Interleukin-8 Receptor 2 (IL-8R2)-Deficient Mice Are More Resistant to Pulmonary Coccidioidomycosis than Control Mice.

The pathology of human coccidioidomycosis is granulomatous inflammation with many neutrophils surrounding ruptured spherules, but the chemotactic pathways that draw neutrophils into the infected tissues are not known. We previously showed that formalin-killed spherules (FKS) stimulate mouse macrophages to secret macrophage inflammatory protein 2 (MIP-2), which suggested that CXC ELR+ chemokines might be involved in neutrophil recruitment in vivo To test that hypothesis, we intranasally infected interleukin-8R2 (IL-8R2) (Cxcr2)-deficient mice on a BALB/c background with Coccidioides immitis RS. IL-8R2-deficient mice had fewer neutrophils in infected lungs than controls, but unexpectedly the IL-8R2-deficient mice had fewer organisms in their lungs than the control mice. Infected IL-8R2-deficient mouse lungs had higher expression of genes associated with lymphocyte activation, including the Th1 and Th17-related cytokines Ifnγ and Il17a and the transcription factors Stat1 and Rorc Additionally, bronchial alveolar lavage fluid from infected IL-8R2-deficient mice contained more IL-17A and interferon-γ (IFN-γ). We postulate that neutrophils in the lung directly or indirectly interfere with the development of a protective Th1/Th17 immune response to C. immitis at the site of infection.

APX001 and Other Gwt1 Inhibitor Prodrugs Are Effective in Experimental Coccidioides immitis Pneumonia.

Coccidioidomycosis is a systemic fungal infection caused by the inhalation of the arthroconidia of either of two closely related dimorphic fungi, Coccidioides immitis and C. posadasii, that are endemic in the southwestern United States and other areas in the Western Hemisphere. Chronic cavitary pulmonary infections and extrapulmonary sites of infection are very difficult to treat and often require lifelong azole therapy. APX001A is the first in a new class of broad-spectrum antifungal agents that inhibit Gwt1, an enzyme which is required for cell wall localization of glycosylphosphatidylinositol (GPI)-anchored mannoproteins in fungi. APX001A and several analogs were highly active against clinical isolates of Coccidioides, inhibiting hyphal growth at low nanogram/ml concentrations. APX001 is the N-phosphonooxymethyl prodrug of APX001A, currently in clinical trials for the treatment of invasive fungal infections. Mice were treated orally once daily with 26 mg/kg/day of APX001 and the prodrug analog APX2097, 2 h after administration of the pan-cytochrome P450 inhibitor 1-aminobenzotriazole, which was used to enhance drug half-life and exposures to more closely mimic human pharmacokinetics of APX001A. Five days of treatment reduced lung colony counts by nearly 3 logs and prevented dissemination, similar to the efficacy of fluconazole dosed orally at 25 mg/kg twice daily. In a survival experiment, both APX001- and APX2097-treated mice survived significantly longer than control and fluconazole-treated mice. APX001 and other members of this new class of antifungal agents may offer great promise as effective therapies for coccidioidomycosis.

Myeloid Differentiation Factor 88 and Interleukin-1R1 Signaling Contribute to Resistance to Coccidioides immitis.

Rodents are a natural host for the dimorphic pathogenic fungi Coccidioides immitis and Coccidioides posadasii, and mice are a good model for human infection. Humans and rodents both express Dectin-1 and Toll-like receptor 2 (TLR2) on myeloid cells, and those receptors collaborate to maximize the cytokine/chemokine responses to spherules (the tissue form of the fungi) and to formalin-killed spherules (FKS). We showed that Dectin-1 is necessary for resistance to pulmonary coccidioidomycosis, but the importance of TLR2 in vivo is uncertain. Myeloid differentiation factor 88 (MyD88) is the adapter protein for TLR2 and -4, interleukin-1R1 (IL-1R1), and IL-18R1. MyD88/TRIF-/- and MyD88-/- mice were equally susceptible to C. immitis infection, in contrast to C57BL/6 (B6) controls. Of the four surface receptors, only IL-1R1 was required for resistance to C. immitis, partially explaining the susceptibility of MyD88-/- mice. We also found that FKS stimulated production of IL-1Ra by bone marrow-derived dendritic cells (BMDCs), independent of MyD88 and Dectin-1. There also was a very high concentration of IL-1Ra in the lungs of infected B6 mice, supporting the potential importance of this regulatory IL-1 family protein in the largely ineffective response of B6 mice to coccidioidomycosis. These results suggest that IL-1R1 signaling is important for defense against C. immitis infection.

MD-2 binds cholesterol.

Cholesterol is a structural component of cellular membranes, which is transported from liver to peripheral cells in the form of cholesterol esters (CE), residing in the hydrophobic core of low-density lipoprotein. Oxidized CE (OxCE) is often found in plasma and in atherosclerotic lesions of subjects with cardiovascular disease. Our earlier studies have demonstrated that OxCE activates inflammatory responses in macrophages via toll-like receptor-4 (TLR4). Here we demonstrate that cholesterol binds to myeloid differentiation-2 (MD-2), a TLR4 ancillary molecule, which is a binding receptor for bacterial lipopolysaccharide (LPS) and is indispensable for LPS-induced TLR4 dimerization and signaling. Cholesterol binding to MD-2 was competed by LPS and by OxCE-modified BSA. Furthermore, soluble MD-2 in human plasma and MD-2 in mouse atherosclerotic lesions carried cholesterol, the finding supporting the biological significance of MD-2 cholesterol binding. These results help understand the molecular basis of TLR4 activation by OxCE and mechanisms of chronic inflammation in atherosclerosis.

Neither dectin-2 nor the mannose receptor is required for resistance to Coccidioides immitis in mice.

We investigated the roles of the mannose receptor (MR) and Dectin-2 in resistance to pulmonary coccidioidomycosis in C57BL/6 (B6) mice and in the interaction of myeloid cells with spherules, using B6 mice with targeted mutations in Mrc1 and Clec4n. Spherules are the tissue form of Coccidioides, and we determined that the MR on bone marrow-derived dendritic cells (BMDC) was important for recognition of spherules (formalin-killed spherules [FKS]) and for secretion of interleukin 10 (IL-10) and proinflammatory cytokines in response to FKS by both elicited macrophages and BMDC. Infected MR knockout (KO) mice produced more IL-10 in their lungs than did B6 mice, and MR KO mice also made more protective Th-17 cytokines. In contrast to the MR, Dectin-2 was not required for recognition of FKS by BMDC or for the production of cytokines by BMDC in response to FKS. However, Dectin-2 KO was required for stimulation of elicited peritoneal macrophages. Despite that, lung cytokine levels were not significantly different in Dectin-2 KO mice and B6 mice 14 days after infection, except for IL-1ß, which was higher in Dectin-2 KO lungs. Although both Dectin-2(-/-) and MR(-/-) myeloid cells had reduced proinflammatory cytokine responses to FKS in vitro, neither MR nor Dectin-2 deficiency reduced the resistance of B6 mice to pulmonary coccidioidomycosis.

Gene expression in human fungal pathogen Coccidioides immitis changes as arthroconidia differentiate into spherules and mature.

BACKGROUND: Coccidioides immitis is a dimorphic fungus that causes disease in mammals, including human beings. It grows as a mycelium containing arthroconidia in the soil and in the host arthroconidia differentiates into a unique structure called a spherule. We used a custom open reading frame oligonucleotide microarray to compare the transcriptome of C. immitis mycelia with early (day 2) and late stage (day 8) spherules grown in vitro. All hybridizations were done in quadruplicate and stringent criteria were used to identify significantly differentially expressed genes. RESULTS: 22% of C. immitis genes were differentially expressed in either day 2 or day 8 spherules compared to mycelia, and about 12% of genes were differentially expressed comparing the two spherule time points. Oxireductases, including an extracellular superoxide dismutase, were upregulated in spherules and they may be important for defense against oxidative stress. Many signal transduction molecules, including pleckstrin domain proteins, protein kinases and transcription factors were downregulated in day 2 spherules. Several genes involved in sulfur metabolism were downregulated in day 8 spherules compared to day 2 spherules. Transcription of amylase and α (1,3) glucan synthase was upregulated in spherules; these genes have been found to be important for differentiation to yeast in Histoplasma. There were two homologs of 4-hydroxyphenylpyruvate dioxygenase (4-HPPD); transcription of one was up- and the other downregulated. We tested the effect of a 4-HPPD inhibitor, nitisinone, on mycelial and spherule growth and found that it inhibited mycelial but not spherule growth. CONCLUSIONS: Transcription of many genes was differentially expressed in the process of arthroconidia to spherule conversion and spherule maturation, as would be expected given the magnitude of the morphologic change. The transcription profile of early stage (day 2) spherules was different than late stage (day 8) endosporulating spherules. In addition, very few genes that are important for spore to yeast conversion in other dimorphic fungi are differentially expressed in C. immitis mycelia and spherules suggesting that dimorphic fungi may have evolved different mechanisms to differentiate from mycelia to tissue invasive forms.

Factors regulated by interferon gamma and hypoxia-inducible factor 1A contribute to responses that protect mice from Coccidioides immitis infection.

BACKGROUND: Coccidioidomycosis results from airborne infections caused by either Coccidioides immitis or C. posadasii. Both are pathogenic fungi that live in desert soil in the New World and can infect normal hosts, but most infections are self-limited. Disseminated infections occur in approximately 5% of cases and may prove fatal. Mouse models of the disease have identified strains that are resistant (e.g. DBA/2) or susceptible (e.g. C57BL/6) to these pathogens. However, the genetic and immunological basis for this difference has not been fully characterized. RESULTS: Microarray technology was used to identify genes that were differentially expressed in lung tissue between resistant DBA/2 and sensitive C57BL/6 mice after infection with C. immitis. Differentially expressed genes were mapped onto biological pathways, gene ontologies, and protein interaction networks, which revealed that innate immune responses mediated by Type II interferon (i.e., IFNG) and the signal transducer and activator of transcription 1 (STAT1) contribute to the resistant phenotype. In addition, upregulation of hypoxia inducible factor 1A (HIF1A), possibly as part of a larger inflammatory response mediated by tumor necrosis factor alpha (TNFA), may also contribute to resistance. Microarray gene expression was confirmed by real-time quantitative PCR for a subset of 12 genes, which revealed that IFNG HIF1A and TNFA, among others, were significantly differentially expressed between the two strains at day 14 post-infection. CONCLUSION: These results confirm the finding that DBA/2 mice express more Type II interferon and interferon stimulated genes than genetically susceptible strains and suggest that differential expression of HIF1A may also play a role in protection.

Decoding Transcription Regulatory Mechanisms Associated with Coccidioides immitis Phase Transition Using Total RNA.

New or emerging infectious diseases are commonly caused by pathogens that cannot be readily manipulated or studied under common laboratory conditions. These limitations hinder standard experimental approaches and our abilities to define the fundamental molecular mechanisms underlying pathogenesis. The advance of capped small RNA sequencing (csRNA-seq) now enables genome-wide mapping of actively initiated transcripts from genes and other regulatory transcribed start regions (TSRs) such as enhancers at a precise moment from total RNA. As RNA is nonpathogenic and can be readily isolated from inactivated infectious samples, csRNA-seq can detect acute changes in gene regulation within or in response to a pathogen with remarkable sensitivity under common laboratory conditions. Studying valley fever (coccidioidomycosis), an emerging endemic fungal infection that increasingly impacts livestock, pet, and human health, we show how csRNA-seq can unravel transcriptional programs driving pathogenesis. Performing csRNA-seq on RNA isolated from different stages of the valley fever pathogen Coccidioides immitis revealed alternative promoter usage, connected cis-regulatory domains, and a WOPR family transcription factor, which are known regulators of virulence in other fungi, as being critical for pathogenic growth. We further demonstrate that a C. immitis WOPR homologue, CIMG_02671, activates transcription in a WOPR motif-dependent manner. Collectively, these findings provide novel insights into valley fever pathogenesis and provide a proof of principle for csRNA-seq as a powerful means to determine the genes, regulatory mechanisms, and transcription factors that control the pathogenesis of highly infectious agents. IMPORTANCE Infectious pathogens like airborne viruses or fungal spores are difficult to study; they require high-containment facilities, special equipment, and expertise. As such, establishing approaches such as genome editing or other means to identify the factors and mechanisms underlying caused diseases, and, thus, promising drug targets, is costly and time-intensive. These obstacles particularly hinder the analysis of new, emerging, or rare infectious diseases. We recently developed a method termed capped small RNA sequencing (csRNA-seq) that enables capturing acute changes in active gene expression from total RNA. Prior to csRNA-seq, such an analysis was possible only by using living cells or nuclei, in which pathogens are highly infectious. The process of RNA purification, however, inactivates pathogens and thus enables the analysis of gene expression during disease progression under standard laboratory conditions. As a proof of principle, here, we use csRNA-seq to unravel the gene regulatory programs and factors likely critical for the pathogenesis of valley fever, an emerging endemic fungal infection that increasingly impacts livestock, pet, and human health.

The role of reactive oxygen intermediates in experimental coccidioidomycois in mice.

BACKGROUND: Coccidioidomycosis is usually a self-limited infection in immunocompentent people. In immunocompentent human beings second infections due to Coccidioides are very rare, indicating that recovery from infection results in protective immunity. In experimental animals, immunization with several different proteins or attenuated mutants protects against a virulent challenge. To explore what mechanisms are responsible for protective immunity, we investigated the course of Coccidioides infection in the gp91phox knock out mouse that has a defect in the oxidative burst that results in chronic granulomatous disease. RESULTS: We found that the gp91phox knock out mice were somewhat more resistant to intraperitoneal infection and equally as resistant to low dose intranasal infection, but slightly more susceptible to high dose intranasal infection compared to control mice. The gp91phox knock out mice made a more robust inflammatory response to infection than controls, as measured by histology and production of inflammatory cytokines. The gp91phox knock out mice were as protected by immunization with the recombinant Coccidioides protein Ag2/PRA as the controls were against either intraperitoneal or intranasal infection. Coccidioides immitis arthroconidia and spherules were significantly more resistant to H2O2 treatment in vitro than Aspergillus fumigatus spores. CONCLUSION: These data suggest that oxidative burst may not be required for protective immunity to coccidioidomycois.

Transcriptional Analysis of Coccidioides immitis Mycelia and Spherules by RNA Sequencing.

Coccidioides immitis and C. posadasii are dimorphic fungi that transform from mycelia with internal arthroconidia in the soil to a tissue form known as a spherule in mammals. This process can be recapitulated in vitro by increasing the temperature, CO2 and changing other culture conditions. In this study, we have analyzed changes in gene expression in mycelia and young and mature spherules. Genes that were highly upregulated in young spherules include a spherule surface protein and iron and copper membrane transporters. Genes that are unique to Coccidioides spp. are also overrepresented in this group, suggesting that they may be important for spherule differentiation. Enriched GO terms in young spherule upregulated genes include oxidation-reduction, response to stress and membrane proteins. Downregulated genes are enriched for transcription factors, especially helix-loop-helix and C2H2 type zinc finger domain-containing proteins, which is consistent with the dramatic change in transcriptional profile. Almost all genes that are upregulated in young spherules remain upregulated in mature spherules, but a small number of genes are differentially expressed in those two stages of spherule development. Mature spherules express more Hsp31 and amylase and less tyrosinase than young spherules. Some expression of transposons was detected and most of the differentially expressed transposons were upregulated in spherules.

Toll-like receptor 4-dependent and -independent cytokine secretion induced by minimally oxidized low-density lipoprotein in macrophages.

OBJECTIVE: Innate immune responses to oxidized low-density lipoprotein LDL (LDL) regulate the development of atherosclerosis. We demonstrated previously that an early form of oxidized LDL, minimally modified LDL (mmLDL), triggers cytoskeletal rearrangements in macrophages via CD14 and Toll-like receptor 4 (TLR4)/MD-2. Because lipopolysaccharide (LPS) activation of TLR4 leads to proinflammatory gene expression, in this study, we asked whether mmLDL also induced proinflammatory signaling. METHODS AND RESULTS: We studied cytokine secretion and signaling in J774 and primary peritoneal macrophages stimulated with mmLDL, which was prepared by incubating LDL with cells expressing human 15-lipoxygenase. MmLDL stimulated robust phosphoinositide 3-kinase (PI3K) activation, and Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, which exceeded that induced by LPS. On the other hand, although mmLDL induced nuclear factor kappaB (NF-kappaB) p65 translocation to the nucleus, there was no detectable NF-kappaB activation. However, mmLDL induced early mRNA and protein expression of the cytokines MIP-2, MCP-1, tumor necrosis factor-alpha, and interleukin-6. Chemokine MIP-2 but not MCP-1 secretion depended on TLR4/MyD88, ERK1/2, and PI3K signaling. In turn, TLR4 regulated phosphorylation of ERK1/2 but not of Akt, suggesting that mmLDL-induced PI3K activation is TLR4 independent. CONCLUSIONS: In macrophages, mmLDL activates TLR4-dependent and -independent signaling pathways, resulting in secretion of proinflammatory cytokines. These results provide new insights into the inflammatory origins of atherosclerosis.

Dectin-1 is required for resistance to coccidioidomycosis in mice.

We assessed the role of Dectin-1 in the immune response to the pathogenic fungus Coccidioides, both in vitro and in vivo, using mice with a targeted mutation in Clec7a. Elicited peritoneal macrophages responded to formalin-killed spherules (FKS) and alkali-treated FKS by secreting proinflammatory cytokines in a Dectin-1- and ß-glucan-dependent manner. The responses of bone marrow-derived dendritic cells (BMDC) to the same stimulants were more complex; interleukin 1ß (IL-1ß) and tumor necrosis factor alpha (TNF-α) secretion was independent of Dectin-1, while IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were largely but not entirely dependent on Dectin-1. After intranasal infection, Dectin-1(-/-) mice had lower concentrations of IL-12p70, gamma interferon (IFN-γ), IL-1ß, and the Th17 cytokines IL-22, IL-23, and 17A in the lung lavage fluid, which may explain why they were significantly more susceptible to pulmonary coccidioidomycosis two weeks after infection. The Dectin-1 mutation was even more deleterious in (B6 × DBA/2)F(2) mice, which are more resistant to coccidioidomycosis than B6 mice by virtue of protective genes from DBA/2, a genetically resistant strain. We also found that two susceptible strains of mice (B6 and BALB/c) expressed much less Dectin-1 in their lungs than did resistant DBA/2 mice. We conclude that Dectin-1 is necessary for resistance to Coccidioides immitis, that Dectin-1 promotes both Th1 and Th17 protective immune responses to this infection, and that there is a correlation between expression of Dectin-1 by the inflammatory infiltrate and resistance to coccidioidomycosis. IMPORTANCE Coccidioidomycosis is a fungal infection endemic in the southwestern United States and neighboring Mexico, causing ~150,000 lung infections in people and resulting in ~17,000 hospitalizations annually in California alone. Very little is known about innate immunity to this fungus. This paper shows that Dectin-1, the primary ß-glucan receptor on myeloid cells, is required for resistance to this pathogen. Dectin-1 is part of the innate immune system, and it is needed to direct the acquired immune response toward into a pathway that will lead to macrophage activation. Lungs from infected mice lacking Dectin-1 had lower concentrations of Th1 and Th17 cytokines, two cytokine pathways that are very important for acquired T cell immunity to Coccidioides spp. This is the first demonstration that Dectin-1 is required for host resistance to a dimorphic, primary pathogenic fungus.

Mutational analysis of membrane and soluble forms of human MD-2.

Toll-like receptor 4 and MD-2 form a receptor for lipopolysaccharide (LPS), a major constituent of Gram-negative bacteria. MD-2 is a 20-25-kDa extracellular glycoprotein that binds to Tolllike receptor 4 (TLR4) and LPS and is a critical part of the LPS receptor. Here we have shown that the level of MD-2 expression regulates TLR4 activation by LPS. Using site-directed mutagenesis, we have found that glycosylation has no effect on MD-2 function as a membrane receptor for LPS. We used alanine-scanning mutagenesis to identify regions of human MD-2 that are important for TLR4 and LPS binding. We found that mutation in the N-terminal 46 amino acids of MD-2 did not substantially diminish LPS activation of Chinese hamster ovary (CHO) cells co-transfected with TLR4 and mutant MD-2. The residues 46-50 were important for LPS activation but not LPS binding. The residues 79-83, 121-124, and 125-129 are identified as important in LPS activation but not surface expression of membrane MD-2. The function of soluble MD-2 is somewhat more sensitive to mutation than membrane MD-2. Our results suggest that the 46-50 and 127-131 regions of soluble MD-2 bind to TLR4. The region 79-120 is not involved in LPS binding but affects monomerization of soluble MD-2 as well as TLR4 binding. We define the LPS binding region of monomeric soluble MD-2 as a cluster of basic residues 125-131. Studies on both membrane and soluble MD-2 suggest that domains of MD-2 for TLR4 and LPS binding are separate as well as overlapping. By mapping these regions on a three-dimensional model, we show the likely binding regions of MD-2 to TLR4 and LPS.

Engagement of CD14 mediates the inflammatory potential of monosodium urate crystals.

Phagocyte ingestion of monosodium urate (MSU) crystals can induce proinflammatory responses and trigger acute gouty inflammation. Alternatively, the uptake of MSU crystals by mature macrophages can be noninflammatory and promote resolution of gouty inflammation. Macrophage activation by extracellular MSU crystals involves apparent recognition and ingestion mediated by TLR2 and TLR4, with subsequent intracellular recognition linked to caspase-1 activation and IL-1beta processing driven by the NACHT-LRR-PYD-containing protein-3 inflammasome. In this study, we examined the potential role in gouty inflammation of CD14, a phagocyte-expressed pattern recognition receptor that functionally interacts with both TLR2 and TLR4. MSU crystals, but not latex beads, directly bound recombinant soluble (s) CD14 in vitro. CD14(-/-) bone marrow-derived macrophages (BMDMs) demonstrated unimpaired phagocytosis of MSU crystals but reduced p38 phosphorylation and approximately 90% less IL-1beta and CXCL1 release. Attenuated MSU crystal-induced IL-1beta release in CD14(-/-) BMDMs was mediated by decreased pro-IL-1beta protein expression and additionally by decreased caspase-1 activation and IL-1beta processing consistent with diminished NACHT-LRR-PYD-containing protein-3 inflammasome activation. Coating of MSU crystals with sCD14, but not sTLR2 or sTLR4, restored IL-1beta and CXCL1 production in CD14(-/-) BMDMs in vitro. Gain of function of CD14 directly enhanced TLR4-mediated signaling in response to MSU crystals in transfected Chinese hamster ovary cells in vitro. Last, MSU crystal-induced leukocyte influx at 6 h was reduced by approximately 75%, and local induction of IL-1beta decreased by >80% in CD14(-/-) mouse s.c. air pouches in vivo. We conclude that engagement of CD14 is a central determinant of the inflammatory potential of MSU crystals.

Toll-like receptor 4 protects against lethal Leptospira interrogans serovar icterohaemorrhagiae infection and contributes to in vivo control of leptospiral burden.

The roles of innate immune responses in protection from or pathogenesis of severe leptospirosis remain unclear. We examined the role of Toll-like receptors (TLRs) in mouse infection and macrophage responses to Leptospira. C3H/HeJ mice (TLR4 deficient) and C3H/HeJ-SCID mice, but not C3H/OuJ mice (TLR4 intact), died after intraperitoneal infection with Leptospira interrogans serovar Icterohaemorrhagiae. Death in both C3H/HeJ mouse strains was associated with jaundice and pulmonary hemorrhage, similar to the patient from whom the isolate was obtained. In chronic sublethal infection, TLR4-deficient mice harbored more leptospires in liver, lung, and kidney than control mice. Heat-killed Leptospira stimulated macrophages to secrete proinflammatory cytokines, tumor necrosis factor alpha, interleukin-6, and macrophage inflammatory protein 2 not inhibited by polymyxin B, suggesting that leptospiral lipopolysaccharide (LPS) did not drive these responses. Anti-TLR4 and anti-MD-2 but not anti-CD14 monoclonal antibodies inhibited cytokine production. Peritoneal macrophages from CD14-/- and TLR2-/- mice exhibited no defect in cytokine responses to Leptospira compared to controls. Macrophages from C3H/HeJ, TLR4-/-, and MyD88-/- mice secreted far-lower levels of cytokines than wild-type macrophages in response to Leptospira. TLR4 plays a crucial role in protection from acute lethal infection and control of leptospiral burden during sublethal chronic infection. Cytokine responses in macrophages correlated with leptospiral clearance. These TLR4-dependent but CD14/TLR2-independent responses are likely mediated by a leptospiral ligand(s) other than LPS.

Characterization of monoclonal antibodies to human soluble MD-2 protein.

Toll-like receptors (TLRs) are mammalian innate immune recognition receptors that are activated by pathogen associated molecular patterns (PAMPs). TLR4 is the signaling molecule of the lipopolysaccharide (LPS) receptor complex. TLR4 associates with its adapter molecule, MD-2, which is absolutely required for LPS-induced activation of TLR4. MD-2 exists as a cell surface protein in association with TLR4 and as secreted forms consisting of MD-2 monomers and multimers. To facilitate the studies of MD-2 distribution, abundance, and function, we produced monoclonal antibodies (MAbs) to baculovirally expressed soluble MD-2 (sMD-2). Eleven MAbs were characterized by enzyme-linked immunosorbent assay (ELISA) with soluble TLR4/MD-2 complex (sTLR4/MD-2) and sMD-2, Western blotting against sMD-2 monomer and multimers, and inhibition of direct LPS binding to sMD-2. Four MAbs preferentially recognized mainly MD-2 oligomers, not monomers, as judged by Western blotting and ELISA. Anti-MD-2 MAbs useful for indirect immunofluorescent staining of cells expressing TLR4 and MD-2 were identified. One MAb that recognized all forms of MD-2 was used in an ELISA to measure sMD-2 in normal human sera as well as sera from intensive care patients with and without sepsis. Serum levels of sMD-2 were undetectable or very low in normal and in nonsepsis patients but significantly (p < 0.05) increased in sepsis patients. These MAbs should therefore be very useful new tools for studies of MD-2 expression and function in health and disease.

Innate immunity to the pathogenic fungus Coccidioides posadasii is dependent on Toll-like receptor 2 and Dectin-1.

Coccidioides posadasii is a pathogenic fungus that causes endemic and epidemic coccidioidomycosis in the deserts of North, Central, and South America. How the innate immune system responds to the organism is not well understood. Here we show that elicited mouse peritoneal macrophages respond to spherules (the tissue form of the fungus) by producing proinflammatory cytokines as measured by quantitative PCR of cellular transcripts and by enzyme-linked immunosorbent assay (ELISA) assays for secreted protein. We examined the contribution of Toll-like receptors (TLR) and MyD88 in macrophage responses to formalin-killed spherules (FKS) by comparing cytokine responses of elicited macrophages from different knockout mice. FKS were added to elicited mouse peritoneal macrophages from wild-type, TLR2-/-, and MyD88-/- cells, and wild-type cells made more tumor necrosis factor alpha, MIP-2, and interleukin 6 than did the mutant macrophages. In contrast, the C3H/HeJ mice, which have a point mutation in TLR4, and TLR4-/- B6 mice exhibited no defect in cytokine production compared to the control mice. We also investigated the role of the macrophage beta-glucan receptor, Dectin-1. RAW 264.7 macrophages overexpressing Dectin-1 produced more cytokines in respond to FKS, live spherules, and purified beta-glucan than did control RAW cells. Blockage of Dectin-1 with antibodies inhibited cytokine production in elicited mouse peritoneal macrophages. Taken together, these results show that cytokine responses in mouse peritoneal macrophages to C. posadasii spherules are dependent on TLR2, MyD88, and Dectin-1.

Membrane-anchored CD14 is required for LPS-induced TLR4 endocytosis in TLR4/MD-2/CD14 overexpressing CHO cells.

Lipopolysaccharide (LPS) induces inflammatory activation through TLR4 (toll-like receptor-4)/MD-2 (myeloid differentiation-2)/CD14 (cluster of differentiation-14) complex. Although optimal LPS signaling is required to activate our innate immune systems against gram-negative bacterium, excessive amount of LPS signaling develops a detrimental inflammatory response in gram-negative bacterial infections. Downregulation of surface TLR4 expression is one of the critical mechanisms that can restrict LPS signaling. Here, we found that membrane-anchored CD14 is required for LPS-induced downregulation of TLR4 and MD-2 in CHO cells. Moreover, pretreatment of the cells with sterol-binding agent filipin reduced LPS-induced TLR4 downregulation, suggesting the involvement of caveolae-mediated endocytosis pathway. Involvement of caveolae in LPS-induced TLR4 endocytosis was further confirmed by immunoprecipitation. Thus, our data indicate that caveolae-dependent endocytosis pathway is involved in LPS-induced TLR4 downregulation and that this is dependent on membrane-anchored CD14 expression.

Lipopolysaccharide (LPS)-binding protein inhibits responses to cell-bound LPS.

Lipopolysaccharide (LPS)-binding protein (LBP) is an acute phase reactant that may play a dual role in vivo, both potentiating and decreasing cell responses to bacterial LPS. Whereas low concentrations of LBP potentiate cell stimulation by transferring LPS to CD14, high LBP concentrations inhibit cell responses to LPS. One inhibitory mechanism involves the ability of LBP to neutralize LPS by transferring it to plasma lipoproteins, whereas other inhibitory mechanisms, such as the one described here, do not require exogenous lipoproteins. Here we show that LBP can inhibit monocyte responses to LPS that has already bound to membrane-bound CD14 (mCD14) on the cell surface. LBP caused rapid dissociation of LPS from mCD14 as measured by the ability of LBP to inhibit cross-linking of a radioiodinated, photoactivatable LPS derivative to mCD14. Whereas LBP removed up to 75% of the mCD14-bound LPS in 10 min, this was not accompanied by extensive release of the LPS from the cells. The cross-linking data suggest that much of the LPS that remained bound to the cells was associated with LBP. The ability of LBP to inhibit cell responses could not be explained by its effect on LPS internalization, because LBP did not significantly increase the internalization of the cell-bound LPS. In cell-free LPS cross-linking experiments, LBP inhibited the transfer of LPS from soluble CD14 to soluble MD-2. Our data support the hypothesis that LBP can inhibit cell responses to LPS by inhibiting LPS transfer from mCD14 to the Toll-like receptor 4-MD-2 signaling receptor.

Minimally modified LDL binds to CD14, induces macrophage spreading via TLR4/MD-2, and inhibits phagocytosis of apoptotic cells.

Minimally modified low density lipoprotein (mmLDL) is a pro-inflammatory and pro-atherogenic lipoprotein that, unlike profoundly oxidized LDL (OxLDL), is not recognized by scavenger receptors and thus does not have enhanced uptake by macrophages. However, here we demonstrate that mmLDL (as well as OxLDL) induces actin polymerization and spreading of macrophages, which results in such pro-atherogenic consequences as inhibition of phagocytosis of apoptotic cells but enhancement of OxLDL uptake. We also demonstrate for the first time that the lipopolysaccharide receptor, CD14, and toll-like receptor-4/MD-2 are involved in these mmLDL effects. Macrophages of the J774 cell line exhibited higher mmLDL binding and F-actin response than its CD14-deficient mutant, LR-9 cells. Similarly, Chinese hamster ovary cells transfected with human CD14 specifically bound mmLDL and responded with higher F-actin compared with control cells. Macrophages from C3H/HeJ mice, which have a point mutation in the Tlr4 gene, responded with lower F-actin to mmLDL and did not spread as well as macrophages from control animals. A significantly higher F-actin response was also observed in Chinese hamster ovary cells transfected with human toll-like receptor-4/MD-2 but not with TLR4 alone or TLR2. Thus, in addition to inhibition of phagocytosis, the recognition of mmLDL by macrophage lipopolysaccharide receptors results in convergence of cellular immune responses to products of microorganisms and to oxidation-specific self-antigens, which could both influence macrophage function and atherogenesis.