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

Wei Cao, Ph.D.

[email protected]

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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 DNA with 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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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.

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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/

http://hmpdacc.org/

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 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.

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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 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.

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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.

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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., Interactions Between the Microbiota and the 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., Interactions Between the Microbiota and the 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 interactions in health and disease.” 2011, Genome Medicine, 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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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-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

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: regulators of barrier function and immune homeostasis”, Nat Rev Immunol. 2014, 14(3):141-53.

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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 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?

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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

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Detecting microbial metagenomes: A complete 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 microbial DNA with 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 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

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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 for all

real time PCR instruments

• 2 hours

• Upload and Analyze Data (FREE)• 15 minutes from raw Ct to fold

change data

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• 2 hours


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• 2 hours


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• 2 hours


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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 downregulated in 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 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

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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 Crosstalk between 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

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


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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 DNA with our new protocol

• Part 3: Microbiome: From identification to characterization

Sample to Insight

Questions?

Thank you for attending


Contact QIAGEN Technical ServiceCall: 1-800-426-8157 for USCall: +49 2103-29-12400 for EUEmail: [email protected]@qiagen.com

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

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