We have previously reported that compromised interleukin 17A (IL-17A) production in the lungs increased susceptibility to infection with the invasive fungal pathogenAspergillus fumigatus. Here we have shown that culturing lung cells fromA. fumigatus-challenged miceex vivodemonstrated Dectin-1-dependent IL-17A production. In this system, neutralization of IL-23 but not IL-6, IL-1β, or IL-18 resulted in attenuated IL-17A production.Il23mRNA expression was found to be lower in lung cells fromA. fumigatus-challenged Dectin-1-deficient mice, whereas bone marrow-derived dendritic cells from Dectin-1-deficient mice failed to produce IL-23 in response toA. fumigatusin vitro. Addition of recombinant IL-23 augmented IL-17A production by wild-type (WT) and Dectin-1-deficient lung cells, although the addition of IL-6 or IL-1β did not augment the effect of IL-23. Intracellular cytokine staining of lung cells revealed lower levels of CD11b+IL-17A+and Ly-6G+IL-17A+cells inA. fumigatus-challenged Dectin-1-deficient mice. Ly-6G+neutrophils purified from the lungs ofA. fumigatus-challenged Dectin-1-deficient mice displayed lowerIl17amRNA expression but surprisingly had intactRorcandRoramRNA expression. We further demonstrated that Ly-6G+neutrophils required the presence of myeloid cells for IL-17A production. Finally, uponin vitrostimulation withA. fumigatus, thioglycolate-elicited peritoneal neutrophils were positive for intracellular IL-17A expression and produced IL-17A in a Dectin-1- and IL-23-dependent manner. In summary, Dectin-1-dependent IL-17A production in the lungs during invasive fungal infection is mediated in part by CD11b+Ly-6G+neutrophils in an IL-23-dependent manner.

Sensitization to fungi, such as the mold Aspergillus fumigatus, is increasingly becoming linked with asthma severity. We have previously shown that lung responses generated via the beta-glucan receptor Dectin-1 are required for lung defense during acute, invasive A. fumigatus infection. Unexpectedly, in an allergic model of chronic lung exposure to live A. fumigatus conidia, beta-glucan recognition via Dectin-1 led to the induction of multiple proallergic (Muc5ac, Clca3, CCL17, CCL22 and IL-33) and proinflammatory (IL-1β and CXCL1) mediators that compromised lung function. Attenuated proallergic and proinflammatory responses in the absence of Dectin-1 was not associated with changes in Ido (indoleamine 2,3-dioxygenase), Il12p35/Ebi3 (IL-35), IL-10 or TGF-β levels. Assessment of T helper responses demonstrated that purified lung CD4+ T cells produced IL-4, IL-13, IFN-γ and IL-17A, but not IL-22, in a Dectin-1 dependent manner. In contrast, we observed robust, Dectin-1 dependent IL-22 production by unfractionated lung digest cells. Intriguingly, the absence of IL-22 alone mimicked the attenuated proallergic and proinflammatory responses observed in the absence of Dectin-1, suggesting that Dectin-1 mediated IL-22 production potentiated responses that led to decrements in lung function. To this end, neutralization of IL-22 improved lung function in normal mice. Collectively, these results indicate that the beta-glucan receptor Dectin-1 contributes to lung inflammation and immunopathology associated with persistent fungal exposure via the production of IL-22.

We have previously reported that mice deficient in the beta-glucan receptor Dectin-1 displayed increased susceptibility to Aspergillus fumigatus lung infection in the presence of lower interleukin 23 (IL-23) and IL-17A production in the lungs and have reported a role for IL-17A in lung defense. As IL-23 is also thought to control the production of IL-22, we examined the role of Dectin-1 in IL-22 production, as well as the role of IL-22 in innate host defense against A. fumigatus. Here, we show that Dectin-1-deficient mice demonstrated significantly reduced levels of IL-22 in the lungs early after A. fumigatus challenge. Culturing cells from enzymatic lung digests ex vivo further demonstrated Dectin-1-dependent IL-22 production. IL-22 production was additionally found to be independent of IL-1␤, IL-6, or IL-18 but required IL-23. The addition of recombinant IL-23 augmented IL-22 production in wild-type (WT) lung cells and rescued IL-22 production by lung cells from Dectin-1-deficient mice. In vivo neutralization of IL-22 in the lungs of WT mice resulted in impaired A. fumigatus lung clearance. Moreover, mice deficient in IL-22 also demonstrated a higher lung fungal burden after A. fumigatus challenge in the presence of impaired IL-1␣, tumor necrosis factor alpha (TNF-␣), CCL3/MIP-1␣, and CCL4/MIP-1␤ production and lower neutrophil recruitment, yet intact IL-17A production. We further show that lung lavage fluid collected from both A. fumigatus-challenged Dectin-1-deficient and IL-22-deficient mice had compromised anti-fungal activity against A. fumigatus in vitro. Although lipocalin 2 production was observed to be Dectin-1 and IL-22 dependent, lipocalin 2-deficient mice did not demonstrate impaired A. fumigatus clearance. Moreover, lung S100a8, S100a9, and Reg3g mRNA expression was not lower in either Dectin-1-deficient or IL-22-deficient mice. Collectively, our results indicate that early innate lung defense against A. fumigatus is mediated by Dectin-1-dependent IL-22 production.

Exposure to the mold Aspergillus fumigatus may result in allergic bronchopulmonary aspergillosis, chronic necrotizing pulmonary aspergillosis, or invasive aspergillosis (IA), depending on the host's immune status. Neutrophil deficiency is the predominant risk factor for the development of IA, the most life-threatening condition associated with A. fumigatus exposure. Here we demonstrate that in addition to neutrophils, eosinophils are an important contributor to the clearance of A. fumigatus from the lung. Acute A. fumigatus challenge in normal mice induced the recruitment of CD11b+ Siglec F+ Ly-6G(lo) Ly-6C(neg) CCR3+ eosinophils to the lungs, which was accompanied by an increase in lung Epx (eosinophil peroxidase) mRNA levels. Mice deficient in the transcription factor dblGATA1, which exhibit a selective deficiency in eosinophils, demonstrated impaired A. fumigatus clearance and evidence of germinating organisms in the lung. Higher burden correlated with lower mRNA expression of Epx (eosinophil peroxidase) and Prg2 (major basic protein) as well as lower interleukin 1β (IL-1β), IL-6, IL-17A, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and CXCL1 levels. However, examination of lung inflammatory cell populations failed to demonstrate defects in monocyte/macrophage, dendritic cell, or neutrophil recruitment in dblGATA1-deficient mice, suggesting that the absence of eosinophils in dlbGATA1-deficient mice was the sole cause of impaired lung clearance. We show that eosinophils generated from bone marrow have potent killing activity against A. fumigtaus in vitro, which does not require cell contact and can be recapitulated by eosinophil whole-cell lysates. Collectively, our data support a role for eosinophils in the lung response after A. fumigatus exposure.

During many chronic infections, the responding CD8 T cells become exhausted as they progressively lose their ability to elaborate key effector functions. Unlike prototypic memory CD8 cells, which rapidly synthesize IFN-γ following activation, severely exhausted T cells fail to produce this effector molecule. Nevertheless, the ontogeny of exhausted CD8 T cells, as well as the underlying mechanisms that account for their functional inactivation, remains ill defined. We have used cytokine reporter mice, which mark the transcription of IFN-γ mRNA by the expression of Thy1.1, to decipher how activation events during the early stages of a chronic infection dictate the development of exhaustion. We show that virus-specific CD8 T cells clearly respond during the early stages of chronic lymphocytic choriomeningitis virus infection, and that this early T cell response is more pronounced than that initially observed in acutely infected hosts. Thus, exhausted CD8 T cells appear to emerge from populations of potently activated precursors. Unlike acute infections, which result in massive expansion of the responding T cells, there is a rapid attenuation of further expansion during chronic infections. The exhausted T cells that subsequently emerge in chronically infected hosts are incapable of producing the IFN-γ protein. Surprisingly, high levels of the IFN-γ transcript are still present in exhausted cells, demonstrating that ablation of IFN-γ production by exhausted cells is not due to transcriptional silencing. Thus, posttranscription regulatory mechanisms likely disable this effector module.

Under selective pressure from host immunity, viruses have retained genes encoding “immunoevasins” - molecules interfering with host viral recognition and clearance. Due to their binding specificities, immunoevasins can be exploited as affinity labels to identify host-encoded molecules of previously unsuspected importance in defense against the relevant class of virus. We previously described an orthopoxvirus MHC class I-like protein (OMCP) that binds with high affinity to the activating receptor NKG2D on NK and T cell subsets, implicating NKG2D in anti-orthopoxvirus immunity. Here, we report that OMCP also binds in an NKG2D-independent manner to B cells and monocytes/macrophages. We identify murine Fc receptor-like 5 (FCRL5), an orphan immunoregulatory protein highly expressed by innate B lymphocytes, as a specific receptor for OMCP. The three N-terminal Ig domains of FCRL5 are required for OMCP binding. The targeting of FCRL5 by an orthopoxvirus immunoevasin strongly implicates it in contributing to host defense against zoonotic orthopoxviruses.

Sensitization to fungi, such as the mold Aspergillus fumigatus, is increasingly becoming linked with asthma severity. Although long considered a Th2 dominated disease, emerging data indicates that IL-17A and IL-22 may also contribute to asthma pathogenesis. Our laboratory has identified protective roles for Dectin-1 mediated beta-glucan recognition and Dectin-1 dependent IL-17A and IL-22 production during invasive A. fumigatus infection. Unexpectedly, in an allergic model of repeated lung exposure to live A. fumigatus conidia, we show that the absence of Dectin-1 resulted in markedly improved respiratory mechanics in the presence of reductions in multiple allergy-associated and inflammatory mediators. Intriguingly, attenuated lung IL-17A and IL-22 production in Dectin-1 deficient mice, but not Th2 cytokines, correlated with a better outcome. IL-22 deficiency reproduced the phenotype observed in Dectin-1 deficient mice after repetitive A. fumigatus exposure. We further identified IL-33 as a Dectin-1 and IL-22 dependent pro-allergic mediator. Collectively, our data suggests that during repetitive fungal exposure, beta-glucan recognition via Dectin-1 results in the generation of IL-17A and IL-22 responses as well as to the induction of IL-33 leading to the initiation of the allergic cascade. These results provide a novel link between infectious agents (fungi), new players (IL-22) and immunopathogenesis (IL-33) in severe asthma.

The ontogeny of exhausted CD8 T cells as well as the underlying mechanisms that account for the functional inactivation of these cells remains ill-defined. We have utilized cytokine reporter mice, which mark the synthesis of interferon-γ by the expression of Thy1.1, to decipher how activation events during the early stages of a chronic infection dictate the development of exhaustion. We show that, during chronic lymphocytic choriomeningitis virus infection, the precursors of exhausted T cells all upregulate interferon-γ mRNA and become susceptible to depletion with anti-Thy1.1 antibodies. This potent phase of hyperactivation which precedes exhaustion is antigen-dependent and is dictated by viral levels and T cell precursor frequencies. Unlike acute infections, which result in massive expansion of the responding T cells, during chronic infections further expansion of the initial response becomes rapidly attenuated. The exhausted T cells which subsequently emerge in chronically infected hosts attain a distinct CD127lo, KLRG-1lo phenotype but do not silence interferon-γ transcription even though protein expression is abolished. Thus ablation of interferon-γ production by exhausted cells is not due to transcriptional silencing, implicating post-transcription regulatory mechanisms in disabling this effector module.