Depletion of Blood-Borne Macrophages Does Not Reduce Demyelination in Mice Infected with a Neurotropic Coronavirus

Mice infected with the neurotropic coronavirus mouse hepatitis virus strain JHM (MHV-JHM) develop a chronic demyelinating disease with symptoms of hindlimb paralysis. Histological examination of the brains and spinal cords of these animals reveals the presence of large numbers of activated macrophages/microglia. In two other experimental models of demyelination, experimental allergic encephalomyelitis and Theiler's murine encephalomyelitis virus-induced demyelination, depletion of hematogenous macrophages abrogates the demyelinating process. In both of these diseases, early events in the demyelinating process are inhibited by macrophage depletion. From these studies, it was not possible to determine whether infiltrating macrophages were required for late steps in the process, such as myelin removal. In this study, we show that when macrophages are depleted with either unmodified or mannosylated liposomes encapsulating dichloromethylene diphosphate, the amount of demyelination detected in MHV-infected mice is not affected. At a time when these cells were completely depleted from the liver, approximately equivalent numbers of macrophages were present in the spinal cords of control and drug-treated animals. These results suggest that blood-borne macrophages are not required for MHV-induced demyelination and also suggest that other cells, such as perivascular macrophages or microglia, perform the function of these cells in the presence of drug.     

SIV Vpx Is Essential for Macrophage Infection but Not for Development of AIDS

Analysis of rhesus macaques infected with a vpx deletion mutant virus of simian immunodeficiency virus mac239 (SIVΔvpx) demonstrates that Vpx is essential for efficient monocyte/macrophage infection in vivo but is not necessary for development of AIDS. To compare myeloid-lineage cell infection in monkeys infected with SIVΔvpx compared to SIVmac239, we analyzed lymphoid and gastrointestinal tissues from SIVΔvpx-infected rhesus (n = 5), SIVmac239-infected rhesus with SIV encephalitis (7 SIV239E), those without encephalitis (4 SIV239noE), and other SIV mutant viruses with low viral loads (4 SIVΔnef, 2 SIVΔ3). SIV+ macrophages and the percentage of total SIV+ cells that were macrophages in spleen and lymph nodes were significantly lower in rhesus infected with SIVΔvpx (2.2%) compared to those infected with SIV239E (22.7%), SIV239noE (8.2%), and SIV mutant viruses (10.1%). In colon, SIVΔvpx monkeys had fewer SIV+ cells, no SIV+ macrophages, and lower percentage of SIV+ cells that were macrophages than the other 3 groups. Only 2 SIVΔvpx monkeys exhibited detectable virus in the colon. We demonstrate that Vpx is essential for efficient macrophage infection in vivo and that simian AIDS and death can occur in the absence of detectable macrophage infection.     

Priming of CD8+ T Cells during Central Nervous System Infection with a Murine Coronavirus Is Strain Dependent

Virus-specific CD8⁺ T cells are critical for protection against neurotropic coronaviruses; however, central nervous system (CNS) infection with the recombinant JHM (RJHM) strain of mouse hepatitis virus (MHV) elicits a weak CD8⁺ T-cell response in the brain and causes lethal encephalomyelitis. An adoptive transfer model was used to elucidate the kinetics of CD8⁺ T-cell priming during CNS infection with RJHM as well as with two MHV strains that induce a robust CD8⁺ T-cell response (RA59 and SJHM/RA59, a recombinant A59 virus expressing the JHM spike). While RA59 and SJHM/RA59 infections resulted in CD8⁺ T-cell priming within the first 2 days postinfection, RJHM infection did not lead to proliferation of naïve CD8⁺ T cells. While all three viruses replicated efficiently in the brain, only RA59 and SJHM/RA59 replicated to appreciable levels in the cervical lymph nodes (CLN), the site of T-cell priming during acute CNS infection. RJHM was unable to suppress the CD8⁺ T-cell response elicited by RA59 in mice simultaneously infected with both strains, suggesting that RJHM does not cause generalized immunosuppression. RJHM was also unable to elicit a secondary CD8⁺ T-cell response in the brain following peripheral immunization against a viral epitope. Notably, the weak CD8⁺ T-cell response elicited by RJHM was unique to CNS infection, since peripheral inoculation induced a robust CD8⁺ T-cell response in the spleen. These findings suggest that the failure of RJHM to prime a robust CD8⁺ T-cell response during CNS infection is likely due to its failure to replicate in the CLN.Members of the family Coronaviridae infect a wide range of mammalian species, including humans, and induce mild to severe disease of the respiratory tract, gastrointestinal tract, and central nervous system (CNS). Mouse hepatitis virus (MHV) infection provides a useful model for the study of acute and chronic CNS disease and specifically the process of demyelination, the hallmark of the human disease multiple sclerosis. Different strains of MHV induce disease with various degrees of severity. For example, CNS infection with the recombinant wild-type A59 (RA59) strain causes acute encephalitis during the first week of infection; a strong CD8⁺ T-cell response is observed in the brain, coinciding with viral clearance. However, despite clearance of infectious RA59 virus, demyelination develops, peaking at approximately 4 weeks postinfection (p.i.) (17, 20). In contrast, infection with the recombinant wild-type JHM (RJHM) strain (derived from the JHM isolate referred to as MHV-4 or JHM.SD [7, 28]) causes severe encephalomyelitis; the virus is not cleared, and mice typically succumb to disease by the end of the first week of infection. Furthermore, RJHM infection of the CNS elicits a very weak virus-specific CD8⁺ T-cell response in the brain (7, 20, 34). However, we have examined only the most virulent strain of JHM. It should be noted that there are other strains of JHM that have deletions and mutations within the spike glycoprotein, rendering them less virulent and sometimes resulting in a change in cell tropism. The ability of neurotropic strains of MHV to replicate within cells of the CNS and cause disease of various degrees is ideal for allowing the dissection of both viral and host determinants of neuropathogenesis.The spike glycoprotein of MHV is a major determinant of neurovirulence (32). It controls virus tropism and spread as it both binds the cellular receptor and induces fusion with target cells. In addition, it encodes neutralizing antibody epitopes and the H-2^b-restricted CD8⁺ T-cell epitopes recognized in C57BL/6 (B6) mice. The A59 spike differs from the JHM spike in that it contains a deletion of 52 amino acids within the hypervariable region. The hypervariable region has been well documented to tolerate mutation, but with attenuating effects on virulence (5, 7). RA59 and RJHM both encode an H-2K^b epitope at positions S598 to S605 (S598); however, due to the deletion, the A59 spike lacks the immunodominant H-2D^b epitope at positions S510 to S519 (S510). We previously selected isogenic recombinant viruses expressing the JHM spike in which all other genes are derived from the A59 strain of MHV (SJHM/RA59). The isogenic SJHM/RA59 virus has a 50% lethal dose (LD₅₀) similar to that of RJHM, demonstrating that the JHM spike is sufficient to generate a highly neurovirulent phenotype and an increased ability to spread within the CNS (32, 33). However, SJHM/RA59-infected mice exhibit slower kinetics of death than RJHM-infected mice, and notably, unlike RJHM, the chimeric SJHM/RA59 virus induces a strong CD8⁺ T-cell response in the brain (14, 34).In addition to the spike, there is increasing evidence that other viral genes play important roles in pathogenesis. We (14, 21) and others (34, 35) have noted that the low CD8⁺ T-cell response observed during RJHM infection is not dependent on the spike, since the SJHM/RA59 recombinant induces a robust virus-specific CD8⁺ T-cell response. The difference between the CD8⁺ T-cell responses elicited by SJHM/RA59 and RJHM may explain why SJHM/RA59 kills mice more slowly than RJHM. Furthermore, the reverse chimeric recombinant virus expressing the A59 spike in the JHM background (SA59/RJHM) is unable to replicate in the liver despite the fact that it expresses the spike from the hepatotropic RA59 strain (27), suggesting that background genes play a significant role in viral tropism.It is well established that virus-specific CD8⁺ T cells play a protective role against MHV and are essential for clearance of infectious virus from the CNS (6, 20, 40, 41). The effector mechanisms exerted by activated, virus-specific CD8⁺ T cells include the ability to secrete cytokines and the ability to lyse target cells. Gamma interferon (IFN-γ) expression is essential for clearance of MHV from the brain (3, 22, 29), and perforin-mediated lysis of infected cells also appears to play a role in viral clearance (6, 31). In contrast to infection with RA59 or the relatively neuroattenuated glial-cell-tropic strains of JHM, CNS infection with the highly neurovirulent RJHM strain results in very low levels of activated, virus-specific CD8⁺ T cells in the spleen and brain (14, 34). Furthermore, RJHM infection induces a different profile of cytokines and chemokines in the brains of infected mice than infection with RA59 (34, 35, 38). One dramatic difference is that RA59 infection results in a robust IFN-γ response whereas RJHM infection results in higher, sustained levels of IFN-β (34). These observations prompted us to address the following questions. (i) Does RJHM elicit a CD8⁺ T-cell response in the brain following intranasal (i.n.) inoculation, a route that requires more virus and results in slower infection than intracranial (i.c.) inoculation? (ii) What are the kinetics of CD8⁺ T-cell priming during CNS infections with RA59, SJHM/RA59, and RJHM? (iii) Is CNS infection with RJHM generally immunosuppressive? (iv) Do RA59, SJHM/RA59, and RJHM replicate efficiently in the draining cervical lymph nodes (CLN)? (v) Can RJHM elicit a secondary CD8⁺ T-cell response in the brain following peripheral immunization against a viral epitope? (vi) Is the low CD8⁺ T-cell response elicited during RJHM infection an inherent characteristic of the viral strain or specific to RJHM infection of the CNS? Our results suggest that RJHM fails to prime a CD8⁺ T-cell response specifically during infection of the CNS without causing generalized immunosuppression and that this lack of priming correlates with a low level of RJHM replication in the draining CLN, the site of CD8⁺ T-cell priming during acute CNS infection.     

Glycerol Monomycolate Is a Novel Ligand for the Human,but Not Mouse Macrophage Inducible C-type Lectin,Mincle

An array of lipidic compounds that constitute the cell wall of mycobacteria is recognized by host receptors. Examples include trehalose dimycolate (TDM), which is a major surface-exposed glycolipid of mycobacteria, that interacts with the macrophage inducible C-type lectin, Mincle, and exerts its highly potent adjuvant functions. Recent evidence has suggested that glycerol monomycolate (GroMM), another mycolate-containing lipid species produced by mycobacteria, can stimulate innate immune cells; however, its specific host receptors have yet to be identified. We here demonstrated that cell transfectants expressing human Mincle (hMincle) reacted to both TDM and GroMM, while those expressing mouse Mincle (mMincle) only reacted to TDM and failed to recognize GroMM. Studies using domain swap chimeras confirmed that the ectodomain of hMincle, but not that of mMincle, interacted with GroMM, and site-directed mutagenesis analyses revealed that short stretches of amino acid residues at positions 174–176 and 195–196 were involved in GroMM recognition. To further substantiate the differential recognition of GroMM by hMincle and mMincle, hMincle transgenic/mMincle knock-out mice (i.e. hMincle⁺ mice) were established and compared with non-transgenic mice (i.e. mMincle⁺ mice). We showed that macrophages derived from hMincle⁺ mice were activated by GroMM and produced inflammatory cytokines, whereas those derived from mMincle⁺ mice did not exhibit any reactivity to GroMM. Furthermore, local inflammatory responses were elicited in the GroMM-injected skin of hMincle⁺, but not mMincle⁺ mice. These results demonstrated that GroMM is a unique ligand for hMincle that is not recognized by mMincle.     

Mouse Dipeptidyl Peptidase 4 Is Not a Functional Receptor for Middle East Respiratory Syndrome Coronavirus Infection

Human dipeptidyl peptidase 4 (hDPP4) was recently identified as the receptor for Middle East respiratory syndrome coronavirus (MERS-CoV) infection, suggesting that other mammalian DPP4 orthologs may also support infection. We demonstrate that mouse DPP4 cannot support MERS-CoV infection. However, employing mouse DPP4 as a scaffold, we identified two critical amino acids (A288L and T330R) that regulate species specificity in the mouse. This knowledge can support the rational design of a mouse-adapted MERS-CoV for rapid assessment of therapeutics.     

Non-Antigen-Specific B-Cell Activation following Murine Gammaherpesvirus Infection Is CD4 Independent In Vitro but CD4 Dependent In Vivo

The murine gammaherpesvirus MHV-68 multiplies in the respiratory epithelium after intranasal inoculation, then spreads to infect B cells in lymphoid germinal centers. Exposing B cells to MHV-68 in vitro caused an increase in cell size, up-regulation of the CD69 activation marker, and immunoglobulin M (IgM) production. The infectious process in vivo was also associated with increased CD69 expression on B cells in the draining lymph nodes and spleen, together with a rise in total serum Ig. However, whereas the in vitro effect on B cells was entirely T-cell independent, evidence of in vivo B-cell activation was minimal in CD4⁺ T-cell-deficient (I-A^b−/−) or CD4⁺ T-cell-depleted mice. Furthermore, the Ig present at high levels in serum was predominantly of the IgG class. Surprisingly, the titer of influenza virus-specific serum IgG in previously immunized mice fell following MHV-68 infection, suggesting that there was relatively little activation of memory B cells. Thus, CD4⁺ T cells seemed both to amplify a direct viral activation of B cells in lymphoid tissue and to promote new Ig class switching despite a lack of obvious cognate antigen.Herpesvirus (HV) infections are often associated with non-antigen-specific B-cell activation (13, 14, 16, 21, 22). Although no definite role has been established for this process in viral pathogenesis, it is of particular interest in gammaherpesvirus (γ-HV) infections, since chronic B-cell stimulation may contribute to the oncogenesis (9, 15) associated with Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV-8) infections. Infection with EBV activates B cells expressing the immunoglobulin (Ig) V_4-34 gene (4), which is also overrepresented in certain lymphomas (6, 25). EBV-activated V_4-34-expressing B cells can undergo somatic mutation and isotype switching, indicating a participation in normal germinal-center interactions (5). The latent membrane protein 1 (LMP-1) of EBV, which has intracellular signaling substrates similar to those of CD40 (12), and LMP-2A, which can trigger lymphocyte activation (2), may both contribute to this process. However, analysis of lymphocyte interactions in vivo has not been possible with the human γ-HVs.The murine γ-HV-68 (MHV-68) is a natural γ-HV of small rodents that is related to EBV (8) and to HHV-8 (33). After intranasal (i.n.) infection of conventional mice, the virus spreads from the lung to the lymphoid tissue (29) and then persists in B lymphocytes (28) and in epithelial cells (27). This persistent infection is associated with an infectious mononucleosis-like illness (7, 20) characterized by a CD4-dependent splenomegaly and an increase in viral load (31). In BALB/c mice, MHV-68 causes an acute and apparently non-antigen-specific rise in total serum IgG (26). The virus-specific serum antibody response is, in contrast, relatively slow in onset and does not reach plateau levels until 2 to 3 months after infection (26). MHV-68-infected C57BL/6J (B6) mice have more IgG⁺ cells and fewer IgM⁺ cells in the spleen (18) than uninfected controls, but to what extent this represents normal immunity is unclear.There is evidence (3) of MHV-68 infection in splenic germinal centers, and both the non-antigen-specific B-cell activation and the CD4-dependent increase in viral load may reflect an exploitation by the virus of normal germinal-center function. The present analysis defines the need, or lack thereof, for CD4⁺ T-cell help to drive B-cell activation following in vitro or in vivo exposure to MHV-68.     

Anti-CD45 Radioimmunotherapy with 90Y but Not 177Lu Is Effective Treatment in a Syngeneic Murine Leukemia Model

Radioimmunotherapy (RIT) for treatment of hematologic malignancies has primarily employed monoclonal antibodies (Ab) labeled with ¹³¹I or ⁹⁰Y which have limitations, and alternative radionuclides are needed to facilitate wider adoption of RIT. We therefore compared the relative therapeutic efficacy and toxicity of anti-CD45 RIT employing ⁹⁰Y and ¹⁷⁷Lu in a syngeneic, disseminated murine myeloid leukemia (B6SJLF1/J) model. Biodistribution studies showed that both ⁹⁰Y- and ¹⁷⁷Lu-anti-murine CD45 Ab conjugates (DOTA-30F11) targeted hematologic tissues, as at 24 hours 48.8±21.2 and 156±14.6% injected dose per gram of tissue (% ID/g) of ⁹⁰Y-DOTA-30F11 and 54.2±9.5 and 199±11.7% ID/g of ¹⁷⁷Lu-DOTA-30F11 accumulated in bone marrow (BM) and spleen, respectively. However, ⁹⁰Y-DOTA-30F11 RIT demonstrated a dose-dependent survival benefit: 60% of mice treated with 300 µCi ⁹⁰Y-DOTA-30F11 lived over 180 days after therapy, and mice treated with 100 µCi ⁹⁰Y-DOTA-30F11 had a median survival 66 days. ⁹⁰Y-anti-CD45 RIT was associated with transient, mild myelotoxicity without hepatic or renal toxicity. Conversely, ¹⁷⁷Lu- anti-CD45 RIT yielded no long-term survivors. Thus, ⁹⁰Y was more effective than ¹⁷⁷Lu for anti-CD45 RIT of AML in this murine leukemia model.     

Infection with Cytotoxic T-Lymphocyte Escape Mutants Results in Increased Mortality and Growth Retardation in Mice Infected with a Neurotropic Coronavirus

C57BL/6 mice infected with mouse hepatitis virus strain JHM (MHV-JHM) develop a chronic demyelinating encephalomyelitis several weeks after inoculation. Previously, we showed that mutations in the immunodominant CD8 T-cell epitope (S-510-518) could be detected in nearly all samples of RNA and virus isolated from these mice. These mutations abrogated recognition by T cells harvested from the central nervous systems of infected mice in direct ex vivo cytotoxicity assays. These results suggested that cytotoxic T-lymphocyte (CTL) escape mutants contributed to virus amplification and the development of clinical disease in mice infected with wild-type virus. In the present study, the importance of these mutations was further evaluated by infecting naive mice with MHV-JHM variants isolated from infected mice and in which epitope S-510-518 was mutated. Compared to mice infected with wild-type virus, variant virus-infected animals showed higher mortality and morbidity manifested by decreased weight gain and neurological signs. Although a delay in the kinetics of virus clearance has been demonstrated in previous studies of CTL escape mutants, this is the first illustration of significant changes in clinical disease resulting from infection with viruses able to evade the CD8 T-cell immune response.     

Endogenous Murine BST-2/Tetherin Is Not a Major Restriction Factor of Influenza A Virus Infection

BST-2 (tetherin, CD317, HM1.24) restricts virus growth by tethering enveloped viruses to the cell surface. The role of BST-2 during influenza A virus infection (IAV) is controversial. Here, we assessed the capacity of endogenous BST-2 to restrict IAV in primary murine cells. IAV infection increased BST-2 surface expression by primary macrophages, but not alveolar epithelial cells (AEC). BST-2-deficient AEC and macrophages displayed no difference in susceptibility to IAV infection relative to wild type cells. Furthermore, BST-2 played little role in infectious IAV release from either AEC or macrophages. To examine BST-2 during IAV infection in vivo, we infected BST-2-deficient mice. No difference in weight loss or in viral loads in the lungs and/or nasal tissues were detected between BST-2-deficient and wild type animals. This study rules out a major role for endogenous BST-2 in modulating IAV in the mouse model of infection.     

Retrotransposition of Long Interspersed Nucleotide Element-1 Is Associated with Colitis but Not Tumors in a Murine Colitic Cancer Model

Long interspersed element-1 (L1) is a transposable element that can move within the genome, potentially leading to genome diversity and modified gene function. Although L1 activity in somatic cells is normally suppressed through DNA methylation, some L1s are activated in tumors including colorectal carcinoma. However, how L1-retrotransposition (L1-RTP) is involved in gastrointestinal disorders remains to be elucidated. We hypothesized that L1-RTP in somatic cells might contribute to colitis-associated cancer (CAC). To address this, we employed an experimental model of CAC using transgenic L1-reporter mice carrying a human L1-EGFP reporter gene. Mice were subjected to repeated cycles of colitis induced by administration of dextran sodium sulfate (DSS) in drinking water with injection of carcinogen azoxymethane (AOM). L1-RTP levels were measured by a quantitative polymerase chain reaction targeting the newly inserted reporter EGFP in various tissues and cell types, including samples obtained by laser microdissection and cell sorting with flow cytometry. DNA methylation levels of the human L1 promoter were analyzed by bisulfite pyrosequencing. AOM+DSS-treated mice exhibited significantly higher levels of L1-RTP in whole colon tissue during the acute phase of colitis when compared with control naïve mice. L1-RTP levels in whole colon tissue were positively correlated with the histological severity of colitis and degree of neutrophil infiltration into the lamina propria (LP), but not with tumor development in the colon. L1-RTP was enriched in LP mesenchymal cells rather than epithelial cells (ECs), myeloid, or lymphoid cells. DNA methylation levels of the human L1 promoter region showed a negative correlation with L1-RTP levels. L1-RTP was absent from most tumors found in 22-week-old mice. In conclusion, we demonstrated that L1-RTP was induced in the mouse CAC mucosa in accordance with the acute inflammatory response; however, retrotransposition appears not to have direct relevance to colitis-induced cancer initiation.     

The Number of Cultural Traits Is Correlated with Female Group Size but Not with Male Group Size in Chimpanzee Communities

What determines the number of cultural traits present in chimpanzee (Pan troglodytes) communities is poorly understood. In humans, theoretical models suggest that the frequency of cultural traits can be predicted by population size. In chimpanzees, however, females seem to have a particularly important role as cultural carriers. Female chimpanzees use tools more frequently than males. They also spend more time with their young, skewing the infants'' potential for social learning towards their mothers. In Gombe, termite fishing has been shown to be transmitted from mother to offspring. Lastly, it is female chimpanzees that transfer between communities and thus have the possibility of bringing in novel cultural traits from other communities. From these observations we predicted that females are more important cultural carriers than males. Here we show that the reported number of cultural traits in chimpanzee communities correlates with the number of females in chimpanzee communities, but not with the number of males. Hence, our results suggest that females are the carriers of chimpanzee culture.     

Mental Rotational Ability Is Correlated with Spatial but Not Verbal Working Memory Performance and P300 Amplitude in Males

This study investigated how both sex and individual differences in a mental rotation test (MRT) influence performance on working memory (WM). To identify the neural substrate supporting these differences, brain electrical activity was measured using the event-related potential technique. No significant sex differences were observed in a test of verbal WM, however males were significantly faster than females to respond to probe stimuli in a test of spatial WM. This difference was no longer significant after controlling for differences in MRT score, suggesting that rotational ability mediates performance in the spatial memory task for both sexes. A posterior P300 was observed in both tasks as participants encoded information into memory, however the amplitude of the P300 correlated with RT in the spatial task but not in the verbal task. Individual differences in the MRT also correlated with RT and with the amplitude of the P300, but again only in the spatial task. After splitting the analysis by sex, partial correlations controlling for MRT revealed that for males, individual differences in rotational ability completely mediated the correlation between the P300 and RT in the spatial task. This mediating effect was not observed for the female participants. The results therefore suggest a relatively stronger association in males between innate mental rotational ability, spatial memory performance, and brain electrophysiological processes supporting spatial memory.     

Infectivity of Dengue Virus Serotypes 1 and 2 Is Correlated with E-Protein Intrinsic Dynamics but Not to Envelope Conformations

1. Download high-res image (256KB)
2. Download full-size image

     

Evidence that TLR4 Is Not a Receptor for Saturated Fatty Acids but Mediates Lipid-Induced Inflammation by Reprogramming Macrophage Metabolism

1. Download high-res image (223KB)
2. Download full-size image

     

Distinct Macrophage Fates after in vitro Infection with Different Species of Leishmania: Induction of Apoptosis by Leishmania (Leishmania) amazonensis,but Not by Leishmania (Viannia) guyanensis

Leishmania is an intracellular parasite in vertebrate hosts, including man. During infection, amastigotes replicate inside macrophages and are transmitted to healthy cells, leading to amplification of the infection. Although transfer of amastigotes from infected to healthy cells is a crucial step that may shape the outcome of the infection, it is not fully understood. Here we compare L. amazonensis and L. guyanensis infection in C57BL/6 and BALB/c mice and investigate the fate of macrophages when infected with these species of Leishmania in vitro. As previously shown, infection of mice results in distinct outcomes: L. amazonensis causes a chronic infection in both strains of mice (although milder in C57BL/6), whereas L. guyanensis does not cause them disease. In vitro, infection is persistent in L. amazonensis-infected macrophages whereas L. guyanensis growth is controlled by host cells from both strains of mice. We demonstrate that, in vitro, L. amazonensis induces apoptosis of both C57BL/6 and BALB/c macrophages, characterized by PS exposure, DNA cleavage into nucleosomal size fragments, and consequent hypodiploidy. None of these signs were seen in macrophages infected with L. guyanensis, which seem to die through necrosis, as indicated by increased PI-, but not Annexin V-, positive cells. L. amazonensis-induced macrophage apoptosis was associated to activation of caspases-3, -8 and -9 in both strains of mice. Considering these two species of Leishmania and strains of mice, macrophage apoptosis, induced at the initial moments of infection, correlates with chronic infection, regardless of its severity. We present evidence suggestive that macrophages phagocytize L. amazonensis-infected cells, which has not been verified so far. The ingestion of apoptotic infected macrophages by healthy macrophages could be a way of amastigote spreading, leading to the establishment of infection.