Inflammasome complexes function as key innate immune effectors that trigger inflammation in response to pathogen- and danger-associated signals. Here, we report that germline mutations in the inflammasome sensor NLRP1 cause two overlapping skin disorders: multiple self-healing palmoplantar carcinoma (MSPC) and familial keratosis lichenoides chronica (FKLC). We find that NLRP1 is the most prominent inflammasome sensor in human skin, and all pathogenic NLRP1 mutations are gain-of-function alleles that predispose to inflammasome activation. Mechanistically, NLRP1 mutations lead to increased self-oligomerization by disrupting the PYD and LRR domains, which are essential in maintaining NLRP1 as an inactive monomer. Primary keratinocytes from patients experience spontaneous inflammasome activation and paracrine IL-1 signaling, which is sufficient to cause skin inflammation and epidermal hyperplasia. Our findings establish a group of non-fever inflammasome disorders, uncover an unexpected auto-inhibitory function for the pyrin domain, and provide the first genetic evidence linking NLRP1 to skin inflammatory syndromes and skin cancer predisposition.

Immunological tolerance is a fundamental tenant of immune homeostasis and overall health. Self-tolerance is a critical component of the immune system that allows for the recognition of self, resulting in hyporeactivity instead of immunogenicity. Dendritic cells are central to the establishment of dominant immune tolerance through the secretion of immunosuppressive cytokines and regulatory polarization of T cells. Cellular metabolism holds the key to determining DC immunogenic or tolerogenic cell fate. Recent studies have demonstrated that dendritic cell maturation leads to a shift toward a glycolytic metabolic state and preferred use of glucose as a carbon source. In contrast, tolerogenic dendritic cells favor oxidative phosphorylation and fatty acid oxidation. This dichotomous metabolic reprogramming of dendritic cells drives differential cellular function and plays a role in pathologies, such as autoimmune disease. Pharmacological alterations in metabolism have promising therapeutic potential.

A complex interaction of anabolic and catabolic metabolism underpins the ability of leukocytes to mount an immune response. Their capacity to respond to changing environments by metabolic reprogramming is crucial to effector function. However, current methods lack the ability to interrogate this network of metabolic pathways at single-cell level within a heterogeneous population. We present Met-Flow, a flow cytometry-based method capturing the metabolic state of immune cells by targeting key proteins and rate-limiting enzymes across multiple pathways. We demonstrate the ability to simultaneously measure divergent metabolic profiles and dynamic remodeling in human peripheral blood mononuclear cells. Using Met-Flow, we discovered that glucose restriction and metabolic remodeling drive the expansion of an inflammatory central memory T cell subset. This method captures the complex metabolic state of any cell as it relates to phenotype and function, leading to a greater understanding of the role of metabolic heterogeneity in immune responses.

25Natural killer (NK) cells are innate effectors, which play a crucial role in controlling 26 viral infections. Administration of IFN-α has shown promising results as a therapeutic, 27 controlling HIV, and chronic viral hepatitis. However the downstream mechanisms by which 28 IFN-α mediates its anti-viral effects is largely unknown. In this investigation, we evaluated 29 the impact of IFN-α on peripheral blood NK cells from healthy donors. High dimensional 30 flow cytometry analysis of NK cell surface receptors following exposure to IFN-α showed an 31 increased expression of the check point inhibitor LAG3. Further characterization revealed 32 that LAG3 was expressed in a subset of NK cells with high expression of activation and 33 maturation markers. Assessment of metabolic pathways showed that LAG3+ NK cells had 34 enhanced rates of glycolysis and glycolytic capacity, suggesting that it is a primed effector 35 subset with enhanced glucose metabolism. Inhibition of LAG3 on NK cells using antibody in 36 vitro resulted in a profound increase in secretion of cytokines IFN-γ, TNF-α, MIP-1α and 37 MIP-1β, without affecting the cytotoxic activity. Taken together, these results showed that 38 LAG3 is a negative regulator of cytokine production by mature NK cells. 39 40 Introduction: 41 2 The plasmacytoid (pDC)-Natural Killer (NK) cell axis acts as a first line of defence by the 42 host against viral infections. pDCs secrete type I interferons upon recognizing virus-43 associated pathogen associated molecular patterns (PAMPs) through pattern recognition 44 receptors (PRRs). Type I interferons (IFN-I) bind to IFN (IFN-/) receptors on the surface 45 of NK cells to prime, activate and initiate mechanisms for the destruction of infected cells. 46 The subsequent stimulation of the IFN signalling pathway results in the increased expression 47 65 expression of LAG3, an inhibitory receptor, commonly associated with T cell exhaustion in 66 chronic infections and cancer. To date, the function of LAG3 in NK cells is not well 67 understood due to contrasting results from mouse and human NK cells. While studies using 68 knockout mouse models suggested a positive role for LAG3 in NK cell cytotoxicity, these 69 could not be successfully reproduced in human NK cells (10,11). High dimensional flow 70 cytometry analysis in these studies revealed that LAG3 expression marks a subset of mature 71 activated NK cells. Metabolic assays additionally showed that NK cells expressing LAG3 72 have enhanced glycolytic activity. Blockade of LAG3 enhanced cytokine production by 73 activated NK cells without altering their cytotoxicity. Taken together, these results 74 demonstrate that LAG3 is a negative regulator of cytokine production and a potential 75 therapeutic target for chronic viral infections. 76 77 78 3 Materials and methods: 79 Isolation and stimulation of NK cells: Blood samples from healthy volunteer donors were 80 obtained after approval by the National Healthcare Group Domain Specific Review Board, 81 Singapore (NHG DSRB Ref: 2000/0...

A complex interaction of anabolic and catabolic metabolism underpins the ability of leukocytes to mount an immune response. Their capacity to respond and adapt to changing environments by metabolic reprogramming is crucial to their effector function. However, current methods lack the ability to interrogate this network of metabolic pathways at the single cell level within a heterogeneous population. Here we present Met-Flow, a novel flow cytometry-based method that captures the metabolic state of immune cells by targeting key proteins and rate-limiting enzymes across multiple pathways. We demonstrate the ability to simultaneously measure divergent metabolic profiles and dynamic remodeling in human peripheral blood mononuclear cells. Using Met-Flow, we discovered that glucose restriction and metabolic remodeling drive the expansion of an inflammatory central memory T cell subset. This method captures the complex metabolic state of any cell as it relates to its phenotype and function, leading to a greater understanding of the role of metabolic heterogeneity in immune responses.

Objectives Platelets and low-density neutrophils (LDNs) are major players in the immunopathogenesis of SLE. Despite evidence showing the importance of platelet–neutrophil complexes (PNCs) in inflammation, little is known about the relationship between LDNs and platelets in SLE. We sought to characterize the role of LDNs and Toll-like receptor 7 (TLR7) in clinical disease. Methods Flow cytometry was used to immunophenotype LDNs from SLE patients and controls. The association of LDNs with organ damage was investigated in a cohort of 290 SLE patients. TLR7 mRNA expression was assessed in LDNs and high-density neutrophils (HDNs) using publicly available mRNA sequencing datasets and our own cohort using RT-PCR. The role of TLR7 in platelet binding was evaluated in platelet–HDN mixing studies using TLR7-deficient mice and Klinefelter syndrome patients. Results SLE patients with active disease have more LDNs, which are heterogeneous and more immature in patients with evidence of kidney dysfunction. LDNs are platelet bound, in contrast to HDNs. LDNs settle in the peripheral blood mononuclear cell (PBMC) layer due to the increased buoyancy and neutrophil degranulation from platelet binding. Mixing studies demonstrated that this PNC formation was dependent on platelet–TLR7 and that the association results in increased NETosis. The neutrophil:platelet ratio is a useful clinical correlate for LDNs, and a higher NPR is associated with past and current flares of LN. Conclusions LDNs sediment in the upper PBMC fraction due to PNC formation, which is dependent on the expression of TLR7 in platelets. Collectively, our results reveal a novel TLR7-dependent crosstalk between platelets and neutrophils that may be an important therapeutic opportunity for LN.

Our objective was to examine differences in cytokine/chemokine response in chronic hepatitis B(CHB) patients to understand the immune mechanism of HBsAg loss (functional cure) during antiviral therapy. We used an unbiased machine learning strategy to unravel the immune pathways in CHB nucleo(t)side analogue-treated patients who achieved HBsAg loss with peg-interferon-α(peg-IFN-α) add-on or switch treatment in a randomised clinical trial. Cytokines/chemokines from plasma were compared between those with/without HBsAg loss, at baseline, before and after HBsAg loss. Peg-IFN-α treatment resulted in higher levels of IL-27, IL-12p70, IL-18, IL-13, IL-4, IL-22 and GM-CSF prior to HBsAg loss. Probabilistic network analysis of cytokines, chemokines and soluble factors suggested a dynamic dendritic cell driven NK and T cell immune response associated with HBsAg loss. Bayesian network analysis showed a dominant myeloid-driven type 1 inflammatory response with a MIG and I-TAC central module contributing to HBsAg loss in the add-on arm. In the switch arm, HBsAg loss was associated with a T cell activation module exemplified by high levels of CD40L suggesting T cell activation. Our findings show that more than one immune pathway to HBsAg loss was found with peg-IFN-α therapy; by myeloid-driven Type 1 response in one instance, and T cell activation in the other.