Respiratory syncytial virus (RSV) and influenza A virus are leading causes of acute lower respiratory infectious disease. Respiratory diseases caused by RSV and influenza A virus result in serious economic burden and life-threatening disease for immunocompromised people. With the revelation that p38 mitogen-activated protein kinase (MAPK) activity in host cells is crucial for infection and replication of RSV and influenza A virus, inhibition of p38 MAPK activity has been suggested as a potential antiviral therapeutic strategy. However, the low selectivity and high toxicity of the p38 MAPK inhibitors necessitate the development of better inhibitors. Herein, we report the synthesis of a novel p38 MAPK inhibitor, NJK14047, with high kinase selectivity. In this work, it was demonstrated that NJK14047 inhibits RSV- and influenza A-mediated p38 MAPK activation in epithelial cells. Subsequently, NJK14047 treatment resulted in decreased viral replication and viral mRNA synthesis. In addition, secretion of interleukin-6 from infected cells was greatly diminished by NJK14047, suggesting that it can ameliorate immunopathological responses to RSV and influenza A. Collectively, the results suggest that NJK14047 has therapeutic potential to treat respiratory viral infection through the suppression of p38 MAPK activation, which is suggested to be an essential step for respiratory virus 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.

Recently, we introduced a distinct Mycobacterium intracellulare INT-5 genotype, distantly related to other genotypes of M. intracellulare (INT-1 to -4). The aim of this study is to determine the exact taxonomic status of the M. intracellulare INT-5 genotype via genome-based phylogenetic analysis. To this end, genome sequences of the two INT-5 strains, MOTT-H4Y and MOTT-36Y were compared with M. intracellulare ATCC 13950(T) and Mycobacterium yongonense DSM 45126(T). Our phylogenetic analysis based on complete genome sequences, multi-locus sequence typing (MLST) of 35 target genes, and single nucleotide polymorphism (SNP) analysis indicated that the two INT-5 strains were more closely related to M. yongonense DSM 45126(T) than the M. intracellulare strains. These results suggest their taxonomic transfer from M. intracellulare into M. yongonense. Finally, we selected 5 target genes (argH, dnaA, deaD, hsp65, and recF) and used SNPs for the identification of M. yongonese strains from other M. avium complex (MAC) strains. The application of the SNP analysis to 14 MAC clinical isolates enabled the selective identification of 4 M. yongonense clinical isolates from the other MACs. In conclusion, our genome-based phylogenetic analysis showed that the taxonomic status of two INT-5 strains, MOTT-H4Y and MOTT-36Y should be revised into M. yongonense. Our results also suggest that M. yongonense could be divided into 2 distinct genotypes (the Type I genotype with the M. parascrofulaceum rpoB gene and the Type II genotype with the M. intracellulare rpoB gene) depending on the presence of the lateral gene transfer of rpoB from M. parascrofulaceum.

Tripartite motif protein 25 (TRIM25), mediates K63-linked polyubiquitination of Retinoic acid inducible gene I (RIG-I) that is crucial for downstream antiviral interferon signaling. Here, we demonstrate that TRIM25 is required for melanoma differentiation-associated gene 5 (MDA5) and MAVS mediated activation of NF-κB and interferon production. TRIM25 is required for the full activation of NF-κB at the downstream of MAVS, while it is not involved in IRF3 nuclear translocation. Mechanical studies showed that TRIM25 is involved in TRAF6-mediated NF-κB activation. These collectively indicate that TRIM25 plays an additional role in RIG-I/MDA5 signaling other than RIG-I ubiquitination via activation of NF-κB.

Respiratory syncytial virus (RSV) causes severe acute lower respiratory tract disease. Retinoic acid-inducible gene-I (RIG-I) serves as an innate immune sensor and triggers antiviral responses upon recognizing viral infections including RSV. Since tripartite motif-containing protein 25 (TRIM25)-mediated K63-polyubiquitination is crucial for RIG-I activation, several viruses target initial RIG-I activation through ubiquitination. RSV NS1 and NS2 have been shown to interfere with RIG-I-mediated antiviral signaling. In this study, we explored the possibility that NS1 suppresses RIG-I-mediated antiviral signaling by targeting TRIM25. Ubiquitination of ectopically expressed RIG-I-2Cards domain was decreased by RSV infection, indicating that RSV possesses ability to inhibit TRIM25-mediated RIG-I ubiquitination. Similarly, ectopic expression of NS1 sufficiently suppressed TRIM25-mediated RIG-I ubiquitination. Furthermore, interaction between NS1 and TRIM25 was detected by a co-immunoprecipitation assay. Further biochemical assays showed that the SPRY domain of TRIM25, which is responsible for interaction with RIG-I, interacted sufficiently with NS1. Suppression of RIG-I ubiquitination by NS1 resulted in decreased interaction between RIG-I and its downstream molecule, MAVS. The suppressive effect of NS1 on RIG-I signaling could be abrogated by overexpression of TRIM25. Collectively, this study suggests that RSV NS1 interacts with TRIM25 and interferes with RIG-I ubiquitination to suppress type-I interferon signaling.

Recently, we introduced the complete genome sequence of Mycobacterium massiliense clinical isolates, Asan 50594 belonging to Type II genotype with rough colony morphology. Here, to address the issue of whether the rough colony morphotype of M. massiliense Type II genotype is genetically determined or not, we compared polymorphisms of the glycopeptidolipid (GPL) gene locus between M. massiliense Type II Asan 50594 and other rapidly growing mycobacteria (RGM) strains via analysis of genome databases.

The TRIpartite Motif (TRIM) family of RING-domain-containing proteins participate in a variety of cellular functions. The β-transducin repeat-containing protein (β-TrCP), a component of the Skp-Cullin-F-box-containing (SCF) E3 ubiquitin ligase complex, recognizes the NF-κB inhibitor IκBα and precursor p100 for proteasomal degradation and processing, respectively. β-TrCP thus plays a critical role in both canonical and non-canonical NF-κB activation. Here we report that TRIM9 is a negative regulator of NF-κB activation. Interaction between the phosphorylated degron motif of TRIM9 and the WD40 repeat region of β-TrCP prevented β-TrCP from binding its substrates, stabilizing IκBα and p100 and thereby blocking NF-κB activation. Consequently, expression or depletion of the TRIM9 gene significantly affected NF-κB-induced inflammatory cytokine production. This study not only elucidates a mechanism for TRIM9-mediated regulation of the β-TrCP SCF complex activity but also identifies TRIM9 as a brain-specific negative regulator of the NF-κB pro-inflammatory signalling pathway.

RIG-I (retinoic acid-inducible gene I) and TRIM25 (tripartite motif protein 25) have emerged as key regulatory factors to induce interferon (IFN)-mediated innate immune responses to limit viral replication. Upon recognition of viral RNA, TRIM25 E3 ligase binds the first caspase recruitment domain (CARD) of RIG-I and subsequently induces lysine 172 ubiquitination of the second CARD of RIG-I, which is essential for the interaction with downstream MAVS/IPS-1/CARDIF/VISA and, thereby, IFN-beta mRNA production. Although ubiquitination has emerged as a major factor involved in RIG-I activation, the potential contribution of other post-translational modifications, such as phosphorylation, to the regulation of RIG-I activity has not been addressed. Here, we report the identification of serine 8 phosphorylation at the first CARD of RIG-I as a negative regulatory mechanism of RIG-I-mediated IFN-beta production. Immunoblot analysis with a phosphospecific antibody showed that RIG-I serine 8 phosphorylation steady-state levels were decreased upon stimulation of cells with IFN-beta or virus infection. Substitution of serine 8 in the CARD RIG-I functional domain with phosphomimetic aspartate or glutamate results in decreased TRIM25 binding, RIG-I ubiquitination, MAVS binding, and downstream signaling. Finally, sequence comparison reveals that only primate species carry serine 8, whereas other animal species carry an asparagine, indicating that serine 8 phosphorylation may represent a primate-specific regulation of RIG-I activation. Collectively, these data suggest that the phosphorylation of RIG-I serine 8 operates as a negative switch of RIG-I activation by suppressing TRIM25 interaction, further underscoring the importance of RIG-I and TRIM25 connection in type I IFN signal transduction.

Even though the rate of new human immunodeficiency virus type 1 (HIV-1) infections is gradually decreasing worldwide, an effective preventive vaccine for HIV-1 is still urgently needed. The recombinant Mycobacterium bovis BCG (rBCG) is promising for the development of an HIV-1 vaccine. Recently, we showed that a recombinant Mycobacterium smegmatis expressing HIV-1 gag in a pMyong2 vector system (rSmeg-pMyong2-p24) increased the efficacy of a vaccine against HIV-1 in mice. Here, we evaluated the potential of an rBCG expressing HIV-1 p24 antigen Gag in pMyong2 (rBCG-pMyong2-p24) in a vaccine application for HIV-1 infection. We found that rBCG-pMyong2-p24 elicited an enhanced HIV-1 p24 Gag expression in rBCG and infected antigen-presenting cells. We also found that compared to rBCG-pAL-p24 in a pAL5000 derived vector system, rBCG-pMyong2-p24 elicited enhanced p24-specific immune responses in vaccinated mice as evidenced by higher levels of HIV-1 Gag-specific CD4 and CD8 T lymphocyte proliferation, gamma interferon ELISPOT cell induction, antibody production, and cytotoxic T lymphocytes (CTL) responses. Furthermore, rBCG-pMyong2-p24 showed a higher level of p24-specific Ab production than rSmeg-pMyong2-p24 in the same pMyong2 vector system. In conclusion, our data indicated that a live recombinant BCG expressing HIV-1 Gag using a pMyong2 vector system, rBCG-pMyong2-p24 elicited an enhanced immune response against HIV-1 infections in a mouse model system. So, rBCG-pMyong2-p24 may have the potential as a prime vaccine in a heterologous prime-boost vaccine strategy for HIV-1 infection.

Medicinal plants have been used as alternative therapeutic tools to alleviate inflammatory diseases. The objective of this study was to evaluate anti-inflammatory properties of Kyungheechunggan-tang- (KCT-) 01, KCT-02, and Injinchunggan-tang (IJCGT) as newly developed decoctions containing 3-11 herbs in LPS-induced macrophages. KCT-01 showed the most potent inhibitory effects on LPS-induced NO, PGE2, TNF-α, and IL-6 production among those three herbal formulas. In addition, KCT-01 significantly inhibited LPS-induced iNOS and COX-2 at protein levels and expression of iNOS, COX-2, TNF-α, and IL-6 at mRNA levels. Molecular data revealed that KCT-01 attenuated the activation of JAK/STAT signaling cascade without affecting NF-κB or AP-1 activation. In ear inflammation induced by croton oil, KCT-01 significantly reduced edema, MPO activity, expression levels of iNOS and COX-2, and STAT3 phosphorylation in ear tissues. Taken together, our findings suggest that KCT-01 can downregulate the expression of proinflammatory genes by inhibiting JAK/STAT signaling pathway under inflammatory conditions. This study provides useful data for further exploration and application of KCT-01 as a potential anti-inflammatory medicine.

We recently showed that Mycobacterium yongonense could be divided into two genotypes: Type I, in which the rpoB gene has been transferred from Mycobacterium parascrofulaceum, and Type II, in which the rpoB gene has not been transferred. Comparative genome analysis of three M. yongonense Type I, two M. yongonense Type II and M. parascrofulaceum type strains were performed in this study to gain insight into gene transfer from M. parascrofulaceum into M. yongonense Type I strains. We found two genome regions transferred from M. parascrofulaceum: one contained 3 consecutive genes, including the rpoBC operon, and the other contained 57 consecutive genes that had been transferred into M. yongonense Type I genomes via homologous recombination. Further comparison between the M. yongonense Type I and II genomes revealed that Type I, but not Type II has a distinct DNA mismatch repair gene (MutS4 subfamily) that was possibly transferred via non-homologous recombination from other actinomycetes. We hypothesized that it could facilitate homologous recombination from the M. parascrofulaceum to the M. yongonense Type I genomes. We therefore generated recombinant Mycobacterium smegmatis containing a MutS4 operon of M. yongonense. We found that the M. tuberculosis rpoB fragment with a rifampin resistance-conferring mutation was more frequently inserted into recombinant M. smegmatis than the wild type, suggesting that MutS4 is a driving force in the gene transfer from M. parascrofulaceum to M. yongonense Type I strains via homologous recombination. In conclusion, our data indicated that MutS4 in M. yongonense Type I genomes may drive gene transfer from M. parascrofulaceum via homologous recombination, resulting in division of M. yongonense into two genotypes, Type I and II.

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.

The hepatic adiponectin and farnesoid X receptor (FXR) signaling pathways play multiple roles in modulating lipid and glucose metabolism, reducing hepatic inflammation and fibrosis, and altering various metabolic targets for the management of non-alcoholic fatty liver disease (NAFLD). Alisma orientale (AO, Ze xie in Chinese and Taeksa in Korean) is an herbal plant whose tubers are enriched with triterpenoids, which have been reported to exhibit various bioactive properties associated with NAFLD. Here, the present study provides a preclinical evaluation of the biological functions and related signaling pathways of AO extract for the treatment of NAFLD in a Western diet (WD)-induced mouse model. The findings showed that AO extract significantly reversed serum markers (liver function, lipid profile, and glucose) and improved histological features in the liver sections of mice fed WD for 52 weeks. In addition, it also reduced hepatic expression of fibrogenic markers in liver tissue and decreased the extent of collagen-positive areas, as well as inhibited F4/80 macrophage aggregation and inflammatory cytokine secretion. The activation of adiponectin and FXR expression in hepatic tissue may be a major mechanistic signaling cascade supporting the promising role of AO in NAFLD pharmacotherapy. Collectively, our results demonstrated that AO extract improves non-alcoholic steatohepatitis (NASH) resolution, particularly with respect to NASH-related fibrosis, along with the regulation of liver enzymes, postprandial hyperglycemia, hyperlipidemia, and weight loss, probably through the modulation of the hepatic adiponectin and FXR pathways.

Serological tests offer the potential in order to improve the diagnosis of tuberculosis (TB). Macrophage migration inhibitory factor (MIF) plays a protective role in infection control in TB; however, to date, no studies on antibody responses to MIF have been reported. We measured immunoglobulin (Ig)A and IgG responses to MIF in individuals with either active tuberculosis (ATB; n = 65), latent tuberculosis (LTBI; n = 53), or in non-infected individuals (NI; n = 62). The QuantiFERON-TB Gold In-Tube (QFT-GIT) assay was used in order to screen for LTBI. The level of IgA against MIF was significantly lower in LTBI and ATB patients than in NI individuals, was significantly related to LTBI and ATB diagnosis, and it could discriminate between LTBI and ATB. In contrast, the level of IgG against MIF was significantly lower in LTBI patients than in NI individuals and was significantly related to LTBI diagnosis. Anti-MIF IgG levels were significantly lower in AFB-negative TB, minimal TB, and new ATB patients, than in the NI group. IgA and IgG levels against MIF both showed significant negative correlations with IFN-γ levels, as assessed using the QFT-GIT test. Although none of the antibodies could achieve high diagnostic predictive power individually, our results suggest the possibility of using IgA antibody responses to MIF in the diagnosis of LTBI and ATB.

Chronic neuroinflammation, aggressive amyloid beta (Aβ) deposition, neuronal cell loss, and cognitive impairment are pathological presentations of Alzheimer's disease (AD). Therefore, resolution of neuroinflammation and inhibition of Aβ-driven pathology have been suggested to be important strategies for AD therapy. Previous efforts to prevent AD progression have identified p38 mitogen-activated protein kinases (MAPKs) as a promising target for AD therapy. Recent studies showed pharmacological inhibition of p38α MAPK improved memory impairment in AD mouse models.

At present, concerns that the recent global emergence of SARS-CoV-2 variants could compromise the current vaccines have been raised, highlighting the urgent demand for new vaccines capable of eliciting T cell-mediated immune responses, as well as B cell-mediated neutralizing antibody production. In this study, we developed a novel recombinant Mycobacterium paragordonae expressing the SARS-CoV-2 receptor-binding domain (RBD) (rMpg-RBD-7) that is capable of eliciting RBD-specific immune responses in vaccinated mice. The potential use of rMpg-RBD-7 as a vaccine for SARS-CoV-2 infections was evaluated in in vivo using mouse models of two different modules, one for single-dose vaccination and the other for two-dose vaccination. In a single-dose vaccination model, we found that rMpg-RBD-7 versus a heat-killed strain could exert an enhanced cell-mediated immune (CMI) response, as well as a humoral immune response capable of neutralizing the RBD and ACE2 interaction. In a two-dose vaccination model, rMpg-RBD-7 in a two-dose vaccination could also exert a stronger CMI and humoral immune response to neutralize SARS-CoV-2 infections in pseudoviral or live virus infection systems, compared to single dose vaccinations of rMpg-RBD or two-dose RBD protein immunization. In conclusion, our data showed that rMpg-RBD-7 can lead to an enhanced CMI response and humoral immune responses in mice vaccinated with both single- or two-dose vaccination, highlighting its feasibility as a novel vaccine candidate for SARS-CoV-2. To the best of our knowledge, this study is the first in which mycobacteria is used as a delivery system for a SARS-CoV-2 vaccine.

Skin vaccination using dissolving microneedle patch (MNP) technology for transdermal delivery is a promising vaccine delivery strategy to overcome the limitations of the existing vaccine administration strategies using syringes. To improve the traditional microneedle mold fabrication technique, we introduced droplet extension (DEN) to reduce drug loss. Tuberculosis remains a major public health problem worldwide, and BCG revaccination had failed to increase the protective efficacy against tuberculosis. We developed an MNP with live Mycobacterium paragordonae (Mpg) (Mpg-MNP) as a candidate of tuberculosis booster vaccine in a heterologous prime-boost strategy to increase the BCG vaccine efficacy.

For complete or functional cure of hepatitis B virus (HBV) infection, application of immunotherapy is now being attempted. Recently, we reported that a 6-mer hepatitis B virus (HBV)-derived peptide, Poly6, exerts a strong anticancer effect in tumor-implanted mice through inducible nitric oxide synthase (iNOS)-producing DCs (Tip-DCs) in a type 1 interferon (IFN-I)-dependent manner, suggesting its potential as a vaccine adjuvant.

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), and its pathogenicity is associated with its ability to evade the host defense system. The secretory form of the chorismate mutase of M. tuberculosis (TBCM, encoded by Rv1885c) is assumed to play a key role in the pathogenesis of TB; however, the mechanism remains unknown.

A peptide vaccine designed to induce T-cell immunity to telomerase, GV1001, has been shown to modulate cellular signaling pathways and confer a direct anti-cancer effect through the interaction with heat shock protein (HSP) 90 and 70. Here, we have found that GV1001 can modulate transactivation protein-mediated human immunodeficiency virus (HIV)-1 transactivation in an HSP90-dependent manner. GV1001 treatment resulted in significant suppression of HIV-1 replication and rescue of infected cells from death by HIV-1. Transactivation of HIV-long terminal repeat (LTR) was inhibited by GV1001, indicating that GV1001 suppressed the transcription from proviral HIV DNA. The anti-HIV-1 activity of GV1001 was completely abrogated by an HSP90-neutralizing antibody, indicating that the antiviral activity depends on HSP90. Further mechanistic studies revealed that GV1001 suppresses basal NF-κB activation, which is required for HIV-1 LTR transactivation in an HSP90-dependent manner. Inhibition of LTR transactivation by GV1001 suggests its potential to suppress HIV-1 reactivation from latency. Indeed, PMA-mediated reactivation of HIV-1 from latent infected cells was suppressed by GV1001. The results suggest the potential therapeutic use of GV1001, a peptide proven to be safe for human use, as an anti-HIV-1 agent to suppress the reactivation from latently infected cells.