• Characterization of the response of GM-CSF supplemented THP-1 human monocytes to LPS of oral microorganisms

      Baqui, A. A. M. Abdullahel; Falkler, William A., Ph.D.; Meiller, Timothy F. (1996)
      The effect of granulocyte macrophage colony stimulating factor (GM-CSF) on the differentiation and activation of a human monocyte cell line (THP-1) in the presence of lipopolysaccharide (LPS) from oral organisms has not been investigated. It was hypothesized that GM-CSF treated THP-1 cells are immunologically and functionally hyperactivated in the presence of LPS of oral microorganisms. A study was undertaken to elucidate the immunological expression of activation antigens and production of cytokines by THP-1 cells after treatment with GM-CSF and in response to LPS of the putative periodontal pathogens, Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum). LPS of F. nucleatum and P. gingivalis was prepared, characterized by SDS PAGE, standardized by protein concentration and tested for endotoxin content. Morphological changes in THP-1 cells were observed with light, immunofluorescence (IF) and transmission electron microscopy (TEM), following treatment with LPS/PMA (phorbol-12-myristate-13 acetate) and/or GM-CSF at various concentrations and time intervals. The expression of seven different activation antigens namely, CD-11b, CD-11c, CD-14, CD-35, CD-68, CD-71 and HLA-DR, in THP-1 cells was evaluated in this experimental model. Direct labeling of THP-1 cell activation antigens was performed using the Vectastain ABC-AP staining kit with light microscopy. Alternatively, LPS/PMA and/or GM-CSF stimulated and unstimulated cells were stained with FITC labelled antibodies for IF and gold-labelled antibodies were used for TEM. To evaluate chemotaxis as a functional variation in THP-1 cells, an assay was performed using a microchemotaxis chamber and polyvinylpyrolidone free polycarbonate membrane filters. To determine phagocytic activity in the experimental model, an assay was performed using FITC labelled S. cereviseae. Phagocytic uptake of cells was determined with the use of a fluorescence microscope. Production of extracellular cytokines, TNF-alpha, IL-1beta, IL-6, IL-8 and IL-12, by THP-1 cells in the experimental model, was measured by ELISA. Reverse transcription polymerase chain reaction (RT-PCR) of the of TNF-alpha, IL-1 beta and IL-6 cytokines was performed to correlate the cytokine gene transcription with cytokine gene translation (ELISA). Three percent of untreated THP-1 cells expressed HLA-DR; 9%, CD-11b; 8%, CD-11c; 22%, CD-14; 9%, CD-35 and 7%, CD-68 antigens. CD-71 was not expressed in untreated THP-1 cells. Treatment with LPS of F. nucleatum and P. gingivalis and PMA increased the expression of activation antigens. Following treatment with combined GM-CSF and LPS of P. gingivalis and F. nucleatum there was a significant (p<0.05) up-regulation and expression of HLA-DR, CD-11b, CD-11c, CD-35 and CD-71 activation antigens over baseline values. Expression of the LPS receptor, CD-14, was significantly (p<0.05) down-regulated by this treatment for 1-2 d and then up-regulated at 2-4 d. Antigens important in phagocytosis, CD-11b and CD-35, were significantly (p<0.05) up-regulated by GM-CSF. The up-regulation was further demonstrated in phagocytosis functional assays. Stimulation with combined GM-CSF and oral LPS resulted in a significant (p<0.05) escalation in phagocytosis by the THP-1 cells. There was a two-fold increase in chemotactic response with GM-CSF treatment by 4 d, which decreased after 7 d. RT-PCR data indicated that TNF-alpha transcripts were constituitively produced in the THP-1 cell but that translation to a high level of production of functional cytokines required the LPS/GM-CSF stimulus. Gene transcription for IL-6 was detected as early as 5 min post stimulation. (Abstract shortened by UMI.)
    • Establishing a Lipid Model of Host-Pathogen Interaction Using Multimodal Mass Spectrometry Imaging in a Francisella Infection

      Scott, Alison June; Ernst, Robert K.; 0000-0001-6969-4707 (2015)
      Host membranes are intimately involved in the immune response to any infection, including formation of lipid docking sites for proteins, organization of immune signaling complexes in lipid rafts, and maintaining a reservoir of fatty acids that contribute to acute inflammation. Francisella species maintain several host immune evasion strategies, one of which involves induction of the immunomodulatory lipid prostaglandin E2 (PGE2). The source of PGE2 is arachidonic acid (AA), a structural component of membrane phospholipids. Mass spectrometry imaging (MSI) was used to map and characterize both host- and pathogen-borne lipids using Francisella infected spleens in a murine model. Here, we identified and mapped the unique bacterial molecule, lipid A within infected mouse spleens by MALDI-MSI, confirming the in vivo structure (m/z 1665.1) in a mammalian infection. Francisella lipid A mapped primarily to the red pulp of the spleen, with signal first appearing between 24 and 36 hours post-infection, corresponding to the onset of bacteremia. Numerous changes in host lipid levels were correlated with progression of the infection. A phosphatidylinositol species, 1-stearoyl, 2-arachidonyl phosphatidylinositol (SAPI) was identified in the periphery of the splenic white pulp, suggesting a cell-specific origin. SAPI abundance peaks at 24 hours and is depleted in the timepoints preceding lethality (48 to 60 hours). In vitro reports demonstrate that SAPI is the earliest source of AA in activated macrophages. We have subsequently linked importation of SAPI into the spleen by monocytic infiltrates, which increases the total SAPI load. Additionally, accumulation of cholesterol was observed by SIMS-Imaging in the infected spleens and may be another indicator of immune infiltration. These data highlight a role for newly immigrant cells in contributing to the pool of total inflammatory lipids. Here, MSI is presented as a new approach to studying lipid-level host-pathogen interactions, facilitating targeted and untargeted discovery.
    • Immune Responses to a Francisella Lipid A Mutant: Characterization and Therapeutic Potential

      Powell, Daniel Alford; Ernst, Robert K. (2012)
      Francisella tularensis tularensis (Ft) is an intracellular Gram-negative bacterium and the causative agent of the severe human disease tularemia with potential for use as a bioweapon. Francisella lipid A, normally the biologically active component of lipopolysaccharide (LPS) has extremely low endotoxic activity. A Francisella tularensis novicida (Fn) lipid A biosynthesis mutant was generated that lacked the 4'-phosphatase enzyme (LpxF). Analysis of lipid A isolated from this mutant strain, compared to WT Fn showed retention of the phosphate moiety at the 4' position and the N-linked fatty acid at the 3' position on the diglucosamine backbone. This mutant was previously shown in our laboratory to be avirulent and confer protective immunity to a lethal WT Fn challenge. Further work has been carried out to elucidate the mechanisms of this protective immunity. The role of B cells was examined using μMT<super>-/-</super> mice, which lack functional B cells. While all mice survived the initial inoculation, all of the μMT<super>-/-</super> mice succumbed to the lethal challenge. This complete lack of protection shows that B cells are absolutely required for the protective response generated by the lpxF-null mutant. While development of an effective vaccine to Francisella remains a priority, we decided to address the therapeutic potential of serum obtained from immunization with the lpxF-null mutant in the possibility of a Francisella outbreak. Mice were infected with Fn and then were treated with serum either from naïve or immune mice. Mice receiving immune serum were completely rescued out to 36 hours post-infection, and were partially rescued at 48 hours, whereas mice receiving naïve serum started succumbing to infection at 60 hours post-infection, indicating the serum has therapeutic potential even late in an infection. The identity of the proteins recognized by this protective serum was further investigated. Our work not only identified known Francisella immunogens but revealed proteins previously unknown to be antigenic. Serum from LVS lpxF-null vaccinated-mice showed a similar protective capacity when given as a therapeutic. Current work is being carried out to generate monoclonal antibodies to these identified proteins and assay their ability to be used in the event of a Francisella outbreak.
    • Role of Lipopolysaccharide and RANKL in Osteoclastogenesis: Potential Inhibitory Effects of C-Phycocyanin on the Respective Molecular Pathways of Osteoclastogenesis

      Al Qranei, Mohammed; Chellaiah, Meenakshi A. (2020)
      Many skeletal diseases are characterized by excessive bone loss. Bone loss is mediated by osteoclasts, which are differentiated from cells of the monocyte/macrophage lineage upon stimulation of two indispensable factors, the RANKL and M-CSF. Lipopolysaccharide (LPS), a bacterial pathogenic factor, has also been shown to engage in osteoclastogenesis during inflammatory events actively. C-phycocyanin (C-PC) is a phycobiliprotein found in the blue-green algae that showed many therapeutic effects, including anti-arthritic and anti-inflammatory properties. However, the exact mechanism by which LPS regulates osteoclastogenesis and also the impact of C-PC on osteoclastogenesis needs further elucidations. We studied the osteoclast differentiation process in vitro using RAW 264.7 macrophage cell line. First, we showed that LPS induced osteoclastogenesis in RANKL-primed cells in vitro. LPS elicited osteoclastogenic mechanism by signaling through the TLR4 receptor, which is expressed in osteoclast precursors. Here we also found that TNF-α secreted by osteoclast precursors in response to TLR4 stimulation regulated the processes of osteoclastogenesis via TNF-R signaling. Second, we tested the inhibitory effect of C-PC on osteoclastogenesis. We showed here that C-PC strongly inhibited the early stage of osteoclast differentiation, thus significantly suppressing RANKL- and LPS- mediated osteoclastogenesis. Nonetheless, osteoblast differentiation and activity were not affected by C-PC. Reactive oxygen species (ROS) are generated during RANKL-mediated osteoclast differentiation. While studying the possible mechanisms of osteoclast differentiation, we found that C-PC a) attenuated RANKL-induced ROS; and b) interfered with RANKL-stimulated NF-κB signaling by preventing the degradation of cytosolic IκB-α; subsequently, these resulted in the loss of sequential activation of the osteoclastogenic downstream markers such as c-Fos and NFATc1. We propose that a unique mechanism of osteoclastogenesis is mediated by bacterial LPS that can be targeted during inflammatory-mediated bone loss. Also, C-PC could potentially be used as a therapeutic compound in osteolytic diseases caused by osteoclast activation without affecting osteoblast function.