Reprogramming of Amino Acid Metabolism Differs between Community-Acquired Pneumonia and Infection-Associated Exacerbation of Chronic Obstructive Pulmonary Disease.

Amino acids and their metabolites are key regulators of immune responses, and plasma levels may change profoundly during acute disease states. Using targeted metabolomics, we evaluated concentration changes in plasma amino acids and related metabolites in community-acquired pneumonia (CAP, n = 29; compared against healthy controls, n = 33) from presentation to hospital through convalescence. We further aimed to identify biomarkers for acute CAP vs. the clinically potentially similar infection-triggered COPD exacerbation (n = 13). Amino acid metabolism was globally dysregulated in both CAP and COPD. Levels of most amino acids were markedly depressed in acute CAP, and total amino acid concentrations on admission were an accurate biomarker for the differentiation from COPD (AUC = 0.93), as were reduced asparagine and threonine levels (both AUC = 0.92). Reduced tryptophan and histidine levels constituted the most accurate biomarkers for acute CAP vs. controls (AUC = 0.96, 0.94). Only kynurenine, symmetric dimethyl arginine, and phenylalanine levels were increased in acute CAP, and the kynurenine/tryptophan ratio correlated best with clinical recovery and resolution of inflammation. Several amino acids did not reach normal levels by the 6-week follow-up. Glutamate levels were reduced on admission but rose during convalescence to 1.7-fold above levels measured in healthy control. Our data suggest that dysregulated amino acid metabolism in CAP partially persists through clinical recovery and that amino acid metabolism constitutes a source of promising biomarkers for CAP. In particular, total amino acids, asparagine, and threonine may constitute plasma biomarker candidates for the differentiation between CAP and infection-triggered COPD exacerbation and, perhaps, the detection of pneumonia in COPD.

Nitric oxide controls proliferation of Leishmania major by inhibiting the recruitment of permissive host cells.

Nitric oxide (NO) is an important antimicrobial effector but also prevents unnecessary tissue damage by shutting down the recruitment of monocyte-derived phagocytes. Intracellular pathogens such as Leishmania major can hijack these cells as a niche for replication. Thus, NO might exert containment by restricting the availability of the cellular niche required for efficient pathogen proliferation. However, such indirect modes of action remain to be established. By combining mathematical modeling with intravital 2-photon biosensors of pathogen viability and proliferation, we show that low L. major proliferation results not from direct NO impact on the pathogen but from reduced availability of proliferation-permissive host cells. Although inhibiting NO production increases recruitment of these cells, and thus pathogen proliferation, blocking cell recruitment uncouples the NO effect from pathogen proliferation. Therefore, NO fulfills two distinct functions for L. major containment: permitting direct killing and restricting the supply of proliferation-permissive host cells.

Characterization of mechanisms positioning costimulatory complexes in immune synapses.

Small immunoglobulin superfamily (sIGSF) adhesion complexes form a corolla of microdomains around an integrin ring and secretory core during immunological synapse (IS) formation. The corolla recruits and retains major costimulatory/checkpoint complexes, such as CD28, making forces that govern corolla formation of particular interest. Here, we investigated the mechanisms underlying molecular reorganization of CD2, an adhesion and costimulatory molecule of the sIGSF family during IS formation. Computer simulations showed passive distal exclusion of CD2 complexes under weak interactions with the ramified F-actin transport network. Attractive forces between CD2 and CD28 complexes relocate CD28 from the IS center to the corolla. Size-based sorting interactions with large glycocalyx components, such as CD45, or short-range CD2 self-attraction successfully explain the corolla 'petals.' This establishes a general simulation framework for complex pattern formation observed in cell-bilayer and cell-cell interfaces, and the suggestion of new therapeutic targets, where boosting or impairing characteristic pattern formation can be pivotal.

Agent-Based Modeling of T Cell Receptor Cooperativity.

Immunological synapse (IS) formation is a key event during antigen recognition by T cells. Recent experimental evidence suggests that the affinity between T cell receptors (TCRs) and antigen is actively modulated during the early steps of TCR signaling. In this work, we used an agent-based model to study possible mechanisms for affinity modulation during IS formation. We show that, without any specific active mechanism, the observed affinity between receptors and ligands evolves over time and depends on the density of ligands of the antigen peptide presented by major histocompatibility complexes (pMHC) and TCR molecules. A comparison between the presence or absence of TCR-pMHC centrally directed flow due to F-actin coupling suggests that centripetal transport is a potential mechanism for affinity modulation. The model further suggests that the time point of affinity measurement during immune synapse formation is critical. Finally, a mathematical model of F-actin foci formation incorporated in the agent-based model shows that TCR affinity can potentially be actively modulated by positive/negative feedback of the F-actin foci on the TCR-pMHC association rate kon.

A dynamic CD2-rich compartment at the outer edge of the immunological synapse boosts and integrates signals.

The CD2-CD58 recognition system promotes adhesion and signaling and counters exhaustion in human T cells. We found that CD2 localized to the outer edge of the mature immunological synapse, with cellular or artificial APC, in a pattern we refer to as a 'CD2 corolla'. The corolla captured engaged CD28, ICOS, CD226 and SLAM-F1 co-stimulators. The corolla amplified active phosphorylated Src-family kinases (pSFK), LAT and PLC-γ over T cell receptor (TCR) alone. CD2-CD58 interactions in the corolla boosted signaling by 77% as compared with central CD2-CD58 interactions. Engaged PD-1 invaded the CD2 corolla and buffered CD2-mediated amplification of TCR signaling. CD2 numbers and motifs in its cytoplasmic tail controlled corolla formation. CD8+ tumor-infiltrating lymphocytes displayed low expression of CD2 in the majority of people with colorectal, endometrial or ovarian cancer. CD2 downregulation may attenuate antitumor T cell responses, with implications for checkpoint immunotherapies.

F-Actin-Driven CD28-CD80 Localization in the Immune Synapse.

During immunological synapse (IS) formation, T cell receptor (TCR) signaling complexes, integrins, and costimulatory molecules exhibit a particular spatial localization. Here, we develop an agent-based model for the IS formation based on TCR peptide-bound major histocompatibility complex (pMHC) and leukocyte-function-associated antigen 1 (LFA-1) intracellular activation molecule 1 (ICAM-1) dynamics, including CD28 binding to a costimulatory ligand, coupling of molecules to the centripetal actin flow, and size-based segregation (SBS). A radial gradient of LFA-1 in the peripheral supramolecular activation cluster (pSMAC) toward the central supramolecular activation cluster (cSMAC) emerged as a combined consequence of actin binding and diffusion and modified the positioning of other molecules. The simulations predict a mechanism of CD28 movement, according to which CD28-CD80 complexes passively follow TCR-pMHC microclusters. However, the characteristic CD28-CD80 localization in a ring pattern around the cSMAC only emerges with a particular CD28-actin coupling strength that induces a centripetal motion. These results have implications for the understanding of T cell activation and fate decisions.

Mathematical Modeling of Synaptic Patterns.

During antigen recognition by T cells, a specific spatial structure is formed at the contact face to an antigen-presenting cell (APC), called an immunological synapse (IS). The IS supports bidirectional signaling and release of effector molecules and is widely studied both biologically and numerically, in order to understand the process of T cell activation and signaling. This specialized structure harbors a central area (central supramolecular activation cluster, cSMAC) populated by T cell receptor-peptide-major histocompatibility complex (TCR-pMHC ) interactions, hedged by a peripheral ring (peripheral supramolecular activation cluster, pSMAC) of integrin lymphocyte function associated-1 interactions with its immunoglobulin superfamily ligand intercellular adhesion molecule-1 (LFA-1-ICAM-1). These two regions form the "bull's eye" pattern characteristic of the mature IS.In theoretical studies, different modeling architectures, including partial differential equations (PDE) and agent-based models , have been developed with the purpose to answer mechanistic questions about the IS dynamics. In this chapter, we explain possible physiological mechanisms that lead to the formation of ISs and technical issues that may occur in the course of development of agent-based models.