Theo N. Kirkland

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.

Comparative genomic analyses of the human fungal pathogens Coccidioides and their relatives.

While most Ascomycetes tend to associate principally with plants, the dimorphic fungi Coccidioides immitis and Coccidioides posadasii are primary pathogens of immunocompetent mammals, including humans. Infection results from environmental exposure to Coccidiodies, which is believed to grow as a soil saprophyte in arid deserts. To investigate hypotheses about the life history and evolution of Coccidioides, the genomes of several Onygenales, including C. immitis and C. posadasii; a close, nonpathogenic relative, Uncinocarpus reesii; and a more diverged pathogenic fungus, Histoplasma capsulatum, were sequenced and compared with those of 13 more distantly related Ascomycetes. This analysis identified increases and decreases in gene family size associated with a host/substrate shift from plants to animals in the Onygenales. In addition, comparison among Onygenales genomes revealed evolutionary changes in Coccidioides that may underlie its infectious phenotype, the identification of which may facilitate improved treatment and prevention of coccidioidomycosis. Overall, the results suggest that Coccidioides species are not soil saprophytes, but that they have evolved to remain associated with their dead animal hosts in soil, and that Coccidioides metabolism genes, membrane-related proteins, and putatively antigenic compounds have evolved in response to interaction with an animal host.

Innate Immunity to the Pathogenic Fungus Coccidioides posadasii Is Dependent on Toll-Like Receptor 2 and Dectin-1

ABSTRACT 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 β-glucan receptor, Dectin-1. RAW 264.7 macrophages overexpressing Dectin-1 produced more cytokines in respond to FKS, live spherules, and purified β-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.

Population genomic sequencing of Coccidioides fungi reveals recent hybridization and transposon control

We have sequenced the genomes of 18 isolates of the closely related human pathogenic fungi Coccidioides immitis and Coccidioides posadasii to more clearly elucidate population genomic structure, bringing the total number of sequenced genomes for each species to 10. Our data confirm earlier microsatellite-based findings that these species are genetically differentiated, but our population genomics approach reveals that hybridization and genetic introgression have recently occurred between the two species. The directionality of introgression is primarily from C. posadasii to C. immitis, and we find more than 800 genes exhibiting strong evidence of introgression in one or more sequenced isolates. We performed PCR-based sequencing of one region exhibiting introgression in 40 C. immitis isolates to confirm and better define the extent of gene flow between the species. We find more coding sequence than expected by chance in the introgressed regions, suggesting that natural selection may play a role in the observed genetic exchange. We find notable heterogeneity in repetitive sequence composition among the sequenced genomes and present the first detailed genome-wide profile of a repeat-induced point mutation (RIP) process distinctly different from what has been observed in Neurospora. We identify promiscuous HLA-I and HLA-II epitopes in both proteomes and discuss the possible implications of introgression and population genomic data for public health and vaccine candidate prioritization. This study highlights the importance of population genomic data for detecting subtle but potentially important phenomena such as introgression.

Toll-Like Receptor 4 Protects against Lethal Leptospira interrogans Serovar Icterohaemorrhagiae Infection and Contributes to In Vivo Control of Leptospiral Burden

ABSTRACT 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.

Isolation and characterization of two chitinase-encoding genes (cts1, cts2) from the fungus Coccidioides immitis

Two chitinase (CTS)-encoding genes (cts) from Coccidioides immitis (Ci), a respiratory fungal pathogen of humans, were cloned and sequenced. Both the genomic and cDNA sequences are presented. The transcription start points and poly(A)-addition sites were confirmed. The cts1 gene contains five introns and a 1281-bp ORF which translates a 427-amino-acid (aa) protein of 47.4 kDa. The cts2 gene contains two introns and a 2580-bp ORF which translates a 860-aa protein of 91.4 kDa. The deduced CTS1 protein showed highest homology to the Aphanocladium album and Trichoderma harzianum CTS (74% and 76%, respectively), while CTS2 showed highest homology to the CTS of Saccharomyces cerevisiae (Sc) and Candida albicans (47% and 51%, respectively). The putative N-terminal sequence of the mature CTS1 protein also showed 89% homology to the reported N-terminal sequence of a 48-kDa complement fixation antigen (CF-Ag) of Ci which has demonstrated chitinase activity. The CF-Ag is a clinically important antigen used in serodiagnosis of this fungal disease. CTS2 showed several of the conserved features of the Sc CTS, including putative catalytic and Ser/Thr-rich domains, and a C-terminal Cys-rich region. We propose that CTS1 and CTS2 of Ci are members of two distinct classes of fungal chitinases, an observation not previously reported for a single fungus.

Cloning and expression of a gene encoding a T-cell reactive protein from coccidioides immitis: homology to 4-hydroxyphenylpyruvate dioxygenase and the mammalian F antigen

The gene which encodes a previously described T-cell reactive protein (TCRP) of the human fungal pathogen Coccidioides immitis (Ci) was cloned and sequenced. Both the genomic and cDNA sequences were determined. The transcription start point was confirmed. The tcrP gene has three introns and a 1197-bp ORF which translates to a 399-amino-acid (aa) protein (45.2 kDa). The predicted protein has approx. 50% aa sequence identity and 70% similarity to mammalian 4-hydroxyphenylpyruvate dioxygenase (HPPD) proteins and mammalian F-antigens. Expression of the Ci tcrP in Escherichia coli resulted in production of a deep brown pigment, consistent with E. coli expression of the bacterial HPPD homolog from Shewanella colwelliana. The TCRP is likely the Ci form of HPPD.

Isolation and characterization of the urease gene (URE) from the pathogenic fungus Coccidioides immitis

The urease (URE)-encoding gene from Coccidioides immitis (Ci), a respiratory fungal pathogen of humans, was cloned, sequenced, chromosome-mapped and expressed. Both the genomic and cDNA sequences are reported. The transcription start point and poly(A)-addition site were confirmed. The URE gene contains eight introns and a 2517-bp ORF that translates a 839-amino-acid (aa) protein of 91.5 kDa and pI of 5.5, as deduced by computer analysis of the nucleotide sequence. The translated protein revealed eight putative N-glycosylation sites. The deduced URE showed comparable levels of homology to reported URE of the jack bean plant (Canavalia ensiformis; 71.8%) and URE of several genera of bacteria (Bp, 71.7%; Hp, 68.3%; Ka, 71.6%; Pm, 71.9%). The URE gene was mapped to chromosome III of Ci and was shown to be a single copy gene by Southern hybridization. Expression of a 1687-bp fragment of the URE gene in E. coli resulted in the production of a 63-kDa recombinant protein that was recognized in an immunoblot by antiserum raised against the Ka URE homolog. This is the first report of a fungal URE gene.

Susceptibility to Coccidioides species in C57BL/6 mice is associated with expression of a truncated splice variant of Dectin-1 (Clec7a).

Coccidioides posadasii spherules stimulate macrophages to make cytokines via TLR-2 and Dectin-1. We used formalin-killed spherules and 1,3-β-glucan purified from spherules to stimulate elicited peritoneal macrophages and myeloid dendritic cells (mDCs) from susceptible (C57BL/6) and resistant (DBA/2) mouse strains. DBA/2 macrophages produced more TNF-α and IL-6 than macrophages from C57BL/6 mice, and the amount of TNF-α made was dependent on both TLR2 and Dectin-1. DCs from C57BL/6 mice made more IL-10 and less IL-23p19 and IL-12p70 than did DBA/2 DC. These responses were inhibited by a monoclonal antibody to Dectin-1. DBA/2 mice expressed full-length Dectin-1, whereas C57BL/6 mice spliced out exon 3, which encodes most of the stalk. RAW cells transduced to express the full-length Dectin-1 responded better to FKS than cells expressing truncated Dectin-1. We compared the isoform of Dectin-1 expressed by 34 C57BL/6 X DBA/2 recombinant inbred (BXD RI) lines with their susceptibility to Coccidioides immitis. In 25 of 34 RI lines susceptibility or resistance corresponded to short or full-length isoforms, respectively. These results suggest that alternative splicing of the Dectin-1 gene contributes to susceptibility of C57BL/6 mice to coccidioidomycosis, and affects the cytokine responses of macrophages and mDCs to spherules.

MD-2 binds to bacterial lipopolysaccharide

Many LPS binding proteins have been described, but the exact nature of the LPS receptors that signal cells remains unclear. MD-2 is a molecule that is found in association with Toll-like receptor 4, which has been shown to be a receptor for LPS. We have produced human MD-2 in baculovirus and tested it for LPS binding. MD-2 binds the lipid A region of LPS without the need for LPS binding protein. These data suggest that MD-2 may be binding LPS as part of the TLR4 receptor complex.