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The role of neutrophils in the course of Leishmania infection remains controversial, displaying tremendous variability depending on the species of parasite, stage of infection, host genetic background, and methodological discrepancies among studies. Although neutrophils have long been categorized as short-lived cells with limited capacity to express proteins de novo, recent advances have revealed significant plasticity in neutrophil transcriptional programmes and intrapopulation heterogeneity, which can be regulated by both intrinsic and extrinsic factors that together determine the profile of neutrophil effector response. In this review, we focus on the current understanding of neutrophil transcriptional plasticity, neutrotime, evidence of Leishmania-mediated alterations in neutrophil transcriptome leading to the rise of subpopulations, and finally, functional implications of those findings to the course of Leishmania infection.

BackgroundVisceral leishmaniasis is an opportunistic disease in HIV-1 infected individuals, although not yet recognized as a determining factor for AIDS diagnosis. The growing geographical overlap of HIV-1 and Leishmania infections is an emerging challenge worldwide, as co-infection increases morbidity and mortality for both. Here, we determined the prevalence of people living with HIV (PWH) with a previous or ongoing infection by Leishmania infantum in Bahia, Brazil and investigated the virological and immunological factors associated with co-infection.Methodology and Principal FindingsWe adopted a two-stage cross-sectional cohort (CSC) design (CSC-I, n=5,346 and CSC-II, n=317) of treatment-naïve HIV-1-infected individuals in Bahia, Brazil. In CSC-I, samples collected at the time of HIV-1 diagnosis between 1998 and 2013 were used for serological screening for leishmaniasis by an in-house immunoassay (ELISA) with SLA (Soluble Leishmania Antigen), resulting in a prevalence of previous or ongoing infection of 16.27%. Next, 317 PWH were prospectively recruited from July 2014 to December 2015 with collection of sociodemographic and clinical data. Serological validation by two different immunoassays confirmed a prevalence of 15.46% and 8.20% by anti-SLA, and anti-HSP70 serology, respectively, whereas 4.73% were double-positive (DP). Stratification of these 317 individuals in DP and double-negative (DN) revealed a significant reduction of CD4+ counts and CD4+/CD8+ ratios and a tendency of increased viral load in the DP group, as compared to DN. No statistical differences in HIV-1 subtype distribution were observed between the two groups. However, we found a significant increase of CXCL10/IP-10 (p=0.0076) and a tendency of increased CXCL9/MIG (p=0.061) in individuals with DP serology for L. infantum, demonstrating intensified immune activation in this group. These findings were corroborated at the transcriptome level in independent Leishmania- and HIV-1-infected cohorts (Swiss HIV Cohort and Piaui Northeast Brazil Cohort), indicating that CXCL10 transcripts are shared by the IFN-dominated immune activation gene signatures of both pathogens and positively correlated to viral load in untreated PWH.Conclusions/SignificanceThis study demonstrated a high prevalence of PWH with L. infantum seropositivity in Bahia, Brazil, linked to IFN-mediated immune activation and a significant decrease in CD4+ levels. Our results highlight the urgent need to increase awareness and define public health strategies for the management and prevention of HIV-1 and L. infantum co-infection.AUTHOR SUMMARYMore than 1 billion people live in areas endemic for leishmaniasis and are at risk of infection, which includes one third of the 38 million people living with HIV (PWH) worldwide. Leishmaniasis is a neglected tropical disease caused by infection with Leishmania parasites, transmitted by bites from infected sand flies. HIV/Leishmania co-infection is increasing worldwide, especially in Southern Europe and Brazil, due to both human and ecological factors (migration, climate change, deforestation). This study demonstrates that a worryingly high number (one out of 6) of PWH in Bahia (Northeast Brazil) show previous or ongoing infection with the parasite Leishmania infantum, determined by antibodies detection against this parasite in PWH. Using a rigorous study design with data collection over 18 years in >5500 PWH, we found similarly high numbers (16.3% and 15.5%) of PWH with antibodies against Leishmania infantum in two consecutive cohorts. Moreover, PWH with antibodies against Leishmania infantum in Bahia, Brazil displayed a worse immune profile, with lower CD4 immune cells numbers and immune activation mediated by interferon, which we confirmed in independent HIV- and Leishmania-infected cohorts from other geographic areas. Our results underscore the need to raise awareness and define public health strategies to prevent HIV/Leishmania co-infection, since therapeutic failure and mortality are strongly increased in co-infected PWH, even with antiretroviral therapy as standard of care.

Leishmania (L.) amazonensis is one of the etiological agents of cutaneous leishmaniasis (CL) in Brazil. Currently, there is no vaccine approved for human use against leishmaniasis, although several vaccine preparations are in experimental stages. One of them is Leishvacin, or LaAg, a first-generation vaccine composed of total L. amazonensis antigens that has consistently shown an increase of mouse resistance against CL when administered intranasally (i.n.). Since Toll-like receptor 9 (TLR9) is highly expressed in the nasal mucosa and LaAg is composed of TLR9-binding DNA CpG motifs, in this study we proposed to investigate the role of TLR9 in both L. amazonensis infection and in LaAg vaccine efficacy in C57BL/6 (WT) mice and TLR9-/- mice. First, we evaluated, the infection of macrophages by L. amazonensis in vitro, showing no significant difference between macrophages from WT and TLR9-/- mice in terms of both infection percentage and total number of intracellular amastigotes, as well as NO production. In addition, neutrophils from WT and TLR9-/- mice had similar capacity to produce neutrophil extracellular traps (NETs) in response to L. amazonensis. L. amazonensis did not activate dendritic cells from WT and TLR9-/- mice, analysed by MHCII and CD86 expression. However, in vivo, TLR9-/- mice were slightly more susceptible to L. amazonensis infection than WT mice, presenting a larger lesion and an increased parasite load at the peak of infection and in the chronic phase. The increased TLR9-/- mice susceptibility was accompanied by an increased IgG and IgG1 production; a decrease of IFN-γ in infected tissue, but not IL-4 and IL-10; and a decreased number of IFN-γ producing CD8+ T cells, but not CD4+ T cells in the lesion-draining lymph nodes. Also, TLR9-/- mice could not control parasite growth following i.n. LaAg vaccination unlike the WT mice. This protection failure was associated with a reduction of the hypersensitivity response induced by immunization. The TLR9-/- vaccinated mice failed to respond to antigen stimulation and to produce IFN-γ by lymph node cells. Together, these results suggest that TLR9 contributes to C57BL/6 mouse resistance against L. amazonensis, and that the TLR9-binding LaAg comprising CpG motifs may be important for intranasal vaccine efficacy against CL., Author summary Leishmaniasis is a major neglected tropical disease, being responsible for more than 20 million deaths per year. The high mortality rate highlights the difficulties and ineffectiveness of the current prophylactic approaches and treatments currently available. Therefore, the development of an effective vaccine would be highly advantageous to circumvent these problems. Despite the many vaccines preparations that have been studied in the last few years, none have shown satisfactory efficacy to be approved for human use. Immune receptors, including the TLR family, are known to be important for host defense during parasitic infections, such as leishmaniasis, and also for vaccine efficacy. In this work, we investigate the role of TLR9 during Leishmania amazonensis infection in vaccinated and non-vaccinated mice. We used a C57BL/6 TLR9-/- mouse model and a first-generation vaccine preparation (LaAg) composed of a total lysate of L. amazonensis. We demonstrate that TLR9 is important for controlling leishmaniasis infection caused by L. amazonensis and is involved in the efficacy of the LaAg vaccine. These findings will certainly help in the development of a better vaccine against leishmaniasis.

Exogenously administered glucocorticoids enhance eosinophil and neutrophil granulocyte production from murine bone-marrow. A hematological response dependent on endogenous glucocorticoids underlies bone-marrow eosinophilia induced by trauma or allergic sensitization/challenge. We detected a defect in granulopoiesis in nonsensitized, perforin-deficient mice. In steady-state conditions, perforin- (Pfp-) deficient mice showed significantly decreased bone-marrow and blood eosinophil and neutrophil counts, and colony formation in response to GM-CSF, relative to wild-type controls of comparable age and/or weight. By contrast, peripheral blood or spleen total cell and lymphocyte numbers were not affected by perforin deficiency. Dexamethasone enhanced colony formation by GM-CSF-stimulated progenitors from wild-type controls, but not Pfp mice. Dexamethasone injection increased bone-marrow eosinophil and neutrophil counts in wild-type controls, but not Pfp mice. Because perforin is expressed in effector lymphocytes, we examined whether this defect would be corrected by transferring wild-type lymphocytes into perforin-deficient recipients. Short-term reconstitution of the response to dexamethasone was separately achieved for eosinophils and neutrophils by transfer of distinct populations of splenic lymphocytes from nonsensitized wild-type donors. Transfer of the same amount of splenic lymphocytes from perforin-deficient donors was ineffective. This demonstrates that the perforin-dependent, granulopoietic response to dexamethasone can be restored by transfer of innate lymphocyte subpopulations.