Biofilm-Mediated Fragmentation and Degradation of Microcrystalline Cellulose by Cellulomonas flavigena KU (ATCC 53703).

Cellulomonas flavigena KU (ATCC 53703) produces an extracellular matrix involved in the degradation of microcrystalline cellulose. This extracellular material is primarily composed of the gel-forming, ß-1,3-glucan known as curdlan and associated, cellulose-degrading enzymes. In this study, the effects of various forms of nutrient limitation on cellulose attachment, cellular aggregation, curdlan production, and biofilm formation were investigated throughout a 7-day incubation period by using phase-contrast microscopy. Compared to cultures grown in non-limiting media, nitrogen-limitation promoted early attachment of C. flavigena KU cells to the cellulose surface, and cellulose attachment was congruent with cellular aggregation and curdlan production. Over the course of the experiment, microcolonies of attached cells grew into curdlan-producing biofilms on the cellulose. By contrast, bacterial cells grown on cellulose in non-limiting media remained unattached and unaggregated throughout most of the incubation period. By 7 days of incubation, bacterial aggregation was ninefold greater in N-limited cultures compared to nutritionally complete cultures. In a similar way, phosphorus- and vitamin-limitation (i.e., yeast extract-limitation) also resulted in early cellulose attachment and biofilm formation. Furthermore, nutrient limitation promoted more rapid and efficient fragmentation and degradation of cellulose, with cellulose fragments in low-N media averaging half the size of those in high-N media after 7 days. Two modes of cellulose degradation are proposed for C. flavigena KU, a "planktonic mode" and a "biofilm mode". Similar observations have been reported for other curdlan-producing cellulomonads, and these differing cellulose degradation strategies may ultimately prove to reflect sequential stages of a multifaceted biofilm cycle important in the bioconversion of this abundant and renewable natural resource.

IL-17A expression by both T cells and non-T cells contribute to HSV-IL-2-induced CNS demyelination.

Previously we reported that a recombinant HSV-1 expressing murine IL-2 (HSV-IL-2) causes CNS demyelination in different strains of mice and in a T cell-dependent manner. Since TH17 cells have been implicated in CNS pathology, in the present study, we looked into the effects of IL-17A-/- and three of its receptors on HSV-IL-2-induced CNS demyelination. IL-17A-/- mice did not develop CNS demyelination, while IL-17RA-/-, IL-17RC-/-, IL-17RD-/- and IL-17RA-/-RC-/- mice developed CNS demyelination. Adoptive transfer of T cells from wild-type (WT) mice to IL-17A-/- mice or T cells from IL-17A-/- mice to Rag-/- mice induced CNS demyelination in infected mice. Adoptive T cell experiments suggest that both T cells and non-T cells expressing IL-17A contribute to HSV-IL-2-induced CNS demyelination with no difference in the severity of demyelination between the two groups of IL-17A producing cells. IL-6, IL-10, or TGFß did not contribute to CNS demyelination in infected mice. Transcriptome analysis between IL-17A-/- brain and spinal cord of infected mice with and without T cell transfer from WT mice revealed that "neuron projection extension involved in neuron projection guidance" and "ensheathment of neurons" pathways were associated with CNS demyelination. Collectively, the results indicate the importance of IL-17A in CNS demyelination and the possible involvement of more than three of IL-17 receptors in CNS demyelination.

Expression, Inducers and Cellular Sources of the Chemokine MIG (CXCL 9), During Primary Herpes Simplex Virus Type-1 Infection of the Cornea.

PURPOSE: To investigate the production of monokine induced by gamma-interferon (MIG) during a primary Herpes simplex virus type 1 (HSV-1) infection of the cornea. We hypothesize that multiple CXCR3 ligands are involved in T cell recruitment during HSV-1 corneal infection and that neutrophils have the potential to contribute to their production. MATERIALS AND METHODS: Levels of MIG were evaluated in an in vivo murine model of HSV-1 corneal infection by quantitative ELISA. Cultured murine corneal fibroblast (MCF) cells and purified neutrophils were stimulated in vitro with IFN-γ and IL-1α to determine inducers of MIG. Cellular sources of MIG production in vivo were investigated via cellular depletion studies. Additionally, MIG production resulting from interaction between resident human corneal cells and neutrophils was evaluated in an ex vivo model of human corneal infection. RESULTS: MIG was significantly elevated on days 2-6 and on day 8 following corneal infection. MCF and neutrophils secreted MIG in response to IFN-γ, but not IL-1α stimulation. Co-stimulation with IFN-γ and IL-1α induced a four-fold increase in MIG production by MCF. However, the same combination led to a three-fold decrease in MIG production by neutrophils. In vivo, a 52% reduction in MIG levels was observed in the neutrophil depleted host. In the human ex vivo model, MIG levels were significantly elevated in response to communication between HSV-1 infected corneal tissue and neutrophils. CONCLUSIONS: Here, we report the evidence for the production of MIG, a second CXCR3 ligand, during the primary immune response to HSV-1 corneal infection. Our results support the hypothesis that both neutrophils and resident corneal cells contribute to MIG production in vivo. However, neutrophils produce MIG in response to communication with HSV-1-infected resident corneal cells more efficiently than by direct interaction with virus. In addition, we found that MIG production by neutrophils and resident corneal cells was differentially regulated by IL-1α.

IL-17 receptor signaling influences virus-induced corneal inflammation.

IL-17 has been associated with selected inflammatory and autoimmune diseases. We characterized the expression of this proinflammatory cytokine following HSV-1 corneal infection and investigated whether IL-17R signaling modulated the host response to the viral pathogen at early time-points postinfection. IL-17 was elevated in the murine cornea 24 h after high-dose virus infection and subsequently persisted at low levels during the first week. Immunofluorescent studies showed that the IL-17R was expressed by cultured mouse corneal fibroblasts. Exposure of corneal cells to IL-17 led to production of IL-6 and MIP-2 in vitro and in vivo, indicating that the IL-17R was functional. Mice lacking IL-17R displayed significantly reduced neutrophil infiltration and corneal opacity. However, this effect was transient, as corneal pathology and neutrophil influx resembled that of wild-type (WT) hosts 4 days postinfection. HSV-1 growth and clearance in IL-17R(-/-) hosts were similar to that of the WT controls. Infection of IFN-gamma gene knockout mice was associated with elevated IL-17 levels and accelerated corneal opacity, suggesting that IFN-gamma negatively regulated IL-17 expression. Collectively, our results establish that IL-17 is rapidly produced in the cornea after HSV-1 infection and is regulated at least in part by IFN-gamma. The absence of IL-17 signaling results in a transient decrease in the expression of proinflammatory mediators, neutrophil migration, and corneal pathology, but control of virus growth in the cornea and trigeminal ganglia is not compromised. Thus, IL-17 actively influences early virus-induced corneal inflammation.