USP12 promotes antiviral responses by deubiquitinating and stabilizing IFI16.

Deubiquitinating enzymes (DUBs) regulate antiviral immune response through targeting DNA sensor signaling pathway members. As one of the DNA sensors, interferon (IFN)-γ inducible protein 16 (IFI16) play a major role in response to virus infections through activating the canonical STING/TBK-1/IRF3 signaling pathway. Only a few studies discuss the function of DUBs in IFI16-mediated antiviral response. Ubiquitin-specific protease 12 (USP12), which is one of the major members of the USP family, participates in various biological functions. However, whether USP12 regulates the nucleic acid sensor to modulate antiviral immune responses has not yet been elucidated. In this study, we found that knockout or knockdown of USP12 impaired the HSV-1-induced expressions of IFN-ß, CCL-5, IL-6, and downstream interferon-stimulated genes (ISGs). Moreover, USP12 deficiency increased HSV-1 replication and host susceptibility to HSV-1 infection. Mechanistically, USP12 inhibited the proteasome-dependent degradation of IFI16 through its deubiquitinase activity, thereby maintaining IFI16 stability and promoting IFI16-STING-IRF3- and p65-mediated antiviral signaling. Overall, our findings demonstrate an essential role of USP12 in DNA-sensing signaling and contribute to the understanding of deubiquitination-mediated regulation of innate antiviral responses.

Correction: USP12 promotes antiviral responses by deubiquitinating and stabilizing IFI16.

[This corrects the article DOI: 10.1371/journal.ppat.1011480.].

NLRC3 expression in dendritic cells attenuates CD4+ T cell response and autoimmunity.

NOD-like receptor (NLR) family CARD domain containing 3 (NLRC3), an intracellular member of NLR family, is a negative regulator of inflammatory signaling pathways in innate and adaptive immune cells. Previous reports have shown that NLRC3 is expressed in dendritic cells (DCs). However, the role of NLRC3 in DC activation and immunogenicity is unclear. In the present study, we find that NLRC3 attenuates the antigen-presenting function of DCs and their ability to activate and polarize CD4+ T cells into Th1 and Th17 subsets. Loss of NLRC3 promotes pathogenic Th1 and Th17 responses and enhanced experimental autoimmune encephalomyelitis (EAE) development. NLRC3 negatively regulates the antigen-presenting function of DCs via p38 signaling pathway. Vaccination with NLRC3-overexpressed DCs reduces EAE progression. Our findings support that NLRC3 serves as a potential target for treating adaptive immune responses driving multiple sclerosis and other autoimmune disorders.

Viperin deficiency promotes dendritic cell activation and function via NF-kappaB activation during Mycobacterium tuberculosis infection.

OBJECTIVES AND DESIGN: Dendritic cells (DCs) are one of the key immune cells in bridging innate and adaptive immune response against Mycobacterium tuberculosis (Mtb) infection. Interferons (IFNs) play important roles in regulating DC activation and function. Virus-inhibitory protein, endoplasmic reticulum-associated, interferon-inducible (Viperin) is one of the important IFN-stimulated genes (ISGs), and elicits host defense against infection. METHODS: We investigated the effects and mechanisms of Viperin on DC activation and function using Viperin deficient bone marrow-derived dendritic cells (BMDCs) during Mtb infection. RESULTS: Viperin deficiency enhanced phagocytic activity and increased clearance of Mtb in DCs, produced higher abundance of NO, cytokine including interleukin-12 (IL-12), Tumor necrosis factor-α (TNF-α), IL-1ß, IL-6 and chemokine including CXCL1, CXCL2 and CXCL10, elevated MHC I, MHC II and co-stimulatory molecules expression, and enhanced CD4+ and CD8+ T cell responses. Mechanistically, Viperin deficiency promoted DC activation and function through NF-κB p65 activation. NF-κB p65 inhibitor prevented cytokine and chemokine production, and co-stimulatory molecules expression promoted by Viperin deficiency. CONCLUSIONS: These results suggest that Mtb induced Viperin expression could impair the activation of host defense function of DCs and DC-T cell cross talk during Mtb infection. This research may provide a potential target for future HDT in TB therapy.

ß-Arrestin 2 Regulates Inflammatory Responses against Mycobacterium tuberculosis Infection through ERK1/2 Signaling.

Mycobacterium tuberculosis, the pathogen that causes tuberculosis, exhibits complex host-pathogen interactions. Pattern recognition receptors and their downstream signaling pathways play crucial roles in determining the outcome of infection. In particular, the scaffold protein ß-arrestin 2 mediates downstream signaling of G protein-coupled receptors. However, the role of ß-arrestin 2 in conferring immunity against M. tuberculosis has not yet been explored. We found that ß-arrestin 2 was upregulated in the lesioned regions of lung tissues in patients with tuberculosis. M. tuberculosis infection upregulated ß-arrestin 2 expression in human macrophages, and silencing of ß-arrestin 2 significantly enhanced bactericidal activity by enhancing the expression of proinflammatory cytokines such as TNF-α. ß-Arrestin 2 was shown to inhibit the activation of the TLR2/ERK1/2 pathway and its transcriptional regulation activity upon M. tuberculosis infection. Furthermore, ß-arrestin 2 transcriptionally regulates TNF-α by binding to CREB1. These observations revealed that the upregulation of ß-arrestin 2 is critical for M. tuberculosis to escape immune surveillance through an unknown mechanism. Our research offers a novel interference modality to enhance the immune response against tuberculosis by targeting ß-arrestin 2 to modulate the TLR2-ß-arrestin 2-ERK1/2-CREB1-TNF-α regulatory axis.

USP12 positively regulates M-MDSC function to inhibit antitumour immunity through deubiquitinating and stabilizing p65.

The relative abundance of myeloid-derived suppressor cells (MDSCs) compared to cytotoxic T cells determines the outcomes of diseases and the efficacy of immunotherapy. Ubiquitin-specific peptidase 12 (USP12), a member of the USP family of deubiquitinases, targets multiple signalling pathways and regulates diverse biological processes, including cell proliferation and survival. It is well known that ubiquitylation is an important mechanism for regulating the immune response. However, it is unclear whether USP12 regulates tumour growth by influencing MDSCs. In the present study, we reported that USP12 deficiency decreased infiltration and impaired the suppressor function of monocytic (M)-MDSCs, resulting in increased CD8+ T-cell response and decelerated tumour growth. USP12-knockout M-MDSCs were less potent in inhibiting the proliferation of CD8+ T cells and their ability to secrete IFN-γ. Furthermore, USP12 deficiency inhibited the suppressor function of M-MDSCs by downregulating the negative regulatory molecules inducible nitric oxide synthase and PD-L1, through deubiquitinating and stabilizing p65. Our results suggest that USP12 is a positive regulator of M-MDSCs and may serve as a potential target for antitumor therapy.

Pam2 lipopeptides enhance the immunosuppressive activity of monocytic myeloid-derived suppressor cells by STAT3 signal in chronic inflammation.

Chronic inflammation develops when the immune system is unable to clear a persistent insult. Unresolved chronic inflammation leads to immunosuppression to maintain the internal homeostatic conditions, which is mediated primarily by myeloid-derived suppressor cells (MDSCs). Toll-like receptors 2 (TLR2) has an important role in chronic inflammation and can be activated by a vast number and diversity of TLR2 ligands, for example Pam2CSK4. However, the regulatory effect of TLR2 signaling on MDSCs in chronic inflammation remains controversial. This study demonstrated that heat-killed Mycobacterium bovis BCG-induced pathology-free chronic inflammation triggered suppressive monocytic MDSCs (M-MDSCs) that expressed TLR2. Activation of TLR2 signaling by Pam2CSK4 treatment enhanced immunosuppression of M-MDSCs by upregulating inducible nitric oxide synthase (iNOS) activity and nitric oxide (NO) production partly through signal transducer and activator of transcription 3 (STAT3) activation. Thus, TLR2 has a fundamental role in promoting the MDSC-mediated immunosuppressive environment during chronic inflammation and might represent a potentially therapeutic target in chronic inflammation disease.

IL-36γ Promotes Killing of Mycobacterium tuberculosis by Macrophages via WNT5A-Induced Noncanonical WNT Signaling.

Mycobacterium tuberculosis, which primarily infects mononuclear phagocytes, remains the leading bacterial cause of enormous morbidity and mortality because of bacterial infections in humans throughout the world. The IL-1 family of cytokines is critical for host resistance to M. tuberculosis As a newly discovered subgroup of the IL-1 family, although IL-36 cytokines have been proven to play roles in protection against M. tuberculosis infection, the antibacterial mechanisms are poorly understood. In this study, we demonstrated that IL-36γ conferred to human monocyte-derived macrophages bacterial resistance through activation of autophagy as well as induction of WNT5A, a reported downstream effector of IL-1 involved in several inflammatory diseases. Further studies showed that WNT5A could enhance autophagy of monocyte-derived macrophages by inducing cyclooxygenase-2 (COX-2) expression and in turn decrease phosphorylation of AKT/mTOR via noncanonical WNT signaling. Consistently, the underlying molecular mechanisms of IL-36γ function are also mediated by the COX-2/AKT/mTOR signaling axis. Altogether, our findings reveal a novel activity for IL-36γ as an inducer of autophagy, which represents a critical inflammatory cytokine that control the outcome of M. tuberculosis infection in human macrophages.

Vitamin B6 prevents excessive inflammation by reducing accumulation of sphingosine-1-phosphate in a sphingosine-1-phosphate lyase-dependent manner.

Vitamin B6 is necessary to maintain normal metabolism and immune response, especially the anti-inflammatory immune response. However, the exact mechanism by which vitamin B6 plays the anti-inflammatory role is still unclear. Here, we report a novel mechanism of preventing excessive inflammation by vitamin B6 via reduction in the accumulation of sphingosine-1-phosphate (S1P) in a S1P lyase (SPL)-dependent manner in macrophages. Vitamin B6 supplementation decreased the expression of pro-inflammatory cytokines by suppressing nuclear factor-κB and mitogen-activated protein kinases signalling pathways. Furthermore, vitamin B6-reduced accumulation of S1P by promoting SPL activity. The anti-inflammatory effects of vitamin B6 were inhibited by S1P supplementation or SPL deficiency. Importantly, vitamin B6 supplementation protected mice from lethal endotoxic shock and attenuated experimental autoimmune encephalomyelitis progression. Collectively, these findings revealed a novel anti-inflammatory mechanism of vitamin B6 and provided guidance on its clinical use.

NLRC3 negatively regulates CD4+ T cells and impacts protective immunity during Mycobacterium tuberculosis infection.

NLRC3, a member of the NLR family, has been reported as a negative regulator of inflammatory signaling pathways in innate immune cells. However, the direct role of NLRC3 in modulation of CD4+ T-cell responses in infectious diseases has not been studied. In the present study, we showed that NLRC3 plays an intrinsic role by suppressing the CD4+ T cell phenotype in lung and spleen, including differentiation, activation, and proliferation. NLRC3 deficiency in CD4+ T cells enhanced the protective immune response against Mycobacterium tuberculosis infection. Finally, we demonstrated that NLRC3 deficiency promoted the activation, proliferation, and cytokine production of CD4+ T cells via negatively regulating the NF-κB and MEK-ERK signaling pathways. This study reveals a critical role of NLRC3 as a direct regulator of the adaptive immune response and its protective effects on immunity during M. tuberculosis infection. Our findings also suggested that NLRC3 serves as a potential target for therapeutic intervention against tuberculosis.

Different Signaling Pathways Define Different Interferon-Stimulated Gene Expression during Mycobacteria Infection in Macrophages.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) represents one of the greatest threats to human health., Interferons (IFNs) in combination with the first-line of anti-TB drugs have been used for treating TB for decades in the clinic, but how Mtb infection regulates interferon-stimulated genes (ISGs) in human macrophages (Mϕs) remains unknown. In this study, we investigated the expression-signature and associated innate signaling mechanisms of ISGs in Mtb-infected human monocyte-derived Mϕs (hMDMs) and THP-1-derived Mϕs (THP-1-Mϕs). Among 28 of the detected ISGs, 90% of them exerted a significant increase in Mtb-infected Mϕs. Additionally, we found that cytosolic cyclic (GMP-AMP) synthase (cGAS), toll-like receptor-2 (TLR-2) and TLR-4 signaling pathways participated in ISG induction. Their downstream elements of TANK-binding kinase 1 (TBK1), nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and Janus kinase-signal transducer and activator of transcription (JAK-STAT) were selectively involved in Mtb-mediated ISG production. Finally, the numerous types of ISG expression in hMDMs of TB patients were more susceptible to restimulation of Mtb infection or/and IFN treatment than that of healthy people. Hence, different signaling pathways define different ISG expression during Mtb infection and this helps to illustrate how ISGs are elucidated and to better understand the host immune responses to Mtb infection in Mϕs.

Novel Function of Cyclooxygenase-2: Suppressing Mycobacteria by Promoting Autophagy via the Protein Kinase B/Mammalian Target of Rapamycin Pathway.

In Mycobacterium tuberculosis-infected macrophages, cyclooxygenase-2 (COX-2) expression considerably increases to defend the body against mycobacteria by regulating adaptive immunity and restoring the mitochondrial inner membrane. Moreover, in cancer cells, COX-2 enhances the autophagy machinery, an important bactericidal mechanism. However, the association between M. tuberculosis-induced COX-2 and autophagy-mediated antimycobacterial response has not been explored. Here, COX-2 expression silencing reduced the autophagy and bactericidal activity against intracellular M. tuberculosis, while COX-2 overexpression reversed the above effects. In addition, enhancement of bactericidal activity was suppressed by inhibiting autophagy in COX-2-overexpressing cells, indicating that COX-2 accelerated mycobacterial elimination by promoting autophagy. Furthermore, the regulatory effects of COX-2 on autophagy were mediated by its catalytic products, which functioned through inhibiting the protein kinase B/mammalian target of rapamycin pathway. Thus, COX-2 contributes to host defense against mycobacterial infection by promoting autophagy, establishing the basis for development of novel therapeutic agents against tuberculosis by targeting COX-2.

The Multiplicity of Infection-Dependent Effects of Recombinant Adenovirus Carrying HGF Gene on the Proliferation and Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells.

Absence of effective therapeutic methods for avascular necrosis of femoral head (ANFH) is still perplexing the world's medical community. Bone marrow mesenchymal stem cells (BMSCs) adoptive cell therapy combined with core decompression is a promising modality, which is highly dependent on the cellular activities of BMSCs. Hepatocyte growth factor (HGF) is a survival factor for BMSCs, yet the underlying mechanism is not fully elucidated. In this study, the effects of multiplicity of infections (MOIs) of recombinant adenovirus carrying HGF gene (rAd-HGF) on human BMSC proliferation and osteogenic differentiation were systemically examined. Infection of rAd-HGF produced secretory HGF and promoted hBMSC proliferation in a MOI-dependent manner, while the osteogenesis was also strengthened as indicated by enhanced calcium nodule formation with the strongest effects achieved at MOI = 250. Blocking the activities of c-MET or its downstream signaling pathways, WNT, ERK1/2, and PI3K/AKT led to differential consequents. Specifically, blockage of the WNT pathway significantly promoted osteogenic differentiation, which also showed additive effects when combined application with rAd-HGF. Our data demonstrated the pro-osteogenic effects of optimized MOIs of rAd-HGF, while inhibition of WNT pathway or activation of PI3K/AKT pathway may act as candidate adjuvant modalities for promoting osteogenic differentiation in rAd-HGF-modified hBMSC treatment on ANFH.

Development of reverse-transcription loop-mediated isothermal amplification assay for rapid detection and differentiation of dengue virus serotypes 1-4.

BACKGROUND: Dengue virus (DENV), the most widely prevalent arbovirus, continues to be a threat to human health in the tropics and subtropics. Early and rapid detection of DENV infection during the acute phase of illness is crucial for proper clinical patient management and preventing the spread of infection. The aim of the current study was to develop a specific, sensitive, and robust reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for detection and differentiation of DENV1-4 serotypes. RESULTS: The method detection primers, which were designed to target the different DENV serotypes, were identified by inspection of multiple sequence alignments of the non-structural protein (NS) 2A of DENV1, NS4B of DENV2, NS4A of DENV3 and the 3' untranslated region of the NS protein of DENV4. No cross-reactions of the four serotypes were observed during the tests. The detection limits of the DENV1-4-specific RT-LAMP assays were approximately 10-copy templates per reaction. The RT-LAMP assays were ten-fold more sensitive than RT-PCR or real-time PCR. The diagnostic rate was 100% for clinical strains of DENV, and 98.9% of the DENV-infected patients whose samples were tested were detected by RT-LAMP. Importantly, no false-positives were detected with the new equipment and methodology that was used to avoid aerosol contamination of the samples. CONCLUSION: The RT-LAMP method used in our study is specific, sensitive, and suitable for further investigation as a useful alternative to the current methods used for clinical diagnosis of DENV1-4, especially in hospitals and laboratories that lack sophisticated diagnostic systems.

TREM-2 Drives Development of Multiple Sclerosis by Promoting Pathogenic Th17 Polarization.

Multiple sclerosis (MS) is a neuroinflammatory demyelinating disease, mediated by pathogenic T helper 17 (Th17) cells. However, the therapeutic effect is accompanied by the fluctuation of the proportion and function of Th17 cells, which prompted us to find the key regulator of Th17 differentiation in MS. Here, we demonstrated that the triggering receptor expressed on myeloid cells 2 (TREM-2), a modulator of pattern recognition receptors on innate immune cells, was highly expressed on pathogenic CD4-positive T lymphocyte (CD4+ T) cells in both patients with MS and experimental autoimmune encephalomyelitis (EAE) mouse models. Conditional knockout of Trem-2 in CD4+ T cells significantly alleviated the disease activity and reduced Th17 cell infiltration, activation, differentiation, and inflammatory cytokine production and secretion in EAE mice. Furthermore, with Trem-2 knockout in vivo experiments and in vitro inhibitor assays, the TREM-2/zeta-chain associated protein kinase 70 (ZAP70)/signal transducer and activator of transcription 3 (STAT3) signal axis was essential for Th17 activation and differentiation in EAE progression. In conclusion, TREM-2 is a key regulator of pathogenic Th17 in EAE mice, and this sheds new light on the potential of this therapeutic target for MS.

ZNFX1 promotes AMPK-mediated autophagy against Mycobacterium tuberculosis by stabilizing Prkaa2 mRNA.

Tuberculosis has the highest mortality rate worldwide for a chronic infectious disease caused by a single pathogen. RNA-binding proteins (RBPs) are involved in autophagy - a key defense mechanism against Mycobacterium tuberculosis (M. tuberculosis) infection - by modulating RNA stability and forming intricate regulatory networks. However, the functions of host RBPs during M. tuberculosis infection remain relatively unexplored. Zinc finger NFX1-type containing 1 (ZNFX1), a conserved RBP critically involved in immune deficiency diseases and mycobacterial infections, is significantly upregulated in M. tuberculosis-infected macrophages. Here, we aimed to explore the immunoregulatory functions of ZNFX1 during M. tuberculosis infection. We observed that Znfx1 knockout markedly compromised the multifaceted immune responses mediated by macrophages. This compromise resulted in reduced phagocytosis, suppressed macrophage activation, increased M. tuberculosis burden, progressive lung tissue injury, and chronic inflammation in M. tuberculosis-infected mice. Mechanistic investigations revealed that the absence of ZNFX1 inhibited autophagy, consequently mediating immune suppression. ZNFX1 critically maintained AMPK-regulated autophagic flux by stabilizing protein kinase AMP-activated catalytic subunit alpha 2 mRNA, which encodes a key catalytic α subunit of AMPK, through its zinc finger region. This process contributed to M. tuberculosis growth suppression. These findings reveal a function of ZNFX1 in establishing anti-M. tuberculosis immune responses, enhancing our understanding of the roles of RBPs in tuberculosis immunity and providing a promising approach to bolster antituberculosis immunotherapy.

RNF157 attenuates CD4+ T cell-mediated autoimmune response by promoting HDAC1 ubiquitination and degradation.

Background: CD4+ T cells play an important role in body development and homeostasis. Quantitative and functional changes in CD4+ T cells result in abnormal immune responses, which lead to inflammation, cancer, or autoimmune diseases, such as multiple sclerosis (MS). Ubiquitination plays an essential role in the differentiation and functioning of CD4+ T cells. However, the function of several E3 ubiquitin ligases in CD4+ T cell differentiation and T cell-mediated pathological diseases remains unclear. Methods: RNA sequencing data were analyzed to identify the E3 ubiquitin ligases that participate in the pathogenesis of MS. Furthermore, conditional knockout mice were generated. Specifically, flow cytometry, qPCR, western blot, CO-IP and cell transfer adoptive experiments were performed. Results: In this study, we identified The RING finger 157 (RNF157) as a vital regulator of CD4+ T cell differentiation; it promoted Th1 differentiation but attenuated Th17 differentiation and CCR4 and CXCR3 expressions in CD4+ T cells, thereby limiting experimental autoimmune encephalomyelitis development. Mechanistically, RNF157 in CD4+ T cells targeted HDAC1 for K48-linked ubiquitination and degradation. Notably, RNF157 expression was significantly decreased and showed a significant negative correlation with RORγt expression in patients with MS. Conclusions: Our study highlights the critical role of RNF157 in regulating CD4+ T cell functions in autoimmune diseases and suggests RNF157 as a potential target in adaptive immune responses against MS and other autoimmune disorders.

USP1-regulated reciprocal differentiation of Th17 cells and Treg cells by deubiquitinating and stabilizing TAZ.

The balance between inflammatory T helper type 17 (Th17) and immunosuppressive regulatory T (Treg) cells is critical for maintaining immune homeostasis in the human body and is tightly regulated under healthy conditions. An increasing number of studies have reported that deubiquitinases (DUBs) play a vital role in regulating Th17- and Treg-cell differentiation. However, the biological functions of only a small fraction of DUBs in Th17- and Treg-cell differentiation are well defined. In this study, we identified ubiquitin-specific peptidase 1 (USP1) as a vital regulator of CD4+ T-cell differentiation. USP1 promoted Th17-cell differentiation but attenuated Treg-cell differentiation, thereby promoting the development of inflammatory diseases. Mechanistically, USP1 in CD4+ T cells enhanced the activity of RORγt but promoted the proteasomal degradation of Foxp3 through deubiquitination and stabilization of TAZ in vitro and in vivo. Notably, ML323, a specific inhibitor of the USP1/UAF1 deubiquitinase complex, inhibited Th17-cell differentiation and promoted Treg-cell differentiation in vitro and in vivo, indicating that ML323 might be a promising candidate for the treatment of diseases associated with an imbalance between Th17 and Treg cells. Our study highlights the critical role of USP1 in regulating adaptive immune responses and suggests that USP1 might be a drug target for the treatment of diseases associated with an imbalance between Th17 and Treg cells.

Functional analysis of the antigen binding sites on the MTB/HIV-1 peptide bispecific T-cell receptor complementarity determining region 3α.

OBJECTIVE: Mycobacterium tuberculosis /human immunodeficiency virus (MTB/HIV) coinfection has become an urgent problem in the field of prevention and control of infectious diseases in recent years. Adoptive cellular immunotherapy using antigen-specific T-cell receptor (TCR) engineered T cells which recognize the specific antigen artificially may have tremendous potential in anti-MTB/HIV coinfection. We have previously successfully identified a MTB Ag85B 199-207 and HIV-1 Env 120-128 peptide-bispecific TCR screened out from peripheral blood mononuclear cells of a HLA-A∗0201 + healthy individual and have further studied that how residues on the predicted complementarity determining region (CDR) 3 of the ß chain contribute to the bispecific TCR contact with the peptide-MHC. However, it is not clear which amino acids in the predicted CDR3α of the bispecific TCR play a crucial role in ligand recognition. METHODS: The variants in the CDR3α of the bispecific TCR were generated using alanine substitution. We then evaluated the immune effects of the five variants on T-cell recognition upon encounter with the MTB or HIV-1 antigen. RESULTS: Mutation of two amino acids (E112A, Y115A) in CDR3α of the bispecific TCR caused a markedly diminished T-cell response to antigen, whereas mutation of the other three amino acids (S113A, P114A, S116A) resulted in completely eliminated response. CONCLUSION: This study demonstrates that Ser 113 , Pro 114 and Ser 116 in CDR3α of the bispecific TCR are especially important for antigen recognition. These results will pave the way for the future development of an improved high-affinity bispecific TCR for use in adoptive cellular immunotherapy for MTB/HIV coinfected patients.

Cisatracurium besylate rescues Mycobacterium Tuberculosis-infected macrophages from necroptosis and enhances the bactericidal effect of isoniazid.

OBJECTIVE: Tuberculosis is the leading killer among the chronic single-source infectious diseases. Mycobacterium tuberculosis can induce necrotic-dominant multiple modes of cell death in macrophages, which accelerates bacterium dissemination and expands tissue injury in host lungs. Mining drugs to counteract Mycobacterium tuberculosis-induced cell death would be beneficial to tuberculosis patients. METHODS: In this study, the protective drug was screened out from the FDA-approved drug library in Mycobacterium tuberculosis-infected macrophages with CCK-8 assay. The death mode regulated by the drug was identified using transcriptomic sequencing, cytomorphological observation, and in the experimental mouse Mycobacterium tuberculosis-infection model. The functional mechanism was explored using western blot, co-immunoprecipitation, and DARTS assay. The intracellular bacterial survival was detected using colony forming unit assays. RESULTS: Cisatracurium besylate was identified to be highly protective for the viability of macrophages during Mycobacterium tuberculosis infection via inhibiting necroptosis. Cisatracurium besylate prevented RIPK3 to be associated with the executive molecule MLKL for forming the necroptotic complex, resulting in the inhibition of MLKL phosphorylation and pore formation on cell membrane. However, Cisatracurium besylate did not interfere with the association between RIPK3 with its upstream kinase RIPK1 or ZBP1 but regulated RIPK3 autophosphorylation. Moreover, Cisatracurium besylate significantly inhibited the expansion of intracellular Mycobacterium tuberculosis both in vitro and in vivo, which also displayed a strong auxiliary bacteriostatic effect to support the therapeutic efficacy of isoniazid and rifampicin, the first-line anti-tubercular drugs. CONCLUSION: Cisatracurium besylate performs anti-Mycobacterium tuberculosis and anti-necroptotic roles, which potentiates its application to be an adjuvant drug for antituberculosis therapy to assist the battle against drug-resistant tuberculosis.