Hyphal growth of phagocytosed fusarium oxysporum causes cell lysis and death of murine macrophages

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Airway macrophage metabolic reprogramming during interstitial lung disease

Airway macrophages (AM) play a key role during the pathogenesis of pulmonary fibrosis. The underlying metabolic alterations driving this phenotype however are little understood. The aim of this thesis was to investigate the metabolic phenotype of AMs during pulmonary fibrosis (PF), to identify factors, which influence the pathogenesis of PF and to investigate the role of the immune modulatory metabolite itaconate during PF. In chapter 3, AM metabolic phenotype was analysed in patients with idiopathic pulmonary fibrosis (IPF) or chronic hypersensitivity pneumonitis (CHP) compared to healthy controls. In IPF AMs increased gene expression of first half TCA cycle genes was detected, as well as a dependency on glycolysis to sustain a pro-fibrotic phenotype, while AMs from CHP patients highly increased glycolysis and OXPHOS utilisation and a more pro-inflammatory macrophages phenotype was observed. These results highlight the influence of the environment and identify distinct AM phenotypes in clinically similar IPF and CHP. Recent paradigm shifting studies have shown that two types of AMs exist in the lung and during the pathogenesis of PF. In chapter 4, the underlying metabolic phenotype of tissue-resident (Tr-AM) and monocyte-recruited AMs (Mo-AM) was investigated using the bleomycin mouse model, which showed an increased utilisation of OXPHOS in Tr-AM but not Mo-AM during peak fibrosis. In chapter 5, the role of immune modulatory metabolite itaconate was investigated during PF. Itaconate has been shown to regulate AM metabolic activity and is anti-inflammatory and anti-microbial, however its role in the context of fibrosis is unknown. Analysis of the metabolic phenotype of AMs from IPF patients indicated that there was decreased expression of ACOD1, a gene which controls the synthesis of itaconate. Furthermore, Acod1-/- mice had more severe fibrosis compared to WT mice in bleomycin models, suggesting an anti-fibrotic role for itaconate. Ultimately, fibrosis was ameliorated by treatment with inhaled itaconate or adoptive transfer of itaconate-expressing Mo-AMs. Collectively, these findings reveal underlying metabolic programmes driving AM phenotype during pulmonary fibrosis and identify new therapeutic targets.Open Acces

Pulmonary dendritic cells and alveolar macrophages are regulated by γδ T cells during the resolution of S. pneumoniae-induced inflammation

γδ T cells commonly associate with mucosal and epithelial sites, fulfilling a variety of immunoregulatory functions. While lung γδ T cells have well-characterized pro-inflammatory activity, their potential role in the resolution of lung inflammation has yet to be explored in any detail. Indeed, given the importance of minimizing inflammation, the cellular mechanisms driving the resolution of lung inflammation are poorly understood. Using a murine model of acute Streptococcus pneumoniae-mediated lung inflammation, we now show that resolution of inflammation following bacterial clearance is associated with a > 30-fold increase in γδ T-cell number. Although inflammation eventually resolves in TCRδ−/− mice, elevated numbers of alveolar macrophages and pulmonary dendritic cells, and the appearance of well-formed granulomas in lungs of TCRδ−/− mice, together indicated a role for γδ T cells in regulating mononuclear phagocyte number. Ex vivo, both alveolar macrophages and pulmonary dendritic cells were susceptible to lung γδ T cell-mediated cytotoxicity, the first demonstration of such activity against a dendritic cell population. These findings support a model whereby expansion of γδ T cells helps restore mononuclear phagocyte numbers to homeostatic levels, protecting the lung from the consequences of inappropriate inflammation. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

Glial cells are functionally impaired in juvenile neuronal ceroid lipofuscinosis and detrimental to neurons.

The neuronal ceroid lipofuscinoses (NCLs or Batten disease) are a group of inherited, fatal neurodegenerative disorders of childhood. In these disorders, glial (microglial and astrocyte) activation typically occurs early in disease progression and predicts where neuron loss subsequently occurs. We have found that in the most common juvenile form of NCL (CLN3 disease or JNCL) this glial response is less pronounced in both mouse models and human autopsy material, with the morphological transformation of both astrocytes and microglia severely attenuated or delayed. To investigate their properties, we isolated glia and neurons from Cln3-deficient mice and studied their basic biology in culture. Upon stimulation, both Cln3-deficient astrocytes and microglia also showed an attenuated ability to transform morphologically, and an altered protein secretion profile. These defects were more pronounced in astrocytes, including the reduced secretion of a range of neuroprotective factors, mitogens, chemokines and cytokines, in addition to impaired calcium signalling and glutamate clearance. Cln3-deficient neurons also displayed an abnormal organization of their neurites. Most importantly, using a co-culture system, Cln3-deficient astrocytes and microglia had a negative impact on the survival and morphology of both Cln3-deficient and wildtype neurons, but these effects were largely reversed by growing mutant neurons with healthy glia. These data provide evidence that CLN3 disease astrocytes are functionally compromised. Together with microglia, they may play an active role in neuron loss in this disorder and can be considered as potential targets for therapeutic interventions

Structural and Functional Determinants of Rodent and Human Surfactant Protein A: A Synthesis of Binding and Computational Data

Surfactant protein A (SP-A) provides surfactant stability, first line host defense, and lung homeostasis by binding surfactant phospholipids, pathogens, alveolar macrophages (AMs), and epithelial cells. Non-primates express one SP-A protein whereas humans express two: SP-A1 and SP-A2 with core intra- and inter-species differences in the collagen-like domain. Here, we used macrophages and solid phase binding assays to discern structural correlates of rat (r) and human (h) SP-A function. Binding assays using recombinant rSP-A expressed in insect cells showed that lack of proline hydroxylation, truncations of amino-terminal oligomerization domains, and site-directed serine (S) or alanine (A) mutagenesis of cysteine 6 (C6S), glutamate 195 (E195A), and glutamate 171 (E171A) in the carbohydrate recognition domain (CRD) all impaired SP-A binding. Replacement of arginine 197 with alanine found in hSP-A (R197A), however, restored the binding of hydroxyproline-deficient rSP-A to the SP-A receptor SP-R210 similar to native rat and human SP-A. In silico calculation of Ca++ coordination bond length and solvent accessibility surface area revealed that the “humanized” R197A substitution alters topology and solvent accessibility of the Ca++ coordination residues of the CRD domain. Binding assays in mouse AMs that were exposed to either endogenous SP-A or hSP-A1 (6A2) and hSP-A2 (1A0) isoforms in vivo revealed that mouse SP-A is a functional hybrid of hSP-A1 and hSP-A2 in regulating SP-A receptor occupancy and binding affinity. Binding assays using neonatal and adult human AMs indicates that the interaction of SP-A1 and SP-A2 with AMs is developmentally regulated. Furthermore, our data indicate that the auxiliary ion coordination loop encompassing the conserved E171 residue may comprise a conserved site of interaction with macrophages, and SP-R210 specifically, that merits further investigation to discern conserved and divergent SP-A functions between species. In summary, our findings support the notion that complex structural adaptation of SP-A regulate conserved and species specific AM functions in vertebrates

An ongoing pulmonary cowpox virus infection suppresses an immune response to OVA peptide delivered to the lungs

Cowpox virus (CPXV), a close relative of variola virus, the orthopoxvirus that causes smallpox, can suppress the immune system through a large array of immunosuppressive gene products. We developed a murine model in which DO11.10 T cells specific for an OVA peptide were transferred into BALB/c mice to assess the impact of a pulmonary CPXV infection on DO11.10 T cell proliferation in lung draining lymph nodes following intranasal OVA peptide delivery. High and low-dose CPXV infections were compared. Both doses lead to clinical illness including ruffled coat and weight loss, but the high dose is lethal and is characterized by viral dissemination to the spleen. A high-dose infection reduced DO11.10 T cell proliferation, but a low-dose infection did not. At the time that proliferation of T cells was assessed (6d post infection), 15±1% of lung dendritic cells (DCs) were infected at the high-dose, but only 5±1% of DCs at the low-dose. At both doses, infected and uninfected lung DCs had decreased expression of MHC class II and the co-stimulatory molecules CD80 and CD86. DCs and T cells were not infected in the lymph nodes at either dose, but lymph node DCs also showed a reduction in antigen-presenting molecules. We speculated that the lung microenvironment created by infection, rather than direct infection of the DCs, suppressed DC antigen-specific T cell activation. In support, we found that alveolar lavage fluid and supernatant derived from lung homogenates from infected mice suppressed the function of uninfected lung DCs in vitro. Furthermore, the suppressive activity was more highly concentrated in lungs from high-dose infected mice. Cytokine analysis revealed the presence of IL-10, an immunosuppressive cytokine, in lung supernatants in mice receiving a high-dose of CPXV. We used IL-10 knockout mice in our adoptive transfer model to examine a role for IL-10 in T cell suppression in the lymph nodes. However, the knockout mice behaved similarly to BALB/c, with lack of DO11.10 T cell proliferation in the high-dose, but not the low-dose, and concluded IL-10 does not prevent T cell proliferation at the high-dose. Finally, we examined a possible virally encoded immunomodulatory protein, a soluble IFNγ receptor (IFNγR), to determine if sequestration of host-produced IFNγ contributed to the immune suppression seen in the high-dose. A mutant virus lacking the IFNγR behaved similarly to wild-type virus, and the survival and day 6 lung titers were comparable. Other virally encoded factors may play a role in suppressing DO11.10 T cell proliferation and should be examined in future experiments. These studies strongly suggest that orthopoxvirus infections create an immunosuppressive microenvironment that compromises the host pulmonary immune responses

Metabolism of tissue macrophages in homeostasis and pathology.

Cellular metabolism orchestrates the intricate use of tissue fuels for catabolism and anabolism to generate cellular energy and structural components. The emerging field of immunometabolism highlights the importance of cellular metabolism for the maintenance and activities of immune cells. Macrophages are embryo- or adult bone marrow-derived leukocytes that are key for healthy tissue homeostasis but can also contribute to pathologies such as metabolic syndrome, atherosclerosis, fibrosis or cancer. Macrophage metabolism has largely been studied in vitro. However, different organs contain diverse macrophage populations that specialize in distinct and often tissue-specific functions. This context specificity creates diverging metabolic challenges for tissue macrophage populations to fulfill their homeostatic roles in their particular microenvironment and conditions their response in pathological conditions. Here, we outline current knowledge on the metabolic requirements and adaptations of macrophages located in tissues during homeostasis and selected diseases.SKW and the project that gave rise to these results received support in the form of a fellowship from the La Caixa Foundation (ID 100010434). The fellowship code is LCF/BQ/ PR20/11770008. GD is supported by a European Molecular Biology Organization Longterm Fellowship (ALTF 379-2019). This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie SkłodowskaCurie grant agreement No. 892965. IHM is supported by a La Caixa INPhINIT fellowship (ID 100010434, fellowship code: LCF/BQ/IN17/11620074). Work in the DS laboratory is funded by the CNIC, by the European Research Council (ERC-2016-Consolidator Grant 725091), by the Agencia Estatal de Investigación (PID2019-108157RB), by the Comunidad de Madrid (B2017/BMD-3733 Immunothercan-CM), by Atresmedia (Constantes y Vitales prize), by the Fondo Solidario Juntos (Banco Santander), and by the Fundació La Marató de TV3 (201723). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the MICINN and the Pro CNIC Foundation.S