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Live-cell imaging allows the study of apoptosis at cellular level, highlighting morphological hallmarks such as nuclear shrinkage, membrane blebbing, and cell disruption. Identifying the exact location and timing of this process is essential to foster the understanding of its spatial-temporal regulation. However, the analysis of live-cell imaging datasets is complex, whereas computational tools tailored to this task are yet scarce. Therefore, we developed ADeS, an Apoptosis Detection System based on deep learning and activity recognition. ADeS uses morpho-dynamic hallmarks to detect the exact location and timing of apoptotic events in different cell types, reaching an accuracy above 97% in the classification of our validation datasets acquiredin vitroandin vivo. Moreover, ADeS is the first successful implementation of a deep learning network for the automatic detection of apoptotic cells in full microscopy movies in an end-to-end fashion, outperforming human in the same task. As a case study, we employed ADeS for the analysis of cell survivalin vitro, and for tissue damage assessmentin vivo, showing its potential application for toxicity assays, treatments evaluation and measuring of tissue dynamics.

Protective immunity to cutaneous leishmaniasis is mediated by IFN-γ–secreting CD4+ Th1 cells. IFN-γ binds to its receptor on Leishmania-infected macrophages, resulting in their activation, production of NO, and subsequent destruction of parasites. This study investigated the role of Semaphorin 3E (Sema3E) in host immunity to Leishmania major infection in mice. We observed a significant increase in Sema3E expression at the infection site at different timepoints following L. major infection. Sema3E-deficient (Sema3E knockout [KO]) mice were highly resistant to L. major infection, as evidenced by significantly (p < 0.05–0.01) reduced lesion sizes and lower parasite burdens at different times postinfection when compared with their infected wild-type counterpart mice. The enhanced resistance of Sema3E KO mice was associated with significantly (p < 0.05) increased IFN-γ production by CD4+ T cells. CD11c+ cells from Sema3E KO mice displayed increased expression of costimulatory molecules and IL-12p40 production following L. major infection and were more efficient at inducing the differentiation of Leishmania-specific CD4+ T cells to Th1 cells than their wild-type counterpart cells. Furthermore, purified CD4+ T cells from Sema3E KO mice showed increased propensity to differentiate into Th1 cells in vitro, and this was significantly inhibited by the addition of recombinant Sema3E in vitro. These findings collectively show that Sema3E is a negative regulator of protective CD4+ Th1 immunity in mice infected with L. major and suggest that its neutralization may be a potential therapeutic option for treating individuals suffering from cutaneous leishmaniasis.

Intravital microscopy, such as 2-photon microscopy, is now a mainstay in immunological research to visually characterize immune cell dynamics during homeostasis and pathogen infections. This approach has been especially beneficial in describing the complex process of host immune responses to parasitic infections in vivo, such as Leishmania. Human-parasite co-evolution has endowed parasites with multiple strategies to subvert host immunity in order to establish chronic infections and ensure human-to-human transmission. While much focus has been placed on viral and bacterial infections, intravital microscopy studies during parasitic infections have been comparatively sparse. In this review, we will discuss how in vivo microscopy has provided important insights into the generation of innate and adaptive immunity in various organs during parasitic infections, with a primary focus on Leishmania. We highlight how microscopy-based approaches may be key to providing mechanistic insights into Leishmania persistence in vivo and to devise strategies for better parasite control.

Despite the generation of a strong T cell response, clearance of Leishmania major is incomplete. Understanding of the persistence mechanisms is lacking, but Leishmania major driven induction of the immunosuppressive microenvironment through recruitment of regulatory T cells has been proposed to prevent parasite clearance in vivo. In the presented study, we used a novel TCR transgenic mouse model, where CD4+ T cells recognize an immunodominant peptide derived from Leishmania-glycosomal phosphoenolpyruvate carboxykinase (PEPCK), to visualize the dynamics of anti-L. major CD4+ T cell responses and to characterize mechanisms which restrain their effector function. We show that macrophage:T cell interaction dynamics were prolonged upon antigen recognition. This leads to a production of high levels of IFNγ and can be significantly suppressed by PEPCK-specific Tregs in vitro, as compared to polyclonal Treg controls. Treg antigen activation leads to a substantial increase in IL-10 levels, while decreasing IL-12, TNF, and IL-2 production. Intravital microscopy studies characterizing PEPCK-specific CD4+ T cell migration within skin lesions in mice show a significant recruitment of adoptively transferred effector T cells to the lesion site in vivo, displaying behaviours consistent with antigen recognition at early stages, but not at chronic stages. Imaging of infected FoxP3-GFP reporter mice show recruitment of Tregs to the lesion site, where they localize in the lesion, unlike the effectors cells which surround it. Collectively, our findings show for the first time that Leishmania-specific Tregs influence effector CD4+ T cell responses and this could be a mechanism that derives antigen persistence in L. major infection. Research funded by a grant from the GSK-CIHR Partnered Chair Program

Summary The long pentraxin 3 (PTX3) plays a critical role in inflammation, tissue repair, and wound healing. Here, we show that PTX3 regulates disease pathogenesis in cutaneous leishmaniasis (CL). PTX3 expression increases in skin lesions in patients and mice during CL, with higher expression correlating with severe disease. PTX3-deficient (PTX3−/−) mice are highly resistant to L. major and L. braziliensis infections. This enhanced resistance is associated with increases in Th17 and IL-17A responses. The neutralization of IL-17A abolishes this enhanced resistance, while rPTX3 treatment results in decrease in Th17 and IL-17A responses and increases susceptibility. PTX3−/− CD4+ T cells display increased differentiation to Th17 and expression of Th17-specific transcription factors. The addition of rPTX3 suppresses the expression of Th17 transcription factors, Th17 differentiation, and IL-17A production by CD4+ T cells from PTX3−/− mice. Collectively, our results show that PTX3 contributes to the pathogenesis of CL by negatively regulating Th17 and IL-17A responses.

T cells actively migrate along reticular networks within lymphoid organs in search for cognate antigen, but how these behaviors impact HIV entry and infection is unclear. Here, we show that migratory T cells in 3D collagen matrix display significantly enhanced infection and integration by cell-free R5-tropic lab adapted and transmitted/founder molecular HIV clones in the absence of exogenous cytokines or cationic polymers. Using two different collagen matrices that either support or restrict T cell migration, we observe high levels of HIV fusion in migratory T cells, whereas non-motile T cells display low viral entry and integration. Motile T cells were less sensitive to combination antiretroviral drugs and were able to freely migrate into regions with high HIV densities, resulting in high infection rates. Together, our studies indicate that the environmental context in which initial HIV-T cell encounters occur modulates HIV-1 entry and integration efficiencies.

The long Pentraxin 3 (PTX3), a soluble pattern recognition molecule, plays a critical role in inflammation, tissue repair and wound healing. Here, we show that PTX3 regulates disease pathogenesis in cutaneous leishmaniasis (CL). PTX3 expression is increased in active skin lesions in patients and mice during CL, with higher levels being expressed in individuals with severe disease. PTX3 deficient (PTX3-/-) mice were highly resistant toL. majorinfection and the enhanced resistance was associated with increased IL-17 response. Neutralization of IL-17A abolished this enhanced resistance while treatment with recombinant PTX3 resulted in reduced IL-17A response and increased susceptibility toL. majorinfection. Naïve CD4+T cells from PTX3-/-mice displayed increased differentiation into Th17 cells, which was reversed in the presence of recombinant PTX3. The enhanced Th17 response observed in PTX3-/-cells was associated with increasedLeishmaniaspecific IL-6 production from dendritic cells along with enhanced expression of Th17-specific transcription factors including RORγt, AhR and STAT3. Addition of recombinant PTX3 significantly inhibited the expression of Th17-specific transcription factors and dramatically reduced the frequency of Th17 cells in Th17-polarizing cultures of PTX3-/-CD4+T cells. Collectively, our results show that PTX3 contributes to the pathogenesis of CL by suppressing Th17 differentiation and IL-17A production.Author SummaryCutaneous leishmaniasis (CL) is caused by several species ofLeishmania. Currently, there is no approved vaccine against human CL because of the poor understanding of the mechanisms that regulate disease pathogenesis and correlates of protective immunity. Because the long pentraxin 3 (PTX3, a soluble pattern recognition molecule that forms an integral part of the host innate immunity), regulates inflammation and tissue repair, which are critical physiological events associated with resolution of skin lesions during CL, we investigated its role in disease pathogenesis.Here, we show that PTX3 levels were elevated in skin-lesions in patients and mice during CL. Using a loss of function approach, we showed that PTX3 contributes to pathogenesis, and this was associated with increased IL-17A responses. Neutralization and recombinant cytokine treatment studies showed that the increased resistance of PTX3 deficient mice toL. majoris due to enhanced Th17 response in these mice. We further show that PTX3 negatively regulates IL-6 production by dendritic cells and the expression of IL-17A-specific transcription factors (including RORγT, STAT3, IRF4, BATF and AhR) in CD4+T cells. Collectively, these findings show that PTX3 is a negative regulator of Th17 response and protective immunity duringL. majorinfection.

The long pentraxin 3 (PTX3) plays a critical role in inflammation, tissue repair, and wound healing. Here, we show that PTX3 regulates disease pathogenesis in cutaneous leishmaniasis (CL). PTX3 expression increases in skin lesions in patients and mice during CL, with higher expression correlating with severe disease. PTX3-deficient (PTX3

Resolution of cutaneous leishmaniasis (CL), caused by the protozoan parasite Leishmania (L) major, relies on the nature of the host immune response. The long pentraxin 3 (PTX3), a soluble pattern recognition molecule, is critical for wound healing by regulating tissue repair and also in innate and adaptive responses during infection and inflammation. Here, we show that PTX3 regulates disease pathogenesis and immunity to CL. PTX3 expression is increased in active skin lesions in patients and mice during CL, with higher levels being expressed in individuals with severe disease. PTX3−/− mice were highly resistant to primary and secondary L. major infection. Interestingly, the enhanced resistance of PTX3−/− mice to primary or secondary L. major infection was not associated with enhanced IFN-γ or decreased IL-10 response. Instead, L. major-infected PTX3−/− mice displayed strong IL-17 response and in vivo neutralization of IL-17A abolished their enhanced resistance. Naïve CD4+ T cells from PTX3−/− mice displayed increased differentiation into Th17 cells and this was associated with increased expression of Th17-specific transcription factors including RORγt, AhR and STAT3. Addition of recombinant PTX3 significantly inhibited the expression of Th17-specific transcription factors and the frequency of Th17 cells in Th17 polarizing cultures of PTX3−/− CD4+ T cells. Similarly, healed PTX3−/− mice had higher frequencies of effector memory CD4+ T cells that rapidly proliferate and differentiate into IL-17A-producing CD4+ T cells upon secondary challenge. Collectively, our results show that PTX3 contributes to pathogenesis of CL by negatively regulating protective Th17 and IL-17 responses.