Th17 cells and neutrophils: Close collaborators in chronic Leishmania mexicana infections leading to disease severity.

Cutaneous leishmaniasis caused by Leishmania mexicana is associated with an important inflammatory response. We here analysed the kinetics of Th17 cells and neutrophils in ear lobe lesions caused by Leishmania mexicana throughout 90 days of disease progression in susceptible BALB/c and semi-resistant C57BL/6 mice infected with 1 × 105 Leishmania mexicana promastigotes. Cells in the lesions were extracted and quantified by flow cytometry, whereas their distribution in the tissues in relation to the parasites was analysed by immunohistochemistry. Our results show that in BALB/c mice, both Th17 cells and neutrophils increase concomitantly and to significantly higher levels on day 90 post-infection, as compared to C57BL/6 mice. Our results provide novel evidence on the cells causing chronic inflammation throughout Leishmania mexicana infections, resulting as a consequence of neutrophil recruitment together with Th17 cell differentiation and recruitment, both of which remain in the infection site throughout the late phase of the infection. We conclude that the more enhanced levels of Th17 cells and neutrophils during chronic inflammatory lesions in BALB/c mice participate in their enhanced susceptibility towards a progressive disease evolution, whereas the more controlled response of these cells in C57BL/6 mice possibly relates to the more resistant profile of this mouse strain.

Leishmania mexicana lipophosphoglycan differentially regulates PKCalpha-induced oxidative burst in macrophages of BALB/c and C57BL/6 mice.

Leishmania are protozoan parasites that infect macrophages and their survival is partially achieved through inhibition of the cellular oxidative burst by parasite lipophosphoglycan (LPG). PKCalpha is the predominant PKC isoenzyme required for macrophage oxidative burst, yet it is not known if different susceptibility of BALB/c and C57BL/6 mice to Leishmania mexicana could be related to PKCalpha. We analysed the effect of L. mexicana promastigotes and parasite LPG on expression of PKCalpha and on its activity in macrophages of both mouse strains. Our data show that expression of the isoenzyme was not altered either by LPG or by L. mexicana promastigotes. Yet LPG exerted opposing effects on PKCalpha activity of macrophages between both strains: in susceptible BALB/c cells, it inhibited PKCalpha activity, whereas in the more resistant strain it augmented enzymatic activity 2.8 times. In addition, LPG inhibited oxidative burst only in susceptible BALB/c macrophages and the degree of inhibition correlated with parasite survival. Promastigotes also inhibited PKCalpha activity and oxidative burst in macrophages of BALB/c mice, whereas in C57BL/6, they enhanced PKCalpha activity and oxidative burst inhibition was less severe. Our data indicate that control of PKCalpha-induced oxidative burst by L. mexicana LPG relates with its success to infect murine macrophages.

Ultrastructural damage in Streptococcus mutans incubated with saliva and histatin 5.

OBJECTIVE: To study the ultrastructural alterations induced in Streptococcus mutans (ATCC 25175) incubated with saliva, saliva plus histatin 5 and histatin 5. METHODS: S. mutans incubated with saliva histatin 5 or a combination of both were morphologically analyzed and counted. The results were expressed as (CFU)ml-1. Ultrastructural damage was evaluated by transmission electron microscopy. Ultrastructural localization of histatin 5 was examined using immunogold labeling. Apoptotic cell death was determined by flow cytometry (TUNEL). RESULTS: A decrease in the bacteria numbers was observed after incubation with saliva, saliva with histatin 5 or histatin 5 compared to the control group (p<0.0001). Ultrastructural damage in S. mutans incubated with saliva was found in the cell wall. Saliva plus histatin 5 induced a cytoplasmic granular pattern and decreased the distance between the plasma membrane bilayers, also found after incubation with histatin 5, together with pyknotic nucleoids. Histatin 5 was localized on the bacterial cell walls, plasma membranes, cytoplasm and nucleoids. Apoptosis was found in the bacteria incubated with saliva (63.9%), saliva plus histatin 5 (71.4%) and histatin 5 (29.3%). Apoptosis in the control bacteria was 0.2%. CONCLUSIONS: Antibacterial activity against S. mutans and the morphological description of damage induced by saliva and histatin 5 was demonstrated. Pyknotic nucleoids observed in S. mutans exposed to saliva, saliva plus histatin 5 and histatin 5 could be an apoptosis-like death mechanism. The knowledge of the damage generated by histatin 5 and its intracellular localization could favor the design of an ideal peptide as a therapeutic agent.

Vaccination with Leishmania mexicana LPG induces PD-1 in CD8⁺ and PD-L2 in macrophages thereby suppressing the immune response: a model to assess vaccine efficacy.

Leishmania lipophosphoglycan (LPG) is a molecule that has been used as a vaccine candidate, with contradictory results. Since unsuccessful protection could be related to suppressed T cell responses, we analyzed the expression of inhibitory receptor PD-1 in CD8(+) and CD4(+) lymphocytes and it is ligand PD-L2 in macrophages of BALB/c mice immunized with various doses of Leishmania mexicana LPG and re-stimulated in vitro with different concentrations of LPG. Vaccination with LPG enhanced the expression of PD-1 in CD8(+) cells. Activation molecules CD137 were reduced in CD8(+) cells from vaccinated mice. In vitro re-stimulation enhanced PD-L2 expression in macrophages of healthy mice in a dose-dependent fashion. The expression of PD-1, PD-L2 and CD137 is modulated according to the amount of LPG used during immunization and in vitro re-stimulation. We analyzed the expression of these molecules in mice infected with 1×10(4) or 1×10(5)L. mexicana promastigotes and re-stimulated in vitro with LPG. Infection with 1×10(5) parasites increased the PD-1 expression in CD8(+) and diminished PD-L2 in macrophages. When these CD8(+) cells were re-stimulated in vitro with LPG, simulating a second exposure to parasite antigens, PD-1 expression increased significantly more, in a dose dependent fashion. We conclude that CD8(+) T lymphocytes and macrophages express inhibition molecules according to the concentrations of Leishmania LPG and to the parasite load. Vaccination with increased amounts of LPG or infections with higher parasite numbers induces enhanced expression of PD-1 and functional inactivation of CD8(+) cells, which can have critical consequences in leishmaniasis, since these cells are crucial for disease control. These results call for pre-vaccination evaluations of potential immunogens, specifically where CD8 cells are required, since inhibiting molecules can be induced after certain thresholds of antigen concentrations. We propose that the analysis of PD-1 and PD-L2 are useful tools to monitor the optimal dose for vaccination candidates.