Centre for Molecular Medicine


Material on offer

September 2019

Dear Colleagues,

An offer of scientific material to colleagues in related fields

Over the last few years I have spent a considerable amount of time and effort on writing applications for funding in various fields of science: Predominantly on monocyte and macrophage function and on the role of innate immunity in the cause of Crohn's disease.

Although these applications were not funded, they have taken a considerable amount of work and I am confident that the science contained within them is sound, interesting and important.

I am at the stage in my life at which I will be unable to fund and complete this work and I am therefore offering these applications for use by the scientific community. They can be used as they are to do the work described,  or can be combined with other, complimentary investigations. 

Feel free to share this information with colleagues that might not have received this letter.

The applications can be accessed at:

The titles of the applications and the abstracts are shown below.

1. Monocyte macrophage dysfunction in the pathogenesis of atherosclerosis

2018 British Heart Foundation 

Atherosclerosis results from the accumulation of cholesterol and other lipids in the intima, initially within macrophages that form foam cells, and subsequently free within the intestitium, leading to inflammation and scarring. While clinical factors such as hyperlipidaemia, smoking and diabetes commonly drive this process, we hypothesise that some patients, especially those without overt risk factors, may be predisposed to atherosclerosis because their macrophages have a reduced ability to digest lipids phagocytosed from lipoproteins and efferocytotic cells. Digestion within mononuclear phagocytes takes place within the phagocytic vacuole and depends upon the enzymes released into it from the cytoplasmic granules, or lysosomes, and the suitability of the conditions within this compartment for their activity. Monocytes and macrophages have more recently been separated into Classical, non-Classical and Intermediate monocytes, M1 and M2 macrophages and at least 3 subtypes of Dendritic cells based upon their surface markers.
In 20 patients with early atherosclerosis in the absence of traditional risk factors and 20 matched controls without atherosclerosis, we will:

  • Separate the monocyte and macrophage subtypes and compare the profiles in cases and controls;
  • Measure the pH within their phagocytic vacuoles using SNARF labelled Candida, a method we developed;
  • Quantify efferocytosis and the digestion of radiolabelled efferocytosed cells;
  • Purify the cytoplasmic granules and analyse their contents by proteomics;

Taken together these parameters will indicate whether or not aberrant mononuclear cell function may be responsible for the accumulation of atherosclerotic plaque in these patients, and as such represent a novel mechanism for predisposition to atherosclerosis. 

2. The effect of pH on phagocytic vacuole biology in monocytes, macrophages and dendritic cells

2018 Wellcome Trust

We developed a novel method for the measurement of pH within the phagocytic vacuole of neutrophils. We found the pH in this compartment to be alkaline, at about 9.0, optimal for neutral proteases which kill and digest microbes. We have applied this measurement of pH to monocytes, macrophages and dendritic cells and found the pH to be alkaline in Classical monocytes and M1 macrophages and acidic in Non-classical monocytes, M2 macrophages and blood and monocyte derived dendritic cells. The pH of the phagocytic vacuoles will have a major impact on the enzymic action within that compartment and on the function of the cell as a whole. Accordingly I wish to identify the constituent proteins in the granules that are released into, and dictate the function of the vacuoles, of these different cell types, and monocyte/macrophage cell lines. The cells will be sorted, granules purified and their components identified using proteomics. In the different cell types we will:

  • Study the cell biology of the enzymes.
  • Investigate bacterial killing.
  • Measure digestion, antigen presentation and efferocytosis.
  • Examine the consequences of targeting genes for the major enzymes on the above processes.
  • Measure pH changes and granule components in patients with SLE.

3. Cause of Crohn's disease

ERC Advanced Grant 2016

Crohn’s disease (CD) is caused by the penetration of intestinal contents into the wall of the bowel as a result of damage to the mucosa by enteric infections. In susceptible individuals the reaction to the faeces in the tissues is abnormal, resulting in impaired clearance of the foreign material and the development of a chronic granulomatous inflammation. 

The incidence of CD is rising rapidly in many developed countries, the cause of which is unclear and has been attributed to the “hygiene hypothesis”. Enteric infections can be sexually transmitted and the epidemiology of CD indicates that the recent rise in its incidence could be due in part to an increase in oral and anal heterosexual practises. This possibility has important social consequences and must be thoroughly investigated.

The dominant constitutional predisposition to CD is a failure of the acute inflammation as a result of impaired secretion of pro-inflammatory cytokines by macrophages. We have undertaken a series of investigations to determine the underlying molecular causes. We found that Optineurin is under-expressed in CD macrophages which, together with NOD2, we will characterise it further. We have also performed genetic studies in large Ashkenazi Jewish families. Exome sequencing identified a validated causal variant in CSF2RB and several strong candidate molecules, particularly DUOX2, LRRK2, NLRP2, and HEATR3. We will knock the human mutations into mice to determine whether they predispose the animals to bowel inflammation, and if they do we will characterise the mechanisms involved. 

Because in most cases the cellular abnormality in CD involves macrophages, their progenitor cells are present in bone marrow. Theoretically it should be feasible to aspirate these cells, edit out the pathological mutation in the DNA by CRISPR-Cas gene editing, and reinject normally functioning cells into conditioned hosts. This will be tested in the mouse models as a prelude to treatment of patients.

4. Mechanisms and molecules involved in the pathogenesis of Crohn's disease

2017 Wellcome Trust

It is increasingly accepted that there is an underlying predisposition to Crohn's disease (CD) that involves innate immunity. We used a genetic approach to identify causal molecules. We concentrated on Ashkenazi Jews (AJs) because they have a fourfold incidence of CD, and may have large families. We identified two such families with ~800 and ~250 individuals with 54 and 26 cases of CD respectively. We have also studied large numbers of sporadic cases of CD in AJs. We have investigated genetic factors in these subjects, including exome sequencing of their DNA. We have identified several genetic variants that are likely to be causally related to CD. A frameshift mutation in CSF2RB has been validated, as have variants in LRRK2, and variants in DUOX2 and NLRP2 are strong candidates.

Key goals are:

  • The further analysis of the large AJ CD families to identify asymptomatic carriers and phenocopies, the macrophage phenotype and their microbiome.
  • To validate candidate variants in CSF2RB, DUOX2 and LRRK2, (+/- NOD2-/-) in Knock-in and Knock-out mice, in the predisposition to bowel inflammation and immune function.
  • To study these molecules, and NLRP2, in cell lines to determine their binding partners and signalling networks.

5. CSF2RB and DUOX2 knock-out/knock-in mice

Details of the generation of CSF2RB S708A and DUOX2 P303R mice, done in collaboration with Professor Werner Muller and Dr Antony Adamson (Genome Editing Unit, Faculty of Biology, Medicine and Health University of Manchester, AV Hill Building, M13 9PT, United Kingdom). If you wish to breed from the frozen embryos please contact Antony.Adamson@manchester.ac.uk.

Good luck!

Yours sincerely,

Tony Segal

AW Segal