Interleukin-1 Receptor but Not Toll-Like Receptor 2 Is Essential for MyD88-Dependent Th17 Immunity to Coccidioides Infection

Interleukin-17A (IL-17A)-producing CD4+ T helper (Th17) cells have been shown to be essential for defense against pulmonary infection with Coccidioides species. However, we have just begun to identify the required pattern recognition receptors and understand the signal pathways that lead to Th17 cell activation after fungal infection. We previously reported that Card9 −/− mice vaccinated with formalin-killed spherules failed to acquire resistance to Coccidioides infection. Here, we report that both MyD88 −/− and Card9 −/− mice immunized with a live, attenuated vaccine also fail to acquire protective immunity to this respiratory disease. Like Card9 −/− mice, vaccinated MyD88 −/− mice revealed a significant reduction in numbers of both Th17 and Th1 cells in their lungs after Coccidioides infection. Both Toll-like receptor 2 (TLR2) and IL-1 receptor type 1 (IL-1r1) upstream of MyD88 have been implicated in Th17 cell differentiation. Surprisingly, vaccinated TLR2 −/− and wild-type (WT) mice showed similar outcomes after pulmonary infection with Coccidioides, while vaccinated IL-1r1 −/− mice revealed a significant reduction in the number of Th17 cells in their infected lungs compared to WT mice. Thus, activation of both IL-1r1/MyD88- and Card9-mediated Th17 immunity is essential for protection against Coccidioides infection. Our data also reveal that the numbers of Th17 cells were reduced in IL-1r1 −/− mice to a lesser extent than in MyD88 −/− mice, raising the possibility that other TLRs are involved in MyD88-dependent Th17 immunity to coccidioidomycosis. An antimicrobial action of Th17 cells is to promote early recruitment of neutrophils to infection sites. Our data revealed that neutrophils are required for vaccine immunity to this respiratory disease.

Related papers:

title: Progress Toward a Human Vaccine Against Coccidioidomycosis
abstract: Coccidioidomycosis (San Joaquin Valley fever) is a human respiratory disease caused by a soil-borne mold, and is recognized as an intransigent microbial infection by physicians who treat patients with the potentially life-threatening, disseminated form of this mycosis. Epidemiological studies based on surveys of skin-test reactivity of people who reside in the endemic regions of the Southwestern US have shown that at least 150,000 new infections occur annually. The clinical spectrum of coccidioidomycosis ranges from an asymptomatic insult to a severe pulmonary disease in which the pathogen may spread from the lungs to the skin, bones, brain and other body organs. Escalation of symptomatic infections and increased cost of long-term antifungal treatment warrant a concerted effort to develop a vaccine against coccidioidomycosis. This review examines recently reported strategies used to generate such a vaccine and summarizes current understanding of the nature of protective immunity to this formidable disease.

title: Coccidioidomycosis: Host Response and Vaccine Development
abstract: Coccidioidomycosis is caused by the dimorphic fungi in the genus Coccidioides. These fungi live as mycelia in the soil of desert areas of the American Southwest, and when the infectious spores, the arthroconidia, are inhaled, they convert into the parasitic spherule/endospore phase. Most infections are mild, but these organisms are frank pathogens and can cause severe lethal disease in fully immunocompetent individuals. While there is increased risk of disseminated disease in certain racial groups and immunocompromised persons, the fact that there are hosts who contain the initial infection and exhibit long-term immunity to reinfection supports the hypothesis that a vaccine against these pathogens is feasible. Multiple studies have shown that protective immunity against primary disease is associated with T-helper 1 (Th-1)-associated immune responses. The single best vaccine in animal models, formalin-killed spherules (FKS), was tested in a human trial but was not found to be significantly protective. This result has prompted studies to better define immunodominant Coccidioides antigen with the thought that a subunit vaccine would be protective. These efforts have defined multiple candidates, but the single best individual immunogen is the protein termed antigen 2/proline-rich antigen (Ag2/PRA). Studies in multiple laboratories have shown that Ag2/PRA as both protein and genetic vaccines provides significant protection against mice challenged systemically with Coccidioides. Unfortunately, compared to the FKS vaccine, it is significantly less protective as measured by both assays of reduction in fungal CFU and assays of survival. The capacity of Ag2/PRA to induce only partial protection was emphasized when animals were challenged intranasally. Thus, there is a need to define new candidates to create a multivalent vaccine to increase the effectiveness of Ag2/PRA. Efforts of genomic screening using expression library immunization or bioinformatic approaches to identify new candidates have revealed at least two new protective proteins, expression library immunization antigen 1 (ELI-Ag1) and a β-1,3-glucanosyltransferase (GEL-1). In addition, previously discovered antigens such as Coccidioides-specific antigen (CSA) should be evaluated in assays of protection. While studies have yet to be completed with combinations of the current candidates, the hypothesis is that with increased numbers of candidates in a multivalent vaccine, there will be increased protection. As the genome sequences of the two Coccidioides strains which are under way are completed and annotated, the effort to find new candidates can increase to provide a complete genomic scan for immunodominant proteins. Thus, much progress has been made in the discovery of subunit vaccine candidates against Coccidioides and there are several candidates showing modest levels of protection, but for complete protection against pulmonary challenge we need to continue the search for additional candidates.

title: Vaccine Immunity to Coccidioidomycosis Occurs by Early Activation of Three Signal Pathways of T Helper Cell Response (Th1, Th2, and Th17)
abstract: We have previously reported that C57BL/6 mice vaccinated with a live, attenuated mutant of Coccidioides posadasii, referred to as the ΔT vaccine, are fully protected against pulmonary coccidioidomycosis. This model was used here to explore the nature of vaccine immunity during the initial 2-week period after intranasal challenge. Elevated neutrophil and eosinophil infiltration into the lungs of nonvaccinated mice contrasted with markedly reduced recruitment of these cells in vaccinated animals. The numbers of lung-infiltrated macrophages and dendritic cells showed a progressive increase in vaccinated mice and corresponded with reduction of the lung infection. Concentrations of selected inflammatory cytokines and chemokines were initially higher in lung homogenates of vaccinated mice but then generally decreased at 14 days postchallenge in correlation with containment of the organism and apparent dampening of the inflammation of host tissue. Profiles of cytokines detected in lung homogenates of ΔT-vaccinated mice were indicative of a mixed T helper 1 (Th1)-, Th2-, and Th17-type immune response, a conclusion which was supported by detection of lung infiltration of activated T cells with the respective CD4+ gamma interferon (IFN-γ)+, CD4+ interleukin-5 (IL-5)+, and CD4+ IL-17A+ phenotypes. While Th1 and Th2 immunity was separately dispensed of by genetic manipulation without loss of ΔT vaccine-mediated protection, loss of functional Th17 cells resulted in increased susceptibility to infection in immunized mice. Characterization of the early events of protective immunity to Coccidioides infection in vaccinated mice contributes to the identification of surrogates of immune defense and provides potential insights into the design of immunotherapeutic protocols for treatment of coccidioidomycosis.

title: Vaccine-induced protection against 3 systemic mycoses endemic to North America requires Th17 cells in mice.
abstract: Worldwide rates of systemic fungal infections, including three of the major pathogens responsible for such infections in North America (Coccidioides posadasii, Histoplasma capsulatum, and Blastomyces dermatitidis), have soared recently, spurring interest in developing vaccines. The development of Th1 cells is believed to be crucial for protective immunity against pathogenic fungi, whereas the role of Th17 cells is vigorously debated. In models of primary fungal infection, some studies have shown that Th17 cells mediate resistance, while others have shown that they promote disease pathology. Here, we have shown that Th1 immunity is dispensable and that fungus-specific Th17 cells are sufficient for vaccine-induced protection against lethal pulmonary infection with B. dermatitidis in mice. Further, vaccine-induced Th17 cells were necessary and sufficient to protect against the three major systemic mycoses in North America. Mechanistically, Th17 cells engendered protection by recruiting and activating neutrophils and macrophages to the alveolar space, while the induction of Th17 cells and acquisition of vaccine immunity unexpectedly required the adapter molecule Myd88 but not the fungal pathogen recognition receptor Dectin-1. These data suggest that human vaccines against systemic fungal infections should be designed to induce Th17 cells if they are to be effective.

title: The role of Syk/CARD9 coupled C-type lectins in antifungal immunity
abstract: Fungal infections are affecting an increasing number of people, and the failure of current therapies in treating systemic infection has resulted in an unacceptably high mortality rate. It is therefore of importance that we understand immune mechanisms operating during fungal infections, in order to facilitate development of adjunctive immunotherapies for the treatment of these diseases. C‐type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that are critical for immune responses to fungi. Many of these receptors are coupled to Syk kinase, which allows these receptors to signal via CARD9 leading to NF‐κB activation, which in turn contributes to the induction of both innate and adaptive immunity. Dectin‐1, Dectin‐2 and Mincle are all CLRs that share this common signalling mechanism and have been shown to play key roles in antifungal immunity. This review aims to update existing paradigms and summarise the most recent findings on these CLRs, their signal transduction mechanisms and the collaborations between these CLRs and other PRRs.

title: MyD88 Signaling Is Required for Efficient Innate and Adaptive Immune Responses to Paracoccidioides brasiliensis Infection
abstract: 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.

title: Adaptive immunity to fungi.
abstract: Only a handful of the more than 100,000 fungal species on our planet cause disease in humans, yet the number of life-threatening fungal infections in patients has recently skyrocketed as a result of advances in medical care that often suppress immunity intensely. This emerging crisis has created pressing needs to clarify immune defense mechanisms against fungi, with the ultimate goal of therapeutic applications. Herein, we describe recent insights in understanding the mammalian immune defenses deployed against pathogenic fungi. The review focuses on adaptive immune responses to the major medically important fungi and emphasizes how dendritic cells and subsets in various anatomic compartments respond to fungi, recognize their molecular patterns, and signal responses that nurture and shape the differentiation of T cell subsets and B cells. Also emphasized is how the latter deploy effector and regulatory mechanisms that eliminate these nasty invaders while also constraining collateral damage to vital tissue.

title: Deep dermatophytosis and inherited CARD9 deficiency.
abstract: Deep dermatophytosis is a severe and sometimes life-threatening fungal infection caused by dermatophytes. It is characterized by extensive dermal and subcutaneous tissue invasion and by frequent dissemination to the lymph nodes and, occasionally, the central nervous system. The condition is different from common superficial dermatophyte infection and has been reported in patients with no known immunodeficiency. Patients are mostly from North African, consanguineous, multiplex families, which strongly suggests a mendelian genetic cause.



We studied the clinical features of deep dermatophytosis in 17 patients with no known immunodeficiency from eight unrelated Tunisian, Algerian, and Moroccan families. Because CARD9 (caspase recruitment domain-containing protein 9) deficiency has been reported in an Iranian family with invasive fungal infections, we also sequenced CARD9 in the patients.



Four patients died, at 28, 29, 37, and 39 years of age, with clinically active deep dermatophytosis. No other severe infections, fungal or otherwise, were reported in the surviving patients, who ranged in age from 37 to 75 years. The 15 Algerian and Tunisian patients, from seven unrelated families, had a homozygous Q289X CARD9 allele, due to a founder effect. The 2 Moroccan siblings were homozygous for the R101C CARD9 allele. Both alleles are rare deleterious variants. The familial segregation of these alleles was consistent with autosomal recessive inheritance and complete clinical penetrance.



All the patients with deep dermatophytosis had autosomal recessive CARD9 deficiency. Deep dermatophytosis appears to be an important clinical manifestation of CARD9 deficiency.

title: Syk- and CARD9-dependent coupling of innate immunity to the induction of T helper cells that produce interleukin 17
abstract: The C-type lectin dectin-1 binds to yeast and signals through the kinase Syk and the adaptor CARD9 to induce production of interleukin 10 (IL-10) and IL-2 in dendritic cells (DCs). However, whether this pathway promotes full DC activation remains unclear. Here we show that dectin-1–Syk–CARD9 signaling induced DC maturation and the secretion of proinflammatory cytokines, including IL-6, tumor necrosis factor and IL-23, but little IL-12. Dectin-1-activated DCs 'instructed' the differentiation of CD4+ IL-17-producing effector T cells (TH-17 cells) in vitro, and a dectin-1 agonist acted as an adjuvant promoting the differentiation of TH-17 and T helper type 1 cells in vivo. Infection with Candida albicans induced CARD9-dependent TH-17 responses to the organism. Our data indicate that signaling through Syk and CARD9 can couple innate to adaptive immunity independently of Toll-like receptor signals and that CARD9 is required for the development of TH-17 responses to some pathogens.

title: C-Type Lectin Receptors Differentially Induce Th17 Cells and Vaccine Immunity to the Endemic Mycosis of North America
abstract: Vaccine immunity to the endemic mycoses of North America requires Th17 cells, but the pattern recognition receptors and signaling pathways that drive these protective responses have not been defined. We show that C-type lectin receptors exert divergent contributions to the development of antifungal Th17 cells and vaccine resistance against Blastomyces dermatitidis, Histoplasma capsulatum, and Coccidioides posadasii. Acquired immunity to B. dermatitidis requires Dectin-2, whereas vaccination against H. capsulatum and C. posadasii infection depends on innate sensing by Dectin-1 and Dectin-2, but not Mincle. Tracking Ag-specific T cells in vivo established that the Card9 signaling pathway acts indispensably and exclusively on differentiation of Th17 cells, while leaving intact their activation, proliferation, survival, and migration. Whereas Card9 signaling is essential, C-type lectin receptors offer distinct and divergent contributions to vaccine immunity against these endemic fungal pathogens. Our work provides new insight into innate immune mechanisms that drive vaccine immunity and Th17 cells.

title: Virulence Mechanisms of Coccidioides
abstract: Coccidioides is a fungal respiratory pathogen of humans that can cause disease in both immunosuppressed and immunocompetent individuals. We describe here three mechanisms by which the pathogen survives in the hostile host environment: production of a dominant spherule outer wall glycoprotein (SOWgp) that modulates host immune response and results in compromised cell‐mediated immunity to coccidioidal infection, depletion of SOWgp presentation on the surface of endospores, which prevents host recognition of the pathogen when the fungal cells are most vulnerable to phagocytic defenses, and induction of elevated production of host arginase I and coccidioidal urease, which contribute to tissue damage at sites of infection. Arginase I competes with inducible nitric oxide synthase (iNOS) in macrophages for the common substrate, L‐arginine, and thereby reduces nitric oxide (NO) production and increases the synthesis of host orinithine and urea. Host‐derived L‐ornithine may promote pathogen growth and proliferation by providing a pool of the monoamine, which could be taken up and used for synthesis of polyamines via metabolic pathways of the parasitic cells. We have shown that high concentrations of Coccidioides‐ and host‐derived urea at infection sites in the presence of urease produced and released by the pathogen, results in secretion of ammonia and contributes to alkalinization of the microenvironment. We propose that ammonia and enzymatically active urease released from spherules during the parasitic cycle of Coccidioides exacerbate the severity of coccidioidal infection by contributing to a compromised immune response to infection and damage of host tissue at foci of infection.

title: A Genetically Engineered Live Attenuated Vaccine of Coccidioides posadasii Protects BALB/c Mice against Coccidioidomycosis
abstract: Coccidioidomycosis (also known as San Joaquin Valley fever) is an occupational disease. Workers exposed to outdoor dust which contains spores of the soil-inhabiting fungus have a significantly increased risk of respiratory infection. In addition, people with compromised T-cell immunity, the elderly, and certain racial groups, particularly African-Americans and Filipinos, who live in regions of endemicity in the southwestern United States have an elevated incidence of symptomatic infection caused by inhalation of spores of Coccidioides posadasii or Coccidioides immitis. Recurring epidemics and escalation of medical costs have helped to motivate production of a vaccine against valley fever. The major focus has been the development of a defined, T-cell-reactive, recombinant protein vaccine. However, none of the products described to date have provided full protection to coccidioidal disease-susceptible BALB/c mice. Here we describe the first genetically engineered, live, attenuated vaccine that protects both BALB/c and C57BL/6 mice against coccidioidomycosis. Two chitinase genes (CTS2 and CTS3) were disrupted to yield the attenuated strain, which was unable to endosporulate and was no longer infectious. Vaccinated survivors mounted an immune response characterized by production of both T-helper-1- and T-helper-2-type cytokines. Histology revealed well-formed granulomas and markedly diminished inflammation. Significantly fewer organisms were observed in the lungs of survivors than in those of nonvaccinated mice. Additional investigations are required to further define the nature of the live, attenuated vaccine-induced immunity against Coccidioides infection.

title: MyD88 is dispensable for resistance to Paracoccidioides brasiliensis in a murine model of blood-borne disseminated infection.
abstract: We have studied the role of MyD88, an adaptor protein of Toll-like receptors (TLRs), in murine defenses against Paracoccidioides brasiliensis in a model of blood-borne disseminated infection. Wild-type (WT) and MyD88-deficient mice infected intravenously with P. brasiliensis yeast cells showed an equivalent fungal burden, as well as similar levels of proinflammatory IL-1beta, IL-6, IL-12p70, tumor necrosis factor (TNF)-alpha and MIP-2, T-helper type 1 (Th1) (IFN-gamma) and Th2 cytokines (IL-4) in tissue homogenates. In vitro production of TNF-alpha, IFN-gamma and IL-12p70, by antigen-stimulated splenocytes from infected animals, was also similar in both types of mice; this production of Th1 cytokines correlated with a similar frequency of IFN-gamma-producing CD4 T cells. Recruitment of neutrophils to the peritoneal cavity of intraperitoneally infected mice was not affected in TLR2-/-, TLR4-/- as compared with WT mice, but significantly decreased in MyD88-deficient mice. In vitro production of TNF-alpha by peritoneal macrophages from MyD88-, TLR2- and TLR4-deficient mice in response to P. brasiliensis yeasts was undiminished, as compared with macrophages from WT mice, and, in addition, laminarin failed to inhibit production of TNF-alpha by WT and MyD88-deficient macrophages. Overall, these data suggest that the response to P. brasiliensis yeasts occurs independently of the adaptor molecule MyD88, and indicate that TLR2, TLR4 and dectin-1 do not play a significant role in recognition of P. brasiliensis yeast cells.

title: Aspergillus fumigatus Induces Innate Immune Responses in Alveolar Macrophages through the MAPK Pathway Independently of TLR2 and TLR41
abstract: Aspergillus fumigatus causes invasive aspergillosis in immunosuppressed patients. In the immunocompetent host, inhaled conidia are cleared by alveolar macrophages. The signaling pathways of the alveolar macrophage involved in the clearance of A. fumigatus are poorly understood. Therefore, we investigated the role of TLRs in the immune response against A. fumigatus and their contribution to the signaling events triggered in murine alveolar macrophages upon infection with A. fumigatus conidia. Specifically, we examined the MAPKs and NF-κB activation and cytokine signaling. Our investigations revealed that immunocompetent TLR2, TLR4, and MyD88 knockout mice were not more susceptible to invasive aspergillosis as compared with wild-type mice and that the in vitro phosphorylation of the MAPKs ERK and p38 was not affected in TLR2, TLR4, or MyD88 knockout mice following stimulation with conidia. In vivo experiments suggest that ERK was an essential MAPK in the defense against A. fumigatus, whereas the activation of NF-κB appeared to play only a secondary role. In conclusion, our findings demonstrate that TLR2/4 recognition and MyD88 signaling are dispensable for the clearance of A. fumigatus under immunocompetent situations. Furthermore, our data stress the important role of ERK activation in innate immunity to A. fumigatus.

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

title: Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function.
abstract: MyD88, originally isolated as a myeloid differentiation primary response gene, is shown to act as an adaptor in interleukin-1 (IL-1) signaling by interacting with both the IL-1 receptor complex and IL-1 receptor-associated kinase (IRAK). Mice generated by gene targeting to lack MyD88 have defects in T cell proliferation as well as induction of acute phase proteins and cytokines in response to IL-1. Increases in interferon-gamma production and natural killer cell activity in response to IL-18 are abrogated. In vivo Th1 response is also impaired. Furthermore, IL-18-induced activation of NF-kappaB and c-Jun N-terminal kinase (JNK) is blocked in MyD88-/- Th1-developing cells. Taken together, these results demonstrate that MyD88 is a critical component in the signaling cascade that is mediated by IL-1 receptor as well as IL-18 receptor.

title: 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.

title: Critical regulation of early Th17 cell differentiation by interleukin-1 signaling.
abstract: T helper (Th) 17 cells have been recently discovered in both mouse and human. Here we show that interleukin-1 (IL-1) signaling on T cells is critically required for the early programming of Th17 cell lineage and Th17 cell-mediated autoimmunity. IL-1 receptor1 expression in T cells, which was induced by IL-6, was necessary for the induction of experimental autoimmune encephalomyelitis and for early Th17 cell differentiation in vivo. Moreover, IL-1 signaling in T cells was required in dendritic cell-mediated Th17 cell differentiation from naive or regulatory precursors and IL-1 synergized with IL-6 and IL-23 to regulate Th17 cell differentiation and maintain cytokine expression in effector Th17 cells. Importantly, IL-1 regulated the expression of the transcription factors IRF4 and RORgammat during Th17 cell differentiation; overexpression of these two factors resulted in IL-1-independent Th17 cell polarization. Our data thus indicate a critical role of IL-1 in Th17 cell differentiation and this pathway may serve as a unique target for Th17 cell-mediated immunopathology.

title: TLR2 Stimulation Drives Human Naive and Effector Regulatory T Cells into a Th17-Like Phenotype with Reduced Suppressive Function
abstract: Naturally occurring CD4+CD25+FOXP3+ regulatory T cells suppress the activity of pathogenic T cells and prevent development of autoimmune responses. There is growing evidence that TLRs are involved in modulating regulatory T cell (Treg) functions both directly and indirectly. Specifically, TLR2 stimulation has been shown to reduce the suppressive function of Tregs by mechanisms that are incompletely understood. The developmental pathways of Tregs and Th17 cells are considered divergent and mutually inhibitory, and IL-17 secretion has been reported to be associated with reduced Treg function. We hypothesized that TLR2 stimulation may reduce the suppressive function of Tregs by regulating the balance between Treg and Th17 phenotype and function. We examined the effect of different TLR2 ligands on the suppressive functions of Tregs and found that activation of TLR1/2 heterodimers reduces the suppressive activity of CD4+CD25hiFOXP3lowCD45RA+ (naive) and CD4+CD25hiFOXP3hiCD45RA− (memory or effector) Treg subpopulations on CD4+CD25−FOXP3−CD45RA+ responder T cell proliferation while at the same time enhancing the secretion of IL-6 and IL-17, increasing RORC, and decreasing FOXP3 expression. Neutralization of IL-6 or IL-17 abrogated Pam3Cys-mediated reduction of Treg suppressive function. We also found that, in agreement with recent observations in mouse T cells, TLR2 stimulation can promote Th17 differentiation of human T helper precursors. We conclude that TLR2 stimulation, in combination with TCR activation and costimulation, promotes the differentiation of distinct subsets of human naive and memory/effector Tregs into a Th17-like phenotype and their expansion. Such TLR-induced mechanism of regulation of Treg function could enhance microbial clearance and increase the risk of autoimmune reactions.

title: An agonist of human complement fragment C5a enhances vaccine immunity against Coccidioides infection.
abstract: Coccidioides is a fungal pathogen and causative agent of a human respiratory disease against which no clinical vaccine exists. In this study we evaluated a novel vaccine adjuvant referred to as EP67, which is a peptide agonist of the biologically active C-terminal region of human complement component C5a. The EP67 peptide was conjugated to live spores of an attenuated vaccine strain (ΔT) of Coccidioides posadasii. The non-conjugated ΔT vaccine provided partial protection to BALB/c mice against coccidioidomycosis. In this report we compared the protective efficacy of the ΔT-EP67 conjugate to the ΔT vaccine in BALB/c mice. Animals immunized subcutaneously with the ΔT-EP67 vaccine showed significant increase in survival and decrease in fungal burden over 75 days postchallenge. Increased pulmonary infiltration of dendritic cells and macrophages was observed on day 7 postchallenge but marked decrease in neutrophil numbers had occurred by 11 days. The reduced influx of neutrophils may have contributed to the observed reduction of inflammatory pathology. Mice immunized with the ΔT-EP67 vaccine also revealed enhanced expression of MHC II molecules on the surface of antigen presenting cells, and in vitro recall assays of immune splenocytes showed elevated Th1- and Th17-type cytokine production. The latter correlated with a marked increase in lung infiltration of IFN-γ- and IL-17-producing CD4(+) T cells. Elevated expression of T-bet and RORc transcription factors in ΔT-EP67-vaccinated mice indicated the promotion of Th1 and Th17 cell differentiation. Higher titers of Coccidioides antigen-specific IgG1 and IgG2a were detected in mice immunized with the EP67-conjugated versus the non-conjugated vaccine. These combined results suggest that the EP67 adjuvant enhances protective efficacy of the live vaccine by augmentation of T-cell immunity, especially through Th1- and Th17-mediated responses to Coccidioides infection.

title: Construction and Evaluation of a Novel Recombinant T Cell Epitope-Based Vaccine against Coccidioidomycosis
abstract: Clinical and animal studies of coccidioidomycosis have demonstrated that activated CD4+ T lymphocytes are essential for protection against this fungal respiratory disease. We previously reported a vaccine against Coccidioides infection which contained three recombinant CD4+ T cell-reactive proteins and induced a robust, protective immune response in mice. Due to the anticipated high cost of production and clinical assessment of this multivalent vaccine, we generated a single protein which contained immunodominant T cell epitopes of the three polypeptides. Epitopes were initially identified by computational prediction of their ability to bind promiscuously to human major histocompatibility complex class II (MHC II) molecules. Cellular immunoassays confirmed the immunogenicity of the synthesized epitope peptides, while in vitro binding assays revealed a range of peptide affinity for MHC II. A DNA construct was synthesized for bacterial expression of a recombinant protein vaccine which contained five epitopes with the highest affinity for human MHC II, each fused with leader and spacer peptides proposed to optimize epitope processing and presentation to T cell receptors. Recall assays of immune T lymphocytes obtained from human MHC II-expressing HLA-DR4 transgenic mice confirmed that 4 of the 5 epitope peptides were processed. Mice immunized with the epitope-based vaccine admixed with a synthetic oligodeoxynucleotide adjuvant or loaded into yeast glucan particles and then challenged intranasally with Coccidioides showed early lung infiltration of activated T helper-1 (Th1), Th2, and Th17 cells, elevated gamma interferon (IFN-γ) and interleukin (IL)-17 production, significant reduction of fungal burden, and prolongation of survival compared to nonvaccinated mice. This is the first report of an epitope-based vaccine against coccidioidomycosis.

title: Use of Ly6G‐specific monoclonal antibody to deplete neutrophils in mice
abstract: The anti‐granulocyte receptor‐1 (Gr‐1) mAb, RB6‐8C5, has been used extensively to deplete neutrophils in mice and to investigate the role of these cells in host defense. RB6‐8C5 binds to Ly6G, which is present on neutrophils, and to Ly6C, which is expressed on neutrophils, dendritic cells, and subpopulations of lymphocytes and monocytes. It is thus likely that in vivo administration of RB6‐8C5 may deplete not only neutrophils but also other Gr‐l+ (Ly6C+) cells. This study describes the use of an Ly6G‐specific mAb, 1A8, as an alternative means to deplete neutrophils. In vivo administration of RB6‐8C5 reduced blood neutrophils and Gr‐1+ monocytes, whereas administration of 1A8 reduced blood neutrophils but not Gr‐1+ monocytes. Plasma TNF‐α in endotoxemia was increased 20‐fold by RB6‐8C5 pretreatment and fourfold by 1A8 pretreatment. In a wound model, pretreatment with either antibody decreased wound neutrophils and macrophages. TNF‐α staining in brefeldin‐treated wound leukocytes was increased by pretreatment with RB6‐8C5, but not 1A8. Neutrophil depletion with 1A8 offers advantages over the use of RB6‐8C5, as it preserves non‐neutrophil Gr‐1+ cells depleted by the anti‐Gr‐1 antibody. The loss of non‐neutrophil Gr‐1+ populations in RB6‐8C5‐treated animals is associated with increased TNF‐α responses, suggesting these cells may function to suppress TNF‐α production.

title: Tc17 Cells Mediate Vaccine Immunity against Lethal Fungal Pneumonia in Immune Deficient Hosts Lacking CD4+ T Cells
abstract: Vaccines may help reduce the growing incidence of fungal infections in immune-suppressed patients. We have found that, even in the absence of CD4+ T-cell help, vaccine-induced CD8+ T cells persist and confer resistance against Blastomyces dermatitidis and Histoplasma capsulatum. Type 1 cytokines contribute to that resistance, but they also are dispensable. Although the role of T helper 17 cells in immunity to fungi is debated, IL-17 producing CD8+ T cells (Tc17 cells) have not been investigated. Here, we show that Tc17 cells are indispensable in antifungal vaccine immunity in hosts lacking CD4+ T cells. Tc17 cells are induced upon vaccination, recruited to the lung on pulmonary infection, and act non-redundantly in mediating protection in a manner that requires neutrophils. Tc17 cells did not influence type I immunity, nor did the lack of IL-12 signaling augment Tc17 cells, indicating a distinct lineage and function. IL-6 was required for Tc17 differentiation and immunity, but IL-1R1 and Dectin-1 signaling was unexpectedly dispensable. Tc17 cells expressed surface CXCR3 and CCR6, but only the latter was essential in recruitment to the lung. Although IL-17 producing T cells are believed to be short-lived, effector Tc17 cells expressed low levels of KLRG1 and high levels of the transcription factor TCF-1, predicting their long-term survival and stem-cell like behavior. Our work has implications for designing vaccines against fungal infections in immune suppressed patients.

title: Absence of phagocyte NADPH oxidase 2 leads to severe inflammatory response in lungs of mice infected with Coccidioides.
abstract: Production of reactive oxygen species (ROS) resulting from phagocytic NADPH oxidase (NOX2) activity has been reported to contribute to host defense against numerous microbial pathogens. In this study we explored the role of NOX2 production in experimental coccidioidomycosis, a human respiratory disease caused by a soil-borne fungal pathogen. Activated and non-activated macrophages isolated from either NOX2(-/-) knock-out or wild type (WT) mice showed comparable ROS production and killing efficiency in vitro when infected with parasitic cells of Coccidioides. Both mouse strains also revealed similar fungal burden in their lungs and spleen at 7 and 11 days after intranasal challenge with Coccidioides spores, although the NOX2(-/-) mice died earlier than the WT strain. Immunization of the NOX2(-/-) and WT mice with a live, attenuated vaccine strain of Coccidioides also resulted in comparable reduction of the fungal burden in both lungs and spleen. These combined results initially suggested that NOX2 activity and ROS production are not essential for protection against Coccidioides infection. However, the reduced survival of non-vaccinated NOX2(-/-) mice correlated with high, sustained numbers of lung-infiltrated neutrophils on days 7 and 11 postchallenge, an expansion of the regulatory T cell population in infected lungs in the knock-out mice, and elevated concentrations of pro-inflammatory cytokines and chemokines in lung homogenates compared to infected WT mice. Although NOX2-derived ROS appeared to be dispensable for both innate and acquired immunity to pulmonary Coccidioides infection, evidence is presented that NOX2 production plays a role in limiting pathogenic inflammation in this murine model of coccidioidomycosis.

title: A TCR Transgenic Mouse Reactive with Multiple Systemic Dimorphic Fungi
abstract: Dimorphic fungi collectively account for 5–10 million new infections annually worldwide. Ongoing efforts seek to clarify mechanisms of cellular resistance to these agents and develop vaccines. A major limitation in studying the development of protective T cells in this group of organisms is the lack of tools to detect, enumerate, and characterize fungus-specific T cells during vaccination and infection. We generated a TCR transgenic mouse (Bd 1807) whose CD4+ T cells respond to a native epitope in Blastomyces dermatitidis and also in Histoplasma capsulatum. In this study, we characterize the mouse, reveal its applications, and extend our analysis showing that 1807 cells also respond to the related dimorphic fungi Coccidioides posadasii and Paracoccidioides lutzii. On adoptive transfer into vaccinated wild-type mice, 1807 cells become activated, proliferate, and expand in the draining lymph nodes, and they differentiate into T1 effectors after trafficking to the lung upon lethal experimental challenge. Bd 1807 cells confer vaccine-induced resistance against B. dermatitidis, H. capsulatum, and C. posadasii. Transfer of naive 1807 cells at serial intervals postvaccination uncovered the prolonged duration of fungal Ag presentation. Using 1807 cells, we also found that the administration of vaccine only once induced a maximal pool of effector/memory CD4+ cells and protective immunity by 4 wk after vaccination. The autologous adoptive transfer system described in this study reveals novel features of antifungal immunity and offers a powerful approach to study the differentiation of Ag-specific T cells responsive to multiple dimorphic fungi and the development of CD4+ T cell memory needed to protect against fungal infection.

title: C-type lectin receptors orchestrate antifungal immunity
abstract: Immunity to pathogens critically requires pattern-recognition receptors (PRRs) to trigger intracellular signaling cascades that initiate and direct innate and adaptive immune responses. For fungal infections, these responses are primarily mediated by members of the C-type lectin receptor family. In this Review, we highlight recent advances in the understanding of the roles and mechanisms of these multifunctional receptors, explore how these PRRs orchestrate antifungal immunity and briefly discuss progress in the use of these receptors as targets for antifungal and other vaccines.

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

title: Dectin-1 Is Required for Resistance to Coccidioidomycosis in Mice
abstract: 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 β-glucan-dependent manner. The responses of bone marrow-derived dendritic cells (BMDC) to the same stimulants were more complex; interleukin 1β (IL-1β) 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-1β, 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)F2 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.  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 β-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. 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 β-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.

title: Toll‐like receptor (TLR) 2 and TLR4 are essential for Aspergillus‐induced activation of murine macrophages
abstract: Aspergillus fumigatius is a ubiquitous saprophytic fungus that has become the most prevalent airborne fungal pathogen for immunocompromised patients during the last two decades. In this report we have analysed how macrophages recognize this microorganism. Using transfected human HEK 293 cells we demonstrate that NF‐κB‐dependent promoter activation triggered by A. fumigatus is mediated by Toll‐like receptors TLR2 and TLR4, whereas no activation was observed in cells overexpressing other distinct TLR proteins (TLR1, TLR3, TLR5–10). Using macrophages derived from mice lacking TLR2 expression, expressing defective TLR4 or both we found that A. fumigatus conidia and hyphae induce NF‐κB translocation, release of pro‐inflammatory molecules, like TNFα, and the chemoattractant MIP‐2 in a TLR2‐ and TLR4‐dependent manner. Recognition of A. niger and A. fumigatus , was similar in terms of the parameters analysed, suggesting that pathogenic and non‐pathogenic aspergilli are sensed by macrophages in a similar fashion. Finally, we found that recruitment of neutrophils is severely impaired in mice lacking both functional TLR2 and TLR4, but is less impaired in single TLR2‐ or TLR4‐deficient mice, providing evidence that both receptors are required for an optimal immune response to Aspergillus in vivo.

title: Innate immune activation and CD4+ T cell priming during respiratory fungal infection.
abstract: Aspergillus fumigatus is a mold that causes a spectrum of diseases, including lethal lung infections in immunocompromised humans and allergic asthma in atopic individuals. T helper 1 (Th1) CD4(+) T cells protect against invasive A. fumigatus infections whereas Th2 CD4(+) T cells exacerbate asthma upon inhalation of A. fumigatus spores. Herein, we demonstrate that A. fumigatus-specific T cells were rapidly primed in lymph nodes draining the lung and fully differentiated into interferon-gamma (IFN-gamma)-producing Th1 CD4(+) T cells upon arrival in the airways. T-bet induction in A. fumigatus-specific CD4(+) T cells was enhanced by MyD88-mediated signals in draining lymph nodes, but T cell proliferation, trafficking, and Th1 differentiation in the airways were Toll-like receptor (TLR) and MyD88 independent. Our studies demonstrate that CD4(+) T cell differentiation during respiratory fungal infection occurs incrementally, with TLR-mediated signals in the lymph node enhancing the potential for IFN-gamma production whereas MyD88-independent signals promote Th1 differentiation in the lung.

title: Innate antifungal immunity: the key role of phagocytes.
abstract: Fungal diseases have emerged as significant causes of morbidity and mortality, particularly in immune-compromised individuals, prompting greater interest in understanding the mechanisms of host resistance to these pathogens. Consequently, the past few decades have seen a tremendous increase in our knowledge of the innate and adaptive components underlying the protective (and nonprotective) mechanisms of antifungal immunity. What has emerged from these studies is that phagocytic cells are essential for protection and that defects in these cells compromise the host's ability to resist fungal infection. This review covers the functions of phagocytes in innate antifungal immunity, along with selected examples of the strategies that are used by fungal pathogens to subvert these defenses.

title: Interleukin-1 (IL-1) Pathway
abstract: IL-1α and IL-1β orchestrate the proinflammatory response by multiple tightly controlled mechanisms. The interleukin-1 (IL-1) family of cytokines comprises 11 proteins (IL-1F1 to IL-1F11) encoded by 11 distinct genes in humans and mice. IL-1–type cytokines are major mediators of innate immune reactions, and blockade of the founding members IL-1α or IL-1β by the interleukin-1 receptor antagonist (IL-1RA) has demonstrated a central role of IL-1 in a number of human autoinflammatory diseases. IL-1α or IL-1β rapidly increase messenger RNA expression of hundreds of genes in multiple different cell types. The potent proinflammatory activities of IL-1α and IL-1β are restricted at three major levels: (i) synthesis and release, (ii) membrane receptors, and (iii) intracellular signal transduction. This pathway summarizes extracellular and intracellular signaling of IL-1α or IL-1β, including positive- and negative-feedback mechanisms that amplify or terminate the IL-1 response. In response to ligand binding of the receptor, a complex sequence of combinatorial phosphorylation and ubiquitination events results in activation of nuclear factor κB signaling and the JNK and p38 mitogen-activated protein kinase pathways, which, cooperatively, induce the expression of canonical IL-1 target genes (such as IL-6, IL-8, MCP-1, COX-2, IκBα, IL-1α, IL-1β, MKP-1) by transcriptional and posttranscriptional mechanisms. Of note, most intracellular components that participate in the cellular response to IL-1 also mediate responses to other cytokines (IL-18 and IL-33), Toll-like-receptors (TLRs), and many forms of cytotoxic stresses.

title: IL-1 family members and STAT activators induce cytokine production by Th2, Th17, and Th1 cells
abstract: Expression of T1ST2, the IL-33R, by Th2 cells requires GATA3. Resting Th2 cells express little GATA3, which is increased by IL-33 and a STAT5 activator, in turn increasing T1ST2 from its low-level expression on resting Th2 cells. Th2 cells that have upregulated T1ST2 produce IL-13, but not IL-4, in response to IL-33 plus a STAT5 activator in an antigen-independent, NF-κB-dependent, cyclosporin A (CsA)-resistant manner. Similarly, Th17 cells produce IL-17A in response to IL-1β and a STAT3 activator and Th1 cells produce IFNγ in response to IL-18 and a STAT4 inducer. Thus, each effector Th cell produces cytokines without antigenic stimulation in response to an IL-1 family member and a specific STAT activator, implying an innate mechanism through which memory CD4 T cells are recruited by an induced cytokine environment.

title: Immunological and inflammatory functions of the interleukin-1 family.
abstract: More than any other cytokine family, the interleukin (IL)-1 family is closely linked to the innate immune response. This linkage became evident upon the discovery that the cytoplasmic domain of the IL-1 receptor type I is highly homologous to the cytoplasmic domains of all Toll-like receptors (TLRs). Thus, fundamental inflammatory responses such as the induction of cyclooxygenase type 2, increased expression of adhesion molecules, or synthesis of nitric oxide are indistinguishable responses of both IL-1 and TLR ligands. Both families nonspecifically affect antigen recognition and lymphocyte function. IL-1beta is the most studied member of the IL-1 family because of its role in mediating autoinflammatory diseases. Although the TLR and IL-1 families evolved to assist in host defense against infection, unlike the TLR family, the IL-1 family also includes members that suppress inflammation, both specifically within the IL-1 family but also nonspecifically for TLR ligands and the innate immune response.

title: IL-1 and T Helper Immune Responses
abstract: CD4 T cells play a critical role in mediating adaptive immunity to a variety of pathogens as well as in tumor immunity. If not adequately regulated, CD4 T cells can be also involved in autoimmunity, asthma, and allergic responses. During TCR activation in a particular cytokine milieu, naïve CD4 T cells may differentiate into one of several lineages of T helper (Th) cells, including Th1, Th2, and Th17, as defined by their pattern of cytokine production and function. IL-1, the prototypic proinflammatory cytokine, has been shown to influence growth and differentiation of immunocompetent lymphocytes. The differential expression of IL-1RI on human CD4 T cell subsets confers distinct capacities to acquire specific effector functions. In this review, we summarize the role of IL-1 on CD4 T cells, in terms of differentiation, activation, and maintenance or survival.

title: Blood leukocyte microarrays to diagnose systemic onset juvenile idiopathic arthritis and follow the response to IL-1 blockade
abstract: Systemic onset juvenile idiopathic arthritis (SoJIA) represents up to 20% of juvenile idiopathic arthritis. We recently reported that interleukin (IL) 1 is an important mediator of this disease and that IL-1 blockade induces clinical remission. However, lack of specificity of the initial systemic manifestations leads to delays in diagnosis and initiation of therapy. To develop a specific diagnostic test, we analyzed leukocyte gene expression profiles of 44 pediatric SoJIA patients, 94 pediatric patients with acute viral and bacterial infections, 38 pediatric patients with systemic lupus erythematosus (SLE), 6 patients with PAPA syndrome, and 39 healthy children. Statistical group comparison and class prediction identified genes differentially expressed in SoJIA patients compared with healthy children. These genes, however, were also changed in patients with acute infections and SLE. An analysis of significance across all diagnostic groups identified 88 SoJIA-specific genes, 12 of which accurately classified an independent set of SoJIA patients with systemic disease. Transcripts that changed significantly in patients undergoing IL-1 blockade were also identified. Thus, leukocyte transcriptional signatures can be used to distinguish SoJIA from other febrile illnesses and to assess response to therapy. Availability of early diagnostic markers may allow prompt initiation of therapy and prevention of disabilities.

title: Regulating human Th17 cells via differential expression of IL-1 receptor.
abstract: In humans, interleukin-1beta (IL-1beta) has been suggested as an essential cytokine for developing IL-17- or IL-17A-producing CD4(+) T helper 17 (Th17) cells. However, little is known about the relationship of IL-1 receptor expression and Th17 cell differentiation. We report here the presence of 2 distinct CD4(+) T-cell populations with and without expression of IL-1RI that correlates with the capacity to produce IL-17 in naive and memory CD4(+) T cells of human peripheral blood. IL-1RI(+) memory CD4(+) T cells had increased gene expression of IL17, RORC, and IRF4 even before T-cell receptor triggering, indicating that the effect of IL-1beta is programmed in these cells via IL-1RI. Although CD4(+) T cells from umbilical cord blood did not express IL-1RI, the cytokines IL-7, IL-15, and transforming growth factor-beta (TGF-beta) up-regulated IL-1RI expression on naive CD4(+) T cells, suggesting that IL-1RI(+) naive CD4(+) T cells develop in periphery. Furthermore, IL-17 production from the cytokine-treated naive CD4(+) T cells was induced by IL-1beta and this induction was blocked by IL-1R antagonist. These results indicate that human Th17 cell differentiation is regulated via differential expression of IL-1RI, which is controlled by IL-7 and IL-15.

title: Evidence for a cross-talk between human neutrophils and Th17 cells.
abstract: Interleukin-17A (IL-17A) and IL-17F are 2 of several cytokines produced by T helper 17 cells (Th17), which are able to indirectly induce the recruitment of neutrophils. Recently, human Th17 cells have been phenotypically characterized and shown to express discrete chemokine receptors, including CCR2 and CCR6. Herein, we show that highly purified neutrophils cultured with interferon-gamma plus lipopolysaccharide produce the CCL2 and CCL20 chemokines, the known ligands of CCR2 and CCR6, respectively. Accordingly, supernatants from activated neutrophils induced chemotaxis of Th17 cells, which was greatly suppressed by anti-CCL20 and anti-CCL2 antibodies. We also discovered that activated Th17 cells could directly chemoattract neutrophils via the release of biologically active CXCL8. Consistent with this reciprocal recruitment, neutrophils and Th17 cells were found in gut tissue from Crohn disease and synovial fluid from rheumatoid arthritis patients. Finally, we report that, although human Th17 cells can directly interact with freshly isolated or preactivated neutrophils via granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-alpha, and interferon-gamma release, these latter cells cannot be activated by IL-17A and IL-17F, because of their lack of IL-17RC expression. Collectively, our results reveal a novel chemokine-dependent reciprocal cross-talk between neutrophils and Th17 cells, which may represent a useful target for the treatment of chronic inflammatory diseases.

title: Human polymorphonuclear-leukocyte inhibition of incorporation of chitin precursors into mycelia of Coccidioides immitis.
abstract: Human polymorphonuclear leukocytes (PMNLs), when added to arthroconidial suspensions of Coccidioides immitis, markedly inhibit fungal incorporation of the cell-wall precursor N-acetylglucosamine. This effect does not require serum but is facilitated by a heat-labile serum component(s), probably by promoting PMNL attachment to the arthroconidia. Inhibition is entirely reversible within 24 hr. In parallel with this finding, PMNLs exhibited virtually no killing of arthroconidia and (as determined by electron microscopy) did not produce damage to the fungal ultrastructure. PMNLs from a patient with chronic granulomatous disease suppressed fungal incorporation of N-acetyl-glucosamine by only 0%-22% under various conditions, as compared with 54%-85% suppression by PMNLs from normal donors. Our studies demonstrate that PMNLs can influence the metabolism of C immitis and raise the possibility that their presence in coccidioidal histopathology may be an important immediate host defense in limiting progression early after infection.

title: Inhibition of different phases of Coccidioides immitis by human neutrophils or hydrogen peroxide.
abstract: As arthroconidia of Coccidioides immitis transform into spherules, the fungal particles progressively become more resistant to the inhibitory effects of human polymorphonuclear leukocytes (PMNLs) as measured by PMNL inhibition of fungal incorporation of N-acetylglucosamine. Similar changes were noted when leucine incorporation was measured. When H2O2, at a concentration of 2.0 mM, was substituted for PMNLs, an equivalent inhibition of arthroconidia was produced and, as with PMNL effects, was lost with spherule maturation. The results from these studies indicate that C. immitis may evade natural inhibition by PMNLs by transformation into the spherule phase.

title: Influence of fungal surface components on the interaction of Coccidioides immitis with polymorphonuclear neutrophils.
abstract: Polymorphonuclear neutrophils (PMNs) possess phagocytic and fungicidal activity against Coccidioides immitis that declines during maturation from arthroconidia to round cells, is lost throughout spherule maturation, and returns when endospores are released from ruptured spherules. Studies of PMN chemiluminescence, iodination, and degranulation give similar results. Phagocytosis of forms other than spherules is strain dependent and enhanced by immune serum. The absence of adequate PMN-spherule interaction may be attributed to the production of an extracellular fibrillar matrix, glycoprotein in composition, that restricts intimate PMN-spherule contact. When the spherule ruptures, PMNs enter to phagocytose endospores that are themselves invested by a matrix derived from the inner spherule wall. The immunochemical relationship between the outer matrix and the inner matrix remains to be discovered. Nevertheless, presence of the outer matrix may help to explain the long-standing histopathologic observation that PMNs fail to attack spherules until they release their endospores.

title: Differences in oxidant release by human polymorphonuclear leukocytes produced by stimulation with different phases of Coccidioides immitis.
abstract: Human polymorphonuclear leukocytes (PMNL) inhibit growth of arthroconidia and young spherules but not mature spherules of Coccidioides immitis. To evaluate these differences, oxidants were measured when PMNL were exposed to C. immitis or other particulate stimuli. Concentrations of arthroconidia, young spherules, mature spherules, and Candida glabrata were adjusted to result in equivalent superoxide release. Hydrogen peroxide and hypochlorous acid were stimulated by arthroconidia or young spherules but were undetectable after exposure to mature spherules. Levels of catalase in mature spherules were too low to account for the low levels of hydrogen peroxide and hypochlorous acid. When mature spherules were added to arthroconidia or zymosan, stimulation of hydrogen peroxide and hypochlorous acid occurred. The results suggest that mature spherules stimulate superoxide release but interfere with hydrogen peroxide and hypochlorous acid production. The low levels of these antimicrobial oxidants may contribute to resistance of C. immitis to human PMNL.

title: Neutrophils in innate and adaptive immunity
abstract: Neutrophils have long been viewed as short-lived cells crucial for the elimination of extracellular pathogens, possessing a limited role in the orchestration of the immune response. This dogma has been challenged by recent lines of evidence demonstrating the expression of an increasing number of cytokines and effector molecules by neutrophils. Moreover, in analogy with their “big brother” macrophages, neutrophils integrate the environmental signals and can be polarized towards an antitumoural or protumoural phenotype. Neutrophils are a major source of humoral fluid phase pattern recognition molecules and thus contribute to the humoral arm of innate immunity. Neutrophils cross talk and shape the maturation and effector functions of other leukocytes in a direct or indirect manner, through cell–cell contact or cytokine production, respectively. Therefore, neutrophils are integrated in the activation and regulation of the innate and adaptive immune system and play an important role in the resolution or exacerbation of diverse pathologies, including infections, chronic inflammation, autoimmunity and cancer.

title: Interleukin 1 enhances vaccine-induced antifungal T-helper 17 cells and resistance against Blastomyces dermatitidis infection.
abstract: Vaccine-induced T-helper 17 (Th17) cells are necessary and sufficient to protect against fungal infection. Although live fungal vaccines are efficient in driving protective Th17 responses and immunity, attenuated fungi may not be safe for human use. Heat-inactivated formulations and subunit vaccines are safer but less potent and require adjuvant to increase their efficacy. Here, we show that interleukin 1 (IL-1) enhances the capacity of weak vaccines to induce protection against lethal Blastomyces dermatitidis infection in mice and is far more effective than lipopolysaccharide. While IL-1 enhanced expansion and differentiation of fungus-specific T cells by direct action on those cells, cooperation with non-T cells expressing IL-1R1 was necessary to maximize protection. Mechanistically, IL-17 receptor signaling was required for the enhanced protection induced by IL-1. Thus, IL-1 enhances the efficacy of safe but inefficient vaccines against systemic fungal infection in part by increasing the expansion of CD4(+) T cells, allowing their entry into the lungs, and inducing their differentiation to protective Th17 cells.

title: Novel Strategies to Enhance Vaccine Immunity against Coccidioidomycosis
abstract: Coccidioidomycosis is a potentially life-threatening respiratory mycosis endemic to the Americas and caused by inhalation of spores produced by the molds Coccidioides immitis and C. posadasii. The dry, air-dispersed infectious spores (arthroconidia) are derived from saprobic-phase filaments that grow in semidesert soil of the southwestern United States and arid regions of Mexico and Central and South America . Coccidioides is a dimorphic ascomycetous fungus with distinct saprobic and parasitic phases and is classified in the order Onygenales together with other genera of pathogenic molds that include Histoplasma, Blastomyces, and Paracoccidioides. The clinical spectrum of disease caused by these environmental pathogens ranges from a mild infection that resolves spontaneously to a disseminated mycosis. Inhaled spores of Coccidioides are small enough to colonize the lowermost reaches of the respiratory tree. Nonhuman primates experimentally challenged by aerosolization with fewer than ten infectious propagules developed a severe form of the disease and died within four to six weeks . The parasitic cycle of Coccidioides is unique among these medically important, dimorphic molds. Spores in the lungs germinate to form multinucleate spherules (.60 mm diameter) that are too large to be engulfed by host phagocytes. The contents of mature spherules differentiate into a multitude of propagative endospores. Production of indigestible spherules that display high fecundity during the endosporulation stage confound phagocytic defenses and augment survival of the fungus in the lungs. Mammalian innate immune defenses are further compromised by the ability of endosporulating spherules to generate an alkaline microenvironment at infection sites due to their release of ammonia, which contributes to the pathogen s virulence .