Manufacture and evaluation of a HER2-positive breast cancer immunotoxin 4D5Fv-PE25.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) positive breast cancer is an aggressive subtype, accounting for around 20% of all breast cancers. The development of HER2-targeted therapy has substantially improved patient outcomes. Nevertheless, the increasing rate of side effects and resistance to targeted drugs limit their efficacy in clinical practice. In this study, we designed and synthesized a new immunotoxin, 4D5Fv-PE25, which targets HER2-positive breast cancer, and evaluated its effectiveness in vitro and in vivo. RESULTS: The 4D5Fv-PE25 was expressed in high-density Escherichia coli (E. coli.) using the fermentor method and refined via hydrophobicity, ion exchange, and filtration chromatography, achieving a 56.06% recovery rate. Additionally, the semi-manufactured product with 96% purity was prepared into freeze-dried powder by the lyophilized process. Flow cytometry was used to detect the expression of HER2 in SK-BR-3, BT-474, MDA-MB-231, and MDA-MB-468 breast cancer cell lines. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method was used for cytotoxicity assay, and the half-maximal inhibitory concentration (IC50) of 4D5Fv-PE25 lyophilized products to HER2-positive cell line SK-BR-3 was 12.53 ng/mL. The 4D5Fv-PE25 was injected into xenograft tumor mice via the tail vein on the 1st, 4th, and 8th day, it indicated that the growth of tumor volume was effectively inhibited for 24 days, although the 4D5Fv-PE25 was metabolized within 60 min by measuring the release of 3 H-Thymidine radiation. CONCLUSION: we succeeded in producing the 4D5Fv-PE25 freeze-dried powder using the prokaryotic expression method, and it could be employed as a potential drug for treating HER2-positive breast cancer.

Mice with double knockout of Egr-1 and RCAN1 exhibit reduced inflammation during Pseudomonas aeruginosa lung infection.

Pseudomonas aeruginosa represents one of the major opportunistic pathogens, which causes nosocomial infections in immunocompromised individuals. The molecular mechanisms controlling the host immune response to P. aeruginosa infections are not completely understood. In our previous study, early growth response 1 (Egr-1) and regulator of calcineurin 1 (RCAN1) were found to positively and negatively regulate the inflammatory responses, respectively, during P. aeruginosa pulmonary infection, and both of them had an impact on activating NF-κB pathway. Herein, we examined the inflammatory responses of Egr-1/RCAN1 double knockout mice using a mouse model of P. aeruginosa acute pneumonia. As a result, the Egr-1/RCAN1 double knockout mice showed reduced production of proinflammatory cytokines (IL-1ß, IL-6, TNF and MIP-2), diminished inflammatory cell infiltration and decreased mortality, which were similar to those of Egr-1-deficienct mice but different from those of RCAN1-deficient mice. In vitro studies demonstrated that Egr-1 mRNA transcription preceded RCAN1 isoform 4 (RCAN1.4) mRNA transcription in macrophages, and the macrophages with Egr-1 deficiency exhibited decreased RCAN1.4 mRNA levels upon P. aeruginosa LPS stimulation. Moreover, Egr-1/RCAN1 double-deficient macrophages had reduced NF-κB activation compared to RCAN1-deficient macrophages. Taken together, Egr-1 predominates over RCAN1 in regulating inflammation during P. aeruginosa acute lung infection, which influences RCAN1.4 gene expression.

Calcineurin Aα Contributes to IgE-Dependent Mast-Cell Mediator Secretion in Allergic Inflammation.

Mast cells (MCs) are key mediators of allergic inflammation through the activation of cross-linked immunoglobulin E (IgE) bound to the high-affinity IgE receptor (FcϵRI) on the cell surface, leading to the release of biologically potent mediators, either from preformed granules or newly synthesized. Pharmacological inhibitors have been developed to target a key signaling protein phosphatase in this pathway, calcineurin, yet there is a lack of genetic and definitive evidence for the various isoforms of calcineurin subunits in FcϵRI-mediated responses. In this study, we hypothesized that deficiency in the calcineurin Aα isoform will result in a decreased allergic immune response by the MCs. In a model of passive cutaneous anaphylaxis, there was a reduction in vascular permeability in MC-deficient mouse tissues reconstituted with calcineurin subunit A (CnAα) gene-knockout (CnAα-/-) MCs, and in vitro experiments identified a significant reduction in release of preformed mediators from granules. Furthermore, released levels of de novo synthesized cytokines were reduced upon FcϵRI activation of CnAα-/- MCs in vitro. Characterizing the mechanisms associated with this deficit response, we found a significant impairment of nuclear factor of kappa light polypeptide gene enhancer in B cell phosphorylation and impaired nuclear factor kappa-light-chain-enhancer of activated B-cell inhibitor alpha (NF-κB) activation. Thus, we concluded that CnAα contributes to the release of preformed mediators and newly synthesized mediators from FcϵRI-mediated activation of MCs, and this regulation includes NF-κB signaling.

PTP1B negatively regulates nitric oxide-mediated Pseudomonas aeruginosa killing by neutrophils.

Neutrophils play a critical role in host defense against Pseudomonas aeruginosa infection. Mechanisms underlying the negative regulation of neutrophil function in bacterial clearance remain incompletely defined. Here, we demonstrate that protein tyrosine phosphatase-1B (PTP1B) is a negative regulator of P. aeruginosa clearance by neutrophils. PTP1B-deficient neutrophils display greatly enhanced bacterial phagocytosis and killing, which are accompanied by increased Toll-like receptor 4 (TLR4) signaling activation and nitric oxide (NO) production following P. aeruginosa infection. Interestingly, PTP1B deficiency mainly upregulates the production of IL-6 and IFN-ß, leads to enhanced TLR4-dependent STAT1 activation and iNOS expression by neutrophils following P. aeruginosa infection. Further studies reveal that PTP1B and STAT1 are physically associated. These findings demonstrate a negative regulatory mechanism in neutrophil underlying the elimination of P. aeruginosa infection though a PTP1B-STAT1 interaction.

Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies.

Pseudomonas aeruginosa is an opportunistic pathogen that is a leading cause of morbidity and mortality in cystic fibrosis patients and immunocompromised individuals. Eradication of P. aeruginosa has become increasingly difficult due to its remarkable capacity to resist antibiotics. Strains of Pseudomonas aeruginosa are known to utilize their high levels of intrinsic and acquired resistance mechanisms to counter most antibiotics. In addition, adaptive antibiotic resistance of P. aeruginosa is a recently characterized mechanism, which includes biofilm-mediated resistance and formation of multidrug-tolerant persister cells, and is responsible for recalcitrance and relapse of infections. The discovery and development of alternative therapeutic strategies that present novel avenues against P. aeruginosa infections are increasingly demanded and gaining more and more attention. Although mostly at the preclinical stages, many recent studies have reported several innovative therapeutic technologies that have demonstrated pronounced effectiveness in fighting against drug-resistant P. aeruginosa strains. This review highlights the mechanisms of antibiotic resistance in P. aeruginosa and discusses the current state of some novel therapeutic approaches for treatment of P. aeruginosa infections that can be further explored in clinical practice.

CXCL4 contributes to host defense against acute Pseudomonas aeruginosa lung infection.

Platelets have been implicated in pulmonary inflammation following exposure to bacterial stimuli. The mechanisms involved in the platelet-mediated host response to respiratory bacterial infection remain incompletely understood. In this study, we demonstrate that platelet-derived chemokine (C-X-C motif) ligand 4 (CXCL4) plays critical roles in a mouse model of acute bacterial pneumonia using Pseudomonas aeruginosa. Platelets are activated during P. aeruginosa infection, and mice depleted of platelets display markedly increased mortality and impaired bacterial clearance. CXCL4 deficiency impairs bacterial clearance and lung epithelial permeability, which correlate with decreased neutrophil recruitment to BALF. Interestingly, CXCL4 deficiency selectively regulates chemokine production, suggesting that CXCL4 has an impact on other chemokine expression. In addition, CXCL4 deficiency reduces platelet-neutrophil interactions in blood following P. aeruginosa infection. Further studies revealed that platelet-derived CXCL4 contributes to the P. aeruginosa-killing of neutrophils. Altogether, these findings demonstrate that CXCL4 is a vital chemokine that plays critical roles in bacterial clearance during P. aeruginosa infection through recruiting neutrophils to the lungs and intracellular bacterial killing.

Regulator of calcineurin 1 differentially regulates TLR-dependent MyD88 and TRIF signaling pathways.

Toll-like receptors (TLRs) recognize the conserved molecular patterns in microorganisms and trigger myeloid differentiation primary response 88 (MyD88) and/or TIR-domain-containing adapter-inducing interferon-ß (TRIF) pathways that are critical for host defense against microbial infection. However, the molecular mechanisms that govern TLR signaling remain incompletely understood. Regulator of calcineurin-1 (RCAN1), a small evolutionarily conserved protein that inhibits calcineurin phosphatase activity, suppresses inflammation during Pseudomonas aeruginosa infection. Here, we define the roles for RCAN1 in P. aeruginosa lipopolysaccharide (LPS)-activated TLR4 signaling. We compared the effects of P. aeruginosa LPS challenge on bone marrow-derived macrophages from both wild-type and RCAN1-deficient mice and found that RCAN1 deficiency increased the MyD88-NF-κB-mediated cytokine production (IL-6, TNF and MIP-2), whereas TRIF-interferon-stimulated response elements (ISRE)-mediated cytokine production (IFNß, RANTES and IP-10) was suppressed. RCAN1 deficiency caused increased IκBα phosphorylation and NF-κB activity in the MyD88-dependent pathway, but impaired ISRE activation and reduced IRF7 expression in the TRIF-dependent pathway. Complementary studies of a mouse model of P. aeruginosa LPS-induced acute pneumonia confirmed that RCAN1-deficient mice displayed greatly enhanced NF-κB activity and MyD88-NF-κB-mediated cytokine production, which correlated with enhanced pulmonary infiltration of neutrophils. By contrast, RCAN1 deficiency had little effect on the TRIF pathway in vivo. These findings demonstrate a novel regulatory role of RCAN1 in TLR signaling, which differentially regulates MyD88 and TRIF pathways.

The calcineurin-NFAT axis contributes to host defense during Pseudomonas aeruginosa lung infection.

Infection with the opportunistic pathogen Pseudomonas aeruginosa is effectively controlled through tightly coordinated inflammation in healthy individuals. Dysregulated inflammation in cystic fibrosis greatly increases susceptibility to P. aeruginosa and lung damage. Recently, we identified regulator of calcineurin-1, a small, conserved protein that suppresses the NFAT pathway by inhibition of calcineurin and functions as a central negative regulator of multiple inflammatory transcription factors after P. aeruginosa lung infection, implying a role for the canonical NFAT pathway in P. aeruginosa infection. Calcineurin is a calcium-calmodulin-responsive phosphatase that dephosphorylates NFAT and promotes NFAT nuclear translocation and transcriptional activity. The contribution of the NFAT pathway to host defense against P. aeruginosa remains poorly characterized. In this study, we found that NFAT was rapidly and transiently activated after P. aeruginosa infection both in vitro and in vivo. Deficiency of calcineurin Aß caused impaired activation of NFAT and decreased inflammatory cytokine production in vivo. Finally, we demonstrated that the cross-talk between the NFAT and NFкB pathways coordinately transactivate host response genes during P. aeruginosa infection. Together, these results demonstrate for the first time that NFAT is activated through calcineurin and interacts with NFкB after P. aeruginosa lung infection, and contributes to the host inflammatory response.

Decidual Stromal Cell Necroptosis Contributes to Polyinosinic-Polycytidylic Acid-Triggered Abnormal Murine Pregnancy.

Infectious agents can reach the placenta either via the maternal blood or by ascending the genito-urinary tract, and then initially colonizing the maternal decidua. Decidual stromal cells (DSCs) are the major cellular component of the decidua. Although DSCs at the maternal-fetal interface contribute to the regulation of immunity in pregnancy in the face of immunological and physiological challenges, the roles of these DSCs during viral infection remain ill defined. Here, we characterized the response of DSCs to a synthetic double-stranded RNA molecule, polyinosinic-polycytidylic acid [poly(I:C)], which is a mimic of viral infection. We demonstrated that both transfection of cells with poly(I:C) and addition of extracellular (non-transfected) poly(I:C) trigger the necroptosis of DSCs and that this response is dependent on RIG-I-like receptor/IPS-1 signaling and the toll-like receptor 3/TIR-domain-containing adapter-inducing interferon-ß pathway, respectively. Furthermore, following poly(I:C) challenge, pregnant mixed lineage kinase domain-like protein-deficient mice had fewer necrotic cells in the mesometrial decidual layer, as well as milder pathological changes in the uterine unit, than did wild-type mice. Collectively, our results establish that necroptosis is a contributing factor in poly(I:C)-triggered abnormal pregnancy and thereby indicate a novel therapeutic strategy for reducing the severity of the adverse effects of viral infections in pregnancy.

IL-17RC is critically required to maintain baseline A20 production to repress JNK isoform-dependent tumor-specific proliferation.

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.

RCAN1 deficiency protects against Salmonella intestinal infection by modulating JNK activation.

OBJECTIVE: RCAN1 (regulator of calcineurin 1) has been shown to be involved in various physiological and pathological processes. However, the biological implications of RCAN1 during gastrointestinal tract infection remain unclear. In this study, we tried to determine the role of RCAN1 in acute Salmonella infectious colitis. METHODS: Wild type and RCAN1-deficient mice or macrophages were used to characterize the impacts of RCAN1 on intestinal inflammation, inflammatory cytokines production, animal survival, and pathogen clearance following Salmonella challenge. RESULTS: Histologic and quantitative assessments showed increased inflammation and elevated proinflammatory cytokines production in RCAN1-deficient mice. The aberrant inflammatory response was recapitulated in primary bone marrow-derived macrophages. In addition, we reveal a novel regulatory role for RCAN1 in the proinflammatory JNK signaling both in vitro and in vivo. Further analysis showed that the increased inflammation in RCAN1-deficient mice contributed to pathogen clearance and host survival. CONCLUSIONS: The present study demonstrates that RCAN1 deficiency protects against Salmonella intestinal infection by enhancing proinflammatory JNK signaling.

Protein tyrosine phosphatase 1B (PTP1B) is dispensable for IgE-mediated cutaneous reaction in vivo.

Mast cells play a critical role in allergic reactions. The cross-linking of FcεRI-bound IgE with multivalent antigen initiates a cascade of signaling events leading to mast cell activation. It has been well-recognized that cross linking of FcεRI mediates tyrosine phosphorylation. However, the mechanism involved in tyrosine dephosphorylation in mast cells is less clear. Here we demonstrated that protein tyrosine phosphatase 1B (PTP1B)-deficient mast cells showed increased IgE-mediated phosphorylation of the signal transducer and activator of transcription 5 (STAT5) and enhanced production of CCL9 (MIP-1γ) and IL-6 in IgE-mediated mast cells activation in vitro. However, IgE-mediated calcium mobilization, ß-hexaosaminidase release (degranulation), and phosphorylation of IκB and MAP kinases were not affected by PTP1B deficiency. Furthermore, PTP1B deficient mice showed normal IgE-dependent passive cutaneous anaphylaxis and late phase cutaneous reactions in vivo. Thus, PTP1B specifically regulates IgE-mediated STAT5 pathway, but is redundant in influencing mast cell function in vivo.

Protein Tyrosine Phosphatase-1B Negatively Impacts Host Defense against Pseudomonas aeruginosa Infection.

Pseudomonas aeruginosa is a major opportunistic pathogen in immune-compromised individuals. Mechanisms governing immune responses to P. aeruginosa infection remain incompletely defined. Herein, we demonstrate that protein tyrosine phosphatase-1B (PTP1B) is a critical negative regulator in P. aeruginosa infection. PTP1B-deficient mice display greatly enhanced bacterial clearance and reduced disease scores, which are accompanied by increased neutrophil infiltration and cytokine production. Interestingly, PTP1B deficiency mainly up-regulates the production of interferon-stimulated response elements-regulated cytokines and chemokines, including chemokine ligand 5 (regulated on activation normal T cell expressed and secreted), CXCL10 (interferon γ-inducible protein 10), and interferon-ß production. Further studies reveal that PTP1B deficiency leads to increased interferon regulatory factor 7 (IRF7) expression and activation. These findings demonstrate a novel regulatory mechanism of the immune response to P. aeruginosa infection through PTP1B-IRF7 interaction. This novel PTP1B-IRF7-interferon-stimulated response elements pathway may have broader implications in Toll-like receptor-mediated innate immunity.

Mast cells protect against Pseudomonas aeruginosa-induced lung injury.

Pseudomonas aeruginosa, an opportunistic pathogen, is the leading cause of morbidity and mortality in immune-compromised individuals. Maintaining the integrity of the respiratory epithelium is critical for an effective host response to P. aeruginosa. Given the close spatial relationship between mast cells and the respiratory epithelium, and the importance of tightly regulated epithelial permeability during lung infections, we examined whether mast cells influence airway epithelial integrity during P. aeruginosa lung infection in a mouse model. We found that mast cell-deficient Kit(W-sh)/Kit(W-sh) mice displayed greatly increased epithelial permeability, bacterial dissemination, and neutrophil accumulation compared with wild-type animals after P. aeruginosa infection; these defects were corrected on reconstitution with mast cells. An in vitro Transwell co-culture model further demonstrated that a secreted mast cell factor decreased epithelial cell apoptosis and tumor necrosis factor production after P. aeruginosa infection. Together, our data demonstrate a previously unrecognized role for mast cells in the maintenance of epithelial integrity during P. aeruginosa infection, through a mechanism that likely involves prevention of epithelial apoptosis and tumor necrosis factor production. Our understanding of mechanisms of the host response to P. aeruginosa will open new avenues for the development of successful preventative and treatment strategies.

Calpain-1 contributes to IgE-mediated mast cell activation.

Mast cells play a central role in allergy through secretion of both preformed and newly synthesized mediators. Mast cell mediator secretion is controlled by a complex network of signaling events. Despite intensive studies, signaling pathways in the regulation of mast cell mediator secretion remain incompletely defined. In this study, we examined the role of calpain in IgE-dependent mast cell activation. IgE-mediated activation of mouse bone marrow-derived mast cells enhanced calpain activity. Inhibition of calpain activity by a number of calpain inhibitors reduced IgE-mediated mast cell degranulation both in vitro and in vivo. Calpain inhibitors blocked IgE-mediated TNF and IL-6 production in vitro and reduced late-phase allergic response in vivo. Importantly, mouse calpain-1 null bone marrow-derived mast cells showed reduced IgE-mediated mast cell degranulation in vitro and in vivo, diminished cytokine and chemokine production in vitro, and impaired late-phase allergic response in vivo. Further studies revealed that calpain-1 deficiency led to specific attenuation of IκB-NF-κB pathway and IKK-SNAP23 pathway, whereas calcium flux, MAPK, Akt, and NFAT pathway proceed normally in IgE-activated calpain-1 null mast cells. Thus, calpain-1 is identified as a novel regulator in IgE-mediated mast cell activation and could serve as a potential therapeutic target for the management of allergic inflammation.

Stem cell factor induces AP-1-dependent mast cell IL-6 production via MAPK kinase 3 activity.

Mast cells are critical immune effectors abundant in tissues interfacing with the environment and have major roles in allergen-induced inflammation and host responses to infection. SCF is a regulator of mast cell function and growth. However, the critical mechanisms in SCF-directed events remain incompletely defined. Here, we have investigated the role of MKK3 in mast cell SCF signaling-dependent functions by using BMMCs from MKK3-deficient mice. MKK3 was phosphorylated rapidly and persistently following SCF-induced activation and contributed to mast cell proliferation but not survival or migration in response to SCF. Analysis of SCF-induced mast cell mediator secretion demonstrated that IL-6 production is specifically dependent on MKK3 signals, both independently and in concert with IgE. Analysis of SCF-induced signaling showed that sustained p38 phosphorylation was impaired in MKK3-deficient mast cells, where as early JNK and IκBα activation were enhanced. Notably, SCF-inducible expression and activation of c-Jun, a component of the AP-1 transcription factor, was significantly dependent on MKK3. Accordingly, AP-1 DNA-binding activity and interaction with the IL6 gene promoter was markedly impaired in MKK3-deficient mast cells, whereas transcription factors of the Egr family, NF-κB, and NFAT retained near-full activity. These results designate MKK3 as a novel, positive regulator of SCF-induced mast cell proliferation and a critical signaling protein for AP-1-dependent IL-6 production.

The emerging potential of autophagy-based therapies in the treatment of cystic fibrosis lung infections.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), a channel that normally transports anions across epithelial cell membranes. The most common manifestation of CF is buildup of mucus in the airways and bacterial colonization of the lower respiratory tract, accompanied by chronic inflammation. Antibiotics are used to control CF-associated opportunistic infections, but lengthy antibiotic treatment risks the emergence of multiple-drug resistant (MDR) strains. New antimicrobial strategies are needed to prevent and treat infections in these high-risk individuals. Autophagy contributes to the control of a variety of microbial infections. For this reason, the recent discovery of functional impairment of autophagy in CF provides a new basis for understanding susceptibility to severe infections. Here, we review the role of autophagy in host defense against CF-associated bacterial and fungal pathogens, and survey pharmacologic approaches to restore normal autophagy function in these individuals. Autophagy restoration therapy may improve pathogen clearance and mitigate lung inflammation in CF airways.

MAPK kinase 3 is a tumor suppressor with reduced copy number in breast cancer.

Cancers are initiated as a result of changes that occur in the genome. Identification of gains and losses in the structure and expression of tumor-suppressor genes and oncogenes lies at the root of the understanding of cancer cell biology. Here, we show that the mitogen-activated protein kinase (MAPK) MKK3 suppresses the growth of breast cancer, in which it varies in copy number. A pervasive loss of MKK3 gene copy number in patients with breast cancer is associated with an impairment of MKK3 expression and protein level in malignant tissues. To assess the functional role of MKK3 in breast cancer, we showed in an animal model that MKK3 activity is required for suppression of tumor growth. Active MKK3 enhanced expression of the cyclin-dependent kinase inhibitors p21(Cip1/Waf1) and p27(Kip1), leading to increased cell-cycle arrest in G1 phase of the cell cycle. Our results reveal the functional significance of MKK3 as a tumor suppressor and improve understanding of the dynamic role of the MAPK pathway in tumor progression.

Calcineurin-Rcan1 interaction contributes to stem cell factor-mediated mast cell activation.

The receptor for stem cell factor (SCF) is expressed on mast cells and hematopoietic progenitors. SCF-induced signaling pathways remain incompletely defined. In this study, we identified calcineurin and regulator of calcineurin 1 (Rcan1) as novel components in SCF signaling. Calcineurin activity was induced in SCF-stimulated primary mouse and human mast cells. NFAT was activated by SCF in bone marrow-derived mast cells (BMMCs) and mouse bone marrow cells, which contain hematopoietic progenitors. SCF-mediated activation also induced expression of Rcan1 in BMMCs. Rcan1-deficient BMMCs showed increased calcineurin activity and enhanced transcriptional activity of NF-κB and NFAT, resulting in increased IL-6 and TNF production following SCF stimulation. These results suggest that Rcan1 suppresses SCF-induced activation of calcineurin and NF-κB. We further demonstrated that SCF-induced Rcan1 expression is dependent on the transcription factor early growth response 1 (Egr1). Interestingly, SCF-induced Egr1 was also suppressed by Rcan1, suggesting a negative regulatory loop between Egr1 and Rcan1. Together, our findings revealed that calcineurin contributes to SCF-induced signaling, leading to NFAT activation, which, together with NF-κB and Egr1, is suppressed by Rcan1. Considering the wide range of biological functions of SCF, these novel regulatory mechanisms in SCF signaling may have broad implications.

Autophagy enhances bacterial clearance during P. aeruginosa lung infection.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen which is the leading cause of morbidity and mortality among cystic fibrosis patients. Although P. aeruginosa is primarily considered an extacellular pathogen, recent reports have demonstrated that throughout the course of infection the bacterium acquires the ability to enter and reside within host cells. Normally intracellular pathogens are cleared through a process called autophagy which sequesters and degrades portions of the cytosol, including invading bacteria. However the role of autophagy in host defense against P. aeruginosa in vivo remains unknown. Understanding the role of autophagy during P. aeruginosa infection is of particular importance as mutations leading to cystic fibrosis have recently been shown to cause a blockade in the autophagy pathway, which could increase susceptibility to infection. Here we demonstrate that P. aeruginosa induces autophagy in mast cells, which have been recognized as sentinels in the host defense against bacterial infection. We further demonstrate that inhibition of autophagy through pharmacological means or protein knockdown inhibits clearance of intracellular P. aeruginosa in vitro, while pharmacologic induction of autophagy significantly increased bacterial clearance. Finally we find that pharmacological manipulation of autophagy in vivo effectively regulates bacterial clearance of P. aeruginosa from the lung. Together our results demonstrate that autophagy is required for an effective immune response against P. aeruginosa infection in vivo, and suggest that pharmacological interventions targeting the autophagy pathway could have considerable therapeutic potential in the treatment of P. aeruginosa lung infection.