 cells, Mtb uptake and intracellular Mtb growth was significantly (P amp;lt; 0.01-0.001 and P amp;lt; 0.05-0.01) lower in the M1 (19.3%) compared with the M2 (82.7%) subsets 4 h post-infection. However, infectivity rapidly and gradually increased in M1 cells at 24-72 h. 1,25(OH)(2)D-3-polarized monocyte-derived cells was the most potent subset to inhibit Mtb growth at both 4 and 72 h (P amp;lt; 0.05-0.01) post-Mtb infection. This ability was associated with high mRNA levels of pro-inflammatory cytokines and the antimicrobial peptide LL-37 but also anti-inflammatory IL-10, while expression of the immunosuppressive enzyme IDO (indoleamine 2,3-dioxygenase) remained low in Mtb-infected 1,25(OH)(2)D-3-polarized cells compared with the other subsets. Conclusions: Mtb infection promoted a mixed M1/M2 macrophage activation, and 1,25(OH)(2)D-3-polarized monocyte-derived cells expressing LL-37 but not IDO, were most effective to control intracellular Mtb growth. Macrophage polarization in the presence of vitamin D may provide the capacity to mount an antimicrobial response against Mtb and simultaneously prevent expression of inhibitory molecules that could accelerate local immunosuppression in the microenvironment of infected tissue.Funding Agencies|Swedish Heart and Lung Foundation (HLF) [2016-0470, 2016-0815]; Swedish Research Council (VR)Swedish Research Council [521-2014-3238]; Foundation to Prevent Antibiotic Resistance (Resist); KID (Karolinska Institutet)Karolinska Institutet</p

Immunomodulatory Agents Combat Multidrug-Resistant Tuberculosis by Improving Antimicrobial Immunity

Background. Multidrug-resistant (MDR) tuberculosis has low treatment success rates, and new treatment strategies are needed. We explored whether treatment with active vitamin D-3 (vitD) and phenylbutyrate (PBA) could improve conventional chemotherapy by enhancing immune-mediated eradication of Mycobacterium tuberculosis. Methods. A clinically relevant model was used consisting of human macrophages infected with M. tuberculosis isolates (n = 15) with different antibiotic resistance profiles. The antimicrobial effect of vitD+PBA, was tested together with rifampicin or isoniazid. Methods included colony-forming units (intracellular bacterial growth), messenger RNA expression analyses (LL-37, beta-defensin, nitric oxide synthase, and dual oxidase 2), RNA interference (LL-37-silencing in primary macrophages), and Western blot analysis and confocal microscopy (LL-37 and LC3 protein expression). Results. VitD+PBA inhibited growth of clinical MDR tuberculosis strains in human macrophages and strengthened intracellular growth inhibition of rifampicin and isoniazid via induction of the antimicrobial peptide LL-37 and I,C3-dependent autophagy. Gene silencing of LL-37 expression enhanced MDR tuberculosis growth in vitD+PBA-treated macrophages. Me combination of vitD+PBA and isoniazid were as effective in reducing intracellular MDR tuberculosis growth as a &amp;gt;125-fold higher dose of isoniazid alone, suggesting potent additive effects of vitD+PBA with isoniazid. Conclusions. Immunomodulatory agents that trigger multiple immune pathways can strengthen standard MDR tuberculosis treatment and contribute to next-generation individualized treatment options for patients with difficult-to-treat pulmonary tuberculosis.Funding Agencies|Swedish Heart and Lung FoundationSwedish Heart-Lung Foundation [2019-0299, 2019-0302, 2017-0358, 2015-0236]; Swedish Research CouncilSwedish Research CouncilEuropean Commission [2019-01744, 201904720, 2016-01496, 2016-02043]; Foundation to Prevent Antibiotic Resistance (Resist); Lars Hierta Memorial Foundation; Karolinska Institutet FoundationsKarolinska Institutet; Karolinska InstitutetKarolinska Institutet</p

Phenylbutyrate induces LL-37-dependent autophagy and intracellular killing of <i>Mycobacterium tuberculosis</i> in human macrophages

<p>LL-37 is a human antimicrobial peptide (AMP) of the cathelicidin family with multiple activities including a mediator of vitamin D-induced autophagy in human macrophages, resulting in intracellular killing of <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>). In a previous trial in healthy volunteers, we have shown that LL-37 expression and subsequent <i>Mtb</i>-killing can be further enhanced by 4-phenylbutyrate (PBA), also an inducer of LL-37 expression. Here, we explore a potential mechanism(s) behind PBA and LL-37-induced autophagy and intracellular killing of <i>Mtb</i>. <i>Mtb</i> infection of macrophages downregulated the expression of both the <i>CAMP</i> transcript and LL-37 peptide as well as certain autophagy-related genes (<i>BECN1</i> and <i>ATG5</i>) at both the mRNA and protein levels. In addition, activation of LC3-II in primary macrophages and THP-1 cells was not detected. PBA and the active form of vitamin D₃ (1,25[OH]₂D₃), separately or particularly in combination, were able to overcome <i>Mtb</i>-induced suppression of LL-37 expression. Notably, reactivation of autophagy occurred by stimulation of macrophages with PBA and promoted colocalization of LL-37 and LC3-II in autophagosomes. Importantly, PBA treatment failed to induce autophagy in <i>Mtb</i>-infected THP-1 cells, when the expression of LL-37 was silenced. However, PBA-induced autophagy was restored when the LL-37 knockdown cells were supplemented with synthetic LL-37. Interestingly, we have found that LL-37-induced autophagy was mediated via P2RX7 receptor followed by enhanced cytosolic free Ca²⁺, and activation of AMPK and PtdIns3K pathways. Altogether, these results suggest a novel activity for PBA as an inducer of autophagy, which is LL-37-dependent and promotes intracellular killing of <i>Mtb</i> in human macrophages.</p

In vivo engineering of mobilized stem cell grafts with the immunomodulatory drug FTY720 for allogeneic transplantation

International audienc

Major alterations in the mononuclear phagocyte landscape associated with COVID-19 severity

10.1073/pnas.2018587118Proceedings of the National Academy of Sciences of the United States of America1186e201858711

NK cell frequencies, function and correlates to vaccine outcome in BNT162b2 mRNA anti-SARS-CoV-2 vaccinated healthy and immunocompromised individuals

Adaptive immune responses have been studied extensively in the course of mRNA vaccination against COVID-19. Considerably fewer studies have assessed the effects on innate immune cells. Here, we characterized NK cells in healthy individuals and immunocompromised patients in the course of an anti-SARS-CoV-2 BNT162b2 mRNA prospective, open-label clinical vaccine trial. See trial registration description in notes. Results revealed preserved NK cell numbers, frequencies, subsets, phenotypes, and function as assessed through consecutive peripheral blood samplings at 0, 10, 21, and 35 days following vaccination. A positive correlation was observed between the frequency of NKG2C+ NK cells at baseline (Day 0) and anti-SARS-CoV-2 Ab titers following BNT162b2 mRNA vaccination at Day 35. The present results provide basic insights in regards to NK cells in the context of mRNA vaccination, and have relevance for future mRNA-based vaccinations against COVID-19, other viral infections, and cancer. Trial registration: The current study is based on clinical material from the COVAXID open-label, non-randomized prospective clinical trial registered at EudraCT and clinicaltrials.gov (no. 2021–000175-37). Description: https://clinicaltrials.gov/ct2/show/NCT04780659?term=2021-000175-37&amp;draw=2&amp;rank=1

Immunodeficiency syndromes differentially impact the functional profile of SARS-CoV-2-specific T cells elicited by mRNA vaccination

Many immunocompromised patients mount suboptimal humoral immunity after SARS-CoV-2 mRNA vaccination. Here, we assessed the single-cell profile of SARS-CoV-2-specific T cells post-mRNA vaccination in healthy individuals and patients with various forms of immunodeficiencies. Impaired vaccine-induced cell-mediated immunity was observed in many immunocompromised patients, particularly in solid-organ transplant and chronic lymphocytic leukemia patients. Notably, individuals with an inherited lack of mature B cells, i.e., X-linked agammaglobulinemia (XLA) displayed highly functional spike-specific T cell responses. Single-cell RNA-sequencing further revealed that mRNA vaccination induced a broad functional spectrum of spike-specific CD4+ and CD8+ T cells in healthy individuals and patients with XLA. These responses were founded on polyclonal repertoires of CD4+ T cells and robust expansions of oligoclonal effector-memory CD45RA+ CD8+ T cells with stem-like characteristics. Collectively, our data provide the functional continuum of SARS-CoV-2-specific T cell responses post-mRNA vaccination, highlighting that cell-mediated immunity is of variable functional quality across immunodeficiency syndromes