The ion transporter Na⁺-K⁺-ATPase enables pathological B cell survival in the kidney microenvironment of lupus nephritis

The kidney is a comparatively hostile microenvironment characterized by highsodium concentrations; however, lymphocytes infiltrate and survive therein in autoimmune diseases such as lupus. The effects of sodium-lymphocyte interactions on tissue injury in autoimmune diseases and the mechanisms used by infiltrating lymphocytes to survive the highsodium environment of the kidney are not known. Here, we show that kidneyinfiltrating B cells in lupus adapt to elevated sodium concentrations and that expression of sodium potassium adenosine triphosphatase (Na+-K+-ATPase) correlates with the ability of infiltrating cells to survive. Pharmacological inhibition of Na+-K+-ATPase and genetic knockout of Na+-K+-ATPase γ subunit resulted in reduced B cell infiltration into kidneys and amelioration of proteinuria. B cells in human lupus nephritis biopsies also had high expression of Na+-K+-ATPase. Our study reveals that kidney-infiltrating B cells in lupus initiate a tissue adaption program in response to sodium stress and identifies Na+-K+-ATPase as an organ-specific therapeutic target

Multiscale Regulation of Lipid Metabolism by Cytokine Signaling in B Lymphocytes

At the onset of an immune response lymphocytes receive activating signals in the form of antigen receptor ligation, co-stimulation, and cytokines that drive cellular growth, proliferation, and differentiation. This activation involves a transition from quiescence and catabolic metabolism to a metabolic state with noted similarities to that of cancer cells such as heavy reliance on aerobic glycolysis for energy demands and increased nutrient requirements for biomass accumulation and division. Specific nutrient requirements may be met either by synthesis from metabolite precursors, or by uptake from the extracellular environment. However, much like the intratumoral environment, dense proliferation of cells in lymphatic tissues may lead to local nutrient depletion and a metabolically challenging microenvironment. The goal of this thesis is to define metabolic requirements for B cells during early activation and understand how these are modulated and met by different activating signals that the cell may receive. Herein, I present a broad introduction to B cell activation and metabolism in Chapter 1. In Chapter 2, I introduce trajectory analysis of single cell transcriptomic data from B cells undergoing early IL-4 dependent activation in response to influenza infection. We use this to model the transcriptional coordinates across which cells progress from naïve to activated states, and then to define transcriptional programs that coincide with IL-4 dependent activation within these coordinates in vivo. Through this, we identify the cholesterol biosynthetic pathway to be both transcriptionally upregulated and confirm that it is functionally important in early B cell expansion. The connection between IL-4 and cholesterol synthesis is expounded in Chapter 2, in which I describe our finding that cholesterol specifically is an essential lipid nutrient that enables activation and proliferation of naïve B cells, but that this requirement can be bypassed by IL-4R signaling that leads to a STAT6-dependent upregulation of the SREBP2 transcriptional program and enhanced cholesterol biosynthesis. In Chapter 3, I detail our investigation into the cell biological mechanism through which cholesterol enables B cell proliferation, and after ruling out a number of potential roles determine that cholesterol licenses expansion of the ER and progression from G0 to G1 phase of cell cycle. How IL-4 more broadly restructures global lipid metabolism in B cells is explored in Chapter 4, through which we found an unexpected role for IL-4 in driving an increase in peroxisome abundance. Finally, I report evidence that these peroxisomes function as a site of non-canonical cholesterol synthesis, on clinical observations that human patients with inherited peroxisome defects exhibit substantial defects in hepatic cholesterol synthesis, accordant with clinical observations that human patients with inherited peroxisome defects exhibit substantial defects in hepatic cholesterol synthesis. In sum, this work defines a novel role for IL-4 in transcriptionally upregulating SREBP2-target cholesterol synthesis genes and LXR-target efflux genes, where the activity of these biosynthetic enzymes is enhanced by an IL-4 dependent accumulation of peroxisomes where they are active. To address the questions that rise from this work I discuss implications and future directions in the final chapter

An in vivo screen of noncoding loci reveals that Daedalus is a gatekeeper of an Ikaros-dependent checkpoint during haematopoiesis.

Haematopoiesis relies on tightly controlled gene expression patterns as development proceeds through a series of progenitors. While the regulation of hematopoietic development has been well studied, the role of noncoding elements in this critical process is a developing field. In particular, the discovery of new regulators of lymphopoiesis could have important implications for our understanding of the adaptive immune system and disease. Here we elucidate how a noncoding element is capable of regulating a broadly expressed transcription factor, Ikaros, in a lymphoid lineage-specific manner, such that it imbues Ikaros with the ability to specify the lymphoid lineage over alternate fates. Deletion of th

Mechanosensation of cyclical force by PIEZO1 is essential for innate immunity

Direct recognition of invading pathogens by innate immune cells is a critical driver of the inflammatory response. However, cells of the innate immune system can also sense their local microenvironment and respond to physiological fluctuations in temperature, pH, oxygen and nutrient availability, which are altered during inflammation. Although cells of the immune system experience force and pressure throughout their life cycle, little is known about how these mechanical processes regulate the immune response. Here we show that cyclical hydrostatic pressure, similar to that experienced by immune cells in the lung, initiates an inflammatory response via the mechanically activated ion channel PIEZO1. Mice lacking PIEZO1 in innate immune cells showed ablated pulmonary inflammation in the context of bacterial infection or fibrotic autoinflammation. Our results reveal an environmental sensory axis that stimulates innate immune cells to mount an inflammatory response, and demonstrate a physiological role for PIEZO1 and mechanosensation in immunity

Skin-resident innate lymphoid cells converge on a pathogenic effector state.

Tissue-resident innate lymphoid cells (ILCs) help sustain barrier function and respond to local signals. ILCs are traditionally classified as ILC1, ILC2 or ILC3 on the basis of their expression of specific transcription factors and cytokines1. In the skin, disease-specific production of ILC3-associated cytokines interleukin (IL)-17 and IL-22 in response to IL-23 signalling contributes to dermal inflammation in psoriasis. However, it is not known whether this response is initiated by pre-committed ILCs or by cell-state transitions. Here we show that the induction of psoriasis in mice by IL-23 or imiquimod reconfigures a spectrum of skin ILCs, which converge on a pathogenic ILC3-like state. Tissue-resident ILCs were necessary and sufficient, in the absence of circulatory ILCs, to drive pathology. Single-cell RNA-sequencing (scRNA-seq) profiles of skin ILCs along a time course of psoriatic inflammation formed a dense transcriptional continuum-even at steady state-reflecting fluid ILC states, including a naive or quiescent-like state and an ILC2 effector state. Upon disease induction, the continuum shifted rapidly to span a mixed, ILC3-like subset also expressing cytokines characteristic of ILC2s, which we inferred as arising through multiple trajectories. We confirmed the transition potential of quiescent-like and ILC2 states using in vitro experiments, single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) and in vivo fate mapping. Our results highlight the range and flexibility of skin ILC responses, suggesting that immune activities primed in healthy tissues dynamically adapt to provocations and, left unchecked, drive pathological remodelling