Brigitte Allard

Dual-Affinity Re-Targeting proteins direct T cell–mediated cytolysis of latently HIV-infected cells

Enhancement of HIV-specific immunity is likely required to eliminate latent HIV infection. Here, we have developed an immunotherapeutic modality aimed to improve T cell-mediated clearance of HIV-1-infected cells. Specifically, we employed Dual-Affinity Re-Targeting (DART) proteins, which are bispecific, antibody-based molecules that can bind 2 distinct cell-surface molecules simultaneously. We designed DARTs with a monovalent HIV-1 envelope-binding (Env-binding) arm that was derived from broadly binding, antibody-dependent cellular cytotoxicity-mediating antibodies known to bind to HIV-infected target cells coupled to a monovalent CD3 binding arm designed to engage cytolytic effector T cells (referred to as HIVxCD3 DARTs). Thus, these DARTs redirected polyclonal T cells to specifically engage with and kill Env-expressing cells, including CD4+ T cells infected with different HIV-1 subtypes, thereby obviating the requirement for HIV-specific immunity. Using lymphocytes from patients on suppressive antiretroviral therapy (ART), we demonstrated that DARTs mediate CD8+ T cell clearance of CD4+ T cells that are superinfected with the HIV-1 strain JR-CSF or infected with autologous reservoir viruses isolated from HIV-infected-patient resting CD4+ T cells. Moreover, DARTs mediated CD8+ T cell clearance of HIV from resting CD4+ T cell cultures following induction of latent virus expression. Combined with HIV latency reversing agents, HIVxCD3 DARTs have the potential to be effective immunotherapeutic agents to clear latent HIV-1 reservoirs in HIV-infected individuals.

Serum amyloid A activates NF-κB and proinflammatory gene expression in human and murine intestinal epithelial cells

Serum amyloid A (SAA) is an acute‐phase protein whose levels positively correlate with disease activity in inflammatory bowel diseases. In this study we investigated the impact of SAA on NF‐κB signaling and proinflammatory gene expression in intestinal epithelial cells (IEC). Human HT‐29 and Caco‐2 monolayers were stimulated with recombinant SAA and NF‐κB activation/NF‐κB‐dependent gene expression measured. Adenoviral dominant negative mutants IκB‐α (Ad5IκBAA) were utilized to determine the contribution of NF‐κB signaling pathway to SAA‐dependent gene expression. Intestinal explant and primary IEC derived from κB‐EGFP transgenic mice were exposed to SAA and NF‐κB‐dependent enhanced green fluorescent protein (EGFP) fluorescence measured. SAA induced IκB‐α degradation, RelA serine 536 (S536) phosphorylation, NF‐κB transcriptional activity, RelA recruitment to the IL‐8 gene promoter and endogenous gene expression (IL‐8, COX‐2) in HT‐29 cells. Further, Ad5IκBAA abrogated SAA‐induced RelA nuclear translocation, NF‐κB transcriptional activity and IL‐8 gene expression. SAA‐dependent IL‐8 gene expression required activation of the MAPK ERK, p38 and JNK in HT‐29 cells. Finally, SAA induced EGFP expression in intestinal explants isolated from κB‐EGFP transgenic mice and enhanced RelA and IκBα phosphorylation in primary IEC. This indicates that SAA potentially participate in the inflammatory process by virtue of its ability to activate proinflammatory signaling in IEC.

Tomato Lycopene Extract Prevents Lipopolysaccharide-Induced NF-κB Signaling but Worsens Dextran Sulfate Sodium-Induced Colitis in NF-κBEGFP Mice

Background The impact of tomato lycopene extract (TLE) on intestinal inflammation is currently unknown. We investigated the effect of TLE on lipopolysaccharide (LPS)-induced innate signaling and experimental colitis. Methodology/Principal Findings Mice were fed a diet containing 0.5 and 2% TLE or isoflavone free control (AIN-76). The therapeutic efficacy of TLE diet was assessed using dextran sulfate sodium (DSS) exposed mice and IL-10−/−;NF-κBEGFP mice, representing an acute and spontaneous chronic colitis model respectively. A mini-endoscope was used to determine the extent of macroscopic mucosal lesions. Murine splenocytes and intestinal epithelial cells were used to determine the in vitro impact of TLE on LPS-induced NF-κB signaling. In vitro, TLE blocked LPS-induced IκBα degradation, RelA translocation, NF-κB transcriptional activity and MIP-2 mRNA accumulation in IEC-18 cells. Moreover, LPS-induced IL-12p40 gene expression was dose-dependently inhibited in TLE-treated splenocytes. Interestingly, DSS-induced acute colitis worsened in TLE-fed NF-κBEGFP mice compared to control diet as measured by weight loss, colonoscopic analysis and histological scores. In contrast, TLE-fed IL-10−/−;NF-κBEGFP mice displayed decreased colonic EGFP expression compared to control diet. IL-6, TNFα, and MCP-1 mRNA expression were increased in the colon of TLE-fed, DSS-exposed NF-κBEGFP mice compared to the control diet. Additionally, caspase-3 activation and TUNEL positive cells were enhanced in TLE diet-fed, DSS-exposed mice as compared to DSS control mice. Conclusions/ Significance These results indicate that TLE prevents LPS-induced proinflammatory gene expression by blocking of NF-κB signaling, but aggravates DSS-induced colitis by enhancing epithelial cell apoptosis.

Wound-induced p38MAPK-dependent histone H3 phosphorylation correlates with increased COX-2 expression in enterocytes

Gastrointestinal epithelial cell damage triggers an important biological response called restitution, a process aimed at re‐epithelializing the wounded areas. Unfortunately, little is known about the intrinsic molecular signaling events implicated in this host response. We hypothesized that wounding intestinal epithelial cells activates signaling pathways leading to chromatin modification and COX‐2 upregulation during restitution. Confluent rat IEC18 cells were mechanically wounded by multiple parallel scratches using a pipet tip. NF‐κB(Ser536), p38, and histone H3(Ser10) (H3S10) phosphorylation were determined by Western blot using specific phospho‐antibodies. COX‐2 gene expression was evaluated by RT‐PCR, Western Blot, and ELISA. Association of phosphorylated H3, RelA (NF‐κB), and RNA polymerase II to the COX‐2 gene promoter was evaluated by chromatin immunoprecipitation (ChIP). The specific inhibitors Bay11‐7082 and SB239063 as well as Ad5IκB‐superrepressor (Ad5IκBAA) and Ad5dnp38 were used to block NF‐κB‐ and p38‐signaling pathways, respectively. Wounding induced a rapid and sustained (24 h) phosphorylation of RelAS536, H3S10, and p38MAPK in enterocytes. ChIP analysis of the COX‐2 gene promoter demonstrated the presence of phospho‐H3S10 and recruitment of RelA and RNA polymerase II, a process blocked by SB239063. Finally, molecular blockade of NF‐κB (Ad5IκBAA) or p38MAPK (Ad5dnp38) signaling strongly inhibited enterocyte restitution. p38MAPK‐dependent histone 3 phosphorylation is an important component of the intestinal wound‐healing response. Targeting‐signaling pathways selectively involved in healing/restitution may provide a novel means to maintain or re‐establish host intestinal barrier integrity. J. Cell. Physiol.

Gnotobiotic IL-10−/−; NF-κBEGFP Mice Develop Rapid and Severe Colitis Following Campylobacter jejuni Infection

Limited information is available on the molecular mechanisms associated with Campylobacter jejuni (C. jejuni) induced food-borne diarrheal illnesses. In this study, we investigated the function of TLR/NF-κB signaling in C. jejuni induced pathogenesis using gnotobiotic IL-10−/−; NF-κBEGFP mice. In vitro analysis showed that C. jejuni induced IκB phosphorylation, followed by enhanced NF-κB transcriptional activity and increased IL-6, MIP-2α and NOD2 mRNA accumulation in infected-mouse colonic epithelial cells CMT93. Importantly, these events were blocked by molecular delivery of an IκB inhibitor (Ad5IκBAA). NF-κB signalling was also important for C.jejuni-induced cytokine gene expression in bone marrow-derived dendritic cells. Importantly, C. jejuni associated IL-10−/−; NF-κBEGFP mice developed mild (day 5) and severe (day 14) ulcerating colonic inflammation and bloody diarrhea as assessed by colonoscopy and histological analysis. Macroscopic analysis showed elevated EGFP expression indicating NF-κB activation throughout the colon of C. jejuni associated IL-10−/−; NF-κBEGFP mice, while fluorescence microscopy revealed EGFP positive cells to be exclusively located in lamina propria mononuclear cells. Pharmacological NF-κB inhibition using Bay 11-7085 did not ameliorate C. jejuni induced colonic inflammation. Our findings indicate that C. jejuni induces rapid and severe intestinal inflammation in a susceptible host that correlates with enhanced NF-κB activity from lamina propria immune cells.

PI3K-dependent GSK3ß(Ser9)-phosphorylation is implicated in the intestinal epithelial cell wound-healing response.

Introduction The ability of the intestinal epithelial barrier to respond to various injurious insults is an essential component of intestinal homeostasis. However, the molecular mechanisms responsible for wound-healing and repair in the intestine are poorly understood. The glycogen synthase kinase 3ß (GSK3ß) has been implicated in various biological processes such as cellular motility, cell spreading and recently inflammation. Aim To investigate the role of GSK3ß in intestinal epithelial cell restitution. Methods Rat intestinal epithelial IEC18 cells were serum-starved for 16 to 24h and wounded by multiple scraping. Akt(Ser473)-, GSK3ß(Ser9)- and RelA(Ser536)-phosphorylation were determined by Western blot using specific phospho-antibodies. The inhibitors AG1478 (1 µM) and Ly294002 (25 µM) were used to block EGF-R autophosphorylation and PI3K-activation, respectively. ß-catenin/LEF/TCF dependent transcription was determined by reporter gene assay (TOP/FOP system). C-myc gene expression was evaluated by real-time RT-PCR. GSK3ß−/− mouse embryonic fibroblasts were used to characterize the role of GSK3ß in wounding-induced cell migration. Results Wounding induced GSK3ß(Ser9) phosphorylation in IEC-18 cells, which led to ß-catenin accumulation as well as nuclear translocation of ß-catenin. ß-catenin stabilization/nuclear translocation led to enhanced LEF-TCF transcriptional activity and subsequent c-myc mRNA accumulation in wounded cell monolayers. Blocking PI3K/Akt signaling with Ly294002 prevented wound-induced GSK3ß(Ser9) phosphorylation as well as ß-catenin nuclear translocation and significantly attenuated restitution. Additionally, wounding induced rapid NF-kB(Ser536) phosphorylation, which was inhibited by AG1478, but not by Ly294002. GSK3ß−/− cells demonstrated significantly attenuated wound-induced restitution compared to wild-type cells. Conclusion We conclude that PI3K-mediated GSK3ß phosphorylation is involved in the intestinal epithelial wound-healing response. Phosphorylation of GSK3ß may be important for intestinal restitution by promoting cell motility in response to wounding.

Interval dosing with the HDAC inhibitor vorinostat effectively reverses HIV latency

BACKGROUND. The histone deacetylase (HDAC) inhibitor vorinostat (VOR) can increase HIV RNA expression in vivo within resting CD4+ T cells of aviremic HIV+ individuals. However, while studies of VOR or other HDAC inhibitors have reported reversal of latency, none has demonstrated clearance of latent infection. We sought to identify the optimal dosing of VOR for effective serial reversal of HIV latency. METHODS. In a study of 16 HIV-infected, aviremic individuals, we measured resting CD4+ T cell–associated HIV RNA ex vivo and in vivo following a single exposure to VOR, and then in vivo after a pair of doses separated by 48 or 72 hours, and finally following a series of 10 doses given at 72-hour intervals. RESULTS. Serial VOR exposures separated by 72 hours most often resulted in an increase in cell-associated HIV RNA within circulating resting CD4+ T cells. VOR was well tolerated by all participants. However, despite serial reversal of latency over 1 month of VOR dosing, we did not observe a measurable decrease (>0.3 log10) in the frequency of latent infection within resting CD4+ T cells. CONCLUSIONS. These findings outline parameters for the experimental use of VOR to clear latent infection. Latency reversal can be achieved by VOR safely and repeatedly, but effective depletion of persistent HIV infection will require additional advances. In addition to improvements in latency reversal, these advances may include the sustained induction of potent antiviral immune responses capable of recognizing and clearing the rare cells in which HIV latency has been reversed. TRIAL REGISTRATION. Clinicaltrials.gov NCT01319383. FUNDING. NIH grants U01 AI095052, AI50410, and P30 CA016086 and National Center for Advancing Translational Sciences grant KL2 TR001109.

1045 Campylobacter Jejuni Induces Rapid and Severe Colitis in Gnotobiotic IL-10-/- Mice Through a MyD88 Independent Pathway

Introduction: The Gram-negative bacterium Campylobacter jejuni (C. jejuni) is the leading cause of food-borne diarrheal illnesses worldwide. Limited information is available on the molecular mechanisms associated with its pathogenesis. Aim: To characterize the host responses associated with C. jejuni-induced intestinal inflammation and to define themolecular mechanisms associated with the pathogenesis. Methods: Germ-free IL-10wt/wt;NF-κBEGFP and IL-10-/-;NF-κBEGFP mice were mono-associated with different amount of C. jejuni (strain 81-176; 102, 104, 106, 109cfu/mouse). IL-10-/and IL-10-/-;Myd88-/were associated with C. jejuni under specific pathogen-free (SPF) conditions (108cfu/mouse). Bacterial colonization and extraintestinal manifestations were confirmed by culturing stool, mesenteric lymph nodes (MLN), spleen and liver collected from each group and by real-time PCR (RT-PCR). Macroscopic and confocal microscopic EGFP imaging, RT-PCR analysis and histological analysis were utilized to characterize the bacteria-induced host responses. Colonoscopy was performed to document inflammation using a mini STORZ endoscope. Results: C. jejuni mono-associated IL-10-/mice developed mild (102), moderate (104) and severe (109) inflammation by day 14 as assayed by colonoscopy. Five days after association all mice had clinical signs of inflammation as evidenced by bloody diarrhea. Macroscopic analysis showed elevated EGFP expression throughout the colon of C. jejuni (109) associated IL-10-/-;NF-κBEGFP mice. All mice were colonized equally with C. jejuni as confirmed by RT-PCR and stool sample cultures. Histological analysis showed severe crypt hyperplasia, goblet cell depletion, ulcers and immune cell infiltration. TNF and IL-12p40 mRNA were significantly induced in the colon of these mice compared to WT mice. C. jejuni-induced IL-12p40, IL-23p19 and TNF gene expression was reduced in BMCD generated from IL-10-/-;Myd88-/compared to IL10-/mice. Interestingly, the onset and extent of C. jejuni-induced colitis is similar between IL-10-/-;Myd88-/and IL-10-/mice with comparable induction of TNF, Nod2, and IL-23p19 gene expression. Conclusion: Our findings indicate that C. jejuni induces rapid and severe intestinal inflammation in a susceptible host, which correlates with enhanced NF-κB activity. C. jejuni-induced colitis in IL-10-/mice appears to be mediated by a Myd88-independent pathway. These findings raise the possibility that Nod proteins are involved in C. jejuniinduced colitis.