Containment of aerogenic Mycobacterium tuberculosis infection in mice does not require MyD88 adaptor function for TLR2, -4 and -9

The role of Toll-like receptors (TLR) and MyD88 for immune responses to Mycobacterium tuberculosis (Mtb) infection remains controversial. To address the impact of TLR-mediated pathogen recognition and MyD88-dependent signaling events on anti-mycobacterial host responses, we analyzed the outcome of Mtb infection in TLR2/4/9 triple- and MyD88-deficient mice. After aerosol infection, both TLR2/4/9-deficient and wild-type mice expressed pro-inflammatory cytokines promoting antigen-specific T cells and the production of IFN-gamma to similar extents. Moreover, TLR2/4/9-deficient mice expressed IFN-gamma-dependent inducible nitric oxide synthase and LRG-47 in infected lungs. MyD88-deficient mice expressed pro-inflammatory cytokines and were shown to expand IFN-gamma-producing antigen-specific T cells, albeit in a delayed fashion. Only mice that were deficient for MyD88 rapidly succumbed to unrestrained mycobacterial growth, whereas TLR2/4/9-deficient mice controlled Mtb replication. IFN-gamma-dependent restriction of mycobacterial growth was severely impaired only in Mtb-infected MyD88, but not in TLR2/4/9-deficient bone marrow-derived macrophages. Our results demonstrate that after Mtb infection neither TLR2, -4, and -9, nor MyD88 are required for the induction of adaptive T cell responses. Rather, MyD88, but not TLR2, TLR4 and TLR9, is critical for triggering macrophage effector mechanisms central to anti-mycobacterial defense.     

Toll-like receptor 2-deficient mice succumb to Mycobacterium tuberculosis infection

Recognition of Mycobacterium tuberculosis by the innate immune system is essential in the development of an adaptive immune response. Mycobacterial cell wall components activate macrophages through Toll-like receptor (TLR) 2, suggesting that this innate immune receptor plays a role in the host response to M. tuberculosis infection. After aerosol infection with either 100 or 500 live mycobacteria, TLR2-deficient mice display reduced bacterial clearance, a defective granulomatous response, and develop chronic pneumonia. Analysis of pulmonary immune responses in TLR2-deficient mice after 500 mycobacterial aerosol challenge showed increased levels of interferon-gamma, tumor necrosis factor-alpha, and interleukin-12p40 as well as increased numbers of CD4(+) and CD8(+) cells. Furthermore, TLR2-deficient mice mounted elevated Ag-specific type 1 T-cell responses that were not protective because all deficient mice succumb to infection within 5 months. Taken together, the data suggests that TLR2 may function as a regulator of inflammation, and in its absence an exaggerated immune inflammatory response develops.     

Increased numbers and functional activity of CD56+ T cells in healthy cytomegalovirus positive subjects

Mycobacterium indicus pranii (MIP) is an atypical mycobacterial species possessing strong immunomodulatory properties. It is a potent vaccine candidate against tuberculosis, promotes Th1 immune response and protects mice from tumours. In previous studies, we demonstrated higher protective efficacy of MIP against experimental tuberculosis as compared with bacillus Calmette–Guérin (BCG). Since macrophages play an important role in the pathology of mycobacterial diseases and cancer, in the present study, we evaluated the MIP in live and killed form for macrophage activation potential, compared it with BCG and investigated the underlying mechanisms. High levels of tumour necrosis factor-α, interleukin-12p40 (IL-12p40), IL-6 and nitric oxide were produced by MIP-stimulated macrophages as compared with BCG-stimulated macrophages. Prominent up-regulation of co-stimulatory molecules CD40, CD80 and CD86 was also observed in response to MIP. Loss of response in MyD88-deficient macrophages showed that both MIP and BCG activate the macrophages in a MyD88-dependent manner. MyD88 signalling pathway culminates in nuclear factor-κB/activator protein-1 (NF-κB/AP-1) activation and higher activation of NF-κB/AP-1 was observed in response to MIP. With the help of pharmacological inhibitors and Toll-like receptor (TLR) -deficient macrophages, we observed the role of TLR2, TLR4 and intracellular TLRs in MIP-mediated macrophage activation. Stimulation of HEK293 cells expressing TLR2 in homodimeric or heterodimeric form showed that MIP has a distinctly higher level of TLR2 agonist activity compared with BCG. Further experiments suggested that TLR2 ligands are well exposed in MIP whereas they are obscured in BCG. Our findings establish the higher macrophage activation potential of MIP compared with BCG and delineate the underlying mechanism.     

Toll-like receptor 2 is required for optimal control of Listeria monocytogenes infection

The control of Listeria monocytogenes infection depends on the rapid activation of the innate immune system, likely through Toll-like receptors (TLR), since mice deficient for the common adapter protein of TLR signaling, myeloid differentiation factor 88 (MyD88), succumb to Listeria infection. In order to test whether TLR2 is involved in the control of infections, we compared the host response in TLR2-deficient mice with that in wild-type mice. Here we show that TLR2-deficient mice are more susceptible to systemic infection by Listeria than are wild-type mice, with a reduced survival rate, increased bacterial burden in the liver, and abundant and larger hepatic microabscesses containing increased numbers of neutrophils. The production of tumor necrosis factor, interleukin-12, and nitric oxide and the expression of the costimulatory molecules CD40 and CD86, which are necessary for the control of infection, were reduced in TLR2-deficient macrophages and dendritic cells stimulated by Listeria and were almost abolished in the absence of MyD88, coincident with the high susceptibility of MyD88-deficient mice to in vivo infection. Therefore, the present data demonstrate a role for TLR2 in the control of Listeria infection, but other MyD88-dependent signals may contribute to host resistance.     

Toll-like receptor 9 contributes to recognition of Mycobacterium bovis Bacillus Calmette-Guérin by Flt3-ligand generated dendritic cells

Recognition of mycobacteria by the innate immune system is essential for the development of an adaptive immune response. Mycobacterial antigens stimulate antigen presenting cells (APCs) through distinct Toll-like receptors (TLRs) resulting in rapid activation of the innate immune system. The role of TLRs during infection with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) has been evaluated for TLR2 and TLR4 only. Surprisingly, despite the fact that immune stimulatory CpG-motifs have been originally derived from BCG, for the vaccine strain the role of TLR9 has not been addressed before. To identify the set of TLRs involved in the recognition of BCG, we infected bone marrow-derived macrophages and bone marrow-derived dendritic cells (Flt3-ligand generated DCs) from TLR2, TLR3, TLR4, TLR7, TLR9, MyD88 knockout, TLR2/4 and TLR2/4/9 multiple knockout mice. The degree of activation and stimulation was determined by TNFalpha, IL-6 and IL-12p40 ELISA. Activation of DCs was measured by surface expression of the costimulatory molecule CD86. We observed the most dramatic reduction of the inflammatory response for TLR2-deficient antigen presenting cells. Both macrophages and DCs produce markedly decreased amounts of TNFalpha and IL-6 in the absence of TLR2 whereas no significant reduction could be observed for TLR3, 4, 7, 9 single TLR-knockouts. However, IL-12 production in DCs appears not exclusively dependent on TLR2 and only in TLR2/4/9-deficient DCs BCG-induced IL-12 is reduced to background levels. Similarly, up-regulation of CD86 is abolished only in TLR2/4/9-deficient DCs supporting a role of TLR9 in the recognition of M. bovis BCG by murine dendritic cells.     

Long-term control of Mycobacterium bovis BCG infection in the absence of Toll-like receptors (TLRs): investigation of TLR2-, TLR6-, or TLR2-TLR4-deficient mice

Live mycobacteria have been reported to signal through both Toll-like receptor 2 (TLR2) and TLR4 in vitro. Here, we investigated the role of TLR2 in the long-term control of the infection by the attenuated Mycobacterium, Mycobacterium bovis BCG, in vivo. We sought to determine whether the reported initial defect of bacterial control (K. A. Heldwein et al., J. Leukoc. Biol. 74:277-286, 2003) resolved in the chronic phase of BCG infection. Here we show that TLR2-deficient mice survived a 6-month infection period with M. bovis BCG and were able to control bacterial growth. Granuloma formation, T-cell and macrophage recruitment, and activation were normal. Furthermore, the TLR2 coreceptor, TLR6, is also not required since TLR6-deficient mice were able to control chronic BCG infection. Finally, TLR2-TLR4-deficient mice infected with BCG survived the 8-month observation period. Interestingly, the adaptive response of TLR2- and/or TLR4-deficient mice seemed essentially normal on day 14 or 56 after infection, since T cells responded normally to soluble BCG antigens. In conclusion, our data demonstrate that TLR2, TLR4, or TLR6 are redundant for the control of M. bovis BCG mycobacterial infection.     

Pathological role of Toll-like receptor signaling in cerebral malaria

Toll-like receptors (TLRs) recognize malaria parasites or their metabolites; however, their physiological roles in malaria infection in vivo are not fully understood. Here, we show that myeloid differentiation primary response gene 88 (MyD88)-dependent TLR signaling mediates brain pathogenesis of severe malaria infection, namely cerebral malaria (CM). A significant number of MyD88-, but not TIR domain containing adaptor-inducing IFN-beta (TRIF)-deficient or wild-type (WT) mice survived CM caused by Plasmodium berghei ANKA (PbA) infection. Although systemic parasitemia was comparable, sequestration of parasite and hemozoin load in the brain blood vessels was significantly lower in MyD88-deficient mice compared with those in TRIF-deficient or WT mice. Moreover, brain-specific pathological changes were associated with MyD88-dependent infiltration of CD8+, CCR5+ T cells and CD11c+ dendritic cells, including CD11c+, NK1.1+ and B220+ cells, and up-regulation of genes such as Granzyme B, Lipocalin 2, Ccl3 and Ccr5. Further studies using mice lacking various TLRs suggest that TLR2 and TLR9, but not TLR4, 5 and 7, were involved in CM. These results strongly suggest that TLR2- and/or TLR9-mediated, MyD88-dependent brain pathogenesis may play a critical role in CM, the lethal complication during PbA infection.     

Toll-like receptor 9 contributes to recognition of Mycobacterium bovis Bacillus Calmette-Guérin by Flt3-ligand generated dendritic cells

Recognition of mycobacteria by the innate immune system is essential for the development of an adaptive immune response. Mycobacterial antigens stimulate antigen presenting cells (APCs) through distinct Toll-like receptors (TLRs) resulting in rapid activation of the innate immune system. The role of TLRs during infection with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) has been evaluated for TLR2 and TLR4 only. Surprisingly, despite the fact that immune stimulatory CpG-motifs have been originally derived from BCG, for the vaccine strain the role of TLR9 has not been addressed before. To identify the set of TLRs involved in the recognition of BCG, we infected bone marrow-derived macrophages and bone marrow-derived dendritic cells (Flt3-ligand generated DCs) from TLR2, TLR3, TLR4, TLR7, TLR9, MyD88 knockout, TLR2/4 and TLR2/4/9 multiple knockout mice. The degree of activation and stimulation was determined by TNFα, IL-6 and IL-12p40 ELISA. Activation of DCs was measured by surface expression of the costimulatory molecule CD86. We observed the most dramatic reduction of the inflammatory response for TLR2-deficient antigen presenting cells. Both macrophages and DCs produce markedly decreased amounts of TNFα and IL-6 in the absence of TLR2 whereas no significant reduction could be observed for TLR3, 4, 7, 9 single TLR-knockouts. However, IL-12 production in DCs appears not exclusively dependent on TLR2 and only in TLR2/4/9-deficient DCs BCG-induced IL-12 is reduced to background levels. Similarly, up-regulation of CD86 is abolished only in TLR2/4/9-deficient DCs supporting a role of TLR9 in the recognition of M. bovis BCG by murine dendritic cells.     

MyD88-dependent responses involving toll-like receptor 2 are important for protection and clearance of Legionella pneumophila in a mouse model of Legionnaires' disease

Legionella pneumophila is a gram-negative facultative intracellular parasite of macrophages. Although L. pneumophila is the causative agent of a severe pneumonia known as Legionnaires' disease, it is likely that most infections caused by this organism are cleared by the host innate immune system. It is predicted that host pattern recognition proteins belonging to the Toll-like receptor (TLR) family are involved in the protective innate immune responses. We examined the role of TLR-mediated responses in L. pneumophila detection and clearance using genetically altered mouse hosts in which the macrophages are permissive for L. pneumophila intracellular replication. Our data demonstrate that cytokine production by bone marrow-derived macrophages (BMMs) in response to L. pneumophila infection requires the TLR adapter protein MyD88 and is reduced in the absence of TLR2 but not in the absence of TLR4. Bacterial growth ex vivo in BMMs from MyD88-deficient mice was not enhanced compared to bacterial growth ex vivo in BMMs from heterozygous littermate controls. Wild-type mice were able to clear L. pneumophila from the lung, whereas respiratory infection of MyD88-deficient mice caused death that resulted from robust bacterial replication and dissemination. In contrast to an infection with virulent L. pneumophila, MyD88-deficient mice were able to clear infections with L. pneumophila dotA mutants, indicating that MyD88-independent responses in the lung are sufficient to clear bacteria that are unable to replicate intracellularly. In vivo growth of L. pneumophila was enhanced in the lungs of TLR2-deficient mice, which resulted in a delay in bacterial clearance. No significant differences were observed in the growth and clearance of L. pneumophila in the lungs of TLR4-deficient mice and heterozygous littermate control mice. Our data indicate that MyD88 is crucial for eliciting a protective innate immune response against virulent L. pneumophila and that TLR2 is one of the pattern recognition receptors involved in initiating this MyD88-dependent response.     

TLR2 as an essential molecule for protective immunity against Toxoplasma gondii infection

To investigate the role of the Toll-like receptor (TLR) family in host defense against Toxoplasma gondii, we infected TLR2-, TLR4- and MyD88-deficient mice with the avirulent cyst-forming Fukaya strain of T. gondii. All TLR2- and MyD88-deficient mice died within 8 days, whereas all TLR4-deficient and wild-type mice survived after i.p. infection with a high dose of T. gondii. Peritoneal macrophages from T. gondii-infected TLR2- and MyD88-deficient mice did not produce any detectable levels of NO. T. gondii loads in the brain tissues of TLR2- and MyD88-deficient mice were higher than in those of TLR4-deficient and wild-type mice. Furthermore, high levels of IFN-gamma and IL-12 were produced in peritoneal exudate cells (PEC) of TLR4-deficient and wild-type mice after infection, but low levels of cytokines were produced in PEC of TLR2- and MyD88-deficient mice. On the other hand, high levels of IL-4 and IL-10 were produced in PEC of TLR2- and MyD88-deficient mice after infection, but low levels of cytokines were produced in PEC of TLR4-deficient and wild-type mice. The most remarkable histological changes with infiltration of inflammatory cells were observed in lungs of TLR2-deficient mice infected with T. gondii, where severe interstitial pneumonia occurred and abundant T. gondii were found.     

MyD88 is critical for the development of innate and adaptive immunity during acute lymphocytic choriomeningitis virus infection

We investigated the roles of Toll-like receptor 2 (TLR2) and myeloid differentiation factor 88 (MyD88) in the course of a lymphocytic choriomeningitis virus (LCMV) infection and revealed the following: (i) studies of transfected cells and murine peritoneal macrophages demonstrated that TLR2 and MyD88 are essential for the initial pro-inflammatory cytokine response (human IL-8, mouse IL-6) to LCMV; (ii) TLR2 knockout (KO) mice and MyD88 KO mice challenged with LCMV produced less IL-6 and monocyte chemotactic protein-1 in the serum than wild-type mice; (iii) in contrast to inflammatory cytokines, the production of type 1 IFN (IFN-alpha) in response to LCMV was MyD88 independent; (iv) MyD88 plays an essential role in antiviral CD8(+) T cell responses, CD8(+) T cells in MyD88 KO mice were defective in their expression of intracellular antiviral cytokines; and (v) the failure of MyD88 KO mice to activate CD8(+) T cells was accompanied by persistent viral infection in MyD88 KO mice. We demonstrate that TLR-mediated responses are important in the innate immune response to LCMV and that MyD88 is essential for the control of the LCMV infection and the maturation/activation of virus-specific CD8(+) T cells.     

The Th2-response in mercuric chloride-induced autoimmunity requires continuing costimulation via CD28

Gamma interferon (IFNgamma) plays a key role in host defense against pulmonary mycobacterial infections. A variety of lymphocyte subsets may participate in producing pulmonary IFNgamma responses, but their relative contributions after mycobacterial infection have not been clearly elucidated. To address this question, C57Bl/6 female mice were infected by intrapulmonary instillation of 2.5 x 104 BCG (Mycobacterium bovis Bacillus Calmette-Guerin). Lymphocyte populations in lung interstitium were examined at different time points after the infection. BCG load in lungs peaked between 4 and 6 weeks post-infection and declined to very low levels by the 12th week of infection. Recovery of lung interstitial lymphocytes doubled by 4-6 weeks after infection and declined thereafter. Flow cytometric analysis of the lung-derived lymphocytes revealed that about 5% of the these cells made IFNgamma in control mice, and this baseline IFNgamma production involved T (CD3+NK1.1-), NK (CD3-NK1.1+) and NKT (CD3+NK1.1+) cells. As the BCG lung infection peaked, the total number of CD3+ T cells in the lungs increased threefold at 5-6 weeks post-infection. There was a marked increase (sixfold) in the number of T cells secreting IFNgamma 5-6 weeks post-infection. Some increase was also noted in the NKT cells making IFNgamma, but the numbers of NK cells making IFNgamma in BCG-infected lungs remained unaltered. Our results suggest that whereas NK and NKT cells contribute to baseline IFNgamma secretion in control lungs, expansion in the IFNgamma-producing T-cell population was essentially responsible for the augmented response seen in lungs of BCG-infected mice.     

MyD88 signalling in myeloid cells is sufficient to prevent chronic mycobacterial infection

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis that is responsible for almost 1.5 million deaths per year. Sensing of mycobacteria by the host's immune system relies on different families of receptors present on innate immune cells. Amongst them, several members of the TLR family are involved in the activation of immune cells by mycobacteria, yet the in vivo contribution of individual TLRs to the protective immune response remains controversial. On the contrary, MyD88, the adaptor molecule for most TLRs, plays a non‐redundant role in the protection against tuberculosis and mice with a complete germline deletion of MyD88 succumb very early to infection. MyD88 is expressed in both immune and non‐immune cells, but it is not clear whether control of mycobacteria requires ubiquitous or cell‐type specific MyD88 expression. Therefore, using novel conditional switch‐on mouse models, we aimed to investigate the importance of MyD88 signalling in DCs and macrophages for the induction of protective effector mechanisms against mycobacterial infection. We conclude that specific reactivation of MyD88 signalling in CD11c‐ or lysozyme M‐expressing myeloid cells during Mycobacterium bovis Bacille Calmette‐Guerin infection is sufficient to restore systemic and local inflammatory cytokine production and to control pathogen burden.     

Cellular responses to bacterial cell wall components are mediated through MyD88-dependent signaling cascades

MyD88 is an adaptor molecule essential for signaling via the Toll-like receptor (TLR)/IL-1 receptor family. TLR4 is a member of the TLR family and a point mutation in the Tlr4 gene causes hyporesponsiveness to lipopolysaccharide (LPS) in C3H/HeJ mice. We have previously shown that both TLR4- and MyD88-deficient mice are hyporesponsive to LPS. In this study we examined the responsiveness of these two knockout mice to various bacterial cell wall components. Cells from TLR4-deficient mice responded to several kinds of LPS, peptidoglycan and crude cell wall preparation from Gram-positive bacteria and mycobacterial lysates. In contrast, macrophages and splenocytes from MyD88-deficient mice did not respond to any of the bacterial components we tested. These results show that MyD88 is essential for the cellular response to bacterial cell wall components.     

MyD88-dependent signaling contributes to protection following Bacillus anthracis spore challenge of mice: implications for Toll-like receptor signaling

Bacillus anthracis is a spore-forming, gram-positive organism that is the causative agent of the disease anthrax. Recognition of Bacillus anthracis by the host innate immune system likely plays a key protective role following infection. In the present study, we examined the role of TLR2, TLR4, and MyD88 in the response to B. anthracis. Heat-killed Bacillus anthracis stimulated TLR2, but not TLR4, signaling in HEK293 cells and stimulated tumor necrosis factor alpha (TNF-alpha) production in C3H/HeN, C3H/HeJ, and C57BL/6J bone marrow-derived macrophages. The ability of heat-killed B. anthracis to induce a TNF-alpha response was preserved in TLR2-/- but not in MyD88-/- macrophages. In vivo studies revealed that TLR2-/- mice and TLR4-deficient mice were resistant to challenge with aerosolized Sterne strain spores but MyD88-/- mice were as susceptible as A/J mice. We conclude that, although recognition of B. anthracis occurs via TLR2, additional MyD88-dependent pathways contribute to the host innate immune response to anthrax infection.     

Involvement of CD14, toll-like receptors 2 and 4, and MyD88 in the host response to the fungal pathogen Cryptococcus neoformans in vivo

The major capsular polysaccharide of Cryptococcus neoformans, glucuronoxylomannan (GXM), is recognized by Toll-like receptor 2 (TLR2), TLR4, and CD14. In these studies, mice deficient in CD14, TLR2, TLR4, and the TLR-associated adaptor protein, MyD88, were utilized to investigate the contribution of TLRs and CD14 to in vivo host defenses against C. neoformans. MyD88(-/-) mice had significantly reduced survival compared with wild-type C57BL/6 mice after intranasal (i.n.) and intravenous (i.v.) infection with live C. neoformans. CD14(-/-) mice had reduced survival when infected i.v., while TLR2(-/-) mice died significantly earlier after i.n. infection. Mortality was similar comparing TLR4 mutant C3H/HeJ mice and control C3H/HeOuJ mice following i.v. or i.n. challenge with C. neoformans. The course of pulmonary cryptococcosis was studied in more detail in the CD14(-/-), TLR2(-/-), and MyD88(-/-) mice. MyD88(-/-) mice infected i.n. had higher numbers of CFU in the lungs as well as higher GXM levels in the sera and lungs 7 days after infection than wild-type mice did. Surprisingly, there were no major differences in the levels of tumor necrosis factor alpha, interleukin-4 (IL-4), IL-10, IL-12p70, or gamma interferon in the lungs of C. neoformans-infected knockout mice compared with wild-type mice. Histopathologic analysis of the lungs on day 7 postinfection revealed minimal inflammation in all mouse groups. These studies demonstrate a major role for MyD88 and relatively minor roles for CD14 and TLR2 in the response to cryptococcal infection, with the decreased survival of MyD88(-/-) mice correlating with increased numbers of lung CFU and serum and lung GXM levels.     

Lethal Mycobacterium bovis Bacillus Calmette Guérin infection in nitric oxide synthase 2-deficient mice: cell-mediated immunity requires nitric oxide synthase 2

The role of nitric oxide (NO) in Mycobacterium bovis Bacillus Calmette Guerin (BCG) infection was investigated using nitric oxide synthase 2 (nos2)-deficient mice, because NO plays a pivotal protective role in M. tuberculosis infection. We demonstrate that nos2-deficient mice were unable to eliminate BCG and succumbed within 8 to 12 weeks to BCG infection (10(6) CFU) with cachexia and pneumonia, whereas all infected wild-type mice survived. The greatest mycobacterial loads were observed in lung and spleen. Nos2-deficient mice developed large granulomas consisting of macrophages and activated T cells and caseous necrotic lesions in spleen. The macrophages in granulomas from nos2-deficient mice had reduced acid phosphatase activities, suggesting that NO is required for macrophage activation. The absence of NOS2 affected the cytokine production of the Th1 type of immune response, except IL-18. Serum amounts of IL-12p40 were increased and IFN-gamma was decreased compared with wild-type mice. The lack of NOS2 resulted in an overproduction of TNF, observed throughout the infection period. Additionally, TNFR1 and TNFR2 shedding was altered compared with wild-type mice. Up-regulation of TNF may be compensatory for the lack of NOS2. The late neutralization of TNF by soluble TNF receptors resulted in heightened disease severity and accelerated death in nos2-deficient mice but had no effect in wild-type mice. In conclusion, the inability of nos2-deficient mice to kill M. bovis BCG resulted in an accumulation of mycobacteria with a dramatic activation of the immune system and overproduction of pro-inflammatory cytokines, which resulted in death.     

Lung epithelium and myeloid cells cooperate to clear acute pneumococcal infection

The Gram-positive bacterium Streptococcus pneumoniae causes life-threatening infections, especially among immunocompromised patients. The host's immune system senses S. pneumoniae via different families of pattern recognition receptors, in particular the Toll-like receptor (TLR) family that promotes immune cell activation. Yet, while single TLRs are dispensable for initiating inflammatory responses against S. pneumoniae, the central TLR adapter protein myeloid differentiation factor 88 (MyD88) is of vital importance, as MyD88-deficient mice succumb rapidly to infection. Since MyD88 is ubiquitously expressed in hematopoietic and non-hematopoietic cells, the extent to which MyD88 signaling is required in different cell types to control S. pneumoniae is unknown. Therefore, we used novel conditional knockin mice to investigate the necessity of MyD88 signaling in distinct lung-resident myeloid and epithelial cells for the initiation of a protective immune response against S. pneumoniae. Here, we show that MyD88 signaling in lysozyme M (LysM)– and CD11c-expressing myeloid cells, as well as in pulmonary epithelial cells, is critical to restore inflammatory cytokine and antimicrobial peptide production, leading to efficient neutrophil recruitment and enhanced bacterial clearance. Overall, we show a novel synergistic requirement of compartment-specific MyD88 signaling in S. pneumoniae immunity.     

The Alveolar Epithelial Cell Chemokine Response to Pneumocystis Requires Adaptor Molecule MyD88 and Interleukin-1 Receptor but Not Toll-Like Receptor 2 or 4

Pneumocystis is an opportunistic fungal pathogen that causes pneumonia in a variety of clinical settings. An early step in Pneumocystis infection involves the attachment of organisms to alveolar epithelial cells (AECs). AECs produce chemokines in response to Pneumocystis stimulation, but the upstream host-pathogen interactions that activate AEC signaling cascades are not well-defined. MyD88 is an adaptor molecule required for activation of proinflammatory signaling cascades following Toll-like receptor (TLR)-dependent recognition of conserved molecular patterns on pathogens. To determine whether the TLR/MyD88 pathway is required for the AEC chemokine response to Pneumocystis, wild-type (WT) and MyD88-deficient AECs were incubated with Pneumocystis. As expected, WT AECs produced CCL2 and CXCL2 following Pneumocystis stimulation. In contrast, MyD88-deficient AECs were severely impaired in their ability to respond to Pneumocystis. MyD88-deficient AECs did not display Pneumocystis-induced Jun N-terminal protein kinase activation and produced much less chemokine than Pneumocystis-stimulated WT AECs. Using a panel of TLR agonists, primary murine AECs were found to respond vigorously to TLR2 and TLR4 agonists. However, the AEC chemokine response to Pneumocystis did not require TLR2 or TLR4. Surprisingly, the interleukin-1 receptor (IL-1R) was required for an AEC chemokine response to Pneumocystis. The role of MyD88 in early responses during Pneumocystis infection was supported by in vivo studies demonstrating that MyD88-deficient mice showed impaired Pneumocystis-stimulated chemokine production and impaired inflammatory cell recruitment. These data indicate an important role for MyD88 in the AEC inflammatory response to Pneumocystis.