CD8- DCs induce IL-12-independent Th1 differentiation through Delta 4 Notch-like ligand in response to bacterial LPS

Toll-like receptor (TLR) ligation is believed to skew T cell responses toward T helper (Th)1 differentiation by inducing interleukin (IL)-12 secretion by CD8(+) dendritic cells (DCs). However, TLR-dependent Th1 responses occur in the absence of IL-12. To determine how DCs induce Th1 differentiation in the absence of IL-12, we examined the response of IL-12-deficient DCs to bacterial lipopolysaccharide (LPS). We find that LPS activates MyD88-dependent Delta 4 Notch-like ligand expression by CD8(-) DCs, and that these cells direct Th1 differentiation by an IL-12-independent and Notch-dependent mechanism in vitro and in vivo. Thus, activation of the two DC subsets by TLR4 leads to Th1 responses by two distinct MyD88-dependent pathways.     

TIR domain--containing adaptors regulate TLR-mediated signaling pathways

Recognition of pathogens by Toll-like receptors (TLRs) triggers innate immune responses via signaling pathways mediated by several Toll/IL-1R (TIR) domain-containing adaptors such as MyD88, TIRAP, and TRIF. MyD88 is a common adaptor that is essential for proinflammatory cytokine production, whereas TRIF mediates the MyD88-independent pathway from TLR3 and TLR4 that is responsible for type I interferon production in response to double-stranded RNA and LPS, respectively. TIRAP specifically participates in the MyD88-dependent pathways shared by TLR2 and TLR4, and TRAM is essential for the TLR4-mediated MyD88-independent pathway. Thus, TIR domain-containing adaptors play an important role in the TLR mediated signaling pathways.     

Fatal Mycobacterium tuberculosis infection despite adaptive immune response in the absence of MyD88

Toll-like receptors (TLRs) such as TLR2 and TLR4 have been implicated in host response to mycobacterial infection. Here, mice deficient in the TLR adaptor molecule myeloid differentiation factor 88 (MyD88) were infected with Mycobacterium tuberculosis (MTB). While primary MyD88(-/-) macrophages and DCs are defective in TNF, IL-12, and NO production in response to mycobacterial stimulation, the upregulation of costimulatory molecules CD40 and CD86 is unaffected. Aerogenic infection of MyD88(-/-) mice with MTB is lethal within 4 weeks with 2 log(10) higher CFU in the lung; high pulmonary levels of cytokines and chemokines; and acute, necrotic pneumonia, despite a normal T cell response with IFN-gamma production to mycobacterial antigens upon ex vivo restimulation. Vaccination with Mycobacterium bovis bacillus Calmette-Guerin conferred a substantial protection in MyD88(-/-) mice from acute MTB infection. These data demonstrate that MyD88 signaling is dispensable to raise an acquired immune response to MTB. Nonetheless, this acquired immune response is not sufficient to compensate for the profound innate immune defect and the inability of MyD88(-/-) mice to control MTB infection.     

Microbe sampling by mucosal dendritic cells is a discrete, MyD88-independent step in DeltainvG S. Typhimurium colitis

Intestinal dendritic cells (DCs) are believed to sample and present commensal bacteria to the gut-associated immune system to maintain immune homeostasis. How antigen sampling pathways handle intestinal pathogens remains elusive. We present a murine colitogenic Salmonella infection model that is highly dependent on DCs. Conditional DC depletion experiments revealed that intestinal virulence of S. Typhimurium SL1344 DeltainvG mutant lacking a functional type 3 secretion system-1 (DeltainvG)critically required DCs for invasion across the epithelium. The DC-dependency was limited to the early phase of infection when bacteria colocalized with CD11c(+)CX3CR1(+) mucosal DCs. At later stages, the bacteria became associated with other (CD11c(-)CX3CR1(-)) lamina propria cells, DC depletion no longer attenuated the pathology, and a MyD88-dependent mucosal inflammation was initiated. Using bone marrow chimeric mice, we showed that the MyD88 signaling within hematopoietic cells, which are distinct from DCs, was required and sufficient for induction of the colitis. Moreover, MyD88-deficient DCs supported transepithelial uptake of the bacteria and the induction of MyD88-dependent colitis. These results establish that pathogen sampling by DCs is a discrete, and MyD88-independent, step during the initiation of a mucosal innate immune response to bacterial infection in vivo.     

MyD88-dependent induction of allergic Th2 responses to intranasal antigen

MyD88 is a common Toll-like receptor (TLR) adaptor molecule found to be essential for induction of adaptive Th1 immunity. Conversely, innate control of adaptive Th2 immunity has been shown to occur in a MyD88-independent manner. In this study, we show that MyD88 is an essential innate component in the induction of TLR4-dependent Th2 responses to intranasal antigen; thus we demonstrate what we believe to be a novel role for MyD88 in pulmonary Th2 immunity. Induction of the MyD88-independent type I IFN response to LPS is defective in the pulmonary environment. Moreover, in the absence of MyD88, LPS-induced upregulation of costimulatory molecule expression on pulmonary DCs is defective, in contrast to what has been observed with bone marrow-derived DCs (BMDCs). Reconstitution of Th2 responses occurs upon adoptive pulmonary transfer of activated BMDCs to MyD88-deficient recipients. Furthermore, the dependence of Th2 responses on MyD88 is governed by the initial route of antigen exposure; this demonstrates what we believe are novel site-specific innate mechanisms for control of adaptive Th2 immunity.     

Toll-like receptor 9 mediates innate immune activation by the malaria pigment hemozoin

Malaria parasites within red blood cells digest host hemoglobin into a hydrophobic heme polymer, known as hemozoin (HZ), which is subsequently released into the blood stream and then captured by and concentrated in the reticulo-endothelial system. Accumulating evidence suggests that HZ is immunologically active, but the molecular mechanism(s) through which HZ modulates the innate immune system has not been elucidated. This work demonstrates that HZ purified from Plasmodium falciparum is a novel non-DNA ligand for Toll-like receptor (TLR)9. HZ activated innate immune responses in vivo and in vitro, resulting in the production of cytokines, chemokines, and up-regulation of costimulatory molecules. Such responses were severely impaired in TLR9-/- and myeloid differentiation factor 88 (MyD88)-/-, but not in TLR2, TLR4, TLR7, or Toll/interleukin 1 receptor domain-containing adaptor-inducing interferon beta-/- mice. Synthetic HZ, which is free of the other contaminants, also activated innate immune responses in vivo in a TLR9-dependent manner. Chloroquine (CQ), an antimalarial drug, abrogated HZ-induced cytokine production. These data suggest that TLR9-mediated, MyD88-dependent, and CQ-sensitive innate immune activation by HZ may play an important role in malaria parasite-host interactions.     

CD14 is a coreceptor of Toll-like receptors 7 and 9

Recognition of pathogens by the innate immune system requires proteins that detect conserved molecular patterns. Nucleic acids are recognized by cytoplasmic sensors as well as by endosomal Toll-like receptors (TLRs). It has become evident that TLRs require additional proteins to be activated by their respective ligands. In this study, we show that CD14 (cluster of differentiation 14) constitutively interacts with the MyD88-dependent TLR7 and TLR9. CD14 was necessary for TLR7- and TLR9-dependent induction of proinflammatory cytokines in vitro and for TLR9-dependent innate immune responses in mice. CD14 associated with TLR9 stimulatory DNA in precipitation experiments and confocal imaging. The absence of CD14 led to reduced nucleic acid uptake in macrophages. Additionally, CD14 played a role in the stimulation of TLRs by viruses. Using various types of vesicular stomatitis virus, we showed that CD14 is dispensable for viral uptake but is required for the triggering of TLR-dependent cytokine responses. These data show that CD14 has a dual role in nucleic acid-mediated TLR activation: it promotes the selective uptake of nucleic acids, and it acts as a coreceptor for endosomal TLR activation.     

Leukotriene B4 amplifies NF-κB activation in mouse macrophages by reducing SOCS1 inhibition of MyD88 expression

Activation of NF-κB and 5-lipoxygenase-mediated (5-LO-mediated) biosynthesis of the lipid mediator leukotriene B4 (LTB4) are pivotal components of host defense and inflammatory responses. However, the role of LTB4 in mediating innate immune responses elicited by specific TLR ligands and cytokines is unknown. Here we have shown that responses dependent on MyD88 (an adaptor protein that mediates signaling through all of the known TLRs, except TLR3, as well as IL-1β and IL-18) are reduced in mice lacking either 5-LO or the LTB4 receptor BTL1, and that macrophages from these mice are impaired in MyD88-dependent activation of NF-κB. This macrophage defect was associated with lower basal and inducible expression of MyD88 and reflected impaired activation of STAT1 and overexpression of the STAT1 inhibitor SOCS1. Expression of MyD88 and responsiveness to the TLR4 ligand LPS were decreased by Stat1 siRNA silencing in WT macrophages and restored by Socs1 siRNA in 5-LO-deficient macrophages. These results uncover a pivotal role in macrophages for the GPCR BLT1 in regulating activation of NF-κB through Stat1-dependent expression of MyD88.     

NOD2 signaling contributes to the innate immune response against helper-dependent adenovirus vectors independently of MyD88 in vivo

We previously demonstrated that Toll-like receptor/myeloid differentiation primary response gene 88 (MyD88) signaling is required for maximal innate and acquired [T helper cell type 1 (Th1)] immune responses following systemic administration of helper-dependent adenoviral vectors (HDAds). However, MyD88-deficient mice injected with HDAdLacZ exhibited only partial reduction of innate immune cytokine expression compared with wild-type mice, suggesting MyD88-independent pathways also respond to HDAds. We now show that NOD2, a nucleotide-binding and oligomerization domain (NOD)-like receptor known to detect muramyl dipeptides in bacterial peptidoglycans, also contributes to innate responses to HDAds, but not to humoral or Th1 immune responses. We established NOD2/MyD88 double-deficient mice that, when challenged with HDAds, showed a significant reduction of the innate response compared with mice deficient for either gene singly, suggesting that NOD2 signaling contributes to the innate response independently of MyD88 signaling following systemic administration of HDAds. In addition, NOD2-deficient mice exhibited significantly higher transgene expression than did wild-type mice at an early time point (before development of an acquired response), but not at a later time point (after development of an acquired response). These results indicate that the intracellular sensor NOD2 is required for innate responses to HDAds and can limit transgene expression during early phases of infection.     

MyD88 but not TRIF is essential for osteoclastogenesis induced by lipopolysaccharide, diacyl lipopeptide, and IL-1alpha

Myeloid differentiation factor 88 (MyD88) plays essential roles in the signaling of the Toll/interleukin (IL)-1 receptor family. Toll-IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF)-mediated signals are involved in lipopolysaccharide (LPS)-induced MyD88-independent pathways. Using MyD88-deficient (MyD88-/-) mice and TRIF-deficient (TRIF-/-) mice, we examined roles of MyD88 and TRIF in osteoclast differentiation and function. LPS, diacyl lipopeptide, and IL-1alpha stimulated osteoclastogenesis in cocultures of osteoblasts and hemopoietic cells obtained from TRIF-/- mice, but not MyD88-/- mice. These factors stimulated receptor activator of nuclear factor-kappaB ligand mRNA expression in TRIF-/- osteoblasts, but not MyD88-/- osteoblasts. LPS stimulated IL-6 production in TRIF-/- osteoblasts, but not TRIF-/- macrophages. LPS and IL-1alpha enhanced the survival of TRIF-/- osteoclasts, but not MyD88-/- osteoclasts. Diacyl lipopeptide did not support the survival of osteoclasts because of the lack of Toll-like receptor (TLR)6 in osteoclasts. Macrophages expressed both TRIF and TRIF-related adaptor molecule (TRAM) mRNA, whereas osteoblasts and osteoclasts expressed only TRIF mRNA. Bone histomorphometry showed that MyD88-/- mice exhibited osteopenia with reduced bone resorption and formation. These results suggest that the MyD88-mediated signal is essential for the osteoclastogenesis and function induced by IL-1 and TLR ligands, and that MyD88 is physiologically involved in bone turnover.     

Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptor-associated factor (TRAF)6

Transition of immature antigen presenting cells (APCs) to the state of professional APCs is essential for initiation of cell-mediated immune responses to pathogens. Signal transduction via molecules of the Toll-like receptor (TLR)/interleukin 1 receptor (IL-1R) pathway is critical for activation of APCs either by pathogen-derived pattern ligands like lipopolysaccharides (LPS) or by CD40 ligation through T helper cells. The capacity of bacterial DNA (CpG-DNA) to induce APCs to differentiate into professional APCs represents an interesting discovery. However, the signaling pathways involved are poorly understood. Here we show that CpG-DNA activates the TLR/IL-1R signaling pathway via the molecules myeloid differentiation marker 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6), leading to activation of kinases of the IkappaB kinase complex and the c-jun NH(2)-terminal kinases. Moreover, cells of TLR2- and TLR4-deficient mice are activated by CpG-DNA, whereas cells of MyD88-deficient mice do not respond. The data suggest that CpG-DNA initiates signaling via the TLR/IL-1R pathway in APCs in a manner similar to LPS and to T helper cell-mediated CD40 ligation. Activation of the TLR/IL-1R signaling pathway by foreign bacterial DNA may be one way to initiate innate defense mechanisms against infectious pathogens in vivo.     

Familial transmission rather than defective innate immunity shapes the distinct intestinal microbiota of TLR-deficient mice

The intestinal microbiota contributes to the development of the immune system, and conversely, the immune system influences the composition of the microbiota. Toll-like receptors (TLRs) in the gut recognize bacterial ligands. Although TLR signaling represents a major arm of the innate immune system, the extent to which TLRs influence the composition of the intestinal microbiota remains unclear. We performed deep 16S ribosomal RNA sequencing to characterize the complex bacterial populations inhabiting the ileum and cecum of TLR- and MyD88-deficient mice. The microbiota of MyD88- and TLR-deficient mouse colonies differed markedly, with each colony harboring distinct and distinguishable bacterial populations in the small and large intestine. Comparison of MyD88-, TLR2-, TLR4-, TLR5-, and TLR9-deficient mice and their respective wild-type (WT) littermates demonstrated that the impact of TLR deficiency on the composition of the intestinal microbiota is minimal under homeostatic conditions and after recovery from antibiotic treatment. Thus, differences between TLR-deficient mouse colonies reflected long-term divergence of the microbiota after extended husbandry in isolation from each other. Long-term breeding of isolated mouse colonies results in changes of the intestinal microbiota that are communicated to offspring by maternal transmission, which account for marked compositional differences between WT and mutant mouse strains.     

Tamm-Horsfall glycoprotein links innate immune cell activation with adaptive immunity via a Toll-like receptor-4-dependent mechanism

Tamm-Horsfall glycoprotein (THP) is expressed exclusively in the kidney and constitutes the most abundant protein in mammalian urine. A critical role for THP in antibacterial host defense and inflammatory disorders of the urogenital tract has been suggested. We demonstrate that THP activates myeloid DCs via Toll-like receptor-4 (TLR4) to acquire a fully mature DC phenotype. THP triggers typical TLR signaling, culminating in activation of NF-kappaB. Bone marrow-derived macrophages from TLR4- and MyD88-deficient mice were nonresponsive to THP in contrast to those from TLR2- and TLR9-deficient mice. In vivo THP-driven TNF-alpha production was evident in WT but not in Tlr4-/- mice. Importantly, generation of THP-specific Abs consistently detectable in urinary tract inflammation was completely blunted in Tlr4-/- mice. These data show that THP is a regulatory factor of innate and adaptive immunity and therefore could have significant impact on host immunity in the urinary tract.     

Haptoglobin activates innate immunity to enhance acute transplant rejection in mice

Immune tolerance to transplanted organs is impaired when the innate immune system is activated in response to the tissue necrosis that occurs during harvesting and implantation procedures. A key molecule in this immune pathway is the intracellular TLR signal adaptor known as myeloid differentiation primary response gene 88 (MyD88). After transplantation, MyD88 induces DC maturation as well as the production of inflammatory mediators, such as IL-6 and TNF-α. However, upstream activators of MyD88 function in response to transplantation have not been identified. Here, we show that haptoglobin, an acute phase protein, is an initiator of this MyD88-dependent inflammatory process in a mouse model of skin transplantation. Necrotic lysates from transplanted skin elicited higher inflammatory responses in DCs than did nontransplanted lysates, suggesting DC-mediated responses are triggered by factors released during transplantation. Analysis of transplanted lysates identified haptoglobin as one of the proteins upregulated during transplantation. Expression of donor haptoglobin enhanced the onset of acute skin transplant rejection, whereas haptoglobin-deficient skin grafts showed delayed acute rejection and antidonor T cell priming in a MyD88-dependent graft rejection model. Thus, our results show that haptoglobin release following skin necrosis contributes to accelerated transplant rejection, with potential implications for the development of localized immunosuppressive therapies.     

MyD88-dependent IL-1 receptor signaling is essential for gouty inflammation stimulated by monosodium urate crystals

While it is known that monosodium urate (MSU) crystals cause the disease gout, the mechanism by which these crystals stimulate this inflammatory condition has not been clear. Here we find that the Toll/IL-1R (TIR) signal transduction adaptor myeloid differentiation primary response protein 88 (MyD88) is required for acute gouty inflammation. In contrast, other TIR adaptor molecules, TIRAP/Mal, TRIF, and TRAM, are not required for this process. The MyD88-dependent TLR1, -2, -4, -6, -7, -9, and -11 and IL-18 receptor (IL-18R) are not essential for MSU-induced inflammation. Moreover, MSU does not stimulate HEK cells expressing TLR1-11 to activate NF-kappaB. In contrast, mice deficient in the MyD88-dependent IL-1R showed reduced inflammatory responses, similar to those observed in MyD88-deficient mice. Similarly, mice treated with IL-1 neutralizing antibodies also showed reduced MSU-induced inflammation, demonstrating that IL-1 production and IL-1R activation play essential roles in MSU-triggered inflammation. IL-1R deficiency in bone marrow-derived cells did not affect the inflammatory response; however, it was required in non-bone marrow-derived cells. These results indicate that IL-1 is essential for the MSU-induced inflammatory response and that the requirement of MyD88 in this process is primarily through its function as an adaptor molecule in the IL-1R signaling pathway.     

The TLR9-MyD88 pathway is critical for adaptive immune responses to adeno-associated virus gene therapy vectors in mice

Recombinant adeno-associated viruses (AAVs) have been used widely for in vivo gene therapy. However, adaptive immune responses to AAV have posed a significant hurdle in clinical application of AAV vectors. Recent advances have suggested a crucial role for innate immunity in shaping adaptive immune responses. How AAV activates innate immunity, and thereby promotes AAV-targeted adaptive immune responses, remains unknown. Here we show that AAV activates mouse plasmacytoid DCs (pDCs) via TLR9 to produce type I IFNs. In vivo, the TLR9-MyD88 pathway was crucial to the activation of CD8⁺ T cell responses to both the transgene product and the AAV capsid, leading to loss of transgene expression and the generation of transgene product–specific and AAV-neutralizing antibodies. We further demonstrate that TLR9-dependent activation of adaptive immunity targeting AAV was mediated by type I IFNs and that human pDCs could be activated in vitro to induce type I IFN production via TLR9. These results reveal an essential role for the TLR9-MyD88–type I IFN pathway in induction of adaptive immune responses to AAV and suggest that strategies that interfere with this pathway may improve the outcome of AAV-mediated gene therapy in humans.     

MyD88 inhibition amplifies dendritic cell capacity to promote pancreatic carcinogenesis via Th2 cells

The transition of chronic pancreatic fibroinflammatory disease to neoplasia is a primary example of the paradigm linking inflammation to carcinogenesis. However, the cellular and molecular mediators bridging these entities are not well understood. Because TLR4 ligation can exacerbate pancreatic inflammation, we postulated that TLR4 activation drives pancreatic carcinogenesis. In this study, we show that lipopolysaccharide accelerates pancreatic tumorigenesis, whereas TLR4 inhibition is protective. Furthermore, blockade of the MyD88-independent TRIF pathway is protective against pancreatic cancer, whereas blockade of the MyD88-dependent pathway surprisingly exacerbates pancreatic inflammation and malignant progression. The protumorigenic and fibroinflammatory effects of MyD88 inhibition are mediated by dendritic cells (DCs), which induce pancreatic antigen-restricted Th2-deviated CD4(+) T cells and promote the transition from pancreatitis to carcinoma. Our data implicate a primary role for DCs in pancreatic carcinogenesis and illustrate divergent pathways in which blockade of TLR4 signaling via TRIF is protective against pancreatic cancer and, conversely, MyD88 inhibition exacerbates pancreatic inflammation and neoplastic transformation by augmenting the DC-Th2 axis.     

Homeostatic MyD88-dependent signals cause lethal inflamMation in the absence of A20

Toll-like receptors (TLRs) on host cells are chronically engaged by microbial ligands during homeostatic conditions. These signals do not cause inflammatory immune responses in unperturbed mice, even though they drive innate and adaptive immune responses when combating microbial infections. A20 is a ubiquitin-modifying enzyme that restricts exogenous TLR-induced signals. We show that MyD88-dependent TLR signals drive the spontaneous T cell and myeloid cell activation, cachexia, and premature lethality seen in A20-deficient mice. We have used broad spectrum antibiotics to demonstrate that these constitutive TLR signals are driven by commensal intestinal flora. A20 restricts TLR signals by restricting ubiquitylation of the E3 ligase tumor necrosis factor receptor-associated factor 6. These results reveal both the severe proinflammatory pathophysiology that can arise from homeostatic TLR signals as well as the critical role of A20 in restricting these signals in vivo. In addition, A20 restricts MyD88-independent TLR signals by inhibiting Toll/interleukin 1 receptor domain-containing adaptor inducing interferon (IFN) beta-dependent nuclear factor kappaB signals but not IFN response factor 3 signaling. These findings provide novel insights into how physiological TLR signals are regulated.