David M. Underhill, Ph.D.

Inflammatory Bowel & Immunobiology Research Institute Cedars-Sinai Medical Center, Los Angeles

& UCLA Dept. of Pathology and Laboratory Medicine David M. Underhill, Ph.D.

http://www.customprobiotics.com/about_probiotics.htm

… but it’s all about bacteria

10 x more microbial cells in the body than human cells

100 x more microbial genes in the body than in the human genome Important for digestion, obesity, immunity, ….

Ringworm, Athlete’s foot, etc. - dermathophytes Pneumocystis pneumonia

Sporotrichosis is a fungal disease, usually of the skin, caused by Sporothrix schenckii.

Blastomycosis affects mainly the lungs and the skin -Blastomyces dermatitidis

mucocutaneous candidiasis, thrush , Vaginal candidiasis, or other skin infections – serious systemic infections

Chromoblastomycosis - rhinocladiella species

Coccidioidomycosis (Coccidioides immitis), San Joaquin Valley fever

cryptococcal meningoencephalitis

Lichens are the combination of a fungus with a green alga or cyanobacteria

Mycorrhizas are associations between fungi and plant roots

Cow Rumen - 1011 _{organisms/gram & around 8%} are fungi.

i.e. Cyllamyces aberensis - an anaerobic gut fungus

Ants and Termites “farm” fungi for food and to decompose plant matter Black truffle -Tuber melanosporum Vittad.-

ectomycorrhizal symbiosis on haselnut tree and oaks

Generally report 0n the order of 0.1% fungal…

Average fungus (1 genome) is 50X larger than an average bacterium and is encased in a cell wall that is harder to extract DNA from than bacterial cell walls

PCR with pan-fungus primers

GREEN = anti-Fungi Blue = DAPI

GREEN = anti-Fungi Blue = DAPI

•The first biomarker that was capable of identifying the majority of patients with

Crohn’s disease.

•50-60% of patients with Crohn’s disease have circulating antibodies to fungi

(ASCA).

•The antigen is yeast mannan and is part of the cell wall of nearly all fungi.

•More recently, anti-glucan and anti-chitin antibodies have also been measured in

serum from IBD patients.

Syk Dectin-1 Src Phagocytosis Respiratory Burst NF-κB, NFAT, SRF

C-type lectin domain

• Expressed by Macrophages, Dendritic cells, Neutrophils • Recognizes Fungal Cell Wall β-glucan

• Dectin-1 deficient mice are highly susceptible to a variety of pathogenic fungi • Dectin-1 deficient people are similarly susceptible to fungal infections

Goodridge et al. Nature. 2011

Yeast

Gantner et al. Embo J. 2005, Gersuk et al. J Immunol. 2006

Dectin-1 Soluble Dectin-1 (sDec)

Zymosan

Candida albicans

RED = Soluble Dectin-1 Probe Blue = DAPI Recombinant, Fluorescently Labeled Soluble Dectin-1

Dectin-1 binding blocked with soluble glucans Fluconazole treatment of mice depletes fungi Both probes show that 5-7% of the intestinal material is fungal

So, there’s lots of

fungus there, does

it matter?

Littermate controls

CD4+ _{T cells from lamina propria} (LI-LP) and mesenteric lymph nodes (MLN) 4 days after the 7 days of DSS treatment

Commensal fungi include over 200species representing over 50 genera

Food Feces

Candida spp.: •Candida albicans •Candida tropicalis •Candida glabrata •Candida parapsilosis •Normal microflora: organism commonly found on mucosal surfaces and on skin •Pathogenic infection especially common in immunosuppressed patients such as in AIDS or cancer chemotherapy Saccharomycopsis fibuligera •Common environmental fungus •Especially good at degrading starches •Industrially used in the

fermentation of starches to produce ethanol

Where to start?

IBD is a complex disease with many flavors

Our mouse data implicate Dectin-1 in a DSS model – a model of ulcerative colitis Our Dectin-1 mice do not spontaneously develop colitis, they simply have a more

severe disease

So, perhaps were interested in UC disease severity…

Haplotype Frequency In: rs2078178 rs16910631 MRUC Non-MRUC N=323 N=493 G G 0.45 0.54 G G G G 0.36 0.28 A G A G 0.092 0.045 A G Other Combinations 0.10 0.14

P

_{logistic regression}

= 0.00027

Dectin-1 Association with MR-UC

Over 20 year, folks with this Dectin-1 variant progress to surgery faster.

Underhill Lab

Christopher Reyes Courtney Becker

Cedars-Sinai Genomics & Bioinformatics Core

Vincent Funari Quoclinh Nguyen Jordan Brown Sam Strom

UCLA Pathology & Lab. Med.

Hanlin Wang

University of Aberdeen

Gordon Brown, PhD

David Underhill, PhD ([email protected])

Iliyan Iliev, PhD

And finally, the people who did the work…

Cedars-Sinai IBD & Immunobiology Institute

Dermot P.B. McGovern Phillip R. Fleshner Marla Dubinsky

Cedars-Sinai Medical Genetics Institute