Michael P. Nelson

Neutrophils Produce Interleukin 17A (IL-17A) in a Dectin-1- and IL-23-Dependent Manner during Invasive Fungal Infection

ABSTRACT We have previously reported that compromised interleukin 17A (IL-17A) production in the lungs increased susceptibility to infection with the invasive fungal pathogen Aspergillus fumigatus. Here we have shown that culturing lung cells from A. fumigatus-challenged mice ex vivo demonstrated 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. Il23 mRNA expression was found to be lower in lung cells from A. fumigatus-challenged Dectin-1-deficient mice, whereas bone marrow-derived dendritic cells from Dectin-1-deficient mice failed to produce IL-23 in response to A. fumigatus in 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 in A. fumigatus-challenged Dectin-1-deficient mice. Ly-6G+ neutrophils purified from the lungs of A. fumigatus-challenged Dectin-1-deficient mice displayed lower Il17a mRNA expression but surprisingly had intact Rorc and Rora mRNA expression. We further demonstrated that Ly-6G+ neutrophils required the presence of myeloid cells for IL-17A production. Finally, upon in vitro stimulation with A. 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.

Dectin-1-Dependent Interleukin-22 Contributes to Early Innate Lung Defense against Aspergillus fumigatus

ABSTRACT 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-1β, 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-1α, tumor necrosis factor alpha (TNF-α), CCL3/MIP-1α, and CCL4/MIP-1β 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.

IL-33 and M2a Alveolar Macrophages Promote Lung Defense against the Atypical Fungal Pathogen Pneumocystis murina

We have recently reported that mice deficient in the myeloid Src-family tyrosine kinases Hck, Fgr, and Lyn (Src triple knockout [TKO]) had augmented innate lung clearance of Pneumocystis murina that correlated with a higher ability of alveolar macrophages (AMs) from these mice to kill P. murina. In this article, we show that despite possessing enhanced killing, AMs from naive Src TKO mice did not demonstrate enhanced inflammatory responses to P. murina. We subsequently discovered that both AMs and lungs from P. murina-infected Src TKO mice expressed significantly greater levels of the M2a markers RELM-α and Arg1, and the M2a-associated chemokines CCL17 and CCL22 than did wild-type mice. IL-4 and IL-13, the primary cytokines that promote M2a polarization, were not differentially produced in the lungs between wild-type and Src TKO mice. P. murina infection in Src TKO mice resulted in enhanced lung production of the novel IL-1 family cytokine IL-33. Immunohistochemical analysis of IL-33 in lung tissue revealed localization predominantly in the nucleus of alveolar epithelial cells. We further demonstrate that experimental polarization of naive AMs to M2a resulted in more efficient killing of P. murina compared with untreated AMs, which was further enhanced by the addition of IL-33. Administration of IL-33 to C57BL/6 mice increased lung RELM-α and CCL17 levels, and enhanced clearance of P. murina, despite having no effect on the cellular composition of the lungs. Collectively, these results indicate that M2a AMs are potent effector cells against P. murina. Furthermore, enhancing M2a polarization may be an adjunctive therapy for the treatment of Pneumocystis.

Eosinophil Deficiency Compromises Lung Defense against Aspergillus fumigatus

ABSTRACT Exposure to the mold Aspergillus fumigatus may result in allergic bronchopulmonary aspergillosis, chronic necrotizing pulmonary aspergillosis, or invasive aspergillosis (IA), depending on the hosts 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-6Glo Ly-6Cneg 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.

The absence of Hck, Fgr, and Lyn tyrosine kinases augments lung innate immune responses to Pneumocystis murina.

ABSTRACT Src family tyrosine kinases (SFKs) phosphorylate immunotyrosine activation motifs in the cytoplasmic tail of multiple immunoreceptors, leading to the initiation of cellular effector functions, such as phagocytosis, reactive oxygen species production, and cytokine production. SFKs also play important roles in regulating these responses through the activation of immunotyrosine inhibitory motif-containing inhibitory receptors. As myeloid cells preferentially express the SFKs Hck, Fgr, and Lyn, we questioned the role of these kinases in innate immune responses to Pneumocystis murina. Increased phosphorylation of Hck was readily detectable in alveolar macrophages after stimulation with P. murina. We further observed decreased phosphorylation of Lyn on its C-terminal inhibitory tyrosine in P. murina-stimulated alveolar macrophages, indicating that SFKs were activated in alveolar macrophages in response to P. murina. Mice deficient in Hck, Fgr, and Lyn exhibited augmented clearance 3 and 7 days after intratracheal administration of P. murina, which correlated with elevated levels of interleukin 1β (IL-1β), IL-6, CXCL1/KC, CCL2/monocyte chemoattractant protein 1, and granulocyte colony-stimulating factor in lung homogenates and a dramatic increase in macrophage and neutrophil recruitment. Augmented P. murina clearance was also observed in Lyn−/− mice 3 days postchallenge, although the level was less than that observed in Hck−/− Fgr−/− Lyn−/− mice. A correlate to augmented clearance of P. murina in Hck−/− Fgr−/− Lyn−/− mice was a greater ability of alveolar macrophages from these mice to kill P. murina in vitro, suggesting that SFKs regulate the alveolar macrophage effector function against P. murina. Mice deficient in paired immunoglobulin receptor B (PIR-B), an inhibitory receptor activated by SFKs, did not exhibit enhanced inflammatory responsiveness to or clearance of P. murina. Our results suggest that SFKs regulate innate lung responses to P. murina in a PIR-B-independent manner.

STAT4-Dependent and -Independent Th2 Responses Correlate with Protective Immunity against Lung Infection with Pneumocystis murina

Although it is clear that the loss of CD4+ T cells is a predisposing factor for the development of Pneumocystis pneumonia, specific Th mechanisms mediating protection are not well understood. Th1, Th2, and Th17 responses have each been implicated in protective responses during infection. As STAT4 may promote Th1 and Th17 development, yet antagonize Th2 development, we investigated its role in Pneumocystis murina host defense. STAT4 was required for Th1 and, unexpectedly, Th2 responses in the lungs of C57BL/6 (BL/6) and BALB/c mice 14 d postchallenge, but only BALB/c Stat4−/− mice demonstrated susceptibility to P. murina lung infection. BL/6 Stat4−/−, but not BALB/c Stat4−/−, mice maintained an enhanced alternatively activated (M2) macrophage signature in the lungs, which we have previously reported to be associated with enhanced P. murina clearance. In addition, anti-P. murina class-switched Abs were increased in BL/6 Stat4−/− mice, but not BALB/c Stat4−/− mice. Supporting our experimental observations, plasma from HIV-infected individuals colonized with Pneumocystis jirovecii contained significantly lower levels of the Th2 cytokines IL-4, IL-5, and IL-13 compared with HIV-infected individuals who were not colonized. Collectively, our data suggest that robust local and systemic Th2-mediated responses are critical for immunity to Pneumocystis.

Aspergillus terreus accessory conidia are multinucleated, hyperpolarizing structures that display differential dectin staining and can induce heightened inflammatory responses in a pulmonary model of aspergillosis

In addition to phialidic conidia (PC), A. terreus produces accessory conidia (AC) both in vitro and in vivo. AC are distinct from PC in cell surface architecture, with the AC surfaces displaying more β-glucan, a molecule that can be a trigger for the induction of inflammatory responses. The present study follows β-glucan cell surface presentation throughout the course of germination of both types of conidia, and analyzes the differential capacity of AC and PC to elicit immune responses. Results show that AC display early, increased dectin-1 labeling on their cell surfaces compared to PC, and this differential dectin-1 labeling is sustained on the cell surface from the time of breaking dormancy through early germ tube emergence. Mouse alveolar macrophages showed a stronger inflammatory cytokine/chemokine response when challenged with AC than with PC in both ex vivo and in vivo experiments, correlating with the greater exposure of β-glucan exhibited by AC. Further, histopathologic staining of the lungs from mice challenged with AC demonstrated heightened cell recruitment and increased inflammatory response compared to the lungs of mice challenged with PC. Our study also demonstrates that AC are multinucleate structures with the ability to germinate rapidly, polarizing in multiple directions and producing several hyphal extensions. We present evidence that A. terreus AC are phenotypically distinct from PC and can be potent activators of the innate immune mechanism thus possibly playing a role in this organisms pathogenesis.

Autophagy-lysosome pathway associated neuropathology and axonal degeneration in the brains of alpha-galactosidase A-deficient mice.

BackgroundMutations in the gene for alpha-galactosidase A result in Fabry disease, a rare, X-linked lysosomal storage disorder characterized by a loss of alpha-galactosidase A enzymatic activity. The resultant accumulation of glycosphingolipids throughout the body leads to widespread vasculopathy with particular detriment to the kidneys, heart and nervous system. Disruption in the autophagy-lysosome pathway has been documented previously in Fabry disease but its relative contribution to nervous system pathology in Fabry disease is unknown. Using an experimental mouse model of Fabry disease, alpha-galactosidase A deficiency, we examined brain pathology in 20-24 month old mice with particular emphasis on the autophagy-lysosome pathway.ResultsAlpha-galactosidase A-deficient mouse brains exhibited enhanced punctate perinuclear immunoreactivity for the autophagy marker microtubule-associated protein light-chain 3 (LC3) in the parenchyma of several brain regions, as well as enhanced parenchymal and vascular immunoreactivity for lysosome-associated membrane protein-1 (LAMP-1). Ultrastructural analysis revealed endothelial cell inclusions with electron densities and a pronounced accumulation of electron-dense lipopigment. The pons of alpha-galactosidase A-deficient mice in particular exhibited a striking neuropathological phenotype, including the presence of large, swollen axonal spheroids indicating axonal degeneration, in addition to large interstitial aggregates positive for phosphorylated alpha-synuclein that co-localized with the axonal spheroids. Double-label immunofluorescence revealed co-localization of phosphorylated alpha-synuclein aggregates with ubiquitin and LC3.ConclusionTogether these findings indicate widespread neuropathology and focused axonal neurodegeneration in alpha-galactosidase A-deficient mouse brain in association with disruption of the autophagy-lysosome pathway, and provide the basis for future mechanistic assessment of the contribution of the autophagy-lysosome pathway to this histologic phenotype.

Experimental Pneumocystis lung infection promotes M2a alveolar macrophage-derived MMP12 production

Among several bacterial and viral pathogens, the atypical fungal organism Pneumocystis jirovecii has been implicated as a contributor to the pathogenesis of chronic obstructive pulmonary disease (COPD). In a previous study, we reported that Pneumocystis-colonized HIV-positive subjects had worse obstruction of airways and higher sputum levels of macrophage elastase/matrix metalloproteinase 12 (MMP12), a protease strongly associated with the development of COPD. Here, we examined parameters of Pneumocystis-induced MMP12 in the lungs of mice and its role in the lung immune response to murine Pneumocystis. Initial studies demonstrated that P. murina exposure induced Mmp12 mRNA expression in whole lungs and alveolar macrophages (AMs), which was dependent on the presence of CD4+ T cells as well as signal transducer and activator of transcription 6. Mmp12 mRNA expression was upregulated in AMs by interleukin (IL)-4 treatment, but downregulated by interferon (IFN)-γ, indicating preferential expression in alternatively activated (M2a) macrophages. IL-4 treatment induced the 54-kDa proenzyme form of MMP12 and the 22-kDa fully processed and active form, whereas IFN-γ failed to induce either. Despite a reported antimicrobial role in macrophage phagolysosomes, mice deficient in MMP12 were not found to be more susceptible to lung infection with P. murina. Collectively, our data indicate that MMP12 induction is a component of the P. murina-induced M2 response and thus provides insight into the link between Pneumocystis colonization/infection and exacerbations in COPD.

Eosinophils Contribute to Early Clearance of Pneumocystis murina Infection

Pneumocystis pneumonia remains a common opportunistic infection in the diverse immunosuppressed population. One clear risk factor for susceptibility to Pneumocystis is a declining CD4+ T cell count in the setting of HIV/AIDS or primary immunodeficiency. Non–HIV-infected individuals taking immunosuppressive drug regimens targeting T cell activation are also susceptible. Given the crucial role of CD4+ T cells in host defense against Pneumocystis, we used RNA sequencing of whole lung early in infection in wild-type and CD4-depleted animals as an unbiased approach to examine mechanisms of fungal clearance. In wild-type mice, a strong eosinophil signature was observed at day 14 post Pneumocystis challenge, and eosinophils were increased in the bronchoalveolar lavage fluid of wild-type mice. Furthermore, eosinophilopoiesis-deficient Gata1tm6Sho/J mice were more susceptible to Pneumocystis infection when compared with BALB/c controls, and bone marrow–derived eosinophils had in vitro Pneumocystis killing activity. To drive eosinophilia in vivo, Rag1−/− mice were treated with a plasmid expressing IL-5 (pIL5) or an empty plasmid control via hydrodynamic injection. The pIL5-treated mice had increased serum IL-5 and eosinophilia in the lung, as well as reduced Pneumocystis burden, compared with mice treated with control plasmid. In addition, pIL5 treatment could induce eosinophilia and reduce Pneumocystis burden in CD4-depleted C57BL/6 and BALB/c mice, but not eosinophilopoiesis-deficient Gata1tm6Sho/J mice. Taken together, these results demonstrate that an early role of CD4+ T cells is to recruit eosinophils to the lung and that eosinophils are a novel candidate for future therapeutic development in the treatment of Pneumocystis pneumonia in the immunosuppressed population.