DOI

10.17077/etd.d088xm24

Document Type

Dissertation

Date of Degree

Fall 2016

Degree Name

PhD (Doctor of Philosophy)

Degree In

Immunology

First Advisor

Mary E. Wilson

First Committee Member

Steven M Varga

Second Committee Member

William M Nauseef

Third Committee Member

Lee-Ann Allen

Fourth Committee Member

Fayyaz Sutterwala

Abstract

The vector-borne protozoan Leishmania spp. cause the spectrum of disease known as leishmaniasis in human and animal hosts. The most common manifestations of leishmaniasis are the chronic, ulcerative skin disease cutaneous leishmaniasis (CL), and the more serious visceral leishmaniasis (VL) in which parasites take up residence in internal organs, causing death if not treated. The role of neutrophils (PMNs) in the immune response to CL and VL is unclear. It is s generally thought that PMNs are only a short-lived effector cell, and have been disregarded as playing a role in chronic Leishmania spp. infection. As both CL and VL are diseases characterized by increased inflammatory immune mediators, we hypothesized that PMNs from human or animal models of chronic leishmaniasis would display different properties from PMNs from healthy controls. We found in a subset of CL and VL patients circulating PMNs expressing HLA-DR, the human form of MHC class II, a molecule thought to be restricted primarily to professional antigen cells. When we examined PMNs recruited to CL skin lesions in human patients, or similar lesions in experimental murine model of CL, we found significantly increased MHC class II+ PMNs. Circulating HLA-DR+ PMNs also expressed the co-stimulatory molecules CD80, CD86 and CD40. While this suggested an antigen-presenting cell-like phenotype by these HLA-DR+ PMNs, compared to conventional HLA-DR- PMNs, HLA-DR+ PMNs showed not only a neutrophil-like appearance and function, but in fact increased activation, degranulation, intracellular MPO and phagocytosis of parasites and zymosan particles. Incubation of healthy control whole blood with inflammatory cytokines resulted in increased HLA-DR+ PMNs and the presence of hladrb1 mRNA, suggesting a connection between neutrophil “priming” and upregulation of HLA-DR.

In addition to HLA-DR+ PMNs in CL patients, we also identified the presence of so-called “low-density” neutrophils (LD-PMNs). These neutrophils, which are enriched in low-density fractions following centrifugation of blood over a density gradient, are reported in numerous disease states, including cancer, HIV, and systemic lupus erythematosus. In some disease states, LD-PMN are reported to be immunosuppressive toward T cell activation and proliferation. However, LD-PMNs from leishmaniasis patients showed no evidence of immunosuppression. Additionally, we found that LD-PMNs show significantly increased surface expression of MHC class II, suggesting a heretofore unappreciated connection between these atypical neutrophil phenotypes. We also investigated the in vitro interactions with different Leishmania infantum life-stages, both those that cause acute infection (promastigotes) and amastigotes, which are found during chronic stages of the disease. We found that PMNs are readily infected by all L. infantum life-stages, but that amastigotes may have different methods of interacting with PMN surface receptors and are better equipped to avoid PMN anti-microbial responses.

These data suggest that circulating PMNs in chronic leishmaniasis may have unique phenotypes and interact differently with the Leishmania spp. life-cycle present during chronic infection. Further investigation of the role of PMNs and atypical PMN phenotypes in chronic disease may help identify new immunomodulatory roles for this cell type.

Public Abstract

Leishmaniasis is a parasitic disease that is common in countries with tropical climates. Microscopic parasites infect humans and animals from the bite of an insect. The Leishmania parasites not only survive inside humans, but they actually become permanent residents inside macrophages, cells in many organs that normally kill the microbial pathogens. Neutrophils are the most abundant white blood cell in humans. It is commonly believed that neutrophils simply migrate to areas of infection, consume pathogens and die soon after. Because they are short-lived, it was unknown how long-term Leishmania infection might change host neutrophils.

I studied neutrophils in patients in Brazil and India who had leishmaniasis. We were surprised to find that neutrophils in these patients had proteins on their surface that made them appear like antigen presenting cells, the type of immune cell that activates T cells. We found that these neutrophils not only had unusual proteins on their surface, but also the proteins and machinery specific to neutrophils. They were more activated than normal neutrophils and had higher concentrations of some microbe-killing compounds inside. These findings suggest that in leishmaniasis, and probably in other diseases, the widespread inflammation causes some neutrophils to become more activated and adopt an unexpected set of surface proteins typical of antigen presenting cells. We speculate that these changes might influence the overall immune response in leishmaniasis. In future studies we would like to find out whether these unusual neutrophil types help to cure, or to exacerbate the disease.

Keywords

HLA-DR, Leishmania, Leishmaniasis, Low-density neutrophil, MHC class II, Neutrophil

Pages

xv, 197 pages

Bibliography

Includes bibliographical references (pages 183-197).

Copyright

Copyright © 2016 Richard Elliot Davis

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