Hemozoin-induced IFN-γ production mediates innate immune protection against sporozoite infection.

Hemozoin is a crystal synthesized by Plasmodium parasites during hemoglobin digestion in the erythrocytic stage. The hemozoin released when the parasites egress from the red blood cell, which is complexed with parasite DNA, is cleared from the circulation by circulating and tissue-resident monocytes and macrophages, respectively. Recently, we reported that intravenous administration of purified hemozoin complexed with Plasmodium berghei DNA (HzPbDNA) resulted in an innate immune response that blocked liver stage development of sporozoites that was dose-dependent and time-limited. Here, we further characterize the organismal, cellular, and molecular events associated with this protective innate response in the liver and report that a large proportion of the IV administered HzPbDNA localized to F4/80+ cells in the liver and that the rapid and strong protection against liver-stage development waned quickly such that by 1 week post-HzPbDNA treatment animals were fully susceptible to infection. RNAseq of the liver after IV administration of HzPbDNA demonstrated that the rapid and robust induction of genes associated with the acute phase response, innate immune activation, cellular recruitment, and IFN-γ signaling observed at day 1 was largely absent at day 7. RNAseq analysis implicated NK cells as the major cellular source of IFN-γ. In vivo cell depletion and IFN-γ neutralization experiments supported the hypothesis that tissue-resident macrophages and NK cells are major contributors to the protective response and the NK cell-derived IFN-γ is key to induction of the mechanisms that block sporozoite development in the liver. These findings advance our understanding of the innate immune responses that prevent liver stage malaria infection.

The role of the BAFF/APRIL system in the T cell-independent specific response to blood stage Plasmodium falciparum hemozoin.

BACKGROUND: The B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) are tumor necrosis factor family members that regulate B cell maturation, proliferation, survival and function. We have previously shown that blood-stage Plasmodium falciparum hemozoin (HZ) can act as a T-independent antigen (TI Ag) that induces the production of specific IgG to soluble crude P. falciparum Ag through the BAFF pathway. However, we have not yet clarified whether HZ need APRIL signaling in the TI response. Here, we aimed to clarify whether both BAFF and APRIL signaling pathways play roles in HZ induction of specific antibody production without T-cell help. METHODS: Normal monocytes alone or co-cultured with naïve B cells were stimulated by HZ (10 µM) in vitro. Naïve B cell cultures, with HZ alone or with exogenous recombinant BAFF (rBAFF) and recombinant APRIL (rAPRIL) plus recombinant IL-4 (rIL-4) for 6 and 10 days were used as controls to investigate activation of B cells. At various times, the levels of sBAFF, sAPRIL, and HZ-specific IgG in the culture supernatants were assessed by enzyme-linked immunosorbent assay. The BAFF and APRIL expression levels on the HZ-stimulated monocytes and their specific receptors on activated B cells, including the BAFF receptor (BAFF-R), the transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) and the B cell maturation antigen (BCMA), were determined by flow cytometry. mRNA expression levels for the receptors were validated using Real-Time quantitative PCR. RESULTS: HZ-activated monocytes released sBAFF and sAPRIL during the 72 h stimulation period. Increased mRNA encoding of their cognate receptors, BAFF-R, TACI, and BCMA, and increased HZ-specific IgG levels were also observed in HZ induction within the monocyte and B cell co-culture. The experiments under control conditions revealed that HZ alone could induce B cell culture to produce a small amount of the specific IgG compared with those in medium alone or rBAFF + rAPRIL + rIL-4. CONCLUSION: Taken together, we suggest that in the TI response HZ stimulates monocyte and B cell co-culture to produce specific IgG through BAFF, APRIL and other independent complimentary signaling pathways.

Immunoprotective effects of a hemin-binding peptide derived from hemagglutinin-2 against infection with Porphyromonas gingivalis.

The principal etiologic agent in periodontal disease, Porphyromonas gingivalis, generates cysteine proteases that bind heme with domains such as hemagglutinin-2 (HA2). High-affinity HA2-hemin binding supplies the porphyrin and ferric iron needed for growth and virulence. The DHYAVMISK peptide, recently identified at the hemin-binding site of HA2, inhibits hemin binding. We now evaluate the protective effect of vaccination with DGFPGDHYAVMISK (termed DK) against P. gingivalis using a rat infection model. Rats immunized with DK generated anti-peptide serum IgGs and salivary sIgAs (as measured by ELISA). In a subcutaneous abscess model, the protective effect of immunization was then investigated by measuring abscess size following subcutaneous injection with P. gingivalis. In an oral infection model, a ligature inoculated with P. gingivalis was used to induce periodontitis. The degree of bone erosion, ordinarily provoked by infection, was then evaluated by micro-computed tomography. We found that anti-peptide antibody titers of serum IgGs and salivary sIgAs for rats immunized with DK and adjuvant were significantly higher than for sham-immunized rats (injected with adjuvant/PBS alone; P < .05). In the subcutaneous abscess model, the DK + adjuvant-vaccinated rats recovered faster than sham-vaccinated animals, with their abscess sizes significantly smaller (P < .05). Further, in the experimental periodontitis model, bone loss at the molar palatal side for DK + adjuvant-vaccinated rats was significantly lower than for sham-vaccinated animals (P < .05). Collectively, these data demonstrate the potential of (DK) peptide immunization in terms of eliciting an immunoprotective effect against infection with P. gingivalis.

Phagocytosis of hemozoin by RAW 264.7 cells, but not THP-1 cells, promotes infection by Leishmania donovani with a nitric oxide-independent mechanism.

During its intra-erythrocytic development, the malaria parasite Plasmodium falciparum synthesizes insoluble hemozoin (HZ) crystals that are released into the circulation upon rupture of parasitized red blood cells, and rapidly phagocytized by host mononuclear cells. Here, HZ persists undigested, causing functional impairment and possibly leading to increased host susceptibility to secondary infections. In patients with malaria and visceral leishmaniasis (VL) co-infections, HZ-loaded macrophages are likely to co-harbor Leishmania donovani parasites, but whether this might influence the course of the Leishmania infection is unknown. In this study, L. donovani amastigote growth was monitored in mouse RAW 264.7 macrophages and PMA-differentiated THP-1 cells previously exposed to increasing amounts of HZ or its synthetic analogue ß-hematin (BH). Latex beads were used as a phagocytic control. Data demonstrate that phagocytosis of HZ and BH by RAW 264.7 cells promoted infection therein by L. donovani parasites in a dose-dependent fashion. Similar results were not observed when using THP-1 cells, despite a clear persistence of undigested heme up to 48h after phagocytosis. Conditioning with lipopolysaccharide (LPS)/interferon (IFN)-γ prior to Leishmania infection triggered the release in RAW 264.7 cells of nitric oxide (NO), a highly leishmanicidal metabolite. However, neither HZ nor BH pre-ingestion were able to inhibit NO production following stimulation with LPS/IFN-γ, suggesting that the HZ- and BH-promoting effect on L. donovani infection occurred with an NO-independent mechanism. In conclusion, these preliminary findings highlight a possible detrimental effect of HZ on the course of VL, warranting further investigation into the clinical relevance of the current models.

Hemozoin Enhances Maturation of Murine Bone Marrow Derived Macrophages and Myeloid Dendritic Cells.

BACKGROUND: Falciparum malaria is a severe health burden worldwide. Antigen presenting cells are reported to be affected by erythrocytic stage of the parasite. Malarial hemozoin (HZ), a metabolite of malaria parasite, has adjuvant properties and may play a role in the induction of immune response against the parasite. OBJECTIVE: To determine the immunological impact of hemozoin on the capacity of innate immune cells maturation. METHODS: Plasmodium falciparum (F32 strain) was cultured in O+ blood group up to 18% parasitemia. Natural hemozoin was extracted from infected red blood cells. Murine bone marrow derived macrophages and myeloid dendritic cells were stimulated with 4 µg/mL or 40 µg/mL of synthetic hemozoin (ß-hematin) or natural hemozoin. We assessed the immunomodulatory role of synthetic or natural hemozoinin vitro by flowcytometric analysis. RESULTS: The maturation markers MHC-II, CD80 and CD86 were significantly upregulated (p<0.05) on the surface of murine bone marrow derived macrophages or myeloid dendritic cells. Data confirmed the potential of macrophages or myeloid dendritic cells, through hemozoin activation, to establish an innate immune response against malaria parasites. CONCLUSION: Both synthetic and natural hemozoin are potent inducers of cellular immunity against malaria infection. However, natural hemozoin is a stronger inducer as compared to synthetic hemozoin.

Innate sensing of malaria parasites.

Innate immune receptors have a key role in immune surveillance by sensing microorganisms and initiating protective immune responses. However, the innate immune system is a classic 'double-edged sword' that can overreact to pathogens, which can have deleterious effects and lead to clinical manifestations. Recent studies have unveiled the complexity of innate immune receptors that function as sensors of Plasmodium spp. in the vertebrate host. This Review highlights the cellular and molecular mechanisms by which Plasmodium infection is sensed by different families of innate immune receptors. We also discuss how these events mediate both host resistance to infection and the pathogenesis of malaria.

Schistosoma mansoni hemozoin modulates alternative activation of macrophages via specific suppression of Retnla expression and secretion.

The trematode Schistosoma mansoni is one of the etiological agents of schistosomiasis, a key neglected tropical disease responsible for an estimated annual loss of 70 million disability-adjusted life years. Hematophagy represents the primary nutrient acquisition pathway of this parasite, but digestion of hemoglobin also liberates toxic heme. Schistosomes detoxify heme via crystallization into hemozoin, which is subsequently regurgitated into the host's circulation. Here we demonstrate that during experimental schistosomiasis, hemozoin accumulating in the mouse liver is taken up by phagocytes at a time coincident with the development of the egg-induced T-helper 2 (Th2) granulomatous immune response. Furthermore, the uptake of hemozoin also coincides with the hepatic expression of markers of alternative macrophage activation. Alternatively activated macrophages are a key effector cell population associated with protection against schistosomiasis, making hemozoin well placed to play an important immunomodulatory role in this disease. To systematically explore this hypothesis, S. mansoni hemozoin was purified and added to in vitro bone marrow-derived macrophage cultures concurrently exposed to cytokines chosen to reflect the shifting state of macrophage activation in vivo. Macrophages undergoing interleukin-4 (IL-4)-induced alternative activation in the presence of hemozoin developed a phenotype specifically lacking in Retnla, a characteristic alternatively activated macrophage product associated with regulation of Th2 inflammatory responses. As such, in addition to its important detoxification role during hematophagy, we propose that schistosome hemozoin also provides a potent immunomodulatory function in the coevolved network of host-parasite relationships during schistosomiasis.

Haemozoin induces early cytokine-mediated lysozyme release from human monocytes through p38 MAPK- and NF-kappaB-dependent mechanisms.

Malarial pigment (natural haemozoin, HZ) is a ferriprotoporphyrin IX crystal produced by Plasmodium parasites after haemoglobin catabolism. HZ-fed human monocytes are functionally compromised, releasing increased amounts of pro-inflammatory molecules, including cytokines, chemokines and cytokine-related proteolytic enzyme Matrix Metalloproteinase-9 (MMP-9), whose role in complicated malaria has been recently suggested. In a previous work HZ was shown to induce through TNFalpha production the release of monocytic lysozyme, an enzyme stored in gelatinase granules with MMP-9. Here, the underlying mechanisms were investigated. Results showed that HZ lipid moiety promoted early but not late lysozyme release. HZ-dependent lysozyme induction was abrogated by anti-TNFalpha/IL-1 beta/MIP-1 alpha blocking antibodies and mimicked by recombinant cytokines. Moreover, HZ early activated either p38 MAPK or NF-kappaB pathways by inducing: p38 MAPK phosphorylation; cytosolic I-kappaB alpha phosphorylation and degradation; NF-kappaB nuclear translocation and DNA-binding. Inhibition of both routes through selected molecules (SB203580, quercetin, artemisinin, parthenolide) prevented HZ-dependent lysozyme release. These data suggest that HZ-triggered overproduction of TNFalpha, IL-1 beta and MIP-1 alpha mediates induction of lysozyme release from human monocytes through activation of p38 MAPK and NF-kappaB pathways, providing new evidence on mechanisms underlying the HZ-enhanced monocyte degranulation in falciparum malaria and the potential role for lysozyme as a new affordable marker in severe malaria.

Characterization of human-type monoclonal antibodies against reduced form of hemin binding protein 35 from Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: The gram-negative anaerobe Porphyromonas gingivalis has been implicated as an important pathogen in the development of adult periodontitis, and its colonization of subgingival sites is critical in the pathogenic process. We previously identified a 35 kDa surface protein (hemin binding protein 35; HBP35) from P. gingivalis that exhibited coaggregation activity, while additional analysis suggested that this protein possessed an ability to bind heme molecules. For development of passive immunotherapy for periodontal diseases, human-type monoclonal antibodies have been prepared using HBP35 as an antigen in TransChromo mice. In the present study, we focused on a single antibody, TCmAb-h13, which is known to inhibit heme binding to recombinant HBP35. The aim of our investigation was to clarify the redox-related function of HBP35 and consider the benefits of human-type monoclonal antibodies. MATERIAL AND METHODS: To examine the antigen recognition capability of TCmAbs with immunoblotting and Biacore techniques, we used the native form as well as several Cys-to-Ser variants of recombinant HBP35. RESULTS: We found that the redox state of recombinant HBP35 was dependent on two Cys residues, (48) C and (51) C, in the thioredoxin active center (WCGxCx). Furthermore, TCmAb-h13 recognized the reduced forms of recombinant HBP35, indicating its inhibitory effect on P. gingivalis growth. CONCLUSION: Hemin binding protein 35 appears to be an important molecule involved in recognition of the redox state of environmental conditions. In addition, TCmAb-h13 had an inhibitory effect on heme binding to recombinant HBP35, thereby interfering with P. gingivalis growth.

Blood stage Plasmodium falciparum antigens induce T cell independent immunoglobulin production via B cell activation factor of the TNF family (BAFF) pathway.

T independent (TI) antigens (Ags) activate monocytes to produce a cytokine, termed B cell activation factor (BAFF), involved in immunoglobulin (Ig) production. This study aimed to investigate whether the soluble schizont fraction of Plasmodium falciparum antigen (sPfAg) and hemozoin (HZ) could act as TI Ag to induce P. falciparum (Pf) specific Ig production via BAFF pathway. Co-cultures of monocytes and naïve B cells from 6 healthy donors were stimulated with sPfAg (10mg/ml) or HZ (10µM). At interval times, the expressions of BAFF on activated monocytes, BAFF receptor (BAFF-R) and proliferation nuclear Ag in activated B cells were determined by flow cytometry. The soluble BAFF (sBAFF), total and specific IgG levels in the supernatants were assessed by enzyme-linked immunosorbent assay (ELISA). The finding revealed both sPfAg and HZ could activate monocytes to express BAFF on surface and release sBAFF in the supernatant within 72h of stimulation. The B cells responded to specific activation, indicated by BAFF-R expression on the surface within 72h, marked proliferation on day 7, and final production of total and specific IgG during days 7-12. Comparing to sPfAg, HZ stimulated monocyte and B cell co-culture to express higher levels of BAFF and sBAFF during 24-48h, more BAFF-R on HZ activated B cells within 24h and induced marked proliferation of B cells with higher Pf specific IgG level. However, stimulation with sPfAg showed a more significant correlation between BAFF expression on the activated monocytes at 72h and the Pf specific IgG level on day 12 (r=0.961, p=0.039, Pearson Correlation). In conclusion, it is possible that both sPfAg and HZ stimulated B cells to produce specific IgG with BAFF involvement.

The IL-12p70/IL-10 interplay is differentially regulated by free heme and hemozoin in murine bone-marrow-derived macrophages.

The outcome of malarial anemia is determined by a complex interplay between pro-inflammatory and anti-inflammatory cytokines, its severity associated with accumulation of hemozoin (Hz) in macrophages, elevated IL-10 responses and impaired IL-12 production. Although free heme contributes to malarial anemia by inducing oxidative damage of red blood cells (RBCs) and enhancing their clearance by phagocytes, its impact on IL-12/IL-10 interactions has not been fully characterized. Herein, the effect of hemin (HE) on IL-12 and IL-10 responses was studied in murine bone marrow-derived macrophages (BMDM) and compared with synthetic Hz. Our data reveal that HE induces modest inhibition of IL-12p70 responses to lipopolysaccharide (LPS) whereas Hz significantly impairs IL-12p70 responses to IFNgamma/LPS through down-regulation of IL-12p35 and p40 gene expression. Although reactive oxygen species (ROS) are generated after short-term exposure to HE and Hz, prolonged exposure to these iron protoporphyrins has opposite effects on the cellular redox status, HE being the only compound able to promote persistent ROS production. Accordingly, the inhibitory effect of HE on IL-12p70 seems sustained by redox-dependent induction of IL-10 and is partially controlled by the p38 mitogen-activated protein kinase (MAPK) signalling pathway. Indeed, treatment with n-acetylcysteine (NAC) or with the p38 MAPK inhibitor SB203580 inhibits IL-10 responses and significantly restores IL-12p70 responses to IFNgamma/LPS in HE-conditioned BMDM. Our results suggest that oxidant stress induced by free heme may potentially contribute to sustained production of IL-10 and down-regulation of IL-12 responses in malaria.

Hemozoin from Schistosoma japonicum does not affect murine myeloid dendritic cell function.

Hemozoin (Hz) formation is a byproduct of hemoglobin digestion in some hematophagous organisms. Although Hz produced by Plasmodium falciparum (PfHz) has been shown to affect development and activities of human dendritic cells (DCs), the effects of Schistosoma Hz on DCs have not been elucidated. Our data presented in this report demonstrated that native Schistosoma japonica Hz (SjHz) did not affect the differentiation of murine bone marrow cells into immature DCs (imDCs). Maturation and stimulatory activities to T cells by imDCs induced by LPS were not altered in the presence of SjHz; whereas purified PfHz induced a slight increase in CD40 expression and enhanced IL-12p40 secretion. Lastly, SjHz treatment did not significantly affect the phagocytic activities of DCs. These data suggested that SjHz failed to exert any significant effects on the development and activities of murine myeloid DCs. The mechanisms of different effects on DCs by SjHz and PfHz remain to be elucidated.

Ingestion of the malaria pigment hemozoin renders human macrophages less permissive to HIV-1 infection.

Few studies have investigated the pathophysiologic mechanisms responsible for what seems to be a possible interaction between Plasmodium falciparum, the causative agent of malaria, and HIV-1 in dually infected patients. It has been shown that Plasmodium parasites detoxify heme molecules into a pigment called hemozoin (HZ), which can significantly modulate the immune system. The primary objective of this study was to determine whether exposure of human primary monocyte-derived macrophages (MDMs) to the malaria pigment influences the process of HIV-1 infection. We report here that HIV-1 replication is significantly diminished in HZ-loaded MDMs. The HZ-mediated reduction in virus replication is due to a block at a step in the virus life cycle occurring between the completion of full-length reverse transcripts and integration of viral DNA within the host chromosome. Understanding the pathological mechanisms involved in P. falciparum and HIV-1 co-infection is of high importance because of possible therapeutic ramifications.

Pure Hemozoin is inflammatory in vivo and activates the NALP3 inflammasome via release of uric acid.

The role of proinflammatory cytokine production in the pathogenesis of malaria is well established, but the identification of the parasite products that initiate inflammation is not complete. Hemozoin is a crystalline metabolite of hemoglobin digestion that is released during malaria infection. In the present study, we characterized the immunostimulatory activity of pure synthetic hemozoin (sHz) in vitro and in vivo. Stimulation of naive murine macrophages with sHz results in the MyD88-independent activation of NF-kappaB and ERK, as well as the release of the chemokine MCP-1; these responses are augmented by IFN-gamma. In macrophages prestimulated with IFN-gamma, sHz also results in a MyD88-dependent release of TNF-alpha. Endothelial cells, which encounter hemozoin after schizont rupture, respond to sHz by releasing IL-6 and the chemokines MCP-1 and IL-8. In vivo, the introduction of sHz into the peritoneal cavity produces an inflammatory response characterized by neutrophil recruitment and the production of MCP-1, KC, IL-6, IL-1alpha, and IL-1beta. MCP-1 and KC are produced independently of MyD88, TLR2/4 and TLR9, and components of the inflammasome; however, neutrophil recruitment, the localized production of IL-1beta, and the increase in circulating IL-6 require MyD88 signaling, the IL-1R pathway, and the inflammasome components ICE (IL-1beta-converting enzyme), ASC (apoptosis-associated, speck-like protein containing CARD), and NALP3. Of note, inflammasome activation by sHz is reduced by allopurinol, which is an inhibitor of uric acid synthesis. These data suggest that uric acid is released during malaria infection and may serve to augment the initial host response to hemozoin via activation of the NALP3 inflammasome.

Diagnostic potential of an iron-regulated hemin-binding protein HbpA that is widely conserved in Leptospira interrogans.

Like most bacteria, Leptospira spp. requires iron for growth. However, they face conditions of iron limitation due to the low solubility of the ferric iron and additionally due to the observation that most of the available iron is held as protein-bound iron by the mammalian host. Our experimental observations with pathogenic Leptospira showed that they do not elaborate siderophores upon iron limitation. In Leptospira interrogans serovar Lai, we demonstrated direct acquisition of iron via an iron-regulated hemin-binding protein HbpA. PCR analysis and Southern hybridization studies revealed that the hbpA gene was conserved in the serovars belonging to L. interrogans species and was absent in the non-pathogenic Leptospira biflexa serovar Patoc and Leptospira meyeri serovar Ranarum. Here, we extended the PCR-based detection of the hbpA gene to clinical isolates and demonstrated the hbpA amplicon in all the serovars belonging to L. interrogans species from a total collection of 91 clinical isolates obtained from different geographical regions. In addition, we detected anti-HbpA antibodies in the serum of patients with leptospirosis. PCR-based detection of hbpA in clinical isolates of serovars and the in vivo expression of HbpA reflects the diagnostic potential of this antigen.

Induction of nitric oxide synthase and activation of signaling proteins in Anopheles mosquitoes by the malaria pigment, hemozoin.

Anopheles stephensi, a major vector for malaria parasite transmission, responds to Plasmodium infection by synthesis of inflammatory levels of nitric oxide (NO), which can limit parasite development in the midgut. We have previously shown that Plasmodium falciparum glycosylphosphatidylinositols (PfGPIs) can induce A. stephensi NO synthase (AsNOS) expression in the midgut epithelium in vivo in a manner similar to the manner in which cytokines and NO are induced by PfGPIs in mammalian cells. In mosquito cells, signaling by PfGPIs and P. falciparum merozoites is mediated through Akt/protein kinase B (Akt/PKB), the mitogen-activated protein kinase kinase DSOR1, and extracellular signal-regulated kinase (ERK). In mammalian cells, a second parasite factor, malaria pigment or hemozoin (Hz), signals NOS induction through ERK- and nuclear factor kappa B-dependent pathways and has been demonstrated to be a novel proinflammatory ligand for Toll-like receptor 9. In this study, we demonstrate that Hz can also induce AsNOS gene expression in immortalized A. stephensi and Anopheles gambiae cell lines in vitro and in A. stephensi midgut tissue in vivo. In mosquito cells, Hz signaling is mediated through transforming growth factor beta-associated kinase 1, Akt/PKB, ERK, and atypical protein kinase C zeta/lambda. Our results show that Hz is a prominent parasite-derived signal for Anopheles and that signaling pathways activated by PfGPIs and Hz have both unique and shared components. Together with our previous findings, our data indicate that parasite signaling of innate immunity is conserved in mosquito and mammalian cells.

The basis of the immunomodulatory activity of malaria pigment (hemozoin).

The most common and deadly form of the malaria parasite, Plasmodium falciparum, is responsible for 1.5-2.7 million deaths and 300-500 million acute illnesses annually [Bremen in J. Trop. Med. Hyg. 64:1-11 (2001); World Health Organization (2002)]. Hemozoin, the biomineral formed to detoxify the free heme produced during parasitic hemoglobin catabolism, has long been suspected of contributing to the pathological immunodeficiencies that occur during malarial infection. While there is a growing consensus in the literature that native hemozoin maintains immunosuppressive activity, there is considerable controversy over the reactivity of the synthetic form, beta-hematin (BH). Given the emerging importance of hemozoin in modulating a host immune response to malarial infection, a careful examination of the effects of the constitutive components of the malaria pigment on macrophage response has been made in order to clarify the understanding of this process. Herein, we present evidence that BH alone is unable to inhibit stimulation of NADPH oxidase and inducible nitric oxide synthase, the key enzymes involved in oxidative burst, and is sensitive to the microbicidal agents of these enzymes both in vitro and in vivo. Further, by systematically examining each of the malaria pigment's components, we were able to dissect their impact on the immune reactivity of a macrophage model cell line. Reactions between BH and red blood cell (RBC) ghosts effectively reconstituted the observed immunomodulatory reactivity of native hemozoin. Together, these results suggest that the interaction between hemozoin and the RBC lipids results in the generation of toxic products and that these products are responsible for disrupting macrophage function in vivo.

A critical role for the host mediator macrophage migration inhibitory factor in the pathogenesis of malarial anemia.

The pathogenesis of malarial anemia is multifactorial, and the mechanisms responsible for its high mortality are poorly understood. Studies indicate that host mediators produced during malaria infection may suppress erythroid progenitor development (Miller, K.L., J.C. Schooley, K.L. Smith, B. Kullgren, L.J. Mahlmann, and P.H. Silverman. 1989. Exp. Hematol. 17:379-385; Yap, G.S., and M.M. Stevenson. 1991. Ann. NY Acad. Sci. 628:279-281). We describe an intrinsic role for macrophage migration inhibitory factor (MIF) in the development of the anemic complications and bone marrow suppression that are associated with malaria infection. At concentrations found in the circulation of malaria-infected patients, MIF suppressed erythropoietin-dependent erythroid colony formation. MIF synergized with tumor necrosis factor and gamma interferon, which are known antagonists of hematopoiesis, even when these cytokines were present in subinhibitory concentrations. MIF inhibited erythroid differentiation and hemoglobin production, and it antagonized the pattern of mitogen-activated protein kinase phosphorylation that normally occurs during erythroid progenitor differentiation. Infection of MIF knockout mice with Plasmodium chabaudi resulted in less severe anemia, improved erythroid progenitor development, and increased survival compared with wild-type controls. We also found that human mononuclear cells carrying highly expressed MIF alleles produced more MIF when stimulated with the malarial product hemozoin compared with cells carrying low expression MIF alleles. These data suggest that polymorphisms at the MIF locus may influence the levels of MIF produced in the innate response to malaria infection and the likelihood of anemic complications.

Phagocytosis of hemozoin enhances matrix metalloproteinase-9 activity and TNF-alpha production in human monocytes: role of matrix metalloproteinases in the pathogenesis of falciparum malaria.

Matrix metalloproteinase-9 (MMP-9), secreted by activated monocytes, degrades matrix proteins, disrupts basal lamina, and activates TNF-alpha from its precursors. In turn, TNF-alpha enhances synthesis of MMP-9 in monocytes. We show here that trophozoite-parasitized RBCs/hemozoin-fed adherent human monocytes displayed increased MMP-9 activity and protein/mRNA expression, produced TNF-alpha time-dependently, and showed higher matrix invasion ability. MMP-9 activation was specific for trophozoite/hemozoin-fed monocytes, was dependent on TNF-alpha production, and abrogated by anti-TNF-alpha Ab and by a specific inhibitor of MMP-9/MMP-13 activity. Hemozoin-induced enhancement of MMP-9 and TNF-alpha production would have a 2-fold effect: to start and feed a cyclic reinforcement loop in which hemozoin enhances production of TNF-alpha, which in turn induces both activation of MMP-9 and shedding of TNF-alpha into the extracellular compartment; and, second, to disrupt the basal lamina of endothelia. Excess production of TNF-alpha and disruption of the basal lamina with extravasation of blood cells into perivascular tissues are hallmarks of severe malaria. Pharmacological inhibition of MMP-9 may offer a new chance to control pathogenic mechanisms in malaria.