Innate Immunity in Disease

Abstract

Cells can innately recognize generic products of viruses, bacteria, fungi, or injured tissue by engagement of pattern recognition receptors. Innate immune cells rapidly respond to this engagement in order to control commensals, thwart pathogens and/or prompt repair. Insufficient or excessive activation of the innate immune response results in disease. This review focuses on pattern recognition receptors and cells of the innate immune system important for intestinal function. Our improving knowledge pertaining to this important aspect of our immune response is opening potential important new therapeutic opportunities for the treatment of disease.

Introduction

The gut is a remarkable organ that functions primarily to allow digestion and absorption of food, and disposal of waste. It is home to millions of bacteria that assist with digestion and which produce some of our essential vitamins. These organisms also tone our immune system and promote the health of the epithelial lining.

The intestine is a vibrant immunological organ containing cellular and molecular components of our immune system, which protect us from the multitude of bacteria, fungi and viruses living within the gut lumen as well as from their toxic byproducts. The intestinal immune system is under exceedingly tight control, a process called “immune modulation”. Modulation regulates the intensity and nature of the mucosal inflammatory response. It allows sufficient inflammation to restrain the intestinal flora without allowing a needlessly forceful inflammatory response that can impair absorption or create local or systemic disease. At the same time, it allows assimilation of nutrients, avoiding injurious allergic or hypersensitivity immune responses.

Findings

Adaptive vs. Innate Immunity

The mucosal immune system can be divided into two overlapping functional components called “innate” and “adaptive” immunity. Adaptive immunity refers to responses that utilize clonally unique, genetically recombined receptors that recognize specific molecules (usually protein antigens) produced by particular microorganisms. This type of immunity is delivered by T and B lymphocytes. Examples include immunity that occurs after previous exposure to, or vaccination with, a disease-causing agent or toxin. Adaptive immune responses may require quite some time to fully develop, since the clonally responsive lymphocytes need several days to expand in number and mature. Furthermore, recall adaptive immune responses are highly specific to particular molecules and may not activate after we encounter organisms or some of their products that are closely related, but not identical to the original sensitizing agent.

Cells of the immune system can sense the presence of many potential pathogens using innately inherited, germ-line encoded receptors that instinctively engage classes of molecules common to many types of bacteria, fungi and/or viruses, and which are not produced by their mammalian hosts. These receptors, which have been characterized over the last few years, are called “pattern recognition receptors (PRR)”. Many of the organisms that normally inhabit our intestines are controlled by innate immune recognition using these receptors ^{1, 2}. In addition, some of these innate PRRs recognize host-derived molecules that are only displayed when host cells have died or are injured. This allows the immune system to quickly recognize and clear damaged tissue to speed organ repair without evoking adaptive immunity and perhaps autoimmune disease ³. All cells express PRRs, but different combinations of these receptors are expressed by different cell types. A partial listing of these receptors and the products they recognize is provided in Table 1. Different receptors often recognize the same molecule. It is now appreciated that this redundancy maintains the host’s potential for responsiveness if an organism can evade a particular PAMP.

Table 1.

Some of the Pattern Recognition Receptors (PAMPs, MAMPs, and DAMPs) used by the Innate Immune System.

Receptor		Classic Target
Toll-like receptor family
	TLR1	triacylated lipopeptides
	TLR2	lipoteichoic acid
	TLR3	dsRNA
	TLR4	lipopolysaccharide
	TLR5	flagellin
	TLR6	diacyl lipopeptides
	TLR7	ssRNA
	TLR8*	ssRNA
	TLR9	unmethylated CpG ssDNA
	TLR10	triacylated lipopeptides
	TLR11**	profilin, S. typhi, E. coli
	TLR12	profilin
	TLR13	bacterial ribosomal RNA
NOD-like receptor family
	NOD1	bacterial D-glutamyl-meso-diaminopimelic acid
	NOD2	bacterial muramyl dipeptide
	NLRP1	bacterial muramyl dipeptide
	NLRP3	crystals (e.g. uric acid)
	NLRP6	not identified but regulates inflammasome function
	NLRP12	not identified but regulates NFκB activation
	NLRC4	flagellin
	NLRC5	dsRNA co-regulator of RIG-I, MDA5
	AIM 2	dsDNA
RIG-like receptor family
	RIG-I	viral and dsRNA
	MDA5	viral and dsRNA
	LGP2	dsRNA co-regulator of RIG-I, MDA5
Immunoglobulin-like receptor family
	RAGE	HMGB1, S100A12, amyloid (alarmins)
C-type lectin family
	CLEC1	not identified but regulates Th17 function
	CLEC2	podoplanin (lung alarmin)
	CLEC4a (DCIR1, CLECSF6)	carbohydrate binding
	CLEC4a4 (DCIR2)	bisecting Man4, GlcNAc5 and GlcNAc7
	CLEC4c (BDCA2)	glycans with galactose residues at the non- reducing ends
	CLEC4e (mincle)	range of carbohydrate structures
	CLEC4k (CD207, Langerin)	mannose binding specificity
	CLEC4L(DC-SIGN, CD209)	mannose-containing glycopeptides
	CLEC5a(MDL-1)	Not identified
	CLEC6a or CLEC4n (Dectin-2)	fungal α-mannans
	CLEC7a (dectin-1)	fungal β-glucans
	CLEC9a (DNGR-1)	exposed-actin (alarmin)
	CLEC10a (Mgl1, CD301a)	terminal GalNAc residues
	CLEC12a (DCLA2, MICL)	not identified
	CLEC13d (MMR, MML, manose binding receptor 1, MRC1, CD206)	various carbohydrates
	LGALS3 (galectin-3)	fungal β-mannans
	CD94 (NKG2)	nonclassical MHC glycoproteins class I
	Ly 75 (CD205, Dec 205)	Not identified
	MBL2 (mannose binding lectin)	Amyloid β protein and several carbohydrate ligands
	MMR2 (CD280)	mannosylated proteins
	Reg1	Not identified
	Reg2	Not identified
	Reg3a	Not identified
	Reg3b	Not identified (Salmonella)
	Reg3g	Gram-negative bacteria peptidoglycans
	Reg4	Gram-positive bacteria peptidoglycans
Formyl peptide family receptor family
	FPR1	fMLF, N-formylated peptides
	FPR2	LXA4, N-formylated peptides
	FPR3	F2L, N-formylated peptides

PAMP=Pathogen-Associated Molecular Pattern receptor, MAMP=Microbe-Associated Molecular Pattern Receptor, DAMP=Damage-associated Molecular Pattern Receptor

^*expressed in humans not mice,

^**expressed in mice not humans

dsRNA=double-stranded RNA, ssRNA=single-stranded RNA, ssDNA=single-stranded DNA, Mn/p=monocytes and macrophages, DC=Dendritic cells, Mast=Mast cells, IEC=intestinal epithelial cells, UBEC=urinary bladder epithelial cells

The cellular components of innate immunity

The immune cells associated with innate immune responses in the mucosa include monocytes (Mn), macrophages (Mp), dendritic cells (DC), natural killer (NK) and innate lymphoid cells (ILC), mast cells, neutrophils and eosinophils. We now know that there are cells within the gut not traditionally considered components of the immune system, like epithelial and Paneth cells, which respond to PRR signals releasing molecules that can kill bacteria and/or modulate immune responses.

The cells of innate immunity naturally reside within many tissues like the gut and liver, and more migrate in as a response to invading organisms or tissue damage. The negatively charged surface of damaged host cells and organisms activates, in tissue and plasma, molecular cascades related to coagulation, complement formation, kinin generation and others. Some of these molecules dilate blood vessels and make them more permeable allowing the easy ingress of neutrophils, macrophages and other cell types into tissue. Other components of some of these molecular cascades bind to receptors (e.g. complement receptor) on cells of innate immunity inducing their activation. As discussed above, immune cells have additional receptors [e.g. C-type lectin and Toll-like (TLR) receptors] that recognize classes of foreign molecules unique to bacteria, fungi and virus. Other such receptors can recognize molecules exposed when host cells are damaged (e.g. actin). Engagement of these receptors also can transmit molecular signals leading to immune cell activation.

Macrophages are bone marrow-derived cells that populate all tissues. Macrophages have the potential to engulf and degrade foreign matter and damaged host cells innately without the participation of T or B cells. They have a critical role in killing invading microorganisms and repairing injured tissue. Macrophages as a group are functionally diverse, behaving differently depending on the challenge. Some types of macrophages also exert “regulation” damping immune responses ^{4, 5}. The inverse also is true; lymphocytes secrete cytokines like IFNγ, IL4, or IL10 that profoundly alter macrophage activity ⁶.

Dendritic cells are mononuclear leukocytes derived from bone-marrow that have a central role in initiating/orchestrating both innate and adaptive immune responses. Several functionally distinct DC subsets have now been described based on unique patterns of cell surface protein expression. DCs are present in tissues that are in contact with the external environment such as the skin and the mucosa of the nose, lungs and gastrointestinal tract. They “sample” antigenic substances approaching or entering mucosal surfaces. They then can present fragments of these antigens to T cells, which express the appropriate antigen receptors, either stimulating or inhibiting their response to the specific antigen. DCs also display receptors associated with innate immunity (e.g. TLR and C-type lectin receptors) ⁷. When DCs encounter ligands that engage their innate receptors or when they present antigen to T cells, dendritic cells selectively secrete various inflammatory mediations that influence the development, maturation and function of T cells, B cells, macrophages and other cells associated with inflammation. DCs have been proposed as key mediators of immunological tolerance. In addition to regulating multiple aspects of T-cell physiology, they can destroy T cells or render them non-responsive to antigen (anergic). They also can promote the production of “regulatory T cells” that can block adaptive and/or innate immune responses ⁸.

Innate lymphoid cells (ILC), residing in the lamina propria, comprise natural killer (NK) cells (discussed separately below), ICL1, nuocytes (ILC2), lymphoid tissue inducer cells (LTi), and IL17 or IL22 producing NK-like cells (ILC3) ⁹. The latter mentioned ILC are newly discovered. These cells share a common lymphoid precursor with classic T and B lymphocytes but lack the clonally unique, genetically recombined receptors that define specific antigen-responsive adaptive immune cells. ILC are copious producers of cytokines that are rapidly elaborated upon stimulation. They provide the initial cytokine response to viral, bacterial, and parasitic challenge. NK cells make IFNγ as do ILC1, ILC2 cells make IL13 and IL5; and ILC3 cells make IL17 and IL22. IL17 induces a neutrophilic type response, while IL13 and IL5 promote mast cell and eosinophilic reactions. Some of these cytokines also drive and shape the subsequent T and B cell response. In addition, LTi cells are required for development of lymph nodes and intestinal Peyer’s patches ¹⁰. ILC2 cells are well described in mice where they mediate early helminth expulsion ¹¹ but until recently have been difficult to identify in humans ¹². Patients with active Crohn’s disease have an increased percentage of IL17-producing, ILC3 cells in the intestinal lamina propria as compared to that of patients with active ulcerative colitis or non-diseased intestinal tissue ¹³.

Natural killer (NK) cells are vital effector cells of the innate immune response. They provide rapid cytotoxic and cytokine-producing responses to virally infected cells and to cells undergoing tumor transformation. NK cells innately express cell surface receptors that either activate or inhibit their cellular attack. Mature NK cells do not kill normal cells that strongly express molecules that engage the NK cell inhibitory receptors like the killer-cell immunoglobulin-like receptor (KIR) which recognizes “self” MHC-I. However, the cellular attack immediately proceeds if the NK cell inhibitory receptors fail to encounter their ligands, and there is engagement of stimulatory receptors like FcγRIII, NGK2, Ly49 amongst others. ¹⁴

The cytotoxic effects of NK cells are mediated via proteins such as perforin and proteases known as granzymes. Once released, perforin creates channels through the cellular membrane allowing granzymes to enter the cytoplasm, causing cell death via apoptosis or osmotic lysis. NK cells also release α-defensin, an antimicrobial, which directly kills bacteria released from lysed cells.

Additionally, infected cells are routine targets for antibody attack. Antibodies recognize specific antigenic targets often incorporated into cell surfaces. The non-antigen recognition end of some of these antibodies can engage immunoglobulin receptors on NK cells (FcγRIII or CD16) resulting in NK activation, release of cytolytic granules and consequent target cell apoptosis. This mechanism is termed antibody-dependent cell-mediated cytotoxicity (ADCC).

Mast cells compose about 2% of the lamina propria cell population. Mast cells express immunoglobulin receptors that bind antibody (often IgE) arming the cells for response to allergens. They also express PRR receptors such as TLRs1-9 ¹⁵. On exposure to helminthes, bacteria, viruses or their products, mast cells can rapidly release histamine, proteases, and TNFα ¹⁶. As measured by immunohistochemistry, mast cells compose about 60% of TNFα+ lamina propria cells, and about 25% of lamina propria mast cells express TNFα ¹⁷. In addition, mast cells make IL10 and TGFβ, and respond to other cytokines. Human mast cells abundantly express IL33 receptor (T1/ST2/IL-1R4) and respond to IL33 with increased production of IL8 (a chemokine that attracts neutrophils) and IL13 (a Th2 cytokine) ¹⁸.

Neutrophils, once considered short-lived “cannon fodder” to control acute bacterial infections, are now understood to be integral components of innate immunity ¹⁹. They express many PRRs ²⁰ and rapidly respond to microbial challenge. In addition to well described activities like phagocytosis, reactive oxygen species (ROS) generation, and bactericidal peptide production; neutrophils can lyse in a characteristic pattern (netosis) that spreads out cellular contents to create a neutrophil extracellular trap (NET) ²¹. NETs contain DNA strands coated with antimicrobial compounds, trapping and killing extracellular organisms. Many patients with systemic lupus erythematosus have impaired clearance of NETs that may underlay development of anti-DNA and anti-histone antibodies ²².

Neutrophils also release cytokines (e.g. IL1, IL12, IL17, IL23, IL27, TGFβ) and chemokines (e.g. CCL2, CCL20, CXCL10) that influence both the activity and migration of other immune cells. For example, neutrophils and Th17 cells co-localize in inflammations such as Crohn’s disease ²³ where they can reciprocally create a pro-inflammatory circuit. Splenic neutrophil subsets also make APRIL and BAFF, assisting in B cell maturation ²⁴. Furthermore, neutrophil products are critical in NK cell development ²⁵. Patients with neutropenia have impaired NK and B cell function.

Other innate cells activate neutrophils. Intestinal epithelial cells and mast cells can release IL8 and other chemokines to attract neutrophils to a site of injury or microbial compromise ²⁶. Intestinal epithelial cells can release eicosinoids like hepoxilin A₃ from their apical surfaces that stimulate neutrophil migration into the intestinal lumen ²⁷. There, neutrophils assist in clearing away epithelial cell-adherent bacteria ²⁶.

Eosinophils are found in the peripheral blood, thymus, lower gastrointestinal tract and other tissues. They are present in the distal esophagus in people with chronic reflux esophagitis and at high frequency throughout the esophagus in some food allergies (eosinophilic esophagitis). Intestinal eosinophilia is associated with idiopathic eosinophilic gastroenteritis, food allergies, intestinal helminthic infections and, not infrequently, inflammatory bowel disease. Considering the wide-spread expression of eosinophils at sites of inflammation, it is surprising that loss of eosinophils has no apparent negative effects on normal health.

Eosinophils are short-lived cells surviving in the gut for just a few days. They grow and differentiate in response to cytokines such as IL3, IL5 and granulocyte macrophage–colony stimulating factor. They migrate into the gut in response to chemical attractants (e.g. eotaxin 1 and 2, monocyte chemoattractant protein-1 and -4, leukotriene B4) released in the intestines during helminth, fungal or some bacterial infections, or during enteric allergic reactions. They produce numerous cytokines, reactive oxygen species, eicosinoids and other mediators associated with host defense and tissue repair ^{28, 29}.

Eosinophils have a role in at least four aspects of innate immunity within the gastrointestinal track including homeostasis of the epithelial barrier, immunity to luminal pathogens, interactions with the enteric nervous system and bridging of innate and adaptive immunity ³⁰. They express receptors of innate immune (e.g. TLR) that can stimulate the release of inflammatory mediators affecting the function of other nearby leukocytes and the epithelium. These cytokines are stored preformed in granules and can be rapidly secreted allowing for an immediate innate immune response ³⁰. Eosinophils can bridge innate and adaptive immunity through their secretion of various cytokines and through antigen presenting functions, enabled by the stimulated expression of MHC class II molecules and co-stimulatory receptors (e.g. CD80, CD86 and CD40L3) ^{31, 32}. They also have a role in wound healing and tissue remodeling. Eosinophils, like neutrophils, display a mechanism of antibacterial activity involving the release of mitochondrial DNA-containing bactericidal “traps” into the extracellular space ³³.

Intestinal epithelial cells (IEC) form a sentient barrier and express PRR that enable response to microbial products. The intestinal barrier to bacterial penetration is composed of three components; 1) epithelial cell tight junctions, 2) dynamic mucus layers and 3) antimicrobial peptides. Epithelial cells are bound together at lateral surface tight junctions (zona occludens), which prevent luminal bacteria, virus and antigenic macromolecules from passing between adjacent epithelial cells to enter the lamina propria. The operation of the epithelial cell tight junction complex is regulated by cytokines such as IL17C ³⁴ which itself is produced in an autocrine fashion by epithelial cells in response to innate TLR4 and TLR5 signaling ³⁵. In mice, engineered loss of specific innate signaling pathways impairs IEC barrier function permitting increased bacterial translocation and intestinal inflammation ^{36, 37}.

Also important is the mucus layer sitting above the epithelium. Goblet cells in the mucosal lining produce this mucus. In the small bowel, there is a single unattached layer of mucus. In the colon, there are two distinct layers; a lower nearly sterile epithelial cell-adherent layer and a non-adherent outer layer inhabited by commensal bacteria ³⁸. The development of separate layers probably results from partial proteolytic cleavage of MUC2 mucin. In mice, loss of MUC2 gene expression worsens experimental (dextran sulfate sodium) colitis. Epithelial cells also release soluble C-type lectin receptors that reside in the lower mucus layers and which kill bacteria approaching the epithelial surface ³⁹.

Paneth cells reside in the epithelial cell layer located deep within the crypts. They produce various antimicrobial peptides and proteins (e.g. defensins, RegIIIA, lysozyme, secretory phospholipase A2) that help maintain aseptic intestinal crypts ⁴⁰. Production of these molecules by Paneth cells is induced by engagement of TLR and NOD2 receptors.

The “Biome” and Innate Immunity

The innate immune system responds rapidly and stereotypically to microbiologic challenge. Innate responses end up shaping the microbiome and this, in turn, helps reshape our innate and adaptive immune systems. In mice, segmented filamentous bacteria help prime the development of intestinal immunity ⁴¹. This has far reaching effects on both intestinal microbiota and systemic immunity. Helminths (parasitic worms) evolved in concert with the earliest mammals ⁴² and have shaped our genomes⁴³. Helminths also shape innate and adaptive immune responses ⁴⁴. Highly hygienic environments alter our microbiome and therefore our immune system. This alteration likely explains the emergence of immune-mediated diseases that now afflict greater than 10% of the population.

Importance

The vast majority of potentially malicious organisms to which we are exposed are not thwarted by adaptive immune recognition. Instead, they are destroyed or restrained through employment of the cells and molecules associated with innate immunity. Primary defects in the innate immunity can shift the balance of the immune response toward dysregulated adaptive immunity, resulting in the development of Crohn’s disease and other immune-mediated diseases of the gastrointestinal tract.

The innate immune system “sees” the world through the expression of genetically predetermined pattern recognition receptors (PRR). Inherited defects that impair expression of specific PRR can increase susceptibility to infection with specific classes of microorganisms ⁴⁵ and contribute to intestinal inflammation ⁴⁶ attesting to their importance. Loss of innate signaling pathways such as those mediated by NOD2, increases the risk of developing Crohn’s disease ⁴⁷.

There are many distinct cells that function in innate immune responses. However, dendritic cells are particularly important since we have learned that they are the critical link between the innate and adaptive immune systems. Also newly discovered are many distinct innate lymphoid cells that could be exceedingly important for initiating immune responses. Even many of the well characterized cells of innate immunity are now appreciated to have much broader ranges of function.

Some of these cells have critical functions in human disease. Mast cell activity appears to orchestrate eosinophilic mucosal disease like eosinophilic esophagitis ⁴⁸ and mast cell production of TGFβ may contribute to esophageal dysmotility ⁴⁹. Small bowel Paneth cell α-defensin production is decreased in patients with Crohn’s disease ⁵⁰ perhaps contributing to alterations in the normal composition of the intestinal flora ⁵¹. Many population genetic studies have noted differences in disease susceptibility resulting from inheritance of particular combinations of killer-cell immunoglobulin-like receptor (KIR) and HLA ligands. Certain combinations of KIR and HLA alleles have been implicated in preeclampsia during pregnancy ⁵², better immune response to the hepatitis C virus ⁵³ and protection against the HIV virus ⁵⁴.

Translation

The growing knowledge of the innate immune system is starting to be translated into clinical medicine. Therapeutic monoclonal antibodies that deplete eosinophils have the potential for wide deployment in the treatment of eosinophil-associated diseases ⁵⁵ like eosinophilic esophagitis or eosinophilic gastroenteritis. Therapeutic antibodies that modulate NK cell function such as rituximab (anti-CD20 mAb), trastuzumab (anti- Her2 mAb), cetuximab (anti-EGFR mAb) and mogamulizumab (anti-CCR4 mAb) are presently being used to treat cancer ⁵⁶. Probiotics, helminths and their biological products are emerging as useful therapeutic agents. They appear to work through interactions with dendritic cells and other cells of innate immunity. Newly developed therapeutics that block the function of molecules which call inflammatory cells into host tissue during the earliest stages of tissue injury are proving useful for the treatment of inflammatory bowel disease.

Also, our growing knowledge regarding the molecules make by cells of innate immunity is being used to develop useful clinical tests. For instance, lactoferrin and calprotectin are antimicrobial proteins released by neutrophils that can be measured in stool to non-invasively confirm active intestinal inflammation ⁵⁷.

Roadblocks and/or limitations

Innate reactions help coordinate adaptive immune responses and vice versa, both functioning through various overlapping immune regulatory pathways. These newly discovered pathways of immune regulation open opportunities for therapeutic intervention. However, blockage of cytokines that have the potential to act on many cell types can result in various unexpected effects. Anti-IL17 antibody (secukinumab) treatment is not helpful for Crohn’s disease ⁵⁸ likely due to far-reaching unexpected effects on intestinal innate defenses. IL6 fuels inflammation, but also drives wound healing. Anti-IL6 receptor antibody (tocilizumab), approved for refractory rheumatoid arthritis, can elicit intestinal ulceration and perforation ⁵⁹. It sometimes appears that cellular crosstalk and factor pleotropic effects conspire for chaos. Instead, these interactive systems normally create a robust homeostatic system. Deeper understanding of innate and adaptive immune interactions, and ever finer targeting of interventions, should eventually permit predictable efficacious intervention in even the most refractory inflammation.

Conclusion

Therapeutic interventions targeted to cells and molecules associated with the innate immune response can have far ranging influences on host immunity. Understanding how the innate immune system reacts to challenge and the interplay between innate and adaptive immunity will improve treatment options for patients in our care.

Glossary

ADCC

antibody-dependent cell-mediated cytotoxicity

DAMPS

damage-associated molecular patterns

DC

dendritic cells

dsRNA

double-stranded RNA

ILC

innate lymphoid cells

IEC

intestinal epithelial cells

KIR

killer-cell immunoglobulin-like receptor

Mp

macrophages

MAMPs

microbe-associated molecular pattern receptors

mAb

monoclonal antibody

Mn

monocytes

NK cells

natural killer cells

NET

neutrophil extracellular trap

PAMPS

pathogen-associated molecular pattern receptors

PRR

pattern recognition receptors

ssDNA

single-stranded DNA

ssRNA

single-stranded RNA

TLR

toll-like receptors

UBEC

urinary bladder epithelial cells