Post on 21-Apr-2017
transcript
Sample to Insight
1
Host - Pathogen Interactions:Molecular Basis and Host Defense Mechanisms
Wei Cao, Ph.D.
Wei.Cao@qiagen.com
Sample to Insight
Welcome!
Host-pathogen interactions 2
Pathogen detection: Microbial DNA isolation
and identification to characterization
• Part 1: Host-pathogen Interactions: Molecular Basis and Host Defense Mechanisms
• Part 2: Microbiome: Isolate and enrich microbial DNA with our new protocol
• Part 3: Microbiome: From identification to characterization
Sample to Insight
Legal disclaimer
Host-pathogen interactions 3
• QIAGEN products shown here are intended for molecular biology
applications. These products are not intended for the diagnosis,
prevention or treatment of a disease.
• For up-to-date licensing information and product-specific
disclaimers, see the respective QIAGEN kit handbook or user
manual. QIAGEN kit handbooks and user manuals are available
at www.QIAGEN.com or can be requested from QIAGEN
Technical Services or your local distributor.
Sample to Insight
Agenda
Introduction to the microbiome
The host-pathogen interaction
The host defense mechanisms
Technologies for exploring host-pathogen interactions
Host-pathogen interactions 4
1
2
3
4
Sample to Insight
Agenda
Introduction to the microbiome
The host-pathogen interaction
The host defense mechanisms
Technologies for exploring host-pathogen interactions
Host-pathogen interactions 5
1
2
3
4
Sample to Insight
Introduction to the microbiome
Host-pathogen interactions 6
Cellular composition of the organism
Human
Microbiota
Estimations of the number of microbial cells that live in and on the human body, human cells are outnumbered by a factor of 10.
35 trillion human cells vs. 350 trillion microbial cells!
What does “microbiome” mean?
Microbes are microscopic organisms that can be either single or multicellular.
Microbiota are the microbes that live in a specific location, e.g. the human body, the gut, skin, soil, etc.
Human microbiome is defined as the collectivegenomes of the complete microbiota present in a human body.
Metagenomics is the study of the collection of genomes derived from a specific sample or community.
6
Sample to Insight
NIH Human Microbiome Project (HMP)
Host-pathogen interactions 7
The Common Fund's (HMP) is developing research resources to enable the study of the microbial communities that live in and on our bodies and the roles they play in human health and disease.
The first phase of HMP (FY2007-2012)• Characterize the composition and diversity of microbial
communities which inhabit major mucosal surfaces of the human body, and evaluated the genetic metabolic potential of these communities.
• ~10,000 organisms live with us (10 times more)
• ~ 8 ×106 genes in this genome (20-60% are not cultivable)
The second phase of HMP (FY2013-2015)• Develop tools and datasets for the research community for
studying the role of these microbes in human health and disease.
The HMP plans to sequence 3000 genomes from both cultured and uncultured bacteria, plus several viral and small eukaryotic microbes isolated from human body site.
http://hmpdacc.org/
Sample to Insight
Microbiome is everywhere
Host-pathogen interactions 8
The Microbiome: The trillions of microorganisms maintain health and cause disease in humans
SnapShot: The human microbiomeThe gut microbiota is profoundly altered during pregnancy• Microbial diversity is changed during pregnancy
• Shift in bacterial diversity is unrelated to health state
• Newborn babies are born with a distinct gut microbiome
Hoffmann A.R., et al. “The Microbiome: The Trillions of Microorganisms That Maintain Health and Cause Disease in Humans and Companion Animals.” Vet Pathol. 2015 Koren O. et al. “Host Remodeling of the Gut Microbiome and Metabolic Changes during Pregnancy”, Cell, 2012, 150, 470
Different body sites have unique communities• Each area of the body has its own
microbiome
The human microbiome plays a roles in many human diseases: diabetes, rheumatoid arthritis, muscular dystrophy, multiple sclerosis, fibromyalgia, CNS-related (such as schizophrenia, depression, bipolar disorder), and some cancers.
Sample to Insight
Agenda
Introduction to the microbiome
The host-pathogen interaction
The host defense mechanisms
Technologies for exploring host-pathogen interactions
Host-pathogen interactions 9
1
2
3
4
Sample to Insight
Host-microbiota interaction
• The host provides the microbiota a niche with a stable nutrient supply
• The microbiota performs essential functions for host physiology, including metabolic, digestive, and immune mechanisms
• Regulate the host’s metabolic function and energy balance
• Provide the host with the capacity to hydrolyze complex plant sugars, synthesize vitamins, and detoxify xenobiotics in a mutualistic context
• Affect the most fundamental of host physiological phenotypes, the rate of aging itself
Host-pathogen interactions 10
Dual necessity: Peacefully coexisting to achieve a mutually beneficial relationship.
Commensal gut microbiota protects the host from infection via both direct and indirect mechanisms.
The symbiosis between microbes and humans provides a stable and common metabolic pattern and well-balanced physiological homeostasis.
Caroline Heintz and William Mair, “You Are What You Host: Microbiome Modulation of the Aging Process”, 2014, Cell, 156:408Kamada N, et al. “Role of the gut microbiota in immunity and inflammatory disease”. 2013, Nat Rev Immunol. 13(5):321-35. Review.
10
Sample to Insight
Host-microbiota interaction
Host-pathogen interactions 11
Direct and indirect protection mechanisms of microbiota
Kamada N, et al. “Role of the gut microbiota in immunity and inflammatory disease”. 2013, Nat Rev Immunol. 13(5):321-35. Review.
Sample to Insight
Host-microbiota interaction
Host-pathogen interactions 12
Direct and indirect protection mechanisms of microbiota
Kamada N, et al. “Role of the gut microbiota in immunity and inflammatory disease”. 2013, Nat Rev Immunol. 13(5):321-35. Review.
Sample to Insight
Host-microbiota interaction
Host-pathogen interactions 13
Direct and indirect protection mechanisms of microbiota
Kamada N, et al. “Role of the gut microbiota in immunity and inflammatory disease”. 2013, Nat Rev Immunol. 13(5):321-35. Review.
Sample to Insight
Host-microbiota interaction
Host-pathogen interactions 14
The big question? How, what, who in the microbiota does what, when and how to the immune system?
Kamada N, et al. “Role of the gut microbiota in immunity and inflammatory disease”. 2013, Nat Rev Immunol. 13(5):321-35. Review.
• Identify microbiota• Virulence factors• Antibiotic resistance genes
Sample to Insight
The crosstalk between the microbiome and the immune system
• Impact lymphoid structure development and
epithelial function
• Enhance innate immunity to pathogens
• Shape T cell subsets
• Provide protective roles against systemic
infection
• Influence invariant T cells and innate lymphoid
cells
• Trigger inflammation in immunocompromised
hosts
• Protect against autoimmune diseases
Host-pathogen interactions 15
How does microbiota shape immunity: The cellular and molecular mediators
Lora V. Hooper et al., Interactions Between the Microbiota and the Immune System. 2012, Science 336:1268
Sample to Insight
The crosstalk between the microbiome and the immune system
“The mammalian immune system plays an essential
role in maintaining homeostasis with resident
microbial communities, thus ensuring that the
mutualistic nature of the host-microbial relationship
is maintained.”
The critical controls of the immune system
1. Exerts control over stratification and compartmentalization of the microbiota
2. Exerts control over microbiota composition
The immune system exerts critical controls over microbiota composition, diversity, and location.
Host-pathogen interactions 16
The control of immune system over microbiota
Lora V. Hooper et al., Interactions Between the Microbiota and the Immune System. 2012, Science 336:1268
Sample to Insight
The importance of microbiota in human health and diseases
Host-pathogen interactions 17
When the mutualistic relationship between the host and microbiota is disrupted, the gut microbiota can cause or contribute to diseases.
Kinross et al. “Gut microbiome-host interactions in health and disease.” 2011, Genome Medicine, 3:14. Review.
Diseases influenced by gut microbial metabolism
Sample to Insight
The host responses
It is important to note that commensal bacteria do not always
protect against pathogenic infection and in certain contexts they
can facilitate it.
Under certain conditions, particular bacterial populations can
acquire pathogenic properties.
Host-pathogen interactions 18
The challenge that the host faces
How does the host discriminate between symbiotic and
pathogenic bacteria to adjust its level of immune response?
Sample to Insight
Agenda
Introduction to the microbiome
The host-pathogen interaction
The host defense mechanisms
Technologies for exploring host-pathogen interactions
Host-pathogen interactions 19
1
2
3
4
Sample to Insight
The immune responses: Innate and adaptive response
Host-pathogen interactions 20
The innate immune system is the first line of defense against pathogens and is initiated by genome-encoded pattern recognition receptors (PRRs) that recognize PAMP
• Non-specific and does not confer long-lasting immunity (memory)
• Immune cells: dendritic cells (DCs) and macrophages, intestinal epithelial cells and myofibroblasts
The adaptive immunity is highly specific, confers long lasting immunity, and is adaptable
• Cooperation between the molecules and cells of the innate immune system mounts an effective immune response
• Key players: T cells – Th1, Th2 or Th17 cell
Sample to Insight
Innate immune responses: Toll-like receptors
Host-pathogen interactions 21
Pattern recognition receptors (PRRs) include Toll-like
receptors (TLRs), NOD-like receptors (NLRs) and RIG1-
like receptors (RLRs), etc.
• PRRs signaling cascades result in nuclear factor (NF)-kB
activation of gene transcription and production of pro-
inflammatory mediators
• PRRs also play a crucial role in the crosstalk between innate
and adaptive immune responses by promoting antigen
presenting cell maturation and T cell activation
• TLRs induce the expression of genes required for the
inflammatory response, including inflammatory cytokines,
chemokines, antimicrobial molecules, and major
histocompatibility (MHC) and costimulatory molecules
important for adaptive immune activation
Download map: https://www.qiagen.com/pathway-details.aspx?pwid=445
Sample to Insight
Adaptive immune responses
Host-pathogen interactions 22
Adaptive responses in the gut
• Th1 and Th2 cells• Th17 cells• Regulatory T cells
During active inflammation, Th0 cells differentiate into T helper cells Th1, Th17 and Th2
• Th1 cells produce interferon (IFN)-γ and tumor necrosis factor (TNF)-α
• Th2 cells are a major source of IL-13• Th17 cells release IL-17 and IL-21
Important adaptive immune cells
pDC detect viral antigen and release type I IFN
• Activation of mDC, mV, CD4 , and CD8 T cells
• FRCs secrete chemokines and T cell survival factors
• FDCs coordinate B cell migration and B cell and CD4 T cell interactions
Sample to Insight
Host defense mechanisms: Summary
Host-pathogen interactions 23
Lance W. Peterson and David Artis, “Intestinal epithelial cells: regulators of barrier function and immune homeostasis”, Nat Rev Immunol. 2014, 14(3):141-53.
Sample to Insight
Agenda
Introduction to the microbiome
The host-pathogen interaction
The host defense mechanisms
Technologies for exploring host-pathogen interactions
Host-pathogen interactions 24
1
2
3
4
Sample to Insight
Technologies for exploring host-pathogen interactions
Host-pathogen interactions 25
Characterize the composition and function of the gut microbiome and the contribution of microbes to human health.• Genomics – characterize DNA
• Transcriptomics – characterize RNA
• Metabolomics – characterize small molecules
• Proteomics – characterize proteins and peptides
• Whether changes in the composition of the microbiome are associated with or cause a disease.
• How do microbial functions change in a disease at the DNA, RNA, protein, and metabolite levels?
• How do metabolic processes change in a disease?
• How does an intervention or a treatment affect the composition and function of the microbial community?
Sample to Insight
Technologies for exploring host-pathogen interactions
Host-pathogen interactions 26
16S rRNA gene sequencing
18S rRNA gene sequencing
Total DNA sequencing(shotgun)
Bacteria and Archaea
Fungus/Yeast
Viruses Gene content
RNA expression profiling (transcriptomics)
Gene expressionMetabolite
characterization
Mass spectroscopy(metabolomics)
Identify relative frequencies and pathways
Human microbiome sample
Extract DNA Extract RNA Extract small molecules
What organisms are present and what is their relative abundance? What are the functions of the community?
Sample to Insight
The research questions
How can we identify the microbiome and
monitor innate and adaptive immune
responses?
Host-pathogen interactions 27
Sample to Insight
Technologies for exploring host-pathogen interactions
Host-pathogen interactions 28
Microbial DNA qPCR Assay/Assay
Detect microbial species, virulence genes, or antibiotic resistance genes
• QIAamp DNA Microbiome Kit• QIAamp Fast DNA Stool Mini Kit• QIAamp UCP Pathogen Mini Kit• QIAamp UCP PurePathogen Blood Kit• QIAamp MinElute Media Kit
RNA expression
Sample to Insight
Allprotect Tissue Reagents
• QIAamp DNA Microbiome Kit• QIAamp UCP Pathogen Mini Kit• QIAamp DNA Stool Mini Kit
REPLI-g Single Cell Kit• GeneRead Library Prep
Kits• GeneRead Size
Selection Kit• GeneRead Library
Quant System
• Microbial DNA qPCR Arrays
• Microbial DNA qPCR Assays
• Custom Microbial DNA qPCR Arrays
Sample to Insight workflow
Detecting microbial metagenomes: A complete solution
Host-pathogen interactions 29
QIAGEN provides next-generation sequencing technologies for metagenomics, as well as qPCR assays and arrays for verification of sequencing results and screening for specific bacterial species, virulence factor genes, and antibiotic resistance genes.
Part 2: Microbiome: isolate and enrich microbial DNA with our new protocol
Sample Collection
DNA purification
DNA amplification
Library preparation
Verification by qPCR
Sample to Insight
QIAGEN’s Microbial DNA qPCR assay pipeline
>600 bacteria identification assay
8 Fungi identification assay
1 Protist identification assay
87 Antibiotic resistance genes
87 Virulence factor genes
18 Arrays
Host-pathogen interactions 30
Reveal the mysteries of the microbiome with over 600 assays that target species-specific or gene-specific microbial DNA
• Antibiotic Resistance Genes• Bacterial Vaginosis• Biodefense• Food testing: Dairy• Food testing: Meat• Food testing: Poultry• Food testing: Seafood• Food testing: Vegetable• Intestinal Infections• Intestinal Infections 2• Metabolic Disorders• Oral Disease• Respiratory Infections• Respiratory viral• Sepsis • Urinary Tract Infections• Vaginal Flora • Water Analysis
Part 3: Microbiome: From Identification to Characterization
DNA Purification
Detection by qPCR
Data analysis
Sample to Insight
Technologies for exploring host-pathogen interactions
Host-pathogen interactions 31
A complete workflow for host responses profiling
• Profiling gene expression from immune cells (especially cytokines, chemokines and other immune molecules) can be interpreted into immune system “status”
• Inflammation?• Early• Chronic• Resolution• Type of response (bacterial, viral, other)?
RT2 Profiler PCR Arrays
Sample Collection
Sample Preparation
DNA Purification
Gene expression
analysis
Data analysis
Sample to Insight
Host response profiling: gene expression
Host-pathogen interactions 32
Profile 84 or 370 genes simultaneously with RT2 Profiler PCR Arrays
• Antifungal Response
• Antiviral Response
• Antibacterial Response
• Innate & Adaptive Immune Responses
• Inflammatory Cytokines & Receptors
• Dendritic & Antigen-Presenting Cells
• Inflammasomes
• Th1 & Th2 Responses
• Toll-Like Receptor Signaling Pathway
• IFN-a/b Response
• NFkB Signaling
• NFkB Signaling Targets
• MAPK Signaling
• PI3K/AKT Signaling
• 140+ pathways, including custom arrays
• Wet-bench validated assays for all genes of 13 species
Sample to Insight
PCR Arrays format and contents
Host-pathogen interactions 33
• 84 pathway-specific genes of interest
• 5 housekeeping genes
• Genomic DNA contamination control (GDC)
• Reverse transcription controls (RTC), n=3
• Positive PCR controls (PPC), n=3
• Customizable – Add 4 genes to a catalog RT2 Profiler PCR Array, or Completely customize based on your research.
Each well contains lyophilized – “Verified qPCR Assay”
ACTB, B2M, GAPDH, HPRT1, RPL0
Sample to Insight
How RT2 Profiler PCR arrays work?
Host-pathogen interactions 34
Control Sample
Isolate total RNA• cDNA Synthesis
• Genomic DNA Removal Step (5 min.)
• Reverse Transcription Step (20 min.)
• Load Plates • One sample per PCR Array
• Two minutes with multi-channel pipet
• Run 40 cycle qPCR Program • Standard cycling conditions for all
real time PCR instruments
• 2 hours
• Upload and Analyze Data (FREE)• 15 minutes from raw Ct to fold
change data
Sample to Insight
How RT2 Profiler PCR arrays work?
Host-pathogen interactions 35
Control Sample
Isolate total RNA• cDNA Synthesis
• Genomic DNA Removal Step (5 min.)
• Reverse Transcription Step (20 min.)
• Load Plates • One sample per PCR Array
• Two minutes with multi-channel pipet
• Run 40 cycle qPCR Program • Standard cycling conditions for all
real time PCR instruments
• 2 hours
• Upload and Analyze Data (FREE)• 15 minutes from raw Ct to fold
change data
Sample to Insight
How RT2 Profiler PCR arrays work?
Host-pathogen interactions 36
Control Sample
Isolate total RNA• cDNA Synthesis
• Genomic DNA Removal Step (5 min.)
• Reverse Transcription Step (20 min.)
• Load Plates • One sample per PCR Array
• Two minutes with multi-channel pipet
• Run 40 cycle qPCR Program • Standard cycling conditions for all
real time PCR instruments
• 2 hours
• Upload and Analyze Data (FREE)• 15 minutes from raw Ct to fold
change data
Sample to Insight
How RT2 Profiler PCR arrays work?
Host-pathogen interactions 37
Control Sample
Isolate total RNA• cDNA Synthesis
• Genomic DNA Removal Step (5 min.)
• Reverse Transcription Step (20 min.)
• Load Plates • One sample per PCR Array
• Two minutes with multi-channel pipet
• Run 40 cycle qPCR Program • Standard cycling conditions for all
real time PCR instruments
• 2 hours
• Upload and Analyze Data (FREE)• 15 minutes from raw Ct to fold
change data
Sample to Insight
RT2 Profiler PCR Array data analysis
Host-pathogen interactions 38
• Free complete and easy analysis with web/excel-based software• Multiple analysis formats to interpret gene expression results
Volcano Plot
Scatter Plot Clustergram
Sample to Insight
Application data
Host-pathogen interactions 39
How are cytokines regulated under PMA-Ionomycin treatment?
Human PBMCs were treated with PMA and ionomycin, and then analyzed using the Common Cytokines RT2 Profiler PCR Array. This volcano plot shows both fold-change and the statistical significance, and demonstrates that 23 genes, including IL-10, IFN-gamma, IL-2, and TNF were upregulated, while IL-1beta and 5 other genes were downregulated in response to treatment.
Next step – verify results by ELISA for only the cytokines which are changing.
Sample to Insight
Host response profiling: Gene expression
Host-pathogen interactions 40
mRNA and lncRNA Expression profiling
T and B cellsT and B lymphocytes tailor their responses to each pathogenic insult as part of the adaptive immune system.
• Tools for T and B cell Research
Cancer Inflammation & Immunity CrosstalkIdentify the mediators of communication between tumor cells and the cellular mediators of inflammation and immunity.
• Exploring the cancer immune responses
Innate Immunity: The first line of defenseThe innate immune response attracts immune cells and activates the adaptive response to control novel foreign pathogens
• Identify an innate immune response
Which toll-like receptors are signaling?Toll-like receptors recognize broad classes of microbe, activating downstream signaling pathways that initiate a tailored immune response.
• Profile TLR activity
Sample to Insight
A new webinar series in March
Host-pathogen interactions 41
• Part 1: Exploring the First Line of Defense: Research Tools for the Innate Immune System
• Part 2: Toll-like Receptors in Inflammation
• Part 3: Study the Adaptive Immune Response: Tools for T and B cell Research
• Part 4: The Crosstalk between Cancer Inflammation and Immunity – Exploring the cancer immune responses
Explore the Host response and Defense Mechanisms - A 4-Part Webinar Series
Sample to Insight
Agenda
Introduction to the microbiome• Human microbiome - microbiota• The complex host-microbe relationship• The impact of microbiome on human health and disease
The host-pathogen interaction• The protection mechanisms of microbiota
• The control of immune system over microbiota
The host defense mechanisms• Innate immune response
• Adaptive immune response
Technologies for exploring host-pathogen interactions• Characterize the composition and function of the gut microbiome
• Profile innate and adaptive immune response
Host-pathogen interactions 42
1
2
3
4
Sample to Insight
Upcoming webinars
Host-pathogen interactions 43
Pathogen detection: Microbial DNA isolation
and identification to characterization
• Part 1: Host-pathogen interactions: Molecular basis and host defense mechanisms
• Part 2: Microbiome: Isolate and enrich microbial DNA with our new protocol
• Part 3: Microbiome: From identification to characterization
Sample to Insight
Questions?
Thank you for attending
Host-pathogen interactions 44
Contact QIAGEN Technical ServiceCall: 1-800-426-8157 for USCall: +49 2103-29-12400 for EUEmail: techservice-na@qiagen.comtechservice-eu@qiagen.com
QIAwebinars@qiagen.com