The frequency and function of nucleoprotein-specific CD8+ T cells are critical for heterosubtypic immunity against influenza virus infection.

Cytotoxic T lymphocytes (CTLs) mediate host defense against viral and intracellular bacterial infections and tumors. However, the magnitude of CTL response and their function needed to confer heterosubtypic immunity against influenza virus infection are unknown. We addressed the role of CD8+ T cells in the absence of any cross-reactive antibody responses to influenza viral proteins using an adenoviral vector expressing a 9mer amino acid sequence recognized by CD8+ T cells. Our results indicate that both CD8+ T cell frequency and function are crucial for heterosubtypic immunity. Low morbidity, lower viral lung titers, low to minimal lung pathology, and better survival upon heterosubtypic virus challenge correlated with the increased frequency of NP-specific CTLs. NP-CD8+ T cells induced by differential infection doses displayed distinct RNA transcriptome profiles and functional properties. CD8+ T cells induced by a high dose of influenza virus secreted significantly higher levels of IFN-γ and exhibited higher levels of cytotoxic function. The mice that received NP-CD8+ T cells from the high-dose virus recipients through adoptive transfer had lower viral titers following viral challenge than those induced by the low dose of virus, suggesting differential cellular programming by antigen dose. Enhanced NP-CD8+ T-cell functions induced by a higher dose of influenza virus strongly correlated with the increased expression of cellular and metabolic genes, indicating a shift to a more glycolytic metabolic phenotype. These findings have implications for developing effective T cell vaccines against infectious diseases and cancer. IMPORTANCE: Cytotoxic T lymphocytes (CTLs) are an important component of the adaptive immune system that clears virus-infected cells or tumor cells. Hence, developing next-generation vaccines that induce or recall CTL responses against cancer and infectious diseases is crucial. However, it is not clear if the frequency, function, or both are essential in conferring protection, as in the case of influenza. In this study, we demonstrate that both CTL frequency and function are crucial for providing heterosubtypic immunity to influenza by utilizing an Ad-viral vector expressing a CD8 epitope only to rule out the role of antibodies, single-cell RNA-seq analysis, as well as adoptive transfer experiments. Our findings have implications for developing T cell vaccines against infectious diseases and cancer.

Black box no more: A cross-sectional multi-disciplinary survey for exploring governance and guiding adoption of AI in medical imaging and radiotherapy in the UK.

BACKGROUND: Medical Imaging and radiotherapy (MIRT) are at the forefront of artificial intelligence applications. The exponential increase of these applications has made governance frameworks necessary to uphold safe and effective clinical adoption. There is little information about how healthcare practitioners in MIRT in the UK use AI tools, their governance and associated challenges, opportunities and priorities for the future. METHODS: This cross-sectional survey was open from November to December 2022 to MIRT professionals who had knowledge or made use of AI tools, as an attempt to map out current policy and practice and to identify future needs. The survey was electronically distributed to the participants. Statistical analysis included descriptive statistics and inferential statistics on the SPSS statistical software. Content analysis was employed for the open-ended questions. RESULTS: Among the 245 responses, the following were emphasised as central to AI adoption: governance frameworks, practitioner training, leadership, and teamwork within the AI ecosystem. Prior training was strongly correlated with increased knowledge about AI tools and frameworks. However, knowledge of related frameworks remained low, with different professionals showing different affinity to certain frameworks related to their respective roles. Common challenges and opportunities of AI adoption were also highlighted, with recommendations for future practice.

Black box no more: a scoping review of AI governance frameworks to guide procurement and adoption of AI in medical imaging and radiotherapy in the UK.

Technological advancements in computer science have started to bring artificial intelligence (AI) from the bench closer to the bedside. While there is still lots to do and improve, AI models in medical imaging and radiotherapy are rapidly being developed and increasingly deployed in clinical practice. At the same time, AI governance frameworks are still under development. Clinical practitioners involved with procuring, deploying, and adopting AI tools in the UK should be well-informed about these AI governance frameworks. This scoping review aimed to map out available literature on AI governance in the UK, focusing on medical imaging and radiotherapy. Searches were performed on Google Scholar, Pubmed, and the Cochrane Library, between June and July 2022. Of 4225 initially identified sources, 35 were finally included in this review. A comprehensive conceptual AI governance framework was proposed, guided by the need for rigorous AI validation and evaluation procedures, the accreditation rules and standards, and the fundamental ethical principles of AI. Fairness, transparency, trustworthiness, and explainability should be drivers of all AI models deployed in clinical practice. Appropriate staff education is also mandatory to ensure AI's safe and responsible use. Multidisciplinary teams under robust leadership will facilitate AI adoption, and it is crucial to involve patients, the public, and practitioners in decision-making. Collaborative research should be encouraged to enhance and promote innovation, while caution should be paid to the ongoing auditing of AI tools to ensure safety and clinical effectiveness.

Quinazolin-derived myeloperoxidase inhibitor suppresses influenza A virus-induced reactive oxygen species, pro-inflammatory mediators and improves cell survival.

Superoxide radicals and other reactive oxygen species (ROS) are implicated in influenza A virus-induced inflammation. In this in vitro study, we evaluated the effects of TG6-44, a novel quinazolin-derived myeloperoxidase-specific ROS inhibitor, on influenza A virus (A/X31) infection using THP-1 lung monocytic cells and freshly isolated peripheral blood mononuclear cells (PBMC). TG6-44 significantly decreased A/X31-induced ROS and virus-induced inflammatory mediators in THP-1 cells (IL-6, IFN-γ, MCP-1, TNF-α, MIP-1ß) and in human PBMC (IL-6, IL-8, TNF-α, MCP-1). Interestingly, TG6-44-treated THP-1 cells showed a decrease in percent cells expressing viral nucleoprotein, as well as a delay in translocation of viral nucleoprotein into the nucleus. Furthermore, in influenza A virus-infected cells, TG6-44 treatment led to suppression of virus-induced cell death as evidenced by decreased caspase-3 activation, decreased proportion of Annexin V+PI+ cells, and increased Bcl-2 phosphorylation. Taken together, our results demonstrate the anti-inflammatory and anti-infective effects of TG6-44.

Influenza Virus Infects and Depletes Activated Adaptive Immune Responders.

Influenza infections cause several million cases of severe respiratory illness, hospitalizations, and hundreds of thousands of deaths globally. Secondary infections are a leading cause of influenza's high morbidity and mortality, and significantly factored into the severity of the 1918, 1968, and 2009 pandemics. Furthermore, there is an increased incidence of other respiratory infections even in vaccinated individuals during influenza season. Putative mechanisms responsible for vaccine failures against influenza as well as other respiratory infections during influenza season are investigated. Peripheral blood mononuclear cells (PBMCs) are used from influenza vaccinated individuals to assess antigen-specific responses to influenza, measles, and varicella. The observations made in humans to a mouse model to unravel the mechanism is confirmed and extended. Infection with influenza virus suppresses an ongoing adaptive response to vaccination against influenza as well as other respiratory pathogens, i.e., Adenovirus and Streptococcus pneumoniae by preferentially infecting and killing activated lymphocytes which express elevated levels of sialic acid receptors. These findings propose a new mechanism for the high incidence of secondary respiratory infections due to bacteria and other viruses as well as vaccine failures to influenza and other respiratory pathogens even in immune individuals due to influenza viral infections.

Influenza virus NS1- C/EBPß gene regulatory complex inhibits RIG-I transcription.

Influenza virus non-structural protein 1 (NS1) counteracts host antiviral innate immune responses by inhibiting Retinoic acid inducible gene-I (RIG-I) activation. However, whether NS1 also specifically regulates RIG-I transcription is unknown. Here, we identify a CCAAT/Enhancer Binding Protein beta (C/EBPß) binding site in the RIG-I promoter as a repressor element, and show that NS1 promotes C/EBPß phosphorylation and its recruitment to the RIG-I promoter as a C/EBPß/NS1 complex. C/EBPß overexpression and siRNA knockdown in human lung epithelial cells resulted in suppression and activation of RIG-I expression respectively, implying a negative regulatory role of C/EBPß. Further, C/EBPß phosphorylation, its interaction with NS1 and occupancy at the RIG-I promoter was associated with RIG-I transcriptional inhibition. These findings provide an important insight into the molecular mechanism by which influenza NS1 commandeers RIG-I transcriptional regulation and suppresses host antiviral responses.

Tropism and Infectivity of a Seasonal A(H1N1) and a Highly Pathogenic Avian A(H5N1) Influenza Virus in Primary Differentiated Ferret Nasal Epithelial Cell Cultures.

Ferrets represent an invaluable animal model to study influenza virus pathogenesis and transmission. To further characterize this model, we developed a differentiated primary ferret nasal epithelial cell (FNEC) culture model for investigation of influenza A virus infection and virus-host interactions. This well-differentiated culture consists of various cell types, a mucociliary clearance system, and tight junctions, representing the nasal ciliated pseudostratified respiratory epithelium. Both α2,6-linked and α2,3-linked sialic acid (SA) receptors, which preferentially bind the hemagglutinin (HA) of human and avian influenza viruses, respectively, were detected on the apical surface of the culture with different cellular tropisms. In accordance with the distribution of SA receptors, we observed that a pre-2009 seasonal A(H1N1) virus infected both ciliated and nonciliated cells, whereas a highly pathogenic avian influenza (HPAI) A(H5N1) virus primarily infected nonciliated cells. Transmission electron microscopy revealed that virions were released from or associated with the apical membranes of ciliated, nonciliated, and mucin-secretory goblet cells. Upon infection, the HPAI A(H5N1) virus replicated to titers higher than those of the human A(H1N1) virus at 37°C; however, replication of the A(H5N1) virus was significantly attenuated at 33°C. Furthermore, we found that infection with the A(H5N1) virus induced higher expression levels of immune mediator genes and resulted in more cell damage/loss than with the human A(H1N1) virus. This primary differentiated FNEC culture model, recapitulating the structure of the nasal epithelium, provides a useful model to bridge in vivo and in vitro studies of cellular tropism, infectivity, and pathogenesis of influenza viruses during the initial stages of infection.IMPORTANCE Although ferrets serve as an important model of influenza virus infection, much remains unknown about virus-host interactions in this species at the cellular level. The development of differentiated primary cultures of ferret nasal epithelial cells is an important step toward understanding cellular tropism and the mechanisms of influenza virus infection and replication in the airway milieu of this model. Using lectin staining and microscopy techniques, we characterized the sialic acid receptor distribution and the cellular composition of the culture model. We then evaluated the replication of and immune response to human and avian influenza viruses at relevant physiological temperatures. Our findings offer significant insight into this first line of defense against influenza virus infection and provide a model for the evaluation of emerging influenza viruses in a well-controlled in vitro environmental setting.

Infection and Replication of Influenza Virus at the Ocular Surface.

Although influenza viruses typically cause respiratory tract disease, some viruses, particularly those with an H7 hemagglutinin, have been isolated from the eyes of conjunctivitis cases. Previous work has shown that isolates of multiple subtypes from both ocular and respiratory infections are capable of replication in human ex vivo ocular tissues and corneal or conjunctival cell monolayers, leaving the determinants of ocular tropism unclear. Here, we evaluated the effect of several variables on tropism for ocular cells cultured in vitro and examined the potential effect of the tear film on viral infectivity. All viruses tested were able to replicate in primary human corneal epithelial cell monolayers subjected to aerosol inoculation. The temperature at which cells were cultured postinoculation minimally affected infectivity. Replication efficiency, in contrast, was reduced at 33°C relative to that at 37°C, and this effect was slightly greater for the conjunctivitis isolates than for the respiratory ones. With the exception of a seasonal H3N2 virus, the subset of viruses studied in multilayer corneal tissue constructs also replicated productively after either aerosol or liquid inoculation. Human tears significantly inhibited the hemagglutination of both ocular and nonocular isolates, but the effect on viral infectivity was more variable, with tears reducing the infectivity of nonocular isolates more than ocular isolates. These data suggest that most influenza viruses may be capable of establishing infection if they reach the surface of ocular cells but that this is more likely for ocular-tropic viruses, as they are better able to maintain their infectivity during passage through the tear film.IMPORTANCE The potential spread of zoonotic influenza viruses to humans represents an important threat to public health. Unfortunately, despite the importance of cellular and tissue tropism to pathogenesis, determinants of influenza virus tropism have yet to be fully elucidated. Here, we sought to identify factors that limit the ability of most influenza viruses to cause ocular infection. Although ocular symptoms in humans caused by avian influenza viruses tend to be relatively mild, these infections are concerning due to the potential of the ocular surface to serve as a portal of entry for viruses that go on to establish respiratory infections. Furthermore, a better understanding of the factors that influence infection and replication in this noncanonical site may point toward novel determinants of tropism in the respiratory tract.

Influenza virus exploits tunneling nanotubes for cell-to-cell spread.

Tunneling nanotubes (TNTs) represent a novel route of intercellular communication. While previous work has shown that TNTs facilitate the exchange of viral or prion proteins from infected to naïve cells, it is not clear whether the viral genome is also transferred via this mechanism and further, whether transfer via this route can result in productive replication of the infectious agents in the recipient cell. Here we present evidence that lung epithelial cells are connected by TNTs, and in spite of the presence of neutralizing antibodies and an antiviral agent, Oseltamivir, influenza virus can exploit these networks to transfer viral proteins and genome from the infected to naïve cell, resulting in productive viral replication in the naïve cells. These observations indicate that influenza viruses can spread using these intercellular networks that connect epithelial cells, evading immune and antiviral defenses and provide an explanation for the incidence of influenza infections even in influenza-immune individuals and vaccine failures.

Constitutively Expressed IFITM3 Protein in Human Endothelial Cells Poses an Early Infection Block to Human Influenza Viruses.

A role for pulmonary endothelial cells in the orchestration of cytokine production and leukocyte recruitment during influenza virus infection, leading to severe lung damage, has been recently identified. As the mechanistic pathway for this ability is not fully known, we extended previous studies on influenza virus tropism in cultured human pulmonary endothelial cells. We found that a subset of avian influenza viruses, including potentially pandemic H5N1, H7N9, and H9N2 viruses, could infect human pulmonary endothelial cells (HULEC) with high efficiency compared to human H1N1 or H3N2 viruses. In HULEC, human influenza viruses were capable of binding to host cellular receptors, becoming internalized and initiating hemifusion but failing to uncoat the viral nucleocapsid and to replicate in host nuclei. Unlike numerous cell types, including epithelial cells, we found that pulmonary endothelial cells constitutively express a high level of the restriction protein IFITM3 in endosomal compartments. IFITM3 knockdown by small interfering RNA (siRNA) could partially rescue H1N1 virus infection in HULEC, suggesting IFITM3 proteins were involved in blocking human influenza virus infection in endothelial cells. In contrast, selected avian influenza viruses were able to escape IFITM3 restriction in endothelial cells, possibly by fusing in early endosomes at higher pH or by other, unknown mechanisms. Collectively, our study demonstrates that the human pulmonary endothelium possesses intrinsic immunity to human influenza viruses, in part due to the constitutive expression of IFITM3 proteins. Notably, certain avian influenza viruses have evolved to escape this restriction, possibly contributing to virus-induced pneumonia and severe lung disease in humans. IMPORTANCE: Avian influenza viruses, including H5N1 and H7N9, have been associated with severe respiratory disease and fatal outcomes in humans. Although acute respiratory distress syndrome (ARDS) and progressive pulmonary endothelial damage are known to be present during severe human infections, the role of pulmonary endothelial cells in the pathogenesis of avian influenza virus infections is largely unknown. By comparing human seasonal influenza strains to avian influenza viruses, we provide greater insight into the interaction of influenza virus with human pulmonary endothelial cells. We show that human influenza virus infection is blocked during the early stages of virus entry, which is likely due to the relatively high expression of the host antiviral factors IFITMs (interferon-induced transmembrane proteins) located in membrane-bound compartments inside cells. Overall, this study provides a mechanism by which human endothelial cells limit replication of human influenza virus strains, whereas avian influenza viruses overcome these restriction factors in this cell type.

Rapamycin Does Not Impede Survival or Induction of Antibody Responses to Primary and Heterosubtypic Influenza Infections in Mice.

Impairment of immune defenses can contribute to severe influenza infections. Rapamycin is an immunosuppressive drug often used to prevent transplant rejection and is currently undergoing clinical trials for treating cancers and autoimmune diseases. We investigated whether rapamycin has deleterious effects during lethal influenza viral infections. We treated mice with two concentrations of rapamycin and infected them with A/Puerto Rico/8/1934 (A/PR8), followed by a heterosubtypic A/Hong Kong/1/68 (A/HK68) challenge. Our data show similar morbidity, mortality, and lung viral titer with both rapamycin treatment doses compared to untreated controls, with a delay in morbidity onset in rapamycin high dose recipients during primary infection. Rapamycin treatment at high dose also led to increase in percent cytokine producing T cells in the spleen. However, all infected animals had similar serum antibody responses against A/PR8. Post-A/HK68 challenge, rapamycin had no impeding effect on morbidity or mortality and had similar serum antibody levels against A/PR8 and A/HK68. We conclude that rapamycin treatment does not adversely affect morbidity, mortality, or antibody production during lethal influenza infections.

An oil-in-water nanoemulsion enhances immunogenicity of H5N1 vaccine in mice.

To enhance the immunogenicity of the Influenza H5N1 vaccine, we developed an oil-in-water nanoemulsion (NE) adjuvant. NE displayed good temperature stability and maintained particle size. More importantly, it significantly enhanced IL-6 and MCP-1 production to recruit innate cells, including neutrophils, monocytes/macrophages and dendritic cells to the local environment. Furthermore, NE enhanced dendritic cell function to induce robust antigen-specific T and B cell immune responses. NE-adjuvanted H5N1 vaccine not only elicited significantly higher and long-lasting antibody responses, but also conferred enhanced protection against homologous clade 1 as well as heterologous clade 2 H5N1 virus challenge in young as well as in aged mice. The pre-existing immunity to seasonal influenza did not affect the immunogenicity of NE-adjuvanted H5N1 vaccine.

Emergence of Highly Pathogenic Avian Influenza A(H5N1) Virus PB1-F2 Variants and Their Virulence in BALB/c Mice.

UNLABELLED: Influenza A viruses (IAVs) express the PB1-F2 protein from an alternate reading frame within the PB1 gene segment. The roles of PB1-F2 are not well understood but appear to involve modulation of host cell responses. As shown in previous studies, we find that PB1-F2 proteins of mammalian IAVs frequently have premature stop codons that are expected to cause truncations of the protein, whereas avian IAVs usually express a full-length 90-amino-acid PB1-F2. However, in contrast to other avian IAVs, recent isolates of highly pathogenic H5N1 influenza viruses had a high proportion of PB1-F2 truncations (15% since 2010; 61% of isolates in 2013) due to several independent mutations that have persisted and expanded in circulating viruses. One natural H5N1 IAV containing a mutated PB1-F2 start codon (i.e., lacking ATG) was 1,000-fold more virulent for BALB/c mice than a closely related H5N1 containing intact PB1-F2. In vitro, we detected expression of an in-frame protein (C-terminal PB1-F2) from downstream ATGs in PB1-F2 plasmids lacking the well-conserved ATG start codon. Transient expression of full-length PB1-F2, truncated (24-amino-acid) PB1-F2, and PB1-F2 lacking the initiating ATG in mammalian and avian cells had no effect on cell apoptosis or interferon expression in human lung epithelial cells. Full-length and C-terminal PB1-F2 mutants colocalized with mitochondria in A549 cells. Close monitoring of alterations of PB1-F2 and their frequency in contemporary avian H5N1 viruses should continue, as such changes may be markers for mammalian virulence. IMPORTANCE: Although most avian influenza viruses are harmless for humans, some (such as highly pathogenic H5N1 avian influenza viruses) are capable of infecting humans and causing severe disease with a high mortality rate. A number of risk factors potentially associated with adaptation to mammalian infection have been noted. Here we demonstrate that the protein PB1-F2 is frequently truncated in recent isolates of highly pathogenic H5N1 viruses. Truncation of PB1-F2 has been proposed to act as an adaptation to mammalian infection. We show that some forms of truncation of PB1-F2 may be associated with increased virulence in mammals. Our data support the assessment of PB1-F2 truncations for genomic surveillance of influenza viruses.

NLRC5 interacts with RIG-I to induce a robust antiviral response against influenza virus infection.

The NLR protein, NLRC5 is an important regulator of MHC class I gene expression, however, the role of NLRC5 in other innate immune responses is less well defined. In the present study, we report that NLRC5 binds RIG-I and that this interaction is critical for robust antiviral responses against influenza virus. Overexpression of NLRC5 in the human lung epithelial cell line, A549, and normal human bronchial epithelial cells resulted in impaired replication of influenza virus A/Puerto Rico/8/34 virus (PR8) and enhanced IFN-ß expression. Influenza virus leads to induction of IFN-ß that drives RIG-I and NLRC5 expression in host cells. Our results suggest that NLRC5 extends and stabilizes influenza virus induced RIG-I expression and delays expression of the viral inhibitor protein NS1. We show that NS1 binds to NLRC5 to suppress its function. Interaction domain mapping revealed that NLRC5 interacts with RIG-I via its N-terminal death domain and that NLRC5 enhanced antiviral activity in an leucine-rich repeat domain independent manner. Taken together, our findings identify a novel role for NLRC5 in RIG-I-mediated antiviral host responses against influenza virus infection, distinguished from the role of NLRC5 in MHC class I gene regulation.

Nadph oxidase regulates alveolar epithelial sodium channel activity and lung fluid balance in vivo via O⁻2 signaling.

To define roles for reactive oxygen species (ROS) and epithelial sodium channel (ENaC) in maintaining lung fluid balance in vivo, we used two novel whole animal imaging approaches. Live X-ray fluoroscopy enabled quantification of air space fluid content of C57BL/6J mouse lungs challenged by intratracheal (IT) instillation of saline; results were confirmed by using conventional lung wet-to-dry weight ratios and Evans blue as measures of pulmonary edema. Visualization and quantification of ROS produced in lungs was performed in mice that had been administered a redox-sensitive dye, hydro-Cy7, by IT instillation. We found that inhibition of NADPH oxidase with a Rac-1 inhibitor, NSC23766, resulted in alveolar flooding, which correlated with a decrease in lung ROS production in vivo. Consistent with a role for Nox2 in alveolar fluid balance, Nox2(-/-) mice showed increased retention of air space fluid compared with wild-type controls. Interestingly, fluoroscopic analysis of C57BL/6J lungs IT instilled with LPS showed an acute stimulation of lung fluid clearance and ROS production in vivo that was abrogated by the ROS scavenger tetramethylpiperidine-N-oxyl (TEMPO). Acute application of LPS increased the activity of 20 pS nonselective ENaC channels in rat type 1 cells; the average number of channel and single-channel open probability (NPo) increased from 0.14 ± 0.04 to 0.62 ± 0.23. Application of TEMPO to the same cell-attached recording caused an immediate significant decrease in ENaC NPo to 0.04 ± 0.03. These data demonstrate that, in vivo, ROS has the capacity to stimulate lung fluid clearance by increasing ENaC activity.

Expression profiling and pathway analysis of Krüppel-like factor 4 in mouse embryonic fibroblasts.

BACKGROUND: Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor with diverse regulatory functions in proliferation, differentiation, and development. KLF4 also plays a role in inflammation, tumorigenesis, and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. To gain insight into the mechanisms by which KLF4 regulates these processes, we conducted DNA microarray analyses to identify differentially expressed genes in mouse embryonic fibroblasts (MEFs) wild type and null for Klf4. METHODS: Expression profiles of fibroblasts isolated from mouse embryos wild type or null for the Klf4 alleles were examined by DNA microarrays. Differentially expressed genes were subjected to the Database for Annotation, Visualization and Integrated Discovery (DAVID). The microarray data were also interrogated with the Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA) for pathway identification. Results obtained from the microarray analysis were confirmed by Western blotting for select genes with biological relevance to determine the correlation between mRNA and protein levels. RESULTS: One hundred and sixty three up-regulated and 88 down-regulated genes were identified that demonstrated a fold-change of at least 1.5 and a P-value < 0.05 in Klf4-null MEFs compared to wild type MEFs. Many of the up-regulated genes in Klf4-null MEFs encode proto-oncogenes, growth factors, extracellular matrix, and cell cycle activators. In contrast, genes encoding tumor suppressors and those involved in JAK-STAT signaling pathways are down-regulated in Klf4-null MEFs. IPA and GSEA also identified various pathways that are regulated by KLF4. Lastly, Western blotting of select target genes confirmed the changes revealed by microarray data. CONCLUSIONS: These data are not only consistent with previous functional studies of KLF4's role in tumor suppression and somatic cell reprogramming, but also revealed novel target genes that mediate KLF4's functions.

Enteric commensal bacteria potentiate epithelial restitution via reactive oxygen species-mediated inactivation of focal adhesion kinase phosphatases.

The mechanisms by which enteric commensal microbiota influence maturation and repair of the epithelial barrier are relatively unknown. Epithelial restitution requires active cell migration, a process dependent on dynamic turnover of focal cell-matrix adhesions (FAs). Here, we demonstrate that natural, commensal bacteria stimulate generation of reactive oxygen species (ROS) in intestinal epithelia. Bacteria-mediated ROS generation induces oxidation of target cysteines in the redox-sensitive tyrosine phosphatases, LMW-PTP and SHP-2, which in turn results in increased phosphorylation of focal adhesion kinase (FAK), a key protein regulating the turnover of FAs. Accordingly, phosphorylation of FAK substrate proteins, focal adhesion formation, and cell migration are all significantly enhanced by bacterial contact in both in vitro and in vivo models of wound closure. These results suggest that commensal bacteria regulate cell migration via induced generation of ROS in epithelial cells.

Flagellin administration protects gut mucosal tissue from irradiation-induced apoptosis via MKP-7 activity.

BACKGROUND AND AIMS: Radiotherapy for neoplastic disease is associated with significant adverse enteric effects associated with excessive cell death. Ionising radiation induces cell death by a mechanism that is dependent on JNK (c-jun N-terminal kinase) pathway signalling. Additionally, it is known that cells exposed to extracellular bacterial products such as flagellin, pleiotropically activate a number of innate immune pathways, including that of JNK. The JNK pathway controls its own activity by inducing the transcription of mitogen-activated protein kinase phosphatase-7 (MKP-7) which directly targets phosphorylated JNK, thus functioning as a negative feedback loop. Previously, it has been shown that flagellin limits ionising radiation-induced mortality in mice, but the cellular mechanism of protection remained unknown. METHODS: Wild-type C57BL/6 or tlr5(-/-) C57BL/6 were injected with flagellin 2 h before exposure to irradiation, and their intestines were examined for apoptosis. Candidate proteins mediating cytoprotection from irradiation were identified by expression profiling. One of these candidates, MKP-7, was cloned and packaged into adenovirus particles, used to infect cultured cells, and examined for the extent to which its activity reduced cellular apoptosis by flow cytometry or immunoblot analysis. RESULTS: Flagellin pretreatment protected mice from radiation-induced intestinal mucosal injury and apoptosis via a Toll-like receptor 5 (TLR5)-dependent mechanism. Expression profiling of flagellin-treated mice showed upregulation of MKP-7, an inducible repressor of the JNK pathway. MKP-7 expression reached a maximum at 2 h after flagellin treatment, coinciding with suppression of phosphorylated JNK and JNK pathway inhibition. Furthermore, constitutive MKP-7 expression protected cultured cells from radiation-induced apoptosis. CONCLUSIONS: Flagellin is a promising adjuvant for suppressing ionising radiation-induced injury. MKP-7 activity exhibits cytoprotective effects, and is thus a candidate cellular molecule for limiting the damaging effect of radiotherapy on the gastreointestinal system.

Promoter activation by repositioning of RNA polymerase.

Spo0A, a classical two-component-type response regulator in Bacillus subtilis, binds to a specific DNA sequence found in many promoters to repress or activate the transcription of over 100 genes. On the spoIIG promoter, one of the Spo0A binding sites, centered at position -40, overlaps a consensus -35 element that may also interact with region 4 of the sigma A (sigma(A)) subunit of RNA polymerase. Molecular modeling corroborated by genetic evidence led us to propose that the binding of Spo0A to this site repositions sigma(A) region 4 on the promoter. Therefore, we used a chemical nuclease, p-bromoacetamidobenzyl-EDTA-Fe, that was covalently tethered to a single cysteine in region 4 of sigma(A) to map the position of sigma(A) on the promoter. The results indicated that in the absence of Spo0A, sigma(A) region 4 of the RNA polymerase was located near the -35 element sequence centered at position -40. However, in the presence of Spo0A, sigma(A) region 4 was displaced downstream from the -35 element by 4 bp. These and other results support the model in which the binding of Spo0A to the spoIIG promoter stimulates promoter utilization by repositioning prebound RNA polymerase and stabilizing the repositioned RNA polymerase-promoter complex at a new position that aligns sigma(A) region 2 with the -10 region sequences of the promoter, thus facilitating open complex formation.