Scrub typhus is an acute febrile disease due to Orientia tsutsugamushi (Ot) infection and can be ... more Scrub typhus is an acute febrile disease due to Orientia tsutsugamushi (Ot) infection and can be life-threatening with organ failure, hemorrhage, and fatality. Yet, little is known as to how the host reacts to Ot bacteria at early stages of infection; no reports have addressed the functional roles of type I versus type II interferon (IFN) responses in scrub typhus. In this study, we used comprehensive intradermal (i.d.) inoculation models and two clinically predominant Ot strains (Karp and Gilliam) to uncover early immune events. Karp infection induced sequential expression of Ifnb and Ifng in inflamed skin and draining lymph nodes at days 1 and 3 post-infection. Using double Ifnar1-/-Ifngr1-/and Stat1-/mice, we found that deficiency in IFN/STAT1 signaling resulted in lethal infection with profound pathology and skin eschar lesions, which resembled to human scrub typhus. Further analyses demonstrated that deficiency in IFN-γ, but not IFN-I, resulted in impaired NK cell and macrophage activation and uncontrolled bacterial growth and dissemination, leading to metabolic dysregulation, excessive inflammatory cell infiltration, and exacerbated tissue damage. NK cells were found to be the major cellular source of innate IFN-γ, contributing to the initial Ot control in the draining lymph nodes. In vitro studies with dendritic cell cultures revealed a superior antibacterial effect offered by IFN-γ than IFN-β. Comparative in vivo studies with Karpand Gilliam-infection revealed a crucial role of IFN-γ signaling in protection against progression of eschar lesions and Ot infection lethality. Additionally, our i.d. mouse models of lethal infection with eschar lesions are promising tools for immunological study and vaccine development for scrub typhus.
Dendritic cells (DCs) discriminate different microbial pathogens and induce T-cell responses of a... more Dendritic cells (DCs) discriminate different microbial pathogens and induce T-cell responses of appropriate effector phenotypes accordingly. Microbial recognition and differentiation are mediated in part by pattern recognition receptors such as Toll-like receptors (TLRs), whereas the development of T-cell effector functions is critically dependent on DC-derived cytokines such as interleukin-12 (IL-12) and IL-10. However, it is not entirely clear to what extent various microbial TLR activators could induce different functional states of DCs that favor different T-cell effector phenotypes. Toward a better understanding of this issue, we examined IL-10 and IL-12 production and T-cell-polarizing potentials of murine bone marrow-derived DCs after stimulation by three microbial TLR activators, namely, lipopolysaccharide (LPS), peptidoglycan (PGN), and zymosan. We found that the three stimuli induced drastically different profiles of IL-10 and IL-12 production in DCs. Further, these stimuli differentially conditioned CD40-dependent IL-10 and IL-12 production by DCs. Finally, LPS-, PGN-, and zymosan-stimulated DCs primed distinct T-cell cytokine profiles. Our results support the notion that microbe-specific information sensed through different TLRs by DCs is linked to differential Th priming through DC-derived cytokines.
In experimental murine cutaneous leishmaniasis, the purified Leishmania pifanoi amastigote protei... more In experimental murine cutaneous leishmaniasis, the purified Leishmania pifanoi amastigote protein P-4 has been shown to induce significant protection against infection. Further, recent studies examining the response of peripheral blood mononuclear cells (PBMC) from Leishmania braziliensis-infected human patients have demonstrated that the P-4 protein selectively elicits a significant T H 1-like response. Because a T H 1-like response is associated with cure, epitope studies were conducted to further evaluate the human response to P-4. PBMC from confirmed cutaneous leishmaniasis patients infected with L. braziliensis in Rio de Janeiro, Brazil, an area where the disease is endemic, were examined for T-cell proliferation and/or cytokine production in response to whole-parasite homogenate, isolated P-4 protein, and/or P-4 peptides. Twenty of the 22 patients (91%) examined responded to the native P-4 protein by proliferation and/or gamma interferon (IFN-␥) production. According to the proliferation data, PBMC from 14 patients (64%) were found to respond to the intact P-4 protein (stimulation index of >2.5). Fifty-seven percent of the P-4-responsive patients studied responded to at least one of the P-4 peptides; 11 individual peptides were found to elicit a proliferative response. Of 17 patients examined for cytokine production, no PBMC produced detectable interleukin-4 in response to P-4 protein or peptides. However, PBMC from 14 patients (82%) produced significant levels of IFN-␥ (>20 pg/ml) in response to native P-4 protein. Nineteen of the 23 peptides were found to elicit an IFN-␥ response from at least two patients. These data indicate that multiple epitopes spanning the entire P-4 molecule are responsible for the T H 1-like immune response observed, indicating that the intact P-4 amastigote molecule, rather than selected peptides, may prove to be the most useful for leishmaniasis vaccine development.
Scrub typhus is an understudied, potentially lethal disease caused by infection with Orientia tsu... more Scrub typhus is an understudied, potentially lethal disease caused by infection with Orientia tsutsugamushi. Despite causing an estimated 1 million cases per year and an increasing global presence, mechanisms of scrub typhus pathogenesis remain unclear. One of the most life-threatening conditions that can arise in scrub typhus patients is acute respiratory distress syndrome (ARDS). The development of ARDS is a complex process; some of its pathological hallmarks, including prolonged recruitment of inflammatory immune cells to the lung and vasculature damage, have been observed in humans and/or animal models of O. tsutsugamushi infection. Although different cell types and mechanisms may contribute to ARDS development during O. tsutsugamushi infection, this review highlights our current evidence of pulmonary endothelial activation and damage, the potential roles of neutrophils and macrophages in the lung, and the knowledge gaps in this field. Continued investigation of the lung microenvironment and cellular interactions will help elucidate disease pathogenesis and possible treatment during scrub typhus.
Leishmania amazonensis parasites can cause diverse forms of leishmaniasis in humans and persisten... more Leishmania amazonensis parasites can cause diverse forms of leishmaniasis in humans and persistent lesions in most inbred strains of mice. In both cases, the infection is characterized by a marked immunosuppression of the host. We previously showed that amastigote forms of the parasite make use of surface-exposed phosphatidylserine (PS) molecules to infect host cells and promote alternative macrophage activation, leading to uncontrolled intracellular proliferation of the parasites. In this study, we demonstrated that treatment of infected mice with an PS-targeting monoclonal antibody ameliorated parasite loads and lesion development, which correlated with increased proliferative responses by lymphocytes. In addition, we observed an enhanced dendritic cell (DC) activation and antigen presentation in vitro. Our data imply that the recognition of PS exposed on the surface of amastigotes plays a role in down-modulating DC functions, in a matter similar to that of apoptotic cell clearance. This study provides new information regarding the mechanism of immune suppression in Leishmania infection.
The recent completion of genome sequencing projects for Leishmania major and near completion for ... more The recent completion of genome sequencing projects for Leishmania major and near completion for two other species, L. infantum and L. braziliensis, has provided the needed genomic information for investigating the proteomes of Leishmania parasites. However, the design of effective 2-DEbased proteome mapping for complex protozoan parasites like Leishmania has proven to be severely compromised due to extensive overcrowding of spots especially in the acidic regions, coupled to a relatively low representation of basic proteins. In the present study, we optimized a liquid-phase IEF in combination with 2-DE for L. amazonensis promastigote as a way of reducing protein complexity and enhancing representation for low-abundance proteins on gels. Of 20 pHbased fractions eluted from Rotofor ™ cells, 5 representative fractions selected from acidic, basic or neutral regions of the proteome and with adequate protein concentration were further analyzed by 2-DE using medium-range IPG strips. On this basis, we were able to generate highresolution 2-DE maps encompassing both the acidic and basic ends of the proteome with enhanced spot representation.
The concerns raised from adenovirus 5 (Ad5)-based HIV vaccine clinical trials, where excess HIV i... more The concerns raised from adenovirus 5 (Ad5)-based HIV vaccine clinical trials, where excess HIV infections were observed in some vaccine recipients, have highlighted the importance of understanding host responses to vaccine vectors and the HIV susceptibility of vector-specific CD4 T cells in HIV vaccination. Our recent study reported that human Ad5-specific CD4 T cells induced by Ad5 vaccination (RV156A trial) are susceptible to HIV. Here we further investigated the HIV susceptibility of vector-specific CD4 T cells induced by ALVAC, a canarypox viral vector tested in the Thai trial RV144, as compared to Ad5 vector-specific CD4 T cells in the HVTN204 trial. We showed that while Ad5 vector-specific CD4 T cells were readily susceptible to HIV, ALVAC-specific CD4 T cells in RV144 PBMC were substantially less susceptible to both R5 and X4 HIV in vitro. The lower HIV susceptibility of ALVAC-specific CD4 T cells was associated with the reduced surface expression of HIV entry co-receptors CCR5 and CXCR4 on these cells. Phenotypic analyses identified that ALVAC-specific CD4 T cells displayed a strong Th1 phenotype, producing higher levels of IFN-γ and CCL4 (MIP-1β) but little IL-17. Of interest, ALVAC and Ad5 vectors induced distinct profiles of vector-specific CD8 vs. CD4 T-cell proliferative responses in PBMC, with ALVAC preferentially inducing CD8 T-cell proliferation, while Ad5 vector induced CD4 T-cell proliferation. Depletion of ALVAC-, but not Ad5-, induced CD8 T cells in PBMC led to a modest increase in HIV infection of vector-specific CD4 T cells, suggesting a role of ALVAC-specific CD8 T cells in protecting ALVAC-specific CD4 T cells from HIV. Taken together, our data provide strong evidence for distinct HIV susceptibility of CD4 T cells induced by different vaccine vectors and highlight the importance of better evaluating anti-vector responses in HIV vaccination.
To understand the integrin requirements of T-helper (T H) effector subsets, we investigated the c... more To understand the integrin requirements of T-helper (T H) effector subsets, we investigated the contribution of CD18 (β 2 integrin) to T H 1 and T H 2 function in vitro and in relevant disease models. CD18-deficient (Itgb2-/-) T cells showed largely normal in vitro function. Compared with wild-type mice, Itgb2-/mice were better able to resolve Leishmania major infection and generated a superior T H 1 immune response, as assessed from draining lymph nodes. In contrast, T H 2-dependent allergic lung disease was markedly impaired in mutant mice. In both models, development of T H 1 and T H 2 cells in spleens was normal, but accumulation of T H 2 (not T H 1) cells at inflammatory sites was reduced. Thus, CD18 is selectively required for T H 2, but not T H 1, homing and has a minimal influence on T-effector development. These findings suggest a new integrin-based therapeutic approach in which the outcomes of diverse diseases may be favorably influenced by altering the homing of T H 2 cells.
Recent studies have shown that histone proteins can act as antimicrobial peptides in host defense... more Recent studies have shown that histone proteins can act as antimicrobial peptides in host defense against extracellular bacteria, fungi, and Leishmania promastigotes. In this study, we used human recombinant histone proteins to further study their leishmaniacidal effects and the underlying mechanisms. We found that the histones H2A and H2B (but not H1 0) could directly and efficiently kill promastigotes of Leishmania amazonensis, L. major, L. braziliensis, and L. mexicana in a treatment dose-dependent manner. Scanning electron microscopy revealed surface disruption of histone-treated promastigotes. More importantly, the preexposure of promastigotes to histone proteins markedly decreased the infectivity of promastigotes to murine macrophages (Ms) in vitro. However, axenic and lesion-derived amastigotes of L. amazonensis and L. mexicana were relatively resistant to histone treatment, which correlated with the low levels of intracellular H2A in treated amastigotes. To understand the mechanisms underlying these differential responses, we investigated the role of promastigote surface molecules in histone-mediated killing. Compared with the corresponding controls, transgenic L. amazonensis promastigotes expressing lower levels of surface gp63 proteins were more susceptible to histone H2A, while L. major and L. mexicana promastigotes with targeted deletion of the lipophosphoglycan 2 (lpg2) gene (but not the lpg1 gene) were more resistant to histone H2A. We discuss the influence of promastigote major surface molecules in the leishmaniacidal effect of histone proteins. This study provides new information on host innate immunity to different developmental stages of Leishmania parasites.
Outer membrane protein A (OmpA) is located in the membrane of Escherichia coli and other gram-neg... more Outer membrane protein A (OmpA) is located in the membrane of Escherichia coli and other gram-negative bacteria and plays a multifunctional role in bacterial physiology and pathogenesis. In enterohemorrhagic E. coli (EHEC), especially serotype O157:H7, OmpA interacts with cultured human intestinal cells and likely acts as an important component to stimulate the immune response during infection. To test this hypothesis, we analyzed the effect of EHEC OmpA on cytokine production by dendritic cells (DCs) and on DC migration across polarized intestinal epithelial cells. OmpA induced murine DCs to secrete interleukin-1 (IL-1), IL-10, and IL-12 in a dose-dependent manner, and this effect was independent of Toll-like receptor 4. Although DCs displayed differential responses to EHEC OmpA and OmpA-specific antibodies enhanced DC cytokine secretion, we cannot discard that other EHEC surface elements were likely to be involved. While OmpA was required for bacterial binding to polarized Caco-2 cells, it was not needed for the induction of cytokine production by Caco-2 cells or for human DC migration across polarized cells.
Leishmaniasis is a complex disease that is caused by parasites of the Leishmania genus. Leishmani... more Leishmaniasis is a complex disease that is caused by parasites of the Leishmania genus. Leishmania are further classified into several complexes, each of which can engage in distinct interactions with mammalian hosts resulting in differing disease presentations. It is therefore not unexpected that host immune responses to Leishmania are variable. The induction of interferon gamma (IFN-γ) and response to it in these infections has received considerable attention. In this review, we summarize our current understanding of some of the host responses during Leishmania infections that are regulated by IFN-γ. In addition, studies that explore the nature of parasite-derived molecular mediators that might affect the host response to IFN-γ are also discussed.
Leishmaniasis is a vector-borne disease found in many countries worldwide. The causative agent of... more Leishmaniasis is a vector-borne disease found in many countries worldwide. The causative agent of the disease, Leishmania spp., lives as an obligate intracellular parasite within mammalian hosts. Since tissue macrophages are major target cells for parasite replication, the outcome of infection depends largely on the activation status of these cells. L-arginine is a crucial amino acid required for both nitric oxide (NO)-mediated parasite killing and polyamine-mediated parasite replication. This review highlights the significance of L-arginine as a factor determining the outcomes of Leishmania infection in vitro and its influences on host immune responses in vivo. Various therapeutic approaches targeting L-arginine metabolic pathways during infections with Leishmania are also discussed.
Intracerebral microhemorrhages (CMHs) are small foci of hemorrhages in the cerebrum. Acute infect... more Intracerebral microhemorrhages (CMHs) are small foci of hemorrhages in the cerebrum. Acute infections induced by some intracellular pathogens, including rickettsia, can result in CMHs. Annexin a2 (ANXA2) has been documented to play a functional role during intracellular bacterial adhesion. Here we report that ANXA2-knockout (KO) mice are more susceptible to CMHs in response to rickettsia and Ebola virus infections, suggesting an essential role of ANXA2 in protecting vascular integrity during these intracellular pathogen infections. Proteomic analysis via mass spectrometry of whole brain lysates and brain-derived endosomes from ANXA2-KO and wild-type (WT) mice post-infection with R. australis revealed that a variety of significant proteins were differentially expressed, and the follow-up function enrichment analysis had identified .
Key Point：PI3K/eNOS pathway-mediated, inflammation-triggered vWF secretion is the target of the p... more Key Point：PI3K/eNOS pathway-mediated, inflammation-triggered vWF secretion is the target of the pharmacological manipulation of the cAMP-EPAC system.
Several Leishmania proteins have been identified and characterized in pursuit of understanding pa... more Several Leishmania proteins have been identified and characterized in pursuit of understanding pathogenesis and protection in cutaneous leishmaniasis. In the present study, we utilized sera from infected BALB/c mice to screen a Leishmania amazonensis amastigote cDNA expression library and obtained the full-length gene that encodes a novel Trp-Asp (WD) protein designated LAWD (for Leishmania antigenic WD protein). The WD family of proteins mediates protein-protein interactions and coordinates the formation of protein complexes. The single-copy LAWD gene is transcribed as a ϳ3.1-kb mRNA in both promastigotes and amastigotes, with homologues being detected in several other Leishmania species. Immunoelectron microscopy revealed a predominant localization of the LAWD protein in the flagellar pocket. Analyses of sera from human patients with cutaneous and mucocutaneous leishmaniasis indicated that these individuals mounted significant humoral responses against LAWD. Given that recombinant LAWD protein elicited the production of high levels of gamma interferon, but no detectable levels of interleukin-10 (IL-10), in CD4 ؉ cells of L. amazonensis-infected mice, we further examined whether LAWD could elicit protective immunity. DNA vaccination with the LAWD and IL-12 genes significantly delayed lesion development, which correlated with a dramatic reduction in parasite burdens. Thus, we have successfully identified a promising vaccine candidate and antigenic vehicle to aid in the dissection of the complicated pathogenic immune response of L. amazonensis.
Induction of c-fos and c-myc expression during B cell activation by IL-4 and immunoglobulin bindi... more Induction of c-fos and c-myc expression during B cell activation by IL-4 and immunoglobulin binding ligands.
The purified membrane-associated Leishmania pifanoi amastigote protein P-4 has been shown to indu... more The purified membrane-associated Leishmania pifanoi amastigote protein P-4 has been shown to induce protective immunity against infection and to elicit preferentially a T helper 1-like response in peripheral blood mononuclear cells of patients with American cutaneous leishmaniasis. As this molecule is potentially important for future vaccine studies, the L. pifanoi gene encoding the P-4 membrane protein was cloned and sequenced. Southern blot analyses indicate the presence of six tandemly arrayed copies of the P-4 gene in L. pifanoi; homologues of the P-4 gene are found in all other species of the genus Leishmania examined. DNA-derived protein sequence data indicated an identity to the P1 zinc-dependent nuclease of Penicillium citrinum (20.8%) and the C-terminal domain of the 3 nucleotidase of Leishmania donovani (33.7%). Consistent with these sequence analyses, purified L. pifanoi P-4 protein possesses single strand nuclease (DNA and RNA) and phosphomonoesterase activity, with a preference for UMP > TMP > AMP >> CMP. Double-labeling immunofluorescence microscopic analyses employing anti-binding protein antibodies revealed that the P-4 protein is localized in the endoplasmic reticulum of the amastigote. Northern blot analyses indicated that the gene is selectively expressed in the intracellular amastigote stage (mammalian host) but not in the promastigote stage (insect) of the parasite. Based upon its subcellular localization and singlestranded specific nuclease activity, possible roles of the P-4 nuclease in the amastigote in RNA stability (gene expression) or DNA repair are discussed.
Scrub typhus is an acute febrile disease due to Orientia tsutsugamushi (Ot) infection and can be ... more Scrub typhus is an acute febrile disease due to Orientia tsutsugamushi (Ot) infection and can be life-threatening with organ failure, hemorrhage, and fatality. Yet, little is known as to how the host reacts to Ot bacteria at early stages of infection; no reports have addressed the functional roles of type I versus type II interferon (IFN) responses in scrub typhus. In this study, we used comprehensive intradermal (i.d.) inoculation models and two clinically predominant Ot strains (Karp and Gilliam) to uncover early immune events. Karp infection induced sequential expression of Ifnb and Ifng in inflamed skin and draining lymph nodes at days 1 and 3 post-infection. Using double Ifnar1-/-Ifngr1-/and Stat1-/mice, we found that deficiency in IFN/STAT1 signaling resulted in lethal infection with profound pathology and skin eschar lesions, which resembled to human scrub typhus. Further analyses demonstrated that deficiency in IFN-γ, but not IFN-I, resulted in impaired NK cell and macrophage activation and uncontrolled bacterial growth and dissemination, leading to metabolic dysregulation, excessive inflammatory cell infiltration, and exacerbated tissue damage. NK cells were found to be the major cellular source of innate IFN-γ, contributing to the initial Ot control in the draining lymph nodes. In vitro studies with dendritic cell cultures revealed a superior antibacterial effect offered by IFN-γ than IFN-β. Comparative in vivo studies with Karpand Gilliam-infection revealed a crucial role of IFN-γ signaling in protection against progression of eschar lesions and Ot infection lethality. Additionally, our i.d. mouse models of lethal infection with eschar lesions are promising tools for immunological study and vaccine development for scrub typhus.
Dendritic cells (DCs) discriminate different microbial pathogens and induce T-cell responses of a... more Dendritic cells (DCs) discriminate different microbial pathogens and induce T-cell responses of appropriate effector phenotypes accordingly. Microbial recognition and differentiation are mediated in part by pattern recognition receptors such as Toll-like receptors (TLRs), whereas the development of T-cell effector functions is critically dependent on DC-derived cytokines such as interleukin-12 (IL-12) and IL-10. However, it is not entirely clear to what extent various microbial TLR activators could induce different functional states of DCs that favor different T-cell effector phenotypes. Toward a better understanding of this issue, we examined IL-10 and IL-12 production and T-cell-polarizing potentials of murine bone marrow-derived DCs after stimulation by three microbial TLR activators, namely, lipopolysaccharide (LPS), peptidoglycan (PGN), and zymosan. We found that the three stimuli induced drastically different profiles of IL-10 and IL-12 production in DCs. Further, these stimuli differentially conditioned CD40-dependent IL-10 and IL-12 production by DCs. Finally, LPS-, PGN-, and zymosan-stimulated DCs primed distinct T-cell cytokine profiles. Our results support the notion that microbe-specific information sensed through different TLRs by DCs is linked to differential Th priming through DC-derived cytokines.
In experimental murine cutaneous leishmaniasis, the purified Leishmania pifanoi amastigote protei... more In experimental murine cutaneous leishmaniasis, the purified Leishmania pifanoi amastigote protein P-4 has been shown to induce significant protection against infection. Further, recent studies examining the response of peripheral blood mononuclear cells (PBMC) from Leishmania braziliensis-infected human patients have demonstrated that the P-4 protein selectively elicits a significant T H 1-like response. Because a T H 1-like response is associated with cure, epitope studies were conducted to further evaluate the human response to P-4. PBMC from confirmed cutaneous leishmaniasis patients infected with L. braziliensis in Rio de Janeiro, Brazil, an area where the disease is endemic, were examined for T-cell proliferation and/or cytokine production in response to whole-parasite homogenate, isolated P-4 protein, and/or P-4 peptides. Twenty of the 22 patients (91%) examined responded to the native P-4 protein by proliferation and/or gamma interferon (IFN-␥) production. According to the proliferation data, PBMC from 14 patients (64%) were found to respond to the intact P-4 protein (stimulation index of >2.5). Fifty-seven percent of the P-4-responsive patients studied responded to at least one of the P-4 peptides; 11 individual peptides were found to elicit a proliferative response. Of 17 patients examined for cytokine production, no PBMC produced detectable interleukin-4 in response to P-4 protein or peptides. However, PBMC from 14 patients (82%) produced significant levels of IFN-␥ (>20 pg/ml) in response to native P-4 protein. Nineteen of the 23 peptides were found to elicit an IFN-␥ response from at least two patients. These data indicate that multiple epitopes spanning the entire P-4 molecule are responsible for the T H 1-like immune response observed, indicating that the intact P-4 amastigote molecule, rather than selected peptides, may prove to be the most useful for leishmaniasis vaccine development.
Scrub typhus is an understudied, potentially lethal disease caused by infection with Orientia tsu... more Scrub typhus is an understudied, potentially lethal disease caused by infection with Orientia tsutsugamushi. Despite causing an estimated 1 million cases per year and an increasing global presence, mechanisms of scrub typhus pathogenesis remain unclear. One of the most life-threatening conditions that can arise in scrub typhus patients is acute respiratory distress syndrome (ARDS). The development of ARDS is a complex process; some of its pathological hallmarks, including prolonged recruitment of inflammatory immune cells to the lung and vasculature damage, have been observed in humans and/or animal models of O. tsutsugamushi infection. Although different cell types and mechanisms may contribute to ARDS development during O. tsutsugamushi infection, this review highlights our current evidence of pulmonary endothelial activation and damage, the potential roles of neutrophils and macrophages in the lung, and the knowledge gaps in this field. Continued investigation of the lung microenvironment and cellular interactions will help elucidate disease pathogenesis and possible treatment during scrub typhus.
Leishmania amazonensis parasites can cause diverse forms of leishmaniasis in humans and persisten... more Leishmania amazonensis parasites can cause diverse forms of leishmaniasis in humans and persistent lesions in most inbred strains of mice. In both cases, the infection is characterized by a marked immunosuppression of the host. We previously showed that amastigote forms of the parasite make use of surface-exposed phosphatidylserine (PS) molecules to infect host cells and promote alternative macrophage activation, leading to uncontrolled intracellular proliferation of the parasites. In this study, we demonstrated that treatment of infected mice with an PS-targeting monoclonal antibody ameliorated parasite loads and lesion development, which correlated with increased proliferative responses by lymphocytes. In addition, we observed an enhanced dendritic cell (DC) activation and antigen presentation in vitro. Our data imply that the recognition of PS exposed on the surface of amastigotes plays a role in down-modulating DC functions, in a matter similar to that of apoptotic cell clearance. This study provides new information regarding the mechanism of immune suppression in Leishmania infection.
The recent completion of genome sequencing projects for Leishmania major and near completion for ... more The recent completion of genome sequencing projects for Leishmania major and near completion for two other species, L. infantum and L. braziliensis, has provided the needed genomic information for investigating the proteomes of Leishmania parasites. However, the design of effective 2-DEbased proteome mapping for complex protozoan parasites like Leishmania has proven to be severely compromised due to extensive overcrowding of spots especially in the acidic regions, coupled to a relatively low representation of basic proteins. In the present study, we optimized a liquid-phase IEF in combination with 2-DE for L. amazonensis promastigote as a way of reducing protein complexity and enhancing representation for low-abundance proteins on gels. Of 20 pHbased fractions eluted from Rotofor ™ cells, 5 representative fractions selected from acidic, basic or neutral regions of the proteome and with adequate protein concentration were further analyzed by 2-DE using medium-range IPG strips. On this basis, we were able to generate highresolution 2-DE maps encompassing both the acidic and basic ends of the proteome with enhanced spot representation.
The concerns raised from adenovirus 5 (Ad5)-based HIV vaccine clinical trials, where excess HIV i... more The concerns raised from adenovirus 5 (Ad5)-based HIV vaccine clinical trials, where excess HIV infections were observed in some vaccine recipients, have highlighted the importance of understanding host responses to vaccine vectors and the HIV susceptibility of vector-specific CD4 T cells in HIV vaccination. Our recent study reported that human Ad5-specific CD4 T cells induced by Ad5 vaccination (RV156A trial) are susceptible to HIV. Here we further investigated the HIV susceptibility of vector-specific CD4 T cells induced by ALVAC, a canarypox viral vector tested in the Thai trial RV144, as compared to Ad5 vector-specific CD4 T cells in the HVTN204 trial. We showed that while Ad5 vector-specific CD4 T cells were readily susceptible to HIV, ALVAC-specific CD4 T cells in RV144 PBMC were substantially less susceptible to both R5 and X4 HIV in vitro. The lower HIV susceptibility of ALVAC-specific CD4 T cells was associated with the reduced surface expression of HIV entry co-receptors CCR5 and CXCR4 on these cells. Phenotypic analyses identified that ALVAC-specific CD4 T cells displayed a strong Th1 phenotype, producing higher levels of IFN-γ and CCL4 (MIP-1β) but little IL-17. Of interest, ALVAC and Ad5 vectors induced distinct profiles of vector-specific CD8 vs. CD4 T-cell proliferative responses in PBMC, with ALVAC preferentially inducing CD8 T-cell proliferation, while Ad5 vector induced CD4 T-cell proliferation. Depletion of ALVAC-, but not Ad5-, induced CD8 T cells in PBMC led to a modest increase in HIV infection of vector-specific CD4 T cells, suggesting a role of ALVAC-specific CD8 T cells in protecting ALVAC-specific CD4 T cells from HIV. Taken together, our data provide strong evidence for distinct HIV susceptibility of CD4 T cells induced by different vaccine vectors and highlight the importance of better evaluating anti-vector responses in HIV vaccination.
To understand the integrin requirements of T-helper (T H) effector subsets, we investigated the c... more To understand the integrin requirements of T-helper (T H) effector subsets, we investigated the contribution of CD18 (β 2 integrin) to T H 1 and T H 2 function in vitro and in relevant disease models. CD18-deficient (Itgb2-/-) T cells showed largely normal in vitro function. Compared with wild-type mice, Itgb2-/mice were better able to resolve Leishmania major infection and generated a superior T H 1 immune response, as assessed from draining lymph nodes. In contrast, T H 2-dependent allergic lung disease was markedly impaired in mutant mice. In both models, development of T H 1 and T H 2 cells in spleens was normal, but accumulation of T H 2 (not T H 1) cells at inflammatory sites was reduced. Thus, CD18 is selectively required for T H 2, but not T H 1, homing and has a minimal influence on T-effector development. These findings suggest a new integrin-based therapeutic approach in which the outcomes of diverse diseases may be favorably influenced by altering the homing of T H 2 cells.
Recent studies have shown that histone proteins can act as antimicrobial peptides in host defense... more Recent studies have shown that histone proteins can act as antimicrobial peptides in host defense against extracellular bacteria, fungi, and Leishmania promastigotes. In this study, we used human recombinant histone proteins to further study their leishmaniacidal effects and the underlying mechanisms. We found that the histones H2A and H2B (but not H1 0) could directly and efficiently kill promastigotes of Leishmania amazonensis, L. major, L. braziliensis, and L. mexicana in a treatment dose-dependent manner. Scanning electron microscopy revealed surface disruption of histone-treated promastigotes. More importantly, the preexposure of promastigotes to histone proteins markedly decreased the infectivity of promastigotes to murine macrophages (Ms) in vitro. However, axenic and lesion-derived amastigotes of L. amazonensis and L. mexicana were relatively resistant to histone treatment, which correlated with the low levels of intracellular H2A in treated amastigotes. To understand the mechanisms underlying these differential responses, we investigated the role of promastigote surface molecules in histone-mediated killing. Compared with the corresponding controls, transgenic L. amazonensis promastigotes expressing lower levels of surface gp63 proteins were more susceptible to histone H2A, while L. major and L. mexicana promastigotes with targeted deletion of the lipophosphoglycan 2 (lpg2) gene (but not the lpg1 gene) were more resistant to histone H2A. We discuss the influence of promastigote major surface molecules in the leishmaniacidal effect of histone proteins. This study provides new information on host innate immunity to different developmental stages of Leishmania parasites.
Outer membrane protein A (OmpA) is located in the membrane of Escherichia coli and other gram-neg... more Outer membrane protein A (OmpA) is located in the membrane of Escherichia coli and other gram-negative bacteria and plays a multifunctional role in bacterial physiology and pathogenesis. In enterohemorrhagic E. coli (EHEC), especially serotype O157:H7, OmpA interacts with cultured human intestinal cells and likely acts as an important component to stimulate the immune response during infection. To test this hypothesis, we analyzed the effect of EHEC OmpA on cytokine production by dendritic cells (DCs) and on DC migration across polarized intestinal epithelial cells. OmpA induced murine DCs to secrete interleukin-1 (IL-1), IL-10, and IL-12 in a dose-dependent manner, and this effect was independent of Toll-like receptor 4. Although DCs displayed differential responses to EHEC OmpA and OmpA-specific antibodies enhanced DC cytokine secretion, we cannot discard that other EHEC surface elements were likely to be involved. While OmpA was required for bacterial binding to polarized Caco-2 cells, it was not needed for the induction of cytokine production by Caco-2 cells or for human DC migration across polarized cells.
Leishmaniasis is a complex disease that is caused by parasites of the Leishmania genus. Leishmani... more Leishmaniasis is a complex disease that is caused by parasites of the Leishmania genus. Leishmania are further classified into several complexes, each of which can engage in distinct interactions with mammalian hosts resulting in differing disease presentations. It is therefore not unexpected that host immune responses to Leishmania are variable. The induction of interferon gamma (IFN-γ) and response to it in these infections has received considerable attention. In this review, we summarize our current understanding of some of the host responses during Leishmania infections that are regulated by IFN-γ. In addition, studies that explore the nature of parasite-derived molecular mediators that might affect the host response to IFN-γ are also discussed.
Leishmaniasis is a vector-borne disease found in many countries worldwide. The causative agent of... more Leishmaniasis is a vector-borne disease found in many countries worldwide. The causative agent of the disease, Leishmania spp., lives as an obligate intracellular parasite within mammalian hosts. Since tissue macrophages are major target cells for parasite replication, the outcome of infection depends largely on the activation status of these cells. L-arginine is a crucial amino acid required for both nitric oxide (NO)-mediated parasite killing and polyamine-mediated parasite replication. This review highlights the significance of L-arginine as a factor determining the outcomes of Leishmania infection in vitro and its influences on host immune responses in vivo. Various therapeutic approaches targeting L-arginine metabolic pathways during infections with Leishmania are also discussed.
Intracerebral microhemorrhages (CMHs) are small foci of hemorrhages in the cerebrum. Acute infect... more Intracerebral microhemorrhages (CMHs) are small foci of hemorrhages in the cerebrum. Acute infections induced by some intracellular pathogens, including rickettsia, can result in CMHs. Annexin a2 (ANXA2) has been documented to play a functional role during intracellular bacterial adhesion. Here we report that ANXA2-knockout (KO) mice are more susceptible to CMHs in response to rickettsia and Ebola virus infections, suggesting an essential role of ANXA2 in protecting vascular integrity during these intracellular pathogen infections. Proteomic analysis via mass spectrometry of whole brain lysates and brain-derived endosomes from ANXA2-KO and wild-type (WT) mice post-infection with R. australis revealed that a variety of significant proteins were differentially expressed, and the follow-up function enrichment analysis had identified .
Key Point：PI3K/eNOS pathway-mediated, inflammation-triggered vWF secretion is the target of the p... more Key Point：PI3K/eNOS pathway-mediated, inflammation-triggered vWF secretion is the target of the pharmacological manipulation of the cAMP-EPAC system.
Several Leishmania proteins have been identified and characterized in pursuit of understanding pa... more Several Leishmania proteins have been identified and characterized in pursuit of understanding pathogenesis and protection in cutaneous leishmaniasis. In the present study, we utilized sera from infected BALB/c mice to screen a Leishmania amazonensis amastigote cDNA expression library and obtained the full-length gene that encodes a novel Trp-Asp (WD) protein designated LAWD (for Leishmania antigenic WD protein). The WD family of proteins mediates protein-protein interactions and coordinates the formation of protein complexes. The single-copy LAWD gene is transcribed as a ϳ3.1-kb mRNA in both promastigotes and amastigotes, with homologues being detected in several other Leishmania species. Immunoelectron microscopy revealed a predominant localization of the LAWD protein in the flagellar pocket. Analyses of sera from human patients with cutaneous and mucocutaneous leishmaniasis indicated that these individuals mounted significant humoral responses against LAWD. Given that recombinant LAWD protein elicited the production of high levels of gamma interferon, but no detectable levels of interleukin-10 (IL-10), in CD4 ؉ cells of L. amazonensis-infected mice, we further examined whether LAWD could elicit protective immunity. DNA vaccination with the LAWD and IL-12 genes significantly delayed lesion development, which correlated with a dramatic reduction in parasite burdens. Thus, we have successfully identified a promising vaccine candidate and antigenic vehicle to aid in the dissection of the complicated pathogenic immune response of L. amazonensis.
Induction of c-fos and c-myc expression during B cell activation by IL-4 and immunoglobulin bindi... more Induction of c-fos and c-myc expression during B cell activation by IL-4 and immunoglobulin binding ligands.
The purified membrane-associated Leishmania pifanoi amastigote protein P-4 has been shown to indu... more The purified membrane-associated Leishmania pifanoi amastigote protein P-4 has been shown to induce protective immunity against infection and to elicit preferentially a T helper 1-like response in peripheral blood mononuclear cells of patients with American cutaneous leishmaniasis. As this molecule is potentially important for future vaccine studies, the L. pifanoi gene encoding the P-4 membrane protein was cloned and sequenced. Southern blot analyses indicate the presence of six tandemly arrayed copies of the P-4 gene in L. pifanoi; homologues of the P-4 gene are found in all other species of the genus Leishmania examined. DNA-derived protein sequence data indicated an identity to the P1 zinc-dependent nuclease of Penicillium citrinum (20.8%) and the C-terminal domain of the 3 nucleotidase of Leishmania donovani (33.7%). Consistent with these sequence analyses, purified L. pifanoi P-4 protein possesses single strand nuclease (DNA and RNA) and phosphomonoesterase activity, with a preference for UMP > TMP > AMP >> CMP. Double-labeling immunofluorescence microscopic analyses employing anti-binding protein antibodies revealed that the P-4 protein is localized in the endoplasmic reticulum of the amastigote. Northern blot analyses indicated that the gene is selectively expressed in the intracellular amastigote stage (mammalian host) but not in the promastigote stage (insect) of the parasite. Based upon its subcellular localization and singlestranded specific nuclease activity, possible roles of the P-4 nuclease in the amastigote in RNA stability (gene expression) or DNA repair are discussed.
Uploads
Papers by Lynn Soong