TLR5 Activation Exacerbates Airway Inflammation in Asthma.

INTRODUCTION: Innate immune activation through exposure to indoor and outdoor pollutants is emerging as an important determinant of asthma severity. For example, household levels of the bacterial product lipopolysaccharide (LPS) are associated with increased asthma severity. We hypothesized that activation of the innate immune receptor TLR5 by its bacterial ligand flagellin will exacerbate airway inflammation and asthma symptoms. METHODS: We determined the effect of flagellin co-exposure with ovalbumin in a murine model of allergic asthma. We evaluated the presence of flagellin activity in house dust of asthma patients. Finally, we analyzed the association of a dominant-negative polymorphism in TLR5 (rs5744168) with asthma symptoms in patients with asthma. RESULTS: We showed that bacterial flagellin can be found in the house dust of patients with asthma and that this bacterial product exacerbates allergic airway inflammation in an allergen-specific mouse model of asthma. Furthermore, a dominant-negative genetic polymorphism in TLR5, the receptor for flagellin, is associated with decreased symptoms in patients with asthma. CONCLUSION: Together, our results reveal a novel genetic protective factor (TLR5 deficiency) and a novel environmental pollutant (microbial flagellin) that influence asthma severity. (Clinical trials NCT01688986 and NCT01087307).

The Ataxia telangiectasia gene product is required for oxidative stress-induced G1 and G2 checkpoint function in human fibroblasts.

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by neuronal degeneration accompanied by ataxia, telangiectasias, acute cancer predisposition, and sensitivity to ionizing radiation (IR). Cells from individuals with AT show unusual sensitivity to IR, severely attenuated cell cycle checkpoint functions, and poor p53 induction in response to IR compared with normal human fibroblasts (NHFs). The gene mutated in AT (ATM) has been cloned, and its product, pATM, has IR-inducible kinase activity. The AT phenotype has been suggested to be a consequence, at least in part, of an inability to respond appropriately to oxidative damage. To test this hypothesis, we examined the ability of NHFs and AT dermal fibroblasts to respond to t-butyl hydroperoxide and IR treatment. AT fibroblasts exhibit, in comparison to NHFs, increased sensitivity to the toxicity of t-butyl hydroperoxide, as measured by colony-forming efficiency assays. Unlike NHFs, AT fibroblasts fail to show G(1) and G(2) phase checkpoint functions or to induce p53 in response to t-butyl hydroperoxide. Treatment of NHFs with t-butyl hydroperoxide activates pATM-associated kinase activity. Our results indicate that pATM is involved in responding to certain aspects of oxidative damage and in signaling this information to downstream effectors of the cell cycle checkpoint functions. Our data further suggest that some of the pathologies seen in AT could arise as a consequence of an inability to respond normally to oxidative damage.

Serum starved v-mos-transformed cells are unable to appropriately downregulate cyclins and CDKs.

Serum deprived v-mos-transformed NIH3T3 cells are unable to enter a true quiescent state, but instead, arrest in the early G1 phase of the cell cycle. We have analysed several cell cycle regulatory proteins in these G1 arrested cells and show altered regulation in the expression and activity of certain cyclins and cyclin-dependent kinases. In particular, p34cdc2, cyclin A, cyclin D and cyclin E are not appropriately down-regulated in serum starved, G1 arrested, v-mos-transformed cells as compared with quiescent NIH3T3 cells. Furthermore, serum starved v-mos-transformed cells have elevated histone H1 kinase activity associated with cyclin A, cyclin E, p33cdk2, and p34cdc2. Using a metallothionein-inducible c-mos(mu) expression system, we show that c-mos(mu) induction in quiescent NIH3T3 cells causes elevated expression of p34cdc2. However, this induction of c-mos(mu) and subsequent expression of p34cdc2 was not sufficient to promote significant entry of cells into S phase. Analysis of extracts from serum starved v-H-ras, v-src, and tpr-met transformed NIH3T3 cells demonstrates that these oncogene-transformed cells also contain elevated levels of p34cdc2. We propose that the altered regulation of these critical cell cycle regulatory molecules, and specifically the inability to fully downregulate their activity, contributes significantly to neoplastic transformation and subsequent unregulated growth of tumor cells.

Defective G2 checkpoint function in cells from individuals with familial cancer syndromes.

The early events in the G2 checkpoint response to ionizing radiation (IR) were analyzed in diploid normal human fibroblasts (NHFs) and fibroblasts from patients with two heritable cancer syndromes. Exposure to gamma-radiation of asynchronously growing NHFs resulted in a rapid reduction in the number of cells in mitosis (G2 delay) and was accompanied by a quantitatively similar reduction in the p34CDC2/cyclin B in vitro histone H1 kinase activity as compared with sham-treated controls. This G2 delay was strong by 1 h following exposure to IR, maximal by 2 h, and was accompanied by an accumulation of tyrosine-phosphorylated p34CDC2 molecules. In contrast, fibroblasts from individuals with ataxia telangiectasia displayed significantly less reduction of the mitotic index or histone H1 kinase activity after IR. Low passage fibroblasts from individuals with Li-Fraumeni syndrome having one wild-type and one mutated p53 allele were similar to NHFs in their immediate G2 checkpoint response to IR, as were NHFs expressing the human papilloma virus type 16 E6 gene product (functionally inactivating p53) and low passage cells from p53-deficient mouse embryos. However, the p53-deficient fibroblasts were genomically unstable and became defective in their early G2 checkpoint response to IR. Furthermore, immortal Li-Fraumeni syndrome fibroblasts lacking wild-type p53 displayed an attenuated G2 checkpoint response. These results link the early events in G2 checkpoint response to IR in NHFs with a rapid inhibition of p34CDC2/cyclin B protein kinase activity and demonstrate that while not required for this immediate G2 delay, lack of p53 can lead to subsequent genetic alterations that result in defective G2 checkpoint function.

v-mos-transformed cells fail to enter quiescence but growth arrest in G1 following serum withdrawal.

The product of the mos protooncogene normally functions in the induction of meiosis and regulation of cell-cycle progression in oocytes. Here we have investigated the cell-cycle progression of NIH3T3 cells transformed by the v-mos gene. Flow cytometric analysis showed that logarithmically growing v-mos-transformed cells do not differ from their nontransformed counterparts in the distribution of cells in the G1, S, and G2/M phases. Likewise, after serum withdrawal for 48 h, both normal and v-mos-transformed NIH3T3 cells have essentially ceased proliferation, as analyzed by flow cytometry, [3H]thymidine and BrdU incorporation into newly synthesized DNA, and mitotic indexes. However, while the normal NIH3T3 cells are arrested in a quiescent state, the v-mos-transformed cells are arrested in early to mid G1, prior to the point where cells require certain amino acids for proliferation (V point). In agreement with these different arrest points, the v-mos-transformed cells enter S phase following serum stimulation within about 8 h, without the additional 4- to 6-h lag period characteristically displayed by the parental NIH3T3 cells. In addition, we show a lack of expression of a growth arrest-specific gene product, gas1, in the serum-arrested v-mos-transformed cells. These data demonstrated that v-mos-transformed cells display growth characteristics that differ fundamentally from those of normal cells or cells transformed by overexpression of myc [1]. Our results suggest that the v-mos oncoprotein transforms cells, at least in part, by preventing exit from the cell cycle into quiescence.

Analysis of Fv-1 restriction in two murine embryonal carcinoma cell lines and a series of differentiated derivatives.

We have used antibiotic-resistant retrovirus vectors rescued by Fv-1-sensitive murine leukaemia viruses (MuLV) to examine the Fv-1 phenotype of two undifferentiated embryonal carcinoma (EC) cell lines derived from teratocarcinomas of mouse strain 129. In addition, a set of EC cell-derived differentiated cell lines was analysed. Restriction of both B-tropic and endogenous N-tropic virus is characteristic of the Nr-type restriction reported in mouse strain 129. However, results indicate that Fv-1 restriction is not expressed in the PCC4.aza1R EC cell line. In contrast, the F9 EC cell line showed a strong restriction of the B-tropic pseudotyped vector but failed to restrict endogenous N-tropic pseudotypes. The Fv-1 gene thus seems to be differentially expressed in two EC cell lines derived from the same mouse strain. Furthermore, the selective restriction of B-tropic but not endogenous N-tropic MuLV in F9 cells suggests that these activities function independently of each other. Analysis of PCC4.aza1R-derived differentiated cell lines revealed that three fibroblast cell lines derived by retinoic acid-induced differentiation were also phenotypically silent for Fv-1. However, a pre-adipocyte line established following simultaneous exposure to retinoic acid and 5-azacytidine showed strong restriction of both B-tropic and endogenous N-tropic MuLV. Although additional data suggest that there is no correlation between the differentiated pre-adipocyte phenotype and Fv-1 expression, our results nonetheless show that Nr restriction can be observed in some derivatives of PCC4.aza1R cells, presumably by activating expression of the Fv-1 gene.

Abrogation of Fv-1 restriction by genome-deficient virions produced by a retrovirus packaging cell line.

The Fv-1b-mediated restriction of N-tropic retrovirus vector infection of BALB/3T3 cells was partially abrogated by prior infection with N-tropic murine leukemia virus. Likewise, abrogation of the Fv-1b restriction of N-tropic murine leukemia virus replication was accomplished by prior infection with genome-deficient virions produced by an N-tropic murine leukemia virus packaging cell line. The latter observation suggests that the Fv-1 target in genome-deficient virions abrogates Fv-1 restriction in the absence of any viral genome-directed processes.

Cationic liposomes (Lipofectin) mediate retroviral infection in the absence of specific receptors.

We have used cationic liposomes (Lipofectin) to facilitate retrovirus infection of cells lacking the homologous viral receptor. Ecotropic murine leukemia virus and packaged retroviral vectors were shown to infect mink cells, and amphotropic packaged retroviral vectors were shown to infect hamster cells in the presence of Lipofectin but not in the presence of Polybrene. Lipofectin-mediated infection of cells lacking the homologous receptor results in a titer approximately 0.1% of the titer in cells with the homologous receptor, using the standard Polybrene protocol. The use of Lipofectin may provide a simple means to experimentally infect a wide variety of cells with viruses not normally infectious for the species, tissue, or cell type of interest.

Development and characterization of an Fv-1-sensitive retrovirus-packaging system: single-hit titration kinetics observed in restrictive cells.

We have constructed an RNA-packaging-deficient mutant of N-tropic murine leukemia virus WN1802N by removal of 330 nucleotides located between the upstream long terminal repeat and the start of the gag gene region. Transfection into mink CCL64 cells produced a cell line capable of packaging retrovirus vectors into ecotropic, Fv-1 N-tropic virions. Using retrovirus vectors that confer resistance to the antibiotic G418, we demonstrated that the magnitude of restriction in BALB/3T3 and SIM.R cells (both Fv-1b/b) and in RFM/3T3 cells (Fv-1nr/nr) is approximately 100-fold compared with that in AKR or NIH 3T3 cells (both Fv-1n/n). Furthermore, titration kinetics were single hit in restrictive cells. Colonies of antibiotic-resistant cells recovered after infection of genotypically restrictive cultures were phenotypically restrictive when reinfected, ruling out selection of stably nonrestrictive subpopulations. These results suggest that the ability to infect some fraction of cells in a genotypically restrictive culture does not require specific abrogation and that multihit kinetics may not be an essential feature of Fv-1 restriction.

Fv-1 N- and B-tropism-specific sequences in murine leukemia virus and related endogenous proviral genomes.

Oligonucleotide probes specific for the Fv-1 N- and B-tropic host range determinants of the gag p30-coding sequence were used to analyze DNA clones of various murine leukemia virus (MuLV) and endogenous MuLV-related proviral genomes and chromosomal DNA from four mouse strains. The group of DNA clones consisted of ecotropic MuLVs of known Fv-1 host range, somatically acquired ecotropic MuLV proviruses, xenotropic MuLV isolates, and endogenous nonecotropic MuLV-related proviral sequences from mouse chromosomal DNA. As expected, the prototype N-tropism determinant is carried by N-tropic viruses of several different origins. All seven endogenous nonecotropic MuLV-related proviral sequence clones derived from RFM/Un mouse chromosomal DNA, although not recognized by the N probe, showed positive hybridization with the prototype B-tropism-specific probe. The two xenotropic MuLV clones derived from infectious virus (one of BALB:virus-2 and one of AKR xenotropic virus) failed to hybridize with the N- and B-tropic oligonucleotide probes tested and with one probe specific for NB-tropic Moloney MuLV. One of two endogenous xenotropic class proviruses derived from HRS/J mouse chromosomal DNA (J. P. Stoye and J. M. Coffin, J. Virol. 61:2659-2669, 1987) also failed to hybridize to the N- and B-tropic probes, whereas the other hybridized to the B-tropic probe. In addition, analysis of mouse chromosomal DNA from four strains indicates that hybridization with the N-tropic probe correlates with the presence or absence of endogenous ecotropic MuLV provirus, whereas the B-tropic probe detects abundant copies of endogenous nonecotropic MuLV-related proviral sequences. These results suggest that the B-tropism determinant in B-tropic ecotropic MuLV may arise from recombination between N-tropic ecotropic MuLV and members of the abundant endogenous nonecotropic MuLV-related classes including a subset of endogenous xenotropic proviruses.

Hematopoietic neoplasias of the RFM/Un mouse contain somatic re-integration of the restriction endogenous ecotropic provirus.

We have examined the spleen DNA of individual mice of the RFM/Un strain for evidence of re-integration of the endogenous ecotropic provirus in radiation-induced and spontaneous neoplasms. The ecotropic env specific probe detects only a single 19 kb EcoRI or a single 7.0 kb HindIII fragment in all DNA preparations from normal tissues of RFM mice, corresponding to the endogenous provirus. Additional DNA restriction fragments containing the ecotropic virus (eco) specific sequence, corresponding to somatically acquired provirus, are detected in two out of five spleen DNA samples from animals with myeloid leukemia and one of three with thymic lymphoma. In addition somatically acquired eco-specific fragments are also detected in greater than 85% of DNA samples from reticulum cell sarcomas, a late occurring spontaneous hematopoietic neoplasm in this mouse strain. These results are consistent with a 'promoter/enhancer insertion' model of leukemogenesis involving the endogenous ecotropic provirus and are of particular interest since the RFM/Un mouse possesses a locus that restricts exogenous infection of cells by the endogenous virus.