Inhaled aerosolized nicotine suppresses the lung eosinophilic response to house dust mite allergen.

Nicotine of unprecedented concentrations and purity is being inhaled by those using commercially available electronic nicotine delivery systems (ENDS). The consequences of this route of self-administration on the immunological response to inhaled allergens are not known. In mice, sensitization and inhalation challenge with the common environmental house dust mite (HDM) allergen is an experimental model of this response. When mice were exposed to aerosolized nicotine base (aeroNic) twice daily, 5 days/wk for 8 wk, the HDM-induced recruitment of eosinophils (EOS) was substantially reduced as measured in bronchial alveolar lavage fluid (BALF). Oral nicotine administration had no effect. HDM challenge in the presence of nicotinic receptor subtype α7 (α7)-specific type-1 positive allosteric modulators (PAMs) was alone sufficient to suppress EOS. RNA analysis of alveolar macrophages (AM) collected from BALF after HDM challenge of aeroNic revealed that α7 activation strongly suppresses initiation of Ccl24 (eotaxin 2) transcription. To examine possible cellular signaling mechanisms coupling α7 to Ccl24 transcription, an AM culture model system was used. In AM cultures of freshly collected BALF, Ccl24 transcription was robustly activated by a mixture of IL-4 and IL-10, and this was suppressed by coapplication of type-1 PAMs through a pathway that requires p38MAPK but is independent of Jak2. These results suggest that the EOS response to HDM inhaled allergen is subject to modulation through activation of the α7 receptor and suggest that the allergic response may be substantially modified in ENDS users.

Lung eosinophilia induced by house dust mites or ovalbumin is modulated by nicotinic receptor α7 and inhibited by cigarette smoke.

Eosinophilia (EOS) is an important component of airway inflammation and hyperresponsiveness in allergic reactions including those leading to asthma. Although cigarette smoking (CS) is a significant contributor to long-term adverse outcomes in these lung disorders, there are also the curious reports of its ability to produce acute suppression of inflammatory responses including EOS through poorly understood mechanisms. One possibility is that proinflammatory processes are suppressed by nicotine in CS acting through nicotinic receptor α7 (α7). Here we addressed the role of α7 in modulating EOS with two mouse models of an allergic response: house dust mites (HDM; Dermatophagoides sp.) and ovalbumin (OVA). The influence of α7 on EOS was experimentally resolved in wild-type mice or in mice in which a point mutation of the α7 receptor (α7E260A:G) selectively restricts normal signaling of cellular responses. RNA analysis of alveolar macrophages and the distal lung epithelium indicates that normal α7 function robustly impacts gene expression in the epithelium to HDM and OVA but to different degrees. Notable was allergen-specific α7 modulation of Ccl11 and Ccl24 (eotaxins) expression, which was enhanced in HDM but suppressed in OVA EOS. CS suppressed EOS induced by both OVA and HDM, as well as the inflammatory genes involved, regardless of α7 genotype. These results suggest that EOS in response to HDM or OVA is through signaling pathways that are modulated in a cell-specific manner by α7 and are distinct from CS suppression.

Prenatal ablation of nicotinic receptor alpha7 cell lineages produces lumbosacral spina bifida the severity of which is modified by choline and nicotine exposure.

Lumbosacral spina bifida is a common debilitating birth defect whose multiple causes are poorly understood. Here, we provide the first genetic delineation of cholinergic nicotinic receptor alpha7 (Chrna7) expression and link the ablation of the Chrna7 cell lineage to this condition in the mouse. Using homologous recombination, an IRES-Cre bi-cistronic cassette was introduced into the 3' noncoding region of Chrna7 (Chrna7:Cre) for identifying cell lineages expressing this gene. This lineage first appears at embryonic day E9.0 in rhombomeres 3 and 5 of the neural tube and extends to cell subsets in most tissues by E14.5. Ablation of the Chrna7:Cre cell lineage in embryos from crosses with conditionally expressed attenuated diphtheria toxin results in precise developmental defects including omphalocele (89%) and open spina bifida (SB; 80%). We hypothesized that like humans, this defect would be modified by environmental compounds not only folic acid or choline but also nicotine. Prenatal chronic oral nicotine administration substantially worsened the defect to often include the rostral neural tube. In contrast, supplementation of the maternal diet with 2% choline decreased SB prevalence to 38% and dramatically reduced the defect severity. Folic acid supplementation only trended towards a reduced SB frequency. The omphalocele was unaffected by these interventions. These studies identify the Chrna7 cell lineage as participating in posterior neuropore closure and present a novel model of lower SB that can be substantially modified by the prenatal environment.

Nicotinic receptor subunit alpha5 modifies assembly, up-regulation, and response to pro-inflammatory cytokines.

In the mammalian brain high affinity nicotine-binding sites are composed of at least the alpha4 and beta2 subunits. Additional nicotinic acetylcholine receptor subunits that are often co-expressed with alpha4+beta2 include alpha5. The introduction of alpha5 into 293 cells expressing alpha4+beta2 strongly favors assembly of alpha4+alpha5+beta2 receptors, increases constitutive ligand binding density as measured using [(3)H]epibatidine, but reduces the magnitude of up-regulation in response to chronic nicotine. In contrast, when beta4 is substituted for beta2, alpha5 interferes with the assembly of these receptors, demonstrating an important role for the beta subunit in this process. When cells co-express alpha4+alpha5+beta2+beta4, over 50% of the subunit associations include all four subunits, but they fail to be detected using [(3)H]epibatidine binding. However, complexes of alpha4+alpha5+beta2 do preferentially emerge from these subunit mixtures, and these mixtures bind ligand. In previous studies of alpha4+beta2+beta4 co-expression by 293 cells, the inflammatory cytokines IL-1beta and TNFalpha influenced the outcome of receptor assembly (Gahring, L. C., Days, E. L., Kaasch, T., González de Mendoza, M., Owen, L., Persiyanov, K., and Rogers, S. W. (2005) J. Neuroimmunol. 166, 88-101). When alpha5 is included in this subunit mixture, and cells are exposed to either inflammatory cytokine, subunit association is no longer altered. These findings suggest that alpha5 is an influential modulator of alpha4+beta2 nicotinic acetylcholine receptor assembly and stabilizes their expression in response to fluctuations in external conditions.

Choline promotes nicotinic receptor alpha4 + beta2 up-regulation.

Neuronal nicotinic acetylcholine receptors (nAChR) composed of alpha4 + beta2 subunits, the high affinity nicotine-binding site in the mammalian brain, up-regulate in response to chronic nicotine exposure. The identities of endogenous mediators of this process are unknown. We find that choline also up-regulates alpha4 + beta2 nAChRs stably expressed by HEK293 cells as measured by increased [(3)H]epibatidine density. Choline-mediated up-regulation is dose-dependent and corresponds with an increase in beta2 subunit protein expression. The choline kinase inhibitor hemicholinium-3 inhibits approximately 60% of choline-mediated up-regulation revealing both an HC3-dependent and -independent pathway. Furthermore, choline-mediated up-regulation is not additive with up-regulation agents such as nicotine, but it is additive with weaker promoters of the up-regulation process. When co-applied with the pro-inflammatory cytokine tumor necrosis factor alpha, choline-mediated up-regulation is increased further through a mechanism that includes an increase in both alpha4 and beta2 protein expression, and this is inhibited by the p38 MAPK inhibitor SB202190. These findings extend the view that up-regulation of alpha4 + beta2 nAChRs is a normal physiological response to altered metabolic and inflammatory conditions.

A candidate gene approach identifies the CHRNA5-A3-B4 region as a risk factor for age-dependent nicotine addiction.

People who begin daily smoking at an early age are at greater risk of long-term nicotine addiction. We tested the hypothesis that associations between nicotinic acetylcholine receptor (nAChR) genetic variants and nicotine dependence assessed in adulthood will be stronger among smokers who began daily nicotine exposure during adolescence. We compared nicotine addiction-measured by the Fagerstrom Test of Nicotine Dependence-in three cohorts of long-term smokers recruited in Utah, Wisconsin, and by the NHLBI Lung Health Study, using a candidate-gene approach with the neuronal nAChR subunit genes. This SNP panel included common coding variants and haplotypes detected in eight alpha and three beta nAChR subunit genes found in European American populations. In the 2,827 long-term smokers examined, common susceptibility and protective haplotypes at the CHRNA5-A3-B4 locus were associated with nicotine dependence severity (p = 2.0x10(-5); odds ratio = 1.82; 95% confidence interval 1.39-2.39) in subjects who began daily smoking at or before the age of 16, an exposure period that results in a more severe form of adult nicotine dependence. A substantial shift in susceptibility versus protective diplotype frequency (AA versus BC = 17%, AA versus CC = 27%) was observed in the group that began smoking by age 16. This genetic effect was not observed in subjects who began daily nicotine use after the age of 16. These results establish a strong mechanistic link among early nicotine exposure, common CHRNA5-A3-B4 haplotypes, and adult nicotine addiction in three independent populations of European origins. The identification of an age-dependent susceptibility haplotype reinforces the importance of preventing early exposure to tobacco through public health policies.

Neuronal nicotinic alpha7 receptors modulate early neutrophil infiltration to sites of skin inflammation.

BACKGROUND: A major site of initiation of inflammatory responses upon physical perturbation(s) and infection by invading organisms is the skin. Control of responses in this organ is, in part, modulated by the neuronal nicotinic acetylcholine receptor (nAChR) alpha7. METHODS: To further investigate the role of alpha7 in skin inflammatory responses, a local inflammatory response was induced by topical application of croton oil to the ear skin of wild-type (alpha7WT) and alpha7 knock-out (alpha7KO) mice. Cells infiltrating the inflamed tissue were characterized by flow cytometry and RNA analysis. RESULTS: Six hours following croton oil application, analysis of infiltrating cells showed that the alpha7KO mice exhibited a significantly enhanced number of cells, and specifically, of Ly6G positive neutrophils. Macrophage and lymphocyte infiltration was equivalent in the alpha7KO and alpha7WT mice. RNA analysis showed that IL-1beta and IL-6 were increased significantly in the infiltrating cells of the alpha7KO mouse, although TNF failed to reach significance. In contrast, resident cells of the skin exhibited no differences in these cytokines between genotypes. Both resident and infiltrating cell populations from alpha7KO mice did show elevated message levels for the adhesion protein ICAM1. Measurement of chemokines revealed enhanced expression of the skin-related CCL27 by resident cells in alpha7KO mice. Further, we demonstrate that the population of Ly6G+ neutrophils at the croton oil-inflamed skin site expresses low levels of CCR10, a receptor for CCL27 normally associated with lymphocytes. CONCLUSION: nAChRalpha7 in the skin can impact on early local inflammatory responses mediated through a novel population of neutrophils that are Ly6G+CCR10lo.

Age-Associated Tooth Loss and Oral Microbial Dysbiosis in a Mouse Genetic Model of Chronic Nicotine Exposure.

Nicotine acts as a potent modulator of normal cellular responses through the nicotinic acetylcholine receptor subtype alpha7. In a mouse genetic model of alpha7 receptor dysfunction, alpha7E260A:G, 85 percent of 18 month-old mice exhibit an age-associated spontaneous loosening or complete loss of 3rd molars that was not present in the control mice. The adjacent soft tissues appeared largely unaffected. Further analysis including micro-CT revealed evidence of bone loss surrounding the 3rd molars with areas of cavitation and/or sponge-like (cancellous) bone remodeling in the mandible. The mandible microbiome was examined using 16S-rRNA sequencing. The results show the alpha7E260A:G oral microbiome included increased landscape complexity indicative of dysbiosis, and a significant increase of some bacteria, particularly Staphylococcus. These results suggest that normal alpha7 function plays a relevant role in maintaining normal gene expression and oral microbiome stasis. Consequently, this mouse model suggests there are consequences to ongoing alpha7 receptor dysfunction and oral health, as can occur from chronic exposure to nicotine as expected from electronic nicotine delivery systems (ENDS or "vaping"), that may not be seen until older age.

Nicotinic acetylcholine receptor expression in the hippocampus of 27 mouse strains reveals novel inhibitory circuitry.

Mouse strains are well-characterized to exhibit differences in their physiological and behavioral responses to nicotine. This report examines the expression of the high-affinity nicotine binding receptor subunit, neuronal nicotinic receptor subunit alpha 4 (nAChR alpha 4), in the dorsal hippocampus of 27 inbred mouse strains. Multiple differences among mouse strains in the cellular expression of nAChR alpha 4 between subregions of the hippocampal field are evident. Differences that we describe in the expression of nAChR alpha 4 suggest mouse strains of diverse genetic origin could exhibit significant variation in how this receptor contributes to modulating intrahippocampal circuitry. These findings define a genetic frame-work in which the strain-specific responses to nicotine include underlying contributions by the varied anatomical context in which nAChRs are expressed.

Mouse chromosome 11 harbors genetic determinants of hippocampal strain-specific nicotinic receptor expression.

Differences between isogenic mouse strains in cellular expression of the neuronal nicotinic acetylcholine (ACh) receptor subunit alpha 4 (nAChR alpha 4) by the dorsal hippocampus are well known. To investigate further the genetic basis of these variations, expression of the nAChR alpha 4 subunit was measured in congenic mouse lines derived from two strains exhibiting notable divergence in the expression of this subunit: C3H and C57BL/6. Congenic lines carrying reciprocally introgressed regions (quantitative trait loci; QTL) from chromosomes 4, 5, and 12 each retained the phenotype most closely associated with the parental strain. However, in congenic lines harboring the reciprocal transfer of a chromosome 11 QTL, a characteristic difference in the ratio of interneurons versus astrocytes expressing nAChR alpha 4 in the CA1 region is reversed relative to the parental strain. These finding suggest that this chromosomal segment harbors genes that regulate strain distinct hippocampal morphology that is revealed by nAChR alpha 4 expression.

Neuronal nicotinic alpha7 receptors modulate inflammatory cytokine production in the skin following ultraviolet radiation.

The anti-inflammatory effects of the neuronal nicotinic receptor alpha7 (nAChRalpha7) are proposed to require acetylcholine release from vagal efferents. The necessity for vagal innervation in this anti-inflammatory pathway was tested in the skin, which lacks parasympathetic innervation, using ultraviolet radiation (UVB) to induce a local pro-inflammatory response. Cytokine responses to UV in mice administered chronic oral nicotine, a nAChR agonist, were reduced. Conversely, nAChRalpha7 knock-out mice exposed to UVB elicit an enhanced pro-inflammatory cytokine response in the skin. Altered pro-inflammatory responses correlated with changes in SOCS3 protein. These results demonstrate that nAChRalpha7 can participate in modulating a local pro-inflammatory response in the absence of parasympathetic innervation.

Lung epithelial response to cigarette smoke and modulation by the nicotinic alpha 7 receptor.

Cigarette smoking (CS) is a principal contributor to a spectrum of devastating lung diseases whose occurrence and severity may vary between individuals and not appear for decades after prolonged use. One explanation for the variability and delay in disease onset is that nicotine, the addictive component of CS, acts through the ionotropic nicotinic acetylcholine receptor (nAChR) alpha7 (α7) to modulate anti-inflammatory protection. In this study we measured the impact α7 signaling has on the mouse distal lung response to side-stream CS exposure for mice of the control genotype (α7G) and those in which the α7-receptor signaling mechanisms are restricted by point mutation (α7E260A:G). Flow cytometry results show that after CS there is an increase in a subset of CD11c (CD11chi) alveolar macrophages (AMs) and histology reveals an increase in these cells within the alveolar space in both genotypes although the α7E260A:G AMs tend to accumulate into large aggregates rather than more widely distributed solitary cells common to the α7G lung after CS. Changes to lung morphology with CS in both genotypes included increased tissue cavitation due to alveolar expansion and bronchial epithelium dysplasia in part associated with altered club cell morphology. RNA-Seq analysis revealed changes in epithelium gene expression after CS are largely independent of the α7-genotype. However, the α7E260A:G genotype did reveal some unique variations to transcript expression of gene sets associated with immune responsiveness and macrophage recruitment, hypoxia, genes encoding mitochondrial respiration complex I and extracellular fibrillary matrix proteins (including alterations to fibrotic deposits in the α7G proximal airway bronchioles after CS). These results suggest α7 has a central role in modulating the response to chronic CS that could include altering susceptibility to associated lung diseases including fibrosis and cancer.

Nicotinic alpha 7 receptor expression and modulation of the lung epithelial response to lipopolysaccharide.

Nicotine modulates multiple inflammatory responses in the lung through the nicotinic acetylcholine receptor subtype alpha7 (α7). Previously we reported that α7 modulates both the hematopoietic and epithelium responses in the lung to the bacterial inflammogen, lipopolysaccharide (LPS). Here we apply immunohistochemistry, flow cytometry and RNA-Seq analysis of isolated distal lung epithelium to further define α7-expression and function in this tissue. Mouse lines were used that co-express a bicistronic tau-green fluorescent protein (tGFP) as a reporter of α7 (α7G) expression and that harbor an α7 with a specific point mutation (α7E260A:G) that selectively uncouples it from cell calcium-signaling mechanisms. The tGFP reporter reveals strong cell-specific α7-expression by alveolar macrophages (AM), Club cells and ATII cells. Ciliated cells do not express detectible tGFP, but their numbers decrease by one-third in the α7E260A:G lung compared to controls. Transcriptional comparisons (RNA-Seq) between α7G and α7E260A:G enriched lung epithelium 24 hours after challenge with either intra-nasal (i.n.) saline or LPS reveals a robust α7-genotype impact on both the stasis and inflammatory response of this tissue. Overall the α7E260A:G lung epithelium exhibits reduced inflammatory cytokine/chemokine expression to i.n. LPS. Transcripts specific to Club cells (e.g., CC10, secretoglobins and Muc5b) or to ATII cells (e.g., surfactant proteins) were constitutively decreased in in the α7E260A:G lung, but they were strongly induced in response to i.n. LPS. Protein analysis applying immunohistochemistry and ELISA also revealed α7-associated differences suggested by RNA-Seq including altered mucin protein 5b (Muc5b) accumulation in the α7E260A:G bronchia, that in some cases appeared to form airway plugs, and a substantial increase in extracellular matrix deposits around α7E260A:G airway bronchia linings that was not seen in controls. Our results show that α7 is an important modulator of normal gene expression stasis and the response to an inhaled inflammogen in the distal lung epithelium. Further, when normal α7 signaling is disrupted, changes in lung gene expression resemble those associated with long-term lung pathologies seen in humans who use inhaled nicotine products.

Mouse strain-specific changes in nicotinic receptor expression with age.

The onset and severity of age-related loss of neuronal nicotinic acetylcholine receptor (nAChR) expression in the mammalian hippocampus can vary considerably between individuals. We have examined the expression of four nAChR subunits (nAChR alpha4, nAChR alpha5, nAChR alpha7 and nAChR beta4) in the dorsal hippocampus of adult (12-14 months) and aged (24-28 month) animals from two-mouse strains (CBA/J and C57BL/6). The expression of nAChR alpha4 was selectively diminished with age in both strains, and there was a significant loss of nAChR alpha7 in CA1 of aged CBA/J, but not C57BL/6. There was no change in nAChR alpha5 expression with age whereas nAChR beta4 preferentially diminished in the C57BL/6 CA1 region and remained the same or slightly increased in the aged CBA/J. Coincident with the loss of neuronal nAChR alpha4 in the CBA/J strain was a significant age-related increase of nAChR alpha4 staining of astrocytes, most notably in the stratum radiatum. These results suggest that mouse strains of different genetic backgrounds undergo dissimilar age-related changes in the expression of nAChRs.

Pro-inflammatory cytokines modify neuronal nicotinic acetylcholine receptor assembly.

We have examined the impact of the inflammatory cytokines interleukin-1 beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) on assembly of nAChRs from subunit mixtures of nAChRalpha4, beta2 and beta4 transiently transfected into 293 cells. In control transfections approximately 55% of alpha4 associated preferentially with beta4, but less than 15% complexed with beta2 and the remainder was associated with both beta subunits. These relative ratios were modified by pro-inflammatory cytokines. IL-1beta strongly enhanced alpha4/beta2 association and decreased alpha4/beta4, whereas TNFalpha promoted mixed alpha4/beta2/beta4 interactions. These results show that the emerging rules governing assembly of nAChRs are subject to modification by the pro-inflammatory cytokine environment.

Autoimmunity to glutamate receptors in the central nervous system.

Autoimmunity to glutamate receptors (GluR) of the central nervous system offers an attractive model to begin the experimental dissection of the etiology of immune-related neurodegenerative diseases. Here we discuss the known mechanisms of generating autoimmunity to different members of the GluR neurotransmitter receptor family within the unique environment of the brain and how autoantibody-receptor interactions contribute to abnormal neurotransmission and eventually pathology.

Crystallization of the SH2-binding site of p130Cas in complex with Lck, a Src-family kinase.

Cas-family proteins serve as docking proteins in integrin-mediated signal transduction. The founding member of this family, p130Cas, becomes tyrosine-phosphorylated in response to extracellular stimuli such as integrin-mediated cell adhesion and ligand engagement of receptor tyrosine kinases. Cas proteins are large multidomain molecules that transmit signals as intermediaries through interactions with signaling molecules such as FAK and other tyrosine kinases, as well as tyrosine phosphatases. After Cas is tyrosine-phosphorylated, it acts as a docking protein for binding SH2 domains of Src-family kinases. In order to examine the structural basis for a key step in propagation of signals by Cas, one of the major SH2-binding sites of Cas has been crystallized in complex with the SH3-SH2 regulatory domains of the Src-family kinase Lck. Crystallization conditions were identified by high-throughput screening and optimized with multiple rounds of seeding. The crystals formed at 295 K in space group P2(1)2(1)2(1), with unit-cell parameters a = 77.4, b = 107.3, c = 166.4 A, and diffract to 2.7 A resolution.

Upregulation of Nicotinic Acetylcholine Receptor alph4+beta2 through a Ligand-Independent PI3Kbeta Mechanism That Is Enhanced by TNFalpha and the Jak2/p38Mapk Pathways.

High affinity nicotine-binding sites in the mammalian brain are neuronal nicotinic acetylcholine receptors (nAChR) assembled from at least alpha4 and beta2 subunits into pentameric ion channels. When exposed to ligands such as nicotine, these receptors respond by undergoing upregulation, a correlate of nicotine addiction. Upregulation can be measured using HEK293 (293) cells that stably express alpha4 and beta2 subunits using quantification of [3H]epibatidine ([3H]Eb) binding to measure mature receptors. Treatment of these cells with choline also produces upregulation through a hemicholinium3 (HC3)-sensitive (choline kinase) and an HC3-insensitive pathway which are both independent of the mechanism used by nicotine for upregulation. In both cases, upregulation is significantly enhanced by the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) which signals through its receptor Tnfr1 to activate p38Mapk. Here we report that the inhibition of class1 phosphoinositide 3-kinases isoform PI3Kbeta using the selective antagonist PI828 is alone sufficient to produce upregulation and enhance both nicotine and choline HC3-sensitive mediated upregulation. Further, these processes are impacted upon by an AG-490 sensitive Jak2-associated pathway. Both PI3Kbeta (negative) and Jak2 (positive) modulation of upregulation converge through p38Mapk and both overlap with TNFalpha enhancement of this process. Upregulation through the PI3Kbeta pathway did not require Akt. Collectively these findings support upregulation of endogenous alpha4beta2 as a balance among cellular signaling networks that are highly responsive to multiple environmental, inflammatory and metabolic agents. The findings also suggest how illness and metabolic stress could alter the expression of this important nicotinic receptor and novel avenues to intercede in modifying its expression.

Age-related onset of obesity corresponds with metabolic dysregulation and altered microglia morphology in mice deficient for Ifitm proteins.

The IfitmDel mouse lacks all five of the Ifitm genes via LoxP deletion. This animal breeds normally with no obvious defect in development. The IfitmDel animals exhibit a steady and significantly enhanced weight gain relative to wild-type controls beginning about three months of age and under normal feeding conditions. The increased weight corresponds with elevated fat mass, and in tolerance tests they are hyporesponsive to insulin but respond normally to glucose. Both young (4 mo) and older (12 mo) IfitmDel mice have enhanced levels of serum leptin suggesting a defect in leptin/leptin receptor signaling. Analysis of the gene expression profiles in the hypothalamus of IfitmDel animals, compared to WT, demonstrated an altered ratio of Pomc and Npy neuropeptide expression, which likely impairs the satiation response of the IfitmDel animal leading to an increased eating behavior. Also elevated in hypothalamus of IfitmDel mice were pro-inflammatory cytokine expression and reduced IL-10. Anatomical analysis of the hypothalamus using immunohistochemistry revealed that microglia exhibit an abnormal morphology in IfitmDel animals and respond abnormally to Poly:IC challenge. These abnormalities extend the phenotype of the IfitmDel mouse beyond abnormal responses to viral challenge to include a metabolic phenotype and weight gain. Further, this novel phenotype for the IfitmDel mouse could be related to abnormal neuropeptide production, inflammatory status and microglia status in the hypothalamus.

The nicotinic receptor Alpha7 impacts the mouse lung response to LPS through multiple mechanisms.

The nicotinic acetylcholine receptor alpha7 (α7) is expressed by neuronal and non-neuronal cells throughout the body. We examined the mechanisms of the lung inflammatory response to intranasal (i.n.) lipopolysaccharide (LPS) regulated by α7. This was done in mice using homologous recombination to introduce a point mutation in the α7 receptor that replaces the glutamate residue 260 that lines the pore with alanine (α7E260A), which has been implicated in controlling the exceptional calcium ion conductance of this receptor. The α7E260A mice exhibit normal inflammatory cell recruitment to the blood in response to i.n. LPS administration. This differs from the α7knock-out (α7KO) in which upstream signaling to initiate the recruitment to the blood following i.n. LPS is significantly impaired. While hematopoietic cells are recruited to the bloodstream in the α7E260A mouse, they fail to be recruited efficiently into both the interstitium and alveolar spaces of the lung. Bone marrow reconstitution experiments demonstrate that the responsiveness of both CD45+ and CD45- cells of the α7E260A mouse are impaired. The expression of several pro-inflammatory cytokine and chemokine RNAs including TNFα, IL-1α, Ccl2 and Cxcl10 are decreased in the α7E260A mouse. However, there is a substantial increase in IL-13 expression by CD45- lung interstitial cells in the α7E260A mouse. Our results support the conclusion that α7 functional pleiotropy contributes to modulating the tissue response to an inflammatory insult through impacting upon a variety of mechanisms reflecting the individual cell composition of the lung.