Robert D. Junkins

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.

IFN regulatory factor 3 contributes to the host response during Pseudomonas aeruginosa lung infection in mice.

Pseudomonas aeruginosa is a major opportunistic pathogen. However, host defense mechanisms involved in P. aeruginosa lung infection remain incompletely defined. The transcription factor IFN regulatory factor 3 (IRF3) is primarily associated with host defense against viral infections, and a role of IRF3 in P. aeruginosa infection has not been reported previously. In this study, we showed that IRF3 deficiency led to impaired clearance of P. aeruginosa from the lungs of infected mice. P. aeruginosa infection induced IRF3 translocation to the nucleus, activation of IFN-stimulated response elements (ISRE), and production of IFN-β, suggesting that P. aeruginosa activates the IRF3–ISRE–IFN pathway. In vitro, macrophages from IRF3-deficient mice showed complete inhibition of CCL5 (RANTES) and CXCL10 (IP-10) production, partial inhibition of TNF, but no effect on CXCL2 (MIP-2) or CXCL1 (keratinocyte-derived chemokine) in response to P. aeruginosa stimulation. In vivo, IRF3-deficient mice showed complete inhibition of CCL5 production and partial or no effects on production of other cytokines and chemokines in the bronchoalveolar lavage fluids and lung tissues. Profiling of immune cells in the airways revealed that neutrophil and macrophage recruitment into the airspace was reduced, whereas B cell, T cell, NK cell, and NKT cell infiltration was unaffected in IRF3-deficient mice following P. aeruginosa lung infection. These data suggest that IRF3 regulates a distinct profile of cytokines and chemokines and selectively modulates neutrophil and macrophage recruitment during P. aeruginosa infection. Thus, IRF3 is an integral component in the host defense against P. aeruginosa lung infection.

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.

Regulator of Calcineurin 1 Suppresses Inflammation during Respiratory Tract Infections

Respiratory tract infection with Pseudomonas aeruginosa is a common cause of hospitalization in immune-compromised individuals. However, the molecular mechanisms involved in the immune response to P. aeruginosa lung infection remain incompletely defined. In this study, we demonstrate that the regulator of calcineurin 1 (RCAN1) is a central negative regulator of inflammation in a mouse model of acute bacterial pneumonia using the opportunistic bacterial pathogen P. aeruginosa. RCAN1-deficient mice display greatly increased mortality following P. aeruginosa lung infection despite enhanced neutrophil recruitment and bacterial clearance. This mortality is associated with higher systemic levels of proinflammatory cytokines in RCAN1-deficient animals. These aberrant inflammatory responses coincide with increased transcriptional activity of proinflammatory RCAN1-target proteins NFAT and NF-κB. In addition, we reveal a novel regulatory role for RCAN1 in the ERK/STAT3 pathway both in vitro and in vivo, suggesting that aberrant STAT3 activity may significantly contribute to delayed resolution of inflammatory responses in our model. Together, these findings demonstrate that RCAN1 is a potent negative regulator of inflammation during respiratory tract infections.

Regulator of Calcineurin 1 (Rcan1) Is Required for the Development of Pulmonary Eosinophilia in Allergic Inflammation in Mice

The presence of eosinophils in the lung is often regarded as a defining feature of asthma. On allergen stimulation, numbers of eosinophils and their progenitors are increased in both the bone marrow and lungs. Eosinophil progenitors provide an ongoing supply of mature eosinophils. Here, we report that deficiency in the regulator of calcineurin 1 gene (Rcan1) leads to a near-complete absence of eosinophilia in ovalbumin-induced allergic asthma in mice. In the absence of Rcan1, bone marrow cells produce significantly fewer eosinophils in vivo and in vitro on interleukin-5 stimulation. Importantly, eosinophil progenitor populations are significantly reduced in both naïve and ovalbumin-challenged Rcan1(-/-) mice. Bone marrow cells from Rcan1(-/-) mice are capable of developing into fully mature eosinophils, suggesting that Rcan1 is required for eosinophil progenitor production but may not be necessary for eosinophil maturation. Thus, Rcan1 represents a novel contributor in the development of eosinophilia in allergic asthma through regulation of eosinophil progenitor production.

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.

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, wheras 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.

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-β (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 (IFNβ, 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 Aβ 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.