Colony-stimulating factor 1 (CSF1) and interleukin 34 (IL34) signal via the CSF1 receptor to regulate macrophage differentiation. Studies in IL34- or CSF1-deficient mice have revealed that IL34 function is limited to the central nervous system and skin during development. However, the roles of IL34 and CSF1 at homeostasis or in the context of inflammatory diseases or cancer in wild-type mice have not been clarified in vivo. By neutralizing CSF1 and/or IL34 in adult mice, we identified that they play important roles in macrophage differentiation, specifically in steady-state microglia, Langerhans cells, and kidney macrophages. In several inflammatory models, neutralization of both CSF1 and IL34 contributed to maximal disease protection. However, in a myeloid cell-rich tumor model, CSF1 but not IL34 was required for tumor-associated macrophage accumulation and immune homeostasis. Analysis of human inflammatory conditions reveals IL34 upregulation that may account for the protection requirement of IL34 blockade. Furthermore, evaluation of IL34 and CSF1 blockade treatment during Listeria infection reveals no substantial safety concerns. Thus, IL34 and CSF1 play non-redundant roles in macrophage differentiation, and therapeutic intervention targeting IL34 and/or CSF1 may provide an effective treatment in macrophage-driven immune-pathologies.

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Summary TGF-β signaling is essential in many processes, including immune surveillance, and its dysregulation controls various diseases, including cancer, fibrosis, and inflammation. Studying the innate host defense, which functions in most cell types, we found that RLR signaling represses TGF-β responses. This regulation is mediated by activated IRF3, using a dual mechanism of IRF3-directed suppression. Activated IRF3 interacts with Smad3, thus inhibiting TGF-β-induced Smad3 activation, and, in the nucleus, disrupts functional Smad3 transcription complexes by competing with co-regulators. Consequently, IRF3 activation by innate antiviral signaling represses TGF-β-induced growth inhibition, gene regulation and epithelial-mesenchymal transition, and the generation of Treg effector lymphocytes from naïve CD4+ lymphocytes. Conversely, silencing IRF3 expression enhances epithelial-mesenchymal transition, TGF-β-induced Treg cell differentiation upon virus infection, and Treg cell generation in vivo. We present a novel mode of regulation of TGF-β signaling by the antiviral defense, with evidence for its role in immune tolerance and cancer cell behavior.

Viral clearance requires effector T-cell egress from the draining lymph node (dLN). The mechanisms that regulate the complex process of effector T-cell egress from the dLN after infection are poorly understood. Here, we visualized endogenous pathogen-specific effector T-cell migration within, and from, the dLN. We used an inducible mouse model with a temporally disrupted sphingosine-1-phosphate receptor-1 (S1PR1) gene specifically in endogenous effector T cells. Early after infection, WT and S1PR1 −/− effector T cells localized exclusively within the paracortex. This localization in the paracortex by CD8 T cells was followed by intranodal migration by both WT and S1PR1−/− T cells to positions adjacent to both cortical and medullary lymphatic sinuses where the T cells exhibited intense probing behavior. However, in contrast to WT, S1PR1 −/− effector T cells failed to enter the sinuses.We demonstrate that, even when LN retention signals such as CC chemokine receptor 7 (CCR7) are down-regulated, T cell intrinsic S1PR1 is the master regulator of effector T-cell emigration from the dLN.n effective immune response depends on the large-scale, but carefully regulated, migration of T cells within and between lymphoid and peripheral tissues. This migration is tightly regulated by several factors, including the highly organized secondary lymphoid structure and the cellular expression of chemokine receptors and compartmentalized secretion of their cognate ligands (1). This balance between the anatomy and the ordered expression of cell surface and soluble proteins dictates the exquisite choreography of T-cell migration, and visualizing these dynamics of T-cell behavior in situ within the lymph nodes (LNs) is essential for understanding the mechanisms that mediate the generation of a productive antimicrobial or antitumoral immune response (1, 2). However, our understanding of the factors that regulate the anatomical program followed by endogenous antigen-specific effector T cells after an infection remains incomplete, especially with respect to the mechanisms that regulate egress kinetics of effector T cells from LN (2, 3).T-cell migration, even at steady state, is a highly regulated process (4). T-cell entry into the LN is controlled by G protein-coupled receptors (GPCRs) (3) such as CC chemokine receptor 7 (CCR7), which is also critical for the localization and retention of T cells within the LN paracortex (5, 6). Egress of naive T cells from the LN via the lymphatic vessels is regulated by the GPCR sphingosine-1-phosphate receptor-1 (S1PR1) (3) and adhesion molecules (4). S1PR1 is among four other GPCRs that bind to sphingosine-1-phosphate (S1P) with high affinity. S1PR1 is abundantly expressed in different cell types and tissues, including immune cells and endothelial cells (7). In addition to mediating lymphocyte egress, binding of S1P to S1PR1 and other receptors (S1PR2 to -5) on the cell surface initiates several signaling cascades that affect the functioning of many organ systems and control a multitude of biological ...

Tumor progression locus 2 (TPL2, also known as COT or MAP3K8) is a mitogen-activated protein kinase kinase (MAP3K) activated downstream of TNFαR, IL1R, TLR, CD40, IL17R, and some GPCRs. TPL2 regulates the MEK1/2 and ERK1/2 pathways to regulate a cascade of inflammatory responses. In parallel to this, TPL2 also activates p38α and p38δ to drive the production of various inflammatory mediators in neutrophils. We discuss the implications of this finding in the context of various inflammatory diseases.

D-Alanine is a central component of the cell wall in most prokaryotes. D-Alanine synthesis in Escherichia coliis carried out by two different alanine racemases encoded by the alr and dadX genes. Deletion of alr and dadX from the E. coli genome results in a D-alanine auxotrophic phenotype. However, we have observed growth of prototrophic phenotypic revertants during routine culturing of a D-alanine auxotrophic strain. We present a detailed comparison of the proteome and transcriptome profiles of the D-alanine auxotroph and a prototrophic revertant strain. Most noticeably, a general upregulation of genes involved in methionine synthesis in the revertant strain was detected. The appearance of the revertant phenotype was genetically linked to point mutations in the methionine repressor gene (metJ). Our results reveal an alternative metabolic pathway which can supply essential D-alanine for peptidoglycan synthesis of alr-and dadX-deficient E. coli mutants and provide evidence for significant alanine racemase coactivity of the E. coli cystathionine beta-lyase (MetC).Alanine racemases (EC 5.1.1.1) are unique prokaryotic enzymes that catalyze the reversible racemization of L-and Dalanine, the latter one being an essential component in the biosynthesis of the bacterial peptidoglycan of Gram-positive and Gram-negative bacteria (47). The bacteria investigated to date have been found to possess either one or two distinct alanine racemase genes. The alr gene encodes a constitutively expressed alanine racemase, which provides D-alanine for sufficient cross-linking of adjacent peptidoglycan strands in the cell wall. The second gene encodes the so-called catabolic alanine racemase, which is essential for L-alanine catabolism (24,28,41,42,48). In Escherichia coli, the alr-encoded alanine racemase is constitutively expressed, whereas the dadX-encoded enzyme is essential only for L-alanine catabolism, providing a substrate for a D-alanine-specific dehydrogenase encoded by the dadA gene (51). The dadX gene product provides a secondary source of D-alanine for cell wall biosynthesis.D-Alanine auxotrophic E. coli, Bacillus subtilis, Corynebacterium glutamicum, Listeria monocytogenes, and Lactobacillus plantarum strains have been generated by inactivating genes encoding alanine racemases (15,17,24,42,43,45). A strong selective pressure for maintenance of an alanine racemase (Dal)-encoding plasmid in a chromosomal dal mutant of Bacillus subtilis was observed upon growth on rich medium. In Lactobacillus plantarum, plasmids encoding alanine racemase (Alr) were efficiently selected in an alr-deficient Lactobacillus plantarum strain (5). In Listeria monocytogenes, two genes, an alanine racemase gene (dal) and a D-amino acid aminotransferase gene (dat), which control the synthesis of D-alanine, had to be inactivated in order to achieve complete D-alanine auxotrophy (46).Under certain circumstances, the D-alanine auxotrophic phenotype was lost, indicating a redundancy of alanine racemase activity in bacteria. The D-alanine auxotrophic phenoty...

Programmed death ligand-1 (PD-L1) is an important negative regulator of T cell immune responses via interactions with PD-1 and CD80. However, PD-L1 can also act as a positive costimulator, but the relevant counterreceptor is not known. We analyzed the role of PD-L1 in CD8-T cell responses to infection with Listeria monocytogenes (LM) or vesicular stomatitis virus (VSV). PD-L1 blockade impaired antigen-specific CD8 effector T cell expansion in response to LM, but not to VSV infection, particularly limiting short-lived effector cell differentiation. Simultaneous CD4-T cell depletion and anti-PD-L1 blockade revealed that PD-L1 provided costimulation even in the absence of CD4-T cells. Most importantly, specific blockade of PD-L1 binding to CD80 or to PD-1 did not recapitulate PDL-1 blockade. The results suggested that PD-L1 plays an important costimulatory role for antigen-specific CD8 T cells during LM infection perhaps through a distinct receptor or interaction epitope.

While immune responses have been rigorously examined after intravenous Listeria monocytogenes (Lm) infection, less is understood about its dissemination from the intestines or the induction of adaptive immunity after more physiologic models of foodborne infection. Consequently, this study focused on early events in the intestinal mucosa and draining mesenteric lymph nodes (MLN) using foodborne infection of mice with Lm modified to invade murine intestinal epithelium (InlA M Lm). InlA M Lm trafficked intracellularly from the intestines to the MLN and were associated with Batf3independent dendritic cells (DC) in the lymphatics. Consistent with this, InlA M Lm initially disseminated from the gut to the MLN normally in Batf3 −/− mice. Activated migratory DC accumulated in the MLN by 3 days post-infection and surrounded foci of InlA M Lm. At this time Batf3 −/− mice displayed reduced InlA M Lm burdens, implicating cDC1 in maximal bacterial accumulation in the MLN. Batf3 −/− mice also exhibited profound defects in the induction and gut-homing of InlA M Lm-specific effector CD8 T cells. Restoration of pathogen burden did not rescue antigen-specific CD8 T cell responses in Batf3 −/− mice, indicating a critical role for Batf3 in generating anti-InlA M Lm immunity following foodborne infection. Collectively, these data suggest that DC play diverse, dynamic roles in the early events following foodborne InlA M Lm infection and in driving the establishment of intestinal Lm-specific effector T cells.