Theme 1: Mechanisms of immune injury in autoimmune vasculitis and glomerulonephritis

Supervisors:

  Professor Stephen Holdsworth
  T: 9594 5525
  E: Stephen.Holdsworth@monash.edu

  Professor Richard Kitching
  T: 9594 5520
  E: Richard.Kitching@monash.edu

Description:

Glomerulonephritis (GN) is the most common cause of end stage renal failure in Australia and world-wide. The overall aim of this theme is to further our understanding of key events in the generation of nephritogenic immune responses, autoimmunity as it pertains to the kidney and effector responses in the kidney, so that potential therapeutic targets can be identified. Research in our laboratories covers a range of key questions as to why the kidney can be a target of immune attack. It uses a variety of techniques that involve models of disease, transgenic and knock out mice, molecular biology, cell culture, analysis of immunological endpoints, and histological and functional readouts.
Our laboratories work to define mechanisms of immune renal injury, with a particular focus on the role of T cells. Glomerulonephritis (GN) is a common cause of end stage renal failure. The overall aim of this theme is to further our understanding of key events in the generation of nephritogenic immune responses, autoimmunity as it pertains to the kidney and effector responses in the kidney, so that potential therapeutic targets can be identified. Research in our laboratories covers a range of key questions as to why the kidney can be a target of immune attack. We are well funded and publish regularly in the best journals in the field.
We perform our research using a range of classical immunological techniques in murine models of renal disease. We use a variety of techniques that involve models of
disease, transgenic and knock out mice, molecular biology,
cell culture, analysis of immunological endpoints, and histological and functional readouts. A variety of projects
are available - examples of specific currently available
projects are given below.
The laboratory is made up of postdoctoral scientists, research assistants, and several PhD students and in most years one
or two BMS honours students. As we don't have a regular BSc Honours stream, we can devote more time and effort
to helping BMS honours students achieve high standards.
A number of our honours students have gone on to undertake PhD studies with us, and two Postdoctoral Career Scientists are BMS graduates.

Theme 1 projects include:

1 The role of Neutrophils in Immunoregulation of Adaptive Immunity inducing Arthritis and Glomerulonephritis

Supervisor Professor Stephen Holdsworth
Email Stephen.holdsworth@monash.edu

Innate immune cells are activated during the development of arthritogenic and nephritogenic immune responses mediating arthritis and kidney failure in glomerulonephritis. We have recent evidence that neutrophils also modulate or limit the extent of adaptive injurious immunity. We have shown neutrophils migrate to lymph nodes with denditric cells (DCs) as adaptive immunity develops. In this project we will use intravital microscopy of lymph nodes to define the physical interactions between DCs, neutrophils and T cells. We will explore the role of neutrophils by in vivo depletion and transfer of genetically manipulated neutrophils deficient in likely candidate molecules mediating immunomodulation of DC induced T cell activation.

2 How do Mast Cells turn off disease inducing Autoimmunity?.

Supervisor Professor Stephen Holdsworth
Email Stephen.holdsworth@monash.edu

Supervisor Dr Shaun Summers
Email Shaun.Summers@monash.edu

We have recently revealed a novel new pathway of immunoregulation. In experimental vasculitis mast cells direct T regulatory cells to turn off autoimmunity. This project will explore the mechanisms of the effects using in vivo co-culture assays of Tregs mast cells and injurious antigen specific T effector cells. The molecular basis of the effect will be defined using mast cells deficient in key immunomodulatory molecules and lymph node. Intravital microscopy will reveal the physical cellular interactions between Tregs, mast cells, dendritic cells and T effectors that facilitate this new pathway of immunomodulation.

3 Restoring the Balance: Generation of Therapeutic T Regulatory cells to treat Autoimmune ANCA associated Vasculitis.

Supervisor Professor Stephen Holdsworth
Email Stephen.holdsworth@monash.edu

We have developed a reliable reproducible animal model of autoimmune anti-myeloperoxidase anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitis to use in experiments aimed at using new biological therapies without the toxicities of currently used immunosuppressive drugs. Dendritic cells drive the generation of immunity by presenting autoantigen to autoreactive T cells. However this is insufficient to initiate autoimmunity. Antigen binding to the T cell receptor must be accompanied by a second signal to ensure the induction of immunity. CD40 expressed by DCs provide this. In the absence of this, signal regulatory cells, not effector cells are generated. We have preliminary data that using isolated DC deficient in CD40 (CD40-/- mice) pulsed with MPO we can inject naïve mice and generate antigen specific T regulatory cells. In this study we will generate therapeutic Tregs and use them to prevent and treat animals with autoimmune ANCA associated vasculitis.

4 Effector Th1 and Th17 responses in glomerulonephritis using a novel TcR transgenic cell transfer model

Supervisor Professor Richard Kitching
Email Richard.kitching@monash.edu

Cell mediated immunity is important in severe rapidly progressive forms of glomerulonephritis. We have recently established a new models of glomerulonephritis where a model antigen, ovalbumin can be planted in the glomerulus and antigen-specific T cells polarized to a Th1 or Th17 transferred in to cause pure Th12 or Th17 cell mediated injury. Further studies currently planned and offered as honours projects include:

a)    Determining whether effector injury can be enhanced in the same manner in Th1 and Th17 mediated injury be TLR2, TL4 or TLR9 agonists?

b)    Do CD4+ and CD8+ cells play synergistic roles in cell mediated injury?

c)    Do Th1 and Th17 synergise in the development of glomerular injury?

d)    Can co-transfer of GFP+ Foxp3 Tregs ameliorate injury?

Techniques involved in this project include: culture of TcR Tg T cells with antigen, cytokines and antibodies; in vivo animal work; molecular biology; flow cytometry, histology, immunohistochemistry and immunofluorescence and ELISA.

5 Can inhibiting IL-12p40 prevent or treat experimental crescentic glomerulonephritis?

Supervisor Professor Richard Kitching
Email Richard.kitching@monash.edu

IL-12p40 forms part of both IL-12 (important for Th1 responses) and IL-23 (important for Th17 responses). Targeting IL-12p40 is an approach to treating proliferative and crescentic forms of GN that could be successful in both Th1 and in Th17 mediated RPGN. Agents targeting IL-12p40 been effective in experimental non-renal inflammatory disease and are in Phase II clinical trials. This project will determine the capacity of apilimod (a small molecule IL-12p40 inhibitor) to inhibit experimental crescentic glomerulonephritis. An initial study will commence treatment at the induction of disease and continue for 21 days, later studies will commence at days 7 or day 10 and continue until day 21 or day 35.

Techniques involved in this project include in vivo animal work; molecular biology; flow cytometry, histology, immunohistochemistry and immunofluorescence and ELISA.

6 Induction of nephritogenic autoimmune anti-myeloperoxidase responses using a Staphylococcus aureus derived peptide

Supervisor Professor Richard Kitching
Email Richard.kitching@monash.edu

Supervisor Dr. Joshua Ooi
Email Joshua.ooi@monash.edu

Experimental data suggests that the loss of tolerance to myeloperoxidase (MPO), which is found prominently in neutrophils, leads to glomerulonephritis; known as MPO-ANCA associated glomerulonephritis. It has also been reported that in some patients a Staphylococcus aureus infection precedes the loss of tolerance to myeloperoxidase. This project will test the hypothesis that molecular mimicry by a Staphylococcus aureus derived peptide can lead to the loss of tolerance to myeloperoxidase and lead to MPO-ANCA associated glomerulonephritis.

Techniques involved in this project include in vivo animal work; lymphocyte proliferation assays, histology, immunohistochemistry and immunofluorescence; and ELISA and ELISPOT

7 The role of renal dendritic cells in a mouse model of kidney transplantation

Supervisor Professor Richard Kitching
Email Richard.kitching@monash.edu

Supervisor Dr. Sarah Snelgrove
Email Sarah.snelgrove@monash.edu

In Australia, approximately two-thirds of organ transplants are kidney transplants. When this procedure is performed, there is a period of time during which there is no blood supply to the kidney (termed ischaemia), followed by restoration of blood flow (reperfusion). Ischaemia/reperfusion injury (IRI) is a major cause of kidney dysfunction and has a crucial impact on graft survival. Renal IRI induces an influx of leukocytes including dendritic cells (DCs) to the kidney. DCs are antigen presenting cells which initiate tolerogenic and immunogenic immune responses. Renal DCs have not been extensively studied, and the importance of these cells in the kidney is only now being recognised. We are using a mouse model of IRI to mimic what happens during a kidney transplant operation. The aim of this project will be to investigate the role of renal DCs following IRI and to characterise the phenotype, recruitment and function of these cells in the kidney. Studies currently planned and offered as honours projects include

a)    the role of renal DCs in IRI under immunosuppression

b)    the effect of IRI on the antigen presenting capacity of renal DCS

c)    Which type of DC adheres best in the kidney

This project will utilize whole animal in vivo work, tissue culture, flow cytometry, cell transfer, working with TcR transgenic T cells and in vivo imaging by multi-photon microscopy.