Dexamethasone impairs the expression of antimicrobial mediators in lipopolysaccharide-activated primary macrophages by inhibiting both expression and function of interferon ß.

Glucocorticoids potently inhibit expression of many inflammatory mediators, and have been widely used to treat both acute and chronic inflammatory diseases for more than seventy years. However, they can have several unwanted effects, amongst which immunosuppression is one of the most common. Here we used microarrays and proteomic approaches to characterise the effect of dexamethasone (a synthetic glucocorticoid) on the responses of primary mouse macrophages to a potent pro-inflammatory agonist, lipopolysaccharide (LPS). Gene ontology analysis revealed that dexamethasone strongly impaired the lipopolysaccharide-induced antimicrobial response, which is thought to be driven by an autocrine feedback loop involving the type I interferon IFNß. Indeed, dexamethasone strongly and dose-dependently inhibited the expression of IFNß by LPS-activated macrophages. Unbiased proteomic data also revealed an inhibitory effect of dexamethasone on the IFNß-dependent program of gene expression, with strong down-regulation of several interferon-induced antimicrobial factors. Surprisingly, dexamethasone also inhibited the expression of several antimicrobial genes in response to direct stimulation of macrophages with IFNß. We tested a number of hypotheses based on previous publications, but found that no single mechanism could account for more than a small fraction of the broad suppressive impact of dexamethasone on macrophage type I interferon signaling, underlining the complexity of this pathway. Preliminary experiments indicated that dexamethasone exerted similar inhibitory effects on primary human monocyte-derived or alveolar macrophages.

PIM1 controls GBP1 activity to limit self-damage and to guard against pathogen infection.

Disruption of cellular activities by pathogen virulence factors can trigger innate immune responses. Interferon-γ (IFN-γ)-inducible antimicrobial factors, such as the guanylate binding proteins (GBPs), promote cell-intrinsic defense by attacking intracellular pathogens and by inducing programmed cell death. Working in human macrophages, we discovered that GBP1 expression in the absence of IFN-γ killed the cells and induced Golgi fragmentation. IFN-γ exposure improved macrophage survival through the activity of the kinase PIM1. PIM1 phosphorylated GBP1, leading to its sequestration by 14-3-3σ, which thereby prevented GBP1 membrane association. During Toxoplasma gondii infection, the virulence protein TgIST interfered with IFN-γ signaling and depleted PIM1, thereby increasing GBP1 activity. Although infected cells can restrain pathogens in a GBP1-dependent manner, this mechanism can protect uninfected bystander cells. Thus, PIM1 can provide a bait for pathogen virulence factors, guarding the integrity of IFN-γ signaling.

Toll-like receptor 4 and macrophage scavenger receptor 1 crosstalk regulates phagocytosis of a fungal pathogen.

The opportunistic fungal pathogen Cryptococcus neoformans causes lethal infections in immunocompromised patients. Macrophages are central to the host response to cryptococci; however, it is unclear how C. neoformans is recognised and phagocytosed by macrophages. Here we investigate the role of TLR4 in the non-opsonic phagocytosis of C. neoformans. We find that loss of TLR4 function unexpectedly increases phagocytosis of non-opsonised cryptococci by murine and human macrophages. The increased phagocytosis observed in Tlr4-/- cells was dampened by pre-treatment of macrophages with oxidised-LDL, a known ligand of scavenger receptors. The scavenger receptor, macrophage scavenger receptor 1 (MSR1) (also known as SR-A1 or CD204) was upregulated in Tlr4-/- macrophages. Genetic ablation of MSR1 resulted in a 75% decrease in phagocytosis of non-opsonised cryptococci, strongly suggesting that it is a key non-opsonic receptor for this pathogen. We go on to show that MSR1-mediated uptake likely involves the formation of a multimolecular signalling complex involving FcγR leading to SYK, PI3K, p38 and ERK1/2 activation to drive actin remodelling and phagocytosis. Altogether, our data indicate a hitherto unidentified role for TLR4/MSR1 crosstalk in the non-opsonic phagocytosis of C. neoformans.

Species- and strain-specific differences in the phagocytosis of Prototheca: insights from live-cell imaging.

The genus Prototheca is an extremely unusual group of achlorophyllic, obligately heterotrophic algae. Six species have been identified as pathogens of vertebrates, including cattle and humans. In cattle, P. bovis is the main infectious pathogen and is associated with bovine mastitis. In contrast, human infections typically involve P. wickerhamii and are associated with a spectrum of varying clinical presentations. Prototheca spp. enter the host from the environment and are therefore likely to be initially recognized by cells of the innate immune system. However, little is known about the nature of the interaction between Prototheca spp. and host phagocytes. In the present study, we adopt a live-cell imaging approach to investigate these interactions over time. Using environmental and clinical strains, we show that P. bovis cells are readily internalized and processed by macrophages, whereas these immune cells struggle to internalize P. wickerhamii. Serum opsonization of P. wickerhamii only marginally improves phagocytosis, suggesting that this species (but not P. bovis) may have evolved mechanisms to evade phagocytosis. Furthermore, we show that inhibition of the kinases Syk or PI3K, which are both critical for innate immune signaling, drastically reduces the uptake of P. bovis. Finally, we show that genetic ablation of MyD88, a signaling adaptor critical for Toll-like receptor signaling, has little impact on uptake but significantly prolongs phagosome maturation once P. bovis is internalized. Together, our data suggest that these two pathogenic Prototheca spp. have very different host-pathogen interactions which have potential therapeutic implications for the treatment of human and animal disease.

Identification of Critical Transcriptomic Signaling Pathways in Patients with H Syndrome and Rosai-Dorfman Disease.

Biallelic mutations in SLC29A3 cause histiocytosis-lymphadenopathy plus syndrome, also known as H syndrome (HS). HS is a complex disorder, with ~ 25% of patients developing autoinflammatory complications consisting of unexplained fevers, persistently elevated inflammatory markers, and unusual lymphadenopathies, with infiltrating CD68+, S100+, and CD1a- histiocytes, resembling the immunophenotype found in Rosai-Dorfman disease (RDD). We investigated the transcriptomic profiles of monocytes, non-activated (M0), classically activated (M1), and alternatively activated macrophages (M2) in two patients with HS, one without autoinflammatory (HS1) and one with autoinflammatory complications (HS2). RNA sequencing revealed a dysregulated transcriptomic profile in both HS patients compared to healthy controls (HC). HS2, when compared to HS1, had several differentially expressed genes, including genes associated with lymphocytic-histiocytic predominance (e.g. NINL) and chronic immune activation (e.g. B2M). The transcriptomic and cytokine profiles of HS patients were comparable to patients with SAID with high levels of TNF. SERPINA1 gene expression was found to be upregulated in all patients studied. Moreover, higher levels of IFNγ were found in the serum of both HS patients when compared to HC. Gene ontology (GO) enrichment analysis of the DEGs in HS patients revealed the terms "type I IFN," "IFNγ signaling pathway," and "immune responses" as the top 3 most significant terms for monocytes. Gene expression analysis of lymph node biopsies from sporadic and H syndrome-associated RDD suggests common underlying pathological process. In conclusion, monocytes and macrophages from both HS patients showed transcriptomic profiles similar to SAIDs and also uniquely upregulated IFNγ signature. These findings may help find better therapeutic options for this rare disorder.

Dysregulated signalling pathways in innate immune cells with cystic fibrosis mutations.

Cystic fibrosis (CF) is one of the most common life-limiting recessive genetic disorders in Caucasians, caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CF is a multi-organ disease that involves the lungs, pancreas, sweat glands, digestive and reproductive systems and several other tissues. This debilitating condition is associated with recurrent lower respiratory tract bacterial and viral infections, as well as inflammatory complications that may eventually lead to pulmonary failure. Immune cells play a crucial role in protecting the organs against opportunistic infections and also in the regulation of tissue homeostasis. Innate immune cells are generally affected by CFTR mutations in patients with CF, leading to dysregulation of several cellular signalling pathways that are in continuous use by these cells to elicit a proper immune response. There is substantial evidence to show that airway epithelial cells, neutrophils, monocytes and macrophages all contribute to the pathogenesis of CF, underlying the importance of the CFTR in innate immune responses. The goal of this review is to put into context the important role of the CFTR in different innate immune cells and how CFTR dysfunction contributes to the pathogenesis of CF, highlighting several signalling pathways that may be dysregulated in cells with CFTR mutations.

Different CFTR modulator combinations downregulate inflammation differently in cystic fibrosis.

Previously, we showed that serum and monocytes from patients with CF exhibit an enhanced NLRP3-inflammasome signature with increased IL-18, IL-1ß, caspase-1 activity and ASC speck release (Scambler et al. eLife 2019). Here we show that CFTR modulators down regulate this exaggerated proinflammatory response following LPS/ATP stimulation. In vitro application of ivacaftor/lumacaftor or ivacaftor/tezacaftor to CF monocytes showed a significant reduction in IL-18, whereas IL-1ß was only reduced with ivacaftor/tezacaftor. Thirteen adults starting ivacaftor/lumacaftor and eight starting ivacaftor/tezacaftor were assessed over three months. Serum IL-18 and TNF decreased significantly with treatments, but IL-1ß only declined following ivacaftor/tezacaftor. In (LPS/ATP-stimulated) PBMCs, IL-18/TNF/caspase-1 were all significantly decreased and IL-10 was increased with both combinations. Ivacaftor/tezacaftor alone showed a significant reduction in IL-1ß and pro-IL-1ß mRNA. This study demonstrates that these CFTR modulator combinations have potent anti-inflammatory properties, in addition to their ability to stimulate CFTR function, which could contribute to improved clinical outcomes.

Evidence of B Cell Clonality and Investigation Into Properties of the IgM in Patients With Schnitzler Syndrome.

The Schnitzler Syndrome (SchS) is an acquired, autoinflammatory condition successfully treated with IL-1 inhibition. The two main defining features of this late-onset condition are neutrophilic urticarial dermatoses (NUD) and the presence of an IgM monoclonal component. While the former aspect has been extensively studied in this disease setting, the enigmatic paraproteinaemia and its potential consequential effects within SchS, has not previously been thoroughly addressed. Previous studies analyzing clonal B cell repertoires have largely focused on autoimmune disorders such as Systemic Lupus Erythematous (SLE) and hematological malignancies such as Chronic Lymphocytic Leukaemia (CLL), where B-cell clonality is central to disease pathology. The present study uses next-generation sequencing to provide detailed insight into aspects of B cell VDJ recombination and properties of the resulting immunoglobulin chains. An overview of IgH regional dynamics in 10 SchS patients, with a particular focus on CDR3 sequences and VDJ gene usage is reported, highlighting the presence of specific B cell expansions. Protein microarray detected a substantial proportion of autoreactive IgM to nuclear target proteins, though a single universal target was not identified. Together, these genetic and functional findings impart new understanding into this rare disorder.

ENaC-mediated sodium influx exacerbates NLRP3-dependent inflammation in cystic fibrosis.

Cystic Fibrosis (CF) is a monogenic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in defective CFTR-mediated chloride and bicarbonate transport, with dysregulation of epithelial sodium channels (ENaC). These changes alter fluid and electrolyte homeostasis and result in an exaggerated proinflammatory response driven, in part, by infection. We tested the hypothesis that NLRP3 inflammasome activation and ENaC upregulation drives exaggerated innate-immune responses in this multisystem disease. We identify an enhanced proinflammatory signature, as evidenced by increased levels of IL-18, IL-1ß, caspase-1 activity and ASC-speck release in monocytes, epithelia and serum with CF-associated mutations; these differences were reversed by pretreatment with NLRP3 inflammasome inhibitors and notably, inhibition of amiloride-sensitive sodium (Na+) channels. Overexpression of ß-ENaC, in the absence of CFTR dysfunction, increased NLRP3-mediated inflammation, indicating that dysregulated, ENaC-dependent signalling may drive exaggerated inflammatory responses in CF. These data support a role for sodium in modulating NLRP3 inflammasome activation.

Metabolic Reprograming of Cystic Fibrosis Macrophages via the IRE1α Arm of the Unfolded Protein Response Results in Exacerbated Inflammation.

Cystic Fibrosis (CF) is a recessive genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR mutations cause dysregulation of channel function with intracellular accumulation of misfolded proteins and endoplasmic reticulum (ER) stress, with activation of the IRE1α-XBP1 pathway that regulates a subset of unfolded protein response (UPR) genes. This pathway regulates a group of genes that control proinflammatory and metabolic responses in different immune cells; however, the metabolic state of immune cells and the role of this pathway in CF remain elusive. Our results indicate that only innate immune cells from CF patients present increased levels of ER stress, mainly affecting neutrophils, monocytes, and macrophages. An overactive IRE1α-XBP1 pathway reprograms CF M1 macrophages toward an increased metabolic state, with increased glycolytic rates and mitochondrial function, associated with exaggerated production of TNF and IL-6. This hyper-metabolic state, seen in CF macrophages, is reversed by inhibiting the RNase domain of IRE1α, thereby decreasing the increased glycolic rates, mitochondrial function and inflammation. Altogether, our results indicate that innate immune cells from CF patients are primarily affected by ER stress. Moreover, the IRE1α-XBP1 pathway of the UPR is responsible for the hyper-metabolic state seen in CF macrophages, which is associated with the exaggerated inflammatory response. Modulating ER stress, metabolism and inflammation, by targeting IRE1α, may improve the metabolic fitness of macrophages, and other immune cells in CF and other immune-related disorders.

TNF receptor signalling in autoinflammatory diseases.

Autoinflammatory syndromes are a group of disorders characterized by recurring episodes of inflammation as a result of specific defects in the innate immune system. Patients with autoinflammatory disease present with recurrent outbreaks of chronic systemic inflammation that are mediated by innate immune cells, for the most part. A number of these diseases arise from defects in the tumour necrosis factor receptor (TNFR) signalling pathway leading to elevated levels of inflammatory cytokines. Elucidation of the molecular mechanisms of these recently defined autoinflammatory diseases has led to a greater understanding of the mechanisms of action of key molecules involved in TNFR signalling, particularly those involved in ubiquitination, as found in haploinsufficiency of A20 (HA20), otulipenia/OTULIN-related autoinflammatory syndrome (ORAS) and linear ubiquitin chain assembly complex (LUBAC) deficiency. In this review, we also address other TNFR signalling disorders such as TNFR-associated periodic syndrome (TRAPS), RELA haploinsufficiency, RIPK1-associated immunodeficiency and autoinflammation, X-linked ectodermal dysplasia and immunodeficiency (X-EDA-ID) and we review the most recent advances surrounding these diseases and therapeutic approaches currently used to target these diseases. Finally, we explore therapeutic advances in TNF-related immune-based therapies and explore new approaches to target disease-specific modulation of autoinflammatory diseases.

Tumour necrosis factor signalling in health and disease.

The master pro-inflammatory cytokine, tumour necrosis factor (TNF), has been shown to modulate multiple signalling pathways, with wide-ranging downstream effects. TNF plays a vital role in the typical immune response through the regulation of a number of pathways encompassing an immediate inflammatory reaction with significant innate immune involvement as well as cellular activation with subsequent proliferation and programmed cell death or necrosis. As might be expected with such a broad spectrum of cellular effects and complex signalling pathways, TNF has also been implicated in a number of disease states, such as rheumatoid arthritis, ankylosing spondylitis, and Crohn's disease. Since the time of its discovery over 40 years ago, TNF ligand and its receptors, TNF receptor (TNFR) 1 and 2, have been categorised into two complementary superfamilies, namely TNF (TNFSF) and TNFR (TNFRSF), and 19 ligands and 29 receptors have been identified to date. There have been significant advances in our understanding of TNF signalling pathways in the last decade, and this short review aims to elucidate some of the most recent advances involving TNF signalling in health and disease.