Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage.

Tissue fibrosis is a major cause of mortality that results from the deposition of matrix proteins by an activated mesenchyme. Macrophages accumulate in fibrosis, but the role of specific subgroups in supporting fibrogenesis has not been investigated in vivo. Here, we used single-cell RNA sequencing (scRNA-seq) to characterize the heterogeneity of macrophages in bleomycin-induced lung fibrosis in mice. A novel computational framework for the annotation of scRNA-seq by reference to bulk transcriptomes (SingleR) enabled the subclustering of macrophages and revealed a disease-associated subgroup with a transitional gene expression profile intermediate between monocyte-derived and alveolar macrophages. These CX3CR1+SiglecF+ transitional macrophages localized to the fibrotic niche and had a profibrotic effect in vivo. Human orthologs of genes expressed by the transitional macrophages were upregulated in samples from patients with idiopathic pulmonary fibrosis. Thus, we have identified a pathological subgroup of transitional macrophages that are required for the fibrotic response to injury

Insights from Transcriptomics: CD163+ Profibrotic Lung Macrophages in COVID-19.

Coronavirus disease (COVID-19) begins with upper airway symptoms but proceeds in a significant proportion of patients to life-threatening infection of the lower respiratory tract, where an exuberant inflammatory response, edema, and adverse parenchymal remodeling impair gas exchange. Respiratory failure is caused initially by flooding of the airspaces with plasma exudate, sloughed epithelium, and inflammatory cells. For many patients with COVID-19, this acute phase has been observed to give way to a prolonged course of acute respiratory distress syndrome, and a significant proportion of patients go on to develop fibroproliferative remodeling of the lung parenchyma, which lengthens the duration of respiratory impairment and mechanical ventilation. Monocyte-derived macrophages have previously been implicated in the fibrotic phase of lung injury in multiple models. From several recent studies that used single-cell genomic techniques, a profile of the transcriptomic state of COVID-19 lung macrophages has emerged. Linkages have been made between these macrophages, which are monocyte-derived and CD163+, and profibrotic macrophages found in other contexts, including animal models of fibrosis and idiopathic pulmonary fibrosis. Here, emerging concepts of macrophage profibrotic function in COVID-19 are highlighted with a focus on gaps in knowledge to be addressed by future research

Insights from Transcriptomics: CD163+ Profibrotic Lung Macrophages in COVID-19.

Coronavirus disease (COVID-19) begins with upper airway symptoms but proceeds in a significant proportion of patients to life-threatening infection of the lower respiratory tract, where an exuberant inflammatory response, edema, and adverse parenchymal remodeling impair gas exchange. Respiratory failure is caused initially by flooding of the airspaces with plasma exudate, sloughed epithelium, and inflammatory cells. For many patients with COVID-19, this acute phase has been observed to give way to a prolonged course of acute respiratory distress syndrome, and a significant proportion of patients go on to develop fibroproliferative remodeling of the lung parenchyma, which lengthens the duration of respiratory impairment and mechanical ventilation. Monocyte-derived macrophages have previously been implicated in the fibrotic phase of lung injury in multiple models. From several recent studies that used single-cell genomic techniques, a profile of the transcriptomic state of COVID-19 lung macrophages has emerged. Linkages have been made between these macrophages, which are monocyte-derived and CD163(+), and profibrotic macrophages found in other contexts, including animal models of fibrosis and idiopathic pulmonary fibrosis. Here, emerging concepts of macrophage profibrotic function in COVID-19 are highlighted with a focus on gaps in knowledge to be addressed by future research

IQGAP1-dependent scaffold suppresses RhoA and inhibits airway smooth muscle contraction.

The intracellular scaffold protein IQGAP1 supports protein complexes in conjunction with numerous binding partners involved in multiple cellular processes. Here, we determined that IQGAP1 modulates airway smooth muscle contractility. Compared with WT controls, at baseline as well as after immune sensitization and challenge, Iqgap1-/- mice had higher airway responsiveness. Tracheal rings from Iqgap1-/- mice generated greater agonist-induced contractile force, even after removal of the epithelium. RhoA, a regulator of airway smooth muscle contractility, was activated in airway smooth muscle lysates from Iqgap1-/- mice. Likewise, knockdown of IQGAP1 in primary human airway smooth muscle cells increased RhoA activity. Immunoprecipitation studies indicated that IQGAP1 binds to both RhoA and p190A-RhoGAP, a GTPase-activating protein that normally inhibits RhoA activation. Proximity ligation assays in primary airway human smooth muscle cells and mouse tracheal sections revealed colocalization of p190A-RhoGAP and RhoA; however, these proteins did not colocalize in IQGAP1 knockdown cells or in Iqgap1-/- trachea. Compared with healthy controls, human subjects with asthma had decreased IQGAP1 expression in airway biopsies. Together, these data demonstrate that IQGAP1 acts as a scaffold that colocalizes p190A-RhoGAP and RhoA, inactivating RhoA and suppressing airway smooth muscle contraction. Furthermore, our results suggest that IQGAP1 has the potential to modulate airway contraction severity in acute asthma

Molecular programs of fibrotic change in aging human lung.

Lung fibrosis is increasingly detected with aging and has been associated with poor outcomes in acute lung injury or infection. However, the molecular programs driving this pro-fibrotic evolution are unclear. Here we profile distal lung samples from healthy human donors across the lifespan. Gene expression profiling by bulk RNAseq reveals both increasing cellular senescence and pro-fibrotic pathway activation with age. Quantitation of telomere length shows progressive shortening with age, which is associated with DNA damage foci and cellular senescence. Cell type deconvolution analysis of the RNAseq data indicates a progressive loss of lung epithelial cells and an increasing proportion of fibroblasts with age. Consistent with this pro-fibrotic profile, second harmonic imaging of aged lungs demonstrates increased density of interstitial collagen as well as decreased alveolar expansion and surfactant secretion. In this work, we reveal the transcriptional and structural features of fibrosis and associated functional impairment in normal lung aging

IQGAP1-dependent scaffold suppresses RhoA and inhibits airway smooth muscle contraction

The intracellular scaffold protein IQGAP1 supports protein complexes in conjunction with numerous binding partners involved in multiple cellular processes. Here, we determined that IQGAP1 modulates airway smooth muscle contractility. Compared with WT controls, at baseline as well as after immune sensitization and challenge, Iqgap1(–/–) mice had higher airway responsiveness. Tracheal rings from Iqgap1(–/–) mice generated greater agonist-induced contractile force, even after removal of the epithelium. RhoA, a regulator of airway smooth muscle contractility, was activated in airway smooth muscle lysates from Iqgap1(–/–) mice. Likewise, knockdown of IQGAP1 in primary human airway smooth muscle cells increased RhoA activity. Immunoprecipitation studies indicated that IQGAP1 binds to both RhoA and p190A-RhoGAP, a GTPase-activating protein that normally inhibits RhoA activation. Proximity ligation assays in primary airway human smooth muscle cells and mouse tracheal sections revealed colocalization of p190A-RhoGAP and RhoA; however, these proteins did not colocalize in IQGAP1 knockdown cells or in Iqgap1(–/–) trachea. Compared with healthy controls, human subjects with asthma had decreased IQGAP1 expression in airway biopsies. Together, these data demonstrate that IQGAP1 acts as a scaffold that colocalizes p190A-RhoGAP and RhoA, inactivating RhoA and suppressing airway smooth muscle contraction. Furthermore, our results suggest that IQGAP1 has the potential to modulate airway contraction severity in acute asthma

Invariant natural killer T cells coordinate removal of senescent cells

BackgroundThe failure of immune surveillance to remove senescent cells drive age-related diseases. Here, we target an endogenous immune surveillance mechanism that can promote elimination of senescent cells and reverse disease progression.MethodsWe identify a class of lipid-activated T cells, invariant natural killer T cells (iNKTs) are involved in the removal of pathologic senescent cells. We use two disease models in which senescent cells accumulate to test whether activation of iNKT cells was sufficient to eliminate senescent cells in vivo.FindingsSenescent preadipocytes accumulate in white adipose tissue of chronic high-fat diet (HFD) fed mice, and activation of iNKT cells with the prototypical glycolipid antigen alpha-galactosylceramide (αGalCer) led to a reduction of these cells with improved glucose control. Similarly, senescent cells accumulate within the lungs of mice injured by inhalational bleomycin, and αGalCer-induced activation of iNKT cells greatly limited this accumulation, decreased the lung fibrosis and improved survival. Furthermore, co-culture experiments showed that the preferential cytotoxic activity of iNKT cells to senescent cells is conserved in human cells.ConclusionsThese results uncover a senolytic capacity of tissue-resident iNKT cells and pave the way for anti-senescence therapies that target these cells and their mechanism of activation