Pyruvate dehydrogenase kinase inhibitor dichloroacetate augments autophagy mediated constraining the replication of Mycobacteroides massiliense in macrophages.

Increasing evidence indicates a strong interaction between cellular metabolism and innate macrophage immunity. Here, we show that the intracellular replication of Mycobacteroides massiliense in macrophages depends on host pyruvate dehydrogenase kinase (PDK) activity. Infection with M. massiliense induced a metabolic switch in macrophages by increasing glycolysis and decreasing oxidative phosphorylation. Treatment with dichloroacetate (DCA), a PDK inhibitor, converts this switch in M. massiliense-infected macrophages and restricts intracellular bacterial replication. Mechanistically, DCA resulted in AMPKα1 activation via increased AMP/ATP ratio, consequently inducing autophagy to constrain bacterial proliferation in the phagolysosome. This study suggests that the pharmacological inhibition of PDK could be a strategy for host-directed therapy to control virulent M. massiliense infections.

Development of a transcriptome-based determination of innate immune suppressor (TDIS) assay as an in vitro test for immunotoxicity.

Immunotoxicity has been an important topic in toxicology since inadvertent exposures to xenobiotics were found to alter immune functions in humans. While rodent toxicity tests can reveal some levels of immunotoxicity, alternative methods must be developed to identify the detailed mechanisms. In this study, a method of in vitro prediction of innate immune suppression by substances was developed using a genomics approach. The primary selection of immune suppressors was based on their ability to downregulate MCP-1, CCL3, TNF, IL-8, and IL-12p40 expression levels in lipopolysaccharide (LPS)-stimulated THP-1 cells. Among 11 substances classified as potent immune suppressors, six including dexamethasone, tacrolimus, tofacitinib, prednisolone, sodium lauryl sulfate, and benzoic acid were used to create a dataset by transcriptomics of chemical-treated THP-1 cells using bulk RNA sequencing. We selected genes that were significantly upregulated by suppressor treatment while filtering out genes also upregulated in LPS-treated THP-1 cells. We identified a 226-gene immunosuppressive gene set (ISG). Innate immune suppressor signature scores were calculated as the median expression of the ISG. In a validation dataset, the signature score predicted acyclovir, cyclosporine, and mercuric chloride as immune suppressors, while selecting genistein as a non-immune suppressor. Although more dataset integration is needed in the future, our results demonstrated the possibility and utility of a novel genomics-based approach, the transcriptome-based determination of innate immune suppressor (TDIS) assay, to evaluate innate immune suppression by different substances. This provides insight into the development of future alternative testing methods because it reflects a comprehensive genetic signature derived from multiple substances rather than one cytokine.

Liposomal Dexamethasone Reduces A/H1N1 Influenza-Associated Morbidity in Mice.

Re-emerging viral threats have continued to challenge the medical and public health systems. It has become clear that a significant number of severe viral infection cases are due to an overreaction of the immune system, which leads to hyperinflammation. In this study, we aimed to demonstrate the therapeutic efficacy of the dexamethasone nanomedicine in controlling the symptoms of influenza virus infection. We found that the A/Wisconsin/WSLH34939/2009 (H1N1) infection induced severe pneumonia in mice with a death rate of 80%, accompanied by significant epithelial cell damage, infiltration of immune cells, and accumulation of pro-inflammatory cytokines in the airway space. Moreover, the intranasal delivery of liposomal dexamethasone during disease progression reduced the death rate by 20%. It also significantly reduced the protein level of tumor necrosis factor-alpha (TNFα), interleukin-1ß (IL-1ß), IL-6, and the C-X-C motif chemokine ligand 2 (CXCL2) as well as the number of infiltrated immune cells in the bronchoalveolar lavage fluids as compared to the control and free dexamethasone. The liposomal dexamethasone was mainly distributed into the monocyte/macrophages as a major cell population for inducing the cytokine storm in the lungs. Taken together, the intranasal delivery of liposomal dexamethasone may serve as a novel promising therapeutic strategy for the treatment of influenza A-induced pneumonia.

Human Cytomegalovirus-Induced Interleukin-10 Production Promotes the Proliferation of Mycobacterium massiliense in Macrophages.

Human cytomegalovirus (HCMV) exploits the interleukin-10 (IL-10) pathway as a part of its infection cycle through the manipulation of the host IL-10 signaling cascade. Based on its immunomodulatory nature, HCMV attenuates the host immune response and facilitates the progression of co-infection with other pathogens in an immune-competent host. To investigate the impact of HCMV infection on the burden of non-tuberculous mycobacteria (NTM), whose prevalence is growing rapidly worldwide, macrophages were infected with HCMV and further challenged with Mycobacterium massiliense in vitro. The results showed that HCMV infection significantly increased host IL-10 synthesis and promoted the proliferation of M. massiliense in an IL-10-dependent manner. Transcriptomic analysis revealed that HCMV infection dampened the regulatory pathways of interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-1 (IL-1), consequently abrogating the immune responses to M. massiliense coinfection in macrophages. These findings provide a mechanistic basis of how HCMV infection may facilitate the development of pathogenic NTM co-infection by upregulating IL-10 expression.

Protein Kinase A Catalytic Subunit Is a Molecular Switch that Promotes the Pro-tumoral Function of Macrophages.

As current therapies benefit only a minority of cancer patients, additional therapeutic targets are needed. Tumor-associated macrophages (TAMs) have attracted attention for improving therapeutic responses, yet regulatory strategies remain elusive. Here, we show that the protein kinase A catalytic subunit (PKA-C) acts as a molecular switch, inducing a pro-tumoral immunosuppressive macrophage phenotype within tumors. In human and murine breast cancer, overactivated PKA in TAMs creates a detrimental microenvironment for cancer progression by inducing vascular endothelial growth factor A (VEGFA), interleukin-10 (IL-10), and macrophage-derived arginase 1 (ARG1) expression. Macrophages with genetic deletion of PKA-C are prone to be pro-inflammatory, suggesting a possible immunotherapeutic target. Delivery of liposomal PKA inhibitor facilitates tumor regression and abrogates pro-tumoral TAM functions in mice. The therapeutic effect of targeting PKA is pronounced when combined with αCTLA-4 antibody, increasing cluster of differentiation 8 (CD8)+GranzymeB+ T cells by about 60-fold. Our findings demonstrate critical roles of TAM PKA-C in tumor progression and suggest that targeting PKA-C efficiently augments cancer treatment responses.

Fimasartan, an angiotensin II receptor antagonist, ameliorates an in vivo zebrafish model of heart failure.

BACKGROUND/AIMS: Angiotensin II in the failing heart initially helps to maintain cardiac output and blood pressure, but ultimately accelerates its deterioration. In this study, we established a model of arrhythmia-induced heart failure (HF) in zebrafish and investigated the role of renin-angiotensin-aldosterone system (RAAS) modulation by using an angiotensin II type 1 receptor blocker, fimasartan, through the assessment of cellular and physiologic responses, morbidity, and mortality. METHODS: HF was induced in zebrafish larvae by exposure to 20 µM terfenadine. Morphologic, physiologic, and functional parameters were assessed in the presence or absence of fimasartan treatment. RESULTS: Zebrafish exposed to terfenadine showed marked dilatation of the ventricle and reduced systolic function. Treatment with terfenadine was associated with 10-fold higher expression of atrial natriuretic peptide (p < 0.001 vs. vehicle), increased p53 mRNA expression, and chromatin fragmentation in the TUNEL assay, all of which were significantly reduced by fimasartan treatment. Moreover, fimasartan improved fractional shortening (terfenadine + fimasartan 16.9% ± 3.1% vs. terfenadine + vehicle 11.4% ± 5.6%, p < 0.05) and blood flow (terfenadine + fimasartan 479.1 ± 124.1 nL/sec vs. terfenadine + vehicle 273.0 ± 109.0 nL/sec, p < 0.05). Finally, treatment with fimasartan remarkably reduced mortality (terfenadine + fimasartan 36.0% vs. terfenadine + vehicle 96.0%, p < 0.001). CONCLUSION: Fimasartan effectively protected against the progression of HF in zebrafish by improving hemodynamic indices, which improved survival. A reduction in apoptotic cell death and an improvement in hemodynamics may be the mechanisms behind these effects. Further human studies are warranted to evaluate the possible role of fimasartan in the treatment of HF.

Optimizing the cutoff for the identification of skin sensitizers by the HaCaSens assay: Introducing an ROC-analysis-based cutoff approach.

The worldwide restricted use of animal testing makes it challenging to identify the skin sensitizing potentials of newly manufactured products. The HaCaSens assay has shown promise as an in vitro skin sensitizing assay comparable to existing assays, and is currently under pre-validation. However, there is little agreement on how to assess the results of the assay to discriminate sensitizers from non-sensitizers as the stimulation index (SI) cutoff value was arbitrarily chosen without appropriate statistical methods. Here, we investigated the SI cutoff values in identifying sensitizers to obtain the optimal value. Sensitivities and specificities were calculated for a set of 30 test substances, and plotted in receiver operator characteristics (ROC) curves. The SI cutoff values with the highest sum of sensitivity and specificity according to LLNA data were 2.2, 1.8 and 3.0 for interleukin 1α (IL-1α), interleukin 6 (IL-6), and the combination of the two cytokines respectively. Also, the same statistical analysis of human data demonstrated optimal SI cutoff values 2.0, 2.0 and 3.2 for the same respective parameters. When considering the predictive capacity of each possible SI cutoff value determined by ROC curves, the optimal value for HaCaSens is 3.0 for the combination of IL-1α and IL-6 as it had the highest sensitivity (90.9%), specificity (75.0%) and accuracy (86.7%) based on LLNA data. Thus, we recommend the wide use of the SI cutoff value of 3.0 to ensure consistent endpoints.

Lactic Acid Upregulates VEGF Expression in Macrophages and Facilitates Choroidal Neovascularization.

Purpose: Lactic acid, the end product of glycolysis, has emerged as an immune-modulating metabolite in various diseases. In this study, we aimed to examine whether lactic acid contributes to the disease pathogenesis of choroidal neovascularization (CNV) and to investigate the role of macrophages in CNV pathogenesis. Methods: CNV was induced by laser photocoagulation in C57BL/6J mice. Lactic acid concentration was measured in the RPE-choroid region. Macrophage infiltration and VEGF were quantified by flow cytometry. VEGF-positive areas and CNV lesions were measured by flat-mount immunofluorescence staining. To inhibit lactic acid uptake in vivo, alpha-cyano-4-hydroxycinnamic acid (α-CHC), a monocarboxylate transporter (MCT) blocker, was injected intravitreally 1 day after laser. VEGF productions were measured in ARPE-19, THP-1 cells, and human umbilical vein endothelial cells (HUVECs) by quantitative PCR and ELISA. Angiogenic activity of lactic acid-treated macrophages was assessed by HUVEC tube formation assay. Results: Lactic acid was significantly increased in the RPE-choroid region of CNV-induced mice. Lactic acid upregulated VEGFA mRNA and VEGF protein expressions in THP-1 macrophages, but did not in ARPE-19 or HUVECs. THP-1 macrophages treated with lactic acid increased the angiogenesis of endothelial cells independent of MCT activity. Intravitreal injection of α-CHC substantially reduced the VEGF-positive area that colocalized with F4/80-positive macrophages. CNV lesions were also significantly reduced following α-CHC injection compared with vehicle-injected controls. Conclusions: To our knowledge, these results show for the first time the role of lactic acid in facilitating neovascularization through macrophage-induced angiogenesis. We suggest that targeting macrophage metabolism can be a promising strategy for CNV treatment.

Intra- and inter-laboratory reproducibility and predictivity of the HaCaSens assay: A skin sensitization test using human keratinocytes, HaCaT.

Due to considerable constraints in using animals for risk assessment, much effort has been directed at developing non-animal test methods. Developing assays for skin sensitization, the leading cause of contact dermatitis, is particularly important, but there are currently no in vitro skin sensitization tests that completely replace animal tests. HaCaSens, a simple skin sensitization test using non-transformed HaCaT cells, predicts keratinocyte activation by skin sensitizers with 75% sensitivity, 83% specificity and 77% accuracy in a previous study using 22 coded substances. Although the data show promising results, the number of tested substances is insufficient to prove predictive capacity. Moreover, reproducibility among different laboratories has not been studied. Here, three laboratories participated in a validation in order to assess HaCaSens feasibility for official validation. To examine transferability, intra- and inter-lab reproducibility and predictive capacity, HaCaSens was assessed on a set of 30 test substances coordinated by the Validation Management Team (VMT). The results showed satisfactory transferability as well as intra- and inter-laboratory reproducibility. Further assessment of its predictive capacity on 20 test substances demonstrated a sensitivity of 81.8% (18/22), specificity of 87.5% (7/8), and accuracy of 83.3% (25/30) in identifying skin sensitizers, which is comparable with presently validated assays, KeratinoSens™ and LuSens. This validation study shows that the HaCaSens assay is easily transferable, reproducible and highly predictable for identifying skin sensitizers.

An in vitro model of granuloma-like cell aggregates substantiates early host immune responses against Mycobacterium massiliense infection.

Mycobacterium massiliense (M. mass), belonging to the M. abscessus complex, is a rapidly growing mycobacterium that is known to cause tuberculous-like lesions in humans. To better understand the interaction between host cells and M. mass, we used a recently developed in vitro model of early granuloma-like cell aggregates composed of human peripheral blood mononuclear cells (PBMCs). PBMCs formed granuloma-like, small and rounded cell aggregates when infected by live M. mass Microscopic examination showed monocytes and macrophages surrounded by lymphocytes, which resembled cell aggregation induced by M. tuberculosis (M. tb). M. mass-infected PBMCs exhibited higher expression levels of HLA-DR, CD86 and CD80 on macrophages, and a significant decrease in the populations of CD4+ and CD8+ T cells. Interestingly, low doses of M. mass were sufficient to infect PBMCs, while active host cell death was gradually induced with highly increased bacterial loads, reflecting host destruction and dissemination of virulent rapid-growing mycobacteria (RGM). Collectively, this in vitro model of M. mass infection improves our understanding of the interplay of host immune cells with mycobacteria, and may be useful for developing therapeutics to control bacterial pathogenesis.

Mycobacterium massiliense Induces Macrophage Extracellular Traps with Facilitating Bacterial Growth.

Human neutrophils have been known to release neutrophil extracellular traps (NETs), antimicrobial DNA structures capable of capturing and killing microbes. Recently, a similar phenomenon has been reported in macrophages infected with various pathogens. However, a role for macrophages extracellular traps (METs) in host defense responses against Mycobacterium massiliense (M. mass) has yet to be described. In this study, we show that M. mass, a rapid growing mycobacterium (RGM), also induces the release of METs from PMA-differentiated THP-1 cells. Intriguingly, this process is not dependent on NADPH oxidase activity, which regulates NET formation. Instead, M. mass-induced MET formation partially depends on calcium influx and requires phagocytosis of high bacterial load. The METs consist of a DNA backbone embedded with microbicidal proteins such as histone, MPO and elastase. Released METs entrap M. mass and prevent their dissemination, but do not have bactericidal activity. Instead, they result in enhanced bacterial growth. In this regard, METs were considered to provide interaction of M. mass with cells and an environment for bacterial aggregation, which may facilitate mycobacterial survival and growth. In conclusion, our results demonstrate METs as an innate defense response against M. mass infection, and suggest that extracellular traps play a multifaceted role in the interplay between host and bacteria.