Anna R. Huppler

Metal binding and base ionization in the U6 RNA intramolecular stem-loop structure.

U6 RNA is a key component of the catalytic core of the spliceosome. A metal ion essential for the first catalytic step of pre-mRNA splicing binds to the U80 Sp phosphate oxygen within the yeast U6 intramolecular stem-loop (ISL). Here we present the first structural data for U6 RNA, revealing the three-dimensional structure of the highly conserved U6 ISL. The ISL binds metal ion at the U80 site with the same stereo specificity as the intact spliceosome. The metal-binding site is adjacent to a readily protonated C·A wobble pair. Protonation of the C·A pair and metal binding are mutually antagonistic. These results support a ribozyme model for U6 RNA function and suggest a possible mechanism for the regulation of RNA splicing.

Oral-resident natural Th17 cells and γδ T cells control opportunistic Candida albicans infections

Conti et al. show that IL-17 is produced by tongue-resident populations of γδ T cells and nTh17 cells in response to oropharyngeal candidiasis in mice.

Th17 cells confer long-term adaptive immunity to oral mucosal Candida albicans infections

Oropharyngeal candidiasis (OPC) is an opportunistic infection caused by Candida albicans. Despite its prevalence, little is known about C. albicans-specific immunity in the oral mucosa. Vaccines against Candida generate both T helper type 1 (Th1) and Th17 responses, and considerable evidence implicates interleukin (IL)-17 in immunity to OPC. However, IL-17 is also produced by innate immune cells that are remarkably similar to Th17 cells, expressing the same markers and localizing to similar mucosal sites. To date, the relative contribution(s) of Th1, Th17, and innate IL-17-producing cells in OPC have not been clearly defined. Here, we sought to determine the nature and function of adaptive T-cell responses to OPC, using a new recall infection model. Mice subjected to infection and re-challenge with Candida mounted a robust and stable antigen-specific IL-17 response in CD4+ but not CD8+ T cells. There was little evidence for Th1 or Th1/Th17 responses. The Th17 response promoted accelerated fungal clearance, and Th17 cells could confer protection in Rag1−/− mice upon adoptive transfer. Surprisingly, CD4 deficiency did not cause OPC but was instead associated with compensatory IL-17 production by Tc17 and CD3+CD4−CD8− cells. Therefore, classic CD4+Th17 cells protect from OPC but can be compensated by other IL-17-producing cells in CD4-deficient hosts.

Mucocutaneous candidiasis: the IL-17 pathway and implications for targeted immunotherapy

IL-17 and related cytokines are direct and indirect targets of selective immunosuppressive agents for the treatment of autoimmune diseases and other diseases of pathologic inflammation. Insights into the potential adverse effects of IL-17 blockade can be drawn from the experience of patients with deficiencies in the IL-17 pathway. A unifying theme of susceptibility to mucocutaneous candidiasis is seen in both mice and humans with a variety of genetic defects that converge on this pathway. Mucocutaneous candidiasis is a superficial infection of mucosal, nail or skin surfaces usually caused by the fungal pathogen Candida albicans. The morbidity of the disease includes significant pain, weight loss and secondary complications, including carcinoma and aneurysms. This review describes the known human diseases associated with chronic mucocutaneous candidiasis (CMC) as well as the known and proposed connections to IL-17 signaling. The human diseases include defects in IL-17 signaling due to autoantibodies (AIRE deficiency), receptor mutations (IL-17 receptor mutations) or mutations in the cytokine genes (IL17F and IL17A). Hyper-IgE syndrome is characterized by elevated serum IgE, dermatitis and recurrent infections, including CMC due to impaired generation of IL-17-producing Th17 cells. Mutations in STAT1, IL12B and IL12RB1 result in CMC secondary to decreased IL-17 production through different mechanisms. Dectin-1 defects and CARD9 defects result in susceptibility to C. albicans because of impaired host recognition of the pathogen and subsequent impaired generation of IL-17-producing T cells. Thus, recent discoveries of genetic predisposition to CMC have driven the recognition of the role of IL-17 in protection from mucosal fungal infection and should guide counseling and management of patients treated with pharmacologic IL-17 blockade.

Performance of Low-Risk Criteria in the Evaluation of Young Infants With Fever: Review of the Literature

OBJECTIVE: The goal was to determine the performance of low-risk criteria for serious bacterial illnesses (SBIs) in febrile infants in prospective studies in which empiric antibiotic treatment was withheld, compared with studies (prospective and retrospective) in which empiric antibiotic treatment was administered. METHODS: A search of the English-language literature was undertaken by using a PubMed database and reference lists of relevant studies of fever, low-risk criteria, and SBIs. Studies of infants >90 days of age, infants with specific infections, or infants with additional risk factors for infection were excluded. Publications were categorized as retrospective, prospective with empiric antibiotic treatment for all patients, or prospective with antibiotics withheld. The relative risk of SBI in high-risk versus low-risk patients was determined for pooled data in each category. The rates of SBIs in low-risk patients in each category were compared. RESULTS: Twenty-one studies met the inclusion criteria. In prospective studies in which patients were cared for without empiric antibiotic treatment, 6 patients assigned to the low-risk category had SBIs; all recovered uneventfully. The rate of SBIs in these low-risk patients was 0.67%. The relative risk of SBIs in high-risk versus low-risk patients in these studies was 30.56 (95% confidence interval: 7.0–68.13). The rate of SBIs in low-risk patients in all studies was 2.23%. The rate of SBIs in low-risk patients in the prospective studies without empiric antibiotic treatment was significantly different from the rate in all other studies (0.67% vs 2.71%; P = .01). CONCLUSIONS: Low-risk criteria perform well in prospective studies in which empiric antibiotic treatment is withheld. These criteria allow ∼30% of young febrile infants to be observed without antibiotic treatment, thus avoiding unnecessary hospitalization, nosocomial infection, injudicious use of antibiotics, and adverse effects of antibiotics.

Role of Neutrophils in IL-17–Dependent Immunity to Mucosal Candidiasis

Oropharyngeal candidiasis (OPC), caused by the commensal fungus Candida albicans, is an opportunistic infection associated with infancy, AIDS, and IL-17–related primary immunodeficiencies. The Th17-associated cytokines IL-23 and IL-17 are crucial for immunity to OPC, but the mechanisms by which they mediate immunity are poorly defined. IL-17RA–deficient humans and mice are strongly susceptible to OPC, with reduced levels of CXC chemokines and concomitantly impaired neutrophil recruitment to the oral mucosa. Paradoxically, humans with isolated neutropenia are typically not susceptible to candidiasis. To determine whether immunity to OPC is mediated via neutrophil recruitment, mice lacking CXCR2 were subjected to OPC and were found to be highly susceptible, although there was no dissemination of fungi to peripheral organs. To assess whether the entire neutrophil response is IL-17 dependent, IL-17RA−/− and IL-23−/− mice were administered neutrophil-depleting Abs and subjected to OPC. These mice displayed increased oral fungal burdens compared with IL-17RA−/− or IL-23−/− mice alone, indicating that additional IL-17–independent signals contribute to the neutrophil response. WT mice treated with anti–Gr-1 Abs exhibited a robust infiltrate of CD11b+Ly-6GlowF4/80− cells to the oral mucosa but were nonetheless highly susceptible to OPC, indicating that this monocytic influx is insufficient for host defense. Surprisingly, Ly-6G Ab treatment did not induce the same strong susceptibility to OPC in WT mice. Thus, CXCR2+ and Gr-1+ neutrophils play a vital role in host defense against OPC. Moreover, defects in the IL-23/17 axis cause a potent but incomplete deficiency in the neutrophil response to oral candidiasis.

The adaptor CARD9 is required for adaptive but not innate immunity to oral mucosal Candida albicans infections

ABSTRACT Oropharyngeal candidiasis (OPC [thrush]) is an opportunistic infection caused by the commensal fungus Candida albicans. OPC is common in individuals with HIV/AIDS, infants, patients on chemotherapy, and individuals with congenital immune defects. Immunity to OPC is strongly dependent on the interleukin-23 (IL-23)/IL-17R axis, as mice and humans with defects in IL-17R signaling (IL17F, ACT1, IL-17RA) or in genes that direct Th17 differentiation (STAT3, STAT1, CARD9) are prone to mucocutaneous candidiasis. Conventional Th17 cells are induced in response to C. albicans infection via signals from C-type lectin receptors, which signal through the adaptor CARD9, leading to production of Th17-inducing cytokines such as IL-6, IL-1β, and IL-23. Recent data indicate that IL-17 can also be made by numerous innate cell subsets. These innate “type 17” cells resemble conventional Th17 cells, but they can be activated without need for prior antigen exposure. Because C. albicans is not a commensal organism in rodents and mice are thus naive to this fungus, we had the opportunity to assess the role of CARD9 in innate versus adaptive responses using an OPC infection model. As expected, CARD9−/− mice failed to mount an adaptive Th17 response following oral Candida infection. Surprisingly, however, CARD9−/− mice had preserved innate IL-17-dependent responses to Candida and were almost fully resistant to OPC. Thus, CARD9 is important primarily for adaptive immunity to C. albicans, whereas alternate recognition systems appear to be needed for effective innate responses.

An essential role of interleukin-17 receptor signaling in the development of autoimmune glomerulonephritis

In recent years, proinflammatory cytokines in the nephritic kidney appear to contribute to the pathogenesis of AGN. The complex inflammatory cytokine network that drives renal pathology is poorly understood. IL‐17, the signature cytokine of Th17 cells, which promotes autoimmune pathology in a variety of settings, is beginning to be identified in acute and chronic kidney diseases as well. However, the role of IL‐17‐mediated renal damage in the nephritic kidney has not been elucidated. Here, with the use of a murine model of experimental AGN, we showed that IL‐17RA signaling is critical for the development of renal pathology. Despite normal systemic autoantibody response and glomerular immune‐complex deposition, IL‐17RA−/− mice exhibit a diminished influx of inflammatory cells and kidney‐specific expression of IL‐17 target genes correlating with disease resistance in AGN. IL‐17 enhanced the production of proinflammatory cytokines and chemokines from tECs. Finally, we were able to show that neutralization of IL‐17A ameliorated renal pathology in WT mice following AGN. These results clearly demonstrated that IL‐17RA signaling significantly contributes to renal tissue injury in experimental AGN and suggest that blocking IL‐17RA may be a promising therapeutic strategy for the treatment of proliferative and crescentic glomerulonephritis.

Neutrophils Do Not Express IL-17A in the Context of Acute Oropharyngeal Candidiasis

IL-17 protects against pathogens by acting on nonhematopoietic cells to induce neutrophil recruitment through upregulation of chemokines and G-CSF. IL-17- and Th17-deficient humans and mice are susceptible to mucosal Candida albicans infections, linked to impaired neutrophil responses. IL-17 production is traditionally associated with CD4+ Th17 cells. However, IL-17 is also expressed during innate responses to facilitate rapid pathogen clearance. Innate IL-17-expressing cells include various lymphocyte-type subsets, including ILC3, NKT, γδ-T and “natural” Th17 (nTh17) cells. Some reports suggest that neutrophils can express IL-17 during fungal infections. Here, we asked whether neutrophils serve as a source of IL-17 during acute oropharyngeal candidiasis (OPC) using an IL-17A fate-tracking reporter mouse. Mice were subjected to OPC for two days, and oral tissue was analyzed by flow cytometry. IL-17A was expressed by γδ-T cells and TCRβ+ natural Th17 (nTh17) cells, as recently reported. Although infiltrating neutrophils were recruited to the tongue following infection, they did not express the IL-17A reporter. Moreover, neutrophil-depleted mice exhibited normal transcription of both Il17a and downstream IL-17-dependent gene targets after Candida challenge. Thus, in acute OPC, neutrophils are not a measurable source of IL-17 production, nor are they necessary to trigger IL-17-dependent gene expression, although they are essential for ultimate pathogen control.

Animal Models for Candidiasis

Multiple forms of candidiasis are clinically important in humans. Established murine models of disseminated, oropharyngeal, vaginal, and cutaneous candidiasis caused by Candida albicans are described in this unit. Detailed materials and methods for C. albicans growth and detection are also described. Curr. Protoc. Immunol. 105:19.6.1‐19.6.17.