Does T Helper Differentiation Correlate with Resistance or Susceptibility to Infection with L. major? Some Insights From the Murine Model

The murine model of Leishmania major infection has been an invaluable tool in understanding T helper differentiation in vivo. The initial evidence for a role of distinct CD4+ T helper subsets in the outcome of infection was first obtained with this experimental model. The development of CD4+ Th1 cells was associated with resolution of the lesion, control of parasite replication, and resistance to re-infection in most of the mouse strains investigated (i.e., C57BL/6). In contrast, differentiation of CD4+ Th2 cells correlated with the development of unhealing lesions, and failure to control parasite load in a few strains (i.e., BALB/c). Since these first reports, an incredible amount of effort has been devoted to understanding the various parameters involved in the differentiation of these, and more recently discovered T helper subsets such as Th17 and T regulatory cells. The discovery of cross-talk between T helper subsets, as well as their plasticity force us to reevaluate the events driving a protective/deleterious T helper immune response following infection with L. major in mice. In this review, we describe the individual contributions of each of these CD4+ T helper subsets following L. major inoculation, emphasizing recent advances in the field, such as the impact of different substrains of L. major on the pathogenesis of disease

The prominent role of neutrophils during the initial phase of infection by Leishmania parasites.

Neutrophils are rapidly and massively recruited to the site of Leishmania inoculation, where they phagocytose the parasites, some of which are able to survive within these first host cells. Neutrophils can thus provide a transient safe shelter for the parasites, prior to their entry into macrophages where they will replicate. In addition, neutrophils release and synthesize rapidly several factors including cytokines and chemokines. The mechanism involved in their rapid recruitment to the site of parasite inoculation, as well as the putative consequences of their massive presence on the microenvironment of the focus of infection will be discussed in the context of the development of the Leishmania-specific immune response

Survival Mechanisms Used by Some Leishmania Species to Escape Neutrophil Killing

Neutrophils are the most abundant leukocytes in human blood. Upon microbial infection, they are massively and rapidly recruited from the circulation to sites of infection where they efficiently kill pathogens. To this end, neutrophils possess a variety of weapons that can be mobilized and become effective within hours following infection. However, several microbes including some Leishmania spp. have evolved a variety of mechanisms to escape neutrophil killing using these cells as a basis to better invade the host. In addition, neutrophils are also present in unhealing cutaneous lesions where their role remains to be defined. Here, we will review recent progress in the field and discuss the different strategies applied by some Leishmania parasites to escape from being killed by neutrophils and as recently described for Leishmania mexicana, even replicate within these cells. Subversion of neutrophil killing functions by Leishmania is a strategy that allows parasite spreading in the host with a consequent deleterious impact, transforming the primary protective role of neutrophils into a deleterious one

The Prominent Role of Neutrophils during the Initial Phase of Infection by Leishmania Parasites

Neutrophils are rapidly and massively recruited to the site of Leishmania inoculation, where they phagocytose the parasites, some of which are able to survive within these first host cells. Neutrophils can thus provide a transient safe shelter for the parasites, prior to their entry into macrophages where they will replicate. In addition, neutrophils release and synthesize rapidly several factors including cytokines and chemokines. The mechanism involved in their rapid recruitment to the site of parasite inoculation, as well as the putative consequences of their massive presence on the microenvironment of the focus of infection will be discussed in the context of the development of the Leishmania-specific immune response

Inactivation of Eα and Eβ expression in inbred and wild mice by multiple distinct mutations, some of which predate speciation within Mus species

The H-2 MHC of mice encodes two functional class II heterodimeric proteins: AαAβ (A) and EαEβ (E). While failure to express the A protein has not been reported, a significant proportion of of H-2 haplotypes In both Inbred and wild mice do not express E proteins. We and others have previously characterized the molecular basis for defective E expression in haplotypes from Mus domestlcus (b, f, q, s, from inbred strains) and Af. castaneus (w17, wild-derived) species, identifying six distinct defects in the genes for Eα or Eβ. In this report we have extended these studies to other E- haplotypes, Including several from f-haplotype-bearlng M. domesticus mice (w29, w57, w302) and one derived from the Asian species M. bactrianus (w301). Analyses at the protein, RNA and DNA levels were employed to Identify the defects in the genes for Ea and Eb. At least one new defect was identified that prevents Eβ expression in a t-associated H-2 haplotypes (w57), bringing the number of distinct mutations causing the Eβ phenotype to seven. Another t-associated haplotype, w302, was found to share the same Eβ defect with mice of the inbred q haplotype and of the w17 haplotype from Af. castaneus, while its Ea gene contains the deletion carried also by the Inbred b and s haplotypes and by a number of wild haplotypes. The mutations in the Ea and Eb genes of the w301 haplotype from M. bactrianus were found to be Identical to those of the Inbred f haplotype. This indicates that the origin of the mutations in the Eb genes of the q, w17 and w302 haplotypes and in the Ea and Eb genes of the f and w301 haplotypes, predated speciation within Mus, thought to have occurred ∼0.35-1 million years ago. Their maintenance in mouse populations suggests that in certain conditions the failure to express EαEβ proteins may be advantageous and selected fo

Exposure of monocytes to heat shock does not increase class II expression but modulates antigen-dependent T cell responses

Expression of heat shock (HS) proteins (HSP) increases after exposure to elevated temperatures or other types of injury, such as oxldative injury. Because of their function as ‘molecular chaperones', HSP are suggested to participate in antigen processing and presentation. We have previously reported that HS modulates antigen presentation in a human EBV-transformed B cell line. Here we investigated the effects of HS on MHC class II expression and on antigen processing and presentation by human monocytes. Monocytes were isolated from peripheral blood of normal human volunteers, purified by adherence, then exposed to temperatures ranging from 37 to 45°C for 20 min, allowed to recover for 2 h at 37°C and used for immunofluorescence or as antigen presenting cells in autologous and heterologous lymphocyte proliferation assays. No increase in class II expression was detected as assessed by flow cytometry. Monocytes (3 × 104) and lymphocytes (1 × 105) were co-cultured for 5 days in the presence of several antigens [diphtheria toxold, tetanus toxold or purified peptlde derivative (PPD)] and labeled with 1 μCI [3H]thymldlne for 16 h. Pre-exposure to HS (44°C) significantly (P < 0.001) increased T cell responses to diphtheria toxold, whereas the effect on the responses to other antigens (tetanus toxold or PPD) were not significant. HS did not increase heterologous T cell responses nor T cell proliferation induced by the non-processed superantigens such as staphylococcal enterotoxln B. The effect of HS was inhibited by actlnomycln B and thus appeared dependent upon HSP synthesis. HSP-mediated increases in antigen processing may potentiate the ongoing immune response at inflammatory site

Resistance of Leishmania (Viannia) Panamensis to Meglumine Antimoniate or Miltefosine Modulates Neutrophil Effector Functions.

Leishmania (Viannia) panamensis (L. (V.) p.) is the main causative agent of cutaneous leishmaniasis in Colombia and is usually treated with either meglumine antimoniate (MA) or miltefosine (MIL). In recent years, there has been increasing evidence of the emergence of drug-resistance against these compounds. Neutrophils are known to play an important role in immunity against Leishmania. These cells are rapidly recruited upon infection and are also present in chronic lesions. However, their involvement in the outcome of infection with drug-resistant Leishmania has not been examined. In this study, human and murine neutrophils were infected in vitro with MA or MIL drug-resistant L. (V.) p. lines derived from a parental L. (V.) p. drug-susceptible strain. Neutrophil effector functions were assessed analyzing the production of reactive oxygen species (ROS), the formation of neutrophil extracellular trap (NET) and the expression of cell surface activation markers. Parasite killing by neutrophils was assessed using L. (V.) p. transfected with a luciferase reporter. We show here that MA and MIL-resistant L. (V.) p. lines elicited significantly increased NET formation and MA-resistant L. (V.) p. induced significantly increased ROS production in both murine and human neutrophils, compared to infections with the parental MIL and MA susceptible strain. Furthermore, neutrophils exposed to drug-resistant lines showed increased activation, as revealed by decreased expression of CD62L and increased expression of CD66b in human neutrophils yet presented higher survival within neutrophils than the drug-susceptible strain. These results provide evidence that parasite drug-susceptibility may influences neutrophil activation and function as well as parasite survival within neutrophils. Further investigaton of the inter-relationship of drug susceptibility and neutrophil effector function should contribute to better understanding of the factors involved in susceptibility to anti-Leishmania drugs

Selective Expression of the Vβ14 T Cell Receptor on Leishmania guyanensis-Specific CD8+ T Cells during Human Infection

Peripheral blood mononuclear cells from subjects never exposed to Leishmania were stimulated with Leishmania guyanensis. We demonstrated that L. guyanensis-stimulated CD8+ T cells produced interferon (IFN)-γ and preferentially expressed the Vb14 T cell receptor (TCR) gene family. In addition, these cells expressed cutaneous lymphocyte antigen and CCR4 surface molecules, suggesting that they could migrate to the skin. Results obtained from the lesions of patients with localized cutaneous leishmaniaisis (LCL) showed that Vβ14 TCR expression was increased in most lesions (63.5%) and that expression of only a small number of Vb gene families (Vβ1, Vβ6, Vβ9, Vβ14, and Vβ24) was increased. The presence of Vβ14 T cells in tissue confirmed the migration of these cells to the lesion site. Thus, we propose the following sequence of events during infection with L. guyanensis. After initial exposure to L. guyanensis, CD8+ T cells preferentially expressing the Vb14 TCR and secreting IFN-γ develop and circulate in the periphery. During the infection, these cells migrate to the skin at the site of the parasitic infection. The role of these Vβ14 CD8+ T cells in resistance to infection remains to be determined conclusivel

IL-4Rα Signaling in Keratinocytes and Early IL-4 Production Are Dispensable for Generating a Curative T Helper 1 Response in Leishmania major-Infected C57BL/6 Mice.

Experimental infection with the protozoan parasite Leishmania major has been extensively used to understand the mechanisms involved in T helper cell differentiation. Following infection, C57BL/6 mice develop a small self-healing cutaneous lesion and they are able to control parasite burden, a process linked to the development of T helper (Th) 1 cells. The local presence of IL-12 has been reported to be critical in driving Th1 cell differentiation. In addition, the early secretion of IL-4 was reported to potentially contribute to Th1 cell differentiation. Following infection with L. major, early keratinocyte-derived IL-4 was suggested to contribute to Th1 cell differentiation. To investigate a putative autocrine role of IL-4 signaling on keratinocytes at the site of infection, we generated C57BL/6 mice deficient for IL-4Rα expression selectively in keratinocytes. Upon infection with L. major, these mice could control their inflammatory lesion and parasite load correlating with the development of Th1 effector cells. These data demonstrate that IL-4 signaling on keratinocytes does not contribute to Th1 cell differentiation. To further investigate the source of IL-4 in the skin during the first days after L. major infection, we used C57BL/6 IL-4 reporter mice allowing the visualization of IL-4 mRNA expression and protein production. These mice were infected with L. major. During the first 3 days after infection, skin IL-4 mRNA expression was observed selectively in mast cells. However, no IL-4 protein production was detectable locally. In addition, early IL-4 blockade locally had no impact on subsequent Th1 cell differentiation and control of the disease. Taken together, the present data rule out a major role for skin IL-4 and keratinocyte IL-4Rα signaling in the development of a Th1 protective immune response following experimental infection with L. major