Systematic perturbation screens identify regulators of inflammatory macrophage states and a role for TNF mRNA m6A modification.

Macrophages exhibit remarkable functional plasticity, a requirement for their central role in tissue homeostasis. During chronic inflammation, macrophages acquire sustained inflammatory 'states' that contribute to disease, but there is limited understanding of the regulatory mechanisms that drive their generation. Here we describe a systematic functional genomics approach that combines genome-wide phenotypic screening in primary murine macrophages with transcriptional and cytokine profiling of genetic perturbations in primary human macrophages to uncover regulatory circuits of inflammatory states. This process identifies regulators of five distinct states associated with key features of macrophage function. Among these regulators, loss of the N6-methyladenosine (m6A) writer components abolishes m6A modification of TNF transcripts, thereby enhancing mRNA stability and TNF production associated with multiple inflammatory pathologies. Thus, phenotypic characterization of primary murine and human macrophages describes the regulatory circuits underlying distinct inflammatory states, revealing post-transcriptional control of TNF mRNA stability as an immunosuppressive mechanism in innate immunity.

Optimized Nonviral Gene Disruption in Primary Murine and Human Myeloid Cells.

Genetically engineered myeloid cells such as monocytes, macrophages, and dendritic cells have broad applications in basic and translational research. Their central roles in innate and adaptive immunity make them attractive as putative therapeutic cell products. However, efficient gene editing of primary myeloid cells presents unique challenges owing to their sensitivity to foreign nucleic acids and poor editing efficiencies using current methodologies (Hornung et al., Science 314:994-997, 2006; Coch et al., PLoS One 8:e71057, 2013; Bartok and Hartmann, Immunity 53:54-77, 2020; Hartmann, Adv Immunol 133:121-169, 2017; Bobadilla et al., Gene Ther 20:514-520, 2013; Schlee and Hartmann, Nat Rev Immunol 16:566-580, 2016; Leyva et al., BMC Biotechnol 11:13, 2011). This chapter describes nonviral CRISPR-mediated gene knockout in primary human and murine monocytes as well as monocyte-derived or bone marrow-derived macrophages and dendritic cells. Electroporation-mediated delivery of recombinant Cas9 complexed with synthetic guide RNAs can be applied for population-level disruption of single or multiple gene targets.

Antigen-derived peptides engage the ER stress sensor IRE1α to curb dendritic cell cross-presentation.

Dendritic cells (DCs) promote adaptive immunity by cross-presenting antigen-based epitopes to CD8+ T cells. DCs process internalized protein antigens into peptides that enter the endoplasmic reticulum (ER), bind to major histocompatibility type I (MHC-I) protein complexes, and are transported to the cell surface for cross-presentation. DCs can exhibit activation of the ER stress sensor IRE1α without ER stress, but the underlying mechanism remains obscure. Here, we show that antigen-derived hydrophobic peptides can directly engage ER-resident IRE1α, masquerading as unfolded proteins. IRE1α activation depletes MHC-I heavy-chain mRNAs through regulated IRE1α-dependent decay (RIDD), curtailing antigen cross-presentation. In tumor-bearing mice, IRE1α disruption increased MHC-I expression on tumor-infiltrating DCs and enhanced recruitment and activation of CD8+ T cells. Moreover, IRE1α inhibition synergized with anti-PD-L1 antibody treatment to cause tumor regression. Our findings identify an unexpected cell-biological mechanism of antigen-driven IRE1α activation in DCs, revealing translational potential for cancer immunotherapy.

Murine Monocyte and Macrophage Culture.

Myeloid progenitors in the bone marrow generate monocytes, macrophages, granulocytes and most dendritic cells. Even though these innate immune cells are part of the same lineage, each cell type plays a specific and critical role in tissue development, host defense and the generation of adaptive immunity. Protocols have been developed in the past to differentiate myeloid cell types from bone marrow cells, enabling functional investigation and furthering our understanding about their contribution to mammalian physiology. In this protocol, we describe a simple and rapid method to isolate monocytes from murine bone marrow, culture them for up to 5 days and lastly, differentiate them into bone marrow derived macrophages (Figure 1). Graphic abstract: Figure 1.Experimental outline depicting steps for murine monocyte and macrophage culture.

Targeting STING with covalent small-molecule inhibitors.

Aberrant activation of innate immune pathways is associated with a variety of diseases. Progress in understanding the molecular mechanisms of innate immune pathways has led to the promise of targeted therapeutic approaches, but the development of drugs that act specifically on molecules of interest remains challenging. Here we report the discovery and characterization of highly potent and selective small-molecule antagonists of the stimulator of interferon genes (STING) protein, which is a central signalling component of the intracellular DNA sensing pathway1,2. Mechanistically, the identified compounds covalently target the predicted transmembrane cysteine residue 91 and thereby block the activation-induced palmitoylation of STING. Using these inhibitors, we show that the palmitoylation of STING is essential for its assembly into multimeric complexes at the Golgi apparatus and, in turn, for the recruitment of downstream signalling factors. The identified compounds and their derivatives reduce STING-mediated inflammatory cytokine production in both human and mouse cells. Furthermore, we show that these small-molecule antagonists attenuate pathological features of autoinflammatory disease in mice. In summary, our work uncovers a mechanism by which STING can be inhibited pharmacologically and demonstrates the potential of therapies that target STING for the treatment of autoinflammatory disease.

Signalling strength determines proapoptotic functions of STING.

Mammalian cells use cytosolic nucleic acid receptors to detect pathogens and other stress signals. In innate immune cells the presence of cytosolic DNA is sensed by the cGAS-STING signalling pathway, which initiates a gene expression programme linked to cellular activation and cytokine production. Whether the outcome of the STING response varies between distinct cell types remains largely unknown. Here we show that T cells exhibit an intensified STING response, which leads to the expression of a distinct set of genes and results in the induction of apoptosis. Of note, this proapoptotic STING response is still functional in cancerous T cells and delivery of small molecule STING agonists prevents in vivo growth of T-cell-derived tumours independent of its adjuvant activity. Our results demonstrate how the magnitude of STING signalling can shape distinct effector responses, which may permit for cell type-adjusted behaviours towards endogenous or exogenous insults.The cGAS/STING signalling pathway is responsible for sensing intracellular DNA and activating downstream inflammatory genes. Here the authors show mouse primary T cells and T leukaemia are hyperresponsive to STING agonist, and this strong STING signalling is associated with apoptosis induction.

Upregulation of mRNA expression of MCP-1 by TGF-beta1 in fibroblast cells from Peyronie's disease.

INTRODUCTION: Peyronie's disease (PD) is a localized connective tissue disorder of the penile tunica albuginea (TA) with a still obscure etiopathology. Recent studies from our laboratory have demonstrated differences in Smad3 and Smad4 gene expression of PD-fibroblasts and non-PD-fibroblasts after stimulation with recombinant TGF-beta1 for 1 h. In the present study, we investigated gene expression of Smad2-Smad4 and Smad7 up to 6 h after stimulation with TGF-beta1. As a positive control, MCP-1 gene expression was monitored. MATERIALS AND METHODS: Cells with fibroblast characteristics were isolated from seven PD plaques and three TA controls. The cells were incubated with recombinant TGF-beta1 for 2-6 h and expression of Smad2-Smad4, Smad7, and monocyte chemotactic protein-1 (MCP-1) was determined by quantitative real-time PCR. RESULTS: TGF-beta1 treatment resulted in a statistically significant up-regulation of Smad7 and MCP-1 gene expression. Smad7 expression was increased after 2 h (P < 0.001) and was still high after 4 h (P < 0.05). No significant differences between fibroblasts from PD-patients compared to non-PD-patients were observed. MCP-1 peaked after 4 h (P < 0.001) and remained high up to 6 h (P < 0.01). PD-fibroblasts revealed a significantly increased MCP-1 gene expression compared to non-PD-fibroblasts (P = 0.013) after 2 h and remained significantly different also after 6 h (P = 0.038). Gene expression of Smad2-Smad4 did not change during stimulation with TGF-beta1. CONCLUSION: In conclusion, analysis of MCP-1 expression might be a useful marker for Peyronie's disease.

Investigation of the antifibrotic effect of IFN-gamma on fibroblasts in a cell culture model of Peyronie's disease.

OBJECTIVE: A broad spectrum of options is available for treatment of Peyronie's disease; however, the effects of minimally invasive therapy are generally inadequate. Although useful, oral drugs must be administered at onset of the disease. Only a few patients request penile surgery. Therefore, new medical treatments for Peyronie's disease are needed. A better understanding of the pathogenesis of Peyronie's disease is required to facilitate development of these new medical treatments. Several studies have described an increased level of TGF-beta in the fibrotic plaques of patients with Peyronie's disease, underscoring this important signalling pathway in the onset and/or development of Peyronie's disease. METHODS: Plaque biopsies were taken from 16 patients with Peyronie's disease. Furthermore, 7 patients without Peyronie's disease were biopsied to provide control material. Fibroblasts were cultured from biopsy tissue, and cultured fibroblasts were stimulated with TGF-beta1, BMP-2, IFN-gamma, and IFN-gamma combined with one of the other stimuli. Protein was extracted from treated fibroblasts and prepared for immunoblots. The membranes were probed for phosphorylated Smad and total Smad to indicate activation of TGF-beta signalling. RESULTS: An agonistic effect of IFN-gamma on TGF-beta signalling was observed. Stimulation with TGF-beta1 increased levels of phospho-Smad2 and phospho-Smad3. After stimulation with TGF-beta1 and IFN-gamma combined, the levels of phospho-Smads were higher than those observed with stimulation withTGF-beta1 alone. CONCLUSIONS: The profibrotic effect of TGF-beta1 is enhanced by IFN-gamma in fibroblasts from patients with Peyronie's disease. The inhibitory effects of IFN-gamma on the TGF-beta pathway do not appear in Peyronie's disease. Therefore, IFN-gamma cannot be taken as a useful tool in the therapy of Peyronie's disease.

Alterations in the transforming growth factor (TGF)-beta pathway as a potential factor in the pathogenesis of Peyronie's disease.

OBJECTIVES: The development of fibrotic diseases is associated with alterations in the transforming growth factor beta (TGF-beta) pathway. We have investigated the expression and activity of Smad transcription factors of the TGF-beta pathway in primary tunical fibroblasts derived from patients with Peyronie's disease and from controls. METHODS: Primary fibroblasts were established from biopsies obtained from plaques of 16 patients with Peyronie's disease or the tunica albuginea of 8 control patients. The expression and activity of Smad transcription factors in control and TGF-beta-stimulated primary fibroblasts were investigated at the RNA and protein level by reverse transcription-polymerase chain reaction, Western blotting, and immunofluorescence. RESULTS: RNA expression levels of Smad3 and Smad4 were significantly increased in fibroblasts from patients with Peyronie's disease. When stimulated with TGF-beta1, fibroblasts showed rapid nuclear translocation of Smad2/3, as soon as 15 min after stimulation. This effect was more pronounced and exhibited an earlier onset in fibroblasts from patients with Peyronie's disease, compared with controls. In addition, an increased nuclear retention time of Smad4 was observed in fibroblasts from patients with Peyronie's disease. CONCLUSIONS: The expression and activity of Smad transcription factors of the TGF-beta pathway is increased in fibroblasts of patients with Peyronie's disease. Alterations in the TGF-beta pathway seem to be a pathogenetic factor in the development of Peyronie's disease.

Alpha-1-antitrypsin levels and genetic variation of the alpha-1-antitrypsin gene in Peyronie's disease.

OBJECTIVES: Alpha-1-antitrypsin (alpha1-antitrypsin) is a major protease inhibitor controlling tissue degradation. Reduced alpha1-antitrypsin levels could result in a change of collagen metabolism. Previous studies have described decreased alpha1-antitrypsin levels in patients with Peyronie's disease. However, only a small number of patients were analyzed, and the reason for the decreased alpha1-antitrypsin levels remained unclear. This study investigated prospectively the levels of alpha1-antitrypsin in patients with Peyronie's disease, as well as genetic variation in the coding region of the alpha1-antitrypsin gene. METHODS: Alpha1-antitrypsin levels were determined prospectively in 94 patients with Peyronie's disease and compared to healthy controls. Analysis of the alpha1-antitrypsin gene (S, Z variants; single nucleotid polymorphisms [SNPs]: T-395A, M2, M3, G6118A) was done in 141 Peyronie's patients including 43 patients with investigated alpha1-antitrypsin serum levels and compared to healthy controls. RESULTS: In patients with Peyronie's disease, the alpha1-antitrypsin levels seemed to be decreased significantly compared to healthy controls. However, in the age matched approach no significant differences occurred. Moreover, a significant (p < 0.002) decrease of the alpha1-antitrypsin level with increasing age was observed, explaining the initial differences between the two groups. In confirmation with these findings, no significant association of the alpha1-antitrypsin gene variants with Peyronie's disease was detectable. CONCLUSIONS: The results of this study do not indicate a significant association between Peyronie's disease and decreased alpha1-antitrypsin levels. Low alpha1-antitrypsin levels in Peyronie's patients are, rather, an age-related phenomenon, as revealed by the comparison with aged matched healthy controls. The decrease of the alpha1-antitrypsin serum level with increasing age has not been described before.