Myeloid Differentiation Factor 88 Knockout Mice Research Articles

MyD88-TLR4-dependent choroid plexus activation precedes perilesional inflammation and secondary brain edema in a mouse model of intracerebral hemorrhage.

The functional neurological outcome of patients with intracerebral hemorrhage (ICH) strongly relates to the degree of secondary brain injury (ICH-SBI) evolving within days after the initial bleeding. Different mechanisms including the incitement of inflammatory pathways, dysfunction of the blood-brain barrier (BBB), activation of resident microglia, and an influx of blood-borne immune cells, have been hypothesized to contribute to ICH-SBI. Yet, the spatiotemporal interplay of specific inflammatory processes within different brain compartments has not been sufficiently characterized, limiting potential therapeutic interventions to prevent and treat ICH-SBI. We used a whole-blood injection model in mice, to systematically characterized the spatial and temporal dynamics of inflammatory processes after ICH using 7-Tesla magnetic resonance imaging (MRI), spatial RNA sequencing (spRNAseq), functional BBB assessment, and immunofluorescence average-intensity-mapping. We identified a pronounced early response of the choroid plexus (CP) peaking at 12-24h that was characterized by inflammatory cytokine expression, epithelial and endothelial expression of leukocyte adhesion molecules, and the accumulation of leukocytes. In contrast, we observed a delayed secondary reaction pattern at the injection site (striatum) peaking at 96h, defined by gene expression corresponding to perilesional leukocyte infiltration and correlating to the delayed signal alteration seen on MRI. Pathway analysis revealed a dependence of the early inflammatory reaction in the CP on toll-like receptor 4 (TLR4) signaling via myeloid differentiation factor 88 (MyD88). TLR4 and MyD88 knockout mice corroborated this observation, lacking the early upregulation of adhesion molecules and leukocyte infiltration within the CP 24h after whole-blood injection. We report a biphasic brain reaction pattern after ICH with a MyD88-TLR4-dependent early inflammatory response of the CP, preceding inflammation, edema and leukocyte infiltration at the lesion site. Pharmacological targeting of the early CP activation might harbor the potential to modulate the development of ICH-SBI.

MyD88 regulates a prolonged adaptation response to environmental dust exposure-induced lung disease

BackgroundEnvironmental organic dust exposures enriched in Toll-like receptor (TLR) agonists can reduce allergic asthma development but are associated with occupational asthma and chronic bronchitis. The TLR adaptor protein myeloid differentiation factor88 (MyD88) is fundamental in regulating acute inflammatory responses to organic dust extract (ODE), yet its role in repetitive exposures is unknown and could inform future strategies.MethodsWild-type (WT) and MyD88 knockout (KO) mice were exposed intranasally to ODE or saline daily for 3 weeks (repetitive exposure). Repetitively exposed animals were also subsequently rested with no treatments for 4 weeks followed by single rechallenge with saline/ODE.ResultsRepetitive ODE exposure induced neutrophil influx and release of pro-inflammatory cytokines and chemokines were profoundly reduced in MyD88 KO mice. In comparison, ODE-induced cellular aggregates, B cells, mast cell infiltrates and serum IgE levels remained elevated in KO mice and mucous cell metaplasia was increased. Expression of ODE-induced tight junction protein(s) was also MyD88-dependent. Following recovery and then rechallenge with ODE, inflammatory mediators, but not neutrophil influx, was reduced in WT mice pretreated with ODE coincident with increased expression of IL-33 and IL-10, suggesting an adaptation response. Repetitively exposed MyD88 KO mice lacked inflammatory responsiveness upon ODE rechallenge.ConclusionsMyD88 is essential in mediating the classic airway inflammatory response to repetitive ODE, but targeting MyD88 does not reduce mucous cell metaplasia, lymphocyte influx, or IgE responsiveness. TLR-enriched dust exposures induce a prolonged adaptation response that is largely MyD88-independent. These findings demonstrate the complex role of MyD88-dependent signaling during acute vs. chronic organic dust exposures.

MyD88 controls airway epithelial Muc5ac expression during TLR activation conditions from agricultural organic dust exposure.

Inflammation from airborne microbes can overwhelm compensatory mucociliary clearance mechanisms, leading to mucous cell metaplasia. Toll-like receptor (TLR) activation via myeloid differentiation factor 88 (MyD88) signaling is central to pathogen responses. We have previously shown that agricultural organic dust extract (ODE), with abundant microbial component diversity, activates TLR-induced airway inflammation. With the use of an established model, C57BL/6J wild-type (WT) and global MyD88 knockout (KO) mice were treated with intranasal inhalation of ODE or saline, daily for 1 wk. ODE primarily increased mucin (Muc)5ac levels relative to Muc5b. Compared with ODE-challenged WT mice, ODE-challenged, MyD88-deficient mice demonstrated significantly increased Muc5ac immunostaining, protein levels by immunoblot, and expression by quantitative PCR. The enhanced Muc5ac levels in MyD88-deficient mice were not explained by differences in the differentiation program of airway secretory cells in naïve mice. Increased Muc5ac levels in MyD88-deficient mice were also not explained by augmented inflammation, IL-17A, or neutrophil elastase levels. Furthermore, the enhanced airway mucins in the MyD88-deficient mice were not due to defective secretion, as the mucin secretory capacity of MyD88-KO mice remained intact. Finally, ODE-induced Muc5ac levels were enhanced in MyD88-deficient airway epithelial cells in vitro. In conclusion, MyD88 deficiency enhances airway mucous cell metaplasia under environments with high TLR activation.

Brain-derived neurotrophic factor is regulated via MyD88/NF-\u03baB signaling in experimental Streptococcus pneumoniae meningitis

Streptococcus pneumoniae meningitis is an intractable disease of the central nervous system (CNS). Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophic family and found to participate in the immune inflammatory response. In this study, we investigated if activation of the classical inflammatory signaling pathway, myeloid differentiation factor 88 (MyD88)/nuclear factor-kappa B (NF-κB), regulates BDNF expression in experimental S. pneumoniae meningitis. MyD88 knockout (myd88−/−) mice and wild-type littermates were infected intracisternally with S. pneumoniae suspension. Twenty-four hours after inoculation, histopathology of brains was evaluated. Cytokine and chemokine in brains and spleens was analyzed using ELISA. NF-κB activation was evaluated using EMSA. Cortical and hippocampal BDNF was assessed using RT-PCR and ELISA, respectively. BDNF promoter activity was evaluated using ChIP-PCR. myd88−/− mice showed an obviously weakened inflammatory host response. This diminished inflammation was consistent with worse clinical parameters, neuron injury, and apoptosis. Deficiency in MyD88 was associated with decreased BDNF expression. Furthermore, we identified a valid κB-binding site in the BDNF promoter, consistent with activation of NF-κB induced by inflammation. To sum up, MyD88/NF-κB signaling has a crucial role in up-regulating BDNF, which might provide potential therapeutic targets for S. pneumoniae meningitis.

Critical role of myeloid differentiation factor 88 in necrotizing enterocolitis

The importance of toll-like receptor 4 in necrotizing enterocolitis (NEC) has been intensively studied, but its downstream signaling and the potential regulatory mechanisms remain unidentified. Our study focused on the role of myeloid differentiation factor 88 (MyD88), the first downstream adaptor of toll-like receptor 4 inflammatory and apoptotic signaling in the pathogenesis of NEC. MyD88 knockout (MyD88(-/-)-Ko) mice and lentivirus-mediated stable MyD88-knockdown cell line (IEC-6) were used. NEC was induced by formula gavage, cold, hypoxia, combined with lipopolysaccharide (LPS) in vivo, or LPS stimulation in vitro. NEC was evaluated by histology and multiple inflammatory cytokines. Enterocyte apoptosis was evaluated by terminal-deoxynucleoitidyl transferase-mediated nick end labeling (TUNEL) or Annexin analysis. Inflammatory or apoptotic molecules including NF-κB, Toll/IL-1R domain-containing adaptor-inducing IFN-β, interferon regulatory factor 3, Bax, Bcl-2, and caspases were examined by quantitative real-time PCR (qRT-PCR). In the MyD88-Ko group, NEC severity and intestinal enterocyte apoptosis rate were reduced, the expression of NF-κB, caspases, and Bax, were all downregulated, while Toll/IL-1R domain-containing adaptor-inducing IFN-β and were upregulated, and antiapoptotic gene Bcl-2 remained stable. Cytokine levels of interleukin (IL)-6, IL-1β, and tumor necrosis factor-α (TNF-α) were also all decreased. MyD88-dependent signaling is the prevailing inflammatory and apoptotic signaling in toll-like receptor 4 downstream signaling; MyD88-Ko resulted in reduced inflammatory severity and apoptosis, though MyD88-independent signaling can also be activated, but is of less dominant for the development of NEC.

Evidence for a Gender-Specific Protective Role of Innate Immune Receptors in a Model of Perinatal Brain Injury

Hypoxia-ischemia is a common cause of neurological impairments in newborns, but little is known about how neuroinflammation contributes to the long-term outcome after a perinatal brain injury. In this study, we investigated the role of the fractalkine receptor chemokine CX3C motif receptor 1 (CX3CR1) and of toll-like receptor (TLR) signaling after a neonatal hypoxic-ischemic brain injury. Mice deficient in the TLR adaptor proteins Toll/interleukin-1 receptor-domain-containing adaptor protein inducing interferon β (TRIF) or myeloid differentiation factor-88 (MyD88) and CX3CR1 knock-out (KO) mice were subjected to hypoxia-ischemia at postnatal day 3. In situ hybridization was used to evaluate the expression of TLRs during brain development and after hypoxic-ischemic insults. Behavioral deficits, hippocampal damage, reactive microgliosis, and subplate injury were compared among the groups. Although MyD88 KO mice exhibited no differences from wild-type animals in long-term structural and functional outcomes, TRIF KO mice presented a worse outcome, as evidenced by increased hippocampal CA3 atrophy in males and by the development of learning and motor deficits in females. CX3CR1-deficient female mice showed a marked increase in brain damage and long-lasting learning deficits, whereas CX3CR1 KO male animals did not exhibit more brain injury than wild-type mice. These data reveal a novel, gender-specific protective role of TRIF and CX3CR1 signaling in a mouse model of neonatal hypoxic-ischemic brain injury. These findings suggest that future studies seeking immunomodulatory therapies for preterm infants should consider gender as a critical variable and should be cautious not to abrogate the protective role of neuroinflammation.

Dendritic Cells Play an Essential Role in Transplantation Responses via Myeloid Differentiation Factor 88 Signaling

BackgroundDCs (dendritic cells), play an important role in allo-recognition, but there is no direct evidence that DCs trigger and control allo-responses. In this study, we investigated the role of DCs by using a self-created MyD88 (myeloid differentiation factor 88) inhibitor and a minor antigen-mismatched (HY-mismatched) skin transplantation model in mice. MethodsAn HY-mismatched skin allograft model (BALB/c ♂ → BALB/c ♀) using MyD88-knockout mice was employed in this study. WT (Wild-type) BALB/c immature BMDCs (bone marrow dendritic cells) were co-cultured with or without TJ-M2010 in vitro for 12 hours before transfusion of DCs (2× 107/mouse) into recipients of skin transplantations. Wild-type BALB/c BMDCs were stimulated by the TLR9 (Toll-like receptor 9) agonist, CpG (cytidine-phosphate-guanosine), in the presence of TJ-M2010 or not for 12 hours in vitro. Costimulatory molecules CD80/CD86 were analyzed by flow cytometry. WT C57BL/6 naïve T cells stained with CFSE (carboxyfluorescein diacetate succinimidyl ester) were co-cultured with WT BALB/c DCs for 3 days in the presence or absence of TJ-M2010. CD3+/CFSE+ cells were analyzed by flow cytometry. ResultsTJ-M2010 inhibited DC maturation and T cell proliferation. Permanent survival of the skin allografts was observed in MyD88 knockout (MyD88- KO) mice. However, skin allografts were rejected by MyD88-KO mice infused with wild-type (WT) DCs. More interestingly, after treating the WT DCs with TJ-M2010, the DC infusion could not reverse the tolerance to skin allografts. ConclusionThis study provided evidence that DCs play an essential role in alloresponses.

Myeloid Differentiation Factor 88–Dependent Signaling Is Critical for Acute Organic Dust–Induced Airway Inflammation in Mice

Organic dust exposure within agricultural environments results in airway diseases. Toll-like receptor 2 (TLR2) and TLR4 only partly account for the innate response to these complex dust exposures. To determine the central pathway in mediating complex organic dust-induced airway inflammation, this study targeted the common adaptor protein, myeloid differentiation factor 88 (MyD88), and investigated the relative contributions of receptors upstream from this adaptor. Wild-type, MyD88, TLR9, TLR4, IL-1 receptor I (RI), and IL-18R knockout (KO) mice were challenged intranasally with organic dust extract (ODE) or saline, according to an established protocol. Airway hyperresponsiveness (AHR) was assessed by invasive pulmonary measurements. Bronchoalveolar lavage fluid was collected to quantitate leukocyte influx and cytokine/chemokine (TNF-α, IL-6, chemokine [C-X-C motif] ligands [CXCL1 and CXCL2]) concentrations. Lung tissue was collected for histopathology. Lung cell apoptosis was determined by a terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and lymphocyte influx and intercellular adhesion molecule-1 (ICAM-1) expression were assessed by immunohistochemistry. ODE-induced AHR was significantly attenuated in MyD88 KO mice, and neutrophil influx and cytokine/chemokine production were nearly absent in MyD88 KO animals after ODE challenges. Despite a near-absent airspace inflammatory response, lung parenchymal inflammation was increased in MyD88 KO mice after repeated ODE exposures. ODE-induced epithelial-cell ICAM-1 expression was diminished in MyD88 KO mice. No difference was evident in the small degree of ODE-induced lung-cell apoptosis. Mice deficient in TLR9, TLR4, and IL-18R, but not IL-1IR, demonstrated partial protection against ODE-induced neutrophil influx and cytokine/chemokine production. Collectively, the acute organic dust-induced airway inflammatory response is highly dependent on MyD88 signaling, and is dictated, in part, by important contributions from upstream TLRs and IL-18R.

The crucial role of the MyD88 adaptor protein in the inflammatory response induced by Bothrops atrox venom

Most snake accidents in North Brazil are attributed to Bothrops atrox, a snake species of the Viperidae family whose venom simultaneously induces local and systemic effects in the victims. The former are clinically more important than the latter, as they cause severe tissue lesions associated with strong inflammatory responses. Although several studies have shown that inflammatory mediators are produced in response to B. atrox venom (BaV), there is little information concerning the molecular pathways involved in innate immune system signaling. Myeloid differentiation factor 88 (MyD88) is an adaptor molecule responsible for transmitting intracellular signals from most toll-like receptors (TLRs) after they interact with pathogen-associated molecular patterns (PAMPs) or other stimuli such as endogenous damage-associated molecular patterns (DAMPs). The MyD88-dependent pathway leads to activation of transcription factors, which in turn induce synthesis of inflammatory mediators such as eicosanoids, cytokines and chemokines. The aim of this study was to investigate the involvement of MyD88 on the acute inflammatory response induced by BaV. Wild-type (WT) C57BL/6 mice and MyD88 knockout (MyD88−/−) mice were intraperitoneally injected with BaV. Compared to WT mice, MyD88−/− animals showed an impaired inflammatory response to BaV, with lower influx of polymorphonuclear and mononuclear cells to the peritoneal cavity. Furthermore, peritoneal leukocytes from BaV-injected MyD88−/− mice did not induce COX-2 or LTB4 protein expression and released low concentrations of PGE2. These mice also failed to produce Th1 and Th17 cytokines and CCL-2, but IL-10 levels were similar to those of BaV-injected WT mice. Our results indicate that MyD88 signaling is required for activation of the inflammatory response elicited by BaV, raising the possibility of developing new therapeutic targets to treat Bothrops sp. poisoning.

MyD88 is required for the recruitment of eosinophils and neutrophils but dispensable for host protective immune responses during Fasciola hepatica infection

Adaptive immunity detects non-self through the recognition of peptide antigens using receptors expressed on the surface of B and T cells, however that how innate immune system recognizes non-self has been remained unclear. Myeloid differentiation factor 88 (MyD88) is a critical adaptor molecule shared by toll-like receptors (TLRs), and has been found crucial on control of Th1 immunity. However, whether a Th2 response against helminth infection requires a signaling through MyD88 has been uncertain. We hypothesized Th2 immune responses induced by helminth require a MyD88-dependent signaling. To test it, a MyD88 knockout mouse model infected with Fasciola hepatica metacercariae was used and unexpected results were exhibited with dispensable role of MyD88 on host protective immune responses marked as increased IgG1, IgE, IL4, IL5, IL9 and IL13 and decreased IgG2a and IFNγ, which could be induced even with intact MyD88 molecule during F. hepatica infection. No differences were found on IL17 and TGFβ production in both infected WT and MyD88 knockout mice. The mRNA levels of transcript factors for Th1 (T-bet), Th2 (GATA3), Th17 (RORγt) and Tregs (FoxP3) were also measured by real-time PCR, showing consistent trends to the productions of Th1 (IFNγ), Th2 (IL4, IL5 and IL13) and IL9 cytokines, and no differences were found on RORγt and FoxP3 in both infected WT and MyD88 knockout mice. However, the numbers and proportions of eosinophils and neutrophils recruited in peritoneal cavity were significantly decreased in mice defecting MyD88 although no differences were found on macrophages, lymphocytes and CD4 positive cells, indicating signaling through MyD88 might be involved in recruitment of immune effector cells during helminth infection. Additionally, no differences were found on the adult worm burden and liver damage in both infected WT and MyD88 knockout mice. Taking together, our experiments showed MyD88 is required for the recruitment of eosinophils and neutrophils but dispensable for host protective immune responses during F. hepatica infection.

Mechanisms of Toll-Like Receptor 4 (TLR4)-Mediated Inflammation After Cold Ischemia/Reperfusion in the Heart

Toll-Like Receptor 4 (TLR4) signaling mediates early inflammation after cold ischemia-reperfusion (I/R). We hypothesized that the TLR4 coreceptor CD14, the intracellular adaptor proteins myeloid differentiation factor 88 (MyD88) and TIR domain-containing-adaptor inducing IFNbeta (TRIF) would be required for cold I/R induced inflammation. High mobility group box 1 (HMGB1) is a putative endogenous activator of TLR4. Therefore, we also assessed the contribution of HMGB1 in cold I/R induced inflammation. Syngeneic heart transplants were performed in mice deficient in CD14, MyD88, TRIF, or wild-type mice. In other experiments, anti-HMGB1 neutralizing antibody or control IgG was administered at reperfusion. Donor hearts were subjected to 2 hr of cold ischemia and retrieved after 3 hr of reperfusion. After cold I/R, grafts revealed striking translocation of HMGB1 out of the nucleus in cardiac myocytes. Administration of an anti-HMGB1 neutralizing antibody resulted in reduced systemic interleukin (IL)-6, tumor necrosis factor alpha (TNFalpha), and intercellular adhesion molecule-1 (ICAM-1) messenger RNA (mRNA) levels (P< or =0.05). Compared with controls, CD14 knock-out (KO) mice exhibited significantly lower (P< or =0.05) systemic IL-6 and JE/monocyte chemotractant protein-1 levels after cold I/R. Intragraft TNFalpha and IL-1beta mRNA levels were also significantly lower (P< or =0.05) in CD14 KO grafts. MyD88 KO mice exhibited significantly lower (P< or=0.05) systemic IL-6 levels compared with control mice after cold I/R. Intragraft TNFalpha, IL-6, and ICAM-1 mRNA levels were also significantly lower (P< or =0.05) in MyD88 KO grafts. Significantly lower levels (P< or =0.05) of serum IL-6, monocyte chemotractant protein-1 as well as intragraft TNFalpha, IL-6, IL-1beta, and ICAM-1 were observed after cold I/R in TRIF deficient animals compared with controls. CD14, MyD88, TRIF, and HMGB1 contribute to the inflammatory response that occurs after cold I/R. These results provide insight into the mechanisms of TLR4-mediated inflammation after cold I/R.

Neuroprotective effects of resident microglia following acute brain injury

Microglia quickly react to various neurodegenerative processes by producing cytokines and eliminating cellular debris via phagocytosis. These events are also associated with an increased proliferation of microglia, which derive from resident progenitors and those present in the bone marrow. However, it is not clear whether the innate immune response by resident or newly differentiated microglia is beneficial or detrimental to the central nervous system. The aim of this study was to determine the impact of an altered immune response following acute excitotoxicity. Sodium nitroprusside (SNP) or kainic acid (KA) was administered in the brain of various groups of mice, and the extent of neurodegeneration, myelin damage, and inflammation was evaluated within a period of 2 weeks. We used synthetic glucocorticoid (GC), myeloid differentiation factor 88 (MyD88)-deficient mice to suppress nuclear factor kappaB (NF-kappaB) signaling and transgenic mice that express the thymidine kinase (TK) protein under the control of the CD11b promoter to determine the role of proliferating and infiltrating microglia in acute models of brain injury. Neurodegeneration was more extensive in GC-treated and MyD88-deficient mice, suggesting that NF-kappaB signaling and microglia activation are potent neuroprotective mechanisms in the presence of SNP. KA was also highly toxic to neurons of the amygdala in MyD88 knockout mice but not in their WT littermates. Although bone marrow-derived cells are clearly attracted to neurodegenerative areas, preventing their infiltration and differentiation did not affect the extent of SNP-related damage. These data indicate that MyD88/NF-kappaB signaling in resident non-proliferating microglia plays a critical role by restricting damage during acute excitotoxicity.

Acute Hypoxia Activates the Neuroimmune System, Which Diabetes Exacerbates

Acute hypoxia is experienced in an array of ailments and conditions, including asthma, chronic obstructive pulmonary disease, heart failure, sleep apnea, acute hypotension, and blast lung injury. Classically, infection activates the neuroimmune system, causing loss of interest in the social environment. We report that the non-infectious stimulus acute hypoxia triggers neuroimmune system activation (NSA), causing loss of interest in the social environment, and that recovery from hypoxia-induced NSA is impaired in a mouse model of type 2 diabetes. Importantly, recovery from the behavioral consequences of hypoxia-induced NSA was nearly ablated in MyD88 (myeloid differentiation factor 88) knock-out mice and in mice intracerebroventricularly administered the caspase-1 inhibitor ac-YVAD-CMK (ac-Tyr-Val-Asp-2,6-dimethylbenzoyloxymethylketone). Diabetic mice had prolonged recovery from NSA that could be halved by administration of subcutaneous interleukin-1 (IL-1) receptor antagonist (RA). These results show that acute hypoxia activates the IL-1beta arm of the neuroimmune system, which diabetes exacerbates and treatment with IL-1RA ameliorates.

Sepsis-induced organ failure is mediated by different pathways in the kidney and liver: Acute renal failure is dependent on MyD88 but not renal cell apoptosis

Toll-like receptors (TLRs) are important in sepsis. Myeloid differentiation factor 88 (MyD88) is a key molecule involved in signal transduction by multiple TLRs. The objective of this study was to investigate the contribution of TLR4 and MyD88 to acute renal failure (ARF) induced by polymicrobial sepsis. Liver dysfunction and apoptosis in the spleen contribute to sepsis severity after cecal ligation and puncture (CLP). Therefore, we also investigated liver injury and splenic apoptosis. We used a mouse model of sepsis-induced ARF using CLP to generate polymicrobial sepsis. Despite fluid and antibiotic resuscitation the mice developed multi-organ failure, including ARF, which resembles human sepsis. We investigated the role of the TLR4 receptor by comparing C3H/HeJ mice (which lack TLR4) with C3H/He0UJ normal controls. The role of MyD88 was investigated by comparing MyD88 knockout mice (MyD88(-/-)) with wild-type controls. Following CLP, mice lacking TLR4 and wild-type mice both developed comparable ARF. However, MyD88(-/-) mice did not develop ARF compared to wild-type controls. In contrast, MyD88(-/-) mice developed liver injury comparable to wild type. After CLP, MyD88(-/-) mice had significantly reduced apoptosis in the spleen compared with wild type. Apoptosis was not detected in the kidney of wild-type or MyD88(-/-) mice after CLP. In summary, ARF induced by polymicrobial sepsis is dependent on MyD88, but not TLR4. The absence of MyD88 dissociates ARF from liver injury; liver injury is MyD88-independent. There was MyD88-dependent apoptosis in the spleen, but no apoptosis in the kidney. MyD88 may be a good drug target for some, but not all, organ dysfunctions following sepsis.

Myeloid differentiation factor-88 (MyD88) is essential for control of primary in vivo Francisella tularensis LVS infection, but not for control of intramacrophage bacterial replication

The means by which Francisella tularensis, the causative agent of tularemia, are recognized by mammalian immune systems are poorly understood. Here we wished to explore the contribution of the MyD88/Toll-like receptor signaling pathway in initiating murine responses to F. tularensis Live Vaccine Strain (LVS). MyD88 knockout (KO) mice, but not TLR2-, TLR4- or TLR9-deficient mice, rapidly succumbed following in vivo bacterial infection via the intradermal route even with a very low dose of LVS (5 × 10 1) that was 100,000-fold less than the LD 50 of normal wild-type (WT) mice. By day 5 after LVS infection, bacterial organ burdens were 5–6 logs higher in MyD88 knockout mice; further, unlike infected WT mice, levels of interferon-γ in the sera of LVS-infected MyD88 KO were undetectable. An in vitro culture system was used to assess the ability of bone marrow macrophages derived from either KO or WT mice to support bacterial growth, or to control intracellular bacterial replication when co-cultured with immune lymphocytes. In this assay, bacterial replication was similar in macrophages derived from either WT or any of the TLR KO mice. Bacterial growth was controlled in co-cultures containing macrophages from MyD88 KO mice or TLR KO mice as well as in co-cultures containing immune WT splenic lymphocytes and WT macrophages. Further, MyD88-deficient LVS-immune splenocytes controlled intracellular growth comparably to those from normal mice. Thus MyD88 is essential for innate host resistance to LVS infection, but is not required for macrophage control of intracellular bacterial growth.

Toll-like receptor 2 pathway drives streptococcal cell wall-induced joint inflammation: critical role of myeloid differentiation factor 88.

The IL-1R/Toll-like receptor (TLR) superfamily of receptors has a key role in innate immunity and inflammation. In this study, we report that streptococcal cell wall (SCW)-induced joint inflammation is predominantly dependent on TLR-2 signaling, since TLR-2-deficient mice were unable to develop either joint swelling or inhibition of cartilage matrix synthesis. Myeloid differentiation factor 88 (MyD88) is a Toll/IL-1R domain containing adaptor molecule known to have a central role in both IL-1R/IL-18R and TLR signaling. Mice deficient for MyD88 did not develop SCW-induced arthritis; both joint swelling and disturbance of cartilage chondrocyte anabolic function was completely abolished. Local levels of proinflammatory cytokines and chemokines in synovial tissue washouts were strongly reduced in MyD88-deficient mice. Histology confirmed the pivotal role of MyD88 in acute joint inflammation. TLR-2-deficient mice still allow influx of inflammatory cells into the joint cavity, although the number of cells was markedly reduced. No influx of inflammatory cells was seen in joints of MyD88-deficient mice. In addition, cartilage matrix proteoglycan loss was completely absent in MyD88 knockout mice. These findings clearly demonstrated that MyD88 is a key component in SCW-induced joint inflammation. Since agonists of the Toll-like pathway are abundantly involved in both septic and rheumatoid arthritis, targeting of MyD88 may be a novel therapy in inflammatory joint diseases.