Dimethyl itaconate is effective in host-directed antimicrobial responses against mycobacterial infections through multifaceted innate immune pathways.

doi:10.21203/rs.3.rs-2249328/v1

Download PDF

Research Article

Dimethyl itaconate is effective in host-directed antimicrobial responses against mycobacterial infections through multifaceted innate immune pathways.

https://doi.org/10.21203/rs.3.rs-2249328/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 08 Mar, 2023

Read the published version in Cell & Bioscience →

You are reading this latest preprint version

Background

Itaconate, a crucial immunometabolite, plays a critical role in linking immune and metabolic functions to influence host defense and inflammation. Because of its insoluble nature, several cell-permeable derivatives are being developed to provide therapeutic opportunities in infectious and inflammatory diseases. Yet, it remains largely uncharacterized whether cell-permeable derivatives have potentials in promoting host-directed therapeutics (HDT) against mycobacterial infections. Here, we report dimethyl itaconate (DMI) as the promising candidate for HDT against both Mycobacterium tuberculosis (Mtb) and nontuberculous mycobacteria by orchestrating multiple innate immune programs.

Results

DMI per se has low bactericidal activity against Mtb, M. bovis Bacillus Calmette–Guérin (BCG), and M. avium (Mav). However, DMI robustly activated intracellular elimination of multiple mycobacterial strains (Mtb, BCG, Mav, and even to multidrug-resistant Mtb) in macrophages and in vivo. DMI significantly suppressed the production of interleukins 6 and 10, whereas it enhanced autophagy and phagosomal maturation, during Mtb infection. DMI-mediated autophagy partly contributed to antimicrobial host defenses in macrophages. Moreover, DMI significantly downregulated the activation of signal transducer and activator of transcription 3 (STAT3) signaling during infection with Mtb, BCG, and Mav.

Conclusion

Together, DMI has potent anti-mycobacterial activities in macrophages and in vivo through promoting multifaceted ways for innate host defenses. DMI may bring light to new candidate for HDT against Mtb and nontuberculous mycobacteria, both of which infections are often intractable with antibiotic resistance.

dimethyl itaconate

Mycobacterium tuberculosis

nontuberculous mycobacteria

innate immunity

autophagy

host-directed therapeutics

antimicrobial responses

Mycobacterium tuberculosis (Mtb), the pathogen of human tuberculosis (TB), is a global health burden with high morbidity and mortality rates (1). Despite the development of new antimicrobial strategies for TB, little progress has been made in improving or replacing short-term chemotherapy (directly observed treatment, short course), which comprises isoniazid (INH), rifampicin, pyrazinamide, and ethambutol during the initial 2 months, followed by INH and rifampicin for 4 months, as the first-line regimen for drug-sensitive TB (2, 3). The treatment of drug-resistant cases is difficult and can require > 2 years of drugs with severe side effects. The increasing incidences of multidrug-resistant (MDR) and extremely drug-resistant TB are worrisome (2–4). Moreover, nontuberculous mycobacteria (NTM) include > 170 species and are major pathogens of emerging respiratory infections in immunocompromised and immunocompetent subjects (5, 6). The prevalence and incidence of NTM pulmonary infections are increasing worldwide and often intractable to treat (7–9). NTM treatment with standard antimicrobial regimens is challenging, takes longer than TB treatment, and has a low cure rate because of antibiotic resistance and toxicity (10–12). There is an urgent need for innovative host-directed therapeutics (HDT) based on deciphering an in-depth molecular mechanisms underlying host–pathogen interactions that could be geared for efficient protective responses against TB and NTM infections.

Immune-metabolic adaptations are rewired in macrophages and immune cells during mycobacterial infections. The remodeling of immunometabolism can shape the host defensive responses to intracellular mycobacteria, thus affecting the outcomes of infections (13–15). Recent studies highlighted the function of the immunometabolite itaconic acid (also known as methylenesuccinic acid) in decarboxylating cis-aconitate, which exerts an antimicrobial effect on Salmonella enterica and Mtb by covalent inhibition of bacterial isocitrate lyase (16, 17). Immune-responsive gene 1 (Irg1), a mitochondrial enzyme that catalyzes the production of itaconate in myeloid cells, controls Mtb infection by preventing excessive neutrophil-driven immunopathology (18). In addition, autocrine/paracrine signaling by tumor necrosis factor (TNF)-α and interleukin (IL)-6, which are generated by bystander cells, leads to the juxtaposition of bacterial phagosomes with mitochondria, thereby activating Irg1 signaling and an itaconate-mediated antimicrobial effect against M. avium (Mav) infection (19). Because itaconate is insoluble and its level is low in a quiescent status (16, 20, 21), efforts have been made to develop several cell-permeable derivatives of itaconate that retain its immunoregulatory function (22–24). For example, dimethyl itaconate (DMI) and 4-octyl itaconate ameliorate excessive inflammation and pathologic responses in models of autoimmune and inflammatory disorders including sepsis, mastitis, neuroinflammation, and psoriasis (22–26). Interestingly, DMI is not directly metabolized into itaconate, but it potentiates an increase in the itaconate level in lipopolysaccharide-stimulated macrophages (27). Despite this, it remains largely undefined whether and how DMI modulates antimicrobial host defense against Mtb, NTM, and drug-resistant mycobacterial infections.

In this study, we aimed to evaluate whether DMI exerts host defensive functions and how it achieved protective immune reactions during mycobacterial infections. We examined whether DMI increases antimicrobial host responses against Mtb, M. bovis Bacillus Calmette–Guérin (BCG), Mav, and multidrug-resistant (MDR) Mtb infections. Although DMI per se did not exhibit direct antimicrobial effects against Mtb and NTM, it showed potent antimicrobial activities in macrophages and in vivo. Mechanistically, DMI played multiple roles in the activation of innate immune defenses, i.e., maintenance of inflammatory homeostasis, signal transducer and activator of transcription 3 (STAT3) signaling, and activation of autophagy. These data offer DMI as an effective therapeutic candidate of HDT against Mtb and NTM infections by modulating multifaceted innate immune pathways.

2.1 Mycobacterial strains and cultivation

Mtb H37Rv was supplied by R.L. Friedmann (University of Arizona, Tucson, AZ). BCG, MDR Mtb (KMRC-00116-00150), and Mav (KMRC-00800-00012 and-00013) were acquired from the Korean Mycobacterium Resource Center in the Korean Institute of Tuberculosis (Osong, South Korea). Mycobacteria were cultured in Middlebrook 7H9 (Difco, 271310) medium supplemented with 10% oleic albumin dextrose catalase (BD Biosciences, San Diego, CA, 212240), 0.5% glycerol, and 0.05% Tween-80 on a rotary shaking incubator (140 rpm) at 37℃ to an OD600 of 0.4–0.6. Mtb expressing red fluorescent protein (Mtb-ERFP) was cultivated in Middlebrook 7H9 medium supplemented with 10% oleic albumin dextrose catalase, 0.5% glycerol, 0.05% Tween-80, and 50 µg/mL kanamycin (Sigma-Aldrich, St. Louis, MO, 60615). Bacterial cultures were harvested, and the pellets were washed with phosphate-buffered saline (PBS; LPS solution, Daejeon, Korea, CBP007B) by sequential centrifugation at 2090 × g (3000 rpm) for 30 min. To separate bacteria into single cells, pellets resuspended in PBS with 0.1% Tween-80 were subjected to repeated rounds of sonication. The resulting bacterial suspensions were aliquoted and stored at − 80℃. Colony-forming units (CFUs) were counted on Middlebrook 7H10 agar (Difco, 262710).

2.2 Mice

Female wild-type (WT) C57BL/6 mice (Samtako Bio, Gyeonggi-do, South Korea) were obtained at 6–7 weeks of age and maintained under a 12 h:12 h light:dark cycle and specific-pathogen-free conditions. Information on Atg7-floxed mice and Atg7-lacking mice is available elsewhere (28). The mice were 6–8 weeks old at the time of the experiments and were matched by sex. The animal experiments and handling were conducted following the ethical guidelines of Chungnam National University School of Medicine and were approved by the Institutional Animal Care and Use Committee (202109A-CNU-180; Daejeon, South Korea) and the South Korean Food and Drug Administration.

2.3 Isolation of bone marrow-derived macrophages

Bone marrow-derived macrophages (BMDMs) were collected from the femur and tibia of 6–8-week-old WT C57BL/6 mice and cultured for 4–5 days in Dulbecco’s modified Eagle’s medium (DMEM; Lonza, Walkersville, USA, BE12-60fF) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, 16000-044) and antibiotics (Lonza, 17-745E) containing 25 ng/mL macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN) at 37℃ in 5% CO2.

2.4 Experimental infection

Bacterial cells stored at − 80℃ were thawed and diluted in Dulbecco’s PBS (DPBS; Cytiva Hyclone™, Marlborough, MA, SH30028.02) containing 0.05% Tween-80. Vial containing bacterial cells was sonicated to a bath sonicator 3 times for 30 sec. Infection of BMDMs and peritoneal macrophages (PMs) at the indicated multiplicities of infection (MOI) of Mtb, BCG, or Mav was conducted for 4 h. The remaining bacteria around cells were removed by washing with DPBS, and infected cells were incubated in fresh DMEM for the indicated times. For in vivo infection, mice were anesthetized and intranasally infected with mycobacteria (Mtb: 5 × 10⁴ CFU/mouse; BCG: 1 × 10⁷ CFU/mouse; Mav: 1 × 10⁶ CFU/mouse; MDR TB: 5 × 10³ CFU/mouse). To estimate the bacterial burden, mice were euthanized after 10 days of Mtb, 7 days of BCG, and 10 or 28 days of MDR Mtb infection, and lungs were harvested, homogenized in DPBS, serially diluted, and spotted on 7H10 agar. After incubation for 2–3 weeks, colonies were counted.

2.5 Reagents and antibodies

DMI (592498) and β-cyclodextrin (H5784) were purchased from Sigma-Aldrich. For Western blotting, anti-pSTAT3 (9145S), anti-STAT3 (9139S), and anti-ACTB (5125S) antibodies were purchased from Cell Signaling Technology (Danvers, MA). For immunofluorescence analysis, an anti-LC3A/B (PM036) antibody was obtained from Medical & Biological Laboratories International, and an anti-LAMP1 (SC-19992) antibody was obtained from Santa Cruz Biotechnology. Alexa Fluor 488-conjugated anti-rabbit IgG (A17041) and Alexa Fluor 594-conjugated anti-rabbit IgG (A21207) antibodies were from Molecular Probes. 4’-6-Diamidino-2-phenylindole dihydrochloride (DAPI; D9542) was obtained from Sigma-Aldrich.

2.6 CFU assay

To analyze bacterial survival in murine macrophages, Mtb-, BCG-, or Mav-infected cells (MOI 1) were incubated for 2 h and washed with DPBS to discard extracellular bacteria. The infected cells were incubated in fresh medium for the indicated periods. Thereafter, the cells were lysed in sterile distilled water for 30 min, and intracellular bacteria were collected. Cell lysates were diluted with DPBS and spotted on Middlebrook 7H10 agar containing 10% oleic albumin dextrose catalase. Colonies were counted to assay intracellular bacterial viability after 3 weeks (Mtb and BCG) or 7 days (Mav).

2.7 Histology and immunohistochemistry

Lungs were removed from Mtb-infected mice, fixed in 10% formalin, and embedded in paraffin wax. Paraffin blocks were sectioned (4 µm) and stained with hematoxylin and eosin as described previously (29). The inflamed area was quantified by total field scanning of lung tissues, and the mean fluorescence intensity of the red threshold was examined using FIJI software (ImageJ-win64).

2.8 Determination of dose–response curves

The half-maximal inhibitory concentration (IC50) values were determined following the CLSI guidelines (30). Mtb, BCG, and Mav were cultured in 7H9 medium supplemented with 10% OADC and 0.02% Tween-80 to an OD600 of 0.4–0.6 followed by harvesting, washing twice with PBST, and vortexing 3 times for 5 sec with 30–50 glass beads of diameter 2 mm. Finally, low-speed centrifugation (250 × g) leaves only single bacterial cells in supernatant. Inoculum was prepared from this supernatant. All wells except the negative control (medium only) contained 7H9-OADC with 0.02% Tween-80 and starting inoculum an OD600 of 0.005/mL. DMI, β-cyclodextrin, and INH were two-fold serially diluted 20 times (DMI, β-cyclodextrin) or 10 times (INH) in flat-bottom clear 96-well plates, ranging from 50 mM to 95 nM (DMI) or from 10 µM to 19.5 nM (INH) containing Mtb, BCG, or Mav in a final volume of 100 µL, and then incubated for 5 to 7 days at 37°C. OD600 values were determined using the VersaMax microplate reader (Molecular devices, Sunnyvale, CA). IC50 values were calculated from the OD600 values using Prism 8.0 software (GraphPad Inc., La Jolla, CA).

2.9 RNA preparation and quantitative real-time PCR (qRT-PCR)

Total RNA was isolated from BMDMs, PMs, or lung tissue homogenates using TRIzol reagent (Invitrogen, Waltham, MA, 15596026) in accordance with the manufacturer’s instructions. cDNA was prepared from total RNA using Reverse Transcription Master Premix (ELPIS Biotech, Daejeon, South Korea, EBT-1515) following the manufacturer’s protocols. qRT-PCR was conducted on the Rotor-Gene A 2plex System (Qiagen, Hilden, Germany, 9001620) using cDNA, primer pairs specific to the genes of interest, and SYBR Green Master Mix (Qiagen, 218073) according to the manufacturer’s instructions. Relative mRNA levels were analyzed by the 2^−ΔΔ threshold cycle method and normalized to those of Gapdh. The primer sequences used are as follows: Il1b forward: 5′-TGACGGACCCCAAAAGATGA-3′, reverse: 5′-AAAGACACAGGTAGCTGCCA-3′; Il6 forward: 5′-ACAAAGCCAGAGTCCTTCAGA-3′, reverse: 5′-TGGTCCTTAGCCACTCCTTC-3′; Il10 forward: 5′-GCTCTTGCACTACCAAAGCC-3′, reverse: 5′-CTGCTGATCCTCATGCCAGT-3′; Tnf forward: 5′-CCCACGTCGTAGCAAACCAC-3′, reverse: 5′-GCAGCCTTGTCCCTTGAAGA-3′; Gapdh forward: 5′-TGGCAAAGTGGAGATTGTTGCC-3′, reverse: 5′-AAGATGGTGATGGGCTTCCCG-3′.

2.10 Enzyme-linked immunosorbent assay

The supernatant of mouse BMDMs or lung lysates was stored at − 80℃. The levels of the proinflammatory cytokines IL-6 (555240) and IL-10 (555252) were estimated using the Mouse BD OptEIA Set ELISA Kit (BD Biosciences) according to the manufacturer’s instructions.

2.11 Immunofluorescence analysis

BMDMs were grown on coverslips in 24-well plates followed by infection with Mtb-ERFP for 4 h. After treatment, the upper medium was discarded, and cells on coverslips were washed three times in DPBS followed by fixation in 4% paraformaldehyde for 10 min. Thereafter, the cells were permeabilized in 0.25% Triton X-100 for 10 min and incubated overnight at 4℃ with anti-LC3 A/B (1:400 diluted) and anti-LAMP1 (1:400 diluted) primary antibodies. The cells were washed three times with DPBS and reacted for 2 h with the secondary antibody at room temperature. Fluoromount-G in DAPI mounting medium (Thermo Fisher Scientific, Waltham, MA, 00-4959-52) was used to stain nuclei for 5 min.

2.12 Transmission electron microscopy (TEM)

Cells were scraped from plates and collected by centrifugation. Samples were sequentially fixed in 3% glutaraldehyde and 1% osmium tetroxide, cooled on ice for 1 h, washed with 0.1 M cacodylate buffer (pH 7.2) containing 0.1% CaCl₂, and dehydrated in an ethanol and propylene oxide series. Next, samples were embedded in Epon 812 mixture and polymerized at 60°C for 36 h. Using the ULTRACUT UC7 ultramicrotome (Leica Biosystems, Vienna, Austria), sections of 70 nm thickness were cut and mounted on 75-mesh copper grids. Sections were counterstained with uranyl acetate and lead citrate for 10 min and 7 min, respectively, and examined using the KBSI Bio-High Voltage EM (JEM-1400 Plus at 120 kV and JEM-1000BEF at 1000 kV; JEOL Ltd., Tokyo, Japan).

2.13 Western blotting

BMDM lysates were collected in Protein 5× Sample Buffer (ELPIS BIOTECH, EBA-1052) diluted with RIPA buffer (150 mM sodium chloride, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 50 mM Tris-HCl at pH 7.5, and 2 mM EDTA) supplemented with protease (04693132001) and phosphatase (11836170001) inhibitor cocktails (Roche, Mannheim, Germany). Samples were boiled for 10 min on a heating block and cooled on ice for 10 min. The samples were resolved by SDS-PAGE and transferred to a nitrocellulose (Pall Corporation, NY, 66485) or PVDF (Millipore, Burlington, MA, IPVH0001) membrane at 200 mA for 2 h. To prevent nonspecific binding, membranes were incubated in blocking solution with 1% BSA in TBS containing 0.1% Tween 20 for 30 min at room temperature and reacted overnight with anti-pSTAT3, -STAT3, or -ACTB primary antibody at 4℃. The membranes were reacted with the appropriate horseradish peroxidase-conjugated secondary antibodies (7076S [anti-mouse IgG], 7074S [anti-rabbit IgG], Cell Signaling Technologies) for 1 h at room temperature. Immunoreactive bands were visualized with ECL reagent from the Chemiluminescence Assay Kit (Millipore, WBKL S0500), and the appropriate bands were detected using the UVitec Alliance mini-chemiluminescence device (UVitec, Rugby, UK). Band intensities were measured using Image J software and normalized to that of β-actin.

2.14 Statistical analysis

Statistical analysis was conducted using Prism 8.0 for Windows (GraphPad Software Inc., San Diego, CA). The unpaired Student’s t-test was used to compare two groups and two-way ANOVA for three or more groups. Data are means ± standard deviation or ± standard error of the mean. Statistical significance is indicated as * P < 0.05, ** P < 0.01, and *** P < 0.001.

3.1 DMI induces antimicrobial activity against Mtb, BCG, and Mav in macrophages and in vivo

Irg1, a mitochondrial enzyme that produces itaconate via decarboxylation of cis-aconitate (16), protects against Mtb infection by controlling excessive pathological inflammatory responses and neutrophil recruitment (18). To assess the antimicrobial effect of DMI on Mtb, BCG, and NTM infections, we infected murine BMDMs with Mtb H37Rv, BCG, or Mav at an MOI of 1 and monitored bacterial survival at 72 h post-infection. DMI (25, 50, and 100 µM) caused dose-dependent suppression of intracellular Mtb, BCG, and Mav (Fig. 1A). To examine whether DMI directly kills mycobacteria, we determined the IC50 values and generated dose–response curves. The IC50 values of DMI for Mtb, BCG, and Mav were 866 µM, 1.2 mM, and 3.8 mM, respectively. The IC50 of INH was 154 nM. Compared with INH, the IC50 values of DMI were not markedly decreased in any of the mycobacteria (Additional file 1: Fig. S1). Therefore, the intracellular antimicrobial responses to DMI may not be caused by its inhibition of mycobacterial growth.

C57BL/6 mice were infected intranasally with Mtb (5 × 10⁴ CFU/mouse), BCG (1 × 10⁷ CFU/mouse), Mav (1 × 10⁶ CFU/mouse), or MDR Mtb (5 × 10³ CFU/mouse) and treated intraperitoneally with 50 mg/kg DMI or the solvent controls (SCs) for 4 days (Fig. 1B, left). Pronounced differences were observed in the in vivo bacterial loads of all four different strains in the lung tissues from the infected mice between DMI-treated and vehicle-treated groups (Fig. 1B). In addition, DMI significantly inhibited granulomatous lesions in the lung tissues of Mtb-infected mice (Fig. 1C and D). Taken together, these data strongly suggest that DMI significantly suppresses the pulmonary bacterial loads and inflammatory lesions in mice during mycobacterial infections.

3.2 DMI suppresses IL-6 and IL-10 generation in macrophages and in the lung tissues from infected mice

Because DMI reduced the bacterial burden and lung pathological lesions at the early (< 7 dpi) stages of infection (Fig. 1), we examined its effect on lung inflammatory responses during mycobacterial infection. The Tnf and Il1b mRNA levels were not reduced by DMI at 7 dpi, but the Il6 and Il10 levels were significantly reduced by Mtb, BCG, and Mav in mouse lungs at 7 dpi (Fig. 2A and B). Similarly, the IL-6 and IL-10 protein levels were markedly reduced in the supernatants of lung lysates from DMI-treated compared with control mice infected with mycobacteria (Fig. 2C).

We examined the effect of DMI on Mtb-induced mRNA and protein levels of pro- and anti-inflammatory cytokines (TNF-α, IL-6, IL-1β, and IL-10). Mtb-induced Tnf expression was significantly increased by DMI at 6 h, but decreased at 18 h, in BMDMs (Additional file 1: Fig. S2A). However, Mtb-induced Il6 and Il1b levels were markedly suppressed by DMI at 3, 6, and 18 h (Fig. 3A). Additionally, the Mav-induced Il6 and Il1b levels were markedly decreased at 3–18 h, as with Mtb infection (Additional file 1: Fig. S2B). Moreover, DMI markedly suppressed IL-6 and IL-10 production in BMDMs infected with Mtb, BCG, or Mav, compared with infection alone (Fig. 3B). Therefore, DMI significantly reduces IL-6 and − 10 expression in mouse lungs and macrophages during mycobacterial infection.

3.3 DMI promotes the activation of autophagy and autophagic flux in BMDMs

DMI induces cellular autophagy to inhibit NLRP3-mediated pyroptosis (31). We thus examined whether DMI activates autophagy to enhance antibacterial responses during infection. DMI robustly increased microtubule-associated protein 1 light chain 3 β (LC3) punctate structures in BMDMs in a time-dependent manner (Fig. 4A). In addition, Western blotting revealed that DMI caused slight but significant conversion of cytosolic LC3-I to autophagosome-associated LC3-II (Fig. 4B). Moreover, pretreatment of BMDMs with bafilomycin A1 significantly increased the level of lipidated LC3-II, indicating that DMI increases autophagic flux (Fig. 4B, lane 4).

Ultrastructural analysis by transmission electron microscopy (TEM) showed that DMI significantly increased the number of autophagic vesicles (autophagosomes and autolysosomes) in BMDMs (Fig. 4C and D). When BMDMs were transduced with adeno-associated virus-mRFP-GFP-LC3 prior to DMI treatment, the number of red puncta denoting autolysosomes were significantly increased. However, bafilomycin A1 prevented this effect in DMI-treated BMDMs (Fig. 4E and F). These data confirm that DMI induces autophagy and autophagic flux in BMDMs.

3.4 DMI-mediated autophagy enhances phagosomal maturation of Mtb

We next examined the effect of DMI on phagosomal maturation in BMDMs. DMI treatment of Mtb-infected BMDMs significantly increased the colocalization of endogenous LC3-positive autophagosomal structures and Mtb phagosomes at 6 (early) and 18 (late) h (Fig. 5A and B). Additionally, DMI significantly increased the colocalization of Mtb phagosomes with lysosomes in BMDMs at the same time points (Fig. 5C and D). TEM showed that Mtb-containing autophagosomal-autolysosomal structures were significantly increased in BMDMs at 18 h (Fig. 5E and F). However, there was no significant difference in cytosolic Mtb between DMI-treated and -untreated conditions (Fig. 5F). Bacteria-containing phagosomes were significantly downregulated in BMDMs treated with DMI (Fig. 5F). Therefore, DMI promotes the association of Mtb with autophagosomes and autolysosomes, thereby promoting phagosomal maturation during infection.

3.5 DMI-induced autophagy is partly required for antimicrobial responses to Mtb, BCG, and Mav infection

We next assessed whether autophagy inhibition affects DMI-mediated antimicrobial responses in BMDMs by comparing the DMI-induced intracellular Mtb and BCG load in BMDMs from WT mice (Atg7^flox/flox) versus mice with myeloid cell-specific disruption of the crucial autophagy gene Atg7 (K14Cre⁺;Atg7^flox/flox; conditional homozygous knockout, cKO) (Fig. 6A). Notably, DMI significantly inhibited intracellular Mtb and BCG growth in Atg7 WT and cKO BMDMs (Fig. 6A). However, Atg7 cKO BMDMs showed significantly increased bacterial growth compared with Atg7 WT BMDMs under all conditions at 3 dpi in the presence or absence of DMI. Similar pattern was observed in Atg7 cKO PMs under all conditions in the presence or absence of DMI (Fig. 6B). Notably, the numbers of intracellular Mtb and BCG CFUs were comparable between Atg7 cKO macrophages treated with 100 µM DMI and untreated Atg7 WT macrophages (Fig. 6A and B for BMDMs and PMs, respectively).

DMI-mediated suppression of intracellular Mav growth was observed in Atg7 WT and Atg7 cKO BMDMs (Fig. 6C). The number of intracellular Mav CFUs was significantly higher in Atg7 cKO than Atg7 WT BMDMs in the presence or absence of DMI (Fig. 6C). As with the Mtb-infected conditions, there was no difference in intracellular Mav growth between Atg7 cKO BMDMs treated with 100 µM DMI and untreated Atg7 WT BMDMs (Fig. 6C). Therefore, autophagy is partly required for DMI-induced antimicrobial responses in macrophages during Mtb, BCG, and Mav infections.

3.6 DMI modulates the activation of STAT3 in mycobacterium-infected macrophages

Given that DMI suppresses IL-6 and IL-10 and induces autophagy in macrophages during infection, we evaluated its effect on activation of the transcription factor STAT3, which modulates gene expression in response to IL-6 and IL-10 (32) and the suppression of autophagy (33, 34). In addition, the STAT3 signaling pathway may play a detrimental role in the host defensive responses through the enhanced intracellular Mtb survival, blockade of apoptosis, and aggravation of inflammatory responses during infection and inflammation (35–37). We thus evaluated the effect of DMI on STAT3 phosphorylation and expression. Although the activation patterns differed over time, Mtb, BCG, or Mav significantly increased the phosphorylation of STAT3 at Tyr-705, which was highly phosphorylated at 18 h in BMDMs (Fig. 7A–C). When BMDMs were exposed to DMI, the phosphorylated STAT3 levels were significantly downregulated from 1 to 6 h of infection, which was sustained until 18 h of infection, during Mtb, BCG, and Mav infections (Fig. 7A–C). These data suggest that DMI significantly suppresses the activation of STAT3 which signaling might be related to detrimental effects upon host responses during Mtb and NTM infections.

Mycobacteria–host interactions are complex and dynamic and influence the outcome of infection. Mtb and NTM have evolved multiple strategies to modulate and evade host innate signaling pathways and immune clearance (38–40). These include interruption of phagosomal maturation (41–43), disruption of apoptosis (44) and autophagy (45), evasion of reactive oxygen species (ROS) (46–48), deleterious type I IFN secretion (49), excessive production of inflammatory cytokines (50), and immunosuppression with increased IL-10 production (51, 52). These events can modulate the host–pathogen relationship to favor mycobacterial survival in macrophages. Several drug candidates for HDT can reportedly overcome the immune-evasion mechanisms of each mycobacterial strain and enhance host defensive mechanisms (15, 53–59). However, little is known about the potential drug candidate(s) that can activate innate host defense against both Mtb and NTM infections. Here, we show that DMI offers a therapeutic promise for HDT against multiple mycobacteria including Mtb, BCG, Mav, and even to MDR-Mtb. Importantly, DMI acts as a potential HDT agent through diverse mechanisms involving the maintenance of inflammatory homeostasis, enhancement of autophagy and phagosomal maturation, and suppression of STAT3 signaling.

The natural form of itaconate is produced by Irg1, which is expressed in murine and human macrophages, and Irg1 expression is induced in host cells by Mtb infection via the Mtb ESX-1 secretion system, and the host STING, type I IFN, and TLR2-dependent signaling pathways (16, 60). Michelucci et al. reported that Irg1 and its product itaconic acid suppress mycobacterial growth by blockade of bacterial isocitrate lyase (16). Irg1 and its product itaconate in myeloid cells are required to control neutrophil infiltration and suppress pathological inflammation, ROS generation, and tissue injury (18). However, whether cell-permeable derivatives of itaconate suppress in vitro and in vivo growth of multiple mycobacteria is largely unclear. Our data is important to show that DMI has an in vivo antimicrobial activity to control bacterial growth in the lung tissues during a variety of infections caused by mycobacteria including Mtb, BCG, and Mav. Given that Irg1 is required to control excessive neutrophil infiltration in the lungs to ameliorate pathological inflammation and progression of Mtb infection (61, 62), further research should examine whether DMI-mediated antimicrobial defense is associated with attenuation of neutrophil recruitment in the lungs of mice infected with Mtb, BCG, or Mav.

Although it reduced the intracellular survival of mycobacteria in macrophages, DMI did not exert a direct antimicrobial effect because a DMI concentration of 1000-fold the IC50 of INH is required to suppress bacterial growth. Rather, DMI appeared to induce multiple host-defense strategies during mycobacterial infection. DMI maintained the homeostasis of inflammation by reducing the IL-6 and IL-10 levels in vivo and suppressed the generation of IL-1β and IL-6 in macrophages. Inflammatory response and production of antimicrobial factors including ROS and antimicrobial peptides are important for host immune defense against intracellular mycobacteria (63, 64). However, the unbalanced inflammatory response to chronic infections with intracellular pathogens causes damage to the host, rendering inflammatory homeostasis as a target for HDT against Mtb and NTM, particularly drug-resistant strains (65). Therefore, new drugs that maintain the balance between inflammatory host defense and prevention of necrotic inflammation are urgently needed. Our data revealed that DMI has an activity to ameliorate the levels of IL-6 and IL-10, which are linked to pathological inflammation and immune suppression, respectively. Thus, the DMI effects may contribute to improve host defense through keeping immune homeostasis, through modulating either pro- or anti-inflammatory immune pathways, against Mtb and NTM infections.

Autophagy, a cell-autonomous defense mechanism, is a possible target of HDT against mycobacterial infection (58, 59, 66). Several autophagy-activating factors enhance innate host defense and lysosomal degradation of intracellular mycobacteria (56–59, 66). DMI robustly activated autophagy, which partly affects intracellular mycobacterial survival in Atg7 cKO macrophages. These data suggest that DMI-mediated autophagy activation is necessary but not sufficient, to suppress intracellular mycobacterial survival. DMI further enhances the colocalization of Mtb phagosomes with lysosomes, indicating that DMI-induced autophagy may promote phagosomal maturation during Mtb infection. These results are in partial agreement with a recent report that 4-octyl itaconate enhances autophagy in chondrocytes by suppressing PI3K/Akt/mTOR signaling (67). However, 4-octyl itaconate also suppresses autophagy and ROS generation to exert an anti-fibrotic effect in renal tissue (68). These data suggest that cell-permeable itaconate modulates host autophagy in a context-dependent manner.

Importantly, DMI significantly reduced the STAT3 phosphorylation levels in macrophages during Mtb, BCG, and Mav infection. STAT3 is a transcription factor activated by cytokines such as IL-6, IL-10, and growth factors, and is implicated in the activation of Th17 cell responses and autoimmune diseases as well as anti-inflammatory responses (32, 69, 70). In mycobacterial infection, the role of STAT3 signaling in the immune response has been debated (71). In human innate immune responses, STAT3 signaling and TLR4 pathway activation are important in the vitamin D-mediated antimicrobial pathways in macrophages (72), although the molecular mechanisms are unknown. However, the pSTAT3 inhibitor AG-490 protected against lung injury in a mouse model of type 2 diabetes-associated TB (35). The inhibition of pSTAT3 by AG-490 improves mouse survival and histopathological findings and ameliorates inflammation, fibrosis, and Mtb growth (35). Therefore, DMI-mediated STAT3 inhibition is likely responsible for the suppression of pathological inflammation during mycobacterial infection. Also, STAT3 signaling activation is associated with anti-apoptotic responses, favoring the intracellular survival of Mtb (36). In addition, Mtb Rv2145c-mediated intracellular bacterial growth is dependent on STAT3-mediated IL-10 production (37), suggesting an immunosuppressive role for STAT3. Indeed, STAT3 signaling suppresses autophagy by multiple molecular mechanisms including transcriptional regulation of autophagy-related genes and IL-10-mediated inhibition of autophagy (33, 34). Together with its suppressive functions upon autophagy pathway, the STAT3 pathway contributes to intracellular Mav survival in macrophages (73). Further research is needed to determine whether DMI-mediated suppression of STAT3 signaling underlies the activation of autophagy induced by DMI treatment.

In summary, the present study has demonstrated that DMI shows potent antimicrobial activities against Mtb, BCG, Mav, and even to MDR-Mtb infections. In addition, DMI functions as an important regulator of pathological inflammation, activation of autophagy, and control of STAT3 signaling. DMI-induced autophagy partly contributes to improve host defenses against mycobacterial infections. Together, we propose DMI as a new promising candidate for HDT against both Mtb and NTM infections through orchestrating multiple innate immune strategies.

BCG, Mycobacterium bovis Bacillus Calmette-Guérin; BMDM, bone marrow-derived macrophage; CFU, colony-forming unit; DMI, dimethyl itaconate; DPBS, Dulbecco’s Phosphate-Buffered Saline; DPBS-T, Dulbecco’s Phosphate-Buffered Saline added with 0.05% Tween-80; ELISA, enzyme-linked immunosorbent assay; HDT, host-directed therapeutics; IC50, half maximal inhibitory concentration; IL, interleukin; INH, Isoniazid; Irg1, immune-responsive gene 1; LC3, microtubule-associated protein 1 light chain 3 β; Mav, M. avium; MDR, multidrug-resistant; MOI, multiplicity of infection; NTM, nontuberculous mycobacteria; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline with 0.1% Tween 80; PM, peritoneal macrophage; pSTAT3, phospho-signal transducer and activator of transcription 3; qRT-PCR, quantitative real-time polymerase chain reaction; SC, solvent control; SEM, standard error of the mean; STAT3, signal transducer and activator of transcription 3; TB, tuberculosis; TNF, tumor necrosis factor; WT, wild-type

6. Ethics approval and consent to participate

The animal experiments and handling were conducted following the ethical guidelines of Chungnam National University School of Medicine and were approved by the Institutional Animal Care and Use Committee (202109A-CNU-180; Daejeon, South Korea) and the South Korean Food and Drug Administration.

7. Consent for publication

Not applicable

8. Availability of data and material

All data generated or analyzed during this study are included in this published article and its supplementary information files.

8. Competing interests

The authors declare that the research was performed in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.

9. Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2017R1A5A2015385), supported by a grant from the Sejong Science fellow ship (NRF-2022R1C1C2010228) through the NRF, and by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI22C1361).

10. Authors’ Contributions

EKJ, YJK, and EJP contributed to the conception and critically revised the manuscript. YJK, EJP, PS, JKK, and SHL carried out the experiments and data analysis. EKJ, EJP, and YJK wrote the manuscript, which was then peer-reviewed by SP, SHL, JKK, JSY, JWh and JCJ. EKJ guided and supervised the work. YJK and EJP contributed equally to this work.

11. Acknowledgements

We are sincerely grateful to Prof. S.-H. P. for hard and deep discussion and Dr. H. -W. Suhr for technical support.

WHO 2021. Global tuberculosis report 2021, WHO.
Tousif S, Singh DK, Ahmad S, Moodley P, Bhattacharyya M, Van Kaer L, et al. Isoniazid induces apoptosis of activated CD4+ T cells: implications for post-therapy tuberculosis reactivation and reinfection. J Biol Chem. 2014;289(44): 30190-5. DOI: 10.1074/jbc.C114.598946
Espinal MA, Laszlo A, Simonsen L, Boulahbal F, Kim SJ, Reniero A, et al. Global trends in resistance to antituberculosis drugs. World Health Organization-international union against tuberculosis and lung disease working group on anti-tuberculosis drug resistance surveillance. N Engl J Med. 2001;344(17): 1294-303.
Nathanson E, Nunn P, Uplekar M, Floyd K, Jaramillo E, Lonnroth K, et al. MDR tuberculosis--critical steps for prevention and control. N Engl J Med. 2010;363(11): 1050-8.
Chai J, Han X, Mei Q, Liu T, Walline JH, Xu J, et al. Clinical characteristics and mortality of non-tuberculous mycobacterial infection in immunocompromised vs. immunocompetent hosts. Front Med. (Lausanne) 2022;9: 884446. DOI: 10.3389/fmed.2022.884446
Sharma SK, Upadhyay V. Epidemiology, diagnosis & treatment of non-tuberculous mycobacterial diseases. Indian J Med Res. 2020;152(3): 185-226.
Shamaei M, Mirsaeidi M. Nontuberculous mycobacteria, macrophages, and host Innate immune response. Infect Immun. 2021;89(8): e0081220. DOI: 10.1128/IAI.00812-20
Dahl VN, Molhave M, Floe A, van Ingen PJ, Schon PT, Lillebaek PT, et al. Global trends of pulmonary infections with nontuberculous mycobacteria: a systematic review. Int J Infect Dis. 2022. DOI: 10.1016/j.ijid.2022.10.013
Tissot A, Thomas MF, Corris PA, Brodlie M. Nontuberculous mycobacteria infection and lung transplantation in cystic fibrosis: a worldwide survey of clinical practice. BMC Pulm Med. 2018;18(1): 86.
Gill LI, Dominic C, Tiberi S. Atypical mycobacterial infections - management and when to treat. Curr Opin Pulm Med. 2021;27(3): 216-23. DOI: 10.1097/MCP.0000000000000764
Abate G, Stapleton JT, Rouphael N, Creech B, Stout JE, El Sahly HM, et al. Variability in the management of adults with pulmonary nontuberculous mycobacterial disease. Clin Infect Dis. 2021;72(7): 1127-37.
Thornton CS, Mellett M, Jarand J, Barss L, Field SK, Fisher DA. The respiratory microbiome and nontuberculous mycobacteria: an emerging concern in human health. Eur Respir Rev. 2021;30(160).
Llibre A, Dedicoat M, Burel JG, Demangel C, O'Shea MK, Mauro C. Host immune-metabolic adaptations upon mycobacterial infections and associated co-morbidities. Front Immunol. 2021;12: 747387. DOI: 10.3389/fimmu.2021.747387
Shi L, Jiang Q, Bushkin Y, Subbian S, Tyagi S. Biphasic dynamics of macrophage immunometabolism during Mycobacterium tuberculosis infection. mBio. 2019;10(2).
Paik S, Jo EK. An interplay between autophagy and immunometabolism for host defense against mycobacterial infection. Front Immunol. 2020;11: 603951. DOI: 10.3389/fimmu.2020.603951
Michelucci A, Cordes T, Ghelfi J, Pailot A, Reiling N, Goldmann O, et al. Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production. Proc Natl Acad Sci U S A. 2013;110(19): 7820-5. DOI: 10.1073/pnas.1218599110
Kwai BXC, Collins AJ, Middleditch MJ, Sperry J, Bashiri G, Leung IKH. Itaconate is a covalent inhibitor of the Mycobacterium tuberculosis isocitrate lyase. RSC Med Chem. 2021;12(1): 57-61. DOI: 10.1039/d0md00301h
Nair S, Huynh JP, Lampropoulou V, Loginicheva E, Esaulova E, Gounder AP, et al. Irg1 expression in myeloid cells prevents immunopathology during M. tuberculosis infection. J Exp Med. 2018;215(4): 1035-45. DOI: 10.1084/jem.20180118
Gidon A, Louet C, Rost LM, Bruheim P, Flo TH. The tumor necrosis factor alpha and interleukin 6 auto-paracrine signaling loop controls Mycobacterium avium infection via induction of IRF1/IRG1 in human primary macrophages. mBio. 2021;12(5): e0212121. DOI: 10.1128/mBio.02121-21
Kim HW, Yu AR, Lee JW, Yoon HS, Lee BS, Park HW, et al. Aconitate decarboxylase 1 deficiency exacerbates mouse colitis induced by dextran sodium sulfate. Int J Mol Sci. 2022;23(8).
He W, Henne A, Lauterbach M, Geissmar E, Nikolka F, Kho C, et al. Mesaconate is synthesized from itaconate and exerts immunomodulatory effects in macrophages. Nat Metab. 2022;4(5): 524-33. DOI: 10.1038/s42255-022-00565-1
Zhao C, Jiang P, He Z, Yuan X, Guo J, Li Y, et al. Dimethyl itaconate protects against lippolysacchride-induced mastitis in mice by activating MAPKs and Nrf2 and inhibiting NF-kappaB signaling pathways. Microb Pathog. 2019;133: 103541. DOI: 10.1016/j.micpath.2019.05.024
Kuo PC, Weng WT, Scofield BA, Paraiso HC, Brown DA, Wang PY, et al. Dimethyl itaconate, an itaconate derivative, exhibits immunomodulatory effects on neuroinflammation in experimental autoimmune encephalomyelitis. J Neuroinflammation. 2020;17(1): 138.
Zhang S, Jiao Y, Li C, Liang X, Jia H, Nie Z, et al. Dimethyl itaconate alleviates the inflammatory responses of macrophages in sepsis. Inflammation. 2021;44(2): 549-57. DOI: 10.1007/s10753-020-01352-4
Bambouskova M, Gorvel L, Lampropoulou V, Sergushichev A, Loginicheva E, Johnson K, et al. Electrophilic properties of itaconate and derivatives regulate the IkappaBzeta-ATF3 inflammatory axis. Nature. 2018;556(7702): 501-4. DOI: 10.1038/s41586-018-0052-z
Liao ST, Han C, Xu DQ, Fu XW, Wang JS, Kong LY. 4-Octyl itaconate inhibits aerobic glycolysis by targeting GAPDH to exert anti-inflammatory effects. Nat Commun. 2019;10(1): 5091.
ElAzzouny M, Tom CT, Evans CR, Olson LL, Tanga MJ, Gallagher KA, et al. Dimethyl itaconate is not metabolized into itaconate intracellularly. J Biol Chem. 2017;292(12): 4766-9. DOI: 10.1074/jbc.C117.775270
Yang CS, Kim JJ, Lee HM, Jin HS, Lee SH, Park JH, et al. The AMPK-PPARGC1A pathway is required for antimicrobial host defense through activation of autophagy. Autophagy. 2014;10(5): 785-802. DOI: 10.4161/auto.28072
Kim JK, Kim YS, Lee HM, Jin HS, Neupane C, Kim S, et al. GABAergic signaling linked to autophagy enhances host protection against intracellular bacterial infections. Nat Commun. 2018;9(1): 4184.
Woods GL, Brown-Elliott BA, Conville PS, Desmond EP, Hall GS, Lin G, et al. Susceptibility testing of mycobacteria, nocardiae, and other aerobic Actinomycetes. nd. Wayne (PA).2011.
Yang S, Zhang X, Zhang H, Lin X, Chen X, Zhang Y, et al. Dimethyl itaconate inhibits LPS induced microglia inflammation and inflammasomemediated pyroptosis via inducing autophagy and regulating the Nrf2/HO1 signaling pathway. Mol Med Rep. 2021;24(3). DOI: 10.3892/mmr.2021.12311
Gao Y, Zhao H, Wang P, Wang J, Zou L. The roles of SOCS3 and STAT3 in bacterial infection and inflammatory diseases. Scand J Immunol. 2018;88(6): e12727.
You L, Wang Z, Li H, Shou J, Jing Z, Xie J, et al. The role of STAT3 in autophagy. Autophagy. 2015;11(5): 729-39.
Shi J, Wang H, Guan H, Shi S, Li Y, Wu X, et al. IL10 inhibits starvation-induced autophagy in hypertrophic scar fibroblasts via cross talk between the IL10-IL10R-STAT3 and IL10-AKT-mTOR pathways. Cell Death Dis. 2016;7: e2133.
Wang X, Lin Y, Liang Y, Ye Y, Wang D, Tai A, et al. Phosphorylated STAT3 suppresses microRNA-19b/1281 to aggravate lung injury in mice with type 2 diabetes mellitus-associated pulmonary tuberculosis. J Cell Mol Med. 2020;24(23): 13763-74. DOI: 10.1111/jcmm.15954
Fu B, Xue W, Zhang H, Zhang R, Feldman K, Zhao Q, et al. MicroRNA-325-3p facilitates immune escape of Mycobacterium tuberculosis through targeting LNX1 via NEK6 accumulation to promote anti-apoptotic STAT3 signaling. mBio. 2020;11(3).
Park HS, Back YW, Jang IT, Lee KI, Son YJ, Choi HG, et al. Mycobacterium tuberculosis Rv2145c promotes intracellular survival by STAT3 and IL-10 receptor signaling. Front Immunol. 2021;12: 666293. DOI: 10.3389/fimmu.2021.666293
Cambier CJ, Falkow S, Ramakrishnan L. Host evasion and exploitation schemes of Mycobacterium tuberculosis. Cell. 2014;159(7): 1497-509.
Goldberg MF, Saini NK, Porcelli SA. Evasion of innate and adaptive immunity by Mycobacterium tuberculosis. Microbiol Spectr. 2014;2(5).
Ernst JD. Mechanisms of M. tuberculosis immune evasion as challenges to TB vaccine design. Cell Host Microbe. 2018;24(1): 34-42.
Mehra A, Zahra A, Thompson V, Sirisaengtaksin N, Wells A, Porto M, et al. Mycobacterium tuberculosis type VII secreted effector EsxH targets host ESCRT to impair trafficking. PLoS Pathog. 2013;9(10): e1003734. DOI: 10.1371/journal.ppat.1003734
Shimada K, Takimoto H, Yano I, Kumazawa Y. Involvement of mannose receptor in glycopeptidolipid-mediated inhibition of phagosome-lysosome fusion. Microbiol Immunol. 2006;50(3): 243-51.
Falkinham JO, 3rd. Environmental sources of nontuberculous mycobacteria. Clin Chest Med. 2015;36(1): 35-41.
Velmurugan K, Chen B, Miller JL, Azogue S, Gurses S, Hsu T, et al. Mycobacterium tuberculosis nuoG is a virulence gene that inhibits apoptosis of infected host cells. PLoS Pathog. 2007;3(7): e110.
Ouimet M, Koster S, Sakowski E, Ramkhelawon B, van Solingen C, Oldebeken S, et al. Mycobacterium tuberculosis induces the miR-33 locus to reprogram autophagy and host lipid metabolism. Nat Immunol. 2016;17(6): 677-86. DOI: 10.1038/ni.3434
Voskuil MI, Bartek IL, Visconti K, Schoolnik GK. The response of Mycobacterium tuberculosis to reactive oxygen and nitrogen species. Front Microbiol. 2011;2: 105. DOI: 10.3389/fmicb.2011.00105
Colangeli R, Haq A, Arcus VL, Summers E, Magliozzo RS, McBride A, et al. The multifunctional histone-like protein Lsr2 protects mycobacteria against reactive oxygen intermediates. Proc Natl Acad Sci. U S A 2009;106(11): 4414-8. DOI: 10.1073/pnas.0810126106
Helguera-Repetto AC, Chacon-Salinas R, Cerna-Cortes JF, Rivera-Gutierrez S, Ortiz-Navarrete V, Estrada-Garcia I, et al. Differential macrophage response to slow- and fast-growing pathogenic mycobacteria. Biomed Res Int. 2014;2014: 916521. DOI: 10.1155/2014/916521
Mayer-Barber KD, Andrade BB, Barber DL, Hieny S, Feng CG, Caspar P, et al. Innate and adaptive interferons suppress IL-1alpha and IL-1beta production by distinct pulmonary myeloid subsets during Mycobacterium tuberculosis infection. Immunity. 2011;35(6): 1023-34.
Roca FJ, Ramakrishnan L. TNF dually mediates resistance and susceptibility to mycobacteria via mitochondrial reactive oxygen species. Cell. 2013;153(3): 521-34. DOI: 10.1016/j.cell.2013.03.022
Bouzeyen R, Haoues M, Barbouche MR, Singh R, Essafi M. FOXO3 transcription factor regulates IL-10 expression in mycobacteria-infected macrophages, tuning their polarization and the subsequent adaptive immune response. Front Immunol. 2019;10: 2922. DOI: 10.3389/fimmu.2019.02922
Redpath S, Ghazal P, Gascoigne NR. Hijacking and exploitation of IL-10 by intracellular pathogens. Trends Microbiol. 2001;9(2): 86-92.
Tiberi S, du Plessis N, Walzl G, Vjecha MJ, Rao M, Ntoumi F, et al. Tuberculosis: progress and advances in development of new drugs, treatment regimens, and host-directed therapies. Lancet Infect Dis. 2018;18(7): e183-98. DOI: 10.1016/S1473-3099(18)30110-5
Kilinc G, Saris A, Ottenhoff THM, Haks MC. Host-directed therapy to combat mycobacterial infections. Immunol Rev. 2021;301(1): 62-83. DOI: 10.1111/imr.12951
Kaufmann SHE, Dorhoi A, Hotchkiss RS, Bartenschlager R. Host-directed therapies for bacterial and viral infections. Nat Rev Drug Discov. 2018;17(1): 35-56. DOI: 10.1038/nrd.2017.162
Periyasamy KM, Ranganathan UD, Tripathy SP, Bethunaickan R. Vitamin D - A host directed autophagy mediated therapy for tuberculosis. Mol Immunol. 2020;127: 238-44. DOI: 10.1016/j.molimm.2020.08.007
Ouyang Q, Zhang K, Lin D, Feng CG, Cai Y, Chen X. Bazedoxifene suppresses intracellular Mycobacterium tuberculosis growth by enhancing autophagy. mSphere. 2020;5(2): e00124-20.
Silwal P, Kim IS, Jo EK. Autophagy and host defense in nontuberculous mycobacterial infection. Front Immunol. 2021;12: 728742. DOI: 10.3389/fimmu.2021.728742
Silwal P, Paik S, Kim JK, Yoshimori T, Jo EK. Regulatory mechanisms of autophagy-targeted antimicrobial therapeutics against mycobacterial infection. Front Cell Infect Microbiol. 2021;11: 633360. DOI: 10.3389/fcimb.2021.633360
Bomfim CCB, Fisher L, Amaral EP, Mittereder L, McCann K, Correa AAS, et al. Mycobacterium tuberculosis induces Irg1 in murine macrophages by a pathway involving both TLR-2 and STING/IFNAR signaling and requiring bacterial phagocytosis. Front Cell Infect Microbiol. 2022;12: 862582. DOI: 10.3389/fcimb.2022.862582
Nandi B, Behar SM. Regulation of neutrophils by interferon-gamma limits lung inflammation during tuberculosis infection. J Exp Med. 2011;208(11): 2251-62. DOI: 10.1084/jem.20110919
Kimmey JM, Huynh JP, Weiss LA, Park S, Kambal A, Debnath J, et al. Unique role for ATG5 in neutrophil-mediated immunopathology during M. tuberculosis infection. Nature. 2015;528(7583): 565-9. DOI: 10.1038/nature16451
Weiss G, Schaible UE. Macrophage defense mechanisms against intracellular bacteria. Immunol Rev. 2015;264(1): 182-203.
Krug S, Parveen S, Bishai WR. Host-directed therapies: modulating inflammation to treat tuberculosis. Front Immunol. 2021;12: 660916.
Mohan M, Bhattacharya D. Host-directed therapy: A new arsenal to come. Comb Chem High Throughput Screen 2021;24(1): 59-70.
Paik S, Kim JK, Chung C, Jo EK. Autophagy: A new strategy for host-directed therapy of tuberculosis. Virulence. 2019;10(1): 448-59.
Pan X, Shan H, Bai J, Gao T, Chen B, Shen Z, et al. Four-octyl itaconate improves osteoarthritis by enhancing autophagy in chondrocytes via PI3K/AKT/mTOR signalling pathway inhibition. Commun Biol. 2022;5(1): 641.
Tian F, Wang Z, He J, Zhang Z, Tan N. 4-Octyl itaconate protects against renal fibrosis via inhibiting TGF-beta/Smad pathway, autophagy and reducing generation of reactive oxygen species. Eur J Pharmacol. 2020;873: 172989. DOI: 10.1016/j.ejphar.2020.172989
Zhou L, Ivanov, II, Spolski R, Min R, Shenderov K, Egawa T, et al. IL-6 programs T(H)-17 cell differentiation by promoting sequential engagement of the IL-21 and IL-23 pathways. Nat Immunol. 2007;8(9): 967-74. DOI: 10.1038/ni1488
Liu X, Lee YS, Yu CR, Egwuagu CE. Loss of STAT3 in CD4+ T cells prevents development of experimental autoimmune diseases. J Immunol. 2008;180(9): 6070-6.
Rottenberg ME, Carow B. SOCS3 and STAT3, major controllers of the outcome of infection with Mycobacterium tuberculosis. Semin Immunol. 2014;26(6): 518-32.
Liang S, Huang G, Wu T, Peng Y, Liu X, Ji X, et al. MIR337-3p enhances mycobacterial pathogenicity involving TLR4/MYD88 and STAT3 signals, impairing VDR antimicrobial response and fast-acting immunity. Front Immunol. 2021;12: 739219. DOI: 10.3389/fimmu.2021.739219
Wang Y, Chen C, Xu XD, Li H, Cheng MH, Liu J, et al. Levels of miR-125a-5p are altered in Mycobacterium avium-infected macrophages and associate with the triggering of an autophagic response. Microbes Infect. 2020;22(1): 31-9. DOI: 10.1016/j.micinf.2019.07.002

Additionalfile1.docx

Download PDF

Journal Publication

published 08 Mar, 2023

Read the published version in Cell & Bioscience →

Editorial decision: Minor revision
10 Jan, 2023
Reviewers agreed at journal
19 Nov, 2022
Reviewers invited by journal
19 Nov, 2022
Editor assigned by journal
15 Nov, 2022
First submitted to journal
07 Nov, 2022

You are reading this latest preprint version

Dimethyl itaconate is effective in host-directed antimicrobial responses against mycobacterial infections through multifaceted innate immune pathways.

Status:

Journal Publication

Version 1

Abstract

Background

Results

Conclusion

Figures

1. Background

2. Materials And Methods

2.1 Mycobacterial strains and cultivation

2.2 Mice

2.3 Isolation of bone marrow-derived macrophages

2.4 Experimental infection

2.5 Reagents and antibodies

2.6 CFU assay

2.7 Histology and immunohistochemistry

2.8 Determination of dose–response curves

2.9 RNA preparation and quantitative real-time PCR (qRT-PCR)

2.10 Enzyme-linked immunosorbent assay

2.11 Immunofluorescence analysis

2.12 Transmission electron microscopy (TEM)

2.13 Western blotting

2.14 Statistical analysis

3. Results

3.1 DMI induces antimicrobial activity against Mtb, BCG, and Mav in macrophages and in vivo

3.2 DMI suppresses IL-6 and IL-10 generation in macrophages and in the lung tissues from infected mice

3.3 DMI promotes the activation of autophagy and autophagic flux in BMDMs

3.4 DMI-mediated autophagy enhances phagosomal maturation of Mtb

3.5 DMI-induced autophagy is partly required for antimicrobial responses to Mtb, BCG, and Mav infection

3.6 DMI modulates the activation of STAT3 in mycobacterium-infected macrophages

4. Discussion

5. Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1