Immune system recognition of Trypanosoma cruzi

Innate and adaptive cellular immune recognition is crucial for control of the protozoan parasite Trypanosoma cruzi. T. cruzi triggers both MyD88-dependent and TRIF-dependent innate activation pathways in macrophages and dendritic cells. TLR-2 and TLR-9 recognize GPI anchors and parasite DNA, respectively; however other, as yet undefined receptors and ligands, also appear to be involved in innate recognition. CD8(+) T cells distinguish T. cruzi-infected host cells primarily via robust recognition of MHC-associated peptide epitopes from the large and highly diverse trans-sialidase family of surface proteins. To date there has been minimal investigation of linkages between innate immune recognition in vivo and the generation of adaptive cellular immune responses.     

Subversion of Innate Immune Signaling Through Molecular Mimicry

Innate immune signaling is mediated by a number of membrane-anchored or cytosolic receptor or sensor molecules. Several receptor families utilize conserved signaling domains such as the Toll/interleukin-1 receptor (TIR) domain and Pyrin domain (PYD) to link microbe recognition to induction of proinflammatory cytokines and interferons. Recent studies have identified a number of bacterial and viral TIR domains and PYD domains that directly target the signaling function of their host homologues. Emerging biochemical and structural studies of these microbial TIR and PYD domains suggest that they are mimics of their host counterparts at the sequence and structure levels. Unraveling the mechanisms of such molecular mimicry is crucial to our understanding and clinical intervention of infectious diseases and inflammatory disorders.     

Role of Complement in Immune Lysis of Trypanosoma cruzi

Studies were performed on the mechanism of immune lysis of culture forms of Trypanosoma (Schizotrypanum) cruzi. Antibody-mediated lysis is caused by complement, which is activated via the classical pathway. The properdin system is not required. The kinetics of the reaction is similar to that followed by immune lysis of sensitized sheep erythrocytes, as is the concentration of divalent cations required for optimal lysis (0.15 mm Ca²⁺ and 0.5 mm Mg²⁺), the occurrence of cell membrane uptake of the complement components C3 and C4, and the development of characteristic ultrastructural modifications on the cell membrane.     

Postnatal Development of Hepatic Innate Immune Response

The liver is an immunocompetent organ that plays a key role in the immune response to infections, and the development of hepatic immune function during early postnatal stages has not been thoroughly characterized. This study analyzed the constitutive expression of complement factors, namely C3 and C9, and pattern recognition receptors, namely CD14, toll-like receptor (TLR)-4, and lipopolysaccharide binding protein (LBP), in the liver of postnatal day (P)1, P21, and P70 rats, and compared the kinetics of induction of cytokines and chemokines in the liver of P 1 and P 21 animals. Our studies found that while the mRNA expression of C3, C9, CD14, and TLR-4 was lower in P1 animals, the mRNA level of LBP was higher in P1 animals as compared to older animals, and that the kinetics of induction of cytokines and chemokines was significantly delayed in P1 as compared to P21 liver following LPS stimulation. Our data suggest that hepatic innate immunity is deficient in the neonates and undergo significant development during early postnatal life.     

Pathogen-Derived Oligosaccharides Improve Innate Immune Response to Intracellular Parasite Infection

Pathogen glycolipids, including Leishmania spp. lipophosphoglycan (LPG) and Mycobacterium tuberculosis mannosylated lipoarabinomannan (ManLAM), modulate essential interactions with host phagocytic cells. Polysaccharide and lipid components promote immunomodulation. Owing to the stereochemistry required to synthesize oligosaccharides, the roles for oligosaccharides in the pathogenesis of infectious diseases have remained largely unknown. Recent advances in carbohydrate chemistry allowed us to synthesize pathogen surface oligosaccharides to discern their immune response–altering activities. Trimannose cap carbohydrates from ManLAM and LPG altered the production of proinflammatory cytokines via a toll-like receptor (TLR2)–mediated mechanism in vitro and in vivo. In vivo treatment with trimannose led to increased Th1-polarizing, IL-12p40–producing cells from the draining lymph nodes of treated Leishmania major–infected mice compared with cells from untreated infected mice. Trimannose treatment increased the production of other Th1 proinflammatory cytokines (ie, interferon-γ, IL-6, and tumor necrosis factor-α) critical for a productive immune response to either pathogen. This significant difference in cytokine production between trimannose cap sugar–treated and control groups was not observed in draining lymph node cells from TLR2^−/− mice. Type of inflammation and rate of bead entry into macrophages and dendritic cells were different for trimannose-coated beads compared with control oligosaccharide-coated beads, indicating selective lectin receptor/oligosaccharide interactions mediating cell entry and cytokine production. These novel findings may prompt the development of targeted oligosaccharide adjuvants against chronic infections.Pathogens that cause chronic infectious diseases often inhabit host phagocytic cells. Detection of pathogens and initiation of the innate immune response involve interactions between conserved motifs in the ligands as pathogen-associated molecular patterns and in pattern recognition receptors, including toll-like receptors (TLRs) and C-type lectin receptors expressed on host phagocytic cells.^1–3 Macrophages serve as a predominant phagocytic cell type specialized for identification, phagocytosis, and destruction of invading pathogens. Binding of microbial ligands to pattern recognition receptor triggers various crucial immune effector functions, including the production of proinflammatory cytokines. Glycolipids are major ligands whose components have been shown to induce immune modulation.^4–6Carbohydrate chains are abundantly expressed on outer surfaces of most bacterial, protozoan, viral, and fungal pathogens. These chains are without peer in structural diversity. Leishmania parasites are covered by a complex glycocalyx whose glycoconjugate components are thought to be important factors in promoting virulence.^7–10 Leishmania metacyclic promastigote glycocalyx contains several phosphoglycans composed of polymeric disaccharide phosphate repeating units.¹¹ One of the most abundant promastigote surface glycolipids on Leishmania spp. is lipophosphoglycan (LPG), containing 15 to 30 repeating units, which distally bears an external cap oligosaccharide and proximally is anchored to the parasite membrane by glycosyl phosphatidyl inositol.¹² Many studies have shown that purified LPG has a significant effect on parasite survival, attributed to suppression of host signaling and evasion of activated complement.^7–9 Multiple elegant genetic studies altering Leishmania glycolipid expression identified that these molecules are important for parasite virulence and persistence, although there are critical species differences regarding the complex multiple enzymes required to assemble a virulent parasite glycocalyx.¹³ Pathogenic species of Mycobacterium evolved similar strategies to establish long-term infection. The unique composition of the mycobacterial multiglycosylated envelope complex lipoglycans mannosylated lipoarabinomannan (ManLAM) and lipomannan, strategically located at the host-pathogen interface, contributes to immune alteration.^14–16 The three distal glycosylated portions of ManLAM were shown to promote mycobacterial binding to the lectin binding receptor DC-SIGN and to promote mycobacterial virulence.¹⁴ LPG and ManLAM, implicated in modulating inflammatory responses and supporting continuation of chronic disease,¹⁷ provide the cap sugars of focus for this study.In this study, we use unit oligosaccharide carbohydrates coating inert latex beads as a novel model system to determine the immunologic activity of subunit sugars. We used five cap sugars found on Mycobacterium tuberculosis and Mycobacterium bovis ManLAM, Leishmania mexicana and Leishmania major promastigote LPG, in vitro noninfective mutant Leishmania donovani LPG, and inert control sugar lactose to identify the ability of different oligosaccharides to alter the innate immune response. We found that trimannose, found on mycobacterial ManLAM and Leishmania spp. LPG, alters the production of IL-12p40 in macrophages in vitro and in vivo by engaging the TLR2 pathway. When introduced into mice, the trimannose-coated bead had increased numbers of IL-12p40–producing cells and production of proinflammatory cytokines in wild-type but not TLR2^−/− mice. Understanding how subunit oligosaccharides, singularly or combined, produce immune alteration aids in the production of responsive targeted immunostimulants against complex pathogens, including M. tuberculosis and Leishmania.     

Bile Acids Repress Hypoxia-Inducible Factor 1 Signaling and Modulate the Airway Immune Response

Gastroesophageal reflux (GER) frequently occurs in patients with respiratory disease and is particularly prevalent in patients with cystic fibrosis. GER is a condition in which the duodenogastric contents of the stomach leak into the esophagus, in many cases resulting in aspiration into the respiratory tract. As such, the presence of GER-derived bile acids (BAs) has been confirmed in the bronchoalveolar lavage fluid and sputum of affected patients. We have recently shown that bile causes cystic fibrosis-associated bacterial pathogens to adopt a chronic lifestyle and may constitute a major host trigger underlying respiratory infection. The current study shows that BAs elicit a specific response in humans in which they repress hypoxia-inducible factor 1α (HIF-1α) protein, an emerging master regulator in response to infection and inflammation. HIF-1α repression was shown to occur through the 26S proteasome machinery via the prolyl hydroxylase domain (PHD) pathway. Further analysis of the downstream inflammatory response showed that HIF-1α repression by BAs can significantly modulate the immune response of airway epithelial cells, correlating with a decrease in interleukin-8 (IL-8) production, while IL-6 production was strongly increased. Importantly, the effects of BAs on cytokine production can also be more dominant than the bacterium-mediated effects. However, the effect of BAs on cytokine levels cannot be fully explained by their ability to repress HIF-1α, which is not surprising, given the complexity of the immune regulatory network. The suppression of HIF-1 signaling by bile acids may have a significant influence on the progression and outcome of respiratory disease, and the molecular mechanism underpinning this response warrants further investigation.     

Use of trans-Sialidase Inhibition Assay in a Population Serologically Negative for Trypanosoma cruzi but at a High Risk of Infection

trans-Sialidase inhibition assay (TIA) was employed in a population at high risk of Trypanosoma cruzi infection. From 20 serum samples that were negative by conventional serologic and parasitologic assays, 18 (90%) were reactive in TIA, providing further evidence of the higher sensitivity of TIA and suggesting that the actual prevalence of T. cruzi infection might be underestimated.     

Effects of Granulocyte-Macrophage Colony-Stimulating Factor and Tumor Necrosis Factor Alpha on Trypanosoma cruzi Trypomastigotes

We have previously shown that the addition of exogenous granulocyte-macrophage colony-stimulating factor (GM-CSF) to nonactivated mouse peritoneal macrophages (MPM) limits Trypanosoma cruzi infections in vitro (E. Olivares Fontt and B. Vray, Parasite Immunol. 17:135–141, 1995). Lower levels of infection were correlated with a higher level of production of tumor necrosis factor alpha (TNF-α) in the absence of nitric oxide (NO) release. These data suggested that GM-CSF and/or TNF-α might have a direct parasitocidal effect on T. cruzi trypomastigotes, independently of NO release. To address this question, T. cruzi trypomastigotes were treated with recombinant murine GM-CSF (rmGM-CSF), recombinant murine TNF-α (rmTNF-α), or both cytokines in a cell-free system. Treatment with rmGM-CSF but not rmTNF-α caused morphological changes in the parasites, and most became spherical after 7 h of incubation. Both cytokines exerted a cytolytic activity on the trypomastigotes, yet the trypanolytic activity of rmTNF-α was more effective than that of rmGM-CSF. Viable rmGM-CSF- and rmTNF-α-treated parasites were less able to infect MPM than untreated parasites, and this reduction in infectivity was greatest for rmGM-CSF. Treatments with both cytokines resulted in more lysis and almost complete inhibition of infection. The direct parasitocidal activity of rmTNF-α was inhibited by carbohydrates and monoclonal antibodies specific for the lectin-like domain of TNF-α. Collectively, these results suggest that cytokines such as GM-CSF and TNF-α may directly control the level of T. cruzi trypomastigotes at least in vitro and so could determine the outcome of infection in vivo.     

Enzyme-Linked Immunoassay Using Recombinant trans-Sialidase of Trypanosoma cruzi Can Be Employed for Monitoring of Patients with Chagas' Disease after Drug Treatment

trans-Sialidase is an enzyme present on the surface of Trypanosoma cruzi and is an important antigen recognized by sera from patients with Chagas' disease. In the present study we investigated whether the benznidazole treatment of patients with Chagas' disease induced changes in the reactivity of serum toward a recombinant form of trans-sialidase in order to develop an assay for monitoring of patients after treatment for Chagas' disease, which is needed at Chagas' disease control centers. By using an enzyme-linked immunosorbent assay containing a recombinant protein corresponding to the catalytic domain of trans-sialidase, we found that the antigen had a high specificity for sera from untreated patients with Chagas' disease. Sera from healthy individuals or patients with active visceral leishmaniasis minimally cross-reacted with the antigen. Anti-trans-sialidase immunoglobulin was detected in 98% of 151 untreated patients with Chagas' disease. Of these, 124 patients were treated for 60 days with benznidazole (5 mg/kg of body weight/day), and their sera were assayed for reactivity with the recombinant trans-sialidase. By using this methodology, three groups of patients could be established. The first group (60 patients), which was considered to have been successfully treated, showed no reactivity after treatment. The second group (46 patients) still showed signs of infection, and after treatment their sera recognized trans-sialidase, but with reduced titers. The third group (18 patients) was considered to be resistant to drug treatment, and their sera presented identical reactivities before and after treatment. These results suggest that determination of the absence of antibodies to recombinant trans-sialidase in treated patients by the present assay is indicative of treatment success, while the presence of antibodies may indicate the persistence of infection. Therefore, this method may be useful for the diagnosis and monitoring of patients undergoing benznidazole treatment.     

Pathogenesis of Necrotizing Enterocolitis: Modeling the Innate Immune Response

Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants. The pathophysiology is likely secondary to innate immune responses to intestinal microbiota by the premature infant''s intestinal tract, leading to inflammation and injury. This review provides an updated summary of the components of the innate immune system involved in NEC pathogenesis. In addition, we evaluate the animal models that have been used to study NEC with regard to the involvement of innate immune factors and histopathological changes as compared to those seen in infants with NEC. Finally, we discuss new approaches to studying NEC, including mathematical models of intestinal injury and the use of humanized mice.Necrotizing enterocolitis (NEC) is a disorder characterized by intestinal necrosis in premature infants that results in significant morbidity and mortality.¹ Approximately 7% of infants with a birth weight between 500 and 1500 g develop NEC.¹ The pathogenesis is characterized by intestinal inflammation that can progress to systemic infection/inflammation, multiorgan failure, and death. The bowel is distended and hemorrhagic on gross inspection. On microscopic examination, signs of inflammation, mucosal edema, epithelial regeneration, bacterial overgrowth, submucosal gas bubbles, and ischemic transmural necrosis are seen (Figure 1, A–E).²Open in a separate windowFigure 1Examples of the various grades of morphological damage in hematoxylin and eosin–stained specimens. A–E: Representative samples of premature infants with necrotizing enterocolitis. A: Age-matched control from patient with jejunal atresia. B: Mild injury with hemorrhagic necrosis of mucosa and loss of villus tip architecture. C: Progressive injury with inflammatory infiltration of muscularis with complete villus destruction. D: Severe muscular and epithelial damage with complete loss of mucosa. E: Perforation with transmural necrosis with complete loss of epithelial and muscular architecture. F–J: Representative samples from intestinal injury secondary to gavage feeding in the setting of hypothermia and hypoxia in neonatal rats. F: Intact morphology, grade 0. G: Sloughing of villus tips, grade 1. H: Mid-villus necrosis, grade 2. I: Loss of villi, grade 3. J: Complete destruction of the mucosa, grade 4. Insets in F–J show higher magnified portions of the same sections, corresponding to the boxed regions. K–O: Representative images of tissue injury secondary to 60 minutes of intestinal ischemia and 90 minutes of reperfusion in 2-week-old mice. K: Sham-operated mice (no ischemia). L: Villus tip necrosis. M: Mid-villus necrosis. N: Loss of villus architecture. O: Complete loss of mucosal architecture. F–J, reprinted with permission from Nature Publishing Group.²⁸ Scale bars = 50 μm (A–E, K–O). Original magnification, ×20 (A–O, main images, and F–J, insets).Currently the pathogenesis of NEC is believed to have multifactorial causes, including intestinal immaturity and microbial dysbiosis. Intestinal immaturity leads to a compromised intestinal epithelial barrier, an underdeveloped immune defense, and altered vascular development and tone. The compromised epithelial barrier and underdeveloped immune system, when exposed to luminal microbiota that have been shaped by formula feedings, antibiotic exposure, and Cesarean delivery, can lead to intestinal inflammation and sepsis. Despite therapeutic success in animal model systems, there are relatively few therapeutic strategies that have allowed for significantly improved outcomes in infants with NEC. Two hurdles that persist are our incomplete understanding of the developing immune system in premature infants and our inability to adequately replicate these complex factors in animal models.^3,4 This review summarizes the complex intestinal immune system in premature infants and details what is known about the involvement of innate immune factors in NEC, both in animal models and in human disease.     

Comparative Proteomic Analysis Reveals Activation of Mucosal Innate Immune Signaling Pathways during Cholera

Vibrio cholerae O1 is a major cause of acute watery diarrhea in over 50 countries. Evidence suggests that V. cholerae O1 may activate inflammatory pathways, and a recent study of a Bangladeshi population showed that variants in innate immune genes play a role in mediating susceptibility to cholera. We analyzed human proteins present in the small intestine of patients infected with V. cholerae O1 to characterize the host response to this pathogen. We collected duodenal biopsy specimens from patients with acute cholera after stabilization and again 30 days after initial presentation. Peptides extracted from biopsy specimens were sequenced and quantified using label-free mass spectrometry and SEQUEST. Twenty-seven host proteins were differentially abundant between the acute and convalescent stages of infection; the majority of these have known roles in innate defense, cytokine production, and apoptosis. Immunostaining confirmed that two proteins, WARS and S100A8, were more abundant in lamina propria cells during the acute stage of cholera. Analysis of the differentially abundant proteins revealed the activation of key regulators of inflammation by the innate immune system, including Toll-like receptor 4, nuclear factor kappa-light-chain-enhancer of activated B cells, mitogen-activated protein kinases, and caspase-dependent inflammasomes. Interleukin-12β (IL-12β) was a regulator of several proteins that were activated during cholera, and we confirmed that IL-12β was produced by lymphocytes recovered from duodenal biopsy specimens of cholera patients. Our study shows that a broad inflammatory response is generated in the gut early after onset of cholera, which may be critical in the development of long-term mucosal immunity against V. cholerae O1.     

The Capsule of Acinetobacter baumannii Protects against the Innate Immune Response

Acinetobacter baumannii is an opportunistic pathogen that has recently emerged as a global threat associated with high morbidity, mortality, and antibiotic resistance. We determined the role of type I interferon (IFN) signaling in A. baumannii infection. We report that A. baumannii can induce a type I IFN response that is dependent upon TLR4-TRIF-IRF3 and phagocytosis of the bacterium. Phase variants of A. baumannii that have a reduced capsule, lead to enhanced TLR4-dependent type I IFN induction. This was also observed in a capsule-deficient strain. However, we did not observe a role for this pathway in vivo. The enhanced signaling could be accounted for by increased phagocytosis in capsule-deficient strains that also lead to enhanced host cell-mediated killing. The increased cytokine response in the absence of the capsule was not exclusive to type I IFN signaling. Several cytokines, including the proinflammatory IL-6, were increased in cells stimulated with the capsule-deficient strain, also observed in vivo. After 4 h in our acute pneumonia model, the burden of a capsule-null strain was significantly reduced, yet we observed increases in innate immune cells and inflammatory markers compared to wild-type A. baumannii. This study underscores the role of phase variation in the modulation of host immune responses and indicates that the capsule of A. baumannii plays an important role in protection against host cell killing and evasion from activation of the innate immune response.     

Susceptibility of radiation chimeras to Trypanosoma cruzi.

Reciprocal bone marrow transfers were performed with C3H/HeJ mice, which are susceptible to infection with the Brazil strain of Trypanosoma cruzi, and resistant F1 (C3H/HeJ X C57BL/6J) mice. Mice reconstituted after lethal irradiation with syngeneic bone marrow displayed the resistance phenotype of the strain used, but neither C3H mice reconstituted with F1 bone marrow cells nor F1 mice reconstituted with C3H bone marrow cells survived challenge. Resistance to T. cruzi appears to be dependent upon factors associated both with host background and with bone marrow-derived cells.     

Genetics of murine resistance to Trypanosoma cruzi.

Resistance to the protozoan parasite Trypanosoma cruzi is governed by multiple genetic factors, including at least one coded for by a locus in or near the major histocompatibility complex of the mouse. The influence of the H-2 locus on resistance was evident when H-2 congenic mice on a strain background of intermediate resistance were challenged or when the survival of H-2 typed F2 mice was followed. The H-2k haplotype of the susceptible C3H/An strain was associated with higher mortality when compared with the H-2b haplotype of the resistant C57BL/10 strain. Genetic studies showed that resistance was a dominant trait and increased with genetic heterozygosity. F1 mice derived from crosses between resistant and susceptible strains, or even between two susceptible strains, were much more resistant than either parent. Crosses between two resistant strains, C57BL/6J and DBA/2J, led to resistant progeny in the F1 and F2 generations; but when recombinant inbred strains derived from these parental strains were challenged, susceptible strains were identified, indicating that different genes were responsible for resistance in the two strains.