Absence of PD-L1 signaling hinders macrophage defense against Mycobacterium tuberculosis via upregulating STAT3/IL-6 pathway.

The blockade of programmed death-ligand 1 (PD-L1) pathway has been clinically used in cancer immunotherapy, while its effects on infectious diseases remain elusive. Roles of PD-L1 signaling in the macrophage-mediated innate immune defense against M.tb is unclear. In this study, the outcomes of tuberculosis (TB) in wild-type (WT) mice treated with anti-PD-1/PD-L1 therapy and macrophage-specific Pdl1-knockout (Pdl1ΔΜΦ) mice were compared. Treatment with anti-PD-L1 or anti-PD-1 benefited protection against M.tb infection in WT mice, while Pdl1ΔΜΦ mice exhibited the increased susceptibility to M.tb infection. Mechanistically, the absence of PD-L1 signaling impaired M.tb killing by macrophages. Furthermore, elevated STAT3 activation was found in PD-L1-deficient macrophages, leading to increased interleukin (IL)-6 production and reduced inducible nitric oxide synthase (iNOS) expression. Inhibiting STAT3 phosphorylation partially impeded the increase in IL-6 production and restored iNOS expression in these PD-L1-deficient cells. These findings provide valuable insights into the complexity and mechanisms underlying anti-PD-L1 therapy in the context of tuberculosis.

Flu-CED: A comparative transcriptomics database of influenza virus-infected human and animal models.

BACKGROUND: The continuing emergence of influenza virus has highlighted the value of public databases and related bioinformatic analysis tools in investigating transcriptomic change caused by different influenza virus infections in human and animal models. METHODS: We collected a large amount of transcriptome research data related to influenza virus-infected human and animal models in public databases (GEO and ArrayExpress), and extracted and integrated array and metadata. The gene expression matrix was generated through strictly quality control, balance, standardization, batch correction, and gene annotation. We then analyzed gene expression in different species, virus, cells/tissues or after antibody/vaccine treatment and imported sample metadata and gene expression datasets into the database. RESULTS: Overall, maintaining careful processing and quality control, we collected 8064 samples from 103 independent datasets, and constructed a comparative transcriptomics database of influenza virus named the Flu-CED database (Influenza comparative expression database, https://flu.com-med.org.cn/). Using integrated and processed transcriptomic data, we established a user-friendly website for realizing the integration, online retrieval, visualization, and exploration of gene expression of influenza virus infection in different species and the biological functions involved in differential genes. Flu-CED can quickly query single and multi-gene expression profiles, combining different experimental conditions for comparative transcriptome analysis, identifying differentially expressed genes (DEGs) between comparison groups, and conveniently finding DEGs. CONCLUSION: Flu-CED provides data resources and tools for analyzing gene expression in human and animal models infected with influenza virus that can deepen our understanding of the mechanisms underlying disease occurrence and development, and enable prediction of key genes or therapeutic targets that can be used for medical research.

Application of combined CRISPR screening for genetic and chemical-genetic interaction profiling in Mycobacterium tuberculosis.

CRISPR screening, including CRISPR interference (CRISPRi) and CRISPR-knockout (CRISPR-KO) screening, has become a powerful technology in the genetic screening of eukaryotes. In contrast with eukaryotes, CRISPR-KO screening has not yet been applied to functional genomics studies in bacteria. Here, we constructed genome-scale CRISPR-KO and also CRISPRi libraries in Mycobacterium tuberculosis (Mtb). We first examined these libraries to identify genes essential for Mtb viability. Subsequent screening identified dozens of genes associated with resistance/susceptibility to the antitubercular drug bedaquiline (BDQ). Genetic and chemical validation of the screening results suggested that it provided a valuable resource to investigate mechanisms of action underlying the effects of BDQ and to identify chemical-genetic synergies that can be used to optimize tuberculosis therapy. In summary, our results demonstrate the potential for efficient genome-wide CRISPR-KO screening in bacteria and establish a combined CRISPR screening approach for high-throughput investigation of genetic and chemical-genetic interactions in Mtb.

The Effects of ATIR Blocker on the Severity of COVID-19 in Hypertensive Inpatients and Virulence of SARS-CoV-2 in Hypertensive hACE2 Transgenic Mice.

Angiotensin-converting enzyme 2 (ACE2) is required for the cellular entry of the severe acute respiratory syndrome coronavirus 2. ACE2, via the Ang-(1-7)-Mas-R axis, is part of the antihypertensive and cardioprotective effects of the renin-angiotensin system. We studied hospitalized COVID-19 patients with hypertension and hypertensive human(h) ACE2 transgenic mice to determine the outcome of COVID-19 with or without AT1 receptor (AT1R) blocker treatment. The severity of the illness and the levels of serum cardiac biomarkers (CK, CK-BM, cTnI), as well as the inflammation markers (IL-1, IL-6, CRP), were lesser in hypertensive COVID-19 patients treated with AT1R blockers than those treated with other antihypertensive drugs. Hypertensive hACE2 transgenic mice, pretreated with AT1R blocker, had increased ACE2 expression and SARS-CoV-2 in the kidney and heart, 1 day post-infection. We conclude that those hypertensive patients treated with AT1R blocker may be at higher risk for SARS-CoV-2 infection. However, AT1R blockers had no effect on the severity of the illness but instead may have protected COVID-19 patients from heart injury, via the ACE2-angiotensin1-7-Mas receptor axis.

Preclinical and clinical progress for HDAC as a putative target for epigenetic remodeling and functionality of immune cells.

Genetic changes are difficult to reverse; thus, epigenetic aberrations, including changes in DNA methylation, histone modifications, and noncoding RNAs, with potential reversibility, have attracted attention as pharmaceutical targets. The current paradigm is that histone deacetylases (HDACs) regulate gene expression via deacetylation of histone and nonhistone proteins or by forming corepressor complexes with transcription factors. The emergence of epigenetic tools related to HDACs can be used as diagnostic and therapeutic markers. HDAC inhibitors that block specific or a series of HDACs have proven to be a powerful therapeutic treatment for immune-related diseases. Here, we summarize the various roles of HDACs and HDAC inhibitors in the development and function of innate and adaptive immune cells and their implications for various diseases and therapies.

[Establishment of an Iron-overloaded Mouse Model with Tuberculosis and Analysis of the Iron Metabolism Index].

Objective To establish a mouse model of exogenous iron overload combined with tuberculosis(TB). Methods C57BL/6N mice were divided into negative control, low-, medium-, and high-dose iron groups and received intraperitoneal injection of iron dextran at 0, 3.75, 7.50, and 15.00 mg/dose(3 times/week for 4 weeks), respectively.After 4 weeks, the organ morphology and body weight of the mice were evaluated.The content of serum iron, ferritin, transferrin, and transferrin receptor was determined by ELISA.Heart, liver, spleen, lung, kidney, and small intestine were analyzed for tissue iron content and iron deposition pathology.Mycobacterium tuberculosis(Mtb)standard strain H37Rv was injected via tail vein to infect the mice receiving moderate-dose iron to establish an iron-overloaded mouse model of active TB.HE staining and Mtb culture were employed to analyze tuberculous lesions and bacterial loads of lung, spleen and liver tissues. Results The weight gain percentages of mice in the negative control, low-, medium-, and high-dose iron groups were 25.47%, 25.22%, 24.74%, and 21.36%, respectively, which was significantly lower in the high-dose group than in the negative control(F=17.235, P=0.027), low-dose(F=15.206, P=0.031), and medium-dose(F=11.061, P=0.036)groups.Liver had the highest iron content, followed by spleen, kidney, and small intestine.The iron content in heart and lung tissues of the low-dose group had no significant difference compared with those of the negative control group(F=19.023, P=0.715;F=23.193, P=0.902).Serum iron and ferritin in the iron-overloaded mice increased in a dose-dependent manner, while transferrin and transferrin receptor had no significant changes.HE and Prussian blue staining showed that the iron-overloaded mice had different degrees of iron deposition in tissues and high-dose iron caused liver and kidney damage.The lung(F=23.227, P=0.017), spleen(F=19.023, P=0.021), and liver(F=17.392, P=0.009)of the iron-overloaded mice with TB had a significantly shorter time of bacterial culture than those of the TB-infected mice without iron overload.The lung(F=21.012, P=0.007), spleen(F=20.173, P=0.002), and liver(F=19.091, P=0.005)of the iron-overloaded mice with TB had significantly higher bacterial loads than those of the TB-infected mice without iron overload. Conclusions The exogenous iron-overloaded mouse model with similar symptoms to patients with clinical iron overload can be established by intraperitoneal injection of medium-dose(7.50 mg/dose, 3 times/week for 4 weeks)iron dextran.Mtb injection through the tail vein can help construct a mouse model of iron overload combined with active TB.

The double-sided effects of Mycobacterium Bovis bacillus Calmette-Guérin vaccine.

Bacillus Calmette-Guérin (BCG), the only vaccine proven to be effective against tuberculosis (TB), is the most commonly used vaccine globally. In addition to its effects on mycobacterial diseases, an increasing amount of epidemiological and experimental evidence accumulated since its introduction in 1921 has shown that BCG also exerts non-specific effects against a number of diseases, such as non-mycobacterial infections, allergies and certain malignancies. Recent Corona Virus Disease 2019 (COVID-19) outbreak has put BCG, a classic vaccine with significant non-specific protection, into the spotlight again. This literature review briefly covers the diverse facets of BCG vaccine, providing new perspectives in terms of specific and non-specific protection mechanisms of this old, multifaceted, and controversial vaccine.

Correction: Genomic Polymorphism of the Pandemic A (H1N1) Influenza Viruses Correlates with Viral Replication, Virulence, and Pathogenicity In Vitro and In Vivo.

[This corrects the article DOI: 10.1371/journal.pone.0020698.].

Whole Genome and Transcriptome Sequencing of Two Multi-Drug Resistant Mycobacterium tuberculosis Strains to Facilitate Illustrating Their Virulence in vivo.

Mycobacterium tuberculosis clinical strains usually possess traits different from the laboratory strains like H37Rv, especially those clinical drug resistant strains. With whole genome and transcriptome sequencing, we depicted the feature of two multi-drug resistant Mtb strains in resistance and virulence. Compared with H37Rv, the differential expressed genes (DEGs) of the MDR strains showed featured enrichment in arginine biosynthesis, fatty acid biosynthesis, and metabolism pathway. In the subset of virulence genes, the overlapping DEGs of the MDR strains exhibited downregulation of the cluster in type VII secretion system. In the mice experiment, the MDR strains showed attenuated but distinct virulence, both in survival rate and pathology. Taken together, the whole genome and transcriptome analysis could help understand the unique feature of the MDR strains both in resistance and virulence.

Downregulation of GPR183 on infection restricts the early infection and intracellular replication of mycobacterium tuberculosis in macrophage.

GPR183/EBI2 is a key chemotactic receptor for the positioning of B cells in lymphoid organs, and also for the migration of T cells and other immune cells. Here, we demonstrate that the downregulation of GPR183 in macrophage induced during Mtb infection restrains the bacterial early infection and intracellular replication. Overexpression of GPR183 or stimulation with its natural ligand favors Mtb replication in macrophage, while treatment with its antagonist represses both Mtb early infection and intracellular replication. With mutational analysis, we find that substitution of Asp-73, Arg-83, Tyr-112, Tyr-256 abolished the promotive effect of GPR183 on Mtb early infection and replication in macrophage. In conclusion, we demonstrated that beside the known role of chemotaxis receptor, GPR183 also functions directly in the interaction between macrophage and Mtb in a cell-autonomous way.

Tuberculosis vaccine development: from classic to clinical candidates.

Bacillus Calmette-Guérin (BCG) has been in use for nearly 100 years and is the only licensed TB vaccine. While BCG provides protection against disseminated TB in infants, its protection against adult pulmonary tuberculosis (PTB) is variable. To achieve the ambitious goal of eradicating TB worldwide by 2050, there is an urgent need to develop novel TB vaccines. Currently, there are more than a dozen novel TB vaccines including prophylactic and therapeutic at different stages of clinical research. This literature review provides an overview of the clinical status of candidate TB vaccines and discusses the challenges and future development trends of novel TB vaccine research in combination with the efficacy of evaluation of TB vaccines, provides insight for the development of safer and more efficient vaccines, and may inspire new ideas for the prevention of TB.

Identification of pyrvinium pamoate as an anti-tuberculosis agent in vitro and in vivo by SOSA approach amongst known drugs.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb) bacteria, is a leading infectious cause of mortality worldwide. The emergence of drug-resistant M. tb has made control of TB more difficult. The selective optimization of side activities (SOSA) approach uses old drugs for new pharmacological targets. In the present study by using SOSA approach, we have successfully identified pyrvinium pamoate (PP) which is capable of inhibiting the growth of mycobacteria, including M. tb H37Rv, Mycobacterium smegmatis, Bacille Calmette-Guérin (BCG), M. tb H37Ra, and drug-resistant M. tb clinical isolates in vitro from 1280 known drugs library. The MIC99 of PP, the minimum inhibitory concentration that inhibits more than 99% of M. tb H37Rv and the drug-resistant M. tb clinical isolates, ranges from 1.55 to 4.8 µg/mL. Importantly, PP could reduce the bacterial colony-forming units (CFUs) in lung, spleen and liver tissues, and effectively inhibit inflammatory response in M. tb H37Rv, multidrug-resistant (MDR) M. tb and extensively drug-resistant (XDR) M.tb-infected mice. Our results clearly show that the PP has the potential application for treatment of TB.

Animal Models for Tuberculosis in Translational and Precision Medicine.

Tuberculosis (TB) is a health threat to the global population. Anti-TB drugs and vaccines are key approaches for TB prevention and control. TB animal models are basic tools for developing biomarkers of diagnosis, drugs for therapy, vaccines for prevention and researching pathogenic mechanisms for identification of targets; thus, they serve as the cornerstone of comparative medicine, translational medicine, and precision medicine. In this review, we discuss the current use of TB animal models and their problems, as well as offering perspectives on the future of these models.

Toll-Like Receptor 8 Agonist Strengthens the Protective Efficacy of ESAT-6 Immunization to Mycobacterium tuberculosis Infection.

Accumulating evidence suggests important functions for human Toll-like receptor 8 in vivo in tuberculosis and autoimmune diseases. However, these studies are limited by the lack of specific agonists and by the fact that the homology of TLR8 in human and mice is not sufficient to rely on mouse models. In this study, we examined the role of human TLR8 in the disease progression of experimental Mycobacterium tuberculosis (Mtb) infection, as well as the benefits provided by a TLR8 agonist against Mtb challenge in a human TLR8 transgenic mouse. We found that the expression of human TLR8 in C57BL/6 mice permits higher bacilli load in tissues. A vaccine formulated with ESAT-6, aluminum hydroxide, and TLR8 agonist provided protection against Mtb challenge, with a high percentage of CD44hiCD62Lhi TCM. Using ovalbumin as a model antigen, we demonstrated that the activation of TLR8 enhanced the innate and adaptive immune response, and provided a sustained TCM formation and Th1 type humoral response, which were mainly mediated by type I IFN signaling. Further research is required to optimize the vaccine formulation and seek optimal combinations of different TLR agonists, such as TLR4, for better adjuvanticity in this animal model.

Inhibition of TLR8 mediated signaling promotes BCG induced apoptosis in THP-1 cells.

Apoptosis was considered as one of the important host defense mechanisms against mycobacteria infection. In macrophage, the main target cell of Mycobacterium tuberculosis, apoptosis after infection could help kill the bacillus inside and process the antigens for further presentation and proper immune response. Here, we identified a role of TLR8 during the apoptosis induced by Bacillus Calmette Guérin (BCG) infection in THP-1 cells. Knockdown TLR8 further increased the apoptosis induced by BCG infection, and this enhanced apoptosis was caspase-dependent. During this process, Erk1/2, JNK and NFκB pathways were negatively affected and contributed to the enhanced apoptosis.

Prophylactic Use of Ganoderma lucidum Extract May Inhibit Mycobacterium tuberculosis Replication in a New Mouse Model of Spontaneous Latent Tuberculosis Infection.

A mouse model of spontaneous latent tuberculosis infection (LTBI) that mimics LTBI in humans is valuable for drug/vaccine development and the study of tuberculosis. However, most LTBI mouse models require interventions, and a spontaneous LTBI mouse model with a low bacterial load is difficult to establish. In this study, mice were IV-inoculated with 100 CFU Mycobacterium tuberculosis H37Rv, and a persistent LTBI was established with low bacterial loads (0.5~1.5log10 CFU in the lung; < 4log10 CFU in the spleen). Histopathological changes in the lung and spleen were mild during the first 20 weeks post-inoculation. The model was used to demonstrate the comparative effects of prophylactic and therapeutic administration of Ganoderma lucidum extract (spores and spores lipid) in preventing H37Rv replication in both lung and spleen. H37Rv was inhibited with prophylactic use of G. lucidum extract relative to that of the untreated control and therapy groups, and observed in the spleen and lung as early as post-inoculation week 3 and week 5 respectively. H37Rv infection in the therapy group was comparable to that of the untreated control mice. No significant mitigation of pathological changes was observed in either the prophylactic or therapeutic group. Our results suggest that this new LTBI mouse model is an efficient tool of testing anti-tuberculosis drug, the use of G. lucidum extract prior to M. tuberculosis infection may protect the host against bacterial replication to some extent.

Experimental Mycobacterium tuberculosis infection in the Chinese tree shrew.

In recent years, the Chinese tree shrew has been considered to be a promising experimental animal for numerous diseases. Yet the susceptibility of Mycobacterium tuberculosis (MTB) in Chinese tree shrew is still unknown. We infected Chinese tree shrews with a high dose (2.5 × 10(6) CFU) or a low dose (2.5 × 10(3) CFU) of the H37Rv strain via the femoral vein to cause severe or mild disease. Disease severity was determined by clinical signs, pathologic changes and bacteria distribution in organs. Furthermore, among lung samples of the uninfected, mildly and seriously ill Chinese tree shrews, differentially expressed protein profiles were analyzed through iTRAQ and validated by qPCR. Tuberculous nodules, skin ulceration, pleural effusion and cerebellum necrosis could be observed in seriously ill animals. Regulation of the actin cytoskeleton was newly defined as a possible MTB-related pathway correlated with disease progression. This comprehensive analysis of the experimental infection and the depiction of the proteomics profiles in the Chinese tree shrew provide a foundation for the establishment of a new animal model of tuberculosis and provide a better understanding of the mechanism of tuberculosis.

An optimized real-time PCR to avoid species-/tissue-associated inhibition for H5N1 detection in ferret and monkey tissues.

The real-time PCR diagnostics for avian influenza virus H5N1 in tissue specimens are often suboptimal, since naturally occurring PCR inhibitors present in samples, or unanticipated match of primer to unsequenced species' genome. With the principal aim of optimizing the SYBR Green real-time PCR method for detecting H5N1 in ferret and monkey (Chinese rhesus macaque) tissue specimens, we screened various H5N1 gene-specific primer pairs and tested their ability to sensitively and specifically detect H5N1 transcripts in the infected animal tissues, then we assessed RNA yield and quality by comparing Ct values obtained from the standard Trizol method, and four commonly used RNA isolation kits with small modifications, including Roche High Pure, Ambion RNAqueous, BioMIGA EZgene, and Qiagen RNeasy. The results indicated that a single primer pair exhibited high specificity and sensitivity for H5N1 transcripts in ferret and monkey tissues. Each of the four kits and Trizol reagent produced high-quality RNA and removed all or nearly all PCR inhibitors. No statistically significant differences were found between the Ct values from the isolation methods. So the optimized SYBR Green real-time PCR could avoid species- or tissue-associated PCR inhibition in detecting H5N1 in ferret and monkey tissues, including lung and small intestine.

Higher Immunological Protection of Pandemic 2009 H1N1 Influenza Live Virus Infection than Split Vaccine Against the Homologous Virus for Long Term Immunization in Ferret.

The study was to evaluate the long term immunological efficacy of pandemic 2009 H1N1 influenza live virus infection and split vaccine against the homologous virus challenge in ferrets. Antibodies in ferrets were monitored by haemagglutination inhibition (HI) assay for 200 days, the HI titers of both infected-only and vaccinated plus infected ferrets could maintain a high level for at least 182 days, without significant difference between the two infected groups. While one-dose and two-dose vaccinated ferrets could last a moderate antibody titers for 81 days, with its peak value at day 7 post immunization. After the virus challenge at day 207, the two groups of vaccinated ferrets shed virus for longer time than the two infected groups, while the latter two groups basically did not shed any virus particles. Furthermore, the vaccinated and infected ferrets which were sacrificed at day 211 exerted moderate immune protection against the challenge by alleviating clinical signs and lung lesion without obvious difference between groups. These data supported that both one-dose and two-dose vaccination of 2009 influenza A (H1N1) split vaccine conferred a moderate protection against challenge after 207 days, and there was no significant difference between the two groups. Either the infected only or vaccinated plus infected ones exerted more effective protective immune than one-dose and two-dose vaccination against the challenge, especially in preventing virus shedding, and vaccination primed before infection had no additional efficacy.