Critical roles of myeloid differentiation factor 88-dependent proinflammatory cytokine release in early phase clearance of Listeria monocytogenes in mice

Listeria monocytogenes (LM), a facultative intracellular Gram-positive bacterium, often causes lethal infection of the host. In this study we investigated the molecular mechanism underlying LM eradication in the early phase of infection. Upon infection with LM, both IL-12 and IL-18 were produced, and then they synergistically induced IFN-gamma production, leading to normal LM clearance in the host. IFN-gamma knockout (KO) mice were highly susceptible to LM infection. IL-12/IL-18 double knockout mice were also highly susceptible. Their susceptibility was less than that of IFN-gamma KO mice, but more than that of single IL-12 or IL-18 KO mice. Mice deficient in myeloid differentiation factor 88 (MyD88), an essential adaptor molecule used by signal transduction pathways of all members of the Toll-like receptor (TLR) family, showed an inability to produce IL-12 and IFN-gamma following LM infection and were most susceptible to LM. Furthermore, MyD88-deficient, but not IFN-gamma-deficient, Kupffer cells could not produce TNF-alpha in response to LM in vitro, indicating the importance of MyD88-dependent TNF-alpha production for host defense. As TLR2 KO, but not TLR4 KO, mice showed partial impairment in their capacity to produce IL-12, IFN-gamma, and TNF-alpha, TLR2 activation partly contributed to the induction of IL-12-mediated IFN-gamma production. These results indicated a critical role for TLRs/MyD88-dependent IL-12/TNF-alpha production and for IL-12- and IL-18-mediated IFN-gamma production in early phase clearance of LM.     

Mycobacterial infection in TLR2 and TLR6 knockout mice

To investigate the role of TLR in the development of murine tuberculosis in vivo, TLR2 and TLR6 knockout (KO) mice were infected with Mycobacterium tuberculosis by placing them in the exposure chamber of an airborne infection apparatus. Both TLR2 and TLR6 KO mice survived until sacrifice at 12 weeks after infection. Infected TLR2 KO mice developed granulomatous pulmonary lesions with neutrophil infiltration, which were slightly larger in size than those in wild-type mice. Pulmonary levels of the mRNAs for inducible nitric oxide synthase (iNOS), TNF-alpha, TGF-beta, IL-1beta, and IL-2 were significantly lower, but levels of the mRNAs for IL-4 and IL-6 were higher, than in wild-type (WT) mice. No significant difference was recognized in cytokine mRNA expression between TLR2 KO and WT mice at 12 weeks after infection. DNA binding by NF-kappaB was low in TLR2 KO mice. On the other hand, TLR6 KO mice were not different from WT mice in terms of pulmonary histopathology, mRNA expression and CFU assay. Therefore, TLR2 does not play an essential role in the pathogenesis of murine tuberculosis, although it is important for defense against mycobacterial infection.     

Central role of MyD88-dependent dendritic cell maturation and proinflammatory cytokine production to control Brucella abortus infection

Brucella abortus is a facultative intracellular bacterium that infects humans and domestic animals. The enhanced susceptibility to virulent B. abortus observed in MyD88 knockout (KO) mice led us to investigate the mechanisms involved in MyD88-dependent immune responses. First, we defined the role of MyD88 in dendritic cell (DC) maturation. In vitro as well as in vivo, B. abortus-exposed MyD88 KO DCs displayed a significant impairment on maturation as observed by expression of CD40, CD86, and MHC class II on CD11c+ cells. In addition, IL-12 and TNF-alpha production was totally abrogated in MyD88 KO DCs and macrophages. Furthermore, B. abortus-induced IL-12 production was found to be dependent on TLR2 in DC, but independent on TLR2 and TLR4 in macrophages. Additionally, we investigated the role of exogenous IL-12 and TNF-alpha administration on MyD88 KO control of B. abortus infection. Importantly, IL-12, but not TNF-alpha, was able to partially rescue host susceptibility in MyD88 KO-infected animals. Furthermore, we demonstrated the role played by TLR9 during virulent B. abortus infection. TLR9 KO-infected mice showed 1 log Brucella CFU higher than wild-type mice. Macrophages and DC from TLR9 KO mice showed reduced IL-12 and unaltered TNF-alpha production when these cells were stimulated with Brucella. Together, these results suggest that susceptibility of MyD88 KO mice to B. abortus is due to impaired DC maturation and lack of IL-12 synthesis. Additionally, DC activation during Brucella infection plays an important regulatory role by stimulating and programming T cells to produce IFN-gamma.     

Distinct roles for IL-4 and IL-10 in regulating T2 immunity during allergic bronchopulmonary mycosis

Pulmonary Cryptococcus neoformans infection of C57BL/6 mice is an established model of an allergic bronchopulmonary mycosis that has also been used to test a number of immunomodulatory agents. Our objective was to determine the role of IL-4 and IL-10 in the development/manifestation of the T2 response to C. neoformans in the lungs and lung-associated lymph nodes. In contrast to wild-type (WT) mice, which develop a chronic infection, pulmonary clearance was significantly greater in IL-4 knockout (KO) and IL-10 KO mice but was not due to an up-regulation of a non-T cell effector mechanism. Pulmonary eosinophilia was absent in both IL-4 KO and IL-10 KO mice compared with WT mice. The production of IL-4, IL-5, and IL-13 by lung leukocytes from IL-4 KO and IL-10 KO mice was lower but IFN-gamma levels remained the same. TNF-alpha and IL-12 production by lung leukocytes was up-regulated in IL-10 KO but not IL-4 KO mice. Overall, IL-4 KO mice did not develop the systemic (lung-associated lymph nodes and serum) or local (lungs) T2 responses characteristic of the allergic bronchopulmonary C. neoformans infection. In contrast, the systemic T2 elements of the response remained unaltered in IL-10 KO mice whereas the T2 response in the lungs failed to develop indicating that the action of IL-10 in T cell regulation was distinct from that of IL-4. Thus, although IL-10 has been reported to down-regulate pulmonary T2 responses to isolated fungal Ags, IL-10 can augment pulmonary T2 responses if they occur in the context of fungal infection.     

Mycobacterial infection in MyD88-deficient mice

MyD88 is an adaptor protein that plays a major role in TLR/IL-1 receptor family signaling. To understand the role of MyD88 in the development of murine tuberculosis in vivo, MyD88 knockout (KO) mice aerially were infected with Mycobacterium tuberculosis. Infected MyD88 mice were not highly susceptible to M. tuberculosis infection, but they developed granulomatous pulmonary lesions with neutrophil infiltration which were larger than those in wild-type (WT) mice (P < 0.01). The pulmonary tissue levels of mRNA for iNOS and IL-18 were slightly lower, but levels of mRNA for IL-1 beta, IL-2, IL-4, IL-6, IL-10, IFN-gamma, and TGF-beta were higher in MyD88 KO mice. IFN-gamma, TNF-alpha, IL-1 beta, and IL-12 also were high in the sera of MyD88 KO mice. There were no statistically significant differences in the expression of TNF-alpha, IL-12, and ICAM-1 mRNA between MyD88 KO and WT mice. Thus, MyD88 deficiency did not influence the development of murine tuberculosis. NF-kappa B activity was similar in the alveolar macrophages from the lung tissues of MyD88 KO and WT mice. Also, there may be a TLR2-specific, MyD88-independent IL-1 receptor/TLR-mediated pathway to activate NF-kappa B in the host defense against mycobacterial infection.     

Toll-like receptor-2, but not Toll-like receptor-4, is essential for development of oviduct pathology in chlamydial genital tract infection

The roles of Toll-like receptor (TLR) 2 and TLR4 in the host inflammatory response to infection caused by Chlamydia trachomatis have not been elucidated. We examined production of TNF-alpha and IL-6 in wild-type TLR2 knockout (KO), and TLR4 KO murine peritoneal macrophages infected with the mouse pneumonitis strain of C. trachomatis. Furthermore, we compared the outcomes of genital tract infection in control, TLR2 KO, and TLR4 KO mice. Macrophages lacking TLR2 produced significantly less TNF-alpha and IL6 in response to active infection. In contrast, macrophages from TLR4 KO mice consistently produced higher TNF-alpha and IL-6 responses than those from normal mice on in vitro infection. Infected TLR2-deficient fibroblasts had less mRNA for IL-1, IL-6, and macrophage-inflammatory protein-2, but TLR4-deficient cells had increased mRNA levels for these cytokines compared with controls, suggesting that ligation of TLR4 by whole chlamydiae may down-modulate signaling by other TLRs. In TLR2 KO mice, although the course of genital tract infection was not different from that of controls, significantly lower levels of TNF-alpha and macrophage-inflammatory protein-2 were detected in genital tract secretions during the first week of infection, and there was a significant reduction in oviduct and mesosalpinx pathology at late time points. TLR4 KO mice responded to in vivo infection similarly to wild-type controls and developed similar pathology. TLR2 is an important mediator in the innate immune response to C. trachomatis infection and appears to play a role in both early production of inflammatory mediators and development of chronic inflammatory pathology.     

Toll-like receptor (TLR) 2-9 agonists-induced cytokines and chemokines: I. Comparison with T cell receptor-induced responses

The cells of innate and adaptive immunity, although activated by different ligands, engage in cross talk to ensure a successful immune outcome. To better understand this interaction, we examined the demographic picture of individual TLR (TLRs 2-9) -driven profiles of eleven cytokines (IFN-alpha/beta, IFN-gamma, IL-12p40/IL-12p70, IL-4, 1L-13, TNF-alpha, IL-1beta, IL-2, IL-10) and four chemokines (MCP-1, MIP1beta, IL-8, and RANTES), and compared them with direct T-cell receptor triggered responses in an assay platform using human PBMCs. We find that T-cell activation by a combination of anti-CD3/anti-CD28/PHA induced a dominant IL-2, IL-13, and Type-II interferon (IFN-gamma) response without major IL-12 and little Type-I interferon (IFN-alphabeta) release. In contrast, TLR7 and TLR9 agonists induced high levels of Type-I interferons. The highest IFN-gamma levels were displayed by TLR8 and TLR7/8 agonists, which also induced the highest levels of pro-inflammatory cytokines IL-12, TNF-alpha, and IL-1beta. Amongst endosomal TLRs, TLR7 displayed a unique profile producing weak IL-12, IFN-gamma, TNF-alpha, IL-1beta, and IL-8. TLR7 and TLR9 resembled each other in their cytokine profile but differed in MIP-1beta and MCP1 chemokine profiles. Gram positive (TLR2, TLR2/6) and gram negative (TLR4) pathogen-derived TLR agonists displayed significant similarities in profile, but not in potency. TLR5 and TLR2/6 agonists paralleled TLR2 and TLR4 in generating pro-inflammatory chemokines MCP-1, MIP-1beta, RANTES, and IL-8 but yielded weak TNF-alpha and IL-1 responses. Taken together, the data show that diverse TLR agonists, despite their operation through common pathways induce distinct cytokine/chemokine profiles that in turn have little or no overlap with TCR-mediated response.     

Circulating soluble CD4 directly prevents host resistance and delayed-type hypersensitivity response to Cryptococcus neoformans in mice

In the present study, we examined the effect of soluble CD4 (sCD4) on host resistance and delayed-type hypersensitivity (DTH) response to Cryptococcus neoformans using a novel mutant mouse that exhibits a defect in the expression of membrane-bound CD4 but secretes high levels of sCD4 in the serum. In these mice, host resistance to this pathogen was impaired as indicated by an increased number of live pathogens in the lung. To elucidate the mechanism of immunodeficiency, three different sets of experiments were conducted. First, administration of anti-CD4 mAb restored the attenuated host defense. Second, in CD4 gene-disrupted (CD4KO) mice, host resistance was not attenuated compared to control mice. Third, implantation of sCD4 gene-transfected myeloma cells rendered the CD4KO mice susceptible to this infection, while similar treatment with mock-transfected cells did not show such an effect. These results indicated that immunodeficiency in the mutant mice was attributed to the circulating sCD4 rather than to the lack of CD4+ T cells. In addition, DTH response to C. neoformans evaluated by footpad swelling was reduced in the mutant mice compared to that in the control, and the reduced response was restored by the administration of anti-CD4 mAb. Finally, serum levels of IFN-gamma, IL-12 and IL-18 in the mutant mice were significantly reduced, while there was no difference in Th2 cytokines, such as IL-4 and IL-10. Considered collectively, our results demonstrated that sCD4 could directly prevent host resistance and DTH response to C. neoformans through interference with the production of Th1-type cytokines.     

Deoxynucleic acids from Cryptococcus neoformans activate myeloid dendritic cells via a TLR9-dependent pathway

The mechanism of host cell recognition of Cryptococcus neoformans, an opportunistic fungal pathogen in immunocompromised patients, remains poorly understood. In the present study, we asked whether the DNA of this yeast activates mouse bone marrow-derived myeloid dendritic cells (BM-DCs). BM-DCs released IL-12p40 and expressed CD40 upon stimulation with cryptococcal DNA, and the response was abolished by treatment with DNase, but not with RNase. IL-12p40 production and CD40 expression were attenuated by chloroquine, bafilomycin A, and inhibitory oligodeoxynucleotides (ODN) that suppressed the responses caused by CpG-ODN. Activation of BM-DCs by cryptococcal DNA was almost completely abrogated in TLR9 gene-disrupted (TLR9(-/-)) mice and MyD88(-/-) mice, similar to that by CpG-ODN. In addition, upon stimulation with whole yeast cells of acapsular C. neoformans, TLR9(-/-) BM-DCs produced a lower amount of IL-12p40 than those from wild-type mice, and TLR9(-/-) mice were more susceptible to pulmonary infection with this fungal pathogen than wild-type mice, as shown by increased number of live colonies in lungs. Treatment of cryptococcal DNA with methylase resulted in reduced IL-12p40 synthesis by BM-DCs. Furthermore, using a luciferase reporter assay, cryptococcal DNA activated NF-kappaB in HEK293 cells transfected with the TLR9 gene. Finally, confocal microscopy showed colocalization of fluorescence-labeled cryptococcal DNA with CpG-ODN and the findings merged in part with the distribution of TLR9 in BM-DCs. Our results demonstrate that cryptococcal DNA causes activation of BM-DCs in a TLR9-dependent manner and suggest that the CpG motif-containing DNA may contribute to the development of inflammatory responses after infection with C. neoformans.     

Toll-like receptor 2 (TLR2) and dectin-1 contribute to the production of IL-12p40 by bone marrow-derived dendritic cells infected with Penicillium marneffei

The present study was designed to elucidate the role of TLR2, TLR4 and dectin-1 in the production of IL-12p40 by bone marrow-derived dendritic cells (BM-DCs) infected with Penicillium marneffei. IL-12p40 production was almost completely abrogated in BM-DCs from TLR2 gene-knockout (KO) and MyD88KO mice, but not from TLR4-defective C3H/HeJ mice compared to those from control mice. Furthermore, BM-DCs from dectin-1KO mice faintly produced IL-12p40 upon stimulation with this fungus. Using a luciferase reporter assay, P. marneffei activated NF-kappaB in HEK293 cells transfected with the TLR2 gene, but not with the dectin-1 gene, and their co-transfection did not lead to further increase in this response. These results indicate that TLR2 and dectin-1 are essential in sensing P. marneffei for the activation of BM-DCs.     

Interleukin-4 weakens host resistance to pulmonary and disseminated cryptococcal infection caused by combined treatment with interferon-gamma-inducing cytokines.

We examined the role of interleukin (IL)-4 in host resistance against infection with Cryptococcus neoformans. First, we examined the effects of a neutralizing anti-IL-4 monoclonal antibody (mAb) on survival of mice infected intratracheally with this fungal pathogen. We also compared the number of live C. neoformans in lungs and brains of treated and untreated mice. Treatment with anti-IL-4 mAb significantly prolonged survival of infected mice and reduced the lung and brain burdens of C. neoformans, which was associated with increased production of IFN-gamma in lungs. In the next experiments, infected mice were treated with two IFN-gamma-inducing cytokines, IL-12 and IL-18, known to enhance protection against infection. We then evaluated the effect of such treatment on the number of live microorganisms and concentration of IL-4 in lungs. These two parameters showed a statistically significant relationship, suggesting a negative regulation of host protection by IL-4. Finally, we examined the effects of IL-4 treatment and administration of neutralizing anti-IL-4 mAbs on host protection against C. neoformans and local production of IFN-gamma in lungs induced by treatment with IL-12/IL-18. The former treatment suppressed host protection and reduced IFN-gamma production, while the latter produced the opposite effects. Our results indicated that IL-4 suppressed the host defense mechanisms against infection with C. neoformans potentiated by IFN-gamma-inducing cytokines probably through the suppression of local production of IFN-gamma.     

Toll-like receptor 4 mediates innate immune responses to Haemophilus influenzae infection in mouse lung.

Toll-like receptors (TLRs) have been implicated in the regulation of host responses to microbial Ags. This study characterizes the role of TLR4 in the innate immune response to intrapulmonary administration of Haemophilus influenzae in the mouse. Two different strains of mice efficiently cleared aerosolized H. influenzae concurrent with a brisk elaboration of IL-1beta, IL-6, TNF-alpha, macrophage-inflammatory protein (MIP)-1alpha, and MIP-2 in bronchoalveolar lavage and a corresponding mobilization of intrapulmonary neutrophils. Congenic strains of mice deficient in TLR4 demonstrated a substantial delay in clearance of H. influenzae with diminished IL-1beta, IL-6, TNF-alpha, MIP-1alpha, and MIP-2 in bronchoalveolar lavage and a notable absence of intrapulmonary neutrophils. In TLR4-expressing animals, but not TLR4-deficient animals, TNF-alpha and MIP-1alpha expression was up-regulated in epithelial cells of the conducting airway in response to H. influenzae which was preceded by an apparent activation of the NF-kappaB pathway in these cells based on the findings of decreased overall IkappaB and an increase in its phosphorylated form. This study demonstrates a critical role of TLR4 in mediating an effective innate immune response to H. influenzae in the lung. This suggests that the airway epithelia might contribute to sensing of H. influenzae infection and signaling the innate immune response.     

The MyD88-dependent, but not the MyD88-independent, pathway of TLR4 signaling is important in clearing nontypeable haemophilus influenzae from the mouse lung

TLRs are important for the recognition of conserved motifs expressed by invading bacteria. TLR4 is the signaling receptor for LPS, the major proinflammatory component of the Gram-negative cell wall, whereas CD14 serves as the ligand-binding part of the LPS receptor complex. Triggering of TLR4 results in the activation of two distinct intracellular pathways, one that relies on the common TLR adaptor MyD88 and one that is mediated by Toll/IL-1R domain-containing adaptor-inducing IFN-beta (TRIF). Nontypeable Haemophilus influenzae (NTHi) is a common Gram-negative respiratory pathogen that expresses both TLR4 (LPS and lipooligosaccharide) and TLR2 (lipoproteins) ligands. To determine the roles of CD14, TLR4, and TLR2 during NTHi pneumonia, the following studies were performed: 1) Alveolar macrophages from CD14 and TLR4 knockout (KO) mice were virtually unresponsive to NTHi in vitro, whereas TLR2 KO macrophages displayed a reduced NTHi responsiveness. 2) After intranasal infection with NTHi, CD14 and TLR4 KO mice showed an attenuated early inflammatory response in their lungs, which was associated with a strongly reduced clearance of NTHi from the respiratory tract; in contrast, in TLR2 KO mice, lung inflammation was unchanged, and the number of NTHi CFU was only modestly increased at the end of the 10-day observation period. 3) MyD88 KO, but not TRIF mutant mice showed an increased bacterial load in their lungs upon infection with NTHi. These data suggest that the MyD88-dependent pathway of TLR4 is important for an effective innate immune response to respiratory tract infection caused by NTHi.     

Immunomodulation via novel use of TLR4 by the filarial nematode phosphorylcholine-containing secreted product, ES-62

Filarial nematodes, parasites of vertebrates, including humans, secrete immunomodulatory molecules into the host environment. We have previously demonstrated that one such molecule, the phosphorylcholine-containing glycoprotein ES-62, acts to bias the immune response toward an anti-inflammatory/Th2 phenotype that is conducive to both worm survival and host health. For example, although ES-62 initially induces macrophages to produce low levels of IL-12 and TNF-alpha, exposure to the parasite product ultimately renders the cells unable to produce these cytokines in response to classic stimulators such as LPS/IFN-gamma. We have investigated the possibility that a TLR is involved in the recognition of ES-62 by target cells, because phosphorylcholine, a common pathogen-associated molecular pattern, appears to be responsible for many of the immunomodulatory properties of ES-62. We now demonstrate that ES-62-mediated, low level IL-12 and TNF-alpha production by macrophages and dendritic cells is abrogated in MyD88 and TLR4, but not TLR2, knockout, mice implicating TLR4 in the recognition of ES-62 by these cells and MyD88 in the transduction of the resulting intracellular signals. We also show that ES-62 inhibits IL-12 induction by TLR ligands other than LPS, bacterial lipopeptide (TLR2) and CpG (TLR9), via this TLR4-dependent pathway. Surprisingly, macrophages and dendritic cells from LPS-unresponsive, TLR4-mutant C3H/HeJ mice respond normally to ES-62. This is the first report to demonstrate that modulation of cytokine responses by a pathogen product can be abrogated in cells derived from TLR4 knockout, but not C3H/HeJ mice, suggesting the existence of a novel mechanism of TLR4-mediated immunomodulation.     

Host Defense and Recruitment of Foxp3+ T Regulatory Cells to the Lungs in Chronic Mycobacterium tuberculosis Infection Requires Toll-like Receptor 2

Acute resistance to low dose M. tuberculosis (Mtb) infection is not dependent on Toll-like receptor (TLR) 2. However, whether TLR2 contributes to resistance in chronic Mtb infection has remained uncertain. Here we report that, following low dose aerosol infection with Mtb, mice lacking TLR2 (TLR2KO), in comparison with wild type (WT) mice, exhibit enhanced cellular infiltration and inflammation in the lungs, and fail to stably control bacterial burden during chronic infection. IFNγ and IL-17 was expressed at equivalent levels in the two groups; however, the characteristic accumulation of Foxp3⁺ T regulatory cells (Tregs) in pulmonary granulomas was significantly reduced in TLR2KO mice. Nonetheless, this reduction in Tregs was independent of whether Tregs expressed TLR2 or not. To directly link the reduced number of Tregs to the increased inflammation present in the TLR2KO mice, we used a macrophage adoptive transfer model. At seven weeks post-Mtb infection, TLR2KO mice, which were adoptively transferred with WT macrophages, displayed enhanced accumulation of Tregs in the lungs and a concomitant reduction in inflammation in contrast with control mice that received TLR2KO macrophages. However, the pulmonary bacterial burden between the two groups remained similar indicating that TLR2''s role in modulating immunopathology is functionally distinct from its role in restricting Mtb growth in chronic infection. Together, these findings unequivocally demonstrate that TLR2 contributes to host resistance against chronic Mtb infection and reveal a novel role for TLR2 in mediating the recruitment of Foxp3⁺ Tregs to the lungs to control inflammation.     

Essential role for the p40 subunit of interleukin-12 in neutrophil-mediated early host defense against pulmonary infection with Streptococcus pneumoniae: involvement of interferon-gamma

Interleukin (IL)-12 is a critical cytokine in the T helper (Th)1 response and host defense against intracellular microorganisms, while its role in host resistance to extracellular bacteria remains elusive. In the present study, we elucidated the role of IL-12 in the early-phase host defense against acute pulmonary infection with Streptococcus pneumoniae, a typical extracellular bacterium, using IL-12p40 gene-disrupted (IL-12p40KO) mice. IL-12p40KO mice were highly susceptible to S. pneumoniae infection, as indicated by the shortened survival time, which was completely restored by the replacement therapy with recombinant (r) IL-12, and increased bacterial counts in the lung. In these mice, recruitment of neutrophils in the lung was significantly attenuated when compared to that in wild-type (WT) mice, which correlated well with the reduced production of macrophage inflammatory protein (MIP-2) and tumor necrosis factor (TNF)-alpha in the infected tissues at the early phase of infection. In vitro synthesis of both cytokines by S. pneumoniae-stimulated lung leukocytes was significantly lower in IL-12p40KO mice than in WT mice, and addition of rIL-12 or interferon (IFN)-gamma restored the reduced production of MIP-2 and TNF-alpha in IL-12p40KO mice. Neutralizing anti-IFN-gamma monoclonal antibody (mAb) significantly decreased the effect of rIL-12. Anti-IFN-gamma mAb shortened the survival time of infected mice and reduced the recruitment of neutrophils and production of MIP-2 and TNF-alpha in the lungs. Our results indicated that IL-12p40 plays a critical role in the early-phase host defense against S. pneumoniae infection by promoting the recruitment of neutrophils to the infected tissues.     

MyD88 is pivotal for the early inflammatory response and subsequent bacterial clearance and survival in a mouse model of Chlamydia pneumoniae pneumonia

Chlamydia pneumoniae is the causative agent of respiratory tract infections and a number of chronic diseases. Here we investigated the involvement of the common TLR adaptor molecule MyD88 in host responses to C. pneumoniae-induced pneumonia in mice. MyD88-deficient mice were severely impaired in their ability to mount an acute early inflammatory response toward C. pneumoniae. Although the bacterial burden in the lungs was comparable 5 days after infection, MyD88-deficient mice exhibited only minor signs of pneumonia and reduced expression of inflammatory mediators. MyD88-deficient mice were unable to up-regulate proinflammatory cytokines and chemokines, demonstrated delayed recruitment of CD8+ and CD4+ T cells to the lungs, and were unable to clear the pathogen from their lungs at day 14. At day 14 the MyD88-deficent mice developed a severe, chronic lung inflammation with elevated IL-1beta and IFN-gamma leading to increased mortality, whereas wild-type mice as well as TLR2- or TLR4-deficient mice recovered from acute pneumonia and did not show delayed bacterial clearance. Thus, MyD88 is essential to recognize C. pneumoniae infection and initiate a prompt and effective immune host response against this organism leading to clearance of bacteria from infected lungs.     

MD-2 is required for the full responsiveness of mast cells to LPS but not to PGN

To address the role played by MD-2 in mast cell recognition of LPS, we examined bone marrow-derived mast cells (BMMCs) from MD-2 gene-targeted mice. BMMCs from MD-2-/- mice showed impaired cytokine production (TNF-alpha, IL-6, IL-13, and IL-1beta) in response to LPS from Escherichia coli, but not to peptidoglycan (PGN) from Staphylococcus aureus. In a mast cell-dependent acute septic model, MD-2 deficiency of mast cell resulted in significantly higher mortality due to defective neutrophil recruitment and the production of cytokines in the peritoneal cavity, which was similar to mice with TLR4-deficient mast cells. The TLR2-dependent activation of skin mast cells by PGN was not altered by the absence of MD-2 in vivo. Collectively, MD-2 is essential for the recognition of LPS by TLR4 but not for that of PGN by TLR2 of mast cells.     

Contributions of the MyD88-dependent receptors IL-18R, IL-1R, and TLR9 to host defenses following pulmonary challenge with Cryptococcus neoformans

Signaling via the adapter protein, MyD88, is important in the host defense against Cryptococcus neoformans infection. While certain Toll-like receptors (TLRs) can enhance the clearance of Cryptococcus, the contributions of MyD88-dependent, TLR-independent pathways have not been fully investigated. We examined the roles of IL-1R and IL-18R in vivo by challenging C57BL/6 mice with a lethal strain of Cryptococcus. We found that the absence of IL-18R, but not IL-1R, causes a shift in the survival curve following pulmonary delivery of a virulent strain of C. neoformans (H99). Specifically, IL-18R-deficient mice have significantly shorter median survival times compared to wild-type mice following infection. Cytokine analysis of lung homogenates revealed that deficiency of IL-IR, IL-18R, or MyD88 is associated with diminished lung levels of IL-1β. In order to compare these findings with those related to TLR-deficiency, we studied the effects of TLR9-deficiency and found that deficiency of TLR9 also affects the survival curve of mice following challenge with C. neoformans. Yet the lungs from infected TLR9-deficient mice have robust levels of IL-1β. In summary, we found that multiple signaling components can contribute the MyD88-dependent host responses to cryptococcal infection in vivo and each drives distinct pulmonary responses.     

Activation of TLR2 and TLR4 by glycosylphosphatidylinositols derived from Toxoplasma gondii

GPIs isolated from Toxoplasma gondii, as well as a chemically synthesized GPI lacking the lipid moiety, activated a reporter gene in Chinese hamster ovary cells expressing TLR4, while the core glycan and lipid moieties cleaved from the GPIs activated both TLR4- and TLR2-expressing cells. MyD88, but not TLR2, TLR4, or CD14, is absolutely needed to trigger TNF-alpha production by macrophages exposed to T. gondii GPIs. Importantly, TNF-alpha response to GPIs was completely abrogated in macrophages from TLR2/4-double-deficient mice. MyD88(-/-) mice were more susceptible to death than wild-type (WT), TLR2(-/-), TLR4(-/-), TLR2/4(-/-), and CD14(-/-) mice infected with the ME-49 strain of T. gondii. The cyst number was higher in the brain of TLR2/4(-/-), but not TLR2(-/-), TLR4(-/-), and CD14(-/-), mice, as compared with WT mice. Upon infection with the ME-49 strain of T. gondii, we observed no decrease of IL-12 and IFN-gamma production in TLR2-, TLR4-, or CD14-deficient mice. Indeed, splenocytes from T. gondii-infected TLR2(-/-) and TLR2/4(-/-) mice produced more IFN-gamma than cells from WT mice in response to in vitro stimulation with parasite extracts enriched in GPI-linked surface proteins. Together, our results suggest that both TLR2 and TLR4 receptors may participate in the host defense against T. gondii infection through their activation by the GPIs and could work together with other MyD88-dependent receptors, like other TLRs or even IL-18R or IL-1R, to obtain an effective host response against T. gondii infection.