Isolation and characterization of intestinal epithelial cells from normal and SIV-infected rhesus macaques

Impairment of intestinal epithelial barriers contributes to the progression of HIV/SIV infection and leads to generalized HIV-induced immune-cell activation during chronic infection. Rhesus macaques are the major animal model for studying HIV pathogenesis. However, detailed characterization of isolated rhesus epithelial cells (ECs) from intestinal tissues is not well defined. It is also not well documented whether isolated ECs had any other cell contaminants from intestinal tissues during the time of processing that might hamper interpretation of EC preparations or cultures. In this study, we identify and characterize ECs based on flow cytometry and immunohistochemistry methods using various enzymatic and mechanical isolation techniques to enrich ECs from intestinal tissues. This study shows that normal healthy ECs differentially express HLA-DR, CD23, CD27, CD90, CD95 and IL-10R markers. Early apoptosis and upregulation of ICAM-1 and HLA-DR in intestinal ECs are thought to be the key features in SIV mediated enteropathy. The data suggest that intestinal ECs might be playing an important role in mucosal immune responses by regulating the expression of different important regulatory and adhesion molecules and their function.     

CpG oligodeoxynucleotides protect newborn mice from a lethal challenge with the neurotropic Tacaribe arenavirus

The innate immune system is key to limiting the early spread of most pathogens and directing the development of Ag-specific immunity. Recently, a number of synthetic molecules that activate the innate immune system by stimulating TLRs have been identified. Among them, synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs (CpG ODNs) were shown to activate TLR9-bearing B cells, macrophages, and dendritic cells to induce a strong proinflammatory milieu and a type 1-biased immune response that protects mice from a variety of parasitic, bacterial, and viral infections. Although the protective effect of CpG ODN in adult mice was well established, its effectiveness in neonates, which have lower numbers of dendritic, B, and T cells and tend to favor Th2 responses, was unclear. This study uses the New World arenavirus Tacaribe, a neurotropic pathogen that is lethal in newborn mice, to explore the effectiveness of TLR-mediated innate immune responses. Neonatal BALB/c mice treated with CpG ODN at the time of infection had reduced viral load (p < 0.01) and increased survival (52%, p < 0.001 i.p.; 36%, p < 0.05 intranasally). Protection was achieved in mice treated no later than 3 days postchallenge and appears to be mediated by an increase in Ag-specific Abs (IgG and IgM) and to require inducible NO synthase expression and NO production. To our knowledge, this is the first study assessing the mechanisms by which CpG ODN can protect mice from a neurotropic viral infection.     

CpG oligodeoxynucleotides enhance neonatal resistance to Listeria infection

Infection by Listeria monocytogenes causes serious morbidity and mortality during the neonatal period. Previous studies established that immunostimulatory CpG oligodeoxynucleotides (ODN) can increased the resistance of adult mice to many infectious pathogens, including Listeria. This work examines the capacity of CpG ODN to stimulate a protective immune response in newborns. Results indicate that dendritic cells, macrophages, and B cells from 3-day-old mice respond to CpG stimulation by secreting IFN-gamma, IL-12, and/or TNF-alpha. Spleen cells from CpG-treated neonates produce large amounts of cytokine and NO when exposed to bacteria in vitro. Newborns treated with CpG ODN are protected from lethal Listeria challenge and generate Ag-specific CD4 and CD8 T cells that afford long-term protection against subsequent infection. These results demonstrate that cellular elements of the neonatal immune system respond to stimulation by CpG ODN, thereby reducing host susceptibility to infectious pathogens.     

CpG oligodeoxynucleotides stimulate protective innate immunity against pulmonary Klebsiella infection

Bacterial pneumonia is a leading cause of mortality in the United States. Innate immune responses, including type-1 cytokine production, are critical to the effective clearance of bacterial pathogens from the lung. Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotide motifs (CpG ODN), which mimic the effects of bacterial DNA, have been shown to enhance type-1 cytokine responses during infection due to intracellular pathogens, resulting in enhanced microbial clearance. The role of CpG ODN in modulating protective innate immunity against extracellular pathogens is unknown. Using a murine model of Gram-negative pneumonia, we found that CpG ODN administration stimulated protective immunity against Klebsiella pneumoniae. Specifically, intratracheal (i.t.) administration of CpG ODN (30 microg) 48 h before i.t. K. pneumoniae challenge resulted in increased survival, compared with animals pretreated with control ODN or saline. Pretreatment with CpG ODN resulted in enhanced bacterial clearance in lung and blood, and higher numbers of pulmonary neutrophils, NKT cells, gammadelta-T cells, and activated NK1.1+ cells and gammadelta-T lymphocytes during infection. Furthermore, pretreatment with CpG ODN enhanced the production of TNF-alpha, and type-1 cytokines, including IL-12, IFN-gamma, and the IFN-gamma-dependent ELR- CXC chemokines IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma in response to Klebsiella challenge, compared with control mice. These findings indicate that i.t. administration of CpG ODN can stimulate multiple components of innate immunity in the lung, and may form the basis for novel therapies directed at enhancing protective immune responses to severe bacterial infections of the lung.     

Rhesus rhadinovirus infection in healthy and SIV-infected macaques at Tulane National Primate Research Center

Rhesus rhadinovirus (RRV) infection was quantified in peripheral blood mononuclear cells (PBMC) from healthy and simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta) at the Tulane National Primate Research Center and in a large collection of simian acquired immunodeficiency syndrome--(SAIDS)-associated lymphomas. Quantification of RRV load was performed by real-time PCR using amplification primers specific for the RRV interleukin-6 homologue (RRV vIL-6). RRV infection was detected infrequently and at low levels in PBMC of randomly selected healthy animals. Examination of longitudinally collected PBMC from 22 SIV-infected animals throughout progression to SAIDS revealed similarly low RRV loads that sometimes increased with advancing disease. RRV infection was detected more frequently in the peripheral blood of SIV-infected animals than in healthy animals. Examination of SAIDS-associated lymphomas showed that RRV is rare within the tumor mass, likely representing infection in an occasional tumor-infiltrating cell or contaminating blood. The results indicate that RRV infection in PBMC is not predictive of, and is apparently not required for, development of lymphoma or hyperplastic lymphadenopathy in SIV-infected animals at TNPRC.     

Suppressive oligodeoxynucleotides protect mice from lethal endotoxic shock

Endotoxic shock is a life-threatening condition caused by exposure to bacterial LPS. LPS triggers the release of acute phase, proinflammatory, and Th1 cytokines that facilitate the development of endotoxic shock. Synthetic oligodeoxynucleotides (ODN) expressing suppressive TTAGGG motifs effectively down-regulate the production of proinflammatory and Th1 cytokines elicited by a variety of immune stimuli. The current results demonstrate that suppressive ODN protect mice from LPS-induced endotoxic shock. Underlying this protective effect is the ability of suppressive ODN to bind to and prevent the phosphorylation of STAT1 and STAT4, thereby blocking the signaling cascade mediated by LPS-induced IFN-beta and IL-12. These findings suggest that suppressive ODN might be of use in the treatment of endotoxic shock.     

Vaccination with Leishmania soluble antigen and immunostimulatory oligodeoxynucleotides induces specific immunity and protection against Leishmania donovani infection

In this report, we investigated the effect of ODN containing immunostimulatory CG motifs as adjuvant with soluble antigen (SA) from Leishmania donovani. BALB/c mice were vaccinated with the soluble antigen with or without CpG-ODN as adjuvant and then challenged with L. donovani metacyclic promastigotes. CpG-ODN alone resulted in partial protection against challenge with L. donovani. Immunization of mice with SA and CpG-ODN showed enhanced reduction in parasite load ( approximately 60%) when compared to SA ( approximately 40%) immunized mice. Immunization with SA by itself resulted in a mixed Th1/Th2 response whereas co-administration of SA with CpG-ODN resulted in a strong Th1 promoting isotype as they together promoted production of immunoglobulin G2a. Leishmania-specific Th1 cytokine response was induced by co-administering CpG-ODN and SA as they together promoted production of IFN-gamma and IL-12. In the present study, we demonstrate that immunostimulatory phosphorothioate-modified ODN are promising immune enhancers for vaccination against visceral leishmaniaisis.     

CpG oligodeoxynucleotides protect mice from lethal challenge with Candida albicans via a pathway involving tumor necrosis factor-alpha-dependent interleukin-12 induction

In this study, we have attempted to determine whether the systemic administration of CpG oligodeoxynucleotide (CpG-ODN) 1826 would protect mice against systemic lethal Candida albicans infection. CpG-ODNs were found completely to protect mice from death and also reduced the growth of C. albicans in the kidneys. The administration of CpG-ODNs resulted in early interleukin (IL)-12 mRNA expression in the kidneys and an increase in serum IL-12 levels. The protective activity of CpG-ODN was abolished in IL-12-deficient (IL-12-/-) mice, thereby indicating the IL-12-dependency inherent to the effects of CpG-ODN. The protective effect of CpG-ODN was not associated with the activity of NF-kappaB. Interestingly, in tumor necrosis factor (TNF)-alpha-deficient (TNF-/-) mice CpG-ODN neither exerted protective effects nor induced IL-12 expression. These data indicate that CpG-ODN protects animals against lethal C. albicans challenge via a pathway that involves the TNF-alpha-dependent induction of IL-12.     

Simian immunodeficiency virus (SIV) infection influences the level and function of regulatory T cells in SIV-infected rhesus macaques but not SIV-infected sooty mangabeys

Differences in clinical outcome of simian immunodeficiency virus (SIV) infection in disease-resistant African sooty mangabeys (SM) and disease-susceptible Asian rhesus macaques (RM) prompted us to examine the role of regulatory T cells (Tregs) in these two animal models. Results from a cross-sectional study revealed maintenance of the frequency and absolute number of peripheral Tregs in chronically SIV-infected SM while a significant loss occurred in chronically SIV-infected RM compared to uninfected animals. A longitudinal study of experimentally SIV-infected animals revealed a transient increase in the frequency of Tregs from baseline values following acute infection in RM, but no change in the frequency of Tregs occurred in SM during this period. Further examination revealed a strong correlation between plasma viral load (VL) and the level of Tregs in SIV-infected RM but not SM. A correlation was also noted in SIV-infected RM that control VL spontaneously or in response to antiretroviral chemotherapy. In addition, immunofluorescent cell count assays showed that while Treg-depleted peripheral blood mononuclear cells from RM led to a significant enhancement of CD4+ and CD8+ T-cell responses to select pools of SIV peptides, there was no detectable T-cell response to the same pool of SIV peptides in Treg-depleted cells from SIV-infected SM. Our data collectively suggest that while Tregs do appear to play a role in the control of viremia and the magnitude of the SIV-specific immune response in RM, their role in disease resistance in SM remains unclear.     

Monoclonal antibodies against defined Leishmania antigens protect mice against infection by different species of Leishmania

Mice monoclonal antibodies (IgG) have been raised against Leishmania infantum promastigotes by fusing SP 2/0 myeloma cells and immunized mice splenic cells. The monoclonal antibodies have been detected by indirect immunofluorescence. In vivo tests showed that some of them could inhibit the life cycle of several Leishmania species from the Old and the New World. Studies of these protective monoclonals by the western blot technique showed the presence of three constant antigens (40 kD, 70 kD and 113 kD) amongst the Leishmania species studied.     

Mechanisms and applications of immune stimulatory CpG oligodeoxynucleotides

Immune stimulation has been widely recognized as an undesirable side effect of certain antisense oligodeoxynucleotides (ODN) which can interfere with their therapeutic application. It is now clear that these dose-dependent immune stimulatory effects primarily result from the presence of an unmethylated CpG dinucleotide in particular base contexts ('CpG motif). The sequence-specific immune activation is not just an experimental artifact, but is actually a highly evolved immune defense mechanism whose actual 'goal' is the detection of microbial nucleic acids. In contrast to vertebrate DNA, in which CpG dinucleotides are 'suppressed' and are highly methylated, microbial genomes do not generally feature CpG suppression or methylation [1]. Immune effector cells such as B cells, macrophages, dendritic cells, and natural killer cells appear to have evolved pattern recognition receptors (PRR) that by binding the microbe-restricted structure of CpG motifs, trigger protective immune responses. Although the specific immune activation appears to have a variety of potential therapeutic applications, it is generally undesirable in antisense ODN. Immune stimulation may be avoided in antisense oligos by the selection of CpG-free target sequences, by the use of ODN backbones that do not support immune stimulation, or by selective modifications of the cytosine in any CpG dinucleotides.     

Vaccination with the Leishmania major ribosomal proteins plus CpG oligodeoxynucleotides induces protection against experimental cutaneous leishmaniasis in mice

In the present work we analyze the antigenicity of Leishmania major ribosomal proteins (LRP) in infected BALB/c mice. We show that BALB/c mice vaccinated with LRP in the presence of CpG oligodeoxynucleotides (CpG-ODN) were protected against the development of dermal pathology and showed a reduction in the parasite load after challenge with L. major. This protection was associated with the induction of an IL-12 dependent specific-IFN-gamma response mediated mainly by CD4(+) T cell, albeit a minor contribution of CD8(+) T cells cannot be ruled out. Induction of Th1 responses against LRP also resulted in a reversion of the Th2 responses associated with susceptibility. A marked reduction of IgG1 antibody titer against parasite antigens besides an impaired IL-4 and IL-10 cytokine production by parasite specific T cells was observed. In addition, we show that the administration of the LRP plus CpG-ODN preparation also conferred protection in the naturally resistant C57BL/6 mice. In this strain protection was associated with a LRP specific IFN-gamma production in lymph nodes draining the challenge site. We believe that these evolutionary conserved proteins, combined with adjuvants that favor Th1 responses, may be relevant components of a pan-Leishmania vaccine.     

Early resolution of acute immune activation and induction of PD-1 in SIV-infected sooty mangabeys distinguishes nonpathogenic from pathogenic infection in rhesus macaques

Primate lentiviruses are typically apathogenic in their evolutionarily coadapted host species but can be lethal when transferred to new host species. Why such infections are pathogenic in humans and rhesus macaques (RMs) but not in sooty mangabeys (SMs), a natural host, remains unclear. Studies of chronically infected animals point to the importance of diminished immune activation in response to the infection in SMs. In this study, we sought the causes and timing of the differences in immune activation in a comparative study of acute SIV infection in RMs and SMs. Surprisingly, we show that in acute infection immune activation is comparable in SMs and RMs but thereafter, SMs quickly resolve immune activation, whereas RMs did not. Early resolution of immune activation in SMs correlated with increased expression of PD-1 and with preservation of CD4(+) T cell counts and lymphatic tissue architecture. These findings point to early control of immune activation by host immunoregulatory mechanisms as a major determinant of the different disease outcomes in SIV infection of natural vs non-natural hosts.     

Adhesion- and migration-related side effects of phosphothioated CpG oligodeoxynucleotides

Nucleic acid oligodeoxynucleotides (ODN) are increasingly used in biological research and in clinics where they are used for both diagnostic and therapeutic purposes. In order to increase the stability and efficacy of ODNs, various chemical modifications have been applied to create nucleic acid derivatives that are not recognized by endogenous nucleic acid cleavage mechanisms. One of the most common and cost-effective modifications is the phosphothioate (PTO) modification. The PTO modification is implemented mainly in antisense ODN, but also in ODN that were shown to activate members of the toll-like receptor (TLR) family such as TLR3 (poly-I:C), TLR8 (ssRNA), and TLR9 (CpG). We recently found that PTO-ODN aimed at activating TLR9 induce a non-TLR9-specific detachment phenotype in a growth-substrate dependent manner. Moreover, we found that unmodified and PTO-modified TLR ligands induce distinct patterns of gene expression in cultured neural cells. These findings suggest that PTO-ODN can cause nonspecific effects on cell adhesion that could compromise interpretation of data from experiments using PTO-ODN.     

Immune response of rhesus macaques to recombinant simian immunodeficiency virus gp130 does not protect from challenge infection.

Simian immunodeficiency virus (SIV) infection of rhesus macaques is a model for human immunodeficiency virus (HIV) infection in humans. Inactivated and modified live whole-virus vaccines have provided limited protective immunity against SIV in rhesus macaques. Because of safety concerns in the use of inactivated and live whole-virus vaccines, we evaluated the protective immunity of vaccinia virus recombinants expressing the surface glycoprotein (gp130) of SIVmac and subunit preparations of gp130 expressed in mammalian cells (CHO). Three groups of animals were immunized with recombinant SIV gp130. The first group received SIV gp130 purified from genetically engineered CHO cells (cSIVgp130), the second group was vaccinated with recombinant vaccinia virus expressing SIVmac gp130 (vSIVgp130), and the third group was first primed with vSIVgp130 and then given a booster immunization with cSIVgp130. Although anti-gp130 binding antibodies were elicited in all three groups, neutralizing antibodies were transient or undetectable. None of the immunized animals resisted intravenous challenge with a low dose of cell-free virus. However, the group primed with vSIVgp130 and then boosted with cSIVgp130 had the lowest antigen load (p27) compared with the other groups. The results of these studies suggest that immunization of humans with HIV type 1 surface glycoprotein may not provide protective immunity against virus infection.     

Binding and uptake of immunostimulatory CpG oligodeoxynucleotides by human neuroblastoma cells

Oligodeoxynucleotides (ODNs) that contain unmethylated CpG dinucleotides (CpG-ODN) trigger a strong innate immune response in vertebrates. They have been used to eradicate experimental neuroblastoma, but a direct interaction of CpG-ODN with neuroblastoma cells has not been investigated. We have analyzed uptake, binding, and intracellular distribution of CpG-ODN in the neuroblastoma cells line SKNSH. Our results indicate that cellular uptake of CpG-ODN is dose, time, temperature, and energy dependent but independent of the CpG motif. After internalization, CpGODN localized to the cytoplasm and showed a typical speckled distribution pattern. The intracellular distribution pattern and binding proteins are CpG motif independent as well. Thus, CpG-ODNs are taken up by neuroblastoma cells by a nonspecific transfer mechanism for oligonucleotides and interact with intracellular proteins. These mechanisms might help us to understand the biodistribution of oligo within tumors and might be helpful in evaluating the therapeutic effects of oligonucleotides and rational drug design.     

Substance P receptor antagonist reverses intestinal pathophysiological alterations occurring in a novel ex-vivo model of Cryptosporidium parvum infection of intestinal tissues derived from SIV-infected macaques

BACKGROUND: Cryptosporidium infection leads to life-threatening diarrhea in AIDS patients. Pathogenesis of cryptosporidiosis is due to intestinal physiological alterations. We devised an ex-vivo model using ex-vivo Cryptosporidium parvum infection of jejunal tissues derived from SIV-infected macaques and studied the role of substance P (SP) in the pathogenesis of cryptosporidiosis. METHODS: We measured jejunal SP protein levels using ELISA, and electrophysiological alterations using the Ussing chamber technique in an ex vivo model of Cryptosporidium infection. Paraformaldehyde-fixed jejunum from SIV-infected macaques with and without naturally occurring cryptosporidiosis was studied for SP protein expression by immunohistochemistry and fluorescence deconvolution microscopy. RESULTS: Ex-vivo Cryptosporidium-infected tissues and tissues from SIV-infected macaques with naturally occurring cryptosporidiosis demonstrated elevated SP protein levels compared with tissues from SIV-infected animals without ex-vivo C. parvum infection or tissues from SIV-infected animals that have no evidence of cryptosporidiosis. In our ex-vivo model of Cryptosporidium infection, we demonstrated pathophysiological alterations that were blocked by SP-receptor antagonist treatment. CONCLUSIONS: These studies suggest that SP-receptor antagonists could prove useful for treatment of AIDS-related cryptosporidiosis.     

Cooperative stimulation of dendritic cells by Cryptococcus neoformans mannoproteins and CpG oligodeoxynucleotides

While mannosylation targets antigens to mannose receptors on dendritic cells (DC), the resultant immune response is suboptimal. We hypothesized that the addition of toll-like receptor (TLR) ligands would enhance the DC response to mannosylated antigens. Cryptococcus neoformans mannoproteins (MP) synergized with CpG-containing oligodeoxynucleotides to stimulate enhanced production of proinflammatory cytokines and chemokines from murine conventional and plasmacytoid DC. Synergistic stimulation required the interaction of mannose residues on MP with the macrophage mannose receptor (MR), CD206. Moreover, synergy with MP was observed with other TLR ligands, including tripalmitoylated lipopeptide (Pam3CSK4), polyinosine-polycytidylic acid (pI:C), and imiquimod. Finally, CpG enhanced MP-specific MHC II-restricted CD4(+) T-cell responses by a mechanism dependent upon DC expression of CD206 and TLR9. These data suggest a rationale for vaccination strategies that combine mannosylated antigens with TLR ligands and imply that immune responses to naturally mannosylated antigens on pathogens may be greatly augmented if TLR and MR are cooperatively stimulated.