Pattern Recognition Receptor Signaling and Cytokine Networks in Microbial Defenses and Regulation of Intestinal Barriers: Implications for Inflammatory Bowel Disease

Abstract

Inflammatory bowel disease (IBD) is characterized by defects in epithelial function and dysregulated inflammatory signaling by lamina propria mononuclear cells including macrophages and dendritic cells in response to microbiota. In this review, we focus on the role of pattern recognition receptors in the inflammatory response as well as epithelial barrier regulation. We explore cytokine networks that increase inflammation, regulate paracellular permeability, cause epithelial damage, upregulate epithelial proliferation, and trigger restitutive processes. We focus on studies using patient samples as well as speculate on pathways that can be targeted to more holistically treat patients with IBD.

Summary

PRRs and their regulation of a variety of cell types can have pro- or anti-inflammatory functions in IBD. This principle is also true with respect to cytokine signaling by immune cells or epithelial cells. Extremes in antagonism of inflammatory pathways may lead to decreased epithelial barrier function as well as microbial invasion. Using combinations of strategies directed at reducing—not eliminating—inflammatory cytokine networks and improving the leak in the epithelial barrier will be essential to achieving improved clinical outcomes in IBD.

Introduction

The high density of microbes within the intestinal lumen provides multiple benefits, including nutrient breakdown and immune education. In the absence of appropriate regulation, however, responses to these microbes can cause disease. Such immune dysregulation occurs, for example, in inflammatory bowel disease (IBD). As the key structure that separates luminal materials from the underlying lamina propria, the intestinal epithelium is a critical determinant of the extent to which immune cells and other cells are exposed to microbes and their products. A broad range of microbial products are sensed by pattern recognition receptors (PRRs; which include toll-like receptors [TLRs], NOD-like receptors [NLRs], and RIG-I-like receptors [RLRs]). PRR signaling is, therefore, carefully regulated, particularly with respect to downstream cytokine secretion. While innate immune cells, e.g., macrophages and dendritic cells, are central to these responses, many other cell types, including epithelial, stromal, endothelial, and adaptive immune cells express PRRs. Moreover, PRR-initiated responses in one cell subset can modulate responses in other cell types via PRR-independent processes, many of which are essential for mucosal homeostasis. Regulation of microbial exposure and sensing is therefore essential for intestinal immune conditioning and responses that limit bacterial burden and prevent excessive inflammatory responses (Figure 1). Here we review mechanisms of cross-talk between immune and epithelial cells within the intestinal environment, the contributions of PRRs and cytokines to these processes, and their contributions to intestinal homeostasis or, when dysregulated, disease.

Figure 1. Upon microbial challenges, balancing PRR responses in innate immune cells is critical for regulating interactions between innate and adaptive immunity, cytokines, and epithelial barrier function.

Either low or high levels of innate immune responses through PRR-initiated pathways can ultimately lead to intestinal inflammation. With initial inadequately low innate immune responses, the low levels of antimicrobial pathways and Th1- and Th17-conditioned adaptive pathways (which are also required for bacterial responses) can lead to high levels of bacterial burden in intestinal tissues resulting in injury and subsequent inflammation. With excessive PRR responses on innate immune cells the high levels of cytokines can similarly lead to intestinal inflammation, which in turn disrupts the epithelial barrier, thereby further amplifying the inflammation. MLN, mesenteric lymph node.

PRRs and shaping of innate immune responses in the intestine

Innate immune cells are key mediators of PRR-initiated responses to microbes. There is a diverse spectrum of innate cells in the intestine; these cells cross-regulate each other and are also modulated by local factors, including the microbiome.^1–5 Intestinal macrophages are one critical innate immune cell subset. Experiments in mice have shown that intestinal macrophages are continuously replenished by peripheral monocytes in a CCR2-dependent manner.⁶ Depending on the intestinal environment into which the peripheral monocytes are recruited, they can give rise to various myeloid cell phenotypes, including anti-inflammatory resident intestinal macrophages, and macrophages that mediate microbial clearance, intestinal injury, or resolution of inflammation after injury.^6–11

Under homeostatic conditions intestinal macrophages are more effective at clearing bacteria but have reduced cytokine secretion relative to peripheral macrophages.^12,13 This allows for microbial clearance with minimal tissue injury. A range of factors within the intestinal environment contribute to these dual outcomes. The prolonged exposure to microbial products occurring in the intestine leads to chronic PRR stimulation that upregulates antimicrobial processes while downregulating inflammatory cytokines.^14–16 The importance of PRRs in conditioning intestinal immune cells is highlighted by the adverse outcomes induced by deletion of specific TLRs, e.g., TLR4 or TLR5, or essential components of TLR signaling pathways. TLR signaling defects can result in an increased bacterial burden, altered microbiota, increased expression of inflammatory mediators, dysregulation of immune cells, and intestinal inflammation (Figure 1).^17–26 The Crohn’s disease (CD) associated PRR NOD2 has been well-studied in this regard.^{15, 27} In the absence of NOD2, antimicrobial pathways are impaired and inflammatory cytokines are not adequately downregulated. As a result, experimental colitis severity can increase in NOD2-deficient mice,^27,28 while mice with NOD2 overexpression have resistance to experimental colitis.²⁹ Combined deficiency of NOD2 with other IBD-associated pathways and/or additional risk factors can further increase colitis severity ^30,31. For example, combined deficiency of NOD2 and gp91phox (NADPH oxidase member) in mice can lead to early-onset spontaneous intestinal inflammation associated with accumulation of Mucispirillium schaedleri ³¹.

The conditioning of intestinal macrophages through PRRs leads to the upregulation of anti-microbial pathways.¹⁴ One such pathway is autophagy,¹⁶ and genetic variants (ATG16L1, IRGM, MTMR3) leading to a loss-of-autophagy increase susceptibility to IBD.³² PRR stimulation also promotes production of reactive oxygen species (ROS) and reactive nitrogen species in macrophages, and these pathways cooperate to limit systemic dissemination of intestinal microbiota in mouse models (Figure 1).³³ The importance of PRR signaling and microbial conditioning for mounting defenses to intestinal infectious challenges extends to additional cell subsets, including neutrophils³⁴ and NK cells.³⁵ Innate lymphoid cells (ILCs) are also important for homeostasis and regulating bacteria. For example, ILCs regulate dissemination of bacteria (e.g. Alcaligenes)³⁶ and commensal bacteria-specific CD4+ T cell responses;³⁷ ILCs are expanded in human IBD and can also contribute to the inflammation in experimental colitis.^38,39 Interestingly, while PRR-initiated signaling in myeloid cells promotes antimicrobial pathways, downstream consequences of PRR signaling (e.g. endosomal acidification) can also serve as cues for upregulating microbial virulence factors and intracellular microbial growth (e.g. SPI2 genes in S. Typhimurium).^40,41 Microbial-dependent dietary products also contribute to beneficial conditioning of intestinal macrophages, as is observed with butyrate-induced autophagy and antimicrobial proteins.^42,43

Intestinal myeloid cells are continuously exposed to microbial products and downregulating inflammatory responses to these products is essential. Mechanisms mediating the downregulation of PRR-initiated inflammatory pathways in myeloid cells include upregulation of inhibitory cell surface molecules (e.g. Tyro, Axl, Mer), inhibitory intracellular molecules (e.g. IRAKM, Tollip, IRF4, ZNRF4, A20), transcriptional repressors (e.g. Twists, NFκB1) and autocrine/paracrine inhibitory secreted mediators (e.g. IL10, TGFβ).^{11–13,15,28,44–53}. Anti-inflammatory mechanisms in myeloid cells can be induced by specific microbes, microbial products and/or metabolites (e.g. Clostridium species, polysaccharide).^54,55 Additional mechanisms contributing to cytokine downregulation in intestinal myeloid cell subsets include the uptake of apoptotic cells (e.g. epithelial cells).^56–58

As autocrine/paracrine cytokine pathways are important drivers of PRR-initiated responses, mechanisms inhibiting inflammatory cytokine-initiated signaling pathways (e.g. SOCS family members) or promoting anti-inflammatory cytokine signaling (e.g. SMAD family members) also contribute to downregulatory mechanisms in innate immune cells. As such the ability of innate immune cells to respond to IL10 is critical to anti-inflammatory conditioning in the intestine and to regulating intestinal inflammation.^59,60 Additional anti-inflammatory mediators such as TGFβ, which can be secreted by intestinal stromal cells, also downregulate PRR-induced inflammatory cytokine secretion from myeloid cells.¹² Mechanisms allowing for increased TGFβ responsivity can be modulated in the intestine. For example, PRRs upregulate the integrin αvβ8 which promotes TGFβ activation, and these pathways are upregulated in intestinal dendritic cells.⁶¹ On the other hand, intestinal myeloid cells from IBD patients and animal models of colitis can demonstrate reduced responsivity to anti-inflammatory cytokines, including to IL10⁶² and to TGFβ due to upregulation of SMAD7.⁶³ Consistently, intestinal myeloid cells from IBD patients demonstrate an inflammatory phenotype with increased production of inflammatory cytokines (e.g. IL23, IL12, TNF, IL1β).⁶⁴ Therefore, on the one hand, microbes and PRR-initiated pathways promote inflammatory cytokines (e.g. IL23, IL12, TNF, IL1β) and colitis.^65,66 On the other hand, intestinal microbiota and PRRs mediate numerous protective effects in intestinal tissues, such that in the absence of PRR signaling colitis can be more severe and the ability of microbial communities to mediate beneficial effects may be impaired.^21–26

PRRs and regulation of adaptive immunity

Adaptive immunity plays an important role in both intestinal immune homeostasis and in the inflammation observed in IBD patients. Th1 (IFNγ, TNF), Th17 (IL17, IL21, IL22), and Th9 (IL9) cells are present in intestinal tissues under homeostatic conditions, but their dysregulation can lead to IBD.^67–72 Regulatory T cells (Tregs) are similarly present in intestinal tissues during homeostasis and are critical for downregulating inflammatory pathways. The proper recognition and response to microbial products through PRRs is required for optimally regulating T and B cell adaptive immune responses; this regulation can occur through pathways both intrinsic and extrinsic to adaptive immune cells.

Direct PRR stimulation of T cells can regulate T cell outcomes which can, in turn, modulate colitis outcomes.^21,73,74 Distinct PRRs can also cross-regulate each other (e.g. RIG-I like receptors can suppress activation by TLRs⁷⁵) to modulate T cells. In contrast, some studies have shown that expression of the TLR adaptor protein MyD88 by T cells is not necessary for either pathogenic or regulatory functions in the intestine.⁶⁶ With respect to adaptive immune cell-extrinsic PRR stimulation, PRR stimulation of innate immune cells, which can serve as antigen presenting cells, enhances antigen uptake and antigen presentation and upregulates costimulatory molecules and cytokine secretion, thereby modulating T cell activation and differentiation.⁷⁶ When PRR pathways are deficient in myeloid cells (e.g. TRAF6, LACC1, IRF5^77–79), Th1 and Th17 cells can be reduced and Th2 cells, in turn, can increase. While this T cell modulation can reduce tissue damage in some situations, it can also reduce efficacy of microbial clearance and increase susceptibility to intestinal injury and subsequent inflammation (Figure 1). The proper recognition and sensing of microbial products can also regulate myeloid cell-dependent trafficking of microbes to mesenteric lymph nodes, thereby regulating both T and B cell responses.⁸⁰ Adaptive immune cells can also cross-regulate myeloid cells and intestinal microbiota. For example, adaptive immune-dependent IL17 can recruit neutrophils which in turn regulate IL22, antimicrobial peptides, and expansion of potentially harmful microbiota in mice (e.g. segmented filamentous bacteria [SFB]).⁸¹

Consistent with the role of PRRs in regulating T cells, specific microbes can promote distinct T cell phenotypes. For example, various Clostridia strains can promote Tregs in colonic mucosa and thereby protect from colitis.⁸² On the other hand, microbial-driven IL23 increases with inflammation and promotes more pathogenic Th17 cells which can drive disease pathology.^68,83,84 Importantly, transfer of microbiota from IBD patients into germ free mice can lead to increased Th1 and Th17 cells and reduced Treg cells, epithelial barrier dysfunction, increased bacterial translocation and more severe experimental colitis.^85–87 Microbes can also regulate intestinal adaptive immune outcomes through PRR-independent mechanisms. For example, intestinal microbiota regulate bile acids (e.g. lithocholic acid derivatives), which in turn, can regulate adaptive immune outcomes (e.g. Th17, Tregs).⁸⁸ Microbial-derived butyrate generated upon fermentation of dietary fiber induces colonic Tregs in mice.⁸⁹

Antibody production to microbial products in the intestine requires PRR signaling.⁹⁰ For example, PRR stimulation (e.g. TLR5) of lamina propria myeloid cells can result in their production of retinoic acid and differentiation of naïve B cells into IgA-producing plasma cells⁹¹ and MyD88 on intestinal T cells coordinates germinal center responses and IgA+ B cells;⁹² this can in turn modulate the ability of bacteria to breach the intestinal epithelial barrier.⁹³ TLRs can also promote IgG responses from B cells which protect from systemic bacteria⁹⁴ and IL10-producing intestinal B cells which are able to reduce colitis severity.⁹⁵ Taken together, PRRs and cytokines cooperate to modulate the spectrum and specificity of adaptive immune responses in the intestine. The coordination of PRRs, cytokines and immune responses, in turn, cross-regulate intestinal epithelial cells.

Epithelial barrier function

The intestinal barrier separates the lamina propria and deeper tissues from the harsh luminal environment. Diminished function of this barrier is commonly cited as both a cause and consequence of disease. Although useful as an overarching term, understanding the mechanisms that lead to altered barrier function in specific settings has profound pathophysiological and therapeutic implications. In general, barrier function is considered to be the opposite of permeability. Although this generally refers to paracellular permeability, there are multiple pathways by which materials can cross the epithelium. These can be divided into transcellular and paracellular routes. Transcellular transport is exemplified by absorption of most nutrients and salts, in which distinct apical and basal transporters ensure vectorial, i.e., directionally-oriented, solute movement. Transcellular transport can also be mediated by vesicular transport, of which IgA transcytosis into the lumen and antigen presentation by M cells are examples. In contrast to transcellular transport, paracellular transport is passive, with the direction of net transepithelial flux determined by existing gradients. Because these gradients can be established by transcellular transport, paracellular transport can be directed by transcellular transport. For example, diarrhea induced by TNF, a prototypic effector of increased paracellular permeability and net fluid efflux, requires both protein kinase C-mediated inhibition of NHE3-mediated Na⁺ absorption, which reduces the osmotic driving force for paracellular water absorption, as well as myosin light chain kinase (MLCK)-induced tight junction permeability increases that mediate paracellular water efflux.⁹⁶

Intestinal permeability is frequently equated with permeability of the tight junction, which seals the space between cells and is the rate-limiting step of paracellular flux. It is, however, important to also consider barrier loss and increased passive flux that occurs at sites of epithelial damage. Flow at these sites does not display the size- and charge-selectivity that characterize trans-tight junction flux and is, therefore, referred to as unrestricted. The unrestricted pathway is the route by which bacterial invasion and massive macromolecular flux occur. This contrasts sharply with the two routes across the tight junction which, as discussed below, are highly selective.

Trans-tight junction routes differ in molecular composition, physical characteristics, and mechanisms of regulation. The pore pathway is created by some members of the claudin protein family and represents physical channels that traverse the tight junction.^97–99 These actively-gated channels create a high-capacity route that is both charge and size-selective. In the gut, the channels are generally cation selective and have maximum diameters of 6 – 8 Å (0.6 – 0.8 nm) that restrict flux to water and small ions.^99–102 This pathway does not, however, have the structural specificity of the transmembrane transporters that drive transcellular transport, and also transports other small cations that transmembrane transporters exclude. The second trans-tight junction flux route, the leak pathway, is regulated by the dense ring of actin and myosin that encircles the tight and adherens junctions. Activation of MLCK, which induces remodeling of the perijunctional ring and triggers endocytic removal of some tight junction proteins, e.g., occludin, increases permeability of the low-capacity, charge-nonselective leak pathway, which accommodates molecules up to ~125 Å (12.5 nm) in diameter, including small proteins.^96,103–106 This size-selectivity prevents bacteria from crossing pore or leak pathways. However, the relative absence of specificity allows the leak pathway to mediate flux of some bacterial products, including small metabolites and lipopolysaccharide (LPS) (Figure 2).

Figure 2. Immune regulation of tight junction-dependent and tight junction-independent passive permeability pathways.

In addition to transcellular transport (not shown), three distinct routes account for intestinal permeability. These are the trans-tight junction pore (green) and leak (blue) pathways and the tight junction-independent unrestricted pathway (red). The immune system can selectively upregulate pore pathway permeability by IL22-induced upregulation of claudin-2 (green) expression. In contrast, TNF primarily upregulates leak pathway permeability by activating myosin light chain kinase transcription and enzymatic activity. Flux via the unrestricted pathway (red) occurs at sites of epithelial damage and is independent of tight junctions. LPS, lipopolysaccharide.

Measuring barrier function in vivo

In vivo, epithelial barrier function has been variably defined and measured using a range of approaches including assessment of mucus thickness, epithelial morphology, tight junction protein expression, recovery of orally-delivered inert probes, or presence of endogenous markers thought to correlate with barrier permeability in serum or urine. As might be expected, these all report on different aspects of barrier function that may be disrupted by a wide range of factors. All of the approaches do, therefore, have utility depending on the question being studied (Table I). However, only recovery of inert probes measures intestinal permeability directly.

Table I.

Molecules that have been used as markers of intestinal permeability changes

Orally-delivered probes
Lactulose, 11.5Å (d)	Gold standard (as part of lactulose:mannitol ratio)
Mannitol, 7Å (d)	Gold standard (as part of lactulose:mannitol ratio). Sometimes present in foods; 13C-mannitol can be used instead
4kD dextran, 28Å (d)	Large quantities required, useful in small animal models
70 kD dextran, 120Å (d)	Large quantities required, useful in small animal models
Albumin, 7Å (d)	Degraded by intestinal proteases
Creatinine, 5.2Å (d)	Can be used in humans and animals
Cr-EDTA, 13.6Å (d)	Data inconsistent with similar sized probes
Erythritol, 7.4Å (d)	>90% absorbed, may compete with other sugars
Inulin, 27.8Å (d)	Too little absorbed to be useful
Ovalbumin, 8Å (d)	Degraded by intestinal proteases
PEG400, 3.4Å (d)	Linear molecule, difficult to analyze and interpret
PEG4000, 31.8Å (d)	Linear molecule, difficult to analyze and interpret
PEG900, 16.8Å (d)	Linear molecule, difficult to analyze and interpret
Rhamnose, 9.8Å (d)	Can be metabolized, has been used in place of mannitol
Sucralose, ~13Å (d)	Alternative to lactulose, not metabolized by colonic bacteria
Tc-DTPA, ~14Å (d)	Limited data, likely similar to Cr-EDTA
Urea, 3.6Å (d)	Active transcellular transporters exist
Serum/plasma markers
16s bacterial DNA	Possible surrogate for LPS
C reactive protein (CRP)	Acute phase reactant
Citrulline	Marker of epithelial damage, not permeability
Claudin-3	Small changes, uncertain mechanism rarely used
Cortisol	Stress marker
Cytokines (IL-1β, IL-6, IL-8, TNF)	Inflammatory marker
FABP (intestinal fatty acid binding protein)	Marker of epithelial damage, not permeability
LPS	Unlikely to be detectable in many cases
LPS binding protein	Acute phase reactant
Soluble CD14	Marker of monocyte activation
Zonulin	Controversial
a-1-acid glycoprotein (α-1AGP)	Acute phase reactant
Fecal/urine markers
Calprotectin (fecal)	Inflammatory marker, neutrophil product
Claudin-3 (urinary)	Small changes, uncertain mechanism, rarely used
Lactoferrin (fecal)	Inflammatory marker, neutrophil product
Lipocalin-2 (fecal)	Inflammatory marker, neutrophil product

In patients, the most commonly-used measure of intestinal barrier function measures urinary recovery of lactulose and mannitol, which have diameters of 11.5 Å and 7 Å, respectively. Both of these cross the leak pathway but are too large to be accommodated by the pore pathway in vitro. It remains to be determined, however, if some mannitol crosses the pore pathway in vivo. Because mammals lack transporters and enzymes necessary for absorption and metabolism of lactulose and mannitol they can only cross the barrier at tight junctions or at sites of epithelial damage. Once absorbed, both probes are small enough to pass through the glomerular filter into the renal tubules and, since they cannot be actively resorbed, recovered in the urine. Of course, factors other than intestinal permeability can affect urinary recovery, including impaired renal perfusion and renal disease. Some investigators have also reported that mannitol in the food chain has become a confounder and have turned to ¹³C-mannitol instead of unlabeled mannitol.¹⁰⁷ Breakdown of lactulose and mannitol by bacteria can skew results in patients with small bowel bacterial overgrowth and also prevents their use to measure colonic permeability. Sucralose, which is slightly larger than lactulose and resistant to bacterial degradation can be used to overcome this problem and is often used in single-probe assessments of colonic permeability. Nevertheless, the lactulose-mannitol dual-probe assay remains the gold-standard for in vivo measurements of small intestinal permeability in human subjects.

Interactions between immune cells and the epithelial barrier

The roles of cytokines in directing epithelial barrier loss are well documented. Cytokines including IL6, IL13, and IL22 upregulate expression of claudin-2, which forms cation-selective paracellular channels that enhance pore pathway permeability (Figure 2).^99,108–110 In contrast, TNF, LIGHT, and IL1β induce epithelial MLCK expression upregulation that triggers occludin removal from the tight junction and increases leak pathway permeability.^{103–106,111–116} TGFβ has been reported to reduce paracellular permeability through incompletely-defined mechanisms.^117–119 γδ T cells may also support maintenance of the epithelial barrier in mice.^120,121 In the context of damage, adaptive and innate immune cells can signal through cytokine networks to promote epithelial proliferation and restitution, which ultimately leads to barrier restoration. These cytokine networks include IL22, IL23, IL33, IL36, IL10, TGFβ, and, perhaps counterintuitively, TNF and IL6.^23,122–129

PRR signaling also contributes to mucus layer maintenance and function of specialized cells, e.g., Paneth cells.^130,131 Macrophages can regulate the epithelial barrier directly by cytokine secretion and via cross-regulation of other immune cells. For example, microbial sensing through MyD88 in CX3CR1+ mononuclear phagocytes cross-regulates ILC3s, which in turn leads to IL22 production, epithelial claudin-2 expression, increased pore pathway permeability, and protection from microbial-induced colitis.^109,132 Stromal cells contribute to epithelial barrier restoration subsequent to damage. PTSG2-expressing stromal cells migrate to the base of crypts in a MyD88-dependent manner, allowing for intestinal epithelial cell (IEC) proliferation after injury.¹³³ The manner in which immune cells regulate the epithelial barrier is also influenced by dietary factors (e.g. fiber, sugars, fat, emulsifiers) and this may be through both direct and indirect mechanisms, including modulation of intestinal microbiota, interactions with PRRs, and cytokine production.^134–144 Therefore, multiple factors modulate the interactions between PRRs and cytokines on immune cell subsets which can contribute to both epithelial health and to the dysregulated barrier observed during disease.

Epithelial PRR functions

Intestinal epithelial cells have multiple mechanisms through which they regulate responses to the high density of microbes in the lumen, including through PRR signaling. PRRs are generally expressed at greater levels in colonic compared to small intestinal epithelia and these PRR responses need to be carefully regulated.^145,146 The TLR and IL1β receptor adapter protein MyD88 is critical to epithelial expression of antimicrobial proteins (e.g. Regenerating family member 3γ [Reg3γ], Reg3β, RELMβ) that limit penetration of resident and pathogenic bacteria.^147,148 MyD88 is also required for optimal induction of autophagy, which limits dissemination of enteric pathogens.¹⁴⁹ The threshold of PRR signaling is also important in epithelial cells as overly robust PRR signaling can lead to impaired intestinal resident microbial colonization, and in turn, increased susceptibility to enteric pathogens.¹⁵⁰ Interestingly, PRR expression changes with development. For example, intestinal epithelial TLR5 expression is much lower in neonates relative to older individuals.¹⁴⁶ This may contribute to the altered microbiome of neonates and susceptibility of neonates to certain infections.

Epithelial cells reduce responses to microbial products through downregulating key PRR-associated signaling molecules (e.g. IRAK-1¹⁵¹) or upregulating molecules inhibiting PRR signaling (e.g. Tollip, SIGIRR).^150,152 In fact, the failure to properly regulate such pathways after birth may contribute to necrotizing enterocolitis pathogenesis.¹⁵¹ Some studies have demonstrated segregation of PRRs on epithelial surfaces and/or differential outcomes depending on the surface mediating the PRR response as another mechanism by which epithelial cells regulate responses to resident microbes.^153,154

Epithelial cross-talk to immune cells and regulated transfer of microbial communication

Contrary to conventional wisdom, increased intestinal permeability is insufficient to cause disease in both human subjects and animal models.^{109,155–162} However, studies using genetically-modified animal models have shown that increases in either pore or leak pathway permeabilities are sufficient to augment immune-mediated systemic and intestinal disease.¹⁶³ Moreover, either pharmacological restoration of pore or leak pathway barrier function is therapeutically effective in experimental IBD.^111,157 Although similar experiments cannot be performed in humans, recent data show that, in healthy first degree relatives of CD patients, increased permeability is a risk-factor for later IBD development.¹⁶² In patients with established CD, increased permeability during remission is a risk-factor for disease reactivation.^164,165 Notably, environmental factors including stress and microbiome perturbations can modulate intestinal permeability.^166–170 Thus, extensive evidence links increased intestinal permeability to pathogenesis and progression of intestinal disease.

Epithelial cells provide instruction to immune cells residing within intestinal lymphoid tissues; this instruction contributes to homeostatic pathways in health but can also promote inflammatory pathways. As an increasing spectrum of epithelial cell subsets is defined through single-cell sequencing and other approaches,¹⁷¹ the specialized functions of these subsets provide insight into important cross-talk mechanisms. Under homeostatic conditions, IECs can condition myeloid cells (e.g. dendritic cells, CX3CR1+ antigen presenting cells) to a more ‘non-inflammatory’ and/or ‘regulatory’ phenotype.^172,173 Epithelial cells secrete cytokines such as IL33 and TSLP, which then regulate underlying immune cells. For example, IL33 can promote colonic Treg function¹⁷⁴ and a feedback loop to ILC2s through the amphiregulin-epidermal growth factor receptor (EGFR) pathway which then reduces inflammation and restores epithelial cell function during injury.¹⁷⁵ TSLP secreted from epithelial cells can condition ‘non-inflammatory’ dendritic cells which secrete IL10 and promote a Th2 response upon microbial challenges; TSLP expression can be reduced in epithelial cells from CD patients.¹⁷² Studies in recently described intestinal tuft cells have shown that they express IL25 which promotes ILC2 homeostasis and during helminth infection allows for ILC2-mediated secretion of Th2-associated cytokines which then promotes differentiation of tuft cells and goblet cells along with mucus production.^176–178 While mucus is traditionally considered to protect the underlying epithelium from luminal bacteria and food antigens, glycans in the mucus can also instruct underlying myeloid cells with tolerogenic signals.¹⁷⁹ Additional epithelial cell mechanisms (e.g. retinoic acid receptor β, APRIL) can promote regulation of Th17 cells, CD4+ T cell homing, and IgA-producing B cells.^180,181 Importantly, increased intestinal permeability can also increase antimicrobial and regulatory mechanisms in both innate and adaptive immune cells, which ultimately can improve outcomes with acute injury.^16,119,182 While increased intestinal epithelial permeability frequently increases recruitment of a range of intestinal immune cells, in some cases this increased permeability can also lead to reduced recruitment of select immune cell subsets (e.g. neutrophils).¹⁸³

Epithelial cells play a key role in transferring microbial products and luminal substances, which then condition the underlying immune system (e.g. SFB and bacterial strains from UC patients can promote Th17 cells).^184,185 Specialized epithelial subsets such as goblet cells can transfer intestinal luminal antigens,¹⁸⁶ with MyD88 regulating the transfer of antigens by goblet cells.¹⁸⁷ In addition, lamina propria myeloid cell extensions between epithelial cells and into the intestinal lumen enable sampling of luminal antigens; these can depend on PRR signaling (e.g. MyD88), and are increased during injury.^188–190 In some cases the epithelial regulation of underlying cells can then lead to communication back to the epithelial cells. For example, during C. rodentium infection, goblet cell-derived RELM-β recruits CD4+ T cells and promotes IL22 production, which in turn, leads to IEC proliferation and reduced mucosal pathology.¹⁹¹ Epithelial cells also communicate with ILC3s, which can in turn modulate epithelial outcomes.¹⁹² In aggregate, the specific PRRs stimulated, the strength and environment of this stimulation, and the cell subsets undergoing this stimulation influence the direct responses, cytokines secreted, and cross-talk between the intestinal epithelium and immune cell subsets.

IBD-associated genes regulating PRRs, cytokines and epithelial cells

The success in IBD genetic discoveries has provided an important opportunity to better understand IBD pathogenesis; a number of IBD-associated genes have been found to regulate PRR-initiated outcomes and cytokines, and in turn, epithelial and immune cell interactions in the intestine. Over 240 genetic loci have been identified to alter IBD susceptibility.^32,193–195 Consistent with the importance of balancing PRR regulation, genetic variants leading to either a relative decrease or increase in PRR signaling can modulate IBD risk (Figure 1). Variants leading to a loss-of-function in the CD-associated gene NOD2 were an early example of risk genes leading to a loss in PRR-initiated pathways;^196,197 they can lead to a loss of both antimicrobial mechanisms and tolerance. Additional IBD-risk genes conferring a loss-of-function in PRR-initiated outcomes include ATG16L1, ICOSL, LACC1, INAVA, IL18RAP, and RNF186.^32,198–206 Further highlighting the importance of antimicrobial mechanisms are numerous rare coding genetic variants associated with early onset IBD and leading to a loss in antimicrobial pathways.^207–212 On the other hand, genetic variants leading to increased PRR-initiated signaling and cytokines from innate cells can also increase risk for IBD, with examples including IL23R, IRF5, TNFSF15, TPL2 (MAP3K8), JAK2, STAT3/5, STAT1/4, IL10R, PTPN2, PTPN22, and MTMR3^32,213–225 Consistent with the important role for myeloid cells in regulating cytokine secretion during intestinal injury, and cytokines in turn regulating barrier function through both direct and indirect mechanisms, IBD genes such as PTPN2, TNFSF15, and IRF5 can regulate barrier function and/or mucosal healing through their role in myeloid cells.^79,226,227

Genes conferring altered risk for IBD can do so through regulating multiple distinct cell types. For example, IBD-associated variants in PTPN2, IL23R, IRF5, ATG16L1, RNF186, and INAVA can regulate myeloid cell, T cell and/or epithelial cell outcomes.^{201,202, 204–206,228–240} In some cases a given gene can differentially regulate inflammatory outcomes in distinct cell types. For example, the increased TPL2 expression in macrophages with the IBD-risk variant leads to increased inflammatory cytokines/inflammation.²¹⁵ In contrast, TPL2 expression in intestinal myofibroblasts is essential for optimal levels of the PGE2 pathway which protects from epithelial injury.²⁴¹ In other cases modulation of the risk gene expression in two different cell types may be cooperative. For example, decreased INAVA expression with the IBD-risk variant in human macrophages leads to decreased PRR-induced outcomes with a reduced ability to clear intracellular bacteria,²⁰¹ and reduced INAVA expression in epithelial cells leads to reduced epithelial barrier function.^206,233

Various rare IBD-risk coding variants can lead to altered enterocyte function (e.g. apoptosis, adhesion, and permeability), including TTC7A and CDH1.^242,243 NOX1 regulates epithelial brush border ROS in colonic crypts.²⁴⁴ In some cases, studies have further defined the regulation of these genes in vivo through epithelial cell-intrinsic regulation of the gene. Epithelial cell-intrinsic XBP1 deletion leads to altered ER stress and dysregulated responses to microbial challenges.^234,245 A number of IBD-associated genes regulate cytokine pathways in epithelial cells, including PTPN2.^246–248 Either ATG16L1 deletion or the ATG16L1 T300A CD risk variant leading to a loss in autophagy can lead to increased ER stress, abnormal morphology, and reduced bacterial clearance in Paneth cells.^{202,234–237} The cooperation of ATG16L1 dysfunction with environmental factors such as viral infection promotes the intestinal inflammation and altered Paneth cell phenotypes observed in mice.^202,249 In human studies, combined expression of ATG16L1 and NOD2 IBD-risk alleles cooperate to promote abnormal Paneth cell phenotypes,²⁵⁰ as do ATG16L1 risk alleles and environmental interactions, such as smoking.²⁵¹ In another example of cooperative interactions, whereas deficiency of either A20 (regulates both PRR and TNF signaling) or ABIN-1 in epithelial cells leads to minimal IEC loss, combined A20 and ABIN-1 deficiency in epithelial cells leads to IEC death and increased lethality in mice.²⁵² Taken together, the IBD genetic discoveries have highlighted pathways leading to crosstalk between PRRs, cytokines and epithelial cells to promote both health and disease.

IBD Therapies and regulation of barrier function and PRRs

Clinical trials targeting pathways implicated in IBD pathogenesis provide an important opportunity to understand how these pathways function in human disease. In some cases, these clinical trials have highlighted potential unintended effects of blocking select cytokines in epithelial function, including in epithelial proliferation, barrier integrity and antimicrobial protein expression.

Mucosal healing and biologic therapy: Pathogenetic mechanisms of barrier dysfunction targeted by therapy

Anti-TNF therapy ushered in the modern era of IBD treatment and demonstrated that effective therapy could, and ideally should, heal mucosal ulcerations and dampen endoscopically-detectable inflammation (Figure 3). More recently, the definition of mucosal healing has been extended to include resolution of histologic inflammation. TNF inhibition promotes mucosal healing by multiple pathways. These include prevention of TNF-induced increases in leak and unrestricted pathway permeabilities. For example, disruption of TNF-induced leak pathway regulation has proven effective, and superior to anti-TNF, in experimental immune-mediated IBD.^111,156

Figure 3. IBD Therapies and regulation of PRRs, cytokines, and barrier function.

Various current (red boxes) and investigational (blue boxes) therapies in IBD patients that target cytokine pathways can also impact epithelial function, in some cases with unintended effects. PRR-initiated signaling leads to cytokine secretion and cytokines, in turn, cooperate with PRR pathways, ultimately affecting a wide range of intestinal cell subsets. To successfully achieve mucosal healing and remission, a multipronged approach aimed at strategically reducing inflammatory cytokine networks while simultaneously improving epithelial barrier function and maintaining beneficial PRR-mediated outcomes is necessary.

TNF has pleiotropic effects including proliferation, differentiation, inflammation and cell death depending on the local concentration, tissue type or cellular context. TNF can promote proliferation and expansion of naïve and pathogenic effector T-cell populations. In epithelial cells, TNF can trigger proliferation, increase paracellular permeability, or induce apoptosis. To some extent, these likely represent activities of different receptors, as TNF can transactivate EGFR and ErbB2 and can also signal directly via 2 distinct TNF receptors.²⁵³ Signaling via TNFR1, the low affinity TNF receptor, is initiated by high local TNF concentrations and induces caspase-3-dependent epithelial apoptosis.^{254, 255} In contrast, signaling via the high affinity TNF receptor TNFR2, which can be activated by low TNF concentrations, activates epithelial proliferation, migration, and barrier regulation.^{156, 256–258} Thus, TNF can increase leak pathway permeability via TNFR2 activation and unrestricted pathway permeability via TNFR1. Effective anti-TNF antibodies in IBD have been suggested to promote expansion of Tregs while inducing apoptosis of lamina propria effector T cells.^259,260 Moreover, similar to various cytokines, TNF amplifies PRR-initiated signaling, such that anti-TNF therapies likely also reduce PRR-initiated inflammatory pathways in intestinal tissues. Despite the efficacy of TNF blockade in IBD, only one third of patients are in remission after one year.⁶⁸ In some of these patients, disease may be driven by oncostatin M (OSM),²⁶¹ an IL6 family member that may amplify production of chemokines, cytokines, and endothelial adhesion proteins, thereby enhancing leukocyte recruitment. This pathway may synergize with those activated by TNF, such that some patients may benefit from combination therapies targeting both TNF and OSM. IBD patients have increased levels of IL1β and its receptor.^262–265 There is an ongoing study of anakinra, an IL1R antagonist, in patients with acute severe UC.²⁶⁶ Some children with very early onset IBD have inherited mutations in the IL10 receptor, make increased levels of IL1β, and respond to IL1 antagonists including anakinra.²⁶⁷ IL18 is an IL1 superfamily member and is also upregulated in lamina propria mononuclear cells (LPMC) and serum of CD patients.^268–271 Blocking IL18²⁷² and epithelial-specific deletion of IL18R or IL18²⁷³ reduced experimental colitis severity. IL18 antagonists are being considered for CD. However, it is important to note that the NLR family has complex roles in the intestine leading to both protective^274,275 and inflammatory^276,277 effects, such that deficiency and/or blockade of NLR family members, as well as IL1β and IL18, can also promote intestinal inflammation due to key roles in regulating intestinal microbiota and epithelial integrity.^278–280 Moreover, the IBD-associated variants in the IL18RAP/IL18R1/IL1R1 region conferring risk for IBD lead to reduced expression of these receptors in myeloid cells,¹⁹⁹ indicating that a reduction in these pathways can promote human IBD. Given the complexity of these pathways in intestinal inflammation, it may be necessary to identify those subsets of patients benefiting from targeting these pathways.

Therapies with adverse effects on mucosal function

The IL23/Th17 pathway has been an important therapeutic target in IBD patients. An antibody to the shared anti-IL12p40 subunit, ustekinumab, is FDA approved for CD and UC patients. Multiple anti-IL23p19 agents are currently in clinical trials for IBD patients and demonstrate efficacy. However, the IL23/Th17 pathway has important roles in epithelial cell regulation. IL23R^−/−RAG^−/− mice demonstrate more severe DSS-induced colitis with delayed epithelial recovery in the context of reduced IL22.²⁸¹ Mice with IL23R deleted from epithelial cells similarly demonstrate more severe experimental colitis with reduced IL22 and reduced antimicrobial proteins (e.g. Reg3β); complementation of either IL22 or Reg3β improved outcomes.²⁸² Therefore, it is possible that in a subset of patients, blocking IL23 may adversely impact its contributions to epithelial cells and thereby reduce therapeutic efficacy. IL17 was hypothesized to be a Th17-associated cytokine mediating the intestinal inflammation in IBD patients. However, trials blocking either IL17 (Secukinumab)²⁸³ or the IL17 receptor (brodalumab)²⁸⁴ did not show efficacy in phase 2 trials in CD patients. Animal studies highlighted important roles for IL17 in epithelial barrier function,^285,286 thereby suggesting that this essential role for IL17 in epithelial cell function outweighed the benefits of blocking IL17.

Administration of exogenous IL22 is also being investigated as a therapy for IBD patients. IL22 can promote repair of epithelial damage by stimulating epithelial transit-amplifying cell proliferation in vitro and in vivo.^287,288 Moreover, IL22-induced upregulation of claudin-2 and, as a consequence, pore pathway permeability is critical to enteric pathogen clearance.¹⁰⁹ However, both claudin-2 upregulation and IL22 therapy have also been shown to promote intestinal inflammation in experimental colitis,^157,289 and sustained high levels of IL22 can both reduce stem cell numbers and promote increased susceptibility to colon cancer.^279,287 These divergent roles of IL22 highlight a complex underlying regulatory network.¹²⁶ It may therefore be critical to carefully titrate IL22 if it is to become an effective therapy.

As multiple different cytokines cooperate to mediate inflammation, another therapeutic approach in IBD patients has been to target signaling pathways shared amongst more than one cytokine. The JAK-STAT pathway is one such shared pathway. JAK blockade leads to decreased T cell activation and differentiation.²⁹⁰ However, JAK blockade also has complex roles in epithelial function and in PRR-initiated pathways in innate cells where autocrine/paracrine cytokines play an important role. These might serve to counteract some of the benefits of its blockade in T cells or lead to unintended consequences. In vitro studies reducing JAK expression in innate immune cells show that low levels of reduction decrease PRR-induced pro-inflammatory and anti-inflammatory cytokines. However, there is a threshold of JAK expression and activation below which the reduction in anti-inflammatory cytokines results in a failure of the negative feedback loop required to suppress pro-inflammatory cytokines.^216,222 Therefore, as JAK inhibitor doses are increased beyond this threshold, the increased inflammatory cytokines secreted from PRR-stimulated innate cells may lead to unfavorable outcomes with increased inflammation.^216,222 In epithelial cells, JAK3 is required for optimal epithelial proliferation in vitro²⁹¹ and enterocytic and secretory epithelial lineage differentiation in vivo.²⁹² As such, JAK3 deficiency can lead to more severe experimental colitis.²⁹² Complete deletion and epithelial-specific deletion in mice of another JAK family member, TYK2, can also increase severity of experimental colitis; TYK2 deficiency leads to reduced epithelial proliferation and anti-microbial protein production in response to IL22, as well as altered intestinal microbial composition.²⁹³ Multiple other proteins regulating epithelial proliferation^{123,253,291,294} can signal through JAK proteins. In contrast to these studies, other studies have shown that pretreatment with JAK inhibitors prevents IFNγ-induced epithelial barrier permeability disruption in vitro²⁹⁵ and that JAK inhibition can improve barrier function in vivo.²⁹⁶ How JAK inhibitors regulate epithelial cell outcomes in IBD patients, and how this might change with specificity of the JAK inhibitors used, has yet to be determined.

TLR antagonists and agonists as therapy for IBD

While PRRs can contribute to inflammatory pathways, they also mediate essential functions in intestinal tissues, such that therapeutic targeting of PRR signaling is challenging. TLR4 antagonists have been developed, primarily as potential treatments for sepsis.²⁹⁷ TLR4 expression is increased in the intestine of IBD patients,^298,299 and TLR4 inhibitors can reduce inflammation in some experimental colitis models but they have detrimental effects on epithelial repair.³⁰⁰ In yet other reports TLR4 agonists can improve experimental colitis outcomes.²³ Likewise, TLR2 has both protective^23,301 and exacerbating³⁰² contributions to experimental colitis. TLR9 has similarly demonstrated mixed outcomes in regulating experimental colitis.^26,303 Studies in LPMCs from UC patients demonstrated that a TLR9 agonist, cobitolimod, modulates the Th17/Treg cell immune imbalance in intestinal inflammation and induces IL10 production by macrophages and T cells thereby reducing inflammation.³⁰⁴ A phase 2b clinical trial of cobitolimod in UC showed efficacy.³⁰⁵ Stimulator of interferon genes (STING) has also been shown to play a role in experimental IBD models;³⁰⁶ STING inhibitors are being developed for cancer and may be considered for therapeutic repurposing for IBD patients. For circumstances where TLR blockade may be beneficial, soluble forms of TLRs may prove to be an additional approach to attenuate signaling through TLR pathways. Signaling molecules shared across PRRs may also be promising targets for IBD,^307,308 as may direct administration of select microbial products. The challenge will be the specificity and threshold at which to modulate PRR pathways for both local intestinal regulation and systemic effects that may alter susceptibility to infections.

Abbreviations:

CD

Crohn’s disease

EGFR

epidermal growth factor receptor

IBD

inflammatory bowel disease

IEC

intestinal epithelial cell

IL

interleukin

ILC

innate lymphoid cell

LPMC

lamina propria mononuclear cells

LPS

lipopolysaccharide

MLCK

myosin light chain kinase

NLR

NOD-like receptor

OSM

oncostatin M

PRR

pattern recognition receptor

PTSG2

prostaglandin-endoperoxide synthase 2

Reg

Regenerating family member

RLR

RIG-I-like receptor

ROS

reactive oxygen species

SFB

segmented filamentous bacteria

SOCS

suppressor of cytokine signaling

STING

stimulator of interferon genes

Th

T helper

TLR

toll-like receptor

Treg

regulatory T cell

UC

ulcerative colitis