Type I interferons produced by hematopoietic cells protect mice against lethal infection by mammalian reovirus

We defined the function of type I interferons (IFNs) in defense against reovirus strain type 1 Lang (T1L), which is a double-stranded RNA virus that infects Peyer's patches (PPs) after peroral inoculation of mice. T1L induced expression of mRNA for IFN-alpha, IFN-beta, and Mx-1 in PPs and caused localized intestinal infection that was cleared in 10 d. In contrast, T1L produced fatal systemic infection in IFNalphaR1 knockout (KO) mice with extensive cell loss in lymphoid tissues and necrosis of the intestinal mucosa. Studies of bone-marrow chimeric mice indicated an essential role for hematopoietic cells in IFN-dependent viral clearance. Dendritic cells (DCs), including conventional DCs (cDCs), were the major source of type I IFNs in PPs of reovirus-infected mice, whereas all cell types expressed the antiviral protein Mx-1. Neither NK cells nor signaling via Toll-like receptor 3 or MyD88 were essential for viral clearance. These data demonstrate a requirement for type I IFNs in the control of an intestinal viral infection and indicate that cDCs are a significant source of type I IFN production in vivo. Therefore, innate immunity in PPs is an essential component of host defense that limits systemic spread of pathogens that infect the intestinal mucosa.     

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.     

Microbiota-induced IL-1β, but not IL-6, is critical for the development of steady-state TH17 cells in the intestine

T(H)17 cells are a lineage of CD4(+) T cells that are critical for host defense and autoimmunity by expressing the cytokines IL-17A, IL-17F, and IL-22. A feature of T(H)17 cells at steady state is their ubiquitous presence in the lamina propria of the small intestine. The induction of these steady-state intestinal T(H)17 (sT(H)17) cells is dependent on the presence of the microbiota. However, the signaling pathway linking the microbiota to the development of intestinal sT(H)17 cells remains unclear. In this study, we show that IL-1β, but not IL-6, is induced by the presence of the microbiota in intestinal macrophages and is required for the induction of sT(H)17 cells. In the absence of IL-1β-IL-1R or MyD88 signaling, there is a selective reduction in the frequency of intestinal sT(H)17 cells and impaired production of IL-17 and IL-22. Myeloid differentiation factor 88-deficient (MyD88(-/-)) and germ-free (GF) mice, but not IL-1R(-/-) mice, exhibit impairment in IL-1β induction. Microbiota-induced IL-1β acts directly on IL-1R-expressing T cells to drive the generation of sT(H)17 cells. Furthermore, administration of IL-1β into GF mice induces the development of retinoic acid receptor-related orphan receptor γt-expressing sT(H)17 cells in the small intestine, but not in the spleen. Thus, commensal-induced IL-1β production is a critical step for sT(H)17 differentiation in the intestine, which may have therapeutic implications for T(H)17-mediated pathologies.     

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.     

Host innate recognition of an intestinal bacterial pathogen induces TRIF-dependent protective immunity

Toll-like receptor 4 (TLR4), which signals through the adapter molecules myeloid differentiation factor 88 (MyD88) and toll/interleukin 1 receptor domain-containing adapter inducing IFN-β (TRIF), is required for protection against Gram-negative bacteria. TRIF is known to be important in TLR3-mediated antiviral signaling, but the role of TRIF signaling against Gram-negative enteropathogens is currently unknown. We show that TRIF signaling is indispensable for establishing innate protective immunity against Gram-negative Yersinia enterocolitica. Infection of wild-type mice rapidly induced both IFN-β and IFN-γ in the mesenteric lymph nodes. In contrast, TRIF-deficient mice were defective in these IFN responses and showed impaired phagocytosis in regional macrophages, resulting in greater bacterial dissemination and mortality. TRIF signaling may be universally important for protection against Gram-negative pathogens, as TRIF-deficient macrophages were also impaired in killing both Salmonella and Escherichia coli in vitro. The mechanism of TRIF-mediated protective immunity appears to be orchestrated by macrophage-induced IFN-β and NK cell production of IFN-γ. Sequential induction of IFN-β and IFN-γ leads to amplification of macrophage bactericidal activity sufficient to eliminate the invading pathogens at the intestinal interface. Our results demonstrate a previously unknown role of TRIF in host resistance to Gram-negative enteropathogens, which may lead to effective strategies for combating enteric infections.     

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.     

Impaired response to Listeria in H2-M3-deficient mice reveals a nonredundant role of MHC class Ib-specific T cells in host defense

The major histocompatibility complex (MHC) class Ib molecule H2-M3 primes the rapid expansion of CD8+ T cells by presenting N-formylated bacterial peptides. However, the significance of H2-M3-restricted T cells in host defense against bacteria is unclear. We generated H2-M3-deficient mice to investigate the role of H2-M3 in immunity against Listeria monocytogenes (LM), a model intracellular bacterial pathogen. H2-M3-deficient mice are impaired in early bacterial clearance during primary infection, with diminished LM-specific CD8+ T cell responses and compromised innate immune functions. Although H2-M3-restricted CD8+ T cells constitute a significant proportion of the anti-listerial CD8+ T cell repertoire, the kinetics and magnitude of MHC class Ia-restricted T cell responses are not altered in H2-M3-deficient mice. The fact that MHC class Ia-restricted responses cannot compensate for the H2-M3-mediated immunity suggests a nonredundant role of H2-M3 in the protective immunity against LM. Thus, the early H2-M3-restricted response temporally bridges the gap between innate and adaptive immune responses, subsequently affecting the function of both branches of the immune system.     

The effects of sodium ricinoleate on small intestinal function and structure.

The mechanism of hydroxy fatty acid-induced secretion was investigated in perfused hamster small intestine in vivo. Sodium ricinoleate at an 8-mM concentration resulted in not only secretion of water and sodium, but an increase in intestinal clearance of inulin and a 16,000 mol wt dextran as well. A concentration of ricinoleate (2 mM) which did not affect water transport, however, did not alter intestinal permeability. Ricinoleate-induced intestinal secretion was also accompanied by increased mucosal cell exfoliation as measured by the appearance of DNA in the perfusate and by apparent injury to epithelial cell membranes as judged by measurement of sucrase activity and phospholipid in cell-free aliquots of luminal fluid. Light and electron microscopic studies demonstrated substantial mucosal architectural changes with 8 mM ricinoleate with villus shortening and injury to epithelial cells at the villus tips. In contrast, cholera enterotoxin caused marked secretion of sodium and water, presumably by a cyclic AMP mechanism, but did not alter inulin clearance or enhance DNA or sucrase appearance in the lumen. These studies suggest that at least a component of ricinoleate-induced intestinal secretion is related to structural alterations of the mucosa.     

Anaerobic environment of the intestine primes pathogenic Shigella for infection

Marteyn et al. have investigated the role of oxygen and the regulator FNR in infection by the intracellular enteric pathogen Shigella flexneri. FNR is active under anaerobic conditions like those present in the lumen of the distal intestine. FNR causes elongation of a secretion apparatus required for bacterial entry into cells and represses secretion of proteins that trigger entry. Higher oxygen levels present at the intestinal cell surface are sufficient to inactivate FNR, thereby derepressing secretion. Thus, bacteria are 'primed' in the anaerobic environment of the lumen, and entry is triggered by the aerobic conditions at the intestinal cell surface. FNR is conserved among many enteric pathogens, suggesting that regulation of virulence in response to oxygen may be widely conserved.     

Studies on susceptibility to infection following ionizing radiation. IV. The pathogenesis of the endogenous bacteremias in mice

In half of the normal mice examined, cultures of mesenteric lymph nodes were positive for enteric bacteria. When a non-pathogenic microorganism, Serratia marcescens, was established in the intestinal tract by administering it to mice in their drinking water, it, too, was recovered from the mesenteric lymph nodes of almost half of the normal mice examined. From these findings it was concluded that bacteria in small numbers were able to pass from the lumen of the unirradiated gut as far as the regional lymph glands. Such bacteria, except the pathogen, Salmonella, were rarely found in liver or spleen, never in the blood of the normal mice. After x-irradiation with 700 r, the incidence of positive cultures showed the liver or spleen became infected with enteric microorganisms before the blood stream was invaded. It appears, therefore, that these elements of the reticulo-endothelial system were able for a time to maintain the sterility of the blood after failure of the more immediate defenses against bacterial invasion from the intestinal tract. It is concluded that if any increased migration of bacteria through the intestinal mucosa resulted from the radiation injury, the increase must have occurred very soon after irradiation.     

The role of intravenous administration of dextran 70 in enteric bacterial translocation after partial hepatectomy in rats

The aim of this study was to assess the effect of intravenous dextran on bacterial translocation and intestinal vascular endothelial and epithelial barrier function after experimental partial hepatectomy. We determined systemic arterial pressure, enteric bacterial growth (proximal and distal small intestine and colon) and bacterial translocation (BT) to mesenteric lymph nodes (MLN), liver, lungs, spleen, kidneys and blood, as well as intestinal vascular endothelial and epithelial barrier permeability, after sham operation or partial hepatectomy (50% and 90%) with preoperative intravenous administration of saline, albumin or dextran 70. Subtotal hepatectomy induced a significant decrease in arterial pressure and an increase in the number of Escherichia coli in the distal small intestine. BT was not observed in sham-operated animals or in rats with 50% hepatectomy administered dextran. The number of positive cultures of enteric bacteria was significantly increased after hepatectomy, whereas dextran treatment decreased the number of animals with BT. Increased permeability of the intestinal vascular endothelial and epithelial barriers was noted in hepatectomized animals, while dextran prevented hepatectomy-induced vascular endothelial barrier injury. Enteric bacterial translocation occurred following partial hepatectomy in the rat, associated with bacterial overgrowth in the distal small intestine. Intravenous administration of dextran 70 prevented bacterial overgrowth and translocation, at least in part, by maintaining gut vascular endothelial barrier integrity.     

MyD88-5 links mitochondria, microtubules, and JNK3 in neurons and regulates neuronal survival

The innate immune system relies on evolutionally conserved Toll-like receptors (TLRs) to recognize diverse microbial molecular structures. Most TLRs depend on a family of adaptor proteins termed MyD88s to transduce their signals. Critical roles of MyD88-1-4 in host defense were demonstrated by defective immune responses in knockout mice. In contrast, the sites of expression and functions of vertebrate MyD88-5 have remained elusive. We show that MyD88-5 is distinct from other MyD88s in that MyD88-5 is preferentially expressed in neurons, colocalizes in part with mitochondria and JNK3, and regulates neuronal death. We prepared MyD88-5/GFP transgenic mice via a bacterial artificial chromosome to preserve its endogenous expression pattern. MyD88-5/GFP was detected chiefly in the brain, where it associated with punctate structures within neurons and copurified in part with mitochondria. In vitro, MyD88-5 co-immunoprecipitated with JNK3 and recruited JNK3 from cytosol to mitochondria. Hippocampal neurons from MyD88-5-deficient mice were protected from death after deprivation of oxygen and glucose. In contrast, MyD88-5-null macrophages behaved like wild-type cells in their response to microbial products. Thus, MyD88-5 appears unique among MyD88s in functioning to mediate stress-induced neuronal toxicity.     

A protective role of gamma/delta T cells in primary infection with Listeria monocytogenes in mice

We have previously reported that T cells bearing T cell receptors (TCRs) of gamma/delta type appear at a relatively early stage of primary infection with Listeria monocytogenes in mice. To characterize the early-appearing gamma/delta T cells during listeriosis, we analyzed the specificity and cytokine production of the gamma/delta T cells in the peritoneal cavity in mice inoculated intraperitoneally with a sublethal dose of L. monocytogenes. The early-appearing gamma/delta T cells, most of which were of CD4-CD8- phenotype, proliferated and secreted IFN-gamma and macrophage chemotactic factor in response to purified protein derivative from Mycobacterium tuberculosis, or recombinant 65-kD heat-shock protein derived from M. bovis but not to heat-killed Listeria. To further elucidate the potential role of the gamma/delta T cells in the host-defense mechanism against primary infection with Listeria, we examined the effects of in vivo administration of monoclonal antibodies (mAbs) against TCR-gamma/delta or TCR-alpha/beta on the bacterial eradication in mice infected with Listeria. Most of alpha/beta T cells or gamma/delta T cells were depleted in the peripheral lymphoid organs at least for 12 d after an intraperitoneal injection of 200 micrograms TCR-alpha/beta mAb or 200 micrograms TCR-gamma/delta mAb, respectively. An exaggerated bacterial multiplication was evident at the early stage of listerial infection in the gamma/delta T cells-depleted mice, whereas the alpha/beta T cell-depleted mice exhibited much the same resistance level as the control mice at this stage although the resistance was severely impaired at the late stage after listerial infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Memory CD8+ T cells mediate antibacterial immunity via CCL3 activation of TNF/ROI+ phagocytes

Cytolysis, interferon gamma and tumor necrosis factor (TNF) alpha secretion are major effector mechanisms of memory CD8+ T cells that are believed to be required for immunological protection in vivo. By using mutants of the intracellular bacterium Listeria monocytogenes, we found that none of these effector activities is sufficient to protect against secondary infection with wild-type (WT) bacteria. We demonstrated that CCL3 derived from reactivated memory CD8+ T cells is required for efficient killing of WT bacteria. CCL3 induces a rapid TNF-alpha secretion by innate inflammatory mononuclear phagocytic cells (MPCs), which further promotes the production of radical oxygen intermediates (ROIs) by both MPCs and neutrophils. ROI generation is the final bactericidal mechanism involved in L. monocytogenes clearance. These results therefore uncover two levels of regulation of the antibacterial secondary protective response: (a) an antigen-dependent phase in which memory CD8+ T cells are reactivated and control the activation of the innate immune system, and (b) an antigen-independent phase in which the MPCs coordinate innate immunity and promote the bactericidal effector activities. In this context, CCL3-secreting memory CD8+ T cells are able to mediate "bystander" killing of an unrelated pathogen upon antigen-specific reactivation, a mechanism that may be important for the design of therapeutic vaccines.     

Augmented nonspecific resistance and simultaneous impairment of specific immunity to Listeria monocytogenes in tumor-bearing mice

Meth A tumor-bearing mice were examined for changes in the host defense mechanism against infection with Listeria monocytogenes. The resistance of tumor-bearing mice to systemic, i.e., intravenous, infection in an early phase of the infection was suppressed for 1-3 days after tumor implantation (5 x 10(5) cells/mouse, subcutaneously), but was augmented thereafter even on the 35th day as compared with normal mice. Suppression and enhancement of the resistance of tumor-bearing mice to primary infection with L. monocytogenes was also observed in tumor-bearing athymic nude mice. Splenic macrophages from tumor-bearing mice on the 14th day after tumor implantation exerted potent bactericidal activity against L. monocytogenes in vitro as compared with those from normal mice. The function of macrophages as nonimmune scavenger cells seemed to be activated in mice bearing a progressing tumor. However, some of the tumor-bearing mice challenged with a sublethal dose of L. monocytogenes showed diminished resistance in the late phase of the infection; moreover listeria-immunized tumor-bearing mice showed less resistance to a secondary challenge with the bacteria than did normal immunized mice. This suppression of the specific immune response of tumor-bearing mice to L. monocytogenes was shown also in the assay of the delayed-type footpad reaction. The bacterial growth-inhibiting function of listeria-immune T lymphocytes, determined by the effect of adoptive transfer of the cells on the growth of Listeria, was also reduced in tumor-bearing mice as compared with normal mice.     

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.     

Mice lacking the type I interferon receptor are resistant to Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen that induces a cytosolic signaling cascade resulting in expression of interferon (IFN)-beta. Although type I IFNs are critical in viral defense, their role in immunity to bacterial pathogens is much less clear. In this study, we addressed the role of type I IFNs by examining the infection of L. monocytogenes in BALB/c mice lacking the type I IFN receptor (IFN-alpha/betaR-/-). During the first 24 h of infection in vivo, IFN-alpha/betaR-/- and wild-type mice were similar in terms of L. monocytogenes survival. In addition, the intracellular fate of L. monocytogenes in macrophages cultured from IFN-alpha/betaR-/- and wild-type mice was indistinguishable. However, by 72 h after inoculation in vivo, IFN-alpha/betaR-/- mice were approximately 1,000-fold more resistant to a high dose L. monocytogenes infection. Resistance was correlated with elevated levels of interleukin 12p70 in the blood and increased numbers of CD11b+ macrophages producing tumor necrosis factor alpha in the spleen of IFN-alpha/betaR-/- mice. The results of this study suggest that L. monocytogenes might be exploiting an innate antiviral response to promote its pathogenesis.     

Bactericidal properties of murine intestinal phospholipase A2.

We purified a molecule from the murine small intestine that killed both Escherichia coli and Listeria monocytogenes, and identified it as intestinal phospholipase A2 (iPLA2) by NH2-terminal sequencing and enzymatic measurements. The ability of iPLA2 to kill. L. monocytogenes was greatly enhanced by 5 mM calcium, inhibited by EGTA and abolished after reduction and alkylation, suggesting that enzymatic activity was required for iPLA2-mediated bactericidal activity. A mouse-avirulent phoP mutant, S. typhimurium 7953S, was 3.5-fold more susceptible to iPLA2 than its isogenic virulent parent, S. typhimurium 14028S (estimated minimal bactericidal concentrations 12.7 +/- 0.5 micrograms/ml vs. 43.9 +/- 4.5 micrograms/ml P < 0.001). Overall, these findings identify iPLA2 as part of the antimicrobial arsenal that equips Paneth cells to protect the small intestinal crypts from microbial invasion. Because iPLA2 is identical to Type 2 phospholipase A2 molecules found in other sites, including spleen, platelets and inflammatory exudate cells, this enzyme may also contribute to antibacterial defenses elsewhere in the body.     

Type I interferon production enhances susceptibility to Listeria monocytogenes infection

Numerous bacterial products such as lipopolysaccharide potently induce type I interferons (IFNs); however, the contribution of this innate response to host defense against bacterial infection remains unclear. Although mice deficient in either IFN regulatory factor (IRF)3 or the type I IFN receptor (IFNAR)1 are highly susceptible to viral infection, we show that these mice exhibit a profound resistance to infection caused by the Gram-positive intracellular bacterium Listeria monocytogenes compared with wild-type controls. Furthermore, this enhanced bacterial clearance is accompanied by a block in L. monocytogenes-induced splenic apoptosis in IRF3- and IFNAR1-deficient mice. Thus, our results highlight the disparate roles of type I IFNs during bacterial versus viral infections and stress the importance of proper IFN modulation in host defense.     

IL-1R-MyD88 signaling in keratinocyte transformation and carcinogenesis

Constitutively active RAS plays a central role in the development of human cancer and is sufficient to induce tumors in two-stage skin carcinogenesis. RAS-mediated tumor formation is commonly associated with up-regulation of cytokines and chemokines that mediate an inflammatory response considered relevant to oncogenesis. In this study, we report that mice lacking IL-1R or MyD88 are less sensitive to topical skin carcinogenesis than their respective wild-type (WT) controls. MyD88(-/-) or IL-1R(-/-) keratinocytes expressing oncogenic RAS are hyperproliferative and fail to up-regulate proinflammatory genes or down-regulate differentiation markers characteristic of RAS-expressing WT keratinocytes. Although RAS-expressing MyD88(-/-) keratinocytes form only a few small tumors in orthotopic grafts, IL-1R-deficient RAS-expressing keratinocytes retain the ability to form tumors in orthotopic grafts. Using both genetic and pharmacological approaches, we find that the differentiation and proinflammatory effects of oncogenic RAS in keratinocytes require the establishment of an autocrine loop through IL-1α, IL-1R, and MyD88 leading to phosphorylation of IκBα and NF-κB activation. Blocking IL-1α-mediated NF-κB activation in RAS-expressing WT keratinocytes reverses the differentiation defect and inhibits proinflammatory gene expression. Collectively, these results demonstrate that MyD88 exerts a cell-intrinsic function in RAS-mediated transformation of keratinocytes.