Abstract

Acute graft-versus-host disease (GVHD) remains the major obstacle to a more favorable therapeutic outcome of allogeneic hematopoietic stem cell transplantation (HSCT). GVHD is characterized by tissue damage in gut, liver, and skin, caused by donor T cells that are critical for antitumor and antimicrobial immunity after HSCT. One obstacle in combating GVHD used to be the lack of understanding the molecular mechanisms that are involved in the initiation phase of this syndrome. Recent research has demonstrated that interactions between microbial-associated molecules (pathogen-associated molecular patterns [PAMPs]) and innate immune receptors (pathogen recognition receptors [PRRs]), such as NOD-like receptors (NLRs) and Toll-like receptors (TLRs), control adaptive immune responses in inflammatory disorders. Polymorphisms of the genes encoding NOD2 and TLR4 are associated with a higher incidence of GVHD in HSC transplant recipients. Interestingly, NOD2 regulates GVHD through its inhibitory effect on antigen-presenting cell (APC) function. These insights identify important mechanisms regarding the induction of GVHD through the interplay of microbial molecules and innate immunity, thus opening a new area for future therapeutic approaches. This review covers current knowledge of the role of PAMPs and PRRs in the control of adaptive immune responses during inflammatory diseases, particularly GVHD.

Introduction

Acute graft-versus-host disease (GVHD) is a potentially lethal complication in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT). It is characterized by damage of epithelial surfaces in target organs caused by alloactivated T cells recognizing host tissue antigens. The severity of GVHD is determined by many factors, including the level of immunogenic disparity,1  the T-cell dose,2  the conditioning regimen,3  and the degree of immunosuppression.4  A common feature of the primary GVHD target tissues is their exposure to microbes and microbial products through the epidermis, intestinal mucosa, and portal circulation. The significance of the intestinal microbial flora for the pathogenesis of GVHD was first discovered in experimental mouse models and confirmed in human trials in the 1970s.5,6  Based on these findings, it became common practice to perform intestinal decontamination using orally administered antibiotics in patients undergoing allogeneic HSCT. Despite a large body of data providing evidence that microbes or microbial products (pathogen-associated molecular patterns [PAMPs]) interact with innate receptors (pathogen recognition receptors [PRRs]), on hematopoietic cells, mucosal cells, and endothelial cells, it took until the mid 1990s before the first PRR was discovered: In 1996 the PRR “Toll,” was discovered and determined to be responsible for innate immune responses to Aspergillus fumigatus in Drosophila.7  Shortly after this publication, the human homologue of Toll was defined and named Toll-like receptor (TLR) and later on TLR4.8  From this point, research in the field of innate immunity has increased substantially and to date hundreds of receptors, proteins, and small molecules, which are involved in antimicrobial immunity as well as inflammatory disorders, have been discovered.

Mucosal surfaces and vascular endothelium, the potential sites of pathogen entry, are rich in resident innate immune cells, such as macrophages and dendritic cells (DCs).9  Signaling through PRRs regulates the activity of DCs, leading to phagocytosis, chemokine receptor expression, cytokine secretion, migration from peripheral tissue to draining lymph nodes, and antigen presentation.10  Several TLRs have been described to recognize different microbial molecules (PAMPs), including lipopolysaccharide (LPS, TLR4), bacterial lipoproteins (TLR2), flagellin (TLR5), RNA (TLR3+7), and cytosine-phosphorothioate-guanine (CpG) DNA (TLR9).11  TLR downstream signaling activates a complex signaling cascade, eventually leading to host resistance against pathogens by increased production of cytokines, chemokines, adhesion molecules, and antimicrobial peptides as well as to enhanced antigen presentation by antigen-presenting cells (APCs).12  However, PAMPs are recognized not only by TLRs, but also by a PRR family of intracellular NOD-like receptors (NLRs) present in various cell types.13,14  NLRs include proteins such as NALPs (NACHT-, LRR-, and PYD-containing proteins), NOD1 (nucleotide-binding oligomerization domain), and NOD2. NLRs are involved in diverse signaling pathways regulating the secretion of inflammatory cytokines, such as interleukin-1β (IL-1β) and IL-18, as well as the induction of cell death.

Recent advances in our understanding as to how the innate immune system senses microbial antigens and controls adaptive immune responses provided an insight in the molecular mechanisms, thus opening a new and exciting field for future therapies of inflammatory diseases. In this text, we review recently gained knowledge on the role of microbe-associated molecules as well as innate immune receptors and discuss these findings in the context of the pathophysiology of GVHD (Figures 12).

Figure 1

Schematic of the GVHD initiation phase. A key event in the initiation of inflammation during GVHD is the activation of pathogen recognition receptors (PRRs), such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs), by microbial products (PAMPs) produced by the intestinal flora.

Figure 1

Schematic of the GVHD initiation phase. A key event in the initiation of inflammation during GVHD is the activation of pathogen recognition receptors (PRRs), such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs), by microbial products (PAMPs) produced by the intestinal flora.

Figure 2

Schematic representation of how microbial products (LPS, MDP) are detected by TLR4 and NOD2. TLR4 and NOD2 are the best studied innate immune receptors present during GVHD. For clarity, the pathways have been simplified. NOD2 sense intracellular MDP, leading to recruitment of the adaptor protein RiCK as well as to activation of the caspase 1 inflammasome, which eventually results in cell death. Extracellular LPS is recognized by TLR4, which signals through its intracellular domain TIR. Subsequent steps involve the adaptor molecules MyD88, TiRAP, TRAM, and TRiF. The activation and translocation of nuclear factor κB (NF-κB) and IRF-3 result in the transcriptional up-regulation of proinflammatory genes. Whether the CARD domain interacts with caspase is controversial as indicated by a dotted line. TIR indicates Toll–IL-1 receptor, the cytoplasmic domain of TLR4; TRAM, Toll–IL-1R domain–containing adaptor inducing interferon-β–related adaptor molecule; TRiF, TIR domain–containing adapter-inducing interferon-β; MyD88, myeloid differentiation primary response gene; TiRAP, Toll–IL-1 receptor (TIR) domain–containing adaptor protein; and IRF-3, interferon regulatory factor 3.

Figure 2

Schematic representation of how microbial products (LPS, MDP) are detected by TLR4 and NOD2. TLR4 and NOD2 are the best studied innate immune receptors present during GVHD. For clarity, the pathways have been simplified. NOD2 sense intracellular MDP, leading to recruitment of the adaptor protein RiCK as well as to activation of the caspase 1 inflammasome, which eventually results in cell death. Extracellular LPS is recognized by TLR4, which signals through its intracellular domain TIR. Subsequent steps involve the adaptor molecules MyD88, TiRAP, TRAM, and TRiF. The activation and translocation of nuclear factor κB (NF-κB) and IRF-3 result in the transcriptional up-regulation of proinflammatory genes. Whether the CARD domain interacts with caspase is controversial as indicated by a dotted line. TIR indicates Toll–IL-1 receptor, the cytoplasmic domain of TLR4; TRAM, Toll–IL-1R domain–containing adaptor inducing interferon-β–related adaptor molecule; TRiF, TIR domain–containing adapter-inducing interferon-β; MyD88, myeloid differentiation primary response gene; TiRAP, Toll–IL-1 receptor (TIR) domain–containing adaptor protein; and IRF-3, interferon regulatory factor 3.

The intestinal microflora during GVHD

Early studies in allogeneic mouse radiation chimeras showed that the mortality due to the “secondary disease”—later called GVHD—is significantly reduced in germ-free mice or when decontamination of the gut flora was performed.15,16  In one of these studies, Jones et al transplanted bone marrow of DBA/2 mice into conventional or germ-free, lethally irradiated C3H/He mice.15  Four months after irradiation, 98% of the germ-free recipients were still alive—in contrast to the conventional mice, which had all died from wasting syndrome and diarrhea associated with intestinal GVHD. The authors proposed a model picturing equally severe intestinal lesions in conventional versus germ-free recipients, but a higher mortality as a result of secondary infections caused by intestinal microorganisms in the non–germ-free recipients. Soon afterward, this model of secondary infection was challenged by Van Bekkum et al, who infused bone marrow cells of C57BL/Rij donor mice into lethally irradiated CBA mice causing 95% mortality after 100 days. Strikingly, GVHD was virtually absent in the control groups on antibiotic prophylaxis or under germ-free conditions.16  When spleen cells were added (to induce a more acute form of GVHD), mortality of the recipient mice under germ-free conditions or with antibiotic prophylaxis was no longer completely prevented, but still significantly delayed, compared with recipient mice held under normal conditions without antibiotics. The same group successfully used fetal gut implants in germ-free mice after HSCT to show that the degree of histologic damage is positively correlated to the presence of intestinal microbes.17 

These findings collectively gave reason to hypothesize that lymphocytes sensitized against microbial antigens cross-react with epithelial antigens in GVHD. This hypothesis became the most widely accepted model of microbial interactions in the pathogenesis of GVHD in the following decades and triggered several clinical studies in patients undergoing HSCT investigating “protected environment” and gastrointestinal decontamination in the 1980s. The majority of these trials showed efficacy of gastrointestinal decontamination and protected environment in the prevention of GVHD,5,6,18-20  and performance of gut decontamination became—and still is—standard practice in many transplant centers.21,22 

A reduction of the bacterial translocation from the bowel lumen to the systemic circulation can be accomplished by administration not only of antibiotics, but also of oral probiotics or by use of antibodies against microorganisms. In a murine GVHD model, characterized by severe damage of the bowel mucosa and elevated serum LPS levels, the administration of the probiotic Lactobacillus rhamonosus GG resulted in reduced bacterial translocation to mesenteric lymph nodes, ameliorated systemic GVHD, and improved survival.23  Interestingly, the presence of antibody titers against a certain Escherichia coli strain (J5) is associated with a reduced incidence of GVHD in patients undergoing allo–bone marrow transplantation (BMT).24  On this basis, an polyclonal antibody against the same strain of E coli was produced and was successfully used to reduce the incidence of GVHD in a randomized clinical trial.25 

TLRs present during GVHD

TLRs are expressed on hematopoietic cells, such as dendritic cells, T cells, and B cells, as well as on nonhematopoietic cells, such as endothelial cells, epithelial cells, and organ parenchyma cells.26  TLRs are involved in maintaining tolerance and eliminating pathogenic microorganisms, but they also play a role in amplifying autoimmune responses that ultimately cause inflammation.27,28  TLR signaling stimulated by PAMPs was found to regulate immune responses during inflammatory diseases with similarities to GVHD, such as systemic lupus erythematosus,29-31  arthritis,32  and inflammatory bowel disease.33-35  In experimental colitis, bacterial products aggravate acute inflammation via TLR2 and TLR4 signaling and direct the recruitment of inflammatory cells to intestinal sites.36  On the other hand, activation of specific TLRs can inhibit autoimmune diseases in certain mouse models: For example, the stimulation of TLR2 or TLR3 protects mice from experimental colitis.37,38  The repeated exposure to a TLR agonist can have anti-inflammatory effects by inducing hyporesponsiveness to subsequent TLR stimulation.39  Corr and coworkers demonstrated that repeated low-dose administration of a synthetic TLR7 agonist induced hyporesponsiveness or tolerance to TLR2, TLR7, and TLR9 activators and limited the course of neural inflammation in an experimental allergic encephalomyelitis model (Hayashi et al).40  TLR5 stimulation as well as TLR9 stimulation have mostly anti-inflammatory effects, as indicated by development of spontaneous intestinal inflammation in TLR5-deficient mice41  and the high susceptibility of TLR9-deficient mice to experimental colitis.42  Interestingly, TLR4 polymorphisms are associated with a higher risk of inflammatory bowel disease, suggesting that TLR4 stimulation is needed for intestinal homeostasis in humans.43,44 

Convincing evidence of the significance of PAMP-TLR interactions in the pathogenesis of GVHD derives from experimental models of GVHD in rodents. Ferrara and coworkers demonstrated a crucial role of the LPS-TLR4 pathway for the pathophysiology of GVHD (Cooke et al).27  LPS is a structural component of Gram-negative bacteria and is a potent stimulator of TLR4. After allo-HSCT, the translocation of LPS and microorganisms from the bowel lumen through the damaged intestinal mucosa to the circulation can occur. LPS can trigger a broad range of inflammatory responses from different immune cells, such as macrophages, neutrophils, monocytes, and T cells. During GVHD, LPS stimulates the secretion of tumor necrosis factor-α by macrophages leading to increased GVHD mortality.45  Using a mouse BMT model, Ferrara and colleagues demonstrated that the transplantation of donor BM cells, which are resistant to LPS stimulation, results in less severe GVHD (Cooke et al).46  Next, they studied the therapeutic efficacy of LPS antagonism during GVHD by administration of a lipid-A analog from day 0 to day +6 after allo-BMT. They found that LPS antagonism resulted in reduced intestinal GVHD as well as reduced systemic GVHD, but did not alter T-cell activity to host antigens.27  In line with these findings are the observations that elevated LPS serum levels after allo-BMT positively correlate with histopathologic target organ damage during GVHD.46,47 

Moreover, LPS was shown to play a role in transplant-related lung injuries after allogeneic HSCT: In a fully major histocompatibility complex (MHC)–mismatched allo-BMT model without systemic GVHD, the inhalation of LPS caused innate immune activation, accumulation of alloreactive T cells, and histologic damage. This effect was dependent on TLR4 signaling, and treatment with a TLR4 antagonist could protect against lung injury after allogeneic HSCT.48  The authors demonstrated that the absence of functional TLR4 in donor-derived hematopoietic cells abrogated the development of pulmonary damage, whereas the presence or absence of TLR4 on recipient structural lung tissue had no impact on lung injury after transplantation. Because the effects were a result of local activation of pulmonary innate immunity, as opposed to systemic innate immunity, the authors concluded that inhalation of LPS can promote the development of alloimmune lung injury after BMT independent from systemic GVHD.

Although the importance of the LPS-TLR4 pathway during GVHD is well documented in murine GVHD models, experiments with TLR4-deficient mice and MyD88-deficient mice show that TLR signaling is not absolutely required for GVHD.49,50  This might be because of the existence of alternative (non-TLR) activation pathways of APCs leading to alloactivation and proliferation of donor T cells in the absence of TLR signaling. The observation that defects in TLR signaling do not completely prevent GVHD is in line with the clinical data, which is described below. In humans, polymorphisms (Asp299Gly and Thr399Ile) affecting the extracellular domain of the TLR4 receptor are associated with a blunted response to inhaled LPS.51  Lorenz et al tested the impact of those 2 TLR4 polymorphisms on the outcome of 237 HLA-identical sibling allo-HSCTs.52  One or 2 polymorphisms were detected in approximately 10% of patients and donors. The authors found a trend toward a reduced incidence of grade II to IV acute GVHD when a TLR4 polymorphism was present (33% vs 47%) but failed to detect statistically significant results. They concluded that a much larger study population would be needed to confirm the role of LPS and TLR4 in the pathogenesis of GVHD in humans. Interestingly, in a subsequent study, Elmaagacli et al also found no correlation of the presence of TLR4 polymorphisms to the overall incidence of GVHD in a mixed cohort of 307 HSC transplants from HLA-identical siblings and matched unrelated donors.53  Surprisingly, they found that the severity GVHD was significantly increased in a univariate analysis if one TLR4 polymorphism (Thr399Ile) was present in both donor and recipient of an allo-HSC transplant compared with wild-type donor/recipient pairs (42% vs 15% severe GVHD). However, the TLR4 polymorphism failed to influence the occurrence of severe GVHD in the multivariate analysis, indicating that its role might not be very pronounced, which is in line with the data of Lorenz et al. The differences of a strong effect of TLR4 signaling in mouse models and a weaker effect in the clinical studies might be caused in part by the routine performance of bacterial gut decontamination in clinical allo-HSCT. Elmaagacli and coworkers used metronidazol and ciprofloxacin, which reduces the concentration of anaerobic bacteria very effectively by several log steps and is associated with a reduced incidence of acute GVHD (Beelen et al).20  It is reasonable to argue that reducing the concentration of intestinal anaerobic bacteria may also reduce the concentration of microbial products, including bacterial cell wall compounds such as LPS, leading to a reduced stimulation of the TLR4 pathway. Another explanation for the different effects of TLR4 stimulation in humans and in mouse models could be a difference in the balance of proinflammatory effects versus tissue-regenerative/anti-inflammatory effects upon TLR4 activation between humans and mice. Taken together, the clinical data on the connection between TLR4 and GVHD do not draw a clear picture yet. Therefore, futures studies addressing the role of TLR4 polymorphisms in allo-HSCT are needed, taking into account the type of bacterial gut decontamination used.

Sykes and coworkers were able to demonstrate that localized tissue inflammation caused by TLR ligands controls the recruitment of alloreactive T cells to GVHD target organs (Chakraverty et al).54  They transferred B6 T cells either to freshly irradiated BALB/c recipients or to B6-BALB/c mixed chimeras (a model for donor lymphocyte infusions). Both models showed a massive GVH reaction, characterized by T-cell expansion and activation, with similar up-regulation of skin- as well as gut-homing molecules, leading to eradication of host hematopoietic tissue. However, exclusively the irradiated BALB/c recipients (not the B6-BALB/c mixed chimeras) developed intestinal and skin GVHD with profound tissue injury caused by alloreactive T cells. Strikingly, innate immune activation was able to overcome the tolerance of B6-BALB/c mixed chimeras to adoptive transfer of B6 T cells: The systemic application of a TLR7 agonist (R-848) induced recruitment of alloreactive T cells and tissue damage in gut, liver, skin, and lung. These observations indicate that tissue inflammation, induced by signaling through PRRs, controls the development of GVHD at the local level. Blazar and colleagues showed that the TLR7/8 agonist (3M-011) can have differential effects on GVHD depending on the timing of administration. When 3M-011 was given after allo-BMT, the GVHD-related mortality rate was increased from 0% to 50% in a major mismatched model (Taylor et al).55  In a subsequent work the same group demonstrated, however, that pretreatment (prior to allo-BMT) with the same TLR7/8 agonist significantly delayed GVHD lethality and reduced GVHD target organ injury.56  Interestingly, pretreatment with 3M-011 resulted in increased levels of the immunosuppressive intracellular enzyme indoleamine 2,3-dioxygenase, which plays a crucial role in GVHD regulation.56,57  Collectively these data suggest that detection of pathogens (viral DNA and RNA) by TLR7 can lead to localized and systemic inflammation as well as to inhibition of inflammation during GVHD.

In a murine GVHD model, Balsari and colleagues demonstrated reduced systemic GVHD leading to improved survival in TLR9−/− allo-BM transplant recipients (Calcaterra et al).50  A possible mechanism was provided by the authors: The stimulatory activity of spleen APCs from irradiated TLR9−/− mice showed a reduced percentage of cells expressing costimulatory molecules and a significantly lower allostimulatory ability leading to less proliferation of allogeneic donor T cells. Experiments performed in BM chimeric mice showed that TLR9 in the nonhematopoietic system influenced GVHD, whereas the presence or absence of TLR9 on hematopoietic cells had no effect on GVHD. The authors argued that nonhematopoietic cells, such as intestinal epithelial cells expressing high levels of TLR9, might directly participate in the process of antigen presentation during GVHD. However, it is well known that TLRs are highly expressed on dendritic cells (DCs) and that antigen presentation by host DCs is central to the pathophysiology of GVHD.58,59  Cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG ODNs) mimic bacterial and viral DNA and stimulate TLR9, leading to innate immune activation. Blazar and colleagues found that ligation of TLR9 with CpG ODNs at the time of allo-BMT enhanced alloreactive T-cell responses, resulting in increased lethal GVHD (Taylor et al).55  The CpG ODN–mediated effects were dependent on TLR9 signaling in host APCs, which underlines the importance of TLR signaling for host APC function during GVHD. Elmaagacli and colleagues analyzed the impact of 2 polymorphisms of the TLR9 gene (T1486C and T1237C), which are associated with a lower TLR9 expression, on the clinical outcome in 413 donors and allo-HSC transplant recipients.53  The TLR9 polymorphisms were not associated with incidence or severity of GVHD. However, patients with the TLR9 1486 polymorphism had a significantly improved survival because of reduction of treatment-related mortality and relapse rate. The authors hypothesized that overwhelming TLR9-mediated immune responses, such as systemic inflammatory response syndrome and sepsis, might have occurred less often in allo-HSC transplant recipients with TLR9 polymorphisms, in comparison with wild-type allo-HSC transplant recipients. This hypothesis is based on experimental results demonstrating that systemic inflammatory response syndrome and sepsis can be prevented by inhibition of TLR9.60 

NLRs present during GVHD

NOD2 is the best studied NLR present during GVHD. NOD2 (CARD15) detects muramyl dipeptide (MDP), a molecule that is produced during the synthesis and degradation of peptidoglycan, which is a cell wall component of most bacteria. Single nucleotide polymorphisms (SNPs) near or within the NOD2 LRR region (G908R, L1007insC, and R702W) are present in approximately 15% of the population and constitute genetic risk factors for the development of Crohn disease.61,62  In addition, polymorphisms in the NACHT region of NOD2 are linked to other inflammatory diseases, such as Blau syndrome63  and early onset sarcoidosis.64 

We performed studies in murine allo-BMT models to determine the role of NOD2 during GVHD.65  We found that NOD2 deficiency of the allo-BM transplant donor (either donor T cells or BM) has no significant impact on the development of GVHD and does not regulate alloactivation of donor T cells. Our data suggest that NOD2 has no cell-intrinsic role in the regulation of T-cell activity during GVHD despite the increasing evidence that innate immune receptors can modulate T-cell function and activation during inflammation.66-68  In NOD2−/− allo-BM transplant recipients, we observed increased GVHD in both MHC-mismatched and MHC-matched models. Using chimeric mice, we demonstrated that NOD2 deficiency in the hematopoietic system, but not in the nonhematopoietic system, aggravates both experimental GVHD as well as experimental trinitrobenzene sulfonate (TNBS) colitis. We studied DCs of NOD2−/− allo-BM transplant recipients and found increased activation and function leading to augmented proliferation as well as activation of allogeneic donor T cells, which resulted in target organ damage. These data support the hypothesis that NOD2 can negatively regulate the activity and function of host DCs, resulting in increased alloactivation and proliferation of donor T cells in NOD2−/− allo-BM transplant recipients. Our results suggest that loss of intestinal epithelial cell function causing impaired antibacterial resistance, which has been proposed as a mechanism for increased inflammation in NOD2−/− mice,69  is not responsible for the increased GVHD and colitis. Our results are in agreement with the findings of Strober and colleagues (Strober et al,70  Watanabe et al,71-73  and Yang et al74 ), who found increased susceptibility to different types of experimental colitis in NOD2−/− mice that was due to enhanced ability of NOD2−/− APCs to trigger inflammatory T-cell responses.

In clinical allo-HSCT, multiple studies have been performed on the relation of NOD2 polymorphisms and the incidence of GVHD. Several studies show a connection between NOD2 SNPs and increased GVHD incidence as well as GVHD severity. Our group (Holler et al) was the first to investigate the impact of NOD2 SNPs (SNPs 8 [Arg702Trp], 12 [Gly908Arg], and 13 [Leu1007fsinsC]) on clinical outcome after allo-HSCT. We screened donors and recipients of an allogeneic HSC transplant in several European transplant centers in search of SNPs in the LRR region of the NOD2 gene and compared the results to clinical outcome. In a first single-center analysis and also in a subsequent multicenter study, we found an association between a higher incidence of GVHD and NOD2 SNPs of the donor or recipient.75,76  These associations were particularly strong in recipients of an HLA-identical sibling allograft, whereas only the donor L1007insC SNP predicted increased nonrelapse mortality in recipients of an HLA-matched unrelated donor allograft. This difference could reflect either a higher baseline mortality after unrelated donor SCT or the higher frequency of both donor and recipient SNPs in relatives.77  Because the lung has also an extensive exposure to bacterial ligands, we tested the hypothesis that a defective inflammatory response might trigger alloreactivity. In a large analysis of more than 400 patients, the cumulative incidence of bronchiolitis obliterans syndrome was 23% in patients with NOD2 SNPs but only 1% in recipients without NOD2 SNPs.78  Interestingly, Donnelly and coworkers confirmed the connection between NOD2 SNPs and GVHD in a smaller study on T cell–depleted allo-HSCT using the same SNPs (8, 12, and 13; van der Velden et al).79  They found that there was significantly more GVHD when NOD2 SNPs were present in the donor or in the recipient. The effect was strongest in allo-HSC transplant recipients with NOD2 SNPs receiving a transplant from a donor with NOD2 SNPs. The authors hypothesized that defective antimicrobial activity due to NOD2 dysfunction in hematopoietic cells and epithelial cells leads to immune dysregulation. In a large single-center analysis, Elmaagacli et al also found increased incidence of GVHD in allo-HSC transplant recipients with NOD2 SNPs receiving grafts from donors with NOD2 SNPs (8, 12, and 13).53  Surprisingly, GVHD was reduced in wild-type recipients of grafts from donors with NOD2 SNPs. The authors hypothesized that the NOD2 SNPs on the donor side could negatively influence alloreactivity of donor T cells, leading to reduced GVHD.

In contrast to the studies described previously, other transplant centers could not find a significant impact of NOD2 SNPs on the incidence of GVHD. Granell et al reported that the NOD2 SNPs 8, 12, and 13 were not associated with acute or chronic GVHD, but still increased mortality because of pulmonary complications, in a small cohort of 85 patients receiving CD34-selected HLA-identical sibling transplants.80  Sairafi et al also found no significant impact of these NOD2 SNPs on incidence of acute GVHD in a series of 198 patients who underwent transplantation in Stockholm.81  In a study by Marsh and coworkers, in a similarly sized patient cohort the incidence of acute GVHD was low and there was no significant difference due to the presence of NOD2 SNPs (8, 12, and 13), but this trial focused on unrelated donor transplants recipients undergoing alemtuzumab prophylaxis (Mayor et al).82  Steinbach and colleagues also found no significant impact of these 3 NOD2 SNPs on the incidence of GVHD in a cohort of 231 children who underwent allo-HSCT (Gruhn et al).83 

The conflicting results between the different clinical studies on the role of NOD2 polymorphisms during GVHD are most likely explained by differences between the study cohorts including the NOD2 SNP frequency, overall incidence of GVHD, T-cell depletion, type of conditioning regimen, intestinal microbial decontamination, donor source, and environmental factors. In conclusion, it appears that polymorphisms in the NOD2 gene locus of the recipient can negatively influence survival after allogeneic HSCT. However, transplantation-specific strategies such as T-cell depletion or gut decontamination may alter the activation of dendritic cells or epithelial cells and the role of NOD2 in these processes. The effect of NOD2 SNPs of the allo-HSC transplant donor on the incidence of GVHD is controversial. Experimental data suggest that there is no major effect of NOD2 on the allo-HSC transplant donor side on the regulation of GVHD.65 

In addition, functional data on the impact of NOD2/CARD15 SNPs in humans are urgently needed: As shown for inflammatory bowel disease, peripheral blood mononuclear cells from recipients and donors with NOD2/CARD15 SNPs have a significantly decreased capacity to release IL-8 after stimulation with the ligand MDP.75,77,78,84,85  More recently, we observed a diminished recruitment of both CD4 cells and neutrophils in intestinal biopsies from patients suffering from gastrointestinal GVHD, indicating that NOD2/CARD15 may play a role in recruitment of these cells via chemokines released by APCs.86  Based on these data, GVHD can be seen as an imbalance in protective CD4 cells that is further enhanced in the presence of NOD2/VCARD15 SNPs.

Table 1 summarizes studies investigating innate immune receptors during GVHD.

Table 1

Studies on innate immune receptors during GVHD

PRR/speciesObservationReference no.
TLR4   
    Human TLR4 polymorphisms on patient and donor side cause more severe GVHD 53  
    Human TLR4 polymorphisms do not cause more severe GVHD 52  
    Mouse LPS antagonism results in less lethal GVHD 27  
    Mouse LPS antagonism protects against lung injury after allo-BMT 48  
TLR7   
    Mouse TLR7 agonism controls trafficking of T cells to GVHD target organs 54  
TLR7/TLR8   
    Mouse TLR7/8 agonism after allo-BMT increases GVHD 55  
    Mouse TLR7/8 agonism before allo-BMT ameliorates GVHD 56  
TLR9   
    Mouse TLR9 agonism increases GVHD 55  
    Mouse Lethal GVHD is reduced in TLR9−/− allo-BM transplant recipients 50  
NOD2   
    Human NOD2 polymorphisms on patient and donor side cause more severe GVHD 75  
    Human NOD2 polymorphisms on patient and donor side cause more severe GVHD 76  
    Human NOD2 polymorphisms on patient and donor side cause more severe GVHD in recipients of a T cell–depleted HSC transplant 79  
    Human NOD2 polymorphisms on patient and donor side cause more severe GVHD; Incidence of GVHD is reduced when NOD2 polymorphisms are on the donor side 53  
    Human Increased pulmonary complications and death in patients with NOD2 polymorphisms after allo-HSCT. No significant difference in GVHD 80  
    Human GVHD is not significantly increased in patients with NOD2 polymorphisms 81  
    Human GVHD is not significantly increased in patients with NOD2 polymorphisms; Leukemia relapse rate is increased after allo-HSCT in patients with NOD2 polymorphisms 82  
    Human GVHD is not significantly increased in children with NOD2 polymorphisms 83  
    Human NOD2 polymorphisms in allo-HSC transplant recipients with GVHD are associated with diminished recruitment of CD4+ T cells and neutrophils in intestinal biopsies 86  
    Mouse Lethal GVHD is increased in NOD2−/− allo-BM transplant recipients; NOD2 negatively regulates antigen-presenting cell (APC) function during GVHD 65  
PRR/speciesObservationReference no.
TLR4   
    Human TLR4 polymorphisms on patient and donor side cause more severe GVHD 53  
    Human TLR4 polymorphisms do not cause more severe GVHD 52  
    Mouse LPS antagonism results in less lethal GVHD 27  
    Mouse LPS antagonism protects against lung injury after allo-BMT 48  
TLR7   
    Mouse TLR7 agonism controls trafficking of T cells to GVHD target organs 54  
TLR7/TLR8   
    Mouse TLR7/8 agonism after allo-BMT increases GVHD 55  
    Mouse TLR7/8 agonism before allo-BMT ameliorates GVHD 56  
TLR9   
    Mouse TLR9 agonism increases GVHD 55  
    Mouse Lethal GVHD is reduced in TLR9−/− allo-BM transplant recipients 50  
NOD2   
    Human NOD2 polymorphisms on patient and donor side cause more severe GVHD 75  
    Human NOD2 polymorphisms on patient and donor side cause more severe GVHD 76  
    Human NOD2 polymorphisms on patient and donor side cause more severe GVHD in recipients of a T cell–depleted HSC transplant 79  
    Human NOD2 polymorphisms on patient and donor side cause more severe GVHD; Incidence of GVHD is reduced when NOD2 polymorphisms are on the donor side 53  
    Human Increased pulmonary complications and death in patients with NOD2 polymorphisms after allo-HSCT. No significant difference in GVHD 80  
    Human GVHD is not significantly increased in patients with NOD2 polymorphisms 81  
    Human GVHD is not significantly increased in patients with NOD2 polymorphisms; Leukemia relapse rate is increased after allo-HSCT in patients with NOD2 polymorphisms 82  
    Human GVHD is not significantly increased in children with NOD2 polymorphisms 83  
    Human NOD2 polymorphisms in allo-HSC transplant recipients with GVHD are associated with diminished recruitment of CD4+ T cells and neutrophils in intestinal biopsies 86  
    Mouse Lethal GVHD is increased in NOD2−/− allo-BM transplant recipients; NOD2 negatively regulates antigen-presenting cell (APC) function during GVHD 65  

Conclusions and future directions

Recent research has illuminated details as to how stimulation of innate immune receptors by microbial-associated molecules significantly contributes to the inflammatory processes that consequently lead to recruitment of alloactivated T cells as well as tissue damage in GVHD target organs. The inflammatory cascade that characterizes GVHD is initiated by PAMPs that activate PRR signaling in resident innate immune cells, epithelial cells, and endothelial cells. The stimulation of PRRs lead to transcription of inflammatory genes and processing of proinflammatory cytokines, resulting in local tissue inflammation, migration of leukocytes, presentation of host antigens, and eventually antihost reactivity of donor T cells.

To date, most therapeutic and prophylactic efforts in GVHD, such as T-cell depletion or inhibition of T-cell activation/proliferation, intervene with the T cell–dependent effector phase. The main drawback of this approach is the inhibition of graft-versus-tumor activity and reduction of antimicrobial immunity mediated by donor T cells, which are major determinants of the overall outcome in HSCT. Finding therapeutic strategies exclusively targeting unwanted GVH reactions toward epithelial surfaces and sparing the beneficial reactivity toward malignant host cells and microbial pathogens could be the key to improve survival in patients undergoing HSCT. One such strategy is the prophylactic or therapeutic intervention in innate immunity aiming at the activation of innate immune cells by PAMPs; an additional strategy would aim for local modulation of epithelial and/or dendritic cells. Hopefully, improved understanding of the cellular players, receptors, and signaling pathways of innate immunity in GVHD will provide the basis for development of new and effective therapeutic tools in the near future.

Acknowledgments

The authors thank Sabine Czylwik and Ulrike Heider for proofreading the paper.

Authorship

Contribution: O.P., E.H., and M.R.M.v.d.B. wrote the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Marcel van den Brink, Departments of Immunology and Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10065; e-mail: vandenbm@mskcc.org.

References

References
1
Uphoff
 
DE
Law
 
LW
Genetic factors influencing irradiation protection by bone marrow: II, the histocompatibility-2 (H-2) locus.
J Natl Cancer Inst
1958
, vol. 
20
 
3
(pg. 
617
-
624
)
2
Korngold
 
B
Sprent
 
J
Lethal graft-versus-host disease after bone marrow transplantation across minor histocompatibility barriers in mice: prevention by removing mature T cells from marrow.
J Exp Med
1978
, vol. 
148
 
6
(pg. 
1687
-
1698
)
3
Xun
 
CQ
Thompson
 
JS
Jennings
 
CD
Brown
 
SA
Widmer
 
MB
Effect of total body irradiation, busulfan-cyclophosphamide, or cyclophosphamide conditioning on inflammatory cytokine release and development of acute and chronic graft-versus-host disease in H-2-incompatible transplanted SCID mice.
Blood
1994
, vol. 
83
 
8
(pg. 
2360
-
2367
)
4
van Bekkum
 
DW
Knaan
 
S
Zurcher
 
C
Effects of cyclosporin A on experimental graft-versus-host disease in rodents.
Transplant Proc
1980
, vol. 
12
 
2
(pg. 
278
-
282
)
5
Storb
 
R
Prentice
 
RL
Buckner
 
CD
, et al. 
Graft-versus-host disease and survival in patients with aplastic anemia treated by marrow grafts from HLA-identical siblings: beneficial effect of a protective environment.
N Engl J Med
1983
, vol. 
308
 
6
(pg. 
302
-
307
)
6
Beelen
 
DW
Haralambie
 
E
Brandt
 
H
, et al. 
Evidence that sustained growth suppression of intestinal anaerobic bacteria reduces the risk of acute graft-versus-host disease after sibling marrow transplantation.
Blood
1992
, vol. 
80
 
10
(pg. 
2668
-
2676
)
7
Lemaitre
 
B
Nicolas
 
E
Michaut
 
L
Reichhart
 
JM
Hoffmann
 
JA
The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults.
Cell
1996
, vol. 
86
 
6
(pg. 
973
-
983
)
8
Medzhitov
 
R
Preston-Hurlburt
 
P
Janeway
 
CA
A human homologue of the Drosophila Toll protein signals activation of adaptive immunity.
Nature
1997
, vol. 
388
 
6640
(pg. 
394
-
397
)
9
Kim
 
TD
Terwey
 
TH
Zakrzewski
 
JL
, et al. 
Organ-derived dendritic cells have differential effects on alloreactive T cells.
Blood
2008
, vol. 
111
 
5
(pg. 
2929
-
2940
)
10
Huang
 
Q
Liu
 
D
Majewski
 
P
, et al. 
The plasticity of dendritic cell responses to pathogens and their components.
Science
2001
, vol. 
294
 
5543
(pg. 
870
-
875
)
11
Cook
 
DN
Pisetsky
 
DS
Schwartz
 
DA
Toll-like receptors in the pathogenesis of human disease.
Nat Immunol
2004
, vol. 
5
 
10
(pg. 
975
-
979
)
12
Poltorak
 
A
He
 
X
Smirnova
 
I
, et al. 
Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene.
Science
1998
, vol. 
282
 
5396
(pg. 
2085
-
2088
)
13
Bertin
 
J
Nir
 
WJ
Fischer
 
CM
, et al. 
Human CARD4 protein is a novel CED-4/Apaf-1 cell death family member that activates NF-kappaB.
J Biol Chem
1999
, vol. 
274
 
19
(pg. 
12955
-
12958
)
14
Inohara
 
N
Ogura
 
Y
Chen
 
FF
Muto
 
A
Nunez
 
G
Human Nod1 confers responsiveness to bacterial lipopolysaccharides.
J Biol Chem
2001
, vol. 
276
 
4
(pg. 
2551
-
2554
)
15
Jones
 
JM
Wilson
 
R
Bealmear
 
PM
Mortality and gross pathology of secondary disease in germfree mouse radiation chimeras.
Radiat Res
1971
, vol. 
45
 
3
(pg. 
577
-
588
)
16
van Bekkum
 
DW
Roodenburg
 
J
Heidt
 
PJ
van der Waaij
 
D
Mitigation of secondary disease of allogeneic mouse radiation chimeras by modification of the intestinal microflora.
J Natl Cancer Inst
1974
, vol. 
52
 
2
(pg. 
401
-
404
)
17
van Bekkum
 
DW
Knaan
 
S
Role of bacterial microflora in development of intestinal lesions from graft-versus-host reaction.
J Natl Cancer Inst
1977
, vol. 
58
 
3
(pg. 
787
-
790
)
18
Skinhøj
 
P
Jacobsen
 
N
Hoiby
 
N
Faber
 
V
Strict protective isolation in allogenic bone marrow transplantation: effect on infectious complications, fever and graft versus host disease.
Scand J Infect Dis
1987
, vol. 
19
 
1
(pg. 
91
-
96
)
19
Vossen
 
JM
Heidt
 
PJ
van den Berg
 
H
Gerritsen
 
EJ
Hermans
 
J
Dooren
 
LJ
Prevention of infection and graft-versus-host disease by suppression of intestinal microflora in children treated with allogeneic bone marrow transplantation.
Eur J Clin Microbiol Infect Dis
1990
, vol. 
9
 
1
(pg. 
14
-
23
)
20
Beelen
 
DW
Elmaagacli
 
A
Muller
 
KD
Hirche
 
H
Schaefer
 
UW
Influence of intestinal bacterial decontamination using metronidazole and ciprofloxacin or ciprofloxacin alone on the development of acute graft-versus-host disease after marrow transplantation in patients with hematologic malignancies: final results and long-term follow-up of an open-label prospective randomized trial.
Blood
1999
, vol. 
93
 
10
(pg. 
3267
-
3275
)
21
Krüger
 
WH
Bohlius
 
J
Cornely
 
OA
, et al. 
Antimicrobial prophylaxis in allogeneic bone marrow transplantation: guidelines of the infectious diseases working party (AGIHO) of the german society of haematology and oncology.
Ann Oncol
2005
, vol. 
16
 
8
(pg. 
1381
-
1390
)
22
Dykewicz
 
CA
Summary of the Guidelines for Preventing Opportunistic Infections among Hematopoietic Stem Cell Transplant Recipients.
Clin Infect Dis
2001
, vol. 
33
 
2
(pg. 
139
-
144
)
23
Gerbitz
 
A
Schultz
 
M
Wilke
 
A
, et al. 
Probiotic effects on experimental graft-versus-host disease: let them eat yogurt.
Blood
2004
, vol. 
103
 
11
(pg. 
4365
-
4367
)
24
Cohen
 
J
Moore
 
RH
Al Hashimi
 
S
Jones
 
L
Apperley
 
JF
Aber
 
VR
Antibody titres to a rough-mutant strain of Escherichia coli in patients undergoing allogeneic bone-marrow transplantation: evidence of a protective effect against graft-versus-host disease.
Lancet
1987
, vol. 
1
 
8523
(pg. 
8
-
11
)
25
Bayston
 
K
Baumgartner
 
JD
Clark
 
P
Cohen
 
J
Anti-endotoxin antibody for prevention of acute GVHD.
Bone Marrow Transplant
1991
, vol. 
8
 
5
(pg. 
426
-
427
)
26
Schnare
 
M
Barton
 
GM
Holt
 
AC
Takeda
 
K
Akira
 
S
Medzhitov
 
R
Toll-like receptors control activation of adaptive immune responses.
Nat Immunol
2001
, vol. 
2
 
10
(pg. 
947
-
950
)
27
Cooke
 
KR
Gerbitz
 
A
Crawford
 
JM
, et al. 
LPS antagonism reduces graft-versus-host disease and preserves graft-versus-leukemia activity after experimental bone marrow transplantation.
J Clin Invest
2001
, vol. 
107
 
12
(pg. 
1581
-
1589
)
28
Iwasaki
 
A
Medzhitov
 
R
Toll-like receptor control of the adaptive immune responses.
Nat Immunol
2004
, vol. 
5
 
10
(pg. 
987
-
995
)
29
Hawn
 
TR
Wu
 
H
Grossman
 
JM
Hahn
 
BH
Tsao
 
BP
Aderem
 
A
A stop codon polymorphism of Toll-like receptor 5 is associated with resistance to systemic lupus erythematosus.
Proc Natl Acad Sci U S A
2005
, vol. 
102
 
30
(pg. 
10593
-
10597
)
30
Leadbetter
 
EA
Rifkin
 
IR
Hohlbaum
 
AM
Beaudette
 
BC
Shlomchik
 
MJ
Marshak-Rothstein
 
A
Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors.
Nature
2002
, vol. 
416
 
6881
(pg. 
603
-
607
)
31
Viglianti
 
GA
Lau
 
CM
Hanley
 
TM
Miko
 
BA
Shlomchik
 
MJ
Marshak-Rothstein
 
A
Activation of autoreactive B cells by CpG dsDNA.
Immunity
2003
, vol. 
19
 
6
(pg. 
837
-
847
)
32
Abdollahi-Roodsaz
 
S
Joosten
 
LA
Roelofs
 
MF
, et al. 
Inhibition of Toll-like receptor 4 breaks the inflammatory loop in autoimmune destructive arthritis.
Arthritis Rheum
2007
, vol. 
56
 
9
(pg. 
2957
-
2967
)
33
Franchimont
 
D
Vermeire
 
S
El Housni
 
H
, et al. 
Deficient host-bacteria interactions in inflammatory bowel disease? the toll-like receptor (TLR)-4 Asp299gly polymorphism is associated with Crohn's disease and ulcerative colitis.
Gut
2004
, vol. 
53
 
7
(pg. 
987
-
992
)
34
Pierik
 
M
Joossens
 
S
Van Steen
 
K
, et al. 
Toll-like receptor-1, -2, and -6 polymorphisms influence disease extension in inflammatory bowel diseases.
Inflamm Bowel Dis
2006
, vol. 
12
 
1
(pg. 
1
-
8
)
35
Sartor
 
RB
Muehlbauer
 
M
Microbial host interactions in IBD: implications for pathogenesis and therapy.
Curr Gastroenterol Rep
2007
, vol. 
9
 
6
(pg. 
497
-
507
)
36
Heimesaat
 
MM
Fischer
 
A
Siegmund
 
B
, et al. 
Shift towards pro-inflammatory intestinal bacteria aggravates acute murine colitis via Toll-like receptors 2 and 4.
PLoS ONE
2007
, vol. 
2
 
7
pg. 
e662
 
37
Podolsky
 
DK
Gerken
 
G
Eyking
 
A
Cario
 
E
Colitis-associated variant of TLR2 causes impaired mucosal repair because of TFF3 deficiency.
Gastroenterology
2009
, vol. 
137
 
1
(pg. 
209
-
220
)
38
Vijay-Kumar
 
M
Wu
 
H
Aitken
 
J
, et al. 
Activation of toll-like receptor 3 protects against DSS-induced acute colitis.
Inflamm Bowel Dis
2007
, vol. 
13
 
7
(pg. 
856
-
864
)
39
Ehlers
 
M
Ravetch
 
JV
Opposing effects of Toll-like receptor stimulation induce autoimmunity or tolerance.
Trends Immunol
2007
, vol. 
28
 
2
(pg. 
74
-
79
)
40
Hayashi
 
T
Gray
 
CS
Chan
 
M
, et al. 
Prevention of autoimmune disease by induction of tolerance to Toll-like receptor 7.
Proc Natl Acad Sci U S A
2009
, vol. 
106
 
8
(pg. 
2764
-
2769
)
41
Vijay-Kumar
 
M
Sanders
 
CJ
Taylor
 
RT
, et al. 
Deletion of TLR5 results in spontaneous colitis in mice.
J Clin Invest
2007
, vol. 
117
 
12
(pg. 
3909
-
3921
)
42
Lee
 
J
Mo
 
JH
Katakura
 
K
, et al. 
Maintenance of colonic homeostasis by distinctive apical TLR9 signalling in intestinal epithelial cells.
Nat Cell Biol
2006
, vol. 
8
 
12
(pg. 
1327
-
1336
)
43
De Jager
 
PL
Franchimont
 
D
Waliszewska
 
A
, et al. 
The role of the Toll receptor pathway in susceptibility to inflammatory bowel diseases.
Genes Immun
2007
, vol. 
8
 
5
(pg. 
387
-
397
)
44
Oostenbrug
 
LE
Drenth
 
JP
de Jong
 
DJ
, et al. 
Association between Toll-like receptor 4 and inflammatory bowel disease.
Inflamm Bowel Dis
2005
, vol. 
11
 
6
(pg. 
567
-
575
)
45
Nestel
 
FP
Price
 
KS
Seemayer
 
TA
Lapp
 
WS
Macrophage priming and lipopolysaccharide-triggered release of tumor necrosis factor alpha during graft-versus-host disease.
J Exp Med
1992
, vol. 
175
 
2
(pg. 
405
-
413
)
46
Cooke
 
KR
Hill
 
GR
Crawford
 
JM
, et al. 
Tumor necrosis factor-alpha production to lipopolysaccharide stimulation by donor cells predicts the severity of experimental acute graft-versus-host disease.
J Clin Invest
1998
, vol. 
102
 
10
(pg. 
1882
-
1891
)
47
Fegan
 
C
Poynton
 
CH
Whittaker
 
JA
The gut mucosal barrier in bone marrow transplantation.
Bone Marrow Transplant
1990
, vol. 
5
 
6
(pg. 
373
-
377
)
48
Garantziotis
 
S
Palmer
 
SM
Snyder
 
LD
, et al. 
Alloimmune lung injury induced by local innate immune activation through inhaled lipopolysaccharide.
Transplantation
2007
, vol. 
84
 
8
(pg. 
1012
-
1019
)
49
Shlomchik
 
WD
Graft-versus-host disease.
Nat Rev
2007
, vol. 
7
 
5
(pg. 
340
-
352
)
50
Calcaterra
 
C
Sfondrini
 
L
Rossini
 
A
, et al. 
Critical role of TLR9 in acute graft-versus-host disease.
J Immunol
2008
, vol. 
181
 
9
(pg. 
6132
-
6139
)
51
Arbour
 
NC
Lorenz
 
E
Schutte
 
BC
, et al. 
TLR4 mutations are associated with endotoxin hyporesponsiveness in humans.
Nat Genet
2000
, vol. 
25
 
2
(pg. 
187
-
191
)
52
Lorenz
 
E
Schwartz
 
DA
Martin
 
PJ
, et al. 
Association of TLR4 mutations and the risk for acute GVHD after HLA-matched-sibling hematopoietic stem cell transplantation.
Biol Blood Marrow Transplant
2001
, vol. 
7
 
7
(pg. 
384
-
387
)
53
Elmaagacli
 
AH
Koldehoff
 
M
Hindahl
 
H
, et al. 
Mutations in innate immune system NOD2/CARD 15 and TLR-4 (Thr399Ile) genes influence the risk for severe acute graft-versus-host disease in patients who underwent an allogeneic transplantation.
Transplantation
2006
, vol. 
81
 
2
(pg. 
247
-
254
)
54
Chakraverty
 
R
Cote
 
D
Buchli
 
J
, et al. 
An inflammatory checkpoint regulates recruitment of graft-versus-host reactive T cells to peripheral tissues.
J Exp Med
2006
, vol. 
203
 
8
(pg. 
2021
-
2031
)
55
Taylor
 
PA
Ehrhardt
 
MJ
Lees
 
CJ
, et al. 
TLR agonists regulate alloresponses and uncover a critical role for donor APCs in allogeneic bone marrow rejection.
Blood
2008
, vol. 
112
 
8
(pg. 
3508
-
3516
)
56
Jasperson
 
LK
Bucher
 
C
Panoskaltsis-Mortari
 
A
Mellor
 
AL
Munn
 
DH
Blazar
 
BR
Inducing the tryptophan catabolic pathway, indoleamine 2,3-dioxygenase (IDO), for suppression of graft-versus-host disease (GVHD) lethality.
Blood
2009
, vol. 
114
 
24
(pg. 
5062
-
5070
)
57
Jasperson
 
LK
Bucher
 
C
Panoskaltsis-Mortari
 
A
, et al. 
Indoleamine 2,3-dioxygenase is a critical regulator of acute graft-versus-host disease lethality.
Blood
2008
, vol. 
111
 
6
(pg. 
3257
-
3265
)
58
Shlomchik
 
WD
Couzens
 
MS
Tang
 
CB
, et al. 
Prevention of graft versus host disease by inactivation of host antigen-presenting cells.
Science
1999
, vol. 
285
 
5426
(pg. 
412
-
415
)
59
Duffner
 
UA
Maeda
 
Y
Cooke
 
KR
, et al. 
Host dendritic cells alone are sufficient to initiate acute graft-versus-host disease.
J Immunol
2004
, vol. 
172
 
12
(pg. 
7393
-
7398
)
60
Duramad
 
O
Fearon
 
KL
Chang
 
B
, et al. 
Inhibitors of TLR-9 act on multiple cell subsets in mouse and man in vitro and prevent death in vivo from systemic inflammation.
J Immunol
2005
, vol. 
174
 
9
(pg. 
5193
-
5200
)
61
Hugot
 
JP
Chamaillard
 
M
Zouali
 
H
, et al. 
Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease.
Nature
2001
, vol. 
411
 
6837
(pg. 
599
-
603
)
62
Ogura
 
Y
Bonen
 
DK
Inohara
 
N
, et al. 
A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease.
Nature
2001
, vol. 
411
 
6837
(pg. 
603
-
606
)
63
Miceli-Richard
 
C
Lesage
 
S
Rybojad
 
M
, et al. 
CARD15 mutations in Blau syndrome.
Nat Genet
2001
, vol. 
29
 
1
(pg. 
19
-
20
)
64
Kanazawa
 
N
Okafuji
 
I
Kambe
 
N
, et al. 
Early-onset sarcoidosis and CARD15 mutations with constitutive nuclear factor-kappaB activation: common genetic etiology with Blau syndrome.
Blood
2005
, vol. 
105
 
3
(pg. 
1195
-
1197
)
65
Penack
 
O
Smith
 
OM
Cunningham-Bussel
 
A
, et al. 
NOD2 regulates hematopoietic cell function during graft-versus-host disease.
J Exp Med
2009
, vol. 
206
 
10
(pg. 
2101
-
2110
28
66
Caron
 
G
Duluc
 
D
Fremaux
 
I
, et al. 
Direct stimulation of human T cells via TLR5 and TLR7/8: flagellin and R-848 up-regulate proliferation and IFN-gamma production by memory CD4+ T cells.
J Immunol
2005
, vol. 
175
 
3
(pg. 
1551
-
1557
)
67
Dabbagh
 
K
Lewis
 
DB
Toll-like receptors and T-helper-1/T-helper-2 responses.
Curr Opin Infect Dis
2003
, vol. 
16
 
3
(pg. 
199
-
204
)
68
MacLeod
 
H
Wetzler
 
LM
T cell activation by TLRs: a role for TLRs in the adaptive immune response.
Sci STKE
2007
, vol. 
2007
 
402
pg. 
pe48
 
69
Kobayashi
 
KS
Chamaillard
 
M
Ogura
 
Y
, et al. 
Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract.
Science
2005
, vol. 
307
 
5710
(pg. 
731
-
734
)
70
Strober
 
W
Murray
 
PJ
Kitani
 
A
Watanabe
 
T
Signalling pathways and molecular interactions of NOD1 and NOD2.
Nat Rev
2006
, vol. 
6
 
1
(pg. 
9
-
20
)
71
Watanabe
 
T
Asano
 
N
Murray
 
PJ
, et al. 
Muramyl dipeptide activation of nucleotide-binding oligomerization domain 2 protects mice from experimental colitis.
J Clin Invest
2008
, vol. 
118
 
2
(pg. 
545
-
559
)
72
Watanabe
 
T
Kitani
 
A
Murray
 
PJ
Strober
 
W
NOD2 is a negative regulator of Toll-like receptor 2-mediated T helper type 1 responses.
Nat Immunol
2004
, vol. 
5
 
8
(pg. 
800
-
808
)
73
Watanabe
 
T
Kitani
 
A
Murray
 
PJ
Wakatsuki
 
Y
Fuss
 
IJ
Strober
 
W
Nucleotide binding oligomerization domain 2 deficiency leads to dysregulated TLR2 signaling and induction of antigen-specific colitis.
Immunity
2006
, vol. 
25
 
3
(pg. 
473
-
485
)
74
Yang
 
Z
Fuss
 
IJ
Watanabe
 
T
, et al. 
NOD2 transgenic mice exhibit enhanced MDP-mediated down-regulation of TLR2 responses and resistance to colitis induction.
Gastroenterology
2007
, vol. 
133
 
5
(pg. 
1510
-
1521
)
75
Holler
 
E
Rogler
 
G
Brenmoehl
 
J
, et al. 
Prognostic significance of NOD2/CARD15 variants in HLA-identical sibling hematopoietic stem cell transplantation: effect on long-term outcome is confirmed in 2 independent cohorts and may be modulated by the type of gastrointestinal decontamination.
Blood
2006
, vol. 
107
 
10
(pg. 
4189
-
4193
)
76
Holler
 
E
Rogler
 
G
Herfarth
 
H
, et al. 
Both donor and recipient NOD2/CARD15 mutations associate with transplant-related mortality and GvHD following allogeneic stem cell transplantation.
Blood
2004
, vol. 
104
 
3
(pg. 
889
-
894
)
77
Holler
 
E
Rogler
 
G
Brenmoehl
 
J
, et al. 
The role of genetic variants of NOD2/CARD15, a receptor of the innate immune system, in GvHD and complications following related and unrelated donor haematopoietic stem cell transplantation.
Int J Immunogen
2008
, vol. 
35
 
4–5
(pg. 
381
-
384
)
78
Hildebrandt
 
GC
Granell
 
M
Urbano-Ispizua
 
A
, et al. 
Recipient NOD2/CARD15 variants: a novel independent risk factor for the development of bronchiolitis obliterans after allogeneic stem cell transplantation.
Biol Blood Marrow Transplant
2008
, vol. 
14
 
1
(pg. 
67
-
74
)
79
van der Velden
 
WJ
Blijlevens
 
NM
Maas
 
FM
, et al. 
NOD2 polymorphisms predict severe acute graft-versus-host and treatment-related mortality in T-cell-depleted haematopoietic stem cell transplantation.
Bone Marrow Transplant
2009
, vol. 
44
 
4
(pg. 
243
-
248
)
80
Granell
 
M
Urbano-Ispizua
 
A
Arostegui
 
JI
, et al. 
Effect of NOD2/CARD15 variants in T-cell depleted allogeneic stem cell transplantation.
Haematologica
2006
, vol. 
91
 
10
(pg. 
1372
-
1376
)
81
Sairafi
 
D
Uzunel
 
M
Remberger
 
M
Ringden
 
O
Mattsson
 
J
No impact of NOD2/CARD15 on outcome after SCT.
Bone Marrow Transplant
2008
, vol. 
41
 
11
(pg. 
961
-
964
)
82
Mayor
 
NP
Shaw
 
BE
Hughes
 
DA
, et al. 
Single nucleotide polymorphisms in the NOD2/CARD15 gene are associated with an increased risk of relapse and death for patients with acute leukemia after hematopoietic stem-cell transplantation with unrelated donors.
J Clin Oncol
2007
, vol. 
25
 
27
(pg. 
4262
-
4269
)
83
Gruhn
 
B
Intek
 
J
Pfaffendorf
 
N
, et al. 
Polymorphism of interleukin-23 receptor gene but not of NOD2/CARD15 is associated with graft-versus-host disease after hematopoietic stem cell transplantation in children.
Biol Blood Marrow Transplant
2009
, vol. 
15
 
12
(pg. 
1571
-
1577
)
84
van Heel
 
DA
Ghosh
 
S
Butler
 
M
, et al. 
Muramyl dipeptide and toll-like receptor sensitivity in NOD2-associated Crohn's disease.
Lancet
2005
, vol. 
365
 
9473
(pg. 
1794
-
1796
)
85
van Heel
 
DA
Hunt
 
KA
King
 
K
, et al. 
Detection of muramyl dipeptide-sensing pathway defects in patients with Crohn's disease.
Inflamm Bowel Dis
2006
, vol. 
12
 
7
(pg. 
598
-
605
)
86
Landfried
 
K
Bataille
 
F
Rogler
 
G
, et al. 
Recipient NOD2/CARD15 status affects cellular infiltrates in human intestinal graft-versus-host disease.
Clin Exp Immunol
2009
, vol. 
159
 
1
(pg. 
87
-
92
)