Diverse macrophage populations contribute to distinct manifestations of human cutaneous graft-versus-host disease.

BACKGROUND: Graft-versus-host disease (GvHD) is a major life-threatening complication of allogeneic haematopoietic stem cell transplantation (HSCT), limiting the broad application of HSCT for haematological malignancies. Cutaneous GvHD is described as a post-transplant inflammatory reaction by skin-infiltrating donor T cells and remaining recipient tissue-resident memory T cells. Despite the major influence of lymphocytes on GvHD pathogenesis, the complex role of mononuclear phagocytes (MNPs) in tissues affected by GvHD is increasingly appreciated. OBJECTIVES: To characterize the identity, origin and functions of MNPs in patients with acute cutaneous GvHD. METHODS: Using single-cell RNA sequencing and multiplex tissue immunofluorescence, we identified an increased abundance of MNPs in skin and blood from 36 patients with acute cutaneous GvHD. In cases of sex-mismatched transplantation, we used expression of X-linked genes to detect rapid tissue adaptation of newly recruited donor MNPs resulting in similar transcriptional states of host- and donor-derived macrophages within GvHD skin lesions. RESULTS: We showed that cutaneous GvHD lesions harbour expanded CD163+ tissue-resident macrophage populations with anti-inflammatory and tissue-remodelling properties including interleukin-10 cytokine production. Cell-cell interaction analyses revealed putative signalling to strengthen regulatory T-cell responses. Notably, macrophage polarization in chronic cutaneous GvHD types was proinflammatory and drastically differed from acute GvHD, supporting the notion of distinct cellular players in different clinical GvHD subtypes. CONCLUSIONS: Overall, our data reveal a surprisingly dynamic role of MNPs after HSCT. Specific and time-resolved targeting to repolarize this cell subset may present a promising therapeutic strategy in combatting GvHD skin inflammation.

Single-cell and spatial transcriptomics reveal aberrant lymphoid developmental programs driving granuloma formation.

Granulomas are lumps of immune cells that can form in various organs. Most granulomas appear unstructured, yet they have some resemblance to lymphoid organs. To better understand granuloma formation, we performed single-cell sequencing and spatial transcriptomics on granulomas from patients with sarcoidosis and bioinformatically reconstructed the underlying gene regulatory networks. We discovered an immune stimulatory environment in granulomas that repurposes transcriptional programs associated with lymphoid organ development. Granuloma formation followed characteristic spatial patterns and involved genes linked to immunometabolism, cytokine and chemokine signaling, and extracellular matrix remodeling. Three cell types emerged as key players in granuloma formation: metabolically reprogrammed macrophages, cytokine-producing Th17.1 cells, and fibroblasts with inflammatory and tissue-remodeling phenotypes. Pharmacological inhibition of one of the identified processes attenuated granuloma formation in a sarcoidosis mouse model. We show that human granulomas adopt characteristic aspects of normal lymphoid organ development in aberrant combinations, indicating that granulomas constitute aberrant lymphoid organs.

Processing human skin samples for single-cell assays.

Characterizing resident immune cells in human skin using single-cell assays provides insight into their role in infections, inflammation, and cancer. We describe an optimized protocol to rapidly isolate viable cells from 6-mm skin punch-biopsies. We provide an example in which we coupled single-cell RNA sequencing (scRNA-seq) with single-cell T-cell receptor sequencing (scTCR-seq) of skin and blood cells to study transcriptional profiles and clonotypes of skin resident and peripheral circulating, memory, and effector T cells. This is an improved protocol based on Saluzzo et al. (2021). For complete details on the use and execution of this protocol, please refer to Saluzzo et al. (2021).

Delayed antiretroviral therapy in HIV-infected individuals leads to irreversible depletion of skin- and mucosa-resident memory T cells.

People living with HIV (PLWH) are at increased risk for developing skin and mucosal malignancies despite systemic reconstitution of CD4+ T cells upon antiretroviral therapy (ART). The underlying mechanism of chronic tissue-related immunodeficiency in HIV is unclear. We found that skin CD4+ tissue-resident memory T (Trm) cells were depleted after HIV infection and replenished only upon early ART initiation. TCR clonal analysis following early ART suggested a systemic origin for reconstituting CD4+ Trm cells. Single-cell RNA sequencing in PLWH that received late ART treatment revealed a loss of CXCR3+ Trm cells and a tolerogenic skin immune environment. Human papilloma virus-induced precancerous lesion biopsies showed reduced CXCR3+ Trm cell frequencies in the mucosa in PLWH versus HIV- individuals. These results reveal an irreversible loss of CXCR3+ Trm cells confined to skin and mucosa in PLWH who received late ART treatment, which may be a precipitating factor in the development of HPV-related cancer.

Beneficial Metabolic Effects of TREM2 in Obesity Are Uncoupled From Its Expression on Macrophages.

Obesity-induced white adipose tissue (WAT) hypertrophy is associated with elevated adipose tissue macrophage (ATM) content. Overexpression of the triggering receptor expressed on myeloid cells 2 (TREM2) reportedly increases adiposity, worsening health. Paradoxically, using insulin resistance, elevated fat mass, and hypercholesterolemia as hallmarks of unhealthy obesity, a recent report demonstrated that ATM-expressed TREM2 promoted health. Here, we identified that in mice, TREM2 deficiency aggravated diet-induced insulin resistance and hepatic steatosis independently of fat and cholesterol levels. Metabolomics linked TREM2 deficiency with elevated obesity-instigated serum ceramides that correlated with impaired insulin sensitivity. Remarkably, while inhibiting ceramide synthesis exerted no influences on TREM2-dependent ATM remodeling, inflammation, or lipid load, it restored insulin tolerance, reversing adipose hypertrophy and secondary hepatic steatosis of TREM2-deficient animals. Bone marrow transplantation experiments revealed unremarkable influences of immune cell-expressed TREM2 on health, instead demonstrating that WAT-intrinsic mechanisms impinging on sphingolipid metabolism dominate in the systemic protective effects of TREM2 on metabolic health.

First-Breath-Induced Type 2 Pathways Shape the Lung Immune Environment.

From birth onward, the lungs are exposed to the external environment and therefore harbor a complex immunological milieu to protect this organ from damage and infection. We investigated the homeostatic role of the epithelium-derived alarmin interleukin-33 (IL-33) in newborn mice and discovered the immediate upregulation of IL-33 from the first day of life, closely followed by a wave of IL-13-producing type 2 innate lymphoid cells (ILC2s), which coincided with the appearance of alveolar macrophages (AMs) and their early polarization to an IL-13-dependent anti-inflammatory M2 phenotype. ILC2s contributed to lung quiescence in homeostasis by polarizing tissue resident AMs and induced an M2 phenotype in transplanted macrophage progenitors. ILC2s continued to maintain the M2 AM phenotype during adult life at the cost of a delayed response to Streptococcus pneumoniae infection in mice. These data highlight the homeostatic role of ILC2s in setting the activation threshold in the lung and underline their implications in anti-bacterial defenses.

Heme drives hemolysis-induced susceptibility to infection via disruption of phagocyte functions.

Hemolysis drives susceptibility to bacterial infections and predicts poor outcome from sepsis. These detrimental effects are commonly considered to be a consequence of heme-iron serving as a nutrient for bacteria. We employed a Gram-negative sepsis model and found that elevated heme levels impaired the control of bacterial proliferation independently of heme-iron acquisition by pathogens. Heme strongly inhibited phagocytosis and the migration of human and mouse phagocytes by disrupting actin cytoskeletal dynamics via activation of the GTP-binding Rho family protein Cdc42 by the guanine nucleotide exchange factor DOCK8. A chemical screening approach revealed that quinine effectively prevented heme effects on the cytoskeleton, restored phagocytosis and improved survival in sepsis. These mechanistic insights provide potential therapeutic targets for patients with sepsis or hemolytic disorders.

Heme oxygenase-1 drives metaflammation and insulin resistance in mouse and man.

Obesity and diabetes affect more than half a billion individuals worldwide. Interestingly, the two conditions do not always coincide and the molecular determinants of "healthy" versus "unhealthy" obesity remain ill-defined. Chronic metabolic inflammation (metaflammation) is believed to be pivotal. Here, we tested a hypothesized anti-inflammatory role for heme oxygenase-1 (HO-1) in the development of metabolic disease. Surprisingly, in matched biopsies from "healthy" versus insulin-resistant obese subjects we find HO-1 to be among the strongest positive predictors of metabolic disease in humans. We find that hepatocyte and macrophage conditional HO-1 deletion in mice evokes resistance to diet-induced insulin resistance and inflammation, dramatically reducing secondary disease such as steatosis and liver toxicity. Intriguingly, cellular assays show that HO-1 defines prestimulation thresholds for inflammatory skewing and NF-κB amplification in macrophages and for insulin signaling in hepatocytes. These findings identify HO-1 inhibition as a potential therapeutic strategy for metabolic disease.