Long-term culture-expanded alveolar macrophages restore their full epigenetic identity after transfer in vivo.

Alveolar macrophages (AMs) are lung tissue-resident macrophages that can be expanded in culture, but it is unknown to what extent culture affects their in vivo identity. Here we show that mouse long-term ex vivo expanded AMs (exAMs) maintained a core AM gene expression program, but showed culture adaptations related to adhesion, metabolism and proliferation. Upon transplantation into the lung, exAMs reacquired full transcriptional and epigenetic AM identity, even after several months in culture and could self-maintain long-term in the alveolar niche. Changes in open chromatin regions observed in culture were fully reversible in transplanted exAMs and resulted in a gene expression profile indistinguishable from resident AMs. Our results indicate that long-term proliferation of AMs in culture did not compromise cellular identity in vivo. The robustness of exAM identity provides new opportunities for mechanistic analysis and highlights the therapeutic potential of exAMs.

Monocytes compensate Kupffer cell loss during bacterial infection.

Liver Kupffer cells (KCs) are self-maintained tissue-resident macrophages. In this issue of Immunity, Blériot et al. demonstrate that bacterial infection leads to KC necroptosis and quantitative replacement by monocyte-derived macrophages that contribute to antibacterial immunity and restoration of tissue integrity.

Superoxide dismutase 1 regulates caspase-1 and endotoxic shock.

Caspase-1 serves an essential function in the initiation of inflammation by proteolytically maturing the cytokines interleukin 1 beta and interleukin 18. Several Nod-like receptors activate caspase-1 in response to microbial and 'danger' signals by assembling cytosolic protein complexes called 'inflammasomes'. We show here that superoxide dismutase 1 (SOD1) regulates caspase-1 activation. In SOD1-deficient macrophages, higher superoxide production decreased the cellular redox potential and specifically inhibited caspase-1 by reversible oxidation and glutathionylation of the redox-sensitive cysteine residues Cys397 and Cys362. Conversely, hypoxic conditions abrogated caspase-1 inhibition. In vivo, SOD1-deficient mice produced less caspase-1-dependent cytokines and were less susceptible to lipopolysaccharide-induced septic shock. Our findings identify a physiological post-translational mechanism in the control of caspase-1-mediated inflammatory processes.

Tissue macrophage identity and self-renewal.

Macrophages are cellular components of the innate immune system that reside in virtually all tissues and contribute to immunity, repair, and homeostasis. The traditional view that all tissue-resident macrophages derive from the bone marrow through circulating monocyte intermediates has dramatically shifted recently with the observation that macrophages from embryonic progenitors can persist into adulthood and self-maintain by local proliferation. In several tissues, however, monocytes also contribute to the resident macrophage population, on which the local environment can impose tissue-specific macrophage functions. These observations have raised important questions: What determines resident macrophage identity and function, ontogeny or environment? How is macrophage proliferation regulated? In this review, we summarize the current knowledge about the identity, proliferation, and turnover of tissue-resident macrophages and how they differ from freshly recruited short-lived monocyte-derived cells. We examine whether macrophage proliferation can be qualified as self-renewal of mature differentiated cells and whether the concepts and molecular pathways are comparable to self-renewal mechanisms in stem cells. Finally, we discuss how improved understanding of macrophage identity and self-renewal could be exploited for therapeutic intervention of macrophage-mediated pathologies by selectively targeting freshly recruited or resident macrophages.

Mutant superoxide dismutase 1-induced IL-1beta accelerates ALS pathogenesis.

ALS is a fatal motor neuron disease of adult onset. Neuroinflammation contributes to ALS disease progression; however, the inflammatory trigger remains unclear. We report that ALS-linked mutant superoxide dismutase 1 (SOD1) activates caspase-1 and IL-1beta in microglia. Cytoplasmic accumulation of mutant SOD1 was sensed by an ASC containing inflammasome and antagonized by autophagy, limiting caspase-1-mediated inflammation. Notably, mutant SOD1 induced IL-1beta correlated with amyloid-like misfolding and was independent of dismutase activity. Deficiency in caspase-1 or IL-1beta or treatment with recombinant IL-1 receptor antagonist (IL-1RA) extended the lifespan of G93A-SOD1 transgenic mice and attenuated inflammatory pathology. These findings identify microglial IL-1beta as a causative event of neuroinflammation and suggest IL-1 as a potential therapeutic target in ALS.

Isolation and Long-term Cultivation of Mouse Alveolar Macrophages.

Alveolar macrophages (AM) are tissue-resident macrophages that colonize the lung around birth and can self-maintain long-term in an adult organism without contribution of monocytes. AM are located in the pulmonary alveoli and can be harvested by washing the lungs using the method of bronchoalveolar lavage (BAL). Here, we compared different conditions of BAL to obtain high yields of murine AM for in vitro culture and expansion of AM. In addition, we describe specific culture conditions, under which AM proliferate long-term in liquid culture in the presence of granulocyte-macrophage colony-stimulating factor. This method can be used to obtain large numbers of AM for in vivo transplantation or for in vitro experiments with primary mouse macrophages.

M-CSF improves protection against bacterial and fungal infections after hematopoietic stem/progenitor cell transplantation.

Myeloablative treatment preceding hematopoietic stem cell (HSC) and progenitor cell (HS/PC) transplantation results in severe myeloid cytopenia and susceptibility to infections in the lag period before hematopoietic recovery. We have previously shown that macrophage colony-stimulating factor (CSF-1; M-CSF) directly instructed myeloid commitment in HSCs. In this study, we tested whether this effect had therapeutic benefit in improving protection against pathogens after HS/PC transplantation. M-CSF treatment resulted in an increased production of mature myeloid donor cells and an increased survival of recipient mice infected with lethal doses of clinically relevant opportunistic pathogens, namely the bacteria Pseudomonas aeruginosa and the fungus Aspergillus fumigatus M-CSF treatment during engraftment or after infection efficiently protected from these pathogens as early as 3 days after transplantation and was effective as a single dose. It was more efficient than granulocyte CSF (G-CSF), a common treatment of severe neutropenia, which showed no protective effect under the tested conditions. M-CSF treatment showed no adverse effect on long-term lineage contribution or stem cell activity and, unlike G-CSF, did not impede recovery of HS/PCs, thrombocyte numbers, or glucose metabolism. These results encourage potential clinical applications of M-CSF to prevent severe infections after HS/PC transplantation.

Lineage-specific enhancers activate self-renewal genes in macrophages and embryonic stem cells.

Differentiated macrophages can self-renew in tissues and expand long term in culture, but the gene regulatory mechanisms that accomplish self-renewal in the differentiated state have remained unknown. Here we show that in mice, the transcription factors MafB and c-Maf repress a macrophage-specific enhancer repertoire associated with a gene network that controls self-renewal. Single-cell analysis revealed that, in vivo, proliferating resident macrophages can access this network by transient down-regulation of Maf transcription factors. The network also controls embryonic stem cell self-renewal but is associated with distinct embryonic stem cell-specific enhancers. This indicates that distinct lineage-specific enhancer platforms regulate a shared network of genes that control self-renewal potential in both stem and mature cells.

Interleukin-1 Antagonist Anakinra in Amyotrophic Lateral Sclerosis--A Pilot Study.

UNLABELLED: Preclinical studies show that blocking Interleukin-1 (IL-1) retards the progression of Amyotrophic Lateral Sclerosis (ALS). We assessed the safety of Anakinra (ANA), an IL-1 receptor antagonist, in ALS patients. In a single arm pilot study we treated 17 ALS patients with ANA (100 mg) daily for one year. We selected patients with dominant or exclusive lower motor neuron degeneration (LMND) presentation, as peripheral nerves may be more accessible to the drug. Our primary endpoint was safety and tolerability. Secondary endpoints included measuring disease progression with the revised ALS functional rating scale (ALSFRSr). We also quantified serum inflammatory markers. For comparison, we generated a historical cohort of 47 patients that fit the criteria for enrollment, disease characteristics and rate of progression of the study group. Only mild adverse events occurred in ALS patients treated with ANA. Notably, we observed lower levels of cytokines and the inflammatory marker fibrinogen during the first 24 weeks of treatment. Despite of this, we could not detect a significant reduction in disease progression during the same period in patients treated with ANA compared to controls as measured by the ALSFRSr. In the second part of the treatment period we observed an increase in serum inflammatory markers. Sixteen out of the 17 patients (94%) developed antibodies against ANA. This study showed that blocking IL-1 is safe in patients with ALS. Further trials should test whether targeting IL-1 more efficiently can help treating this devastating disease. TRIAL REGISTRATION: ClinicalTrials.gov NCT01277315.

Progressive replacement of embryo-derived cardiac macrophages with age.

Cardiac macrophages (cMΦ) are critical for early postnatal heart regeneration and fibrotic repair in the adult heart, but their origins and cellular dynamics during postnatal development have not been well characterized. Tissue macrophages can be derived from embryonic progenitors or from monocytes during inflammation. We report that within the first weeks after birth, the embryo-derived population of resident CX3CR1(+) cMΦ diversifies into MHCII(+) and MHCII(-) cells. Genetic fate mapping demonstrated that cMΦ derived from CX3CR1(+) embryonic progenitors persisted into adulthood but the initially high contribution to resident cMΦ declined after birth. Consistent with this, the early significant proliferation rate of resident cMΦ decreased with age upon diversification into subpopulations. Bone marrow (BM) reconstitution experiments showed monocyte-dependent quantitative replacement of all cMΦ populations. Furthermore, parabiotic mice and BM chimeras of nonirradiated recipient mice revealed a slow but significant donor contribution to cMΦ. Together, our observations indicate that in the heart, embryo-derived cMΦ show declining self-renewal with age and are progressively substituted by monocyte-derived macrophages, even in the absence of inflammation.

Transcriptional control of macrophage identity, self-renewal, and function.

Macrophages not only are prominent effector cells of the immune system that are critical in inflammation and innate immune responses but also fulfill important functions in tissue homeostasis. Transcription factors can define macrophage identity and control their numbers and functions through the induction and maintenance of specific transcriptional programs. Here, we review the mechanisms employed by lineage-specific transcription factors to shape macrophage identity during the development from hematopoietic stem and progenitor cells. We also present current insight into how specific transcription factors control macrophage numbers, by regulating coordinated proliferation and differentiation of myeloid progenitor cells and self-renewal of mature macrophages. We finally discuss how functional specialization of mature macrophages in response to environmental stimuli can be induced through synergistic activity of lineage- and stimulus-specific transcription factors that plug into preexisting transcriptional programs. Understanding the mechanisms that define macrophage identity, numbers, and functions will provide important insights into the differential properties of macrophage populations under various physiological and pathological conditions.

Identification of cellular interaction partners of the influenza virus ribonucleoprotein complex and polymerase complex using proteomic-based approaches.

Cellular factors that associate with the influenza A viral ribonucleoprotein (vRNP) are presumed to play important roles in the viral life cycle. To date, interaction screens using individual vRNP components, such as the nucleoprotein or viral polymerase subunits, have revealed few cellular interaction partners. To improve this situation, we performed comprehensive, proteomics-based screens to identify cellular factors associated with the native vRNP and viral polymerase complexes. Reconstituted vRNPs were purified from human cells using Strep-tagged viral nucleoprotein (NP-Strep) as bait, and co-purified cellular factors were identified by mass spectrometry (MS). In parallel, reconstituted native influenza A polymerase complexes were isolated using tandem affinity purification (TAP)-tagged polymerase subunits as bait, and co-purified cellular factors were again identified by MS. Using these techniques, we identified 41 proteins that co-purified with NP-Strep-enriched vRNPs and four cellular proteins that co-purified with the viral polymerase complex. Two of the polymerase-associated factors, importin-beta3 and PARP-1, represent novel interaction partners. Most cellular proteins previously shown to interact with either viral NP and/or vRNP were also identified using our method, demonstrating its sensitivity. Co-immunoprecipitation studies in virus-infected cells using selected novel interaction partners, including nucleophosmin (NPM), confirmed their association with vRNP. Immunofluorescence analysis further revealed that NPM is recruited to sites of viral transcription and replication in infected cells. Additionally, overexpression of NPM resulted in increased viral polymerase activity, indicating its role in viral RNA synthesis. In summary, the proteomics-based approaches used in this study represent powerful tools to identify novel vRNP-associated cellular factors for further characterization.