Adam J. MacNeil

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.

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.

MAPK Kinase 3 Specifically Regulates FcεRI-Mediated IL-4 Production by Mast Cells

Mast cells play a central role in allergic inflammation and are activated through cross-linking of FcεRI receptor-bound IgE, initiating a signaling cascade resulting in production of biologically potent mediators. Signaling pathways in the regulation of specific mediators remain incompletely defined. In this study, we examined the role of MAPK kinase 3 (MKK3) in IgE-dependent mast cell activation. In an in vivo model of passive cutaneous anaphylaxis, MKK3-deficient mice showed a deficit in late-phase IgE-dependent inflammation. To characterize the mechanism of this deficiency, we cultured bone marrow-derived mast cells (BMMCs) from wild-type and MKK3-deficient mice. We found that FcεRI-mediated mast cell activation induced rapid MKK3 phosphorylation by 5 min, diminishing slowly after 6 h. In MKK3-deficient BMMCs, phosphorylation of p38 was reduced at early and later time points. Among 40 cytokines tested using a protein array, IL-4 was the only cytokine specifically downregulated in MKK3-deficient BMMCs. Reduced IL-4 expression was seen in the local skin of MKK3-deficient mice following passive cutaneous allergic reaction. Furthermore, early growth response-1 (Egr1) bound to the promoter of IL-4 in FcεRI-activated mast cells, and Egr1 transcription factor activity was diminished in MKK3-deficient BMMCs. Finally, mast cell-deficient mice reconstituted with MKK3-deficient BMMCs displayed a significantly impaired late-phase allergic inflammatory response. Thus, mast cell MKK3 signaling contributes to IgE-dependent allergic inflammation and is a specific regulator of FcεRI-induced IL-4 production.

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.

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.

Male homosexuality and maternal immune responsivity to the Y-linked protein NLGN4Y

Significance Gay men have, on average, a greater number of older brothers than do heterosexual men, a well-known finding within sexual science. This finding has been termed the fraternal birth order effect. Strong scientific interest in sexual orientation exists because it is a fundamental human characteristic, and because its origins are often the focal point of considerable social controversy. Our study is a major advance in understanding the origins of sexual orientation in men by providing support for a theorized but previously unexamined biological mechanism—a maternal immune response to a protein important in male fetal brain development—and by beginning to explain one of the most reliable correlates of male homosexuality: older brothers. We conducted a direct test of an immunological explanation of the finding that gay men have a greater number of older brothers than do heterosexual men. This explanation posits that some mothers develop antibodies against a Y-linked protein important in male brain development, and that this effect becomes increasingly likely with each male gestation, altering brain structures underlying sexual orientation in their later-born sons. Immune assays targeting two Y-linked proteins important in brain development—protocadherin 11 Y-linked (PCDH11Y) and neuroligin 4 Y-linked (NLGN4Y; isoforms 1 and 2)—were developed. Plasma from mothers of sons, about half of whom had a gay son, along with additional controls (women with no sons, men) was analyzed for male protein-specific antibodies. Results indicated women had significantly higher anti-NLGN4Y levels than men. In addition, after statistically controlling for number of pregnancies, mothers of gay sons, particularly those with older brothers, had significantly higher anti-NLGN4Y levels than did the control samples of women, including mothers of heterosexual sons. The results suggest an association between a maternal immune response to NLGN4Y and subsequent sexual orientation in male offspring.

Rosemary extract reduces Akt/mTOR/p70S6K activation and inhibits proliferation and survival of A549 human lung cancer cells.

Compounds of plant origin and food components have attracted scientific attention for use as agents for cancer prevention and treatment. Rosemary extract contains polyphenols that were shown to have anti-cancer and other health benefits. The survival pathways of Akt, mammalian target of rapamycin (mTOR) and p70S6K, and the apoptotic protein poly ADP ribose polymerase (PARP) are key modulators of cancer cell growth and survival. In this study, we examined the effects of rosemary extract on proliferation, survival and apoptosis of human non-small cell lung cancer (NSCLC) cells and its influence on signaling events. Human NSCLC adenocarcinoma A549 cells were used. Cell proliferation and clonogenic survival were assessed using specific assays. Immunoblotting was used to examine total and phosphorylated levels of Akt, mTOR and p70S6K, and cleavage of PARP. Rosemary extract dose-dependently inhibited cell proliferation and reduced clonogenic survival of A549 cells, while PARP cleavage, an indicator of apoptosis, was enhanced. Rosemary extract significantly reduced total and phosphorylated/activated Akt, mTOR and p70S6K levels. In conclusion, rosemary extract inhibited proliferation, blocked clonogenic survival, and enhanced apoptosis of A549 lung cancer cells. These effects were associated with inhibition of Akt and downstream mTOR and p70S6K activity. Our data suggest that rosemary extract may have considerable anti-tumor and chemoprevention properties in lung cancer and deserves further systematic investigation in animal models of lung cancer.

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.

Getting a GRASP on CASP: properties and role of the cytohesin-associated scaffolding protein in immunity.

Cytohesin‐associated scaffolding protein (CASP) is a novel human adaptor protein that participates in the assembly and recruitment of protein complexes associated with intracellular trafficking and signaling. Owing to its exclusive expression in cells of hematopoietic origin, CASP has attracted attention from many groups of researchers as a potential key contributor to molecular mechanisms governing cells of the immune system. The functional characterization of CASP has involved a wide range of experimental approaches and provided broad and interesting insights that, collectively, distinguish CASP as an important contributor for a fully functioning and rapidly responsive immune system. Protein interaction studies have demonstrated that CASP interacts with members of the ADP ribosylation factor (ARF)‐activating cytohesin family and with a unique PDZ domain‐containing member of the sorting nexin family of endocytic trafficking proteins. Physiological knockout studies, however, have revealed that CASP may not be an essential protein in immunity under normal conditions, but rather a streamlining protein that greatly ameliorates the efficiency of the immune system under circumstances of significant stress. Interestingly, an evolutionarily related neuronal protein, called GRP1‐associated scaffolding protein, may further participate in CASP‐related functions in immune cells, conferring a level of redundancy in associated molecular pathways. In this review, we summarize and critically review the current literature, bringing together common themes from a variety of studies that, when considered together, provide new insights into the nature and significance of CASP function in the broad context of immunity.

Gene Duplication in Early Vertebrates Results in Tissue-Specific Subfunctionalized Adaptor Proteins: CASP and GRASP

CASP and GRASP are small cytoplasmic adaptor proteins that share highly similar protein structures as well as an association with the cytohesin/ARNO family of guanine nucleotide exchange factors within the immune and nervous systems respectively. Each contains an N-terminal PDZ domain, a central coiled-coil motif, and a carboxy-terminal PDZ-binding motif (PDZbm). We set out to further characterize the relationship between CASP and GRASP by comparing both their gene structures and their functional motifs across several vertebrate organisms. CASP and GRASP not only share significant protein structure but also share remarkably similar gene structure, with six of eight exons of equal length and relative position. We report on the addition of a unique amino acid within the coiled-coil motif of CASP proteins in several species. We also examine the Class I PDZbm, which is highly conserved across all classes of vertebrates but shows a functionally relevant mutation in the CASP proteins of several species of fish. Further, we determine the evolutionary relationship of these proteins both by use of phylogenetics and by comparative analysis of the conservation of genes near each locus in various chordates including amphioxus. We conclude that CASP and GRASP are the products of a relatively recent gene duplication event in early vertebrate organisms and that the evolution of the adaptive immune system and complex brain most likely contributed to the apparent subfunctionalization of these proteins into tissue-specific roles.