Histocompatibility and Immunogenetics E-Book
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- Herausgeber: Wiley-VCH
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
The EFI-, ASHI-, and APHIA-endorsed professional guide for laboratory diagnostics in transplantation medicine
In Histocompatibility and Immunogenetics: A Guide for Practitioners, expert authors from across the globe deliver an authoritative introduction to the concepts, methods, and clinical applications of matching donors with acceptors of transplanted cells and organs. Fundamental concepts and limitations of current immunogenetic as well as histocompatibility diagnostic approaches are explained, and best practices to improving the outcomes of transplantation therapies are discussed. Readers will also find:
- A holistic view of transplantation diagnostics, including drug and disease associations with histocompatibility markers
- Options for the medical treatment of organ rejection
- Practical discussions of specific methods in immunogenetics and histocompatibility, such as crossmatching in transplantation and KIR genotyping
- A thorough introduction to the regulatory aspects of histocompatibility and immunogenetics
Perfect for clinical geneticists and immunologists, Histocompatibility and Immunogenetics will also benefit medical and research professionals working in medical laboratories and hospitals.
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Veröffentlichungsjahr: 2025
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Table of Contents
Cover
Table of Contents
Title Page
Copyright
Dedication
Preface
Part I: Biological Basics
1 Innate Immune Defence
1.1 Introduction
1.2 Anatomical Barriers and Initial Chemical Defenses
1.3 The Complement System
1.4 Danger Recognition
1.5 Inflammatory Reaction and Effector Cells of the Innate Immune Response
1.6 Bridging Innate and Adaptive Immunity
1.7 Conclusion
1.8 Perspective: The Concept of Trained Immunity
Abbreviations
References
2 Specific Immune Defence
2.1 Introduction
2.2 Components of the Adaptive Immune System
2.3 The Major Histocompatibility Complex and Adaptive Immune Responses
2.4 T Cell‐Mediated Immune Response
2.5 B Cell‐Mediated Immune Response
2.6 Receptors of the Specific Immune System
2.7 Immunological Memory
2.8 Conclusion/Summary/Future
References
3 The HLA/MHC System
3.1 Introduction
3.2 IPD‐IMGT/HLA Database
3.3 Limits to HLA Variation
3.4 Summary
References
4 The HNA System
4.1 Neutrophils and Associated Functions
4.2 Human Neutrophil Antigens (HNA)
4.3 Clinical Conditions Associated with HNA Specific Antibodies
References
5 Natural Killer Cell Receptors
5.1 Introduction
5.2 NK Cells
5.3 “Missing Self” Hypothesis and Hybrid Resistance
5.4 NK Cell Receptors
5.5 Killer Immunoglobulin‐Like Receptors (KIRs)
5.6 CD94‐NKG2 Receptors
References
6 The HPA System
6.1 Platelet Antigens
6.2 Human Platelet Antigens
References
7 Other Polymorphic Systems
7.1 Introduction
7.2 The Structure of HLA‐like MICA and MICB Molecules
7.3 The Functions of HLA‐like MICA and MICB Molecules
7.4 The Expression of HLA‐like MICA and MICB Molecules
7.5 The Polymorphisms of HLA‐like MICA and MICB Molecules and Functional Consequence
7.6 Nonclassical HLA Class I
7.7 Key Differences Between Nonclassical HLA Class I and Classical HLA Class I Molecules
7.8 The Expression of HLA‐E Molecules
7.9 The HLA‐E Peptide Presentation Controls the Functional Interaction with NK and T Cells
7.10 The Polymorphisms of HLA‐E and Its Influence on Gene Expression
7.11 The Expression of HLA‐G
7.12 The Unique Structural Features of HLA‐G
7.13 The Functions of HLA‐G are Directed by the Structural Features
7.14 The Polymorphisms of HLA‐G and Its Impact on Gene Expression
7.15 The Expression of HLA‐F
7.16 The Functions of HLA‐F are Directed by Its Structural Features
7.17 The Polymorphisms of HLA‐F and Its Impact on Gene Expression
7.18 Summary
References
Part II: Clinical Applications
8 Basic Concepts of Organ Transplantation
8.1 Introduction
8.2 Overview of the Decision‐Making Procedure
8.3 Registration and Waiting List
8.4 Procurement of the Organs
8.5 Postmortem Allocation
8.6 The Crossmatch
8.7 Prospective Outlooks
References
Note
9 Basic Concepts of Organ Rejection
9.1 Introduction
9.2 The Immune System: Balancing Self and Foreign Recognition
9.3 Antigens: Self and Foreign?
9.4 Immune Tolerance: Maintaining the Balance
9.5 Autoimmunity
9.6 Foreign Recognition Leading to Rejection
9.7 GvHD
9.8 Conclusion
References
10 Treatment of Organ Rejection
10.1 Introduction
10.2 Prevention of Rejection: Induction and Maintenance Immunosuppression
10.3 Treatment of Rejection
10.4 Conclusions
References
11 Stemcell Transplantation
11.1 State‐of‐the‐Art of Allogeneic Hematopoietic Cell Transplantation
11.2 Haploidentical HCT
11.3 Immunobiology of Haploidentical Related Donor Transplantation
11.4 Selection of Related Donors for Transplantation: Non‐HLA Factors
11.5 Concluding Remarks
References
12 Stemcell Transplantation from Unrelated Donors
12.1 Introduction
12.2 The Search for Unrelated Donors
12.3 Laboratory Work
12.4 Clinical Outcomes of Unrelated Stem Cell Transplantation
12.5 Challenges in Unrelated Stem Cell Transplantation
12.6 Future Directions and Emerging Technologies
12.7 Conclusion
References
13 Chimerism Testing in Stem Cell Transplantation
13.1 Introduction
13.2 Methods for Molecular Genetic Chimerism Testing
13.3 Sensitivity of Molecular Genetic Chimerism Testing
13.4 Choice of Markers for Quantification
13.5 Chimerism Testing for Nonmalignant Disorders
13.6 Reporting Chimerism
13.7 Time Course of Chimerism Analyses
13.8 Advantages and Disadvantages of Highly Sensitive Chimerism Testing
13.9 Cell Sources for Identifying Recipient‐ and Donor‐Specific Markers
13.10 Cell Sources for Chimerism Monitoring
13.11 Intervals for Chimerism Testing
13.12 Chimerism‐Related Diagnostics
References
14 Disease Associations with HLA Alleles
14.1 HLA and Disease Association
14.2 Mechanisms of HLA and Disease Associations
14.3 Autoimmune Diseases Associated with HLA
14.4 Other Diseases Associated with HLA
14.5 Laboratory Diagnostics
14.6 Concluding Remarks
References
15 Drug Associations with HLA Alleles
15.1 Introduction
15.2 Classification of Drug Hypersensitivity Syndromes
15.3 Immunopathogenesis of Drug Hypersensitivity Syndromes
15.4 HLA and Hypersensitivity Reactions
15.5 Clinical Implications of HLA Testing
15.6 Future Directions
15.7 Conclusion
References
16 Diseases in the Field of Transfusion Medicine
16.1 Introduction
16.2 Platelet Transfusion Refractoriness
16.3 Transfusion‐Related Adverse Events
16.4 Neonatal Alloimmune Neutropenia
16.5 Fetal and Neonatal Alloimmune Thrombocytopenia
16.6 Conclusions
References
Part III: Methods in Immunogentics and Histocompatibility
17 Isolation and Purification of Test Material
17.1 DNA Extraction
17.2 RNA Extraction
17.3 cfDNA Extraction
17.4 Cell Preparation
Abbreviations
References
18 HLA‐Antibody Determination
18.1 Overview and Characteristics of Different HLA Antibody Detection Assays
18.2 The Multiplex Bead Array Platform (Luminex
®
)
18.3 Additional Methods/Tools
References
19 Low/Medium‐Resolution HLA Typing
19.1 Introduction
19.2 Cellular and Serological Tests for HLA Typing
19.3 DNA‐based Methods for HLA Low‐Resolution Typing
References
20 High‐Resolution HLA Typing
20.1 Introduction
20.2 HLA Genes
20.3 PCR Amplification
20.4 Gene Capture Method
20.5 SBT by Sanger Sequencing
20.6 HLA Typing by NGS SBT
20.7 HLA Typing by Short‐read Sequencing
20.8 HLA Typing by Long‐read Sequencing
20.9 Ambiguities
20.10 Phasing
20.11 Data Analysis
20.12 Special Cases
Abbreviations
References
21 Crossmatching in Transplantation
21.1 Introduction
21.2 The Mechanism of Crossmatching
21.3 Crossmatch Methods
21.4 Summary
References
22 Determination of Antibodies and Typing in the HPA System
22.1 Introduction to Platelet Antibody and Antigen Testing
22.2 Principles of Antibody Testing
22.3 Additional Techniques
22.4 Protocol: Platelet Suspension Immunofluorescence Test
22.5 Protocol: Isolation of Platelets for Serological Testing
22.6 Protocol: Monoclonal Antibody Immobilization of Platelet Antigens Assay (MAIPA)
22.7 New Developments for Platelet Antibody Detection
22.8 HPA‐Antigen Testing
22.9 Concluding Remarks
References
23 KIR Genotyping
23.1 Complexity of
KIR
Genomic Region
23.2 KIR Genotyping
References
24 Determination of Antibodies and Typing in the HNA System
24.1 Introduction to Granulocyte Antibody and Antigen Testing
24.2 Principles of Antibody Testing
24.3 HNA Typing Techniques
References
25 Determination and Typing of Other Polymorphic Systems
25.1 Introduction
25.2
MICA/B
Allele Typing Methods
25.3
MICA/B
Single Nucleotide Variant Genotyping Methods
25.4
HLA‐G
Allele Typing Methods
25.5
HLA‐E
Typing Methods
25.6
HLA‐F
Typing Methods
25.7 Technical Aspects for the Detection of Anti‐MICA Antibodies in Transplantation Patients
References
Part IV: Regulatory Aspects
26 Regulations in Histocompatibility and Immunogenetics: Accreditation, External Proficiency Testing, Quality Management
26.1 Introduction
26.2 Legal Requirements
26.3 National Guidelines
26.4 Standards
26.5 Elements of Quality Management Systems
26.6 Practical Considerations for a QMS
26.7 Licensing, Accreditation, and Certification
26.8 EFI Accreditation Program
26.9 ISO Standards
26.10 Future Perspectives
Table of Acronyms and Abbreviations
References
26.A Central Elements of Quality Management Systems
26.B Comparison Chart of EFI-Standards, ISO 9001, and ISO 15189
26.C International Cooperation in Laboratory Accreditation
26.D Accreditation, Certification, and Licensing. A Synopsis
Index
End User License Agreement
List of Tables
Chapter 1
Table 1.1 PRRs and their main ligands.
Chapter 3
Table 3.1 Number of Unique HLA alleles and Proteins. The number of ...
Table 3.2 Examples of the HLA nomenclature. The table shows example...
Table 3.3 Growth of alleles over twenty‐five years. The growt...
Table 3.4 Alternative ways of encoding an ambiguous HLA typing resu...
Chapter 4
Table 4.1 Nucleotide and amino acid positions that form the differe...
Table 4.2 HNA phenotype frequencies in different populations.
Chapter 6
Table 6.1 Human platelet antigens and carrier glycoprotein.
Table 6.2 Human platelet antigens and frequency distribution in peo...
Chapter 8
Table 8.1 Count of nations having allocation organizations.
Chapter 10
Table 10.1 Risk factors for rejection [53, 54].
Chapter 11
Table 11.1 Glossary of transplant terms.
Table 11.2 Glossary of genetic terms.
Table 11.3 Genetic factors associated with clinical outcome in hap...
Table 11.4 Factors influencing the selection of haploidentical don...
Chapter 13
Table 13.1 Comparison of methods for molecular genetic chimerism t...
Table 13.2 Proposed time intervals for chimerism testing.
Chapter 15
Table 15.1 Revised Gell–Coombs classification of hypersensitivity ...
Table 15.2 HLA association with various phenotypes of drug hyperse...
Table 15.3 Pharmacogenetic important HLA P groups; assigned as of ...
Chapter 16
Table 16.1 Immune and nonimmune causes of platelet refractoriness....
Table 16.2 Drugs reported to cause drug‐dependent platelet antibod...
Table 16.3 Clinical data from two case series.
Chapter 18
Table 18.1 Summary of major advantages and limitations of differen...
Chapter 21
Table 21.1 Comparison of antibody testing methods.
Table 21.2 Comparison of crossmatching tests.
Chapter 22
Table 22.1 Development and principles of platelet antibody assays,...
Table 22.2 Clones of capturing monoclonal antibodies used for allo...
Chapter 24
Table 24.1 Reaction patterns summarized for GIFT/LIFT.
Table 24.2 An example of an HNA‐1b specific antibody detected usin...
Table 24.3 Example capture antibodies utilized in MAIGA for each g...
Table 24.4 HNA genotyping: known mutations.
Chapter 25
Table 25.1 Nonconservative and conservative amino acid changes in ...
Table 25.2 Nonconservative and conservative amino acid changes in ...
Table 25.3 DNA‐based methods developed for
MICA/B
typing.
Table 25.4 Nonconservative and conservative amino acid changes in ...
Table 25.5 DNA‐based methods developed for
HLA‐G
typing.
Table 25.6 Nonconservative and conservative amino acid changes in ...
Table 25.7 DNA‐based methods developed for
HLA‐E
typing.
Table 25.8 Nonconservative and conservative amino acid changes in ...
Table 25.9 DNA‐based methods developed for
HLA‐F
typing.
List of Illustrations
Chapter 1
Figure 1.1 The complement system. The complement cascade is schema...
Figure 1.2 PRR signaling. TLRs and CLRs expressed on the plasma me...
Figure 1.3 Missing self and allorecognition models. Under steady‐s...
Figure 1.4 The inflammatory reaction. (1–3) Inflammation is induce...
Figure 1.5 Simplified overview of the innate immune response. (1) ...
Chapter 3
Figure 3.1 Illustration of the structure of the HLA class I and II...
Figure 3.2 Illustration of the genetic composition of the known HL...
Figure 3.3 The extent of HLA diversity in 2025. The number of uniq...
Figure 3.4 The figure details how the initial four‐digit name has ...
Figure 3.5 A simplified timeline of key events in the implementati...
Chapter 5
Figure 5.1 KIR protein structures. The structural characteristics ...
Figure 5.2 Genome organization of KIR. The coding regions of the e...
Figure 5.3 KIR nomenclature.
Figure 5.4 KIR haplotypes defined by family studies and genomic se...
Figure 5.5 KIR haplotype model.
Figure 5.6 Genomic organization of the C‐type lectin receptors on ...
Figure 5.7 HLA‐E is the ligand for inhibitory CD94‐NKG2A and activ...
Figure 5.8 (a) Overview of the CD94‐NKG2A receptor docking onto HL...
Figure 5.9 CD94‐NKG2A expressing NK cells from TT individuals are ...
Figure 5.10 CMV has opposing effects on different NK cell subsets...
Chapter 6
Figure 6.1 (a) GPIIb/IIIa carrying the HPA1 and HPA3 system and ma...
Chapter 8
Figure 8.1 Flowchart illustrating the process from decision‐making...
Figure 8.2 Flowchart presents a succinct graphic representation de...
Chapter 9
Figure 9.1 Mechanisms of allorecognition in kidney transplantation
Chapter 10
Figure 10.1 Three‐signal model of allorecognition. Signal 1 impli...
Figure 10.2 The calcineurin pathway. Created in BioRender.com.
Figure 10.3 Mechanism of action of azathioprine. After conversion...
Figure 10.4 The mTOR pathway. The mammalian target of rapamycin (...
Figure 10.5 Different techniques of extracorporeal removal. Plasm...
Chapter 11
Figure 11.1 A Family Study. Six‐locus HLA‐A, ‐C, ‐B, ‐DRB1, ‐DQB1...
Chapter 12
Figure 12.1 Workflow of the donor search in unrelated stemcell tr...
Chapter 13
Figure 13.1 A hypothetic STR marker in three different allelic ma...
Figure 13.2 (A) Pipetting scheme for a qPCR assay. In order to in...
Figure 13.3 Droplet 1 contains templates neither of the marker no...
Figure 13.4 Idealized example of NGS chimerism assay with InDel m...
Figure 13.5 X‐axis, days after transplantation. Y‐axis, percentag...
Figure 13.6 Different scenarios of inside HLA loss of leukemic ce...
Chapter 15
Figure 15.1 Mechanisms of immunopathogenesis in adverse drug reac...
Figure 15.2 Global distribution of clinically important human leu...
Figure 15.3 Factors leading to hypersensitivity reactions.
Chapter 17
Figure 17.1 Automated DNA extractor, in which 24 samples can be p...
Figure 17.2 DNA measurement with Nanodrop. The photo shows the co...
Figure 17.3 Separation of leukocytes with Ficoll–Paque solution. ...
Figure 17.4 Cell separation from venous blood using Ficoll–Paque ...
Figure 17.5 Cell separation from spleen using Ficoll–Paque medium...
Chapter 18
Figure 18.1 Evolution and the analytical principleof HLA antibody...
Figure 18.2 The principle of the Luminex® single antigen bead ass...
Figure 18.3 Technical caveats of single antigen bead analysis lea...
Figure 18.4 Size proportion of HLA/HLA–antibody complex, HLA epit...
Figure 18.5 Antibody‐binding curves demonstrating the impact of c...
Figure 18.6 SAB‐binding curves from dilutions of three patient se...
Figure 18.7 Technology‐dependent affinity results. (a) Affi...
Figure 18.8 The principle of detection of HLA‐specific memory B c...
Chapter 19
Figure 19.1 Luminex‐based PCR‐SSOP method. Luminex xMAP technolog...
Figure 19.2 PCR‐SSP gel electrophoresis. HLA typing is performed ...
Figure 19.3 Real‐time PCR‐SSP TaqMan assay. HLA typing is perform...
Chapter 20
Figure 20.1 Amplification strategy for 11 HLA loci. For the Class...
Figure 20.2 Alignment of the exons of HLA‐A*26:01:01:01 and HLA‐Y...
Figure 20.3 Example of homology between two HLA genes. Identical ...
Figure 20.4 A screenshot of an HLA‐B typing using SBT by Sanger s...
Figure 20.5 The basic steps to perform SBT by NGS.
Figure 20.6 Short reads overlapping throughout the amplicon. Read...
Figure 20.7 The most common short‐read sequencers. (a): Illumina ...
Figure 20.8 A fragment with adapter and indices. The original DNA...
Figure 20.9 Reads of two different alleles have been sequenced (g...
Figure 20.10 The most common long‐read sequencers. (a): The PacB...
Figure 20.11 Sequences of the exons 1, 2, and 3 of the alleles o...
Figure 20.12 Details of the nucleotide content of two genotypes....
Figure 20.13 Phasing counts for positions 560 and 570. There are...
Figure 20.14 Phasing information between positions that do not d...
Figure 20.15 Content of a fastq file. Each read consists of 4 li...
Figure 20.16 Typing result of a sample. For each locus, the HLA ...
Figure 20.17 Example of an HLA‐DPB1 genotype ambiguity. The comb...
Figure 20.18 The results of an HLA‐C analysis. The red marking i...
Figure 20.19 A partial sequence of exon 2 of HLA‐C. The phasing ...
Chapter 22
Figure 22.1 Principle of the MAIPA assay: A detail of the platele...
Chapter 23
Figure 23.1 KIR haplotype model. Upper: The
KIR‐A
haplotype...
Figure 23.2 Killer‐cell immunoglobulin‐like receptor (KIR)‐typing...
Figure 23.3 Schematic view of the KIR multiplex qPCR assay qKAT. ...
Figure 23.4 Overview of KIR genotyping using NGS [59]. (Step 1–3)...
Chapter 24
Figure 24.1 Outline of the steps involved to perform the granuloc...
Figure 24.2 HNA‐1b reactivity pattern in the granulocyte agglutin...
Figure 24.3 Principle of the MAIGA assay depicting the various st...
Chapter 25
Figure 25.1 Localization, organization, and structure of the clas...
Figure 25.2 Schematic representation of methods developed for MIC...
Figure 25.3. Schematic representation of HLA‐G gene organization ...
Figure 25.4 Schematic representation of HLA‐E gene organization a...
Figure 25.5 Schematic representation of HLA‐F gene organization a...
Chapter 26
Figure 26.1 Clinical (red), H&I (green), and regulatory (blue) mi...
Figure 26.2 Gap analysis of local regulations for your laboratory...
Figure 26.3 Deming‐ or PDCA‐Cycle Model [12, 13].
Guide
Cover
Table of Contents
Title Page
Copyright
Dedication
Preface
Begin Reading
Index
End User License Agreement
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Histocompatibility and Immunogenetics
A Guide for Practitioners
Edited by Volker Oberle
Editor
Dr. Volker OberleJena University HospitalAm Klinikum 1JenaGermany
Cover Images: © Lightspring/Shutterstock, © Alila Medical Media/Shutterstock, © Fuse/Getty Images,© Volker Oberle
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Dedicated to my mentors Ulrich Rothe, Dick Hoekstra, Wolfgang Lösche, and Sabine Schröder and to my sons, Oskar, Artur, and Bruno.
Preface
Dear readers, we are proud to present to you the world's first comprehensive textbook on H&I. This has not been an easy task because there are fields of study that are highly specialized and therefore particularly challenging, as they are at the intersection of much broader disciplines. One such field is H&I, histocompatibility and immunogenetics. Mastering this subject requires the integration of knowledge from immunology, genetics, clinical medicine, and laboratory sciences. Due to the sheer amount of knowledge involved, specific textbooks for these fields are rarely available. This makes it difficult for beginners, clinicians who rely on this knowledge, and interested individuals from other fields to obtain a quick and comprehensive guide to the subject.
During the yearly EFI Congress 2023 in Nantes, where the Education Committee presented a new subject‐specific curriculum, the question arose as to what reading materials students should use to learn. Until now, in most cases, teachers have compiled and made available to H&I students only reviews and scientific publications. From a didactic and efficiency point of view, this was certainly not the optimal solution. This made us realize that there was an urgent need for adequate teaching materials. With this in mind, we developed the idea for this innovative textbook.
We are deeply grateful to more than 40 renowned authors from around the world, all experts in their field, who have come together and worked with great enthusiasm and effort to provide a comprehensive overview of H&I. The book covers topics ranging from the basics of polymorphic systems in leukocytes, killer cells, and platelets to their clinical relevance in transplantation and transfusion. It examines the implications for HLA‐determined diseases and the laboratory methods needed to study these systems and to diagnose the effects of clinically relevant processes. It integrates these topics in a unique way for the first time.
We are also grateful to Wiley for not only supporting H&I on a scientific level through relevant journals, but also for giving us the opportunity to develop the field didactically with a dedicated textbook.
With this book, we wish readers an educational and engaging experience, whether they are beginning their journey into the field or seeking to broaden their perspective beyond their own area of expertise to gain a deeper understanding of H&I.
Part IBiological Basics
1Innate Immune Defence
Margot Lepage and Valérie Dubois
Etablissement Français du Sang (EFS) Auvergne‐Rhône‐Alpes, Department of Biology and Therapies, Histocompatibility Laboratory, 111 Rue Elisée Reclus 69150 Décines‐Charpieu, France
1.1 Introduction
The innate immune system is the highly evolutionarily conserved first line of defense of the body. It is characterized by its prompt and efficient response involving non‐specific mechanisms that may be constitutive, such as physical‐chemical barriers and the complement system, or rapidly inducible, such as the inflammatory reaction.
In this chapter, we will begin by describing the anatomical barriers that protect the host against infection and examine the immediate innate defenses provided by various secreted soluble proteins, known as antimicrobial enzymes and peptides. We will then discuss the complement system, which directly kills some microorganisms and interacts with others to promote their removal by phagocytic cells.
Next, we will explore the induced mechanisms of the innate immune response. We will start by detailing the different strategies of danger recognition used by immune cells, with a particular focus on pattern recognition receptors (PRRs), whose discovery has significantly improved our understanding of immune pathophysiology. We will then discuss the inflammatory reaction, outlining the mechanisms involved in the initiation of inflammation and providing an overview of the different phagocytic cells and the steps involved in the process of phagocytosis. Additionally, we will introduce other cells of the innate immune system, known as innate and innate‐like lymphoid cells, including natural killer cells (NK cells). These lymphoid cells contribute to innate host defenses against viruses and other intracellular pathogens.
Finally, we will discuss the role of innate immunity in the initiation and polarization of the next phase of the immune response: adaptive immunity.
1.2 Anatomical Barriers and Initial Chemical Defenses
The first phase of host defense, called immediate innate defenses, consists of non‐specific mechanisms that are always active and ready to respond to any pathogen. Epithelial surfaces act as a physical barrier against microbial colonization and invasion, but also as a chemical barrier, producing a variety of antimicrobial molecules.
1.2.1 Skin and Mucosal Barriers
Anatomical barriers provide the crucial first line of defense by preventing exposure of internal tissues to microorganisms. These barriers include epithelia of the skin on the outer surfaces of the body along with mucosal surfaces of the respiratory, gastrointestinal, and urogenital tracts. Epithelial cells are held together by tight junctions, which effectively form a seal against the external environment [1]. The internal epithelia secrete a viscous fluid called mucus, which contains glycoproteins called mucins, presenting several protective functions. There are two types of mucins, gel‐forming and transmembrane. All are characterized by large, highly O‐glycosylated mucin domains that are diversely modified by Golgi glycosyltransferases to become extended rodlike structures [2]. The general function of mucus on internal epithelial surfaces is to prevent microorganisms from adhering to the epithelium. In the respiratory tract, microorganisms can be expelled in the outward flow of mucus driven by vigorous mucociliary clearance mechanisms. The latter function is most evident in the large intestine, where the inner mucus layer separates the numerous commensal bacteria from the epithelial cells. Also, in the gut, peristalsis is an important mechanism for keeping both food and infectious agents moving through the body. The extreme susceptibility to infection observed in patients suffering from severe cutaneous burns or primary ciliary dyskinesia demonstrates the great importance of this first barrier and the inability of an intact immune system to compensate for its loss [3].
1.2.2 Antimicrobial Enzymes and Peptides
At anatomic barriers, additional resistance mechanisms participate to further strengthen host defenses. Most epithelia produce a wide variety of chemical substances that are microbicidal or that inhibit microbial growth. For example, the acid pH of the stomach and the digestive enzymes, bile salts, fatty acids, and lysolipids present in the upper gastrointestinal tract create a substantial chemical barrier to infection. One important group of antimicrobial proteins includes lysozyme and secretory phospholipase A2, antibacterial enzymes secreted in tears and saliva that attack the chemical characteristics of bacterial cell walls [3].
The second category of antimicrobial agents secreted by epithelial cells and phagocytes consists of antimicrobial peptides such as defensins, cathelicidins, and histatins. These peptides are secreted by epithelial cells at the mucosal surface and by phagocytes in tissues. Defensins are notably an ancient and evolutionarily conserved class of antimicrobial peptides produced by many eukaryotic organisms, including mammals, insects, and plants. Defensins are short cationic peptides (30–40 amino acids) generated by proteolytic processing from inactive propeptides. They act within minutes to disrupt the cell membranes of bacteria and fungi, as well as the membrane envelopes of some viruses. Three subfamilies of defensins (α, β, and θ) are distinguished by their amino acid sequences. Humans only present α‐ and β‐defensins as the human gene homologous to the θ‐defensin gene described in rhesus macaques contains a premature stop codon. To date, six human α‐defensins have been identified, which are further divided into two major classes according to their expression patterns and gene structures: myeloid defensins or human neutrophil peptides (HNPs) 1–4 and human (enteric) defensins (HDs) 5 and 6. HNPs are stored in the azurophilic granules of human neutrophils, along with several other antimicrobial agents. These peptides are typically directed to fuse with phagolysosomes but can also be released into the extracellular medium through the degranulation of activated neutrophils (see part 1.5). HD5 and HD6 are constitutively expressed and secreted by Paneth cells at the bottom of the small intestinal crypt, contributing to the physiological maintenance of the digestive barrier. Although more than 30 β‐defensin genes exist in the human genome, only a few have been extensively characterized at the genomic and functional levels. Their expression is restricted to keratinocytes of the skin and epithelial cells. Human β‐defensin 1 (HBD1) is constitutively expressed in various epithelial and mucosal tissues. HBD2 and HBD3 are induced by microbial aggressions and pro‐inflammatory cytokines, primarily in epithelia of the respiratory and urogenital tracts, skin, and tongue. β‐Defensins produced by keratinocytes in the epidermis and by type II pneumocytes in the lungs are packaged into lamellar bodies, lipid‐rich secretory organelles that release their contents into the extracellular space to form a watertight lipid sheet in the epidermis and the pulmonary surfactant layer in the lung [4].
Another type of bactericidal protein synthesized by epithelia is carbohydrate‐binding proteins, or lectins. C‐type lectins require calcium for the binding activity of their carbohydrate‐recognition domain, which provides a variable interface for binding carbohydrate structures. C‐type lectins of the RegIII family include several bactericidal proteins expressed by intestinal epithelium in humans and mice. Human RegIIIα (also called HIP/PAP for hepatocarcinoma‐intestine‐pancreas/pancreatitis‐associated protein) preferentially kills Gram‐positive bacteria directly by forming a hexameric pore in the bacterial membrane [5].
Finally, peptides S100A8 and S100A9, which heterodimerize to form calprotectin, are produced in high amounts by neutrophils, T cells, and intestinal epithelia. Calprotectin acts to sequester magnesium and iron required by microorganisms and exerts a local antimicrobial effect [5].
Of note, most healthy epithelial surfaces are also associated with a large population of nonpathogenic bacteria, known as commensal bacteria or microbiota. These bacteria help to reinforce the barrier functions of epithelia and can themselves produce antimicrobial substances such as lactic acid by vaginal lactobacilli or bacteriocins by other bacterial strains. Commensal microorganisms also exert an indirect influence by stimulating the epithelial cells to produce antimicrobial peptides. This illustrates how the elimination of commensal microorganisms by antibiotic treatment can pave the way for pathogens to proliferate and cause other diseases [3].
1.3 The Complement System
The complement system is a highly conserved protein interaction platform of the innate immune system, working in coordination with other effectors of immunity to protect the body against infection and other insults.
The complement system is an enzymatic cascade made up of numerous soluble and membrane proteins involved in immune surveillance both in physiological and pathological conditions. These proteins interact with non‐immune cells (epithelial cells, osteoclasts, etc.), innate immune cells (macrophages, dendritic cells, neutrophils, mast cells, eosinophils, basophils), and adaptive immune cells (B and T cells). The complement system has multiple functions. It triggers an immune response to foreign pathogens, has pro‐inflammatory activity, regulates cytokines production, and helps to remove immune complexes and dead or modified cells (following injury, hypoxia, after viral infection, or tumor‐caused modification) [6].
Complement activation is mediated by finely regulated cascades of proteolytic reactions through three main pathways, each initiated by different stimuli: the classical pathway, the lectin pathway, and the alternative pathway (Figure 1.1).
The classical pathway (CP) is activated by antibodies once those bind to antigens. When this complex is formed, the fraction fields (Fc) of IgM, IgG3, and IgG1 interact with the collagen‐like tail of C1q. C1q is part of the C1 complex, which comprises a single C1q molecule bound to two molecules of each C1r and C1s. Once activated, the C1s enzyme cleaves C4 and then C2.
The lectin pathway (LP) is activated by the binding of C‐type lectin and mannose‐binding lectin (MBL), or proteins termed ficolins (L‐ficolin, H‐ficolin, and M‐ficolin), to some carbohydrates on the pathogen surface that are rich in mannose and beta glycans, respectively. MBL and ficolins may form a complex with MBL‐associated serine proteases (MASPs) and with a smaller molecule called MAp19. When the MBL complex binds to a pathogen surface, MASP‐1 and MASP‐2 are activated to cleave C4 and C2.
The alternative pathway can be initiated by many microbial surfaces. While the classical and lectin pathways are activated exclusively after danger recognition through binding antibodies or proteins, the alternative pathway is also constantly active at a low level at the steady state. It can be activated by spontaneous hydrolysis of C3 to give C3 (H
2
O), which binds factor B and enables the bound factor B to be cleaved by factor D.
All pathways converge to the assembly of multi‐protein enzymatic complexes termed C3 and C5 convertases. C3 and C5 convertases cleave C3 and C5, respectively, into their bioactive fragments that mediate a wide array of functions on complement receptor‐expressing cells [2, 7].
There are various types of C3 convertase, depending on the complement pathway activated. In the classical and lectin pathways, the C3 convertase is formed from membrane‐bound C4b complexed with C2b (C4b2b). In the alternative pathway, a homologous C3 convertase is formed from membrane‐bound C3b complexed with Bb (C3bBb). The alternative pathway can act as an amplification loop for all three pathways, as it is initiated by the binding of C3b. C3 is a key mediator in the activation of all complement activation pathways. Once C3 is cleaved by a C3 convertase, it is divided into C3a and C3b. C3a is an anaphylatoxin. It plays a role in innate immune cell recruitment and has antifungal and antimicrobial activity [8]. C3b and some other complement cleavage products belong to the family of opsonins, which can bind to the surface of pathogens and mark them for phagocytosis by immune cells. This opsonization improves the efficiency of pathogens' elimination by phagocytic cells.
Figure 1.1The complement system. The complement cascade is schematized here, with a highlight on the three activation pathways. All of them lead to C3 molecule cleavage, which is a key factor in the complement action. During the complement cascade, small peptides named anaphylatoxins are secreted, which contribute to the recruitment of immune cells. C3b coats the microbial or apoptotic cell surface, and the cascade can progress, while C5b starts the terminal pathway. Once C5b is released, it binds to C6 on the target cell surface, where other components of the MAC will join them.
Source: “Created with BioRender.com”.
C3b also binds the C3 convertase to form a C5 convertase (C4b2b3b or C3bBb3b) that cleaves C5 into C5a and C5b. C5a, just like C3a, is an anaphylactic peptide that attracts and activates neighboring cells to release inflammatory mediators. On the other hand, C5b initiates the “late” events of complement activation called the terminal pathway. It leads to the formation of the membrane attack complex (MAC), which is a cell membrane‐perforating complex derived from the assembly of C5b to C9 components (C5b6789). It induces osmotic changes in the target cell through the entry of water, ions, and other molecules. This will lead to cell lysis, and the production of cytokines, prostaglandins, and other pro‐inflammatory molecules [2, 7].
The complement system is strictly regulated to prevent inappropriate self‐directed activation and tissue damage. Most of these regulatory proteins belong to the regulators of complement activation (RCA) gene family. This family includes soluble regulators such as factor H and C4b‐binding protein (C4BP) as well as membrane‐bound regulators, such as membrane cofactor protein (MCP or CD46), complement receptor type 1 (CR1), and the glycosylphosphatidylinositol (GPI)‐anchored protein decay‐accelerating factor (DAF or CD55) [9].
Of note, the complement system can also regulate tolerance to self‐antigens and influence adaptive immunity by promoting antigen presentation, activating T and B cells, and regulating antibody production [10]. Other physiological functions attributed to complement are angiogenesis, mobilization of hematopoietic stem/progenitor cells, lipid metabolism, coagulation pathway, calcium metabolism, organ regeneration, and neuroprotection (including migration of neurons and synapse elimination).
1.4 Danger Recognition
Beyond immediate immune defenses, the next phase of the innate immune response is to induce an inflammatory response to recruit new phagocytic cells and circulating effector molecules to the site of infection. This process involves the rapid detection of “immunological danger” through different mechanisms.
1.4.1 Pattern Recognition Receptors (PRRs)
Innate immunity relies upon a limited repertoire of germline‐encoded invariant receptors called PRRs. First, PRRs recognize “microbial non‐self” represented by conserved molecular structures expressed by a large variety of microbes and grouped together under the term pathogen‐associated molecular patterns (PAMPs). These structures include mannose‐rich oligosaccharides, peptidoglycans, and lipopolysaccharides of the bacterial cell wall, as well as unmethylated CpG DNA common to many pathogens. Secondly, “damaged self”‐derived host molecules that indicate cellular infection, stress, or transformation can also be recognized by some innate receptors. Such indicator molecules have been termed damage‐associated molecular patterns (DAMPs), and some of them can be recognized by PRRs. DAMPs represent common metabolites and mediator molecules that are upregulated and released during the cell lysis and tissue damage that occurs in the context of both infectious and sterile inflammation [11]. As an example, in the context of organ transplantation, the release of DAMPs by graft cells under conditions of hypoxia and redox stress triggers the innate immune response, contributing to the pathogenesis of early peri‐transplant ischemia‐reperfusion injury [12].
Currently, four different classes of PRR families have been identified. These families include transmembrane proteins such as the Toll‐like receptors (TLRs) and C‐type lectin receptors (CLRs), as well as cytoplasmic proteins such as the retinoic acid‐inducible gene (RIG‐I)‐like receptors (RLRs) and nucleotide oligomerization domain (NOD)‐like receptors (NLRs). These PRRs are expressed in macrophages and dendritic cells, but also in various other immune and non‐immune cells. Except for some NLRs, the sensing of PAMPs or DAMPs by PRRs upregulates the transcription of genes involved in inflammatory responses. These genes encode proinflammatory cytokines, type I interferons (IFNs), chemokines, and antimicrobial proteins; proteins involved in the modulation of PRR signaling; and many uncharacterized proteins. The expression patterns of inducible genes differ according to activated PRRs [11].
1.4.1.1 Toll‐like Receptors (TLRs)
The TLR families are transmembrane glycoproteins responsible for sensing invading pathogens outside of the cell and in intracellular endosomes and lysosomes. TLRs are characterized by an extracellular region containing N‐terminal leucine‐rich repeats (LRRs), which are responsible for the recognition of specific ligands and perform extracellular pattern recognition. The cytoplasmic domain contains the same Toll/IL‐1R (TIR) domain as IL‐1R, which plays a role in signal transduction. To date, 10 functional TLRs (TLR1–10) have been found in humans. Different TLRs recognize the different molecular patterns of microorganisms and self‐components (Table 1.1). The cellular localization of TLRs determines the types of ligands recognized and the recognition mechanism [2, 13].
Some TLRs (TLR1, 2, 4, 5, 6, 10) are expressed on the cell surface as heterodimers or homodimers and mainly recognize the membrane components of pathogenic microorganisms, such as lipids, lipoproteins, and proteins. Mammalian TLR1, TLR2, and TLR6 are, for example, cell‐surface receptors that are activated by various ligands, including lipoteichoic acid and the diacyl and triacyl lipoproteins of Gram‐negative bacteria. They are found in macrophages, dendritic cells, eosinophils, basophils, and mast cells. Ligand binding induces heterodimerization of TLR2 and TLR1 or TLR2 and TLR6. TLR4 recognizes lipopolysaccharide (LPS) together with myeloid differentiation factor 2 (MD2) on the cell surface. LPS is a component derived from the outer membrane of Gram‐negative bacteria and is known to cause septic shock in the context of uncontrolled bacterial infection. In this case, LPS triggers an excessive secretion of cytokines, particularly tumor necrosis factor α (TNFα), leading to systemic vascular permeability – a side effect of its usual role in containing local infections. Structurally, two complexes of TLR4‐MD2‐LPS interact symmetrically to form a TLR4 homodimer. TLR4 is also involved in the recognition of viruses by binding to viral envelope proteins and DAMPs (endogenous oxidized phospholipids). TLR5 is expressed on the cell surface of macrophages, dendritic cells, and intestinal epithelial cells. It recognizes flagellin, a protein subunit of bacterial flagella. It is activated only by monomeric flagellin, which is produced by the breakdown of flagellated bacteria in the extracellular space [2, 13, 14].
Table 1.1 PRRs and their main ligands.
Receptor
Localization
Ligand
Source of the ligand
Toll‐like receptors
TLR1
Plasma membrane
Triacyl lipoprotein
Bacteria
TLR2
Plasma membrane
Lipoprotein
Bacteria, fungi, mycobacterium
TLR3
Endosome
dsRNA
Viruses
TLR4
Plasma membrane
LPS
Bacteria
TLR5
Plasma membrane
Flagellin
Bacteria
TLR6
Plasma membrane
Diacyl lipoprotein
Bacteria, viruses
TLR7
Endosome
ssRNA
Viruses
TLR8
Endosome
ssRNA
Viruses
TLR9
Endosome
CpG‐DNA
Viruses, bacteria, parasites, self
TLR10
Plasma membrane
Unknown
Unknown
C‐type lectin receptors
Dectin‐1/2
Plasma membrane
β‐Glucan, α‐mannan
Fungi
RIG‐I‐like receptors
RIG‐I
Cytosol
Short dsRNA, 5′‐triphosphate RNA
Viruses
MDA5
Cytosol
Long dsRNA
Viruses
NOD‐like receptors and inflammasomes
NOD1
Cytosol
iE‐DAP
Bacteria
NOD2
Cytosol
MDP
Bacteria
NLRP3
Cytosol
Ion flux, organelle dysfunction, nucleic acids
Bacteria, viruses, self
cGAS‐STING pathway
cGAS
Cytosol
dsDNA
Viruses, bacteria, parasites, self
Other TLRs (TLR3, 7, 8, 9) are located intracellularly in the membrane of endosomes and expressed as homodimers, which recognize nucleic acids derived from viruses and bacteria as well as endogenous nucleic acids in pathological contexts. TLR3, for example, is expressed by macrophages, conventional dendritic cells, and intestinal epithelial cells. It recognizes double‐stranded RNA (dsRNA), which is a replicative intermediate of many types of viruses. TLR7, TLR8, and TLR9 are also endosomal nucleotide sensors involved in the recognition of viruses. TLR7 and TLR9 are expressed by plasmacytoid dendritic cells, B cells, and eosinophils. TLR8 is expressed primarily by monocytes and macrophages. TLR7 and TLR8 are activated by single‐stranded RNA (ssRNA), which is a component of healthy mammalian cells (but normally confined to the nucleus and cytoplasm and not present in endosomes) and of many virus genomes. In abnormal settings, TLR7 can be activated by self‐derived ssRNA from apoptotic cells, and polymorphisms in the human TLR7 gene have been associated with an increased risk of the autoimmune disease systemic lupus erythematosus. TLR9 recognizes unmethylated CpG dinucleotides. In mammalian genomes, CpG dinucleotides in genomic DNA are heavily methylated on the cytosine by DNA methyltransferases. However, in the genomes of bacteria and many viruses, CpG dinucleotides remain unmethylated and represent another PAMP [2, 13–15].
Signaling by mammalian TLRs in various cell types induces a diverse range of intracellular responses that together result in the production of inflammatory cytokines, chemotactic factors, antimicrobial peptides, and antiviral cytokines IFNα and IFNβ from the type I IFNs family. The signaling pathways of TLRs depend on the nature of the stimulus, the specific TLR activated, and the downstream adaptor molecule involved. The two main signaling adaptor molecules associated with mammalian TLRs are MyD88 (myeloid differentiation factor 88) and TRIF (TIR domain‐containing adaptor‐inducing IFNβ). The MyD88‐dependent pathway is utilized by all TLRs except TLR3 and leads to the production of inflammatory cytokines such as pro‐interleukin‐1β (pro‐IL‐1β), interleukin‐6 (IL‐6), and TNFα through activation of the transcription factor nuclear factor‐κB (NFκB) or through the mitogen‐activated protein (MAP) kinase pathway. TLR7 and TLR9 signaling also triggers the production of type I IFNs in a MyD88‐dependent manner, along with other NFκB‐dependent cytokines. Conversely, the TRIF‐dependent pathway is used by TLR3 and TLR4 and is associated with the production of inflammatory cytokines through NFκB signaling as well as the production of type‐I IFNs through interferon regulatory factors (IRFs) signaling (Figure 1.2). The collective ability of TLRs to activate both NFκB and IRFs means that they can stimulate either antibacterial or antiviral responses as needed [11].
Figure 1.2PRR signaling. TLRs and CLRs expressed on the plasma membrane (or in endosomes) detect extracellular danger, while cytosolic receptors such as RLRs, NLRs, and cGAS detect intracellular danger. Upon activation, PRRs stimulate transcription factors, including NFκB and IRFs. These transcription factors translocate to the nucleus to activate the expression of genes encoding pro‐inflammatory cytokines (such as IL‐6, TNFα, pro‐IL‐1β, and others) or type I IFNs. Some cytokines are then matured through the inflammasome, which itself results from the activation of a specific subtype of NLRs. The inflammasome is also responsible for the cleavage of gasdermin D (GSDMD), which promotes pore formation in the cell plasma membrane and pyroptosis. The cytokines, IFNs, and DAMPs produced will interact in an autocrine and paracrine manner to recruit and activate the effector cells of the innate immune response.
Source: “Created with BioRender.com”.
1.4.1.2 C‐type Lectin Receptors (CLRs)
CLRs are a family of phagocytic PRRs expressed on macrophages, dendritic cells, and certain tissue cells. They recognize carbohydrates on the surface of self and non‐self structures through their carbohydrate recognition domain (CRD) in a calcium‐dependent manner (C‐type). Depending on the location of the protein on the cell membrane, CLRs are divided into secretory and transmembrane receptors.
The main representative of secretory receptors is the collagen lectin family, including MBL, that circulates in blood and extracellular fluids and can activate complement (see parts 1.2 and 1.3).
Transmembrane receptors can be divided into type I and type II according to their structure: the N‐terminal of type I receptors points to extracellular and contains multiple CRDs, while the N‐terminal of type II receptors points to intracellular and contains only one CRD. The dendritic cell‐associated C‐type lectins (Dectin‐1 and ‐2) are typical representatives of membrane CLRs and recognize polysaccharide structures (glucans, mannans) present in the cell walls of yeast and fungi (Table 1.1). When binding to their ligand, membrane CLRs generate an intracellular activation signal and endocytosis into a cytoplasmic vesicle for direct digestion and elimination.
The CLR family is highly diverse, and the ligands of many of them are not well characterized. Of note, it has been shown that the vast majority of CLRs are involved in the presentation of antigens as active membrane‐associated receptors [2, 11, 13].
1.4.1.3 RIG‐I‐like Receptors (RLRs)
RLRs represent a family of cytosolic PRRs, primarily composed of the retinoic acid‐inducible gene I (RIG‐I) and melanoma differentiation‐associated protein 5 (MDA5) receptors. Expressed directly in the cytoplasm, they sense viral RNAs produced within a cell (in contrast to TLR3, 7, 8, 9, which detect extracellular viral RNAs and DNAs that enter the cell from the endocytic pathway).
RIG‐I recognizes relatively short dsRNA (up to 1 kb), while MDA5 detects long dsRNA (more than 2 kb). In addition, RIG‐I detects the unmodified 5′‐triphosphate RNA end of viral ssRNA, which differs from the capped 5′‐triphosphate RNA end of self ssRNA also present in the cytosol (Table 1.1). RIG‐I and MDA5 bind to viral RNAs using an RNA helicase‐like domain in their C‐terminal part. They also contain two N‐terminal caspase recruitment domains (CARDs) that, when activated, interact with adaptor proteins called mitochondrial antiviral signaling proteins (MAVS) attached to the outer mitochondrial membrane. The signal causes activation of transcription factors IRF3 and IRF7 to produce anti‐viral type‐I IFNs as well as NFκB to produce pro‐inflammatory cytokines (Figure 1.2). In some cell types, RIG‐I, but not MDA5, interacts with apoptosis‐associated speck‐like protein containing a CARD (ASC), thereby inducing the inflammasome, which contributes to inflammatory responses [16].
1.4.1.4 NOD‐like Receptors (NLRs) and the Inflammasome
NLRs are another family of cytosolic PRRs. NLRs are composed of three domains: the central nucleotide‐binding domain (NBD) for nucleic acid binding and oligomerization of NLRs, LRRs at the C‐terminus for ligand recognition, and the N‐terminal effector domain, which is the protein interaction domain. According to the different N‐terminal effector domains, the NLR family can be divided into five subfamilies:
NLRC subfamily, which contains CARDs;
NLRP subfamily, which contains pyrin domains (PYDs);
NLRB subfamily, which contains baculovirus inhibitor of apoptosis protein repeats;
NLRA subfamily, which contains acidic activation domains;
NLRX subfamily containing other NLR effector domains.
Some NLRs activate NFκB to initiate the same inflammatory responses as the TLRs. It is the case of the most extensively studied NLRs, which are nucleotide‐binding oligomerization domain‐containing proteins 1 and 2 (NOD1 and NOD2), from the NLRC subfamily (Figure 1.2). NOD proteins recognize fragments of bacterial cell‐wall peptidoglycans: γ‐glutamyl diaminopimelic acid (iE‐DAP) for NOD1 and muramyl dipeptide (MDP) for NOD2 (Table 1.1). When NOD1 or NOD2 recognizes its ligand, it recruits a CARD‐containing kinase, which results in activation of NFκB signaling, inducing the expression of genes for inflammatory cytokines and for enzymes involved in the production of nitric oxide (NO) (Figure 1.2). NOD1 is an important activator of responses against bacterial infections and may also function as a systemic activator of innate immunity. NOD2 seems to have a more specialized role, being strongly expressed in the Paneth cells of the gut, where it regulates the expression of potent antimicrobial peptides such as the α‐ and β‐defensins [17].
Other NLRs, as the NLRP subfamily, trigger a distinct pathway that forms multimeric protein complexes called inflammasomes, leading to cell death and the production of pro‐inflammatory cytokines (Figure 1.2). The most well‐characterized inflammasome is induced through NLRP3. The NLRP3 inflammasome can be activated through three pathways: canonical, non‐canonical, and alternative pathways. The canonical NLRP3 inflammasome activation is a two‐step process with a priming step relying typically on TLR stimulation and an activation step mediated by an array of stimuli, including ATP, nigericin, gramicidin, bacterial toxins, or particulate matter. So the NLRP3 inflammasome can be triggered either by pathogens or cell damage. NLRP3 activation leads to the recruitment and oligomerization of an adaptor ASC protein, which interacts with the CARD of pro‐caspase 1. After assembly, the NLRP3 inflammasome enables the proteolytic activation of caspase 1. This protease promotes the processing and release of mature IL‐1β and IL‐18, as well as the cleavage of gasdermin D (GSDMD), which promotes pore formation in the cell plasma membrane, leading to a lytic form of cell death called pyroptosis. This process releases various intracellular molecules (ATP, small and large DAMPs such as HMGB1, etc.) that also contribute to inflammation [18].
1.4.2 cGAS‐STING Pathway
In addition to TLR9 for extracellular CpG DNA detection, the major intracellular sensor of DNA that triggers the innate immune response is cyclic guanosine monophosphate (GMP)‐adenosine monophosphate (AMP) (cGAMP) synthase (cGAS). It senses cytosolic DNA from diverse classes of pathogens that contain DNA or generate DNA in their life cycles (DNA viruses, retroviruses, bacteria, and parasites) as well as self‐DNA (genomic and mitochondrial DNA) that can enter the cytosol in response to certain cellular stress or environmental insults (Table 1.1). Thus, the cGAS pathway is also involved in “microbial non‐self” and “damaged self” recognition. Beyond host defense, this pathway has been shown to play an important role in inflammatory diseases, antitumor immunity, and cellular senescence [19].
The binding of DNA to cGAS induces a conformational change and the generation of the second messenger cGAMP from adenosine and guanosine triphosphate (ATP and GTP) substrates. Then, cGAMP activates the stimulator of interferon genes (STING) at the membrane of the endoplasmic reticulum, which activates a signaling cascade leading to the activation of IRF3 and NFκB and the expression of type‐I IFNs and inflammatory cytokines [20] (Figure 1.2).
1.4.3 “Missing Self” and Allorecognition Receptors
Another innate immune recognition strategy involves innate immune receptors that can detect “missing self,” i.e. molecules constitutively expressed by normal healthy cells but not expressed by infected, damaged, or transformed cells (or expressed as a variant form in allogenic cells). Recognition of these signals usually delivers an inhibitory signal to prevent activation of the immune response against host tissues, and their absence can thus trigger innate immunity (Figure 1.3).
The inhibitory system is particularly well documented concerning natural killer (NK) cells: inhibitory killer cell immunoglobulin‐like receptors (KIRs) that recognize self‐major histocompatibility complex (MHC) class I molecules notably play a central role in missing‐self recognition by NK cells. This mechanism prevents NK cells from killing normal host cells and drives them to target infected/tumor cells that downregulate their MHC class I proteins [21]. More precisely, it is the balance between activating and inhibitory signals that together control NK cells' cytotoxic activity and cytokines production and determine whether an individual NK cell will be triggered to kill a particular target cell (see part 1.5 and Chapter 5).
Figure 1.3Missing self and allorecognition models. Under steady‐state conditions, NK cells (KIR/MHC class I interaction model, left panel) or monocytes (SIRPα/CD47 interaction model, right panel) receive equal stimulatory and inhibitory signals from their receptors. Under pathological conditions or in the context of allogenic transplantation, the imbalance between stimulatory and inhibitory signals leads either to cell inhibition or activation (with different effector mechanisms).
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Another inhibitory receptor of growing interest is the signal regulatory protein α (SIRPα, also known as CD172a). SIRPα is a polymorphic immunoglobulin superfamily receptor expressed mainly on myeloid cells, including macrophages, dendritic cells, and neutrophils. It binds to the ubiquitously expressed CD47 protein. CD47 binding to SIRPα delivers an inhibitory signal that represses phagocytosis by macrophages and inhibits multiple aspects of dendritic cell activation. This mechanism prevents macrophages from engulfing normal host cells, and it is often mentioned as a “do‐not‐eat‐me” signal. In some contexts, this interaction is bidirectional with SIRPα and CD47 engaging each other in opposite directions to deliver stimulatory and inhibitory signals. This is the case in immune cells where SIRPα binding to CD47 has stimulatory functions. It provides costimulatory signals to T cells and enhances dendritic cell homeostasis and migration. Signaling through the CD47‐SIRPα axis plays a role in various homeostatic processes, such as the maintenance of erythrocytes, innate immune cells, and T cells. CD47 expression varies according to immune status or pathological condition. Notably, tumor cells and virus‐ or bacteria‐infected cells can upregulate CD47 protein expression to prevent their clearance by macrophages [22, 23].
Of note, in the context of transplantation, these receptors are considered crucial for the mechanisms of innate allorecognition [24–26]. NK cells sense allogenic non‐self through the complete absence of KIRs interaction with mismatched non‐self MHC class I molecules, whereas monocytes sense subtle variations in the binding affinity of mismatched non‐self SIRPα to CD47 (Figure 1.3).
1.5 Inflammatory Reaction and Effector Cells of the Innate Immune Response
Processes activated upon PRR engagement are rapid, considered to be non‐specific, and include mechanisms such as phagocytosis, cell locomotion, killing of pathogens or cells, and cytokines production. These innate immune mechanisms are usually very effective in eliminating invading pathogens.
The inflammatory reaction is a complex immune system response to various stimuli, such as infection, injury, or irritants. The effector mechanisms of this reaction can be divided into several key stages (Figure 1.4
