Senescent CD8+ T and NK cells contribute to immunopathogy duting cutaneous leishmaniasis

Parasites belonging to the genus Leishmania are among the most diverse of human pathogens, both in terms of geographical distribution and in the variety of clinical syndromes caused by them. Currently, 12 million people are infected worldwide in 88 tropical/subtropical countries, 2 million new infections are reported annually, and 350 million people are under infection risk. In the past decade, the number of cases in endemic areas has increased sharply. In addition, leishmaniasis is spreading to several non-endemic areas of the world due to coinfections with HIV. Control measures currently available are case detection and treatment with drugs, which are expensive, not always available and cannot be self-administered. The problem is further aggravated by the surge of drug resistance parasites necessitating the development of an anti-Leishmania vaccine or other immunological therapies urgent.

Research has demonstrated the importance of the immunity in controlling both cutaneous and mucocutaneous leishmaniasis, however the severe skin lesions that develop at the infected site is largely due to non-specific tissue damage but the reasons for this are unclear. The majority of this work has been performed on blood samples taken from patients and not from the site of infection at the skin. In this context, a unique feature of the current proposal is that we will be investigating the blood as well as the skin from the same patients, taken from both lesional and non-lesional sites.

The main hypothesis of this grant application is that the severity of cutaneous immunopathology in infected individuals is due to non-specific damage mediated by both two types of immune cell namely the natural killer (NK) cells and a type of T cell that represent the "Dad's Army" of the immune system. Both these cell types are probably not triggered by the parasite in the skin lesions, instead they recognize decoy molecules in non-infected skin cells that cause them to be destroyed. This results in the large skin lesions that contain few parasites but huge numbers of dangerous white cells.

In this study we will investigate the nature of the white cells in the skin lesions of patients. We will identify the decoy receptors that promote the non-specific tissue damage. We will also look at the wide array of genes that are activated in the skin to identify clue of how the interactions between the immune system and the skin are organized. We will also identify genes that will give us clues as to what other processes may be occurring that do not allow the lesions to resolve. In other parts of the study we will mimic the interaction of cell that are in the skin by growing them in a test tube to probe their interactions and find a way to block them. Finally we will investigate the involvement of a set off molecules known as sestrins, that we previously showed were involved in setting up the decoy mechanisms that may lead to skin lesion formation in patients with cutaneous leishmaniasis.

As added value to this proposal, The Federal University of Espirito Santo will contribute 1 Technician and 1 PhD student to work on aspects of this grant.

Leishmania (Viannia) brasiliensis causes a wide spectrum of cutaneous disease ranging from self-healing cutaneous leishmaniasis (CL) to tissue destructive mucocutaneous leishmaniasis (MCL) in humans. During infection CD4+ and CD8+ naïve T cells are driven to expand into different subsets including memory compartments which are crucial to infection control and tissue damage observed during CL. Although Leishmania-reactive memory T cells have been characterised in blood from CL patients, little is known about the differentiation/function of these cells in the skin/lesion site. Factors such as persistent antigen stimulation and inflammatory state have been found to play a key role in directing the development of NK cells as well as CD4+ and CD8+ differentiation into effector memory (EM) T cells (CD28-CD27-CD45RA-) and also EM T cells that re-express CD45RA (CD28-CD27-CD45RA+; EMRA). These highly differentiated CD8+ T cells cell which exhibit terminal differentiation and senescence characteristics contribute to diminished immune responses due to their increased expression of surface inhibitory receptors, DNA damage and reduced replicative capacity. Mechanisms of memory T cell and NK terminal differentiation during leishmania infection are still unknown, however this may assist the understanding of immune response diversity during CL. In this context, the present study aims to investigate the frequency, differentiation and effector function of NK and CD8+ memory T cell from blood and skin of CL patients caused by L. brasiliensis. In this study we will investigate isolated leukocytes from the blood and biopsies taken from skin lesions of the same patients. We will perform extensive flow cytometric and immunohistological analyses of these samples. We will also investigate gene expression in skin lesions by RNAseq analyses. Age and gender matched healthy controls will be recruited from the same district of Vitoria as the patients.

The main aim of this study is to establish a roadmap to understanding both the nature of the defect in antigen specific immunity during leishmaniasis and whether this is due in part to the high level of pro-inflammatory responses induced by senescent cells in these subjects in vivo. These studies have the potential to identify ways to enhance the Leishmania immunity in humans by manipulating T cell development and function in a highly targeted fashion and also contribute to the improved design of future vaccines, adjuvants and drugs development. We are investigating immune responses to localized cutaneous leishmaniasis (LCL). However, any mechanisms that we may identify will be relevant to the understanding of all different forms of Leishmania immunity and to a wide range of other infectious organisms. This valuable data on immunity in human tissues will be made freely accessible to other scientists, the academic beneficiaries, who will be able to use this information to further extend our knowledge on other aspects of leishmaniasis and also reduce the current spending on patient treatments.
Engagement with industrial collaborators: A second objective and its impact is the potential collaboration with pharmaceutical companies. The work that is proposed in this grant is of considerable interest to Pharmaceutical companies and the data generated will impact directly on strategy for intervening in the interaction between senescent cells and leukocytes that recognize and kill them. Both PI's (Akbar and Gomes) involved in this work have experience and successful collaborations with industry generating products currently marketed or in authorization stage. In the 5th aim of the study, if sestrin inhibition reduces NKR on leukocytes or NKR ligands on stromal cells, this may be a strategy to reduce non-specific immunopathology in CL and also in other diseases where the tissue destruction occurs is overwhelmingly excessive relative to the burden of infectious agent present. This would form the basis for discussions with pharma.

The reduction of the use of animals in experimentation: As our studies will all be performed in human leukocytes and human samples, there is no need for extra animal testing. In fact this proposal makes use of the availability of unique human tissue samples that will be analysed with state of the art novel technology to investigate immunity during cutaneous and mucocutaneous leishmaniasis.

International dimension of the investigation: This collaboration adds value to the current application and the interchange of ideas, reagents and students between both groups. In addition, the Akbar Lab has hosted visiting scientists from Cuba, Italy, Portugal, Netherlands, USA, Austria and from Spain who have spent between 3 months to 3 years assimilating technology for use in their home institutions. This has established an international network of collaborators that the Akbar Lab is able to draw upon for expertise and reagents in a reciprocal manner. This will offer an opportunity to strengthen the new (started in 2014) collaboration between the Akbar lab and a Brazilian group after numerous visits for collaborative work made by Prof. Daniel Gomes (CoPI) and Dr. Luciana Covre (named PDRA).

Developing new expertise: This study involves an exciting multidisciplinary approach using human experimental models for blood and skin biopsies functional responses analyses. UFES has allocated 1 PhD student and 1 technician to this project. Members of the Akbar lab, including postdoctoral researchers and PhD students from UCL will also have the opportunity to visit both host sites and to be involved in aspects of this work. The UFES individuals will liaise and assimilate expertise form members of the Akbar group. This will nurture a new generation of researchers at both institutions who will be well versed in the investigation of cellular, molecular and systems biology aspects of immunity in humans during leishmaniasis.