Cystic fibrosis macrophage function and clinical outcomes after elexacaftor/tezacaftor/ivacaftor.

BACKGROUND: Abnormal macrophage function caused by dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) is a critical contributor to chronic airway infections and inflammation in people with cystic fibrosis (PWCF). Elexacaftor/tezacaftor/ivacaftor (ETI) is a new CFTR modulator therapy for PWCF. Host-pathogen and clinical responses to CFTR modulators are poorly described. We sought to determine how ETI impacts macrophage CFTR function, resulting effector functions and relationships to clinical outcome changes. METHODS: Clinical information and/or biospecimens were obtained at ETI initiation and 3, 6, 9 and 12 months post-ETI in 56 PWCF and compared with non-CF controls. Peripheral blood monocyte-derived macrophages (MDMs) were isolated and functional assays performed. RESULTS: ETI treatment was associated with increased CF MDM CFTR expression, function and localisation to the plasma membrane. CF MDM phagocytosis, intracellular killing of CF pathogens and efferocytosis of apoptotic neutrophils were partially restored by ETI, but inflammatory cytokine production remained unchanged. Clinical outcomes including increased forced expiratory volume in 1 s (+10%) and body mass index (+1.0 kg·m-2) showed fluctuations over time and were highly individualised. Significant correlations between post-ETI MDM CFTR function and sweat chloride levels were observed. However, MDM CFTR function correlated with clinical outcomes better than sweat chloride. CONCLUSION: ETI is associated with unique changes in innate immune function and clinical outcomes.

Fibrosis quística: patogenia bacteriana y moduladores del CFTR (regulador de conductancia transmembranal de la fibrosis quística).

Cystic fibrosis is an autosomal recessive inherited disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). CFTR is a protein that transports ions across the membrane of lung epithelial cells. Loss of its function leads to the production of thick sticky mucus, where various bacterial pathogens can establish and adapt, contributing to the gradual loss of lung function. In this review, evidence of the molecular mechanisms used by Pseudomonas aeruginosa and Burkholderia cenocepacia to survive and persist in the pulmonary environment will be provided. Additionally, new therapeutic strategies based on CFTR function modulators will be described.

La fibrosis quística es una enfermedad hereditaria autosómica recesiva que se origina por mutaciones en el gen regulador de conductancia transmembranal de la fibrosis quística (CFTR, cystic fibrosis transmembrane conductance regulator). El CFTR es una proteína que transporta iones a través de la membrana de las células epiteliales pulmonares. La pérdida de su función conlleva la producción de un moco pegajoso y espeso, donde se pueden establecer y adaptar diversos patógenos bacterianos que contribuyen a la pérdida gradual de la función pulmonar. En este artículo de revisión se dará evidencia de los mecanismos moleculares que utilizan Pseudomonas aeruginosa y Burkholderia cenocepacia para sobrevivir y persistir en el ambiente pulmonar. Adicionalmente, se describirán las nuevas estrategias de terapia a base de moduladores de la función del CFTR.

Disruption of Nurse-like Cell Differentiation as a Therapeutic Strategy for Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia, but, despite advances in treatment, many patients still experience relapse. CLL cells depend on interactions with supportive cells, and nurse-like cells (NLCs) are the major such cell type. However, little is known about how NLCs develop. Here, we performed DNA methylation analysis of CLL patient-derived NLCs using the 850K Illumina array, comparing CD14+ cells at day 1 (monocytes) versus day 14 (NLCs). We found a strong loss of methylation in AP-1 transcription factor binding sites, which may be driven by MAPK signaling. Testing of individual MAPK pathways (MEK, p38, and JNK) revealed a strong dependence on MEK/ERK for NLC development, because treatment of patient samples with the MEK inhibitor trametinib dramatically reduced NLC development in vitro. Using the adoptive transfer Eµ-TCL1 mouse model of CLL, we found that MEK inhibition slowed CLL progression, leading to lower WBC counts and to significantly longer survival time. There were also lower numbers of mouse macrophages, particularly within the M2-like population. In summary, NLC development depends on MEK signaling, and inhibition of MEK leads to increased survival time in vivo. Hence, targeting the MEK/ERK pathway may be an effective treatment strategy for CLL.

Fibrosis quística: patogenia bacteriana y moduladores del CFTR (regulador de conductancia transmembranal de la fibrosis quística) / Cystic fibrosis: bacterial pathogenesis and CFTR (cystic fibrosis transmembrane conductance regulator) modulators

Resumen La fibrosis quística es una enfermedad hereditaria autosómica recesiva que se origina por mutaciones en el gen regulador de conductancia transmembranal de la fibrosis quística (CFTR, cystic fibrosis transmembrane conductance regulator). El CFTR es una proteína que transporta iones a través de la membrana de las células epiteliales pulmonares. La pérdida de su función conlleva la producción de un moco pegajoso y espeso, donde se pueden establecer y adaptar diversos patógenos bacterianos que contribuyen a la pérdida gradual de la función pulmonar. En este artículo de revisión se dará evidencia de los mecanismos moleculares que utilizan Pseudomonas aeruginosa y Burkholderia cenocepacia para sobrevivir y persistir en el ambiente pulmonar. Adicionalmente, se describirán las nuevas estrategias de terapia a base de moduladores de la función del CFTR.

Abstract Cystic fibrosis is an autosomal recessive inherited disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). CFTR is a protein that transports ions across the membrane of lung epithelial cells. Loss of its function leads to the production of thick sticky mucus, where various bacterial pathogens can establish and adapt, contributing to the gradual loss of lung function. In this review, evidence of the molecular mechanisms used by Pseudomonas aeruginosa and Burkholderia cenocepacia to survive and persist in the pulmonary environment will be provided. Additionally, new therapeutic strategies based on CFTR function modulators will be described.

CFTR Modulators Restore Acidification of Autophago-Lysosomes and Bacterial Clearance in Cystic Fibrosis Macrophages.

Cystic fibrosis (CF) human and mouse macrophages are defective in their ability to clear bacteria such as Burkholderia cenocepacia. The autophagy process in CF (F508del) macrophages is halted, and the underlying mechanism remains unclear. Furthermore, the role of CFTR in maintaining the acidification of endosomal and lysosomal compartments in CF cells has been a subject of debate. Using 3D reconstruction of z-stack confocal images, we show that CFTR is recruited to LC3-labeled autophagosomes harboring B. cenocepacia. Using several complementary approaches, we report that CF macrophages display defective lysosomal acidification and degradative function for cargos destined to autophagosomes, whereas non-autophagosomal cargos are effectively degraded within acidic compartments. Notably, treatment of CF macrophages with CFTR modulators (tezacaftor/ivacaftor) improved the autophagy flux, lysosomal acidification and function, and bacterial clearance. In addition, CFTR modulators improved CFTR function as demonstrated by patch-clamp. In conclusion, CFTR regulates the acidification of a specific subset of lysosomes that specifically fuse with autophagosomes. Therefore, our study describes a new biological location and function for CFTR in autophago-lysosomes and clarifies the long-standing discrepancies in the field.

Neutrophil-Macrophage Imbalance Drives the Development of Renal Scarring during Experimental Pyelonephritis.

BACKGROUND: In children, the acute pyelonephritis that can result from urinary tract infections (UTIs), which commonly ascend from the bladder to the kidney, is a growing concern because it poses a risk of renal scarring and irreversible loss of kidney function. To date, the cellular mechanisms underlying acute pyelonephritis-driven renal scarring remain unknown. METHODS: We used a preclinical model of uropathogenic Escherichia coli-induced acute pyelonephritis to determine the contribution of neutrophils and monocytes to resolution of the condition and the subsequent development of kidney fibrosis. We used cell-specific monoclonal antibodies to eliminate neutrophils, monocytes, or both. Bacterial ascent and the cell dynamics of phagocytic cells were assessed by biophotonic imaging and flow cytometry, respectively. We used quantitative RT-PCR and histopathologic analyses to evaluate inflammation and renal scarring. RESULTS: We found that neutrophils are critical to control bacterial ascent, which is in line with previous studies suggesting a protective role for neutrophils during a UTI, whereas monocyte-derived macrophages orchestrate a strong, but ineffective, inflammatory response against uropathogenic, E. coli-induced, acute pyelonephritis. Experimental neutropenia during acute pyelonephritis resulted in a compensatory increase in the number of monocytes and heightened macrophage-dependent inflammation in the kidney. Exacerbated macrophage-mediated inflammatory responses promoted renal scarring and compromised renal function, as indicated by elevated serum creatinine, BUN, and potassium. CONCLUSIONS: These findings reveal a previously unappreciated outcome for neutrophil-macrophage imbalance in promoting host susceptibility to acute pyelonephritis and the development of permanent renal damage. This suggests targeting dysregulated macrophage responses might be a therapeutic tool to prevent renal scarring during acute pyelonephritis.

Whole-blood transcriptomic responses to lumacaftor/ivacaftor therapy in cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) remains without a definitive cure. Novel therapeutics targeting the causative defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are in clinical use. Lumacaftor/ivacaftor is a CFTR modulator approved for patients homozygous for the CFTR variant p.Phe508del, but there are wide variations in treatment responses preventing prediction of patient responses. We aimed to determine changes in gene expression related to treatment initiation and response. METHODS: Whole-blood transcriptomics was performed using RNA-Seq in 20 patients with CF pre- and 6 months post-lumacaftor/ivacaftor (drug) initiation and 20 non-CF healthy controls. Correlation of gene expression with clinical variables was performed by stratification via clinical responses. RESULTS: We identified 491 genes that were differentially expressed in CF patients (pre-drug) compared with non-CF controls and 36 genes when comparing pre-drug to post-drug profiles. Both pre- and post-drug CF profiles were associated with marked overexpression of inflammation-related genes and apoptosis genes, and significant under-expression of T cell and NK cell-related genes compared to non-CF. CF patients post-drug demonstrated normalized protein synthesis expression, and decreased expression of cell-death genes compared to pre-drug profiles, irrespective of clinical response. However, CF clinical responders demonstrated changes in eIF2 signaling, oxidative phosphorylation, IL-17 signaling, and mitochondrial function compared to non-responders. Top overexpressed genes (MMP9 and SOCS3) that decreased post-drug were validated by qRT-PCR. Functional assays demonstrated that CF monocytes normalized calcium (increases MMP9 expression) concentrations post-drug. CONCLUSIONS: Transcriptomics revealed differentially regulated pathways in CF patients at baseline compared to non-CF, and in clinical responders to lumacaftor/ivacaftor.

Caspase-11 Mediates Neutrophil Chemotaxis and Extracellular Trap Formation During Acute Gouty Arthritis Through Alteration of Cofilin Phosphorylation.

Gout is characterized by attacks of arthritis with hyperuricemia and monosodium urate (MSU) crystal-induced inflammation within joints. Innate immune responses are the primary drivers for tissue destruction and inflammation in gout. MSU crystals engage the Nlrp3 inflammasome, leading to the activation of caspase-1 and production of IL-1ß and IL-18 within gout-affected joints, promoting the influx of neutrophils and monocytes. Here, we show that caspase-11-/- mice and their derived macrophages produce significantly reduced levels of gout-specific cytokines including IL-1ß, TNFα, IL-6, and KC, while others like IFNγ and IL-12p70 are not altered. IL-1ß induces the expression of caspase-11 in an IL-1 receptor-dependent manner in macrophages contributing to the priming of macrophages during sterile inflammation. The absence of caspase-11 reduced the ability of macrophages and neutrophils to migrate in response to exogenously injected KC in vivo. Notably, in vitro, caspase-11-/- neutrophils displayed random migration in response to a KC gradient when compared to their WT counterparts. This phenotype was associated with altered cofilin phosphorylation. Unlike their wild-type counterparts, caspase-11-/- neutrophils also failed to produce neutrophil extracellular traps (NETs) when treated with MSU. Together, this is the first report demonstrating that caspase-11 promotes neutrophil directional trafficking and function in an acute model of gout. Caspase-11 also governs the production of inflammasome-dependent and -independent cytokines from macrophages. Our results offer new, previously unrecognized functions for caspase-11 in macrophages and neutrophils that may apply to other neutrophil-mediated disease conditions besides gout.

CD31 Acts as a Checkpoint Molecule and Is Modulated by FcγR-Mediated Signaling in Monocytes.

Monocytes and macrophages express FcγR that engage IgG immune complexes such as Ab-opsonized pathogens or cancer cells to destroy them by various mechanisms, including phagocytosis. FcγR-mediated phagocytosis is regulated by the concerted actions of activating FcγR and inhibitory receptors, such as FcγRIIb and SIRPα. In this study, we report that another ITIM-containing receptor, PECAM1/CD31, regulates FcγR function and is itself regulated by FcγR activation. First, quantitative RT-PCR and flow cytometry analyses revealed that human monocyte FcγR activation leads to a significant downregulation of CD31 expression, both at the message level and at surface expression, mainly mediated through FcγRIIa. Interestingly, the kinetics of downregulation between the two varied, with surface expression reducing earlier than the message. Experiments to analyze the mechanism behind this discrepancy revealed that the loss of surface expression was because of internalization, which depended predominantly on the PI3 kinase pathway and was independent of FcγR internalization. Finally, functional analyses showed that the downregulation of CD31 expression in monocytes by small interfering RNA enhanced FcγR-mediated phagocytic ability but have little effect on cytokine production. Together, these results suggest that CD31 acts as a checkpoint receptor that could be targeted to enhance FcγR functions in Ab-mediated therapies.

Pharmacological inhibition of Notch signaling regresses pre-established abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is characterized by transmural infiltration of myeloid cells at the vascular injury site. Previously, we reported preventive effects of Notch deficiency on the development of AAA by reduction of infiltrating myeloid cells. In this study, we examined if Notch inhibition attenuates the progression of pre-established AAA and potential implications. Pharmacological Notch inhibitor (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester; DAPT) was administered subcutaneously three times a week starting at day 28 of angiotensin II (AngII) infusion. Progressive increase in pulse wave velocity (PWV), maximal intra-luminal diameter (MILD) and maximal external aortic diameter (MEAD) were observed at day 56 of the AngII. DAPT prevented such increase in MILD, PWV and MEAD (P < 0.01). Histologically, the aortae of DAPT-treated Apoe-/- mice had significant reduction in inflammatory response and elastin fragmentation. Naked collagen microfibrils and weaker banded structure observed in the aortae of Apoe-/- mice in response to AngII, were substantially diminished by DAPT. A significant decrease in the proteolytic activity in the aneurysmal tissues and vascular smooth muscle cells (vSMCs) was observed with DAPT (P < 0.01). In human and mouse AAA tissues, increased immunoreactivity of activated Notch signaling correlated strongly with CD38 expression (R2 = 0.61). Collectively, we propose inhibition of Notch signaling as a potential therapeutic target for AAA progression.

Taenia crassiceps-Excreted/Secreted Products Induce a Defined MicroRNA Profile that Modulates Inflammatory Properties of Macrophages.

Helminth parasites modulate immune responses in their host to prevent their elimination and to establish chronic infections. Our previous studies indicate that Taenia crassiceps-excreted/secreted antigens (TcES) downregulate inflammatory responses in rodent models of autoimmune diseases, by promoting the generation of alternatively activated-like macrophages (M2) in vivo. However, the molecular mechanisms triggered by TcES that modulate macrophage polarization and inflammatory response remain unclear. Here, we found that, while TcES reduced the production of inflammatory cytokines (IL-6, IL-12, and TNFα), they increased the release of IL-10 in LPS-induced bone marrow-derived macrophages (BMDM). However, TcES alone or in combination with LPS or IL-4 failed to increase the production of the canonical M1 or M2 markers in BMDM. To further define the anti-inflammatory effect of TcES in the response of LPS-stimulated macrophages, we performed transcriptomic array analyses of mRNA and microRNA to evaluate their levels. Although the addition of TcES to LPS-stimulated BMDM induced modest changes in the inflammatory mRNA profile, it induced the production of mRNAs associated with the activation of different receptors, phagocytosis, and M2-like phenotype. Moreover, we found that TcES induced upregulation of specific microRNAs, including miR-125a-5p, miR-762, and miR-484, which are predicted to target canonical inflammatory molecules and pathways in LPS-induced BMDM. These results suggest that TcES can modulate proinflammatory responses in macrophages by inducing regulatory posttranscriptional mechanisms and hence reduce detrimental outcomes in hosts running with inflammatory diseases.

AR-13 reduces antibiotic-resistant bacterial burden in cystic fibrosis phagocytes and improves cystic fibrosis transmembrane conductance regulator function.

BACKGROUND: There are no effective treatments for Burkholderia cenocepacia in patients with cystic fibrosis (CF) due to bacterial multi-drug resistance and defective host killing. We demonstrated that decreased bacterial killing in CF is caused by reduced macrophage autophagy due to defective cystic fibrosis transmembrane conductance regulator (CFTR) function. AR-12 is a small molecule autophagy inducer that kills intracellular pathogens such as Francisella. We evaluated the efficacy of AR-12 and a new analogue AR-13 in reducing bacterial burden in CF phagocytes. METHODS: Human CF and non-CF peripheral blood monocyte-derived macrophages, neutrophils, and nasal epithelial cells were exposed to CF bacterial strains in conjunction with treatment with antibiotics and/or AR compounds. RESULTS: AR-13 and not AR-12 had growth inhibition on B. cenocepacia and methicillin-resistantStaphylococcus aureus (MRSA) in media alone. There was a 99% reduction in MRSA in CF macrophages, 71% reduction in Pseudomonas aeruginosa in CF neutrophils, and 70% reduction in non-CF neutrophils using AR-13. Conversely, there was no reduction in B. cenocepacia in infected CF and non-CF macrophages using AR-13 alone, but AR-13 and antibiotics synergistically reduced B. cenocepacia in CF macrophages. AR-13 improved autophagy in CF macrophages and CF patient-derived epithelial cells, and increased CFTR protein expression and channel function in CF epithelial cells. CONCLUSIONS: The novel AR-12 analogue AR-13, in combination with antibiotics, reduced antibiotic-resistant bacterial burden in CF phagocytes, which correlated with increased autophagy and CFTR expression. AR-13 is a novel therapeutic for patients infected with B. cenocepacia and other resistant organisms that lack effective therapies.

Dysregulated Calcium Homeostasis in Cystic Fibrosis Neutrophils Leads to Deficient Antimicrobial Responses.

Cystic fibrosis (CF), one of the most common human genetic diseases worldwide, is caused by a defect in the CF transmembrane conductance regulator (CFTR). Patients with CF are highly susceptible to infections caused by opportunistic pathogens (including Burkholderia cenocepacia), which induce excessive lung inflammation and lead to the eventual loss of pulmonary function. Abundant neutrophil recruitment into the lung is a key characteristic of bacterial infections in CF patients. In response to infection, inflammatory neutrophils release reactive oxygen species and toxic proteins, leading to aggravated lung tissue damage in patients with CF. The present study shows a defect in reactive oxygen species production by mouse Cftr-/- , human F508del-CFTR, and CF neutrophils; this results in reduced antimicrobial activity against B. cenocepacia Furthermore, dysregulated Ca2+ homeostasis led to increased intracellular concentrations of Ca2+ that correlated with significantly diminished NADPH oxidase response and impaired secretion of neutrophil extracellular traps in human CF neutrophils. Functionally deficient human CF neutrophils recovered their antimicrobial killing capacity following treatment with pharmacological inhibitors of Ca2+ channels and CFTR channel potentiators. Our findings suggest that regulation of neutrophil Ca2+ homeostasis (via CFTR potentiation or by the regulation of Ca2+ channels) can be used as a new therapeutic approach for reestablishing immune function in patients with CF.

Angiotensin II receptor I blockade prevents stenosis of tissue engineered vascular grafts.

We previously developed a tissue-engineered vascular graft (TEVG) made by seeding autologous cells onto a biodegradable tubular scaffold, in an attempt to create a living vascular graft with growth potential for use in children undergoing congenital heart surgery. Results of our clinical trial showed that the TEVG possesses growth capacity but that its widespread clinical use is not yet advisable due to the high incidence of TEVG stenosis. In animal models, TEVG stenosis is caused by increased monocytic cell recruitment and its classic ("M1") activation. Here, we report on the source and regulation of these monocytes. TEVGs were implanted in wild-type, CCR2 knockout ( Ccr2-/-), splenectomized, and spleen graft recipient mice. We found that bone marrow-derived Ly6C+hi monocytes released from sequestration by the spleen are the source of mononuclear cells infiltrating the TEVG during the acute phase of neovessel formation. Furthermore, short-term administration of losartan (0.6 g/L, 2 wk), an angiotensin II type 1 receptor antagonist, significantly reduced the macrophage populations (Ly6C+/-/F480+) in the scaffolds and improved long-term patency in TEVGs. Notably, the combined effect of bone marrow-derived mononuclear cell seeding with short-term losartan treatment completely prevented the development of TEVG stenosis. Our results provide support for pharmacologic treatment with losartan as a strategy to modulate monocyte infiltration into the grafts and thus prevent TEVG stenosis.-Ruiz-Rosado, J. D. D., Lee, Y.-U., Mahler, N., Yi, T., Robledo-Avila, F., Martinez-Saucedo, D., Lee, A. Y., Shoji, T., Heuer, E., Yates, A. R., Pober, J. S., Shinoka, T., Partida-Sanchez, S., Breuer, C. K. Angiotensin II receptor I blockade prevents stenosis of tissue engineered vascular grafts.

Liver X Receptor Nuclear Receptors Are Transcriptional Regulators of Dendritic Cell Chemotaxis.

The liver X receptors (LXRs) are ligand-activated nuclear receptors with established roles in the maintenance of lipid homeostasis in multiple tissues. LXRs exert additional biological functions as negative regulators of inflammation, particularly in macrophages. However, the transcriptional responses controlled by LXRs in other myeloid cells, such as dendritic cells (DCs), are still poorly understood. Here we used gain- and loss-of-function models to characterize the impact of LXR deficiency on DC activation programs. Our results identified an LXR-dependent pathway that is important for DC chemotaxis. LXR-deficient mature DCs are defective in stimulus-induced migration in vitro and in vivo Mechanistically, we show that LXRs facilitate DC chemotactic signaling by regulating the expression of CD38, an ectoenzyme important for leukocyte trafficking. Pharmacological or genetic inactivation of CD38 activity abolished the LXR-dependent induction of DC chemotaxis. Using the low-density lipoprotein receptor-deficient (LDLR-/-) LDLR-/- mouse model of atherosclerosis, we also demonstrated that hematopoietic CD38 expression is important for the accumulation of lipid-laden myeloid cells in lesions, suggesting that CD38 is a key factor in leukocyte migration during atherogenesis. Collectively, our results demonstrate that LXRs are required for the efficient emigration of DCs in response to chemotactic signals during inflammation.

TRPM2 channel-mediated regulation of autophagy maintains mitochondrial function and promotes gastric cancer cell survival via the JNK-signaling pathway.

A lack of effective treatment is one of the main factors contributing to gastric cancer-related death. Discovering effective targets and understanding their underlying anti-cancer mechanism are key to achieving the best response to treatment and to limiting side effects. Although recent studies have shown that the cation channel transient receptor potential melastatin-2 (TRPM2) is crucial for cancer cell survival, the exact mechanism remains unclear, limiting its therapeutic potential. Here, using molecular and functional assays, we investigated the role of TRPM2 in survival of gastric cancer cells. Our results indicated that TRPM2 knockdown in AGS and MKN-45 cells decreases cell proliferation and enhances apoptosis. We also observed that the TRPM2 knockdown impairs mitochondrial metabolism, indicated by a decrease in basal and maximal mitochondrial oxygen consumption rates and ATP production. These mitochondrial defects coincided with a decrease in autophagy and mitophagy, indicated by reduced levels of autophagy- and mitophagy-associated proteins (i.e. ATGs, LC3A/B II, and BNIP3). Moreover, we found that TRPM2 modulates autophagy through a c-Jun N-terminal kinase (JNK)-dependent and mechanistic target of rapamycin-independent pathway. We conclude that in the absence of TRPM2, down-regulation of the JNK-signaling pathway impairs autophagy, ultimately causing the accumulation of damaged mitochondria and death of gastric cancer cells. Of note, by inhibiting cell proliferation and promoting apoptosis, the TRPM2 down-regulation enhanced the efficacy of paclitaxel and doxorubicin in gastric cancer cells. Collectively, we provide compelling evidence that TRPM2 inhibition may benefit therapeutic approaches for managing gastric cancer.

The expression of Mirc1/Mir17-92 cluster in sputum samples correlates with pulmonary exacerbations in cystic fibrosis patients.

INTRODUCTION: Cystic fibrosis (CF) is a multi-organ disorder characterized by chronic sino-pulmonary infections and inflammation. Many patients with CF suffer from repeated pulmonary exacerbations that are predictors of worsened long-term morbidity and mortality. There are no reliable markers that associate with the onset or progression of an exacerbation or pulmonary deterioration. Previously, we found that the Mirc1/Mir17-92a cluster which is comprised of 6 microRNAs (Mirs) is highly expressed in CF mice and negatively regulates autophagy which in turn improves CF transmembrane conductance regulator (CFTR) function. Therefore, here we sought to examine the expression of individual Mirs within the Mirc1/Mir17-92 cluster in human cells and biological fluids and determine their role as biomarkers of pulmonary exacerbations and response to treatment. METHODS: Mirc1/Mir17-92 cluster expression was measured in human CF and non-CF plasma, blood-derived neutrophils, and sputum samples. Values were correlated with pulmonary function, exacerbations and use of CFTR modulators. RESULTS: Mirc1/Mir17-92 cluster expression was not significantly elevated in CF neutrophils nor plasma when compared to the non-CF cohort. Cluster expression in CF sputum was significantly higher than its expression in plasma. Elevated CF sputum Mirc1/Mir17-92 cluster expression positively correlated with pulmonary exacerbations and negatively correlated with lung function. Patients with CF undergoing treatment with the CFTR modulator Ivacaftor/Lumacaftor did not demonstrate significant change in the expression Mirc1/Mir17-92 cluster after six months of treatment. CONCLUSIONS: Mirc1/Mir17-92 cluster expression is a promising biomarker of respiratory status in patients with CF including pulmonary exacerbation.

Human Cystic Fibrosis Macrophages Have Defective Calcium-Dependent Protein Kinase C Activation of the NADPH Oxidase, an Effect Augmented by Burkholderia cenocepacia.

Macrophage intracellular pathogen killing is defective in cystic fibrosis (CF), despite abundant production of reactive oxygen species (ROS) in lung tissue. Burkholderia species can cause serious infection in CF and themselves affect key oxidase components in murine non-CF cells. However, it is unknown whether human CF macrophages have an independent defect in the oxidative burst and whether Burkholderia contributes to this defect in terms of assembly of the NADPH oxidase complex and subsequent ROS production. In this article, we analyze CF and non-CF human monocyte-derived macrophages (MDMs) for ROS production, NADPH assembly capacity, protein kinase C expression, and calcium release in response to PMA and CF pathogens. CF MDMs demonstrate a nearly 60% reduction in superoxide production after PMA stimulation compared with non-CF MDMs. Although CF MDMs generally have increased total NADPH component protein expression, they demonstrate decreased expression of the calcium-dependent protein kinase C conventional subclass α/ß leading to reduced phosphorylation of NADPH oxidase components p47 phox and p40 phox in comparison with non-CF MDMs. Ingestion of B. cenocepacia independently contributes to and worsens the overall oxidative burst deficits in CF MDMs compared with non-CF MDMs. Together, these results provide evidence for inherent deficits in the CF macrophage oxidative burst caused by decreased phosphorylation of NADPH oxidase cytosolic components that are augmented by Burkholderia These findings implicate a critical role for defective macrophage oxidative responses in persistent bacterial infections in CF and create new opportunities for boosting the macrophage immune response to limit infection.

MIF Promotes Classical Activation and Conversion of Inflammatory Ly6C(high) Monocytes into TipDCs during Murine Toxoplasmosis.

Macrophage migration inhibitory factor (MIF) mediates immunity against Toxoplasma gondii infection by inducing inflammatory cytokines required to control the parasite replication. However, the role of this inflammatory mediator in the cell-mediated immune response against this infection is still poorly understood. Here, we used T. gondii-infected WT and Mif (-/-) mice to analyze the role of MIF in the maturation of CD11b(+) and CD8α (+) dendritic cells (DCs). We found that MIF promotes maturation of CD11b(+) but not CD8α (+) DCs, by inducing IL-12p70 production and CD86 expression. Infected Mif (-/-) mice showed significantly lower numbers of TNF and inducible nitric oxide synthase- (iNOS-) producing DCs (TipDCs) compared to infected WT mice. The adoptive transfer of Ly6C(high) monocytes into infected WT or Mif (-/-) mice demonstrated that MIF participates in the differentiation of Ly6C(high) monocytes into TipDCs. In addition, infected Mif (-/-) mice display a lower percentage of IFN-γ-producing natural killer (NK) cells compared to WT mice, which is associated with reducing numbers of TipDCs in Mif (-/-) mice. Furthermore, administration of recombinant MIF (rMIF) into T. gondii-infected Mif (-/-) mice restored the numbers of TipDCs and reversed the susceptible phenotype of Mif (-/-) mice. Collectively, these results demonstrate an important role for MIF inducing cell-mediated immunity to T. gondii infection.

Novel Markers to Delineate Murine M1 and M2 Macrophages.

Classically (M1) and alternatively activated (M2) macrophages exhibit distinct phenotypes and functions. It has been difficult to dissect macrophage phenotypes in vivo, where a spectrum of macrophage phenotypes exists, and also in vitro, where low or non-selective M2 marker protein expression is observed. To provide a foundation for the complexity of in vivo macrophage phenotypes, we performed a comprehensive analysis of the transcriptional signature of murine M0, M1 and M2 macrophages and identified genes common or exclusive to either subset. We validated by real-time PCR an M1-exclusive pattern of expression for CD38, G-protein coupled receptor 18 (Gpr18) and Formyl peptide receptor 2 (Fpr2) whereas Early growth response protein 2 (Egr2) and c-Myc were M2-exclusive. We further confirmed these data by flow cytometry and show that M1 and M2 macrophages can be distinguished by their relative expression of CD38 and Egr2. Egr2 labeled more M2 macrophages (~70%) than the canonical M2 macrophage marker Arginase-1, which labels 24% of M2 macrophages. Conversely, CD38 labeled most (71%) in vitro M1 macrophages. In vivo, a similar CD38+ population greatly increased after LPS exposure. Overall, this work defines exclusive and common M1 and M2 signatures and provides novel and improved tools to distinguish M1 and M2 murine macrophages.