Inflammation drives dysbiosis and bacterial invasion in murine models of ileal Crohn's disease

Background and Aims

Understanding the interplay between genetic susceptibility, the microbiome, the environment and the immune system in Crohn’s Disease (CD) is essential for developing optimal therapeutic strategies. We sought to examine the dynamics of the relationship between inflammation, the ileal microbiome, and host genetics in murine models of ileitis.

Methods

We induced ileal inflammation of graded severity in C57BL6 mice by gavage with Toxoplasma gondii, Giardia muris, low dose indomethacin (LDI;0.1 mg/mouse), or high dose indomethacin (HDI;1 mg/mouse). The composition and spatial distribution of the mucosal microbiome was evaluated by 16S rDNA pyrosequencing and fluorescence in situ hybridization. Mucosal E. coli were enumerated by quantitative PCR, and characterized by phylogroup, genotype and pathotype.

Results

Moderate to severe ileitis induced by T. gondii (day 8) and HDI caused a consistent shift from >95% Gram + Firmicutes to >95% Gram - Proteobacteria. This was accompanied by reduced microbial diversity and mucosal invasion by adherent and invasive E. coli, mirroring the dysbiosis of ileal CD. In contrast, dysbiosis and bacterial invasion did not develop in mice with mild ileitis induced by Giardia muris. Superimposition of genetic susceptibility and T. Gondii infection revealed greatest dysbiosis and bacterial invasion in the CD-susceptible genotype, NOD2^−/−, and reduced dysbiosis in ileitis-resistant CCR2^−/− mice. Abrogating inflammation with the CD therapeutic anti-TNF-α-mAb tempered dysbiosis and bacterial invasion.

Conclusions

Acute ileitis induces dysbiosis and proliferation of mucosally invasive E. coli, irrespective of trigger and genotype. The identification of CCR2 as a target for therapeutic intervention, and discovery that host genotype and therapeutic blockade of inflammation impact the threshold and extent of ileal dysbiosis are of high relevance to developing effective therapies for CD.     

An in vivo polymicrobial biofilm wound infection model to study interspecies interactions

Chronic wound infections are typically polymicrobial; however, most in vivo studies have focused on monospecies infections. This project was designed to develop an in vivo, polymicrobial, biofilm-related, infected wound model in order to study multispecies biofilm dynamics and in relation to wound chronicity. Multispecies biofilms consisting of both Gram negative and Gram positive strains, as well as aerobes and anaerobes, were grown in vitro and then transplanted onto the wounds of mice. These in vitro-to-in vivo multi-species biofilm transplants generated polymicrobial wound infections, which remained heterogeneous with four bacterial species throughout the experiment. We observed that wounded mice given multispecies biofilm infections displayed a wound healing impairment over mice infected with a single-species of bacteria. In addition, the bacteria in the polymicrobial wound infections displayed increased antimicrobial tolerance in comparison to those in single species infections. These data suggest that synergistic interactions between different bacterial species in wounds may contribute to healing delays and/or antibiotic tolerance.     

Systematic analysis of cell cycle effects of common drugs leads to the discovery of a suppressive interaction between gemfibrozil and fluoxetine

Screening chemical libraries to identify compounds that affect overall cell proliferation is common. However, in most cases, it is not known whether the compounds tested alter the timing of particular cell cycle transitions. Here, we evaluated an FDA-approved drug library to identify pharmaceuticals that alter cell cycle progression in yeast, using DNA content measurements by flow cytometry. This approach revealed strong cell cycle effects of several commonly used pharmaceuticals. We show that the antilipemic gemfibrozil delays initiation of DNA replication, while cells treated with the antidepressant fluoxetine severely delay progression through mitosis. Based on their effects on cell cycle progression, we also examined cell proliferation in the presence of both compounds. We discovered a strong suppressive interaction between gemfibrozil and fluoxetine. Combinations of interest among diverse pharmaceuticals are difficult to identify, due to the daunting number of possible combinations that must be evaluated. The novel interaction between gemfibrozil and fluoxetine suggests that identifying and combining drugs that show cell cycle effects might streamline identification of drug combinations with a pronounced impact on cell proliferation.     

Dynamic measurements of black oystercatcher (Haematopus bachmani) predation on mussels (Mytilus californianus)

Intertidal zone mussels can face threats from a variety of predatory species during high and low tides, and they must balance the threat of predation against other needs such as feeding and aerobic respiration. Black oystercatchers (Haematopus bachmani) on the Pacific coast of North America can depend on the mussel Mytilus californianus for a substantial portion of their diet. Observations suggest that oystercatchers tend to focus on mussels beginning to gape their valves during rising tides, following periods of aerial emersion. We present detailed, autonomous field measurements of the dynamics of three such predation events in the rocky intertidal zone. We measured accelerations of up to 4 g imposed on mussels, with handling times of 115–290 s required to open the shell and remove the majority of tissue. In each case a single oystercatcher attacked a mussel that had gaped the shell valves slightly wider than its neighbors as the rising tide began to splash the mussel bed, but no other obvious characteristic of the mussels, such as body temperature or orientation, could be linked to the oystercatcher's individual prey choice.     

Impact of short-term acidification on nitrification and nitrifying bacterial community dynamics in soilless cultivation media

Soilless medium-based horticulture systems are highly prevalent due to their capacity to optimize growth of high-cash crops. However, these systems are highly dynamic and more sensitive to physiochemical and pH perturbations than traditional soil-based systems, especially during nitrification associated with ammonia-based fertilization. The objective of this study was to assess the impact of nitrification-generated acidification on ammonia oxidation rates and nitrifying bacterial community dynamics in soilless growth media. To achieve this goal, perlite soilless growth medium from a commercial bell pepper greenhouse was incubated with ammonium in bench-scale microcosm experiments. Initial quantitative real-time PCR analysis indicated that betaproteobacterial ammonia oxidizers were significantly more abundant than ammonia-oxidizing archaea, and therefore, research focused on this group. Ammonia oxidation rates were highest between 0 and 9 days, when pH values dropped from 7.4 to 4.9. Pyrosequencing of betaproteobacterial ammonia-oxidizing amoA gene fragments indicated that r-strategist-like Nitrosomonas was the dominant ammonia-oxidizing bacterial genus during this period, seemingly due to the high ammonium concentration and optimal growth conditions in the soilless media. Reduction of pH to levels below 4.8 resulted in a significant decrease in both ammonia oxidation rates and the diversity of ammonia-oxidizing bacteria, with increased relative abundance of the r-strategist-like Nitrosospira. Nitrite oxidizers (Nitrospira and Nitrobacter) were on the whole more abundant and less sensitive to acidification than ammonia oxidizers. This study demonstrates that nitrification and nitrifying bacterial community dynamics in high-N-load intensive soilless growth media may be significantly different from those in in-terra agricultural systems.     

Effect of elicitors on the production of gossypol and methylated gossypol in cotton hairy roots

The effect of two chemical elicitors, salicylic acid and methyl jasmonate, on the production of gossypol, 6-methoxygossypol, and 6,6′-dimethoxygossypol in Gossypium barbadense hairy roots was examined. Methyl jasmonate, but not salicylic acid, was found to increase the production of gossypol and its methylated forms, but with a concomitant reduction in culture growth. The optimal methyl jasmonate dose was between 100 and 300 μM for hairy roots harvested 7 days after elicitation. After 20 d of induction with 100 μM methyl jasmonate, an eightfold increase in the level of gossypol was observed in elicited cultures compared with control cultures, double the highest gossypol levels previously reported for any cotton tissue. A two to threefold increase in the level of 6-methoxygossypol and a slight increase in the levels of 6,6′-dimethoxygossypol were also observed. Although methyl jasmonate stimulated the production of both optical forms of gossypol, the distribution of the enantiomers was different between elicited and control cultures.     

Maximum vehicle cabin temperatures under different meteorological conditions

A variety of studies have documented the dangerously high temperatures that may occur within the passenger compartment (cabin) of cars under clear sky conditions, even at relatively low ambient air temperatures. Our study, however, is the first to examine cabin temperatures under variable weather conditions. It uses a unique maximum vehicle cabin temperature dataset in conjunction with directly comparable ambient air temperature, solar radiation, and cloud cover data collected from April through August 2007 in Athens, GA. Maximum cabin temperatures, ranging from 41–76°C, varied considerably depending on the weather conditions and the time of year. Clear days had the highest cabin temperatures, with average values of 68°C in the summer and 61°C in the spring. Cloudy days in both the spring and summer were on average approximately 10°C cooler. Our findings indicate that even on cloudy days with lower ambient air temperatures, vehicle cabin temperatures may reach deadly levels. Additionally, two predictive models of maximum daily vehicle cabin temperatures were developed using commonly available meteorological data. One model uses maximum ambient air temperature and average daily solar radiation while the other uses cloud cover percentage as a surrogate for solar radiation. From these models, two maximum vehicle cabin temperature indices were developed to assess the level of danger. The models and indices may be useful for forecasting hazardous conditions, promoting public awareness, and to estimate past cabin temperatures for use in forensic analyses.     

Structural studies of the N-terminus of Connexin 32 using H NMR spectroscopy

The amino terminus of gap junction proteins, connexins, plays a fundamental role in voltage gating and ion permeation. We have previously shown with ¹H NMR that the structure of the N-terminus of a representative connexin molecule contains a flexible turn around glycine 12 [P.E. Purnick, D.C. Benjamin, V.K. Verselis, T.A. Bargiello, T.L. Dowd, Arch. Biochem. Biophys. 381 (2000) 181-190] allowing the N-terminus to reside at the cytoplasmic entry of the channel forming a voltage-sensor. Previous functional studies or neuropathies have shown that the mutation G12Y and G12S form non-functional channels while functional channels are formed from G12P. Using 2D ¹H NMR we show that similar to G12, the structure of the G12P mutant contains a more flexible turn around residue 12, whereas the G12S and G12Y mutants contain tighter, helical turns in this region. These results suggest an unconstrained turn is required around residue 12 to position the N-terminus within the pore allowing the formation of the cytoplasmic channel vestibule, which appears to be critical for proper channel function.     

Poorly neutralizing cross-reactive antibodies against the fusion loop of West Nile virus envelope protein protect in vivo via Fcgamma receptor and complement-dependent effector mechanisms

The human antibody response to flavivirus infection is dominantly directed against a cross-reactive epitope on the fusion loop of domain II (DII-FL) of the envelope (E) protein. Although antibodies against this epitope fail to recognize fully mature West Nile virus (WNV) virions and accordingly neutralize infection poorly in vitro, their functional properties in vivo remain less well understood. Here, we show that while passive transfer of poorly neutralizing monoclonal antibodies (MAb) and polyclonal antibodies against the DII-FL epitope protect against lethal WNV infection in wild-type mice, they fail to protect mice lacking activating Fcγ receptors (FcγR) and the complement opsonin C1q. Consistent with this, an aglycosyl chimeric mouse-human DII-FL MAb (E28) variant that lacks the ability to engage FcγR and C1q also did not protect against WNV infection in wild-type mice. Using a series of immunodeficient mice and antibody depletions of individual immune cell populations, we demonstrate that the nonneutralizing DII-FL MAb E28 does not require T, B, or NK cells, inflammatory monocytes, or neutrophils for protection. Rather, E28 treatment decreased viral load in the serum early in the course of infection, which resulted in blunted dissemination to the brain, an effect that required phagocytic cells, C1q, and FcγRIII (CD16). Overall, these studies enhance our understanding of the functional significance of immunodominant, poorly neutralizing antibodies in the polyclonal human anti-flavivirus response and highlight the limitations of current in vitro surrogate markers of protection, such as cell-based neutralization assays, which cannot account for the beneficial effects conferred by these antibodies.