Aggregatibacter actinomycetemcomitans Leukotoxin (LtxA) Requires Death Receptor Fas, in Addition to LFA-1, To Trigger Cell Death in T Lymphocytes.

Leukotoxin (LtxA) (trade name, Leukothera) is a protein secreted by the oral bacterium Aggregatibacter actinomycetemcomitansA. actinomycetemcomitans is an oral pathogen strongly associated with development of localized aggressive periodontitis. LtxA acts as a virulence factor for A. actinomycetemcomitans by binding to the ß2 integrin lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) on white blood cells (WBCs) and causing cell death. In addition, because of its specificity for malignant and activated WBCs, LtxA is being investigated as a therapeutic agent for treatment of hematological malignancies and autoimmune diseases. Here, we report the successful generation and characterization of Jurkat T lymphocytes with deletions in CD18, CD11a, and Fas that were engineered using CRISPR/Cas9 gene editing. Using these clones, we demonstrate the specificity of LtxA for cells expressing LFA-1. We also demonstrate the requirement of the cell death receptor Fas for LtxA-mediated cell death in T lymphocytes. We show that LFA-1 and Fas are early events in the LtxA-mediated cell death cascade as caspase activation and mitochondrial perturbation do not occur in the absence of either receptor. To our knowledge, LtxA is the first molecule, other than FasL, known to require the Fas death receptor to initiate cell death. Knowledge of the mechanism of cell death induced by LtxA will facilitate the understanding of LtxA as a bacterial virulence factor and development of it as a potential therapeutic agent.

Mechanisms of LtxA (Leukotoxin), a Potent New Anti-Inflammatory Agent for the Treatment of Alopecia Areata.

Alopecia areata is an autoimmune condition where activated, pro-inflammatory white blood cells (WBCs) attack the hair follicles, resulting in hair loss. Migration of these activated WBCs from the blood stream and into the follicle tissue requires interaction between the integrin, lymphocyte function-associated antigen-1 (LFA-1) on WBCs, and ICAM-1 on vascular endothelial cells. High levels of active LFA-1 are uniquely expressed on WBCs that are involved in autoimmune and inflammatory conditions. The natural biologic agent LtxA (Leukothera) preferentially targets and depletes disease activated and malignant WBCs by binding to active LFA-1. The experimental drug has demonstrated significant therapeutic efficacy against autoimmune/inflammatory conditions such as psoriasis and allergic asthma in mouse models for these diseases. In addition, when injected into rodents, rhesus macaques, and dogs, LtxA was demonstrated to be physiologically active, biologically specific, and extremely well-tolerated. LFA-1 is an attractive target for therapy because it is only normally present on WBCs and has been shown to be activated and overexpressed on WBCs that are responsible for autoimmune/inflammatory conditions.

Aggregatibacter actinomycetemcomitans leukotoxin utilizes a cholesterol recognition/amino acid consensus site for membrane association.

Aggregatibacter actinomycetemcomitans produces a repeats-in-toxin (RTX) leukotoxin (LtxA) that selectively kills human immune cells. Binding of LtxA to its ß2 integrin receptor (lymphocyte function-associated antigen-1 (LFA-1)) results in the clustering of the toxin·receptor complex in lipid rafts. Clustering occurs only in the presence of LFA-1 and cholesterol, and LtxA is unable to kill cells lacking either LFA-1 or cholesterol. Here, the interaction of LtxA with cholesterol was measured using surface plasmon resonance and differential scanning calorimetry. The binding of LtxA to phospholipid bilayers increased by 4 orders of magnitude in the presence of 40% cholesterol relative to the absence of cholesterol. The affinity was specific to cholesterol and required an intact secondary structure. LtxA contains two cholesterol recognition/amino acid consensus (CRAC) sites; CRAC(336) ((333)LEEYSKR(339)) is highly conserved among RTX toxins, whereas CRAC(503) ((501)VDYLK(505)) is unique to LtxA. A peptide corresponding to CRAC(336) inhibited the ability of LtxA to kill Jurkat (Jn.9) cells. Although peptides corresponding to both CRAC(336) and CRAC(503) bind cholesterol, only CRAC(336) competitively inhibited LtxA binding to this sterol. A panel of full-length LtxA CRAC mutants demonstrated that an intact CRAC(336) site was essential for LtxA cytotoxicity. The conservation of CRAC(336) among RTX toxins suggests that this mechanism may be conserved among RTX toxins.

Aggregatibacter actinomycetemcomitans leukotoxin (LtxA; Leukothera) induces cofilin dephosphorylation and actin depolymerization during killing of malignant monocytes.

Leukotoxin (LtxA; Leukothera), a protein toxin secreted by the oral bacterium Aggregatibacter actinomycetemcomitans, specifically kills white blood cells (WBCs). LtxA binds to the receptor known as lymphocyte function associated antigen-1 (LFA-1), a ß2 integrin expressed only on the surface of WBCs. LtxA is being studied as a virulence factor that helps A. actinomycetemcomitans evade host defences and as a potential therapeutic agent for the treatment of WBC diseases. LtxA-mediated cell death in monocytes involves both caspases and lysosomes; however, the signalling proteins that regulate and mediate cell death remain largely unknown. We used a 2D-gel proteomics approach to analyse the global protein expression changes that occur in response to LtxA. This approach identified the protein cofilin, which underwent dephosphorylation upon LtxA treatment. Cofilin is a ubiquitous actin-binding protein known to regulate actin dynamics and is regulated by LIM kinase (LIMK)-mediated phosphorylation. LtxA-mediated cofilin dephosphorylation was dependent on LFA-1 and cofilin dephosphorylation did not occur when LFA-1 bound to its natural ligand, ICAM-1. Treatment of cells with an inhibitor of LIMK (LIMKi) also led to cofilin dephosphorylation and enhanced killing by LtxA. This enhanced sensitivity to LtxA coincided with an increase in lysosomal disruption, and an increase in LFA-1 surface expression and clustering. Both LIMKi and LtxA treatment also induced actin depolymerization, which could play a role in trafficking and surface distribution of LFA-1. We propose a model in which LtxA-mediated cofilin dephosphorylation leads to actin depolymerization, LFA-1 overexpression/clustering, and enhanced lysosomal-mediated cell death.

Therapeutic Applications of Aggregatibacter actinomycetemcomitans Leukotoxin.

Aggregatibacter actinomycetemcomitans is a Gram-negative oral bacterium that has been primarily studied for its role in causing periodontal disease. The bacterium has also been implicated in several systemic diseases such as endocarditis and soft tissue abscesses. Leukotoxin (LtxA) is perhaps the best studied protein virulence factor from A. actinomycetemcomitans. The protein can rapidly destroy white blood cells (WBCs), helping the bacterium to subvert the host immune system. The functional receptor for LtxA is lymphocyte function associated antigen-1 (LFA-1), which is expressed exclusively on the surfaces of WBCs. Bacterial expression and secretion of the protein are highly regulated and controlled by a number of genetic and environmental factors. The mechanism of LtxA action on WBCs varies depending on the type of cell that is being killed, and the protein has been shown to activate numerous cell death pathways in susceptible cells. In addition to serving as an important virulence factor for the bacterium, because of its exquisite specificity and rapid activity, LtxA is also being investigated as a therapeutic agent that may be used to treat diseases such as hematological malignancies and autoimmune/inflammatory diseases. It is our hope that this review will inspire an increased intensity of research related to LtxA and its effect on Aggressive Periodontitis, the disease that led to its initial discovery.

Leukotoxin (Leukothera®) targets active leukocyte function antigen-1 (LFA-1) protein and triggers a lysosomal mediated cell death pathway.

Leukotoxin (LtxA) is a protein toxin that is secreted from the oral bacterium, Aggregatibacter actinomycetemcomitans. LtxA targets specifically the ß(2) integrin, leukocyte function antigen-1 (LFA-1) on white blood cells (WBCs) and causes cell death. LtxA preferentially targets activated WBCs and is being developed as a therapeutic agent for the treatment of WBC diseases such as hematologic malignancies and autoimmune/inflammatory diseases. However, the mechanism by which interaction between LtxA and LFA-1 results in cell death is not well understood. Furthermore, how LtxA preferentially recognizes activated WBCs is not known. We show here that LtxA interacts specifically with LFA-1 in the active (exposed) conformation. In THP-1 monocytes, LtxA caused rapid activation of caspases, but LtxA could overcome the inhibition of caspases and still intoxicate. In contrast, inhibiting the vesicular trafficking pathway or cathepsin D release from the lysosome resulted in significant inhibition of LtxA-mediated cytotoxicity, indicating a more potent, lysosomal mediated cell death pathway. LtxA caused rapid disruption of the lysosomal membrane and release of lysosomal contents into the cytosol. Binding of LtxA to LFA-1 resulted in the internalization of both LtxA and LFA-1, with LtxA localizing specifically to the lysosomal compartment. To our knowledge, LtxA represents the first bacterial toxin shown to localize to the lysosome where it induces rapid cell death.

Effects of Aggregatibacter actinomycetemcomitans leukotoxin on endothelial cells.

Aggregatibacter actinomycetemcomitans is a human pathogen that produces leukotoxin (LtxA) as a major virulence factor. In this study the effect of LtxA on microvascular endothelial cell viability and phenotype was studied. High doses of single LtxA treatment (500 ng/ml to 5 µg/ml) significantly and irreversibly decreased cell proliferation and induced apoptosis, as assessed by tetrazolium salt and annexin V assay, respectively. Apoptosis was partially inhibited by the pan-caspase inhibitor, z-VAD-fmk. LtxA caused a cell cycle arrest in the G2/M phase after 72 h. Between 500 ng/ml and 5 µg/ml, after long- or short-term stimulation LtxA increased the expression of ICAM-1 and VCAM-1, as well as the percentages of endothelial cells expressing these adhesion molecules. Thus, A. actinomycetemcomitans LtxA has substantial pro-inflammatory effects on human brain endothelial cells by upregulation of ICAM-1 and VCAM-1. Furthermore, LtxA in higher concentration was found to decrease proliferation and induces apoptosis in microvascular endothelial cells.

Prophage induction in lysogenic Aggregatibacter actinomycetemcomitans cells co-cultured with human gingival fibroblasts, and its effect on leukotoxin release.

Lysogeny is common among strains of the periodontal pathogen Aggregatibacter actinomycetemcomitans. Since lysogenic induction is known to result in the increased synthesis and release of bacterial toxins from lysogens, it would be important to elucidate the conditions under which induction of these bacteria may occur. Co-cultures of A. actinomycetemcomitans strains (either lysogenic or non-lysogenic) and human cells (either gingival fibroblasts or pharyngeal epithelial cells) were prepared. Following incubation, bacteriophage titers of up to 6.2 × 10(7) pfu/ml were detected in the cell-free, spent culture media from the co-cultures of the lysogenic A. actinomycetemcomitans strains and the fibroblasts. Little (maximum of 2 × 10(0) pfu/ml) or no titers of phage could be detected in the mono-cultures of the lysogenic A. actinomycetemcomitans strains alone. In contrast, no phage were detectable in the cell-free spent culture media of the lysogens cocultured with the epithelial cells. Futhermore, co-culture of the A. actinomycetemcomitans lysogens with the fibroblasts resulted in enhanced release of the A. actinomycetemcomitans leukotoxin into the culture medium, in comparison with the spent culture media from mono-cultures of the lysogens alone. These results are consistent with the concept that interaction with fibroblasts may mediate prophage induction in lysogenic strains of A. actinomycetemcomitans, and that leukotoxin release is greatly augmented following induction of the lysogens.

The Widespread Colonization Island of Actinobacillus actinomycetemcomitans.

Genomic islands, such as pathogenicity islands, contribute to the evolution and diversification of microbial life. Here we report on the Widespread Colonization Island, which encompasses the tad (tight adherence) locus for colonization of surfaces and biofilm formation by the human pathogen Actinobacillus actinomycetemcomitans. At least 12 of the 14 genes at the tad locus are required for tenacious biofilm formation and synthesis of bundled Flp pili (fibrils) that mediate adherence. The pilin subunit, Flp1, remains inside the cell in tad-locus mutants, indicating that these genes encode a secretion system for export and assembly of fibrils. We found tad-related regions in a wide variety of Bacterial and Archaeal species, and their sequence characteristics indicate possible horizontal transfer. To test the hypothesis of horizontal transfer, we compared the phylogeny of the tad locus to a robust organismal phylogeny using statistical tests of congruence and tree reconciliation techniques. Our analysis strongly supports a complex history of gene shuffling by recombination and multiple horizontal transfers, duplications and losses. We present evidence for a specific horizontal transfer event leading to the establishment of this region as a determinant of disease.

Genome sequence of Kingella kingae septic arthritis isolate PYKK081.

Kingella kingae is a human oral bacterium that can cause infections of the skeletal system in children. The bacterium is also a cardiovascular pathogen causing infective endocarditis in children and adults. We report herein the draft genome sequence of septic arthritis K. kingae strain PYKK081.

Broad-spectrum biofilm inhibition by Kingella kingae exopolysaccharide.

Cell-free extracts prepared from Kingella kingae colony biofilms were found to inhibit biofilm formation by Aggregatibacter actinomycetemcomitans, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Candida albicans, and K. kingae. The extracts evidently inhibited biofilm formation by modifying the physicochemical properties of the cell surface, the biofilm matrix, and the substrate. Chemical and biochemical analyses indicated that the biofilm inhibition activity in the K. kingae extract was due to polysaccharide. Structural analyses showed that the extract contained two major polysaccharides. One was a linear polysaccharide with the structure →6)-α-d-GlcNAcp-(1→5)-ß-d-OclAp-(2→, which was identical to a capsular polysaccharide produced by Actinobacillus pleuropneumoniae serotype 5. The second was a novel linear polysaccharide, designated PAM galactan, with the structure →3)-ß-d-Galf-(1→6)-ß-d-Galf-(1→. Purified PAM galactan exhibited broad-spectrum biofilm inhibition activity. A cluster of three K. kingae genes encoding UDP-galactopyranose mutase (ugm) and two putative galactofuranosyl transferases was sufficient for the synthesis of PAM galactan in Escherichia coli. PAM galactan is one of a growing number of bacterial polysaccharides that exhibit antibiofilm activity. The biological roles and potential technological applications of these molecules remain unknown.

Leukotoxin (LtxA/Leukothera) induces ATP expulsion via pannexin-1 channels and subsequent cell death in malignant lymphocytes.

Leukotoxin (LtxA) (Trade name, Leukothera) is a protein that is secreted from the oral bacterium Aggregatibacter actinomycetemcomitans, which targets and kills activated white blood cells (WBCs) by binding to lymphocyte function associated antigen-1 (LFA-1). Interaction between LtxA and Jurkat T-cells results in cell death and is characterized by increased intracellular Ca2+, activation of caspases, clustering of LtxA and LFA-1 within lipid rafts, and involvement of the Fas death receptor. Here, we show that LtxA can kill malignant lymphocytes via apoptotic and necrotic forms of cell death. We show that LtxA causes activation of caspases and PARP, cleavage of pannexin-1 (Panx1) channels, and expulsion of ATP, ultimately leading to cell death via apoptosis and necrosis. CRISPR-Cas9 mediated knockout (K/O) of Panx1 in Jurkat cells prevented ATP expulsion and resulted in resistance to LtxA for both apoptotic and necrotic forms of death. Resistance to necrosis could only be overcome when supplementing LtxA with endogenous ATP (bzATP). The combination of LtxA and bzATP promoted only necrosis, as no Panx1 K/O cells stained positive for phosphatidylserine (PS) exposure following the combined treatment. Inhibition of LtxA/bzATP-induced necrosis was possible when pretreating Jurkat cells with oATP, a P2X7R antagonist. Similarly, blockage of P2X7Rs with oATP prevented the intracellular mobilization of Ca2+, an important early step in LtxA induced cell death. We show that LtxA is able to kill malignant lymphocytes through an apoptotic death pathway which is potentially linked to a Panx1/P2X7R mediated necrotic form of death. Thus, inhibition of ATP release appears to significantly delay the onset of LtxA induced apoptosis while completely disabling the necrotic death pathway in T-lymphocytes, demonstrating the crucial role of ATP release in LtxA-mediated cell death.

Significance of wall structure, macromolecular composition, and surface polymers to the survival and transport of Cryptosporidium parvum oocysts.

The structure and composition of the oocyst wall are primary factors determining the survival and hydrologic transport of Cryptosporidium parvum oocysts outside the host. Microscopic and biochemical analyses of whole oocysts and purified oocyst walls were undertaken to better understand the inactivation kinetics and hydrologic transport of oocysts in terrestrial and aquatic environments. Results of microscopy showed an outer electron-dense layer, a translucent middle layer, two inner electron-dense layers, and a suture structure embedded in the inner electron-dense layers. Freeze-substitution showed an expanded glycocalyx layer external to the outer bilayer, and Alcian Blue staining confirmed its presence on some but not all oocysts. Biochemical analyses of purified oocyst walls revealed carbohydrate components, medium- and long-chain fatty acids, and aliphatic hydrocarbons. Purified walls contained 7.5% total protein (by the Lowry assay), with five major bands in SDS-PAGE gels. Staining of purified oocyst walls with magnesium anilinonaphthalene-8-sulfonic acid indicated the presence of hydrophobic proteins. These structural and biochemical analyses support a model of the oocyst wall that is variably impermeable and resistant to many environmental pressures. The strength and flexibility of oocyst walls appear to depend on an inner layer of glycoprotein. The temperature-dependent permeability of oocyst walls may be associated with waxy hydrocarbons in the electron-translucent layer. The complex chemistry of these layers may explain the known acid-fast staining properties of oocysts, as well as some of the survival characteristics of oocysts in terrestrial and aquatic environments. The outer glycocalyx surface layer provides immunogenicity and attachment possibilities, and its ephemeral nature may explain the variable surface properties noted in oocyst hydrologic transport studies.

Aggregatibacter actinomycetemcomitans Leukotoxin (LtxA; Leukothera®): Mechanisms of Action and Therapeutic Applications.

Aggregatibacter actinomycetemcomitans is an oral pathogen that produces the RTX toxin, leukotoxin (LtxA; Leukothera®). A. actinomycetemcomitans is strongly associated with the development of localized aggressive periodontitis. LtxA acts as a virulence factor for A. actinomycetemcomitans to subvert the host immune response by binding to the ß2 integrin lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) on white blood cells (WBCs), causing cell death. In this paper, we reviewed the state of knowledge on LtxA interaction with WBCs and the subsequent mechanisms of induced cell death. Finally, we touched on the potential therapeutic applications of LtxA (trade name Leukothera®) toxin therapy for the treatment of hematological malignancies and immune-mediated diseases.

Screen for leukotoxin mutants in Aggregatibacter actinomycetemcomitans: genes of the phosphotransferase system are required for leukotoxin biosynthesis.

Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans is a pathogen that causes localized aggressive periodontitis and extraoral infections including infective endocarditis. Recently, we reported that A. actinomycetemcomitans is beta-hemolytic on certain growth media due to the production of leukotoxin (LtxA). Based on this observation and our ability to generate random transposon insertions in A. actinomycetemcomitans, we developed and carried out a rapid screen for LtxA mutants. Using PCR, we mapped several of the mutations to genes that are known or predicted to be required for LtxA production, including ltxA, ltxB, ltxD, and tdeA. In addition, we identified an insertion in a gene previously not recognized to be involved in LtxA biosynthesis, ptsH. ptsH encodes the protein HPr, a phosphocarrier protein that is part of the sugar phosphotransferase system. HPr results in the phosphorylation of other proteins and ultimately in the activation of adenylate cyclase and cyclic AMP (cAMP) production. The ptsH mutant showed only partial hemolysis on blood agar and did not produce LtxA. The phenotype was complemented by supplying wild-type ptsH in trans, and real-time PCR analysis showed that the ptsH mutant produced approximately 10-fold less ltxA mRNA than the wild-type strain. The levels of cAMP in the ptsH mutant were significantly lower than in the wild-type strain, and LtxA production could be restored by adding exogenous cAMP to the culture.

A bioluminescent HL-60 cell line to assay anti-leukaemia therapeutics under physiological conditions.

Screens for compounds and proteins with anti-cancer activity employ viability assays using relevant cancer cell lines. For leukaemia studies, the human leukaemia cell line, HL-60, is often used as a model system. To facilitate the discovery and investigation of anti-leukaemia therapeutics under physiological conditions, we have engineered HL-60 cells that stably express firefly luciferase and produce light that can be detected using an in vivo imaging system (IVIS). Bioluminescent HL-60luc cells could be rapidly detected in whole blood with a sensitivity of approximately 1000 viable cells/200 microl blood. Treatment of HL-60luc cells with the drug chlorambucil revealed that the bioluminescent viability assay is able to detect cell death earlier than the Trypan blue dye exclusion assay. HL-60luc cells administered intraperitoneally (i.p.) or intravenously (i.v.) were visualized in living mice. The rapidity and ease of detecting HL-60luc cells in biological fluid indicates that this cell line could be used in high-throughput screens for the identification of drugs with anti-leukaemia activity under physiological conditions.

Aggregatibacter actinomycetemcomitans Leukotoxin Is Delivered to Host Cells in an LFA-1-Indepdendent Manner When Associated with Outer Membrane Vesicles.

The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, has been associated with localized aggressive periodontitis (LAP). In particular, highly leukotoxic strains of A. actinomycetemcomitans have been more closely associated with this disease, suggesting that LtxA is a key virulence factor for A. actinomycetemcomitans. LtxA is secreted across both the inner and outer membranes via the Type I secretion system, but has also been found to be enriched within outer membrane vesicles (OMVs), derived from the bacterial outer membrane. We have characterized the association of LtxA with OMVs produced by the highly leukotoxic strain, JP2, and investigated the interaction of these OMVs with host cells to understand how LtxA is delivered to host cells in this OMV-associated form. Our results demonstrated that a significant fraction of the secreted LtxA exists in an OMV-associated form. Furthermore, we have discovered that in this OMV-associated form, the toxin is trafficked to host cells by a cholesterol- and receptor-independent mechanism in contrast to the mechanism by which free LtxA is delivered. Because OMV-associated toxin is trafficked to host cells in an entirely different manner than free toxin, this study highlights the importance of studying both free and OMV-associated forms of LtxA to understand A. actinomycetemcomitans virulence.

TdeA, a TolC-like protein required for toxin and drug export in Aggregatibacter (Actinobacillus) actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans is an oral bacterium that causes localized aggressive periodontitis (LAP) and extra-oral infections such as sub-acute infective endocarditis. As part of its array of virulence factors, A. actinomycetemcomitans produces leukotoxin (LtxA), a member of the RTX family of toxins. LtxA kills human leukocytes and we have recently shown that the toxin is required for beta-hemolysis by A. actinomycetemcomitans on solid medium. In other RTX toxin-producing bacteria, an outer membrane channel-forming protein, TolC, is required for toxin secretion and drug export. We have identified an ORF in A. actinomycetemcomitans that encodes a putative protein having predicted structural properties similar to TolC. Inactivation of this ORF resulted in a mutant that was no longer beta-hemolytic and did not secrete LtxA. This mutant was significantly more sensitive to antimicrobial agents compared to the wild type strain and was unable to export the antimicrobial agent berberine. Thus, this ORF was named tdeA for "toxin and drug export". Examination of the DNA sequence surrounding tdeA revealed two upstream ORFs that encode proteins similar to the drug efflux proteins, MacA and MacB. Inactivation of macB in A. actinomycetemcomitans did not alter the drug sensitivity profile or the hemolytic activity of the mutant. The genes macA, macB and tdeA are organized as an operon and are constitutively expressed as a single transcript. These results show that A. actinomycetemcomitans indeed requires a TolC-like protein for LtxA secretion and that this protein, TdeA, also functions as part of a drug efflux system.

Expression and targeting of lymphocyte function-associated antigen 1 (LFA-1) on white blood cells for treatment of allergic asthma.

Allergic asthma is a chronic respiratory disease that results from an exaggerated inflammatory response in the airways. Environment stimuli, such as pollen and HDM, cause activation and migration of inflammatory WBCs into the respiratory tract, where they cause lung damage. Migration of these WBCs is dependent on the active configuration of the ß2 integrin LFA-1. The experimental therapeutic agent LtxA specifically targets active LFA-1 and causes cell death. We investigated the association between LFA-1 and allergic asthma and hypothesized that targeting LFA-1 with LtxA could be an attractive strategy for treatment of the condition. We examined LFA-1 (CD11a) levels on PBMCs from patients with allergic asthma compared with healthy controls. Patients exhibited a significantly higher percentage of PBMCs expressing LFA-1 than healthy controls. Furthermore, the level of LFA-1 expression on patient PBMCs was greater than on healthy PBMCs. We identified a unique cellular population in patients that consisted of CD4(-) CD11a(hi) cells. We also evaluated LtxA in a HDM extract-induced mouse model for allergic asthma. LtxA caused resolution of disease in mice, as demonstrated by a decrease in BALF WBCs, a reduction in pulmonary inflammation and tissue remodeling, and a decrease in proinflammatory cytokines IL-4, IL-5, IL-9, IL-17F, and IL-23α in lung tissue. LFA-1 may serve as an important marker in allergic asthma, and the elimination of activated WBCs by use of LtxA could be a viable therapeutic strategy for treating patients with this condition.

LFA-1-targeting Leukotoxin (LtxA; Leukothera®) causes lymphoma tumor regression in a humanized mouse model and requires caspase-8 and Fas to kill malignant lymphocytes.

Leukotoxin (LtxA) is a protein secreted from the oral bacterium Aggregatibacter actinomycetemcomitans. LtxA binds to the ß2 integrin lymphocyte-associated function antigen-1 (LFA-1) on human white blood cells (WBCs), resulting in cell death. LtxA is currently under investigation as a novel therapy (Leukothera(®)) for treating hematologic malignancies and autoimmune diseases. We show here that LtxA has potent in vivo anti-lymphoma activity in mice. LtxA caused complete regression of B-cell tumors and promoted long-term survival of mice. The mechanism of LtxA-mediated killing of malignant lymphocytes was further examined. We found that LtxA kills malignant lymphocytes by a novel mechanism requiring the death receptor Fas and caspase-8, but not Fas ligand (FasL) or caspase-9. We also determined that LFA-1 and Fas are closely associated on the cell surface and this proximity of LFA-1 and Fas could explain how signaling through an integrin can lead to cell death. In addition to LFA-1, this work reveals a second surface protein, Fas, that is critical for LtxA-mediated cell death. Knowledge of the mechanism of cell death induced by LtxA will facilitate the development and understanding of this potent experimental therapeutic agent.