Armcx1 Reduces Neurological Damage Via a Mitochondrial Transport Pathway Involving Miro1 After Traumatic Brain Injury.

Armcx1 is a member of the ARMadillo repeat-Containing protein on the X chromosome (ARMCX) family, which is recognized to have evolutionary conserved roles in regulating mitochondrial transport and dynamics. Previous research has shown that Armcx1 is expressed at higher levels in mice after axotomy and in adult retinal ganglion cells after crush injury, and this protein increases neuronal survival and axonal regeneration. However, its role in traumatic brain injury (TBI) is unclear. Therefore, the aim of this study was to assess the expression of Armcx1 after TBI and to explore possible related mechanisms by which Armcx1 is involved in TBI. We used C57BL/6 male mice to model TBI and evaluated the role of Armcx1 in TBI by transfecting mice with Armcx1 small interfering RNA (siRNA) to inhibit Armcx1 expression 24 h before TBI modeling. Western blotting, immunofluorescence, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, Nissl staining, transmission electron microscopy, adenosine triphosphate (ATP) level measurement, neuronal apoptosis analysis, neurological function scoring and the Morris water maze were performed. The results demonstrated that Armcx1 protein expression was elevated after TBI and that the Armcx1 protein was localized in neurons and astroglial cells in cortical tissue surrounding the injury site. In addition, inhibition of Armcx1 expression further led to impaired mitochondrial transport, abnormal morphology, reduced ATP levels, aggravation of neuronal apoptosis and neurological dysfunction, and decrease Miro1 expression. In conclusion, our findings indicate that Armcx1 may exert neuroprotective effects by ameliorating neurological injury after TBI through a mitochondrial transport pathway involving Miro1.

Macrophage targeted iron oxide nanodecoys augment innate immunological and drug killings for more effective Mycobacterium Tuberculosis clearance.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) infection, is still one of the top killers worldwide among infectious diseases. The escape of Mtb from immunological clearance and the low targeting effects of anti-TB drugs remain the substantial challenges for TB control. Iron is particularly required for Mtb growth but also toxic for Mtb in high dosages, which makes iron an ideal toxic decoy for the 'iron-tropic' Mtb. Here, a macrophage-targeted iron oxide nanoparticles (IONPs)-derived IONPs-PAA-PEG-MAN nanodecoy is designed to augment innate immunological and drug killings against intracellular Mtb. IONPs-PAA-PEG-MAN nanodecoy exhibits preferential uptake in macrophages to significantly increase drug uptake with sustained high drug contents in host cells. Moreover, it can serve as a specific nanodecoy for the 'iron-tropic' Mtb to realize the localization of Mtb contained phagosomes surrounding the drug encapsulated nanodecoys and co-localization of Mtb with the drug encapsulated nanodecoys in lysosomes, where the incorporated rifampicin (Rif) can be readily released under acidic lysosomal condition for enhanced Mtb killing. This drug encapsulated nanodecoy can also polarize Mtb infected macrophages into anti-mycobacterial M1 phenotype and enhance M1 macrophage associated pro-inflammatory cytokine (TNF-α) production to trigger innate immunological responses against Mtb. Collectively, Rif@IONPs-PAA-PEG-MAN nanodecoy can synergistically enhance the killing efficiency of intracellular Mtb in in vitro macrophages and ex vivo monocyte-derived macrophages, and also significantly reduce the mycobacterial burdens in the lung of infected mice with alleviated pathology. These results indicate that Rif@IONPs-PAA-PEG-MAN nanodecoy may have a potential for the development of more effective therapeutic strategy against TB by manipulating augmented innate immunity and drug killings.

Circular RNA circTRAPPC6B Enhances IL-6 and IL-1ß Expression and Repolarizes Mycobacteria Induced Macrophages from M2- to M1-Like Phenotype by Targeting miR-892c-3p.

Mycobacterium tuberculosis (Mtb) infection elicits macrophage polarization into M2 phenotype to block the host's protective immune response. However, it remains unclear how Mtb regulates macrophage polarization. Recent studies have suggested that noncoding RNA may play a role in macrophage polarization. In this study, we investigated the potential involvement of circTRAPPC6B, a circular RNA that is downregulated in tuberculosis (TB) patients, in regulating macrophage polarization. We found that Mtb infection downregulated M1-related IL-6 and IL-1ß while highly expressed M2-related CCL22 and CD163. Overexpressed circTRAPPC6B had switched Mtb-infected macrophages from M2- to M1-like phenotype, accompanied by upregulation of IL-6 and IL-1ß. Meanwhile overexpressed circTRAPPC6B significantly inhibited Mtb growth in macrophages. Our findings suggest that circTRAPPC6B may regulate macrophage polarization by targeting miR-892c-3p, which is highly expressed in TB patients and M2-like macrophages. And miR-892c-3p inhibitor decreased intracellular Mtb growth in macrophages. Thus, TB-inhibited circTRAPPC6B could specifically induce IL-6 and IL-1ß expression to switch/antagonize Mtb-induced macrophage polarization from M2- to M1-like phenotype by targeting miR-892c-3p, leading to enhanced host clearance of Mtb. Our results reveal a potential role for circTRAPPC6B in regulating macrophage polarization during Mtb infection and provide new insights into the molecular mechanisms underlying host defense against Mtb.

Dietary palmitoleic acid reprograms gut microbiota and improves biological therapy against colitis.

Magnitude and diversity of gut microbiota and metabolic systems are critical in shaping human health and diseases, but it remains largely unclear how complex metabolites may selectively regulate gut microbiota and determine health and diseases. Here, we show that failures or compromised effects of anti-TNF-α therapy in inflammatory bowel diseases (IBD) patients were correlated with intestinal dysbacteriosis with more pro-inflammatory bacteria, extensive unresolved inflammation, failed mucosal repairment, and aberrant lipid metabolism, particularly lower levels of palmitoleic acid (POA). Dietary POA repaired gut mucosal barriers, reduced inflammatory cell infiltrations and expressions of TNF-α and IL-6, and improved efficacy of anti-TNF-α therapy in both acute and chronic IBD mouse models. Ex vivo treatment with POA in cultured inflamed colon tissues derived from Crohn's disease (CD) patients reduced pro-inflammatory signaling/cytokines and conferred appreciable tissue repairment. Mechanistically, POA significantly upregulated the transcriptional signatures of cell division and biosynthetic process of Akkermansia muciniphila, selectively increased the growth and abundance of Akkermansia muciniphila in gut microbiota, and further reprogrammed the composition and structures of gut microbiota. Oral transfer of such POA-reprogrammed, but not control, gut microbiota induced better protection against colitis in anti-TNF-α mAb-treated recipient mice, and co-administration of POA with Akkermansia muciniphila showed significant synergistic protections against colitis in mice. Collectively, this work not only reveals the critical importance of POA as a polyfunctional molecular force to shape the magnitude and diversity of gut microbiota and therefore promote the intestinal homeostasis, but also implicates a new potential therapeutic strategy against intestinal or abenteric inflammatory diseases.

Structural insights of the elongation factor EF-Tu complexes in protein translation of Mycobacterium tuberculosis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is the second-deadliest infectious disease worldwide. Emerging evidence shows that the elongation factor EF-Tu could be an excellent target for treating Mtb infection. Here, we report the crystal structures of Mtb EF-Tu•EF-Ts and EF-Tu•GDP complexes, showing the molecular basis of EF-Tu's representative recycling and inactive forms in protein translation. Mtb EF-Tu binds with EF-Ts at a 1:1 ratio in solution and crystal packing. Mutation and SAXS analysis show that EF-Ts residues Arg13, Asn82, and His149 are indispensable for the EF-Tu/EF-Ts complex formation. The GDP binding pocket of EF-Tu dramatically changes conformations upon binding with EF-Ts, sharing a similar GDP-exchange mechanism in E. coli and T. ther. Also, the FDA-approved drug Osimertinib inhibits the growth of M. smegmatis, H37Ra, and M. bovis BCG strains by directly binding with EF-Tu. Thus, our work reveals the structural basis of Mtb EF-Tu in polypeptide synthesis and may provide a promising candidate for TB treatment.

MIR337-3p Enhances Mycobacterial Pathogenicity Involving TLR4/MYD88 and STAT3 Signals, Impairing VDR Antimicrobial Response and Fast-Acting Immunity.

Active form of vitamin D (VitD) enhances human innate immunity against Mycobacterium tuberculosis (Mtb) infection. Our previous studies showed that MIR337-3p was highly expressed in lymphocytes of tuberculosis (TB) patients. Here, we identified the mechanism of MIR337-3p in the regulation of fast-acting anti-TB immunity by inhibiting VitD-dependent antimicrobial response pathways. While high-level MIR337-3p expression was induced by mycobacterial infection in cellular models and mice, TB patients exhibited significantly increased MIR337-3p in CD14+ monocytes/macrophages, innate-like Vγ2+ T cells, and CD8+ lymphocytes containing natural killer (NK)/innate lymphoid cells. MIR337-3p promoted the mycobacterial entry/infection and replication/growth in host target cells: macrophages and lung epithelial cells. Such MIR337-3p-enhanced pathogenicity coincided with the MIR337-3p depression of VitD-dependent antimicrobial response of cytochrome P450, family 27, subfamily b, polypeptide 1 (CYP27B1)/Beta-defensin 4 (DEFB4A)/ cathelicidin antimicrobial peptide CAMP pathways. Surprisingly, single MIR337-3p species could specifically target both the Toll-like receptor 4 (TLR4) and signal transducer and activator of transcription 3 (STAT3) 3'-untranslated regions (UTRs) to depress the TLR4/MYD88 and STAT3 signals and impair either of the two signals inhibiting the VitD-dependent antimicrobial pathways in macrophages. Concurrently, human peripheral blood mononuclear cells (PBMCs) expressing high-level MIR337-3p exhibited a reduced ability of innate cell populations to mount fast-acting cellular immunity against intracellular mycobacterial infection. Furthermore, a higher expression of Mir337-3p after mycobacterial infection of mice coincided with much greater colony-forming unit (CFU) counts in lungs and even the death of infected animals, whereas Mir337-3p inhibitor treatment of infected mice reduced Mir337-3p levels and reversed Mir337-3p-mediated increases in CFU counts. Thus, TB-driven single MIR337-3p species could specifically target/impair both TLR4/MYD88 and STAT3 activation signals, inhibiting VitD-dependent antimicrobial response and fast-acting anti-TB immunity, leading to enhanced pathogenicity.

A CD4+CD161+ T-Cell Subset Present in Unexposed Humans, Not Tb Patients, Are Fast Acting Cells That Inhibit the Growth of Intracellular Mycobacteria Involving CD161 Pathway, Perforin, and IFN-γ/Autophagy.

It remains undefined whether a subset of CD4+ T cells can function as fast-acting cells to control Mycobacterium tuberculosis (Mtb) infection. Here we show that the primary CD4+CD161+ T-cell subset, not CD4+CD161-, in unexposed healthy humans fast acted as unconventional T cells capable of inhibiting intracellular Mtb and BCG growth upon exposure to infected autologous and allogeneic macrophages or lung epithelial A549 cells. Such inhibition coincided with the ability of primary CD4+CD161+ T cells to rapidly express/secrete anti-TB cytokines including IFN-γ, TNF-α, IL-17, and perforin upon exposure to Mtb. Mechanistically, blockades of CD161 pathway, perforin or IFN-γ by blocking mAbs abrogated the ability of CD4+CD161+ T cells to inhibit intracellular mycobacterial growth. Pre-treatment of infected macrophages with inhibitors of autophagy also blocked the CD4+CD161+ T cell-mediated growth inhibition of mycobacteria. Furthermore, adoptive transfer of human CD4+CD161+ T cells conferred protective immunity against mycobacterial infection in SCID mice. Surprisingly, CD4+CD161+ T cells in TB patients exhibited a loss or reduction of their capabilities to produce perforin/IFN-γ and to inhibit intracellular growth of mycobacteria in infected macrophages. These immune dysfunctions were consistent with PD1/Tim3 up-regulation on CD4+CD161+ T cells in active tuberculosis patients, and the blockade of PD1/Tim3 on this subset cells enhanced the inhibition of intracellular mycobacteria survival. Thus, these findings suggest that a fast-acting primary CD4+CD161+T-cell subset in unexposed humans employs the CD161 pathway, perforin, and IFN-γ/autophagy to inhibit the growth of intracellular mycobacteria, thereby distinguishing them from the slow adaptive responses of conventional CD4+ T cells. The presence of fast-acting CD4+CD161+ T-cell that inhibit mycobacterial growth in unexposed humans but not TB patients also implicates the role of these cells in protective immunity against initial Mtb infection.

Construction of Lycetin Nanocarriers and Its Effect on the Proliferation and Apoptosis of Hepatocellular Carcinoma Cells by Regulating Nuclear Factor E2 Related Factor/Antioxidant Response Element Pathway.

This article explores the role of lysin nanocarriers in inducing apoptosis of human hepatocellular carcinoma cells and the possible molecular mechanisms. Cytotoxicity tests were performed in human fibroblast cell line MRC-5. Anti-cancer activity was tested in liver cancer cell lines HepG2 and HCCLM3. The results show that nanocarriers have a targeting effect on cancer cells, have high safety, and are good delivery vehicles for drugs. In this paper, the stability of lycopene and its degradation in aqueous solutions at different temperatures were studied, and the structure and mechanism of degradation products were determined. A new type of mesoporous silica nanocarrier was synthesized as a delivery carrier of lysin and its derivatives, which has a targeting effect on cancer cells and has a slow-release effect. Surface modification can improve circulation time and stability for future resistance in vivo. The cancer experiment laid the foundation. The results showed that the lysin nanocarriers inhibited the proliferation of HepG2 and HCCLM3 human liver cancer cells in a dependent manner. After the lysin nanocarriers acted on HepG2 human hepatocellular carcinoma cells for 48 h, the cell apoptosis rate was significantly increased by flow cytometry analysis. The carrier can significantly increase the levels of reactive oxygen species and malondialdehyde, and reduce the content of reduced glutathione and superoxide dismutase. At the same time, the lysin nanocarrier can down-regulate the expression of Nrf2 and HO-1 proteins, and inhibit the occurrence of Nrf2 Nuclear displacement. The lycopene nanocarrier inhibits the proliferation of HepG2, HCCLM3 human liver cancer cells, induces apoptosis, regulates the oxidative stress response in the cell, and regulates the Nrf2/AREE antioxidant signaling pathway, thereby promoting tumor cell apoptosis.

Long-term efficacy and advantages of minimally invasive hepatectomy for hepatolithiasis: A protocol for systematic review and meta-analysis.

BACKGROUND: Hepatolithiasis commonly occurs in the bile duct proximal to the confluence of the right and left hepatic ducts, regardless of the coexistence of gallstones in gallbladder or the common bile duct. Clinical research proves that minimally invasive surgery is effective in the treatment of hepatolithiasis. Although previous meta-analysis also shows that it could reduce intraoperative bleeding and blood transfusion, and shorten hospital stay time, there are few meta-analyses on its long-term efficacy. We conducted the meta-analysis and systematic review to systematically evaluate the long-term efficacy and advantages of minimally invasive hepatectomy in the treatment of hepatolithiasis. METHODS: Articles of randomized controlled trials will be searched in the PubMed, Medline, Embase, Cochrane Library, China National Knowledge Infrastructure, Chongqing VIP Chinese Science and Technology Periodical Database, Chinese Biological and Medical database, and Wanfang database until September, 2020. Literature extraction and risk of bias assessment will be completed by 2 reviewers independently. Statistical analysis will be conducted in RevMan 5.3. RESULTS: This study will summarize the present evidence by exploring the long-term efficacy and advantages of minimally invasive hepatectomy in the treatment of hepatolithiasis CONCLUSIONS:: The findings of the study will help to determine potential long-term efficacy and advantages of minimally invasive hepatectomy in the treatment of hepatolithiasis. ETHICS AND DISSEMINATION: The private information from individuals will not be published. This systematic review also will not involve endangering participant rights. Ethical approval is not required. The results may be published in a peer-reviewed journal or disseminated in relevant conferences. OSF REGISTRATION NUMBER: DOI 10.17605/OSF.IO/H6WRV.

Saikosaponin a attenuates hyperlipidemic pancreatitis in rats via the PPAR-γ/NF-κB signaling pathway.

The therapeutic effect of saikosaponin a (SSa) on hyperlipidemic pancreatitis (HP) is not completely understood. The aim of the present study was to investigate the therapeutic effect and the underlying mechanism of SSa using a rat model of HP. Following successful establishment of the HP rat model, different doses of SSa (low dose group, 10 mg/kg or high dose group, 20 mg/kg) were administrated. Histopathological examination, the wet/dry (W/D) ratio and myeloperoxidase (MPO) activity of the pancreatic tissues were assessed. The lipid, amylase (AMY), lipase and proinflammatory cytokine profiles in serum, as well as the expression of peroxisome proliferator-activated receptor (PPAR)-γ and the NF-κB signaling pathway-related proteins in pancreatic tissues were evaluated. The results showed that SSa effectively attenuated pancreatic pathological injury and reduced both the W/D ratio and MPO activity compared to the HP model rats. SSa also improved lipid metabolism by significantly decreasing the serum levels of total cholesterol and triglycerides (P<0.05). Following the administration of SSa, the activity of AMY and lipase, as well as the levels of the proinflammatory cytokines tumor necrosis factor-α, interleukin (IL)-1ß and IL-6 were reduced, particularly in the high dosage group (P<0.05). Furthermore, SSa activated PPAR-γ expression and suppressed the NF-κB signaling pathway in pancreatic tissues. The present study suggested that SSa attenuated HP in rats by increasing lipid metabolism and inhibiting the release of proinflammatory cytokines via the NF-κB inflammatory pathway. The results from the present study indicated that SSa might be a promising therapeutic agent for the treatment of HP.

Macrophage-Targeted Isoniazid-Selenium Nanoparticles Promote Antimicrobial Immunity and Synergize Bactericidal Destruction of Tuberculosis Bacilli.

Pathogenesis hallmarks for tuberculosis (TB) are the Mycobacterium tuberculosis (Mtb) escape from phagolysosomal destruction and limited drug delivery into infected cells. Several nanomaterials can be entrapped in lysosomes, but the development of functional nanomaterials to promote phagolysosomal Mtb clearance remains a big challenge. Here, we report on the bactericidal effects of selenium nanoparticles (Se NPs) against Mtb and further introduce a novel nanomaterial-assisted anti-TB strategy manipulating Ison@Man-Se NPs for synergistic drug-induced and phagolysosomal destruction of Mtb. Ison@Man-Se NPs preferentially entered macrophages and accumulated in lysosomes releasing Isoniazid. Surprisingly, Ison@Man-Se/Man-Se NPs further promoted the fusion of Mtb into lysosomes for synergistic lysosomal and Isoniazid destruction of Mtb. Concurrently, Ison@Man-Se/Man-Se NPs also induced autophagy sequestration of Mtb, evolving into lysosome-associated autophagosomal Mtb degradation linked to ROS-mitochondrial and PI3K/Akt/mTOR signaling pathways. This novel nanomaterial-assisted anti-TB strategy manipulating antimicrobial immunity and Mtb clearance may potentially serve in more effective therapeutics against TB and drug-resistant TB.

Histone deacetylase inhibitors impair the host immune response against Mycobacterium tuberculosis infection.

Histone deacetylase inhibitors (HDACi), a novel class of anti-cancer drug, have been recently reported to suppress host immunity and increase susceptibility to infection. Tuberculosis, a leading infectious disease killer caused by Mycobacterium tuberculosis (M.tb), is basically the product of the interaction between bacterial virulence and host resistance. However, the effects of HDACi in host immunity against M.tb is largely unknown. In this study, we found that HDACi including Trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) significantly impaired phagocytosis and killing activity of macrophage. In line with these findings, we noted that M.tb induced reactive oxygen species (ROS) production and autophagy are significantly suppressed by TSA. Transcriptome analysis revealed that the suppression of autophagy by TSA might due to its inhibiting autophagy-regulating genes such as CACNA2D3, which regulates intracellular Ca2+ levels. Finally, we confirmed that HDACi including TSA and SAHA significantly exacerbated the histopathological damage and M.tb load in the lung of M.tb infected mice. Taken together, our results indicated that HDACi at least TSA and SAHA significantly impaired macrophage immunity against M.tb and therefore increase susceptibility to TB, our findings raised the concern that the potential side effects of HDACi on latent TB reactivation should be considered in clinic.

Mannosylated graphene oxide as macrophage-targeted delivery system for enhanced intracellular M.tuberculosis killing efficiency.

Tuberculosis (TB), caused by M.tuberculosis (Mtb), has become a top killer among infectious diseases. Enhancing the ability of anti-TB drugs to kill intracellular Mtb in host cells remains a big challenge. Here, an innovative nano-system was developed to increase drug delivery and Mtb-killing efficacy in Mtb-infected macrophages. We employed mannose surface decoration to develop mannosylated and PEGylated graphene oxide (GO-PEG-MAN). Such nano-platform exhibited increased uptake by macrophages via mannose receptor-mediated endocytosis in vitro. Interestingly, drug-loaded GO-PEG-MAN was preferentially up-taken by mannose receptor-expressing mucosal CD14+ macrophages isolated from Mtb-infected rhesus macaques than drug-loaded GO-PEG. Consistently, the drug concentration was also significantly higher in macrophages than that in T and B cells expressing no or low mannose receptor, implicating a useful macrophage/mannose receptor-targeted drug-delivery system relevant to the in vivo settings. Concurrently, rifampicin-loaded GO-PEG-MAN (Rif@GO-PEG-MAN) significantly increased rifampicin uptake, inducing long-lasting higher concentration of rifampicin in macrophages. Such innovative Rif@GO-PEG-MAN could readily get into the lysosomes of the Mtb host cells, where rifampicin underwent an accelerated release in acidic lysosomic condition, leading to explosive rifampicin release after cell entry for more effective killing of intracellular Mtb. Most importantly, Rif@GO-PEG-MAN-enhanced intracellular rifampicin delivery and pharmacokinetics significantly increased the efficacy of rifampicin-driven killing of intracellular BCG and Mtb bacilli in infected macrophages both in vitro and ex vivo. Such innovative nanocarrier approach may potentially enhance anti-TB drug efficacy and reduce drug side effects.

["Multi-component-multi-target-multi-pathway" mechanism of Kuihua Hugan Tablets based on network pharmacology].

To predict the targets of active ingredients of Kuihua Hugan Tablets by network pharmacology, and explore the "multi-component-multi-target-multi-pathway" hepatoprotective mechanism of action. First, through traditional Chinese medicine systems pharmacology(TCMSP) and TCM Database@Taiwan Database, main active ingredients of Kuihua Hugan Tablets were screened out based on oral bioavailability(OB), drug-likeness(DL) and effective half-lives(HL). The targets of active ingredients of Kuihua Hugan Tablets were predicted based on the PharmMapper method. Then, the prediction was conducted by screening the target genes associated with chronic hepatitis and early cirrhosis through CooLGeN and GeneCards databases. Target gene functions and signal pathways were analyzed by bioinformatics annotation database Metascape. Cytoscape software was used to construct the Kuihua Hugan Tablets ingredient-target and ingredient-target-pathway network. String database combined with Cytoscape software was used to construct the networks of component-target and component-target-pathway. STRING database was combined with Cytoscape software to draw protein-protein interaction(PPI) network and conduct network topology analysis. Finally, Systems Dock Web Site software was applied in verifying the molecular docking between active ingredients and potential protein targets. A total of 26 compounds and 509 potential targets were screened out from Kuihua Hugan Tablets in the experiment. The results of PPI network analysis indicated that albumin(ALB), insulin-like growth factor 1(IGF1), matrix metalloproteinase-9(MMP9), matrix metalloproteinase-2(MMP2), non-receptor tyrosine kinase proto-oncogene(SRC), estrogen receptor 1(ESR1) and cancer-signal transduction-inflammation-drugs metabolism-related biological processes and metabolic pathways were closely associated with the active ingredients in Kuihua Hugan Tablets. The effects of Kuihua Hugan Tablets in alleviating chronic hepatitis and early cirrhosis indicated the multi-component, multi-target, and multi-pathway characteristics of traditional Chinese medicines, providing new ideas for further research and development of Kuihua Hugan Tablets.

IL-12 Expands and Differentiates Human Vγ2Vδ2 T Effector Cells Producing Antimicrobial Cytokines and Inhibiting Intracellular Mycobacterial Growth.

While IL-12 plays a key role in differentiation of protective CD4+ Th1 response, little is known about mechanisms whereby IL-12 differentiates other T-cell populations. Published studies suggest that predominant Vγ2Vδ2 T cells in humans/nonhuman primates (NHP) are a fast-acting T-cell subset, with capacities to rapidly expand and produce Th1 and cytotoxic cytokines in response to phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) produced by Mycobacterium tuberculosis (Mtb) or others. However, whether IL-12 signaling pathway mediates fast-acting and Th1 or anti-microbial features of Vγ2Vδ2 T cells remains poorly defined. Here, we show that IL-12, but not other IL-12 family members IL-27/IL-35, apparently expanded HMBPP-activated Vγ2Vδ2 T cells. Although IL-12 and IL-2 similarly expanded HMBPP-activated Vγ2Vδ2 T-cell clones, the IL-12-induced expansion did not require endogenous IL-2 or IL-2 co-signaling during HMBPP + IL-12 co-treatment. IL-12-induced expansion of Vγ2Vδ2 T cells required the PI3K/AKT and STAT4 activation pathways and endogenous TNF-α signaling but did not involve p38/MAPK or IFN-γ signals. IL-12-expanded Vγ2Vδ2 T cells exhibited central/effector memory phenotypes and differentiated into polyfunctional effector cell subtypes which expressed TBX21/T-bet, antimicrobial cytokines IFN-γ, TNF-α, GM-CSF, and cytotoxic granule molecules. Furthermore, the IL-12-expanded Vγ2Vδ2 T cells inhibited the growth of intracellular mycobacteria in IFN-γ- or TNF-α-dependent fashion. Our findings support the concept that IL-12 drives early development of fast-acting Vγ2Vδ2 T effector cells in antimicrobial immune responses.

Multidrug-resistant tuberculosis (MDR-TB) strain infection in macaques results in high bacilli burdens in airways, driving broad innate/adaptive immune responses.

Tuberculosis (TB) has become the most deadly infectious diseases due to epidemics of HIV/AIDS and multidrug-resistant/extensively drug-resistant TB (MDR-/XDR-TB). Although person-to-person transmission contributes to MDR-TB, it remains unknown whether infection with MDR strains resembles infection with drug-sensitive (DS) TB strains, manipulating limited or broad immune responses. To address these questions, macaques were infected with MDR strain V791 and a drug-sensitive Erdman strain of TB. MDR bacilli burdens in the airway were significantly higher than those of the Erdman control after pulmonary exposure. This productive MDR strain infection upregulated the expression of caspase 3 in macrophages/monocytes and induced appreciable innate-like effector responses of CD3-negative lymphocytes and Ag-specific γδ T-cell subsets. Concurrently, MDR strain infection induced broad immune responses of T-cell subpopulations producing Th1, Th17, Th22, and CTL cytokines. Furthermore, MDR bacilli, like the Erdman strain, were capable of inducing typical TB disease characterized by weight loss, lymphocytopenia, and severe TB lesions. For the first time, our results suggest that MDR-TB infection acts like DS to induce high bacterial burdens in the airway (transmission advantage), innate/adaptive immune responses, and disease processes. Because nonhuman primates are biologically closer to humans than other species, our data may provide useful information for predicting the effects of primary MDR strain infection after person-to-person transmission. The findings also support the hypothesis that a vaccine or host-directed adjunctive modality that is effective for drug-sensitive TB is likely to also impact MDR-TB.

Characteristics of peripheral Vγ2Vδ2 T cells in interferon-γ release assay negative pulmonary tuberculosis patients.

BACKGROUND: It is not fully explained why some active tuberculosis patients show negative interferon-γ release assays (IGRAs). In this study, we tried to explore associations of IGRAs with the characteristics of peripheral Vγ2Vδ2 T cells and their functions of producing cytokines. METHODS: 32 pulmonary tuberculosis patients were enrolled and divided into two groups according to their IGRAs results: 16 with IGRA-negative as test group and 16 with IGRA-positive as control group. Chest X-rays and T-SPOT.TB tests were performed and the severity of the lung lesions was scored. The amount of Vγ2Vδ2T cell and their expression levels of the apoptosis-related membrane surface molecule Fas and FasL in peripheral blood were analyzed by flow cytometry, and the function of secreting cytokines (IFN-γ, TNF-α and IL-17A) of Vγ2Vδ2 T cell were determined by intracellular cytokine staining. RESULTS: The IGRA-negative TB patients had more lesion severity scores and displayed reduced peripheral blood Vγ2Vδ2 T cell counts (p = 0.009) as well as higher Fas and FasL expression in peripheral blood Vγ2Vδ2 T cells (p = 0.043, 0.026). A high lesion severity score was correlated with a decreased Vδ2+ T cell number and increased Vγ2Vδ2 T cells Fas/FasL expression leve in the peripheral blood (p = 0.00, P < 0.01). The function of secreting cytokines was slightly impaired in IGRA-negative TB patients (p = 0.402). There is no significant differences in expression levels of Fas and FasL in CD4+ T cells (p = 0.224, 0.287) or CD8+ T cells (p = 0.184, 0.067) between test and control groups. CONCLUSION: Compared with IGRA-positive TB patients, the IGRA-negative TB patients had more lesion severity scores, the number of Vγ2Vδ2 T cells decreased and the function of secreting cytokines impaired. In addition, we suggest that increased expression of Fas/FasL triggers Vγ2Vδ2 T cell apoptosis.

Assessment of recombinant plasmid expressing fusion antigen Ag85B-Rv3425 in management of acute tuberculosis infection in mice.

The emergence of drug-resistant tuberculosis (TB) and HIV-TB co-infection fuels an urgent need to develop novel therapeutic approaches, including therapeutic vaccines. Therapeutic vaccines have been proven to be a good strategy by inducing antigen specific immune responses against TB infection. In the present study, a recombinant plasmid based on lentiviral vector expressing fusion antigen Ag85B-Rv3425 (A3), and was constructed the immunogenicity and treatment effects in TB mice were assessed. The results showed that A3 delivered by the plasmid could be expressed appropriately in vivo and induced higher production of tumor necrosis factor-α and interleukin-2 compared with A3 recombinant protein in mice. Moreover, the recombinant plasmid expressing A3 confered resistance to acute TB infection in mice, characterized by a reduction in the bacterial load in the lungs and spleen, as well as attenuated TB lesions in lung tissues. These results implicated that the recombinant plasmid based on lentiviral vector expressing A3 is a potent and promising therapeutic agent to treat acute TB infection.

IL-12+IL-18 Cosignaling in Human Macrophages and Lung Epithelial Cells Activates Cathelicidin and Autophagy, Inhibiting Intracellular Mycobacterial Growth.

The ability of Mycobacterium tuberculosis to block host antimicrobial responses in infected cells provides a key mechanism for disease pathogenesis. The immune system has evolved to overcome this blockade to restrict the infection, but it is not clear whether two key innate cytokines (IL-12/IL-18) involved in host defense can enhance antimycobacterial mechanisms. In this study, we demonstrated that the combination of IL-12 and IL-18 triggered an antimicrobial response against mycobacteria in infected macrophages (THP-1 and human primary monocyte-derived macrophages) and pulmonary epithelial A549 cells. The inhibition of intracellular bacterial growth required p38-MAPK and STAT4 pathways, the vitamin D receptor, the vitamin D receptor-derived antimicrobial peptide cathelicidin, and autophagy, but not caspase-mediated apoptosis. Finally, the ability of IL-12+IL-18 to activate an innate antimicrobial response in human primary macrophages was dependent on the autonomous production of IFN-γ and the CAMP/autophagy pathway. Together, these data suggest that IL-12+IL-18 cosignaling can trigger the antimicrobial protein cathelicidin and autophagy, resulting in inhibition of intracellular mycobacteria in macrophages and lung epithelial cells.

Deficiency of the AIM2-ASC Signal Uncovers the STING-Driven Overreactive Response of Type I IFN and Reciprocal Depression of Protective IFN-γ Immunity in Mycobacterial Infection.

The nucleic acids of Mycobacterium tuberculosis can be detected by intracellular DNA sensors, such as cyclic GMP-AMP synthase and absent in melanoma 2 (AIM2), which results in the release of type I IFN and the proinflammatory cytokine IL-1ß. However, whether cross-talk occurs between AIM2-IL-1ß and cyclic GMP-AMP synthase-type I IFN signaling upon M. tuberculosis infection in vivo is unclear. In this article, we demonstrate that mycobacterial infection of AIM2-/- mice reciprocally induces overreactive IFN-ß and depressive IFN-γ responses, leading to higher infection burdens and more severe pathology. We also describe the underlying mechanism whereby activated apoptosis-associated speck-like protein interacts with a key adaptor, known as stimulator of IFN genes (STING), and inhibits the interaction between STING and downstream TANK-binding kinase 1 in bone marrow-derived macrophages and bone marrow-derived dendritic cells, consequently reducing the induction of type I IFN. Of note, apoptosis-associated speck-like protein expression is inversely correlated with IFN-ß levels in PBMCs from tuberculosis patients. These data demonstrate that the AIM2-IL-1ß signaling pathway negatively regulates the STING-type I IFN signaling pathway by impeding the association between STING and TANK-binding kinase 1, which protects the host from M. tuberculosis infection. This finding has potential clinical significance.