Host-pathogen Interactions, Molecular Basis and Host Defense: Pathogen Detection Webinar Series Part 1

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Host - Pathogen Interactions:
Molecular Basis and Host Defense Mechanisms
1
Wei Cao, Ph.D.
Wei.Cao@qiagen.com

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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 DNAwith our new
protocol
• Part 3: Microbiome: From identification to characterization

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Legal disclaimer
• 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.

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Agenda
Introduction to the microbiome
The host-pathogen interaction
The host defense mechanisms
Technologies for exploring host-pathogen interactions
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Agenda
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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 collective
genomes 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.
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NIH Human Microbiome Project (HMP)
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/

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Microbiome is everywhere
The Microbiome: The trillions of microorganisms maintain health and cause disease in
humans
SnapShot: The human microbiome
The 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: TheTrillionsof MicroorganismsThat Maintain Health and Cause Disease in Humansand Companion Animals.” Vet Pathol. 2015
Koren O. et al. “Host Remodeling of the Gut Microbiome and Metabolic Changesduring 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.

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Agenda
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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
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 andWilliam Mair, “You Are What You Host: MicrobiomeModulationof the AgingProcess”, 2014, Cell, 156:408
Kamada N, et al. “Role of the gut microbiota inimmunity and inflammatory disease”. 2013, Nat Rev Immunol. 13(5):321-35. Review.
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Direct and indirect protection mechanisms of microbiota

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The big question? How, what, who in the microbiota does what, when and how to
the immune system?
• Identify microbiota
• Virulence factors
• Antibiotic resistance genes

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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
How does microbiota shape immunity: The cellular and molecular mediators
Lora V. Hooper et al., InteractionsBetween the Microbiota andthe Immune System. 2012,Science 336:1268

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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.
The control of immune system over microbiota
Lora V. Hooper et al., InteractionsBetween the Microbiota andthe Immune System. 2012,Science 336:1268

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The importance of microbiota in human health and diseases
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 interactionsin healthand disease.” 2011, GenomeMedicine, 3:14.Review.
Diseases influenced by gut microbial metabolism

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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.
The challenge that the host faces
How does the host discriminate between symbiotic and
pathogenic bacteria to adjust its level of immune response?

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Agenda
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The immune responses: Innate and adaptive response
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

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Innate immune responses: Toll-like receptors
Pattern recognition receptors(PRRs) include Toll-like
receptors (TLRs), NOD-likereceptors (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

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Adaptive immune responses
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

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Host defense mechanisms: Summary
Lance W. Peterson and David Artis, “Intestinal epithelial cells: regulatorsof barrier functionand immune homeostasis”, Nat Rev Immunol. 2014, 14(3):141-53.

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Agenda
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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?

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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 expression
Metabolite
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?

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The research questions
How can we identify the microbiome and
monitor innate and adaptive immune
responses?

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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

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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: Acomplete solution
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 microbialDNAwith our new protocol
Sample
Collection
DNA
purification
DNA
amplification
Library
preparation
Verification
by qPCR

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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
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

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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

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Host response profiling: gene expression
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 SignalingPathway
• IFN-a/b Response
• NFkB Signaling
• NFkB Signaling Targets
• MAPK Signaling
• PI3K/AKT Signaling
• 140+ pathways, including customarrays
• Wet-bench validated assays for all genes of 13 species

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PCR Arrays format and contents
• 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

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How RT2 Profiler PCR arrays work?
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 forall real
time PCR instruments
• 2 hours
• Upload and Analyze Data (FREE)
• 15 minutes from raw Ct to fold
change data

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Control Sample
Isolate total RNA
• cDNA Synthesis
• Load Plates
• 2 hours
change data

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RT2 Profiler PCR Array data analysis
• Free complete and easy analysis with web/excel-based software
• Multiple analysis formats to interpret gene expression results
Volcano Plot
Scatter Plot Clustergram

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Application data
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 downregulatedin response to treatment.
Next step – verify results by ELISA for only the cytokines which are changing.

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Host response profiling: Gene expression
mRNA and lncRNA
Expression profiling
T and B cells
T 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 Crosstalk
Identify 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 defense
The 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

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A new webinar series in March
• 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 Crosstalkbetween Cancer Inflammation and
Immunity – Exploring the cancer immune responses
Explore the Host response and Defense Mechanisms - A 4-
Part Webinar Series

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Agenda
• Human microbiome - microbiota
• The complex host-microbe relationship
• The impact of microbiome on human health and disease
• The protection mechanisms of microbiota
• The control of immune system over microbiota
• Innate immune response
• Adaptive immune response
• Characterize the composition and function of the gut microbiome
• Profile innate and adaptive immune response
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Upcoming webinars
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 DNAwith our new
protocol
• Part 3: Microbiome: From identification to characterization

Sample to Insight
Questions?
Thank you for attending
Contact QIAGEN Technical Service
Call: 1-800-426-8157 for US
Call: +49 2103-29-12400 for EU
Email:
techservice-na@qiagen.com
techservice-eu@qiagen.com
QIAwebinars@qiagen.com

Host-pathogen Interactions, Molecular Basis and Host Defense: Pathogen Detection Webinar Series Part 1

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