Discovery of Species-Specific Proteotypic Peptides To Establish a Spectral Library Platform for Identification of Nontuberculosis Mycobacteria from Mass Spectrometry-Based Proteomics.

Nontuberculous mycobacteria are opportunistic bacteria pulmonary and extra-pulmonary infections in humans that closely resemble Mycobacterium tuberculosis. Although genome sequencing strategies helped determine NTMs, a common assay for the detection of coinfection by multiple NTMs with M. tuberculosis in the primary attempt of diagnosis is still elusive. Such a lack of efficiency leads to delayed therapy, an inappropriate choice of drugs, drug resistance, disease complications, morbidity, and mortality. Although a high-resolution LC-MS/MS-based multiprotein panel assay can be developed due to its specificity and sensitivity, it needs a library of species-specific peptides as a platform. Toward this, we performed an analysis of proteomes of 9 NTM species with more than 20 million peptide spectrum matches gathered from 26 proteome data sets. Our metaproteomic analyses determined 48,172 species-specific proteotypic peptides across 9 NTMs. Notably, M. smegmatis (26,008), M. abscessus (12,442), M. vaccae (6487), M. fortuitum (1623), M. avium subsp. paratuberculosis (844), M. avium subsp. hominissuis (580), and M. marinum (112) displayed >100 species-specific proteotypic peptides. Finally, these peptides and corresponding spectra have been compiled into a spectral library, FASTA, and JSON formats for future reference and validation in clinical cohorts by the biomedical community for further translation.

The Cyclooxygenase 2 Inhibitor Etoricoxib as Adjunctive Therapy in Tuberculosis Impairs Macrophage Control of Mycobacterial Growth.

BACKGROUND: Current tuberculosis treatment regimens could be improved by adjunct host-directed therapies (HDT) targeting host responses. We investigated the antimycobacterial capacity of macrophages from patients with tuberculosis in a phase 1/2 randomized clinical trial (TBCOX2) of the cyclooxygenase-2 inhibitor etoricoxib. METHODS: Peripheral blood mononuclear cells from 15 patients with tuberculosis treated with adjunctive COX-2i and 18 controls (standard therapy) were collected on day 56 after treatment initiation. The ex vivo capacity of macrophages to control mycobacterial infection was assessed by challenge with Mycobacterium avium, using an in vitro culture model. Macrophage inflammatory responses were analyzed by gene expression signatures, and concentrations of cytokines were analyzed in supernatants by multiplex. RESULTS: Macrophages from patients receiving adjunctive COX-2i treatment had higher M. avium loads than controls after 6 days, suggesting an impaired capacity to control mycobacterial infection compared to macrophages from the control group. Macrophages from the COX-2i group had lower gene expression of TNF, IL-1B, CCL4, CXCL9, and CXCL10 and lowered production of cytokines IFN-ß and S100A8/A9 than controls. CONCLUSIONS: Our data suggest potential unfavorable effects with impaired macrophage capacity to control mycobacterial growth in patients with tuberculosis receiving COX-2i treatment. Larger clinical trials are required to analyze the safety of COX-2i as HDT in patients with tuberculosis. CLINICAL TRIALS REGISTRATION: NCT02503839.

Development of a Spectral Library for the Discovery of Altered Genomic Events in Mycobacterium avium Associated With Virulence Using Mass Spectrometry-Based Proteogenomic Analysis.

Mycobacterium avium is one of the prominent disease-causing bacteria in humans. It causes lymphadenitis, chronic and extrapulmonary, and disseminated infections in adults, children, and immunocompromised patients. M. avium has ∼4500 predicted protein-coding regions on average, which can help discover several variants at the proteome level. Many of them are potentially associated with virulence; thus, identifying such proteins can be a helpful feature in developing panel-based theranostics. In line with such a long-term goal, we carried out an in-depth proteomic analysis of M. avium with both data-dependent and data-independent acquisition methods. Further, a set of proteogenomic investigations were carried out using (i) a protein database for Mycobacterium tuberculosis, (ii) an M. avium genome six-frame-translated database, and (iii) a variant protein database of M. avium. A search of mass spectrometry data against M. avium protein database resulted in identifying 2954 proteins. Further, proteogenomic analyses aided in identifying 1301 novel peptide sequences and correcting translation start sites for 15 proteins. Ultimately, we created a spectral library of M. avium proteins, including novel genome search-specific peptides and variant peptides detected in this study. We validated the spectral library by a data-independent acquisition of the M. avium proteome. Thus, we present an M. avium spectral library of 29,033 peptide precursors supported by 0.4 million fragment ions for further use by the biomedical community.

Glycine inhibits NINJ1 membrane clustering to suppress plasma membrane rupture in cell death.

First recognized more than 30 years ago, glycine protects cells against rupture from diverse types of injury. This robust and widely observed effect has been speculated to target a late downstream process common to multiple modes of tissue injury. The molecular target of glycine that mediates cytoprotection, however, remains elusive. Here, we show that glycine works at the level of NINJ1, a newly identified executioner of plasma membrane rupture in pyroptosis, necrosis, and post-apoptosis lysis. NINJ1 is thought to cluster within the plasma membrane to cause cell rupture. We demonstrate that the execution of pyroptotic cell rupture is similar for human and mouse NINJ1 and that NINJ1 knockout functionally and morphologically phenocopies glycine cytoprotection in macrophages undergoing lytic cell death. Next, we show that glycine prevents NINJ1 clustering by either direct or indirect mechanisms. In pyroptosis, glycine preserves cellular integrity but does not affect upstream inflammasome activities or accompanying energetic cell death. By positioning NINJ1 clustering as a glycine target, our data resolve a long-standing mechanism for glycine-mediated cytoprotection. This new understanding will inform the development of cell preservation strategies to counter pathologic lytic cell death.

Pyruvate Supports RET-Dependent Mitochondrial ROS Production to Control Mycobacterium avium Infection in Human Primary Macrophages.

Macrophages deploy a variety of antimicrobial programs to contain mycobacterial infection. Upon activation, they undergo extensive metabolic reprogramming to meet an increase in energy demand, but also to support immune effector functions such as secretion of cytokines and antimicrobial activities. Here, we report that mitochondrial import of pyruvate is linked to production of mitochondrial ROS and control of Mycobacterium avium (M. avium) infection in human primary macrophages. Using chemical inhibition, targeted mass spectrometry and single cell image analysis, we showed that macrophages infected with M. avium switch to aerobic glycolysis without any major imbalances in the tricarboxylic acid cycle volume or changes in the energy charge. Instead, we found that pyruvate import contributes to hyperpolarization of mitochondria in infected cells and increases production of mitochondrial reactive oxygen species by the complex I via reverse electron transport, which reduces the macrophage burden of M. avium. While mycobacterial infections are extremely difficult to treat and notoriously resistant to antibiotics, this work stresses out that compounds specifically inducing mitochondrial reactive oxygen species could present themself as valuable adjunct treatments.

A modular map of Bradykinin-mediated inflammatory signaling network.

Bradykinin, a member of the kallikrein-kinin system (KKS), is associated with an inflammatory response pathway with diverse vascular permeability functions, including thrombosis and blood coagulation. In majority, bradykinin signals through Bradykinin Receptor B2 (B2R). B2R is a G protein-coupled receptor (GPCR) coupled to G protein family such as Gαqs, Gαq/Gα11, Gαi1, and Gß1γ2. B2R stimulation leads to the activation of a signaling cascade of downstream molecules such as phospholipases, protein kinase C, Ras/Raf-1/MAPK, and PI3K/AKT and secondary messengers such as inositol-1,4,5-trisphosphate, diacylglycerol and Ca2+ ions. These secondary messengers modulate the production of nitric oxide or prostaglandins. Bradykinin-mediated signaling is implicated in inflammation, chronic pain, vasculopathy, neuropathy, obesity, diabetes, and cancer. Despite the biomedical importance of bradykinin, a resource of bradykinin-mediated signaling pathway is currently not available. Here, we developed a pathway resource of signaling events mediated by bradykinin. By employing data mining strategies in the published literature, we describe an integrated pathway reaction map of bradykinin consisting of 233 reactions. Bradykinin signaling pathway events included 25 enzyme catalysis reactions, 12 translocations, 83 activation/inhibition reactions, 11 molecular associations, 45 protein expression and 57 gene regulation events. The pathway map is made publicly available on the WikiPathways Database with the ID URL: https://www.wikipathways.org/index.php/Pathway:WP5132 . The bradykinin-mediated signaling pathway map will facilitate the identification of novel candidates as therapeutic targets for diseases associated with dysregulated bradykinin signaling.

In Vivo Microdialysis in Mice Captures Changes in Alzheimer's Disease Cerebrospinal Fluid Biomarkers Consistent with Developing Pathology.

BACKGROUND: Preclinical models of Alzheimer's disease (AD) can provide valuable insights into the onset and progression of the disease, such as changes in concentrations of amyloid-ß (Aß) and tau in cerebrospinal fluid (CSF). However, such models are currently underutilized due to limited advancement in techniques that allow for longitudinal CSF monitoring. OBJECTIVE: An elegant way to understand the biochemical environment in the diseased brain is intracerebral microdialysis, a method that has until now been limited to short-term observations, or snapshots, of the brain microenvironment. Here we draw upon patient-based findings to characterize CSF biomarkers in a commonly used preclinical mouse model for AD. METHODS: Our modified push-pull microdialysis method was first validated ex vivo with human CSF samples, and then in vivo in an AD mouse model, permitting assessment of dynamic changes of CSF Aß and tau and allowing for better translational understanding of CSF biomarkers. RESULTS: We demonstrate that CSF biomarker changes in preclinical models capture what is observed in the brain; with a decrease in CSF Aß observed when plaques are deposited, and an increase in CSF tau once tau pathology is present in the brain parenchyma. We found that a high molecular weight cut-off membrane allowed for simultaneous sampling of Aß and tau, comparable to CSF collection by lumbar puncture in patients. CONCLUSION: Our approach can further advance AD and other neurodegenerative research by following evolving neuropathology along the disease cascade via consecutive sampling from the same animal and can additionally be used to administer pharmaceutical compounds and assess their efficacy.

The Tumor Necrosis Factor Alpha and Interleukin 6 Auto-paracrine Signaling Loop Controls Mycobacterium avium Infection via Induction of IRF1/IRG1 in Human Primary Macrophages.

Macrophages sense and respond to pathogens by induction of antimicrobial and inflammatory programs to alert other immune cells and eliminate the infectious threat. We have previously identified the transcription factor IRF1 to be consistently activated in macrophages during Mycobacterium avium infection, but its precise role during infection is not clear. Here, we show that tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) autocrine/paracrine signaling contributes to controlling the intracellular growth of M. avium in human primary macrophages through activation of IRF1 nuclear translocation and expression of IRG1, a mitochondrial enzyme that produces the antimicrobial metabolite itaconate. Small interfering RNA (siRNA)-mediated knockdown of IRF1 or IRG1 increased the mycobacterial load, whereas exogenously provided itaconate was bacteriostatic at high concentrations. While the overall level of endogenous itaconate was low in M. avium-infected macrophages, the repositioning of mitochondria to M. avium phagosomes suggests a mechanism by which itaconate can be delivered directly to M. avium phagosomes in sufficient quantities to inhibit growth. Using mRNA hybridization, we further show that uninfected bystander cells actively contribute to the resolution of infection by producing IL-6 and TNF-α, which, via paracrine signaling, activate IRF1/IRG1 and strengthen the antimicrobial activity of infected macrophages. This mechanism contributes to the understanding of why patients on anti-inflammatory treatment, e.g., with tocilizumab or infliximab, can be more susceptible to mycobacterial disease. IMPORTANCE The prevalence of lung diseases caused by nontuberculous mycobacteria, such as Mycobacterium avium, is increasing in countries where tuberculosis is not endemic, most likely because of an aging population that is immunocompromised from underlying disease or immunosuppressive therapy. Our study contributes to the understanding of mycobacterial survival and killing in human macrophages and, more broadly, to the impact of immunometabolism during infection. We show evidence of an antimicrobial program in human primary macrophages where activation of the transcription factor IRF1 and expression of the mitochondrial enzyme IRG1 restrict the intracellular growth of M. avium, possibly by directed delivery of itaconate to M. avium phagosomes. The study also sheds light on why patients on immunosuppressive therapy are more susceptible to mycobacterial infections, since TNF-α and IL-6 contribute to driving the described antimycobacterial program.

Frontline Science: Antibiotic treatment routes Mycobacterium avium to phagolysosomes without triggering proinflammatory cytokine production in human Mϕs.

Mycobacterium avium (Mav) causes chronic infections in immunocompromised patients that require long-term antibiotic treatment. We have previously shown that Mav takes residence in host Mϕs and establishes a compartment (MavC) in which it is hidden from host defenses. Failure to establish the MavC traps Mav in Lamp1+ phagolysosomes where growth is prevented, and inflammatory signaling activated through TLRs 7/8. To elucidate how antibiotic treatment affects mycobacterial trafficking and host defenses, we infected human primary Mϕs with Mav for 4 days prior to treatment with a macrolide, aminoglycoside, and ethambutol. We show that Mav is killed and the MavC fuses with Lamp1+ lysosomes following antibiotic treatment. However, this does not result in nuclear translocation of NF-κB or production of inflammatory cytokines, suggesting different Lamp1+ lysosomal compartments can form that differ in their innate signaling capabilities. Thus, we show that upon antibiotic treatment of a chronic infection, Mav is quietly disposed of by Mϕs.

Mycobacterium smegmatis Vaccine Vector Elicits CD4+ Th17 and CD8+ Tc17 T Cells With Therapeutic Potential to Infections With Mycobacterium avium.

Mycobacterium avium (Mav) complex is increasingly reported to cause non-tuberculous infections in individuals with a compromised immune system. Treatment is complicated and no vaccines are available. Previous studies have shown some potential of using genetically modified Mycobacterium smegmatis (Msm) as a vaccine vector to tuberculosis since it is non-pathogenic and thus would be tolerated by immunocompromised individuals. In this study, we used a mutant strain of Msm disrupted in EspG3, a component of the ESX-3 secretion system. Infection of macrophages and dendritic cells with Msm ΔespG3 showed increased antigen presentation compared to cells infected with wild-type Msm. Vaccination of mice with Msm ΔespG3, expressing the Mav antigen MPT64, provided equal protection against Mav infection as the tuberculosis vaccine, Mycobacterium bovis BCG. However, upon challenge with Mav, we observed a high frequency of IL-17-producing CD4+ (Th17 cells) and CD8+ (Tc17 cells) T cells in mice vaccinated with Msm ΔespG3::mpt64 that was not seen in BCG-vaccinated mice. Adoptive transfer of cells from Msm ΔespG3-vaccinated mice showed that cells from the T cell compartment contributed to protection from Mav infection. Further experiments revealed Tc17-enriched T cells did not provide prophylactic protection against subsequent Mav infection, but a therapeutic effect was observed when Tc17-enriched cells were transferred to mice already infected with Mav. These initial findings are important, as they suggest a previously unknown role of Tc17 cells in mycobacterial infections. Taken together, Msm ΔespG3 shows promise as a vaccine vector against Mav and possibly other (myco)bacterial infections.

Sensing of HIV-1 by TLR8 activates human T cells and reverses latency.

During HIV infection, cell-to-cell transmission results in endosomal uptake of the virus by target CD4+ T cells and potential exposure of the viral ssRNA genome to endosomal Toll-like receptors (TLRs). TLRs are instrumental in activating inflammatory responses in innate immune cells, but their function in adaptive immune cells is less well understood. Here we show that synthetic ligands of TLR8 boosted T cell receptor signaling, resulting in increased cytokine production and upregulation of surface activation markers. Adjuvant TLR8 stimulation, but not TLR7 or TLR9, further promoted T helper cell differentiation towards Th1 and Th17. In addition, we found that endosomal HIV induced cytokine secretion from CD4+ T cells in a TLR8-specific manner. TLR8 engagement also enhanced HIV-1 replication and potentiated the reversal of latency in patient-derived T cells. The adjuvant TLR8 activity in T cells can contribute to viral dissemination in the lymph node and low-grade inflammation in HIV patients. In addition, it can potentially be exploited for therapeutic targeting and vaccine development.

The Proteomic Landscape of Resting and Activated CD4+ T Cells Reveal Insights into Cell Differentiation and Function.

CD4+ T cells (T helper cells) are cytokine-producing adaptive immune cells that activate or regulate the responses of various immune cells. The activation and functional status of CD4+ T cells is important for adequate responses to pathogen infections but has also been associated with auto-immune disorders and survival in several cancers. In the current study, we carried out a label-free high-resolution FTMS-based proteomic profiling of resting and T cell receptor-activated (72 h) primary human CD4+ T cells from peripheral blood of healthy donors as well as SUP-T1 cells. We identified 5237 proteins, of which significant alterations in the levels of 1119 proteins were observed between resting and activated CD4+ T cells. In addition to identifying several known T-cell activation-related processes altered expression of several stimulatory/inhibitory immune checkpoint markers between resting and activated CD4+ T cells were observed. Network analysis further revealed several known and novel regulatory hubs of CD4+ T cell activation, including IFNG, IRF1, FOXP3, AURKA, and RIOK2. Comparison of primary CD4+ T cell proteomic profiles with human lymphoblastic cell lines revealed a substantial overlap, while comparison with mouse CD+ T cell data suggested interspecies proteomic differences. The current dataset will serve as a valuable resource to the scientific community to compare and analyze the CD4+ proteome.

Author Correction: Molecular basis of mycobacterial survival in macrophages.

One of the author affiliations was missed to include in the original publication. The correct information is given below.

Correction: Persistent mycobacteria evade an antibacterial program mediated by phagolysosomal TLR7/8/MyD88 in human primary macrophages.

[This corrects the article DOI: 10.1371/journal.ppat.1006551.].

Persistent mycobacteria evade an antibacterial program mediated by phagolysosomal TLR7/8/MyD88 in human primary macrophages.

Pathogenic mycobacteria reside in macrophages where they avoid lysosomal targeting and degradation through poorly understood mechanisms proposed to involve arrest of phagosomal maturation at an early endosomal stage. A clear understanding of how this relates to host defenses elicited from various intracellular compartments is also missing and can only be studied using techniques allowing single cell and subcellular analyses. Using confocal imaging of human primary macrophages infected with Mycobacterium avium (Mav) we show evidence that Mav phagosomes are not arrested at an early endosomal stage, but mature to a (LAMP1+/LAMP2+/CD63+) late endosomal/phagolysosomal stage where inflammatory signaling and Mav growth restriction is initiated through a mechanism involving Toll-like receptors (TLR) 7 and 8, the adaptor MyD88 and transcription factors NF-κB and IRF-1. Furthermore, a fraction of the mycobacteria re-establish in a less hostile compartment (LAMP1-/LAMP2-/CD63-) where they not only evade destruction, but also recognition by TLRs, growth restriction and inflammatory host responses that could be detrimental for intracellular survival and establishment of chronic infections.

N-3 PUFAs induce inflammatory tolerance by formation of KEAP1-containing SQSTM1/p62-bodies and activation of NFE2L2.

Inflammation is crucial in the defense against infections but must be tightly controlled to limit detrimental hyperactivation. Our diet influences inflammatory processes and omega-3 polyunsaturated fatty acids (n-3 PUFAs) have known anti-inflammatory effects. The balance of pro- and anti-inflammatory processes is coordinated by macrophages and macroautophagy/autophagy has recently emerged as a cellular process that dampens inflammation. Here we report that the n-3 PUFA docosahexaenoic acid (DHA) transiently induces cytosolic speckles of the autophagic receptor SQSTM1/p62 (sequestosome 1) (described as SQSTM1/p62-bodies) in macrophages. We suggest that the formation of SQSTM1/p62-bodies represents a fast mechanism of NFE2L2/Nrf2 (nuclear factor, erythroid 2 like 2) activation by recruitment of KEAP1 (kelch like ECH associated protein 1). Further, the autophagy receptor TAX1BP1 (Tax1 binding protein 1) and ubiquitin-editing enzyme TNFAIP3/A20 (TNF α induced protein 3) could be identified in DHA-induced SQSTM1/p62-bodies. Simultaneously, DHA strongly dampened the induction of pro-inflammatory genes including CXCL10 (C-X-C motif chemokine ligand 10) and we suggest that formation of SQSTM1/p62-bodies and activation of NFE2L2 leads to tolerance towards selective inflammatory stimuli. Finally, reduced CXCL10 levels were related to the improved clinical outcome in n-3 PUFA-supplemented heart-transplant patients and we propose CXCL10 as a robust marker for the clinical benefits mobilized by n-3 PUFA supplementation.

Fecal neutrophil gelatinase-associated lipocalin as a biomarker for inflammatory bowel disease.

BACKGROUND AND AIM: Accurate, noninvasive biomarkers are needed to diagnose and monitor inflammatory bowel disease (IBD). Neutrophil gelatinase-associated lipocalin (NGAL), also known as lipocalin 2, is expressed in inflamed colonic epithelium and neutrophilic granulocytes. This study explores its properties as a biomarker in feces and plasma and, for the first time, compares fecal NGAL systematically with the existing fecal biomarker calprotectin. METHODS: Neutrophil gelatinase-associated lipocalin was measured in feces from 73 patients with IBD, 21 patients with infectious enterocolitis, 21 patients with irritable bowel syndrome, and 23 healthy subjects using ELISA. The results were correlated to calprotectin, clinical score, endoscopic score, and high-sensitive C-reactive protein. Plasma from 119 patients with IBD and 28 healthy controls was analyzed for NGAL. RESULTS: Fecal NGAL levels (median and interquartile range) were significantly elevated in active ulcerative colitis (UC) 6.05 (3.6-15.1) mg/kg and Crohn's disease (CD) 4.9 (1.5-7.7) mg/kg, compared with patients with inactive UC 1.3 (0.4-2.6) mg/kg, inactive CD 1.5 (0.5-1.7) mg/kg, irritable bowel syndrome 0.4 (0.2-0.6) mg/kg, and healthy controls (HC) 0.3 (0.1-0.4) mg/kg. Patients with infectious enterocolitis had significantly higher fecal-NGAL levels, 2.7 (1.4-5.6) mg/kg than HC. Sensitivity and specificity was 94.7% and 95.7%, respectively, for distinguishing between active IBD and HC. Stability of NGAL in stool was excellent for 7 days in room temperature. Plasma NGAL was significantly elevated in UC and CD compared with HC. CONCLUSIONS: Fecal NGAL is a promising biomarker for IBD. As existing biomarkers are expressed mainly in granulocytes, NGAL's epithelial localization may give supplementary diagnostic information.

Molecular basis of mycobacterial survival in macrophages.

Macrophages play an essential role in the immune system by ingesting and degrading invading pathogens, initiating an inflammatory response and instructing adaptive immune cells, and resolving inflammation to restore homeostasis. More interesting is the fact that some bacteria have evolved to use macrophages as a natural habitat and tools of spread in the host, e.g., Mycobacterium tuberculosis (Mtb) and some non-tuberculous mycobacteria (NTM). Mtb is considered one of humanity's most successful pathogens and is the causal agent of tuberculosis, while NTMs cause opportunistic infections all of which are of significant public health concern. Here, we describe mechanisms by which intracellular pathogens, with an emphasis on mycobacteria, manipulate macrophage functions to circumvent killing and live inside these cells even under considerable immunological pressure. Such macrophage functions include the selective evasion or engagement of pattern recognition receptors, production of cytokines, reactive oxygen and nitrogen species, phagosome maturation, as well as other killing mechanisms like autophagy and cell death. A clear understanding of host responses elicited by a specific pathogen and strategies employed by the microbe to evade or exploit these is of significant importance for the development of effective vaccines and targeted immunotherapy against persistent intracellular infections like tuberculosis.

Seeing a Mycobacterium-Infected Cell in Nanoscale 3D: Correlative Imaging by Light Microscopy and FIB/SEM Tomography.

Mycobacteria pose a threat to the world health today, with pathogenic and opportunistic bacteria causing tuberculosis and non-tuberculous disease in large parts of the population. Much is still unknown about the interplay between bacteria and host during infection and disease, and more research is needed to meet the challenge of drug resistance and inefficient vaccines. This work establishes a reliable and reproducible method for performing correlative imaging of human macrophages infected with mycobacteria at an ultra-high resolution and in 3D. Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) tomography is applied, together with confocal fluorescence microscopy for localization of appropriately infected cells. The method is based on an Aclar poly(chloro-tri-fluoro)ethylene substrate, micropatterned into an advantageous geometry by a simple thermomoulding process. The platform increases the throughput and quality of FIB/SEM tomography analyses, and was successfully applied to detail the intracellular environment of a whole mycobacterium-infected macrophage in 3D.

Keap1 regulates inflammatory signaling in Mycobacterium avium-infected human macrophages.

Several mechanisms are involved in controlling intracellular survival of pathogenic mycobacteria in host macrophages, but how these mechanisms are regulated remains poorly understood. We report a role for Kelch-like ECH-associated protein 1 (Keap1), an oxidative stress sensor, in regulating inflammation induced by infection with Mycobacterium avium in human primary macrophages. By using confocal microscopy, we found that Keap1 associated with mycobacterial phagosomes in a time-dependent manner, whereas siRNA-mediated knockdown of Keap1 increased M. avium-induced expression of inflammatory cytokines and type I interferons (IFNs). We show evidence of a mechanism whereby Keap1, as part of an E3 ubiquitin ligase complex with Cul3 and Rbx1, facilitates ubiquitination and degradation of IκB kinase (IKK)-ß thus terminating IKK activity. Keap1 knockdown led to increased nuclear translocation of transcription factors NF-κB, IFN regulatory factor (IRF) 1, and IRF5 driving the expression of inflammatory cytokines and IFN-ß. Furthermore, knockdown of other members of the Cul3 ubiquitin ligase complex also led to increased cytokine expression, further implicating this ligase complex in the regulation of the IKK family. Finally, increased inflammatory responses in Keap1-silenced cells contributed to decreased intracellular growth of M. avium in primary human macrophages that was reconstituted with inhibitors of IKKß or TANK-binding kinase 1 (TBK1). Taken together, we propose that Keap1 acts as a negative regulator for the control of inflammatory signaling in M. avium-infected human primary macrophages. Although this might be important to avoid sustained or overwhelming inflammation, our data suggest that a negative consequence could be facilitated growth of pathogens like M. avium inside macrophages.