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Manganese (Mn) is an essential micronutrient critical for the pathogenesis of , a significant cause of human morbidity and mortality. Paradoxically, excess Mn is toxic; therefore, maintenance of intracellular Mn homeostasis is required for survival. Here we describe a Mn exporter in , MntE, which is a member of the cation diffusion facilitator (CDF) protein family and conserved among Gram-positive pathogens. Upregulation of transcription in response to excess Mn is dependent on the presence of MntR, a transcriptional repressor of the Mn uptake system. Inactivation of or leads to reduced growth in media supplemented with Mn, demonstrating MntE is required for detoxification of excess Mn. Inactivation of results in elevated levels of intracellular Mn, but reduced intracellular iron (Fe) levels, supporting the hypothesis that MntE functions as a Mn efflux pump and Mn efflux influences Fe homeostasis. Strains inactivated for are more sensitive to the oxidants NaOCl and paraquat, indicating Mn homeostasis is critical for resisting oxidative stress. Furthermore, and are required for full virulence of during infection, suggesting experiences Mn toxicity Combined, these data support a model in which MntR controls Mn homeostasis by balancing transcriptional repression of and induction of , both of which are critical for pathogenesis. Thus, Mn efflux contributes to bacterial survival and virulence during infection, establishing MntE as a potential antimicrobial target and expanding our understanding of Mn homeostasis. Manganese (Mn) is generally viewed as a critical nutrient that is beneficial to pathogenic bacteria due to its function as an enzymatic cofactor and its capability of acting as an antioxidant; yet paradoxically, high concentrations of this transition metal can be toxic. In this work, we demonstrate utilizes the cation diffusion facilitator (CDF) family protein MntE to alleviate Mn toxicity through efflux of excess Mn. Inactivation of leads to a significant reduction in resistance to oxidative stress and mediated mortality within a mouse model of systemic infection. These results highlight the importance of MntE-mediated Mn detoxification in intracellular Mn homeostasis, resistance to oxidative stress, and virulence. Therefore, this establishes MntE as a potential target for development of anti- therapeutics.
Copyright © 2019 Grunenwald et al.
Small-molecule inhibitors of the mTORC2 kinase (torkinibs) have shown efficacy in early clinical trials. However, the torkinibs under study also inhibit the other mTOR-containing complex mTORC1. While mTORC1/mTORC2 combined inhibition may be beneficial in cancer cells, recent reports describe compensatory cell survival upon mTORC1 inhibition due to loss of negative feedback on PI3K, increased autophagy, and increased macropinocytosis. Genetic models suggest that selective mTORC2 inhibition would be effective in breast cancers, but the lack of selective small-molecule inhibitors of mTORC2 have precluded testing of this hypothesis to date. Here we report the engineering of a nanoparticle-based RNAi therapeutic that can effectively silence the mTORC2 obligate cofactor Rictor. Nanoparticle-based Rictor ablation in HER2-amplified breast tumors was achieved following intratumoral and intravenous delivery, decreasing Akt phosphorylation and increasing tumor cell killing. Selective mTORC2 inhibition , combined with the HER2 inhibitor lapatinib, decreased the growth of HER2-amplified breast cancers to a greater extent than either agent alone, suggesting that mTORC2 promotes lapatinib resistance, but is overcome by mTORC2 inhibition. Importantly, selective mTORC2 inhibition was effective in a triple-negative breast cancer (TNBC) model, decreasing Akt phosphorylation and tumor growth, consistent with our findings that RICTOR mRNA correlates with worse outcome in patients with basal-like TNBC. Together, our results offer preclinical validation of a novel RNAi delivery platform for therapeutic gene ablation in breast cancer, and they show that mTORC2-selective targeting is feasible and efficacious in this disease setting. This study describes a nanomedicine to effectively inhibit the growth regulatory kinase mTORC2 in a preclinical model of breast cancer, targeting an important pathogenic enzyme in that setting that has been undruggable to date. .
©2018 American Association for Cancer Research.
BACKGROUND - IL-33 is one of the most consistently associated gene candidates for asthma identified by using a genome-wide association study. Studies in mice and in human cells have confirmed the importance of IL-33 in inducing type 2 cytokine production from both group 2 innate lymphoid cells (ILC2s) and T2 cells. However, there are no pharmacologic agents known to inhibit IL-33 release from airway cells.
OBJECTIVE - We sought to determine the effect of glucagon-like peptide 1 receptor (GLP-1R) signaling on aeroallergen-induced airway IL-33 production and release and on innate type 2 airway inflammation.
METHODS - BALB/c mice were challenged intranasally with Alternaria extract for 4 consecutive days. GLP-1R agonist or vehicle was administered starting either 2 days before the first Alternaria extract challenge or 1 day after the first Alternaria extract challenge.
RESULTS - GLP-1R agonist treatment starting 2 days before the first Alternaria extract challenge decreased IL-33 release in the bronchoalveolar lavage fluid and dual oxidase 1 (Duox1) mRNA expression 1 hour after the first Alternaria extract challenge and IL-33 expression in lung epithelial cells 24 hours after the last Alternaria extract challenge. Furthermore, GLP-1R agonist significantly decreased the number of ILC2s expressing IL-5 and IL-13, lung protein expression of type 2 cytokines and chemokines, the number of perivascular eosinophils, mucus production, and airway responsiveness compared with vehicle treatment. GLP-1R agonist treatment starting 1 day after the first Alternaria extract challenge also significantly decreased eosinophilia and type 2 cytokine and chemokine expression in the airway after 4 days of Alternaria extract challenge.
CONCLUSION - These results reveal that GLP-1R signaling might be a therapy to reduce IL-33 release and inhibit the ILC2 response to protease-containing aeroallergens, such as Alternaria.
Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
The TGF- and Wnt/-catenin pathways have important roles in modulating CKD, but how these growth factors affect the epithelial response to CKD is not well studied. TGF- has strong profibrotic effects, but this pleiotropic factor has many different cellular effects depending on the target cell type. To investigate how TGF- signaling in the proximal tubule, a key target and mediator of CKD, alters the response to CKD, we injured mice lacking the TGF- type 2 receptor specifically in this epithelial segment. Compared with littermate controls, mice lacking the proximal tubular TGF- receptor had significantly increased tubular injury and tubulointerstitial fibrosis in two different models of CKD. RNA sequencing indicated that deleting the TGF- receptor in proximal tubule cells modulated many growth factor pathways, but Wnt/-catenin signaling was the pathway most affected. We validated that deleting the proximal tubular TGF- receptor impaired -catenin activity and Genetically restoring -catenin activity in proximal tubules lacking the TGF- receptor dramatically improved the tubular response to CKD in mice. Deleting the TGF- receptor alters many growth factors, and therefore, this ameliorated response may be a direct effect of -catenin activity or an indirect effect of -catenin interacting with other growth factors. In conclusion, blocking TGF- and -catenin crosstalk in proximal tubules exacerbates tubular injury in two models of CKD.
Copyright © 2017 by the American Society of Nephrology.
Constitutive WNT activity drives the growth of various human tumors, including nearly all colorectal cancers (CRCs). Despite this prominence in cancer, no WNT inhibitor is currently approved for use in the clinic largely due to the small number of druggable signaling components in the WNT pathway and the substantial toxicity to normal gastrointestinal tissue. We have shown that pyrvinium, which activates casein kinase 1α (CK1α), is a potent inhibitor of WNT signaling. However, its poor bioavailability limited the ability to test this first-in-class WNT inhibitor in vivo. We characterized a novel small-molecule CK1α activator called SSTC3, which has better pharmacokinetic properties than pyrvinium, and found that it inhibited the growth of CRC xenografts in mice. SSTC3 also attenuated the growth of a patient-derived metastatic CRC xenograft, for which few therapies exist. SSTC3 exhibited minimal gastrointestinal toxicity compared to other classes of WNT inhibitors. Consistent with this observation, we showed that the abundance of the SSTC3 target, CK1α, was decreased in WNT-driven tumors relative to normal gastrointestinal tissue, and knocking down CK1α increased cellular sensitivity to SSTC3. Thus, we propose that distinct CK1α abundance provides an enhanced therapeutic index for pharmacological CK1α activators to target WNT-driven tumors.
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
The IL-17/IL-17R axis has controversial roles in cancer, which may be explained by tumor-specific results. Here, we describe a novel molecular mechanism underlying IL-17RC-controlled tumor-specific proliferation. Triggered by IL-17RC knockdown (KD), B16 melanoma and 4T1 carcinoma cells inversely altered homeostatic tumor proliferation and tumor growth in vitro and in vivo. In contrast to the existing dogma that IL-17RC-dependent signaling activates the JNK pathway, IL-17RC KD in both tumor cell lines caused aberrant expression and activation of different JNK isoforms along with markedly diminished levels of the ubiquitin-editing enzyme A20. We demonstrated that differential up-regulation of JNK1 and JNK2 in the two tumor cell lines was responsible for the reciprocal regulation of c-Jun activity and tumor-specific proliferation. Furthermore, we showed that A20 reconstitution of IL-17RCKD clones with expression of full-length A20, but not a truncation-mutant, reversed aberrant JNK1/JNK2 activities and tumor-specific proliferation. Collectively, our study reveals a critical role of IL-17RC in maintaining baseline A20 production and a novel role of the IL-17RC-A20 axis in controlling JNK isoform-dependent tumor-specific homeostatic proliferation.
The magnitude of the LPS-elicited cytokine response is commonly used to assess immune function in critically ill patients. A suppressed response, known as endotoxin tolerance, is associated with worse outcomes, yet endotoxin tolerance-inducing TLR4 ligands are known to protect animals from infection. Thus, it remains unknown whether the magnitude of the LPS-elicited cytokine response provides an accurate assessment of antimicrobial immunity. To address this, the ability of diverse TLR ligands to modify the LPS-elicited cytokine response and resistance to infection were assessed. Priming of mice with LPS, monophosphoryl lipid A (MPLA), or poly(I:C) significantly reduced plasma LPS-elicited proinflammatory cytokines, reflecting endotoxin tolerance, whereas CpG-ODN-primed mice showed augmented cytokine production. In contrast, LPS, MPLA, and CpG-ODN, but not poly(I:C), improved the host response to a infection. Mice primed with protective TLR ligands, including CpG-ODN, showed reduced plasma cytokines during infection. The protection imparted by TLR ligands persisted for up to 15 d yet was independent of the adaptive immune system. In bone marrow-derived macrophages, protective TLR ligands induced a persistent metabolic phenotype characterized by elevated glycolysis and oxidative metabolism as well as augmented size, granularity, phagocytosis, and respiratory burst. Sustained augmentation of glycolysis in TLR-primed cells was dependent, in part, on hypoxia-inducible factor 1-α and was essential for increased phagocytosis. In conclusion, the magnitude of LPS-elicited cytokine production is not indicative of antimicrobial immunity after exposure to TLR ligands. Additionally, protective TLR ligands induce sustained augmentation of phagocyte metabolism and antimicrobial function.
Copyright © 2017 by The American Association of Immunologists, Inc.
BACKGROUND - Patients experiencing large thermal injuries are susceptible to opportunistic infections that can delay recovery and lead to sepsis. Dendritic cells (DC) are important for the detection of pathogens and activation of the innate and acquired immune responses. DCs are significantly decreased in burn patients early after injury, and the development of sepsis is associated with persistent DC depletion. In a murine model of burn wound infection, the enhancement of DCs after injury by treatment with the DC growth factor Fms-like tyrosine kinase-3 ligand (FL) enhances neutrophil migration to infection, improves bacterial clearance, and increases survival in a DC-dependent manner. FL expands the production of both conventional DCs (cDC) and plasmacytoid DCs (pDC). It has been established that cDCs are required for some of the protective effects of FL after burn injury. This study was designed to determine the contribution of the pDC subset.
METHODS - Mice were subjected to full-thickness scald burns under deep anesthesia and were provided analgesia. pDCs were depleted by injection of anti-plasmacytoid dendritic cell antigen-1 antibodies. Survival, bacterial clearance, and neutrophil responses in vivo and in vitro were measured.
RESULTS - Depletion of preexisting pDCs, but not FL-expanded pDCs, abrogated the beneficial effects of FL on survival, bacterial clearance, and neutrophil migration in response to burn wound infection. This requisite role of pDCs for FL-mediated enhancement of neutrophil migratory capacity is not due to direct effects of pDCs on neutrophils. cDCs, but not pDCs, directly increased neutrophil migratory capacity after co-culture.
CONCLUSIONS - The protective effects of FL treatment after burn injury are mediated by both pDCs and cDCs. Pharmacological enhancement of both DC subtypes by FL is a potential therapeutic intervention to enhance immune responses to infection and improve outcome after burn injury.
The 2-component leukotoxin LukAB is critical for Staphylococcus aureus targeting and killing of human neutrophils ex vivo and is produced in the setting of human infection. We report 3 LukAB-specific human monoclonal antibodies (mAbs) with distinct mechanisms of toxin neutralization and in vivo efficacy. Three hybridomas secreting mAbs with anti-LukAB activity (designated SA-13, -15, and -17) were generated from B cells obtained from a 12-year-old boy with S. aureus osteomyelitis. Each of the 3 mAbs neutralized LukAB-mediated neutrophil toxicity, exhibited differing levels of potency, recognized different antigenic sites on the toxin, and displayed at least 2 distinct mechanisms for cytotoxic inhibition. SA-15 bound exclusively to the dimeric form of the toxin, suggesting that human B cells recognize epitopes on the dimerized form of LukAB during natural infection. Both SA-13 and SA-17 bound the LukA monomer and the LukAB dimer. Although all 3 mAbs potently neutralized cytotoxicity, only SA-15 and SA-17 significantly inhibited toxin association with the cell surface. Treatment with a 1:1 mixture of mAbs SA-15 and SA-17 resulted in significantly lower bacterial colony counts in heart, liver, and kidneys in a murine model of S. aureus sepsis. These data describe the isolation of diverse and efficacious antitoxin mAbs.
© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: firstname.lastname@example.org.
BACKGROUND - The role of the chemokine CCL2 in breast cancer is controversial. While CCL2 recruits and activates pro-tumor macrophages, it is also reported to enhance neutrophil-mediated anti-tumor activity. Moreover, loss of CCL2 in early development enhances breast cancer progression.
METHODS - To clarify these conflicting findings, we examined the ability of CCL2 to alter naïve and tumor entrained neutrophil production of ROS, release of granzyme-B, and killing of tumor cells in multiple mouse models of breast cancer. CCL2 was delivered intranasally in mice to elevate CCL2 levels in the lung and effects on seeding and growth of breast tumor cells were evaluated. The TCGA data base was queried for relationship between CCL2 expression and relapse free survival of breast cancer patients and compared to subsets of breast cancer patients.
RESULTS - Even though each of the tumor cell lines studied produced approximately equal amounts of CCL2, exogenous delivery of CCL2 to co-cultures of breast tumor cells and neutrophils enhanced the ability of tumor-entrained neutrophils (TEN) to kill the less aggressive 67NR variant of 4T1 breast cancer cells. However, exogenous CCL2 did not enhance naïve or TEN neutrophil killing of more aggressive 4T1 or PyMT breast tumor cells. Moreover, this anti-tumor activity was not observed in vivo. Intranasal delivery of CCL2 to BALB/c mice markedly enhanced seeding and outgrowth of 67NR cells in the lung and increased the recruitment of CD4+ T cells and CD8+ central memory T cells into lungs of tumor bearing mice. There was no significant increase in the recruitment of CD19+ B cells, or F4/80+, Ly6G+ and CD11c + myeloid cells. CCL2 had an equal effect on CD206+ and MHCII+ populations of macrophages, thus balancing the pro- and anti-tumor macrophage cell population. Analysis of the relationship between CCL2 levels and relapse free survival in humans revealed that overall survival is not significantly different between high CCL2 expressing and low CCL2 expressing breast cancer patients grouped together. However, examination of the relationship between high CCL2 expressing basal-like, HER2+ and luminal B breast cancer patients revealed that higher CCL2 expressing tumors in these subgroups have a significantly higher probability of surviving longer than those expressing low CCL2.
CONCLUSIONS - While our in vitro data support a potential anti-tumor role for CCL2 in TEN neutrophil- mediated tumor killing in poorly aggressive tumors, intranasal delivery of CCL2 increased CD4+ T cell recruitment to the pre-metastatic niche of the lung and this correlated with enhanced seeding and growth of tumor cells. These data indicate that effects of CCL2/CCR2 antagonists on the intratumoral leukocyte content should be monitored in ongoing clinical trials using these agents.