Endotoxin-induced uveitis is primarily dependent on radiation-resistant cells and on MyD88 but not TRIF

TLR4 activation by LPS (endotoxin) is mediated by the MyD88 and TRIF intracellular signaling pathways. We determined the relative activation of these pathways in murine ocular tissue after LPS exposure. Additionally, we explored whether BM-derived or non-BM-derived cells were the major contributors to EIU. Mice deficient in TRIF or MyD88 and their congenic (WT) controls received 250 ng ultrapure LPS ivt at 0 h. Ocular inflammation was assessed by histological analysis at 4, 6, and 24 h, and additionally, in MyD88(-/-) mice, intravital microscopy was performed at 4 h and 6 h to assess adherent, rolling, and infiltrating cells in the iris vasculature and tissue. Cytokines associated with the MyD88 and TRIF intracellular signaling pathways were analyzed in ocular tissue at 4 h. BM chimeric mice (WT→WT, TLR4(-/-)→WT, WT→TLR4(-/-)) received 250 ng LPS by ivt injection, and ocular tissues were examined by histology at 6 h. Lack of MyD88 resulted in a markedly diminished cellular response and reduced production of MyD88-related cytokines 4 h post-LPS treatment. In contrast, lack of TRIF led to reduced production of TRIF-related cytokines and no change in the cellular response to LPS. Therefore, the MyD88 pathway appears to be the dominant TLR4 pathway in EIU. Only WT → TLR4(-/-) chimeric mice were resistant to EIU, and this suggests, surprisingly, that non-BM-derived (radiation-resistant) cells in the eye play a greater role than BM-derived cells.     

Monocyte and neutrophil recruitment during oral Salmonella infection is driven by MyD88‐derived chemokines

Oral Salmonella infection recruits phagocytes to Peyer's patches (PP) and MLN. The chemokines induced in infected PP and MLN, the cellular sources during infection and the TLR signaling pathways involved in vivo are not known. Here, we show that CCL2, CXCL9 and CXCL2 mRNA are up‐regulated in PP and MLN coincident with the first arrival of monocytes and neutrophils. Laser capture microdissection microscopy revealed that chemokine mRNA up‐regulation was differently distributed in PP. Despite this, recruited monocytes and neutrophils formed inflammatory cell clusters throughout PP. Monocytes and neutrophils purified from infected mice preferentially produced CXCL2 and small amounts of CCL2, and neutrophils from infected mice migrated towards CXCL2 and CCL3. Furthermore, phagocyte recruitment to PP and MLN was intact in mice lacking TLR4 alone and when signaling through TLR4 and TLR5 was simultaneously absent; however, recruitment was compromised in MyD88^?/? and more so in MyD88^?/?TLR4^?/? double knockout mice. Phagocyte release into the blood, however, was only marginally reduced in MyD88^?/?TLR4^?/? mice. Defective phagocyte recruitment to PP and MLN of MyD88^?/?TLR4^?/? mice was paralleled by low chemokine induction. These data provide insight into the chemokines and TLR signaling pathways that orchestrate the early phagocyte response to oral Salmonella infection.     

MyD88-dependent recruitment of monocytes and dendritic cells required for protection from pulmonary Burkholderia mallei infection

The Gram-negative bacterium Burkholderia mallei causes rapidly fatal illness in equines and humans when contracted by inhalation and also has the potential to be used as a bioweapon. However, little is known regarding the early innate immune responses and signaling mechanisms required to generate protection from pneumonic B. mallei infection. We showed previously that monocyte chemoattractant protein 1 (MCP-1) was a critical chemokine required for protection from pneumonic B. mallei infection. We have now extended those studies to identify key Toll-like receptor (TLR) signaling pathways, effector cells, and cytokines required for protection from respiratory B. mallei infection. We found that MyD88-/- mice were highly susceptible to pulmonary challenge with B. mallei and had significantly short survival times, increased bacterial burdens, and severe organ pathology compared to wild-type mice. Notably, MyD88-/- mice had significantly fewer monocytes and dendritic cells (DCs) in lung tissues and airways than infected wild-type mice despite markedly higher bacterial burdens. The MyD88-/- mice were also completely unable to produce gamma interferon (IFN-γ) at any time points following infection. In wild-type mice, NK cells were the primary cells producing IFN-γ in the lungs following B. mallei infection, while DCs and monocytes were the primary cellular sources of interleukin-12 (IL-12) production. Treatment with recombinant IFN-γ (rIFN-γ) was able to significantly restore protective immunity in MyD88-/- mice. Thus, we conclude that the MyD88-dependent recruitment of inflammatory monocytes and DCs to the lungs and the local production of IL-12, followed by NK cell production of IFN-γ, are the key initial cellular responses required for early protection from B. mallei infection.     

Sequential MyD88-independent and -dependent activation of innate immune responses to intracellular bacterial infection

Microbial infections induce chemokine and cytokine cascades that coordinate innate immune defenses. Infection with the intracellular bacterial pathogen Listeria monocytogenes induces CCR2-dependent monocyte recruitment and activation, an essential response for host survival. Herein we show that invasive L. monocytogenes, but not killed or noninvasive bacteria, induce secretion of MCP-1, the requisite chemokine for monocyte recruitment. Induction of MCP-1, but not TNF or IL-12, following L. monocytogenes infection is MyD88 independent. Consistent with these results, MyD88 deficiency does not impair monocyte recruitment to L. monocytogenes infected spleens, but prevents monocyte activation. Our results indicate that distinct microbial signals activate innate immune responses in an ordered, step-wise fashion, providing a mechanism to specify and modulate antimicrobial effector functions.     

MyD88 signaling is required for efficient innate and adaptive immune responses to Paracoccidioides brasiliensis infection

The mechanisms that govern the initial interaction between Paracoccidioides brasiliensis, a primary dimorphic fungal pathogen, and cells of the innate immunity need to be clarified. Our previous studies showed that Toll-like receptor 2 (TLR2) and TLR4 regulate the initial interaction of fungal cells with macrophages and the pattern of adaptive immunity that further develops. The aim of the present investigation was to assess the role of MyD88, an adaptor molecule used by TLRs to activate genes of the inflammatory response in pulmonary paracoccidioidomycosis. Studies were performed with normal and MyD88(-/-) C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells. MyD88(-/-) macrophages displayed impaired interaction with fungal yeast cells and produced low levels of IL-12, MCP-1, and nitric oxide, thus allowing increased fungal growth. Compared with wild-type (WT) mice, MyD88(-/-) mice developed a more severe infection of the lungs and had marked dissemination of fungal cells to the liver and spleen. MyD88(-/-) mice presented low levels of Th1, Th2, and Th17 cytokines, suppressed lymphoproliferation, and impaired influx of inflammatory cells to the lungs, and this group of cells comprised lower numbers of neutrophils, activated macrophages, and T cells. Nonorganized, coalescent granulomas, which contained high numbers of fungal cells, characterized the severe lesions of MyD88(-/-) mice; the lesions replaced extensive areas of several organs. Therefore, MyD88(-/-) mice were unable to control fungal growth and showed a significantly decreased survival time. In conclusion, our findings demonstrate that MyD88 signaling is important in the activation of fungicidal mechanisms and the induction of protective innate and adaptive immune responses against P. brasiliensis.     

Role of TLRs/MyD88 in host resistance and pathogenesis during protozoan infection: lessons from malaria

Toll-like receptors (TLRs) are important to initiate the innate immune response to a wide variety of pathogens. The protective role of TLRs during infection with protozoan parasites has been established. In this regard, malaria represents an exception where activation of TLRs seems to be deleterious to the host. In this article, we review the recent findings indicating the contrasting role of Myeloid Differentiation Primary-Response gene 88 (MyD88) and TLRs during malaria and infection with other protozoa. These findings suggest that MyD88 may represent an Achilles’ heel during Plasmodium infection.     

Functional aspects of Toll‐like receptor/MyD88 signalling during protozoan infection: focus on Toxoplasma gondii

Toll‐like receptor (TLR)/MyD88 signalling has emerged as a major pathway of pathogen recognition in the innate immune system. Here, we review recent data that begin to show how this pathway controls the immune response to protozoan infection, with particular emphasis on the opportunistic pathogen Toxoplasma gondii. The various ways that the parasite activates and suppresses TLR/MyD88 signalling defines several key principals that illuminate the complexities of the host–pathogen interaction. We also speculate how TLR/MyD88 signalling might be exploited to provide protection against Toxoplasma, as well as other protozoa and infection in general.     

SHPS-1 and a synthetic peptide representing its ITIM inhibit the MyD88, but not TRIF, pathway of TLR signaling through activation of SHP and PI3K in THP-1 cells

Background

Src homology 2 domain-containing protein tyrosine phosphatase substrate (SHPS)-1 is known to have regulatory effects on myeloid cells. However, its role in macrophage activation is not clearly understood.

Methods and results

In order to investigate the role of SHPS-1 in Toll-like receptor (TLR)-mediated activation, human monocytic cell lines were treated with anti-SHPS-1 monoclonal antibody. The triggering of SHPS-1 blocked the expression of IL-8 and TNF-α in cells treated with a TLR4 ligand that induces a signaling pathway involving myeloid differentiation factor 88 (MyD88) and Toll-interleukin-1 receptor (TIR)-domain-containing adapter-inducing interferon-β (TRIF). Interestingly, SHPS-1 inhibited TLR9/MyD88-mediated, but not TLR3/TRIF-mediated, expression of IL-8. Accordingly, a synthetic peptide representing the immunoreceptor tyrosine-based inhibition motif (ITIM) of SHPS-1 suppressed only the MyD88 pathway. Utilization of specific inhibitors and Western blot analysis indicated that the inhibitory effects were mediated by Src homology 2 domain-containing phosphatases (SHPs) and phosphoinositide 3-kinase (PI3K).

Conclusion

SHPS-1 negatively regulates the MyD88-dependent TLR signaling pathway through the inhibition of NF-κB activation.     

Local and remote tissue injury upon intestinal ischemia and reperfusion depends on the TLR/MyD88 signaling pathway

Innate immune responses against microorganisms may be mediated by Toll-like receptors (TLRs). Intestinal ischemia–reperfusion (i-I/R) leads to the translocation of bacteria and/or bacterial products such as endotoxin, which activate TLRs leading to acute intestinal and lung injury and inflammation observed upon gut trauma. Here, we investigated the role of TLR activation by using mice deficient for the common TLR adaptor protein myeloid differentiation factor 88 (MyD88) on local and remote inflammation following intestinal ischemia. Balb/c and MyD88^?/? mice were subjected to occlusion of the superior mesenteric artery (45 min) followed by intestinal reperfusion (4 h). Acute neutrophil recruitment into the intestinal wall and the lung was significantly diminished in MyD88^?/? after i-I/R, which was confirmed microscopically. Diminished neutrophil recruitment was accompanied with reduced concentration of TNF-α and IL-1β level. Furthermore, diminished microvascular leak and bacteremia were associated with enhanced survival of MyD88^?/? mice. However, neither TNF-α nor IL-1β neutralization prevented neutrophil recruitment into the lung but attenuated intestinal inflammation upon i-I/R. In conclusion, our data demonstrate that disruption of the TLR/MyD88 pathway in mice attenuates acute intestinal and lung injury, inflammation, and endothelial damage allowing enhanced survival.     

Monocyte and dendritic cell recruitment and activation during oral Salmonella infection

Immunity to bacterial infection involves the joint effort of the innate and adaptive immune systems. The innate immune response is triggered when the body senses bacterial components, such as lipopolysaccharide, that alarm the body of the invader. An array of cell types function in the innate response. These cells are rapidly recruited to the infection site and activated to optimally perform their functions. The adaptive immune response follows the innate response, and one cell type in particular, dendritic cells (DCs), are the critical link between the innate and adaptive responses. This review will summarize recent data concerning the events that occur early during oral infection with the intracellular pathogen Salmonella, with emphasis on the phagocytic cells involved in combating the infection in the gut-associated lymphoid tissues. In particular, recent findings concerning the recruitment and activation of mononuclear phagocyte populations and dendritic cell subsets will be presented after an overview of the Salmonella infection model.     

MyD88 deficiency leads to decreased NK cell gamma interferon production and T cell recruitment during Chlamydia muridarum genital tract infection, but a predominant Th1 response and enhanced monocytic inflammation are associated with infection resolution

We have previously shown that MyD88 knockout (KO) mice exhibit delayed clearance of Chlamydia muridarum genital infection compared to wild-type (WT) mice. A blunted Th1 response and ineffective suppression of the Th2 response were also observed in MyD88 KO mice. The goal of the present study was to investigate specific mechanisms whereby absence of MyD88 leads to these effects and address the compensatory mechanisms in the genital tract that ultimately clear infection in the absence of MyD88. It was observed that NK cells recruited to the genital tract in MyD88 KO mice failed to produce gamma interferon (IFN-γ) mRNA and protein. This defect was associated with decreased local production of interleukin-17 (IL-17), IL-18, and tumor necrosis factor alpha (TNF-α) but normal levels of IL-12p70. Additionally, recruitment of CD4 T cells to the genital tract was reduced in MyD88 KO mice compared to that in WT mice. Although chronic infection in MyD88 KO mice resulted in oviduct pathology comparable to that of WT mice, increased histiocytic inflammation was observed in the uterine horns. This was associated with increased CCL2 levels and recruitment of macrophages as a potential compensatory mechanism. Further deletion of TLR4-TRIF signaling in MyD88 KO mice, using TLR4/MyD88 double-KO mice, did not further compromise host defense against chlamydiae, suggesting that compensatory mechanisms are Toll-like receptor (TLR) independent. Despite some polarization toward a Th2 response, a Th1 response remained predominant in the absence of MyD88, and it provided equivalent protection against a secondary infection as observed in WT mice.     

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.     

High levels of susceptibility and T helper 2 response in MyD88-deficient mice infected with Leishmania major are interleukin-4 dependent

Myeloid differentiation protein 88 (MyD88) is a general adaptor for the signaling cascade through receptors of the Toll/IL-1R family. When infected with Leishmania major parasites, MyD88-deficient mice displayed a dramatically enhanced parasite burden in their tissues similar to that found in susceptible BALB/c mice. In contrast, MyD88 knockout mice did not develop ulcerating lesions despite a lack of interleukin-12 (IL-12) production and a predominant T helper 2 cell response. Blockade of IL-4 produced early (day 1) after infection restored a protective T helper 1 response in MyD88 knockout mice.     

Staphylococcal enterotoxin A induction of pro-inflammatory cytokines and lethality in mice is primarily dependent on MyD88

Toll‐like receptors (TLRs) are germline‐encoded innate immune receptors that recognize invading micro‐organisms and induce immune and inflammatory responses. Deregulation of TLRs is known to be closely linked to various immune disorders and inflammatory diseases. Cells at sites of inflammation are exposed to hypoxic stress, which further aggravates inflammatory processes. We have examined if hypoxic stress modulates the TLR activity of macrophages. Hypoxia and CoCl₂ (a hypoxia mimetic) enhanced the expression of TLR4 messenger RNA and protein in macrophages (RAW264.7 cells), whereas the messenger RNA of other TLRs was not increased. To determine the underlying mechanism, we investigated the role of hypoxia‐inducible factor 1 (HIF‐1) in the regulation of TLR4 expression. Knockdown of HIF‐1α expression by small interfering RNA inhibited hypoxia‐induced and CoCl₂‐induced TLR4 expression in macrophages, while over‐expression of HIF‐1α potentiated TLR4 expression. Chromatin immunoprecipitation assays revealed that HIF‐1α binds to the TLR4 promoter region under hypoxic conditions. In addition, deletion or mutation of a putative HIF‐1‐binding motif in the TLR4 promoter greatly attenuated HIF‐1α‐induced TLR4 promoter reporter expression. Up‐regulation of TLR4 expression by hypoxic stress enhanced the response of macrophages to lipopolysaccharide, resulting in increased expression of cyclooxygenase‐2, interleukin‐6, regulated on activation normal T cell expressed and secreted, and interferon‐inducible protein‐10. These results demonstrate that TLR4 expression in macrophages is up‐regulated via HIF‐1 in response to hypoxic stress, suggesting that hypoxic stress at sites of inflammation enhances susceptibility to subsequent infection and inflammatory signals by up‐regulating TLR4.     

Downregulation of Paralemmin-3 Ameliorates Lipopolysaccharide-Induced Acute Lung Injury in Rats by Regulating Inflammatory Response and Inhibiting Formation of TLR4/MyD88 and TLR4/TRIF Complexes

Previous studies have demonstrated paralemmin-3 (PALM3) participates in Toll-like receptor (TLR) signaling. This study investigated the effect of PALM3 knockdown on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and its underlying mechanisms. We constructed a recombinant adenoviral vector containing short hairpin RNA for PALM3 to knockdown PALM3 expression. A transgene-free adenoviral vector was used as a negative control. The ALI rat model was established by LPS peritoneal injection at 48-h post-transfection. Results showed that downregulation of PALM3 improved the survival rate, attenuated lung pathological changes, alleviated pulmonary edema, lung vascular leakage and neutrophil infiltration, inhibited the production of proinflammatory cytokines and activation of nuclear factor κB and interferon β regulatory factor 3, and promoted the secretion of anti-inflammatory cytokine interleukin-10 and expression of suppressor of cytokine signaling-3 in the ALI rat model. However, PALM3 knockdown had no effect on TLR4, myeloid differentiation factor 88 (MyD88), and Toll-interleukin-1 receptor domain-containing adaptor inducing interferon β (TRIF) expression. Moreover, PALM3 knockdown reduced the interaction of TLR4 with MyD88 or TRIF induced by LPS in rat lungs. Therefore, the downregulation of PALM3 protected rats from LPS-induced ALI and its mechanisms were partially associated with the modulation of inflammatory responses and inhibition of TLR4/MyD88 and TLR4/TRIF complex formation.     

CD300a and CD300f differentially regulate the MyD88 and TRIF-mediated TLR signalling pathways through activation of SHP-1 and/or SHP-2 in human monocytic cell lines

CD300a, a membrane protein expressed on myeloid lineages and specific subsets of CD4(+) T cells, has been reported to have inhibitory activities in cellular activation. However, the role of CD300a in Toll-like receptor (TLR) -mediated macrophage activation has not been investigated. The human monocytic cell lines THP-1 and U937 were stimulated with various TLR ligands after triggering of CD300a with specific monoclonal antibody. Interestingly, CD300a blocked TLR4-mediated and TLR9-mediated expression of pro-inflammatory mediators without affecting TLR3-mediated events. In contrast, CD300f, another member of the CD300 family, blocked the activation of cells induced by all TLR ligands. A transient transfection assay using luciferase reporter gene under the regulation of nuclear factor-κB binding sites indicated that co-transfection of CD300f blocked reporter expression induced by over-expression of both myeloid differentiation factor 88 (MyD88) and toll-interleukin 1 receptor-domain-containing adapter-inducing interferon-β (TRIF), whereas CD300a blocked only MyD88-induced events. Synthetic peptides representing immunoreceptor tyrosine-based inhibitory motifs of CD300a or CD300f mimicked the differential inhibition patterns of their original molecules. The use of various signalling inhibitors and Western blotting analysis revealed that TLR9/MyD88-mediated signalling was regulated mainly by SH2-containing tyrosine phosphatase 1 (SHP-1), which could be activated by CD300a or CD300f. In contrast, regulation of the TLR3/TRIF-mediated pathway required the combined action of SHP-1 and SHP-2, which could be accomplished by CD300f but not CD300a. These data indicate that CD300a and CD300f regulate the MyD88 and TRIF-mediated TLR signalling pathways through differential activation of SHP-1 and SHP-2.     

IMQ‐induced skin inflammation in mice is dependent on IL‐1R1 and MyD88 signaling but independent of the NLRP3 inflammasome

The pathogenesis of inflammatory skin diseases such as psoriasis involves the release of numerous proinflammatory cytokines, including members of the IL‐1 family. Here we report overexpression of IL‐1α, IL‐1β, and IL‐1 receptor antagonist mRNA, associated to expression of IL‐23p19, IL‐17A, and IL‐22 in skin cells, upon topical application of the TLR7 agonist imiquimod (IMQ) in C57BL/6J mice. IMQ‐induced skin inflammation was partially reduced in mice deficient for both IL‐1α/IL‐1β or for IL‐1 receptor type 1 (IL‐1R1), but not in IL‐1α‐ or IL‐1β‐deficient mice, demonstrating the redundant activity of IL‐1α and IL‐1β for skin inflammation. NLRP3 or apoptosis‐associated Speck‐like protein containing a Caspase recruitment domain‐deficient mice had no significant reduction of skin inflammation in response to IMQ treatment, mainly due to the redundancy of IL‐1α. However, IMQ‐induced skin inflammation was abolished in the absence of MyD88, the adaptor protein shared by IL‐1R and TLR signaling pathways. These results are consistent with the TLR7 dependence of IMQ‐induced skin inflammation. Thus, IL‐1R1 contributes to the IMQ‐induced skin inflammation, and disruption of MyD88 signaling completely abrogates this response.