Stimulation Of Pattern Recognition Receptors Research Articles

Lactobacillus reuteri I5007 Modulates Intestinal Host Defense Peptide Expression in the Model of IPEC-J2 Cells and Neonatal Piglets.

Modulation of the synthesis of endogenous host defense peptides (HDPs) by probiotics represents a novel antimicrobial approach for disease control and prevention, particularly against antibiotic-resistant infections in human and animals. However, the extent of HDP modulation by probiotics is species dependent and strain specific. In the present study, The porcine small intestinal epithelial cell line (IPEC-J2) cells and neonatal piglets were used as in-vitro and in-vivo models to test whether Lactobacillus reuteri I5007 could modulate intestinal HDP expression. Gene expressions of HDPs, toll-like receptors, and fatty acid receptors were determined, as well as colonic short chain fatty acid concentrations and microbiota. Exposure to 108 colony forming units (CFU)/mL of L. reuteri I5007 for 6 h significantly increased the expression of porcine β-Defensin2 (PBD2), pBD3, pBD114, pBD129, and protegrins (PG) 1-5 in IPEC-J2 cells. Similarly, L. reuteri I5007 administration significantly increased the expression of jejunal pBD2 as well as colonic pBD2, pBD3, pBD114, and pBD129 in neonatal piglets (p < 0.05). This was probably associated with the increase in colonic butyric acid concentration and up-regulating expression of Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ) and G Protein-Coupled Receptor 41 (GPR41) (p < 0.05), but not with stimulation of Pattern-Recognition Receptors. Additionally, supplementation with L. reuteri I5007 in the piglets did not affect the colonic microbiota structure. Our findings suggested that L. reuteri I5007 could modulate intestinal HDP expression and improve the gut health of neonatal piglets, probably through the increase in colonic butyric acid concentration and the up-regulation of the downstream molecules of butyric acid, PPAR-γ and GPR41, but not through modifying gut microbiota structure.

An inflammatory bowel disease-risk variant in INAVA decreases pattern recognition receptor-induced outcomes.

Inflammatory bowel disease (IBD) is characterized by dysregulation in both cytokines and responses to intestinal microbes, and proper regulation of pattern recognition receptor (PRR) signaling is critical for intestinal immune homeostasis. Altered functions for the IBD risk locus containing rs7554511, which encompasses the C1orf106 gene (recently named INAVA), and roles for the protein encoded by the INAVA gene are unknown. Here, we investigated the role of INAVA and INAVA genotype in regulating PRR-initiated outcomes in primary human cells. Both peripheral and intestinal myeloid cells expressed INAVA. Upon PRR stimulation, INAVA was required for optimal MAPK and NF-κB activation, cytokine secretion, and intracellular bacterial clearance. INAVA recruited 14-3-3τ, thereby contributing to recruitment of a signaling complex that amplified downstream signals and cytokines. Further, INAVA enhanced bacterial clearance by regulating reactive oxygen, reactive nitrogen, and autophagy pathways. Macrophages from rs7554511 C risk carriers expressed lower levels of INAVA RNA and protein. Lower expression was attributed in part to decreased transcription mediated directly by the intronic region containing the rs7554511 C variant. In rs7554511 C risk carrier macrophages, lower INAVA expression led to decreased PRR-induced activation of MAPK and NF-κB pathways, cytokines, and bacterial clearance pathways. Thus, IBD-associated polymorphisms in INAVA modulate PRR-initiated signaling, cytokines, and intracellular bacterial clearance, likely contributing to intestinal immune homeostasis.

JAK2 Disease-Risk Variants Are Gain of Function and JAK Signaling Threshold Determines Innate Receptor-Induced Proinflammatory Cytokine Secretion in Macrophages.

JAK2 genetic variants are associated with inflammatory bowel disease (IBD) and JAK inhibitors are being evaluated for therapy targeting immune-mediated diseases, including IBD. As JAK pathway-mediated cytokine regulation varies across cell types and stimulation conditions, we examined how JAK signaling and IBD-associated JAK2 variants regulate distinct acute and chronic microbial product exposure outcomes in human myeloid cells, consistent with the conditions of initial entry and ongoing intestinal tissue residence, respectively. Macrophages from controls and ulcerative colitis patients carrying the IBD-risk rs10758669 CC genotype showed increased JAK2 expression and nucleotide-binding oligomerization domain 2-induced JAK2 phosphorylation relative to AA carriers. Interestingly, the threshold of JAK2 expression and signaling determined pattern-recognition receptor (PRR)-induced outcomes; whereas anti-inflammatory cytokines progressively decreased with lower JAK2 expression, proinflammatory cytokines switched from decreased to increased secretion below a certain JAK2 expression threshold. Low JAK2-expressing rs10758669 AA macrophages were above this threshold; consequently, both PRR-induced pro- and anti-inflammatory cytokines were decreased. However, relative to rs10758669 CC risk carriers, AA carrier macrophages switched to increased nucleotide-binding oligomerization domain 2-induced proinflammatory cytokines at lower therapeutically used JAK inhibitor doses. Importantly, JAK inhibitors increased proinflammatory cytokines secreted by peripheral macrophages following chronic PRR stimulation and by human intestinal myeloid cells following exposure to intestinal pathogens. Mechanistically, the decreased response to and secretion of autocrine/paracrine IL-10, IL-4, IL-22 and thymic stromal lymphopoietin regulated these JAK-dependent outcomes in myeloid cells. Taken together, the JAK signaling threshold determines whether PRR-induced pro- and anti-inflammatory cytokines are reciprocally regulated in myeloid cells; consideration of JAK2 genotype and targeting of specific cell types might improve JAK-targeted therapy in immune-mediated diseases.

CCL19-CCR7-dependent reverse transendothelial migration of myeloid cells clears Chlamydia muridarum from the arterial intima.

Regions of the normal arterial intima predisposed to atherosclerosis are sites of ongoing monocyte trafficking and also contain resident myeloid cells with features of dendritic cells. However, the pathophysiological roles of these cells are poorly understood. Here we found that intimal myeloid cells underwent reverse transendothelial migration (RTM) into the arterial circulation after systemic stimulation of pattern-recognition receptors (PRRs). This process was dependent on expression of the chemokine receptor CCR7 and its ligand CCL19 by intimal myeloid cells. In mice infected with the intracellular pathogen Chlamydia muridarum, blood monocytes disseminated infection to the intima. Subsequent CCL19-CCR7-dependent RTM was critical for the clearance of intimal C. muridarum. This process was inhibited by hypercholesterolemia. Thus, RTM protects the normal arterial intima, and compromised RTM during atherogenesis might contribute to the intracellular retention of pathogens in atherosclerotic lesions.

Powerful Complex Immunoadjuvant Based on Synergistic Effect of Combined TLR4 and NOD2 Activation Significantly Enhances Magnitude of Humoral and Cellular Adaptive Immune Responses.

Binding of pattern recognition receptors (PRRs) by pathogen-associated molecular patterns (PAMPs) activates innate immune responses and contributes to development of adaptive immunity. Simultaneous stimulation of different types of PRRs can have synergistic immunostimulatory effects resulting in enhanced production of molecules that mediate innate immunity such as inflammatory cytokines, antimicrobial peptides, etc. Here, we evaluated the impact of combined stimulation of PRRs from different families on adaptive immunity by generating alum-based vaccine formulations with ovalbumin as a model antigen and the Toll-like receptor 4 (TLR4) agonist MPLA and the Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) agonist MDP adsorbed individually or together on the alum-ovalbumin particles. Multiple in vitro and in vivo readouts of immune system activation all showed that while individual PRR agonists increased the immunogenicity of vaccines compared to alum alone, the combination of both PRR agonists was significantly more effective. Combined stimulation of TLR4 and NOD2 results in a stronger and broader transcriptional response in THP-1 cells compared to individual PRR stimulation. Immunostimulatory composition containing both PRR agonists (MPLA and MDP) in the context of the alum-based ovalbumin vaccine also enhanced uptake of vaccine particles by bone marrow derived dendritic cells (BMDCs) and promoted maturation (up-regulation of expression of CD80, CD86, MHCII) and activation (production of cytokines) of BMDCs. Finally, immunization of mice with vaccine particles containing both PRR agonists resulted in enhanced cellular immunity as indicated by increased proliferation and activation (IFN-γ production) of splenic CD4+ and CD8+ T cells following in vitro restimulation with ovalbumin and enhanced humoral immunity as indicated by higher titers of ovalbumin-specific IgG antibodies. These results indicate that combined stimulation of TLR4 and NOD2 receptors dramatically enhances activation of both the humoral and cellular branches of adaptive immunity and suggests that inclusion of agonists of these receptors in standard alum-based adjuvants could be used to improve the effectiveness of vaccination.

Antidepressant therapies inhibit inflammation and microglial M1-polarization.

Macrophages and their counterparts in the central nervous system, the microglia, detect and subsequently clear microbial pathogens and injured tissue. These phagocytic cells alter and adapt their phenotype depending on their prime activity, i.e., whether they participate in acute defence against pathogenic organisms ('M1'-phenotype) or in clearing damaged tissues and performing repair activities ('M2'-phenotype). Stimulation of pattern recognition receptors by viruses (vaccines), bacterial membrane components (e.g., LPS), alcohol, or long-chain saturated fatty acids promotes M1-polarization. Vaccine or LPS administration to healthy human subjects can result in sickness symptoms and low mood. Alcohol abuse and abdominal obesity are recognized as risk factors for depression. In the M1-polarized form, microglia and macrophages generate reactive oxygen and nitrogen radicals to eradicate microbial pathogens. Inadvertently, also tetrahydrobiopterin (BH4) may become oxidized. This is an irreversible reaction that generates neopterin, a recognized biomarker for depression. BH4 is a critical cofactor for the synthesis of dopamine, noradrenaline, and serotonin, and its loss could explain some of the symptoms of depression. Based on these aspects, the suppression of M1-polarization would limit the inadvertent catabolism of BH4. In the current review, we evaluate the evidence that antidepressant treatments (monoamine reuptake inhibitors, PDE4 inhibitors, lithium, valproate, agomelatine, tianeptine, electroconvulsive shock, and vagus nerve stimulation) inhibit LPS-induced microglia/macrophage M1-polarization. Consequently, we propose that supplementation with BH4 could limit the reduction in central monoamine synthesis and might represent an effective treatment for depressed mood.

β-Glucan–Activated Human B Lymphocytes Participate in Innate Immune Responses by Releasing Proinflammatory Cytokines and Stimulating Neutrophil Chemotaxis

B lymphocytes play an essential regulatory role in the adaptive immune response through Ab production during infection. A less known function of B lymphocytes is their ability to respond directly to infectious Ags through stimulation of pattern recognition receptors expressed on their surfaces. β-Glucans are carbohydrates present in the cell wall of many pathogenic fungi that can be detected in the peripheral blood of patients during infection. They have been shown to participate in the innate inflammatory response, as they can directly activate peripheral macrophages and dendritic cells. However, their effect as direct stimulators of B lymphocytes has not been yet fully elucidated. The aim of this study was to examine the molecular mechanisms and cytokine profiles generated following β-glucan stimulation of B lymphocytes, compared with the well-established TLR-9 agonist CpG oligodeoxynucleotide (CpG), and study the participation of β-glucan-stimulated B cells in the innate immune response. In this article, we demonstrate that β-glucan-activated B lymphocytes upregulate proinflammatory cytokines (TNF-α, IL-6, and IL-8). Of interest, β-glucan, unlike CpG, had no effect on B lymphocyte proliferation or IgM production. When compared with CpG (TLR9 agonist), β-glucan-activated cells secreted significantly higher levels of IL-8. Furthermore, IL-8 secretion was partially mediated by Dectin-1 and required SYK, MAPKs, and the transcription factors NF-κB and AP-1. Moreover, we observed that conditioned media from β-glucan-stimulated B lymphocytes elicited neutrophil chemotaxis. These studies suggest that β-glucan-activated B lymphocytes have an important and novel role in fungal innate immune responses.

TLR and RLR Signaling Are Reprogrammed in Opposite Directions after Detection of Viral Infection

Innate immune recognition of RNA is key for the initiation of immunity in response to viral infection. Although the factors controlling the detection of viral RNA by innate immune receptors in host cells are increasingly well understood, little is known about the dynamic changes in signaling after the initial triggering of these receptors. In this study, we report that preconditioning with the synthetic dsRNA polyinosinic-polycytidylic acid [poly(I:C)], a mimetic of viral RNA, rapidly reprograms murine APCs by simultaneously augmenting sensitivity of endosomal TLRs and inhibiting activation of RIG-I-like receptors (RLRs) in an IFN-β-dependent manner. These changes in receptor sensitivity were also seen in vivo after treatment of mice with poly(I:C). Mechanistically, the increased sensitivity of the TLR pathway was associated with elevated MAPK and NF-κB activity. The RLR response was inhibited downstream of TANK-binding kinase-1, resulting in decreased IFN regulatory factor 3 phosphorylation. Reprogramming of pattern-recognition receptor signaling also occurred after viral infection, because infection of host cells with Sendai virus or their exposure to supernatant from virus-infected cells induced the same changes in TLR and RLR sensitivity as poly(I:C). Thus, innate recognition of viral infection critically modifies responses to pattern-recognition receptor stimulation. These dynamic adaptations to infection may reinforce antiviral immunity and at the same time serve to limit pathological inflammation.

Twist1 and Twist2 Contribute to Cytokine Downregulation following Chronic NOD2 Stimulation of Human Macrophages through the Coordinated Regulation of Transcriptional Repressors and Activators.

Proper regulation of microbial-induced cytokines is critical to intestinal immune homeostasis. Acute stimulation of nucleotide-binding oligomerization domain 2 (NOD2), the Crohn's disease-associated sensor of bacterial peptidoglycan, induces cytokines. However, chronic NOD2 stimulation in macrophages decreases cytokines upon pattern recognition receptor (PRR) restimulation; cytokine attenuation to PRR stimulation is similarly observed in intestinal macrophages. The role for the transcriptional repressors Twist1 and Twist2 in regulating PRR-induced cytokine outcomes is poorly understood and has not been reported for NOD2. We found that Twist1 and Twist2 were required for optimal cytokine downregulation during acute and, particularly, chronic NOD2 stimulation of human macrophages. Consistently, Twist1 and Twist2 expression was increased after chronic NOD2 stimulation; this increased expression was IL-10 and TGF-β dependent. Although Twist1 and Twist2 did not coregulate each other's expression, they cooperated to enhance binding to cytokine promoters after chronic NOD2 stimulation. Moreover, Twist1 and Twist2 contributed to enhance expression and promoter binding of the proinflammatory inhibitor c-Maf and the transcriptional repressor Bmi1. Restoring c-Maf and Bmi1 expression in Twist-deficient macrophages restored NOD2-induced cytokine downregulation. Furthermore, with chronic NOD2 stimulation, Twist1 and Twist2 contributed to the decreased expression and cytokine promoter binding of the transcriptional activators activating transcription factor 4, C/EBPα, Runx1, and Runx2. Knockdown of these transcriptional activators in Twist-deficient macrophages restored cytokine downregulation after chronic NOD2 stimulation. Finally, NOD2 synergized with additional PRRs to increase Twist1 and Twist2 expression and Twist-dependent pathways. Therefore, after chronic NOD2 stimulation Twist1 and Twist2 coordinate the regulation of both transcriptional activators and repressors, thereby mediating optimal cytokine downregulation.

TAM receptor-dependent regulation of SOCS3 and MAPKs contributes to proinflammatory cytokine downregulation following chronic NOD2 stimulation of human macrophages.

Microbial-induced cytokine regulation is critical to intestinal immune homeostasis. Acute stimulation of nucleotide-binding oligomerization domain 2 (NOD2), the Crohn's disease-associated sensor of bacterial peptidoglycan, induces cytokines. However, cytokines are attenuated after chronic NOD2 and pattern recognition receptor stimulation of macrophages; similar attenuation is observed in intestinal macrophages. The role of Tyro3, Axl, and Mer (TAM) receptors in regulating chronic pattern recognition receptor stimulation and NOD2-induced outcomes has not been examined. Moreover, TAM receptors have been relatively less investigated in human macrophages. Whereas TAM receptors did not downregulate acute NOD2-induced cytokines in primary human macrophages, they were essential for downregulating signaling and proinflammatory cytokine secretion after chronic NOD2 and TLR4 stimulation. Axl and Mer were similarly required in mice for cytokine downregulation after chronic NOD2 stimulation in vivo and in intestinal tissues. Consistently, TAM expression was increased in human intestinal myeloid-derived cells. Chronic NOD2 stimulation led to IL-10- and TGF-β-dependent TAM upregulation in human macrophages, which, in turn, upregulated suppressor of cytokine signaling 3 expression. Restoring suppressor of cytokine signaling 3 expression under TAM knockdown conditions restored chronic NOD2-mediated proinflammatory cytokine downregulation. In contrast to the upregulated proinflammatory cytokines, attenuated IL-10 secretion was maintained in TAM-deficient macrophages upon chronic NOD2 stimulation. The level of MAPK activation in TAM-deficient macrophages after chronic NOD2 stimulation was insufficient to upregulate IL-10 secretion; however, full restoration of MAPK activation under these conditions restored c-Fos, c-Jun, musculoaponeurotic fibrosarcoma oncogene homolog K, and PU.1 binding to the IL-10 promoter and IL-10 secretion. Therefore, TAM receptors are critical for downregulating proinflammatory cytokines under the chronic NOD2 stimulation conditions observed in the intestinal environment.

Activation of Pattern Recognition Receptors Up-Regulates Metallothioneins, Thereby Increasing Intracellular Accumulation of Zinc, Autophagy, and Bacterial Clearance by Macrophages

Continuous stimulation of pattern recognition receptors (PRRs), including nucleotide-binding oligomerization domain-2 (NOD2) (variants in NOD2 have been associated with Crohn's disease), alters the phenotype of myeloid-derived cells, reducing production of inflammatory cytokines and increasing microbe clearance. We investigated the mechanisms by which microbial clearance increases in macrophages under these conditions. Monocytes were purified from human peripheral blood mononuclear cells and differentiated to monocyte-derived macrophages (MDMs). We also isolated human intestinal macrophages. Bacterial clearance by MDMs was assessed in gentamicin protection assays. Effects of intracellular zinc and autophagy were measured by flow cytometry, immunoblot, reverse-transcription polymerase chain reaction, and microscopy experiments. Small interfering RNAs were used to knock down specific proteins in MDMs. NOD2-/- and C57BL/6J mice, maintained in a specific pathogen-free facility, were given antibiotics, muramyl dipeptide (to stimulate NOD2), or dextran sodium sulfate; intestinal lamina propria cells were collected and analyzed. Chronic stimulation of human MDMs through NOD2 up-regulated the expression of multiple genes encoding metallothioneins, which bind and regulate levels of intracellular zinc. Intestinal myeloid-derived cells are stimulated continually through PRRs; metallothionein expression was up-regulated in human and mouse intestinal myeloid-derived cells. Continuous stimulation of NOD2 increased the levels of intracellular zinc, thereby increasing autophagy and bacterial clearance. The metal-regulatory transcription factor-1 (MTF-1) was required for regulation of metallothionein genes in human MDMs. Knockdown of MTF-1 did not affect baseline clearance of bacteria by MDMs. However, the increase in intracellular zinc, autophagy, and bacterial clearance observed with continuous NOD2 stimulation was impaired in MDMs upon MTF-1 knockdown. The addition of zinc or induction of autophagy restored bacterial clearance to MDMs after metallothionein knockdown. NOD2 synergized with the PRRs Toll-like receptors 5 and 9 increase the effects of metallothioneins in MDMs. In mice, the intestinal microbiota contributed to the regulation in expression of metallothioneins, levels of zinc, autophagy, and bacterial clearance by intestinal macrophages. In studies of human MDMs and in mice, continuous stimulation of PRRs induces expression of metallothioneins. This leads to increased levels of intracellular zinc and enhanced clearance of bacteria via autophagy in macrophages.

Innate signaling in the inflammatory immune disorders

Pattern recognition receptors (PRRs) of innate immune cells recognize the conserved molecular signatures on pathogens, termed pathogen-associated molecular patterns. PRRs also recognize endogenous damage-associated molecular patterns. Following pathogen infection or tissue damage, the stimulation of PRRs activates distinct but shared signaling pathways that lead to effector mechanisms in innate host defense. PRR signaling is strictly and finely tuned to ensure the appropriate duration and strength to prevent damaging inflammation to the host. Here we attempt to provide a brief background on the agonists and signal transduction pathways of PRRs and summarize the mechanisms underlying the control of PRR signaling, with a particular focus on the recent progress of the involvement of PRR signaling in the inflammatory immune disorders.

Trained immunity or tolerance: opposing functional programs induced in human monocytes after engagement of various pattern recognition receptors.

Upon priming with Candida albicans or with the fungal cell wall component β-glucan, monocytes respond with an increased cytokine production upon restimulation, a phenomenon termed “trained immunity.” In contrast, the prestimulation of monocytes with lipopolysaccharide has long been known to induce tolerance. Because the vast majority of commensal microorganisms belong to bacterial or viral phyla, we sought to systematically investigate the functional reprogramming of monocytes induced by the stimulation of pattern recognition receptors (PRRs) with various bacterial or viral ligands. Monocytes were functionally programmed for either enhanced (training) or decreased (tolerance) cytokine production, depending on the type and concentration of ligand they encountered. The functional reprogramming of monocytes was also associated with cell shape, granulocity, and cell surface marker modifications. The training effect required p38- and Jun N-terminal protein kinase (JNK)-mediated mitogen-activated protein kinase (MAPK) signaling, with specific signaling patterns directing the functional fate of the cell. The long-term effects on the function of monocytes were mediated by epigenetic events, with both histone methylation and acetylation inhibitors blocking the training effects. In conclusion, our experiments identify the ability of monocytes to acquire adaptive characteristics after prior activation with a wide variety of ligands. Trained immunity and tolerance are two distinct and opposing functional programs induced by the specific microbial ligands engaging the monocytes.

Hepcidin Induction by Pathogens and Pathogen-Derived Molecules Is Strongly Dependent on Interleukin-6

Hepcidin, the iron-regulatory hormone, is increased during infection or inflammation, causing hypoferremia. This response is thought to be a host defense mechanism that restricts iron availability to invading pathogens. It is not known if hepcidin is differentially induced by bacterial versus viral infections, whether the stimulation of pattern recognition receptors directly regulates hepcidin transcription, or which of the proposed signaling pathways are essential for hepcidin increase during infection. We analyzed hepcidin induction and its dependence on interleukin-6 (IL-6) in response to common bacterial or viral infections in mice or in response to a panel of pathogen-derived molecules (PAMPs) in mice and human primary hepatocytes. In wild-type (WT) mice, hepcidin mRNA was induced several hundred-fold both by a bacterial (Streptococcus pneumoniae) and a viral infection (influenza virus PR8) within 2 to 5 days. Treatment of mice and human primary hepatocytes with most Toll-like receptor ligands increased hepcidin mRNA within 6 h. Hepcidin induction by microbial stimuli was IL-6 dependent. IL-6 knockout mice failed to increase hepcidin in response to S. pneumoniae or influenza infection and had greatly diminished hepcidin response to PAMPs. In vitro, hepcidin induction by PAMPs in primary human hepatocytes was abolished by the addition of neutralizing IL-6 antibodies. Our results support the key role of IL-6 in hepcidin regulation in response to a variety of infectious and inflammatory stimuli.

Functional RIG-I-like receptors control the survival of mesenchymal stem cells

Because of their potent regenerative and immunomodulatory properties, mesenchymal stem cells (MSCs) have promising therapeutic benefits in clinical treatment of inflammatory and infectious diseases. Recent studies suggest that many biological activities of MSCs are largely determined by pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs). However, the role of PRRs in regulating the survival of MSCs remains unknown. In the present study, we examined the viability of MSCs after stimulation of distinct PRRs. Activation of TLRs by direct addition with their respective ligands showed no significant effect on the survival of MSCs, whereas transfection with double-stranded RNA (dsRNA) resulted in marked cell death in MSCs. Transfection of dsRNA upregulated cytosolic retinoic acid inducible gene I (RIG-I)-like receptors (RLRs), including RIG-I and melanoma differentiation-associated antigen 5 (MDA5). Moreover, transfection of dsRNA activated downstream transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor κB (NF-κB), as well as induced the expression of interferon-β (IFN-β) and pro-inflammatory cytokine interleukin 6 (IL-6) via RLR signaling. Furthermore, we found that transfection of dsRNA triggered both extrinsic and intrinsic apoptotic responses via RLRs. However, ectopic expression of RIG-I or MDA5 was not sufficient to induce apoptosis of MSCs without dsRNA transfection. Our study also revealed that IκB kinase α/β (IKKα/β) was required for RLR-mediated apoptosis in MSCs, while TANK-binding kinase 1 (TBK1)/IKKɛ served a pro-survival role. Moreover, neither overexpression of B-cell lymphoma 2 (Bcl2) nor neutralizing autocrined IFN-β reduced RLR-mediated apoptosis. In addition, autophagy was induced upon activation of RLRs, however, blocking autophagy did not rescue MSCs from the dsRNA-induced cell death. To the best of our knowledge, this is the first study to explore the role of RLRs in controlling the survival of MSCs, which may provide a clue to understand the pathogenesis of viral infection in MSCs.

Pattern recognition receptor-mediated cytokine response in infants across 4 continents⋆

BackgroundSusceptibility to infection as well as response to vaccination varies among populations. To date, the underlying mechanisms responsible for these clinical observations have not been fully delineated. Because innate immunity instructs adaptive immunity, we hypothesized that differences between populations in innate immune responses may represent a mechanistic link to variation in susceptibility to infection or response to vaccination.ObjectiveDetermine whether differences in innate immune responses exist among infants from different continents of the world.MethodsWe determined the innate cytokine response following pattern recognition receptor (PRR) stimulation of whole blood from 2-year-old infants across 4 continents (Africa, North America, South America, and Europe).ResultsWe found that despite the many possible genetic and environmental exposure differences in infants across 4 continents, innate cytokine responses were similar for infants from North America, South America, and Europe. However, cells from South African infants secreted significantly lower levels of cytokines than did cells from infants from the 3 other sites, and did so following stimulation of extracellular and endosomal but not cytosolic PRRs.ConclusionsSubstantial differences in innate cytokine responses to PRR stimulation exist among different populations of infants that could not have been predicted. Delineating the underlying mechanism(s) for these differences will not only aid in improving vaccine-mediated protection but possibly also provide clues for the susceptibility to infection in different regions of the world.

Mitogen-activated protein kinases in innate immunity

Following pathogen infection or tissue damage, the stimulation of pattern recognition receptors on the cell surface and in the cytoplasm of innate immune cells activates members of each of the major mitogen-activated protein kinase (MAPK) subfamilies--the extracellular signal-regulated kinase (ERK), p38 and Jun N-terminal kinase (JNK) subfamilies. In conjunction with the activation of nuclear factor-κB and interferon-regulatory factor transcription factors, MAPK activation induces the expression of multiple genes that together regulate the inflammatory response. In this Review, we discuss our current knowledge about the regulation and the function of MAPKs in innate immunity, as well as the importance of negative feedback loops in limiting MAPK activity to prevent host tissue damage. We also examine how pathogens have evolved complex mechanisms to manipulate MAPK activation to increase their virulence. Finally, we consider the potential of the pharmacological targeting of MAPK pathways to treat autoimmune and inflammatory diseases.

Microbiota metabolite regulation of pattern recognition receptor activation in dendritic cells (P1253)

Abstract The microbiota is a vast community of microbial cells living in symbiosis with the host. The intestinal microbiota stands out due to its direct link with overall host health. The molecular complexity of the microbiota suggests broad activation of multiple pattern recognition receptors on intestinal epithelial cells and dendritic cells. Indeed, mutations in pattern recognition receptors are correlated with increased inflammatory bowel disease incidence. Clearly, the interaction between microbiota and host cells is integral to a healthy host, but what regulates this interaction is poorly understood. In our previous work we reported that indole, a tryptophan metabolite present in the GI tract and strictly produced by the microbiota (not host cells), downregulates NFκB activation and inflammation in intestinal epithelial cells. We hypothesize that indole limits inflammation by modulating pattern recognition receptor-mediated activation of dendritic cells, key sentinels of gut homeostasis. Here, we demonstrate that dendritic cells conditioned with indole at physiologic concentrations have decreased production of pro-inflammatory cytokines after differential pattern recognition receptor stimulation. Notably, indole decreases the severity of DSS-induced colitis. This suggests an anti-inflammatory role for indole in maintenance of homeostasis in the GI tract and reveals a new therapeutic strategy for the treatment of inflammatory bowel disease.

Immunoregulation in Idiopathic Inflammatory Myopathies: From Dendritic Cells to Immature Regenerating Muscle Cells

Idiopathic inflammatory myopathies (IIM) are a group of chronic muscle disorders of unknown origin that lead to muscle destruction. The original classification of IIM proposed by Bohan and Peter in 1975 including polymyositis (PM) and dermatomyositis (DM) was recently revised with the development of histopathological approaches and the discovery of myositis-specific and myositis-associated antibodies that led to the characterization of new entities. In addition to PM and adult and juvenile DM, these novel classifications distinguish inclusion-body myositis (IBM), immune-mediated necrotizing myopathy, overlap myositis, and cancer-associated myositis1,2. While distinct immunopathogenic mechanisms may occur in each subset of myositis, all IIM share a common inflammatory background associated with clinical, histological, and serological overlap. Several molecular pathways, such as adaptive and innate immune responses, autoimmunity, and nonimmune mechanisms, could influence the pathogenesis of IIM. However, the exact contribution of each one in the development of distinct phenotypes remains unclear. In this issue of The Journal , Gendek-Kubiak and Gendek highlight the contribution of dendritic cells (DC)3. We summarize here recent insights in myositis immunoregulation from DC that are central in the development of adaptive and innate response to nonimmune mechanisms. ### Dendritic cells DC are crucial for the development of adaptive and innate immune responses. DC, subdivided into myeloid DC (mDC) and plasmacytoid DC (pDC), are present in lymphatic and blood systems and peripheral organs. They are equipped with a range of pattern-recognition receptors (PRR) and serve as sentinels of the immune system. Their activation after stimulation of PRR, such as Toll-like receptors (TLR) or C-type lectin receptors (CLR), by microbial components and/or endogenous ligands leads to their maturation and the development of an effector T cell immune response. In muscle biopsies from IIM, both myeloid and plasmacytoid DC have been detected with differences according to IIM subtypes. The DC … Address correspondence to Dr. A. Tournadre, Department of Rheumatology, Gabriel Montpied Hospital, CHU Clermont-Ferrand, 58 rue Montalembert BP69, 63003 Clermont-Ferrand Cedex 1, France. E-mail: atournadre{at}chu-clermontferrand.fr

Pro-inflammatory cytokine and caspase-1 responses to pattern recognition receptor activation of neutrophils and dendritic cells in Behcet's disease

Activated innate immunity is implicated in the pathogenesis of Behcet's disease (BD). To clarify the mechanisms of innate immune responses, we investigated inflammasome activation in dendritic cells (DCs) and neutrophils, following stimulation with two different pattern recognition receptors (PRRs) RIG-1-like (RLR) and NOD-like (NLR) in patients with BD. Sixteen active BD patients with mucocutaneous lesions and 17 healthy controls (HCs) were included in this study. DCs were generated from monocytes. DCs and isolated neutrophils were activated by RLR and NLR ligands. Caspase-1 activation and expression of p38 and RIP2 were determined by flow cytometry. Levels of IL-1β, IL-6, TNF-α, IFN-α and IL-18 in culture supernatants were measured by ELISA. Activation of caspase-1 following intracellular PRR stimulation was found to be of similar levels in DCs and neutrophils of BD patients compared with HCs. However, activation of DCs from BD patients to NOD2 stimulus measured by the expression of RIP2 and p38 as well as IL-18 levels was found to be slightly defective (P < 0.05). In neutrophil cultures, IL-6 levels were lower in response to all stimuli in patients with BD compared with HCs (P < 0.01). Inflammasome formation following stimulation with NOD1/NOD2 and RIG measured by caspase-1 activation, cytokine levels and expression of RIP2 and p38 seems to be functionally normal in DCs and neutrophils of BD patients, although slightly defective responses in some pathways and cytokine levels were observed. These results may suggest that caspase-1-independent pathways such as toll-like receptors may be more prominent in BD pathogenesis.