CD116+ fetal precursors migrate to the perinatal lung and give rise to human alveolar macrophages

Despite their importance in lung health and disease, it remains unknown how human alveolar macrophages develop early in life. Here we define the ontogeny of human alveolar macrophages from embryonic progenitors in vivo, using a humanized mouse model expressing human cytokines (MISTRG mice). We identified alveolar macrophage progenitors in human fetal liver that expressed the GM-CSF receptor CD116 and the transcription factor MYB. Transplantation experiments in MISTRG mice established a precursor–product relationship between CD34−CD116+ fetal liver cells and human alveolar macrophages in vivo. Moreover, we discovered circulating CD116+CD64−CD115+ macrophage precursors that migrated from the liver to the lung. Similar precursors were present in human fetal lung and expressed the chemokine receptor CX3CR1. Fetal CD116+CD64− macrophage precursors had a proliferative gene signature, outcompeted adult precursors in occupying the perinatal alveolar niche, and developed into functional alveolar macrophages. The discovery of the fetal alveolar macrophage progenitor advances our understanding of human macrophage origin and ontogeny.

CRISPR/Cas9 knock-in strategy to evaluate phospho-regulation of SAMHD1

Sterile α motif (SAM) and HD domain-containing protein 1 (SAMHD1) is a potent restriction factor for immunodeficiency virus 1 (HIV-1), active in myeloid and resting CD4+ T cells. As a dNTP triphosphate triphosphohydrolase (dNTPase), SAMHD1 is proposed to limit cellular dNTP levels correlating with inhibition of HIV-1 reverse transcription. The anti-viral activity of SAMHD1 is regulated by dephosphorylation of the residue T592. However, the impact of T592 phosphorylation on dNTPase activity is still under debate. Whether additional cellular functions of SAMHD1 impact anti-viral restriction is also not completely understood. We use BlaER1 cells as a novel human macrophage transdifferentiation model combined with CRISPR/Cas9 knock-in (KI) to study SAMHD1 mutations in a physiological context. Transdifferentiated BlaER1 cells, resembling primary human macrophages, harbor active dephosphorylated SAMHD1 that blocks HIV-1 reporter virus infection. Co-delivery of Vpx or CRISPR/Cas9-mediated SAMHD1 knock-out relieves the block to HIV-1. Using CRISPR/Cas9-mediated homologous recombination, we introduced specific mutations into the genomic SAMHD1 locus. Homozygous T592E mutation, but not T592A, leads to loss of HIV-1 restriction, confirming the role of T592 dephosphorylation in the regulation of anti-viral activity. However, T592E KI cells retain wild type dNTP levels, suggesting the antiviral state might not only rely on dNTP depletion. In conclusion, the role of the T592 phospho-site for anti-viral restriction was confirmed in an endogenous physiological context. Importantly, loss of restriction in T592E mutant cells does not correlate with increased dNTP levels, indicating that the regulation of anti-viral and dNTPase activity of SAMHD1 might be uncoupled.

Cell-based screen identifies human type I interferon-stimulated regulators of Toxoplasma gondii Infection

Macrophages activated with interferons (IFNs) respond with transcriptional changes that enhance clearance of intracellular pathogens such as Toxoplasma, a ubiquitous apicomplexan parasite that infects more than a billion people worldwide. Although IFNs generally inhibit Toxoplasma, the parasite can also induce components of the host IFN signalling pathway to enhance survival in host cells. Compared to the type II IFN gamma (IFNγ), the role of type I IFNs in macrophage response to Toxoplasma is relatively not well characterized. Here, using fluorescent Toxoplasma and a CRISPR/Cas9 knockout library that only targets interferon-stimulated genes (ISGs), we adapted a loss-of-function flow cytometry-based approach to systematically identify type I ISGs that control Toxoplasma growth in THP-1 cells, a human macrophage cell line. The system enabled the rapid screening of more than 1900 ISGs for type I (IFNα)-induced inhibitors and enhancers of Toxoplasma growth in THP-1 cells. We identified 26 genes that are associated with Toxoplasma growth arrest out of which we confirmed MAX, SNX5, F2RL2, and SSB, as potent IFNα-induced inhibitors of Toxoplasma in THP1 cells. These findings provide a genetic and experimental roadmap to elucidate type I IFN-induced cell-autonomous responses to Toxoplasma.

Anti-Inflammatory Activity of Fucoidan Extracts In Vitro

Fucoidans are sulfated, complex, fucose-rich polymers found in brown seaweeds. Fucoidans have been shown to have multiple bioactivities, including anti-inflammatory effects, and are known to inhibit inflammatory processes via a number of pathways such as selectin blockade and enzyme inhibition, and have demonstrated inhibition of inflammatory pathologies in vivo. In this current investigation, fucoidan extracts from Undaria pinnatifida, Fucus vesiculosus, Macrocystis pyrifera, Ascophyllum nodosum, and Laminaria japonica were assessed for modulation of pro-inflammatory cytokine production (TNF-α, IL-1β, and IL-6) by human peripheral blood mononuclear cells (PBMCs) and in a human macrophage line (THP-1). Fucoidan extracts exhibited no signs of cytotoxicity in THP-1 cells after incubation of 48 h. Additionally, all fucoidan extracts reduced cytokine production in LPS stimulated PBMCs and human THP-1 cells in a dose-dependent fashion. Notably, the 5–30 kDa subfraction from Macrocystis pyrifera was a highly effective inhibitor at lower concentrations. Fucoidan extracts from all species had significant anti-inflammatory effects, but the lowest molecular weight subfractions had maximal effects at low concentrations. These observations on various fucoidan extracts offer insight into strategies that improve their efficacy against inflammation-related pathology. Further studies should be conducted to elucidate the mechanism of action of these extracts.

Macrophage cell lines and murine infection by Salmonella Typhi L-form bacteria

Antibiotic resistance of pathogenic bacteria has emerged as a major threat to public health worldwide. While stable resistance due to the acquisition of genomic mutations or plasmids carrying antibiotic-resistance genes is well-established, much less is known about the temporary and reversible resistance induced by antibiotic treatment, such as the one due to treatment with bacterial cell-wall inhibiting antibiotics like ampicillin. Typically, ampicillin concentration in the blood and other tissues gradually increases over time after initiation of the treatment. As a result, the bacterial population is exposed to a concentration gradient of ampicillin. This is different from in vitro drug testing where the organism is exposed to fixed drug concentrations from the beginning till the end. To mimic the mode of antibiotic exposure of microorganisms in the tissues, we cultured the wild type, ampicillin-sensitive Salmonella Typhi Ty2 strain (S. Typhi Ty2) in the presence of increasing concentrations of ampicillin over a period of 14 days. This resulted in the development of a strain that exhibited several features of the so-called L-form of bacteria, such as the absence of cell wall, altered shape and slower growth rate compared with the parental strain. Studies on the pathogenesis of S. Typhi L-form showed efficient infection of the murine and human macrophage cell lines. More importantly, S. Typhi L-form was also able to establish infection in a mouse model to the extent comparable to its parental strain. These results suggested that L-form generation following initiation of antibiotic treatment could lead to drug escape of S. Typhi and direct spread to new cells (macrophages), which sustain the infection. Oral infection by the L-form bacteria underscores the potential of rapid disease transmission through faeco-oral route, highlighting the need for new approaches to decrease the reservoir of infection.

A Novel Role for the Regulatory Nod-Like Receptor NLRP12 in Anti-Dengue Virus Response

Dengue Virus (DENV) infection can cause severe illness such as highly fatality dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Innate immune activation by Nod-like receptors (NLRs) is a critical part of host defense against viral infection. Here, we revealed a key mechanism of NLRP12-mediated regulation in DENV infection. Firstly, NLRP12 expression was inhibited in human macrophage following DENV or other flaviviruses (JEV, YFV, ZIKV) infection. Positive regulatory domain 1 (PRDM1) was induced by DENV or poly(I:C) and suppressed NLRP12 expression, which was dependent on TBK-1/IRF3 and NF-κB signaling pathways. Moreover, NLRP12 inhibited DENV and other flaviviruses (JEV, YFV, ZIKV) replication, which relied on the well-conserved nucleotide binding structures of its NACHT domain. Furthermore, NLRP12 could interact with heat shock protein 90 (HSP90) dependent on its Walker A and Walker B sites. In addition, NLRP12 enhanced the production of type I IFNs (IFN-α/β) and interferon-stimulated genes (ISGs), including IFITM3, TRAIL and Viperin. Inhibition of HSP90 with 17-DMAG impaired the upregulation of type I IFNs and ISGs induced by NLRP12. Taken together, we demonstrated a novel mechanism that NLRP12 exerted anti-viral properties in DENV and other flaviviruses (JEV, YFV, ZIKV) infection, which brings up a potential target for the treatment of DENV infection.

Macrophages in dermal disease progression of phospholipase D4–deficient Fleckvieh calves

A new gene defect in Fleckvieh calves leads to a syndrome with partial phenotype overlap with bovine hereditary zinc deficiency. A mutation in a gene encoding phospholipase D4 ( PLD4), an endosomal exonuclease, causes the disorder. In mice, PLD4 activity indirectly regulates the Toll-like receptor 9 (TLR9) pathway via degradation of microbial DNA. PLD4 absence thus results in visceral macrophage activation comparable to human macrophage activation syndrome. In this study, disease progression and the role of macrophages in affected calves were monitored clinically, clinicopathologically, and histologically over time. Breeding data identified 73 risk matings of heterozygous carriers resulting in 54 potentially PLD4-deficient calves born on farms. PLD4 status was examined via 5′-exonuclease assay, detecting 6 calves carrying the defect. These were purchased and monitored daily until final necropsy. The calves developed progressive skin lesions starting with small scaling areas terminating in severe crusting dermatitis, especially in areas with mechanical exposure. Histological and immunohistochemical analyses indicated that macrophages with cytoplasmic vacuolation increased considerably in skin sections obtained weekly during the disease course. Macrophage increase correlated with increased dermal lesion severity. Macrophage activation was confirmed by prominent phagocytic activity in the superficial dermis using electron microscopy. Dermal mRNA abundance of CCL2 and CCL3 measured by quantitative polymerase chain reaction verified macrophage activation. Further increase in mRNA of downstream molecule MyD88 and cytokine IL12b connected bovine PLD4 deficiency to increased TLR9 pathway activation. In contrast to human macrophage activation syndrome, the main feature of bovine PLD4 deficiency was local disease in organs with contact to microbial DNA (skin, intestine, lungs).

Pharmacological Poly (ADP-Ribose) Polymerase Inhibitors Decrease Mycobacterium tuberculosis Survival in Human Macrophages

Diabetes mellites (DM) is correlated with increased susceptibility to and disease progression of tuberculosis (TB), and strongly impairs effective global TB control measures. To better control the TB-DM co-epidemic, unravelling the bidirectional interactivity between DM-associated molecular processes and immune responses to Mycobacterium tuberculosis (Mtb) is urgently required. Since poly (ADP-ribose) polymerase (PARP) activation has been associated with DM and with Mtb infection in mouse models, we have investigated whether PARP inhibition by pharmacological compounds can interfere with host protection against Mtb in human macrophage subsets, the predominant target cell of Mtb. Pharmacological inhibition of PARP decreased intracellular Mtb and MDR-Mtb levels in human macrophages, identifying PARP as a potential target for host-directed therapy against Mtb. PARP inhibition was associated with modified chemokine secretion and upregulation of cell surface activation markers by human macrophages. Targeting LDH, a secondary target of the PARP inhibitor rucaparib, resulted in decreased intracellular Mtb, suggesting a metabolic role in rucaparib-induced control of Mtb. We conclude that pharmacological inhibition of PARP is a potential novel strategy in developing innovative host-directed therapies against intracellular bacterial infections.