Two subsets of stem-like CD8+ memory T cell progenitors with distinct fate commitments in humans.

T cell memory relies on the generation of antigen-specific progenitors with stem-like properties. However, the identity of these progenitors has remained unclear, precluding a full understanding of the differentiation trajectories that underpin the heterogeneity of antigen-experienced T cells. We used a systematic approach guided by single-cell RNA-sequencing data to map the organizational structure of the human CD8+ memory T cell pool under physiological conditions. We identified two previously unrecognized subsets of clonally, epigenetically, functionally, phenotypically and transcriptionally distinct stem-like CD8+ memory T cells. Progenitors lacking the inhibitory receptors programmed death-1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) were committed to a functional lineage, whereas progenitors expressing PD-1 and TIGIT were committed to a dysfunctional, exhausted-like lineage. Collectively, these data reveal the existence of parallel differentiation programs in the human CD8+ memory T cell pool, with potentially broad implications for the development of immunotherapies and vaccines.

Riboflavin-LSD1 axis participates in the in vivo tumor-associated macrophage morphology in human colorectal liver metastases.

Tumor-associated macrophages (TAMs) are key components of the tumor microenvironment (TME). In colorectal liver metastasis (CLM), TAM morphology correlates with prognosis, with smaller TAMs (S-TAMs) conferring a more favorable prognosis than larger TAMs (L-TAMs). However, the metabolic profile of in vivo human TAM populations remains unknown. Multiparametric flow cytometry was used to freshly isolate S- and L-TAMs from surgically resected CLM patients (n = 14S-, 14L-TAMs). Mass spectrometry-based metabolomics analyses were implemented for the metabolic characterization of TAM populations. Gene expression analysis and protein activity were used to support the biochemical effects of the enzyme-substrate link between riboflavin and (lysine-specific demethylase 1A, LSD1) with TAM morphologies. L-TAMs were characterized by a positive correlation and a strong association between riboflavin and TAM morphologies. Riboflavin in both L-TAMs and in-vitro M2 polarized macrophages modulates LSD1 protein expression and activity. The inflammatory stimuli promoted by TNFα induced the increased expression of riboflavin transporter SLC52A3 and LSD1 in M2 macrophages. The modulation of the riboflavin-LSD1 axis represents a potential target for reprogramming TAM subtypes, paving the way for promising anti-tumor therapeutic strategies.

Myocardial hypoxic stress mediates functional cardiac extracellular vesicle release.

AIMS: Increased shedding of extracellular vesicles (EVs)-small, lipid bilayer-delimited particles with a role in paracrine signalling-has been associated with human pathologies, e.g. atherosclerosis, but whether this is true for cardiac diseases is unknown. METHODS AND RESULTS: Here, we used the surface antigen CD172a as a specific marker of cardiomyocyte (CM)-derived EVs; the CM origin of CD172a+ EVs was supported by their content of cardiac-specific proteins and heart-enriched microRNAs. We found that patients with aortic stenosis, ischaemic heart disease, or cardiomyopathy had higher circulating CD172a+ cardiac EV counts than did healthy subjects. Cellular stress was a major determinant of EV release from CMs, with hypoxia increasing shedding in in vitro and in vivo experiments. At the functional level, EVs isolated from the supernatant of CMs derived from human-induced pluripotent stem cells and cultured in a hypoxic atmosphere elicited a positive inotropic response in unstressed CMs, an effect we found to be dependent on an increase in the number of EVs expressing ceramide on their surface. Of potential clinical relevance, aortic stenosis patients with the highest counts of circulating cardiac CD172a+ EVs had a more favourable prognosis for transcatheter aortic valve replacement than those with lower counts. CONCLUSION: We identified circulating CD172a+ EVs as cardiac derived, showing their release and function and providing evidence for their prognostic potential in aortic stenosis patients.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

TNF-Stimulated Gene-6 Is a Key Regulator in Switching Stemness and Biological Properties of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are well established to have promising therapeutic properties. TNF-stimulated gene-6 (TSG-6), a potent tissue-protective and anti-inflammatory factor, has been demonstrated to be responsible for a significant part of the tissue-protecting properties mediated by MSCs. Nevertheless, current knowledge about the biological function of TSG-6 in MSCs is limited. Here, we demonstrated that TSG-6 is a crucial factor that influences many functional properties of MSCs. The transcriptomic sequencing analysis of wild-type (WT) and TSG-6-/- -MSCs shows that the loss of TSG-6 expression leads to the perturbation of several transcription factors, cytokines, and other key biological pathways. TSG-6-/- -MSCs appeared morphologically different with dissimilar cytoskeleton organization, significantly reduced size of extracellular vesicles, decreased cell proliferative rate, and loss of differentiation abilities compared with the WT cells. These cellular effects may be due to TSG-6-mediated changes in the extracellular matrix (ECM) environment. The supplementation of ECM with exogenous TSG-6, in fact, rescued cell proliferation and changes in morphology. Importantly, TSG-6-deficient MSCs displayed an increased capacity to release interleukin-6 conferring pro-inflammatory and pro-tumorigenic properties to the MSCs. Overall, our data provide strong evidence that TSG-6 is crucial for the maintenance of stemness and other biological properties of murine MSCs.

Macrophage ferroportin is essential for stromal cell proliferation in wound healing.

Iron recycling by macrophages is essential for erythropoiesis, but may also be relevant for iron redistribution to neighboring cells at the local tissue level. Using mice with iron retention in macrophages due to targeted inactivation of the iron exporter ferroportin, we investigated the role of macrophage iron release in hair follicle cycling and wound healing, a complex process leading to major clinical problems, if impaired. Genetic deletion of ferroportin in macrophages resulted in iron deficiency and decreased proliferation in epithelial cells, which consequently impaired hair follicle growth and caused transient alopecia. Hair loss was not related to systemic iron deficiency or anemia, thus indicating the necessity of local iron release from macrophages. Inactivation of macrophage ferroportin also led to delayed skin wound healing with defective granulation tissue formation and diminished fibroplasia. Iron retention in macrophages had no impact on the inflammatory processes accompanying wound healing, but affected stromal cell proliferation, blood and lymphatic vessel formation, and fibrogenesis. Our findings reveal that iron/ferroportin plays a largely underestimated role in macrophage trophic function in skin homeostasis and repair.

MicroRNA-127-3p controls murine hematopoietic stem cell maintenance by limiting differentiation.

The balance between self-renewal and differentiation is crucial to ensure the homeostasis of the hematopoietic system, and is a hallmark of hematopoietic stem cells. However, the underlying molecular pathways, including the role of micro-RNA, are not completely understood. To assess the contribution of micro-RNA, we performed micro-RNA profiling of hematopoietic stem cells and their immediate downstream progeny multi-potent progenitors from wild-type control and Pbx1-conditional knockout mice, whose stem cells display a profound self-renewal defect. Unsupervised hierarchical cluster analysis separated stem cells from multi-potent progenitors, suggesting that micro-RNA might regulate the first transition step in the adult hematopoietic development. Notably, Pbx1-deficient and wild-type cells clustered separately, linking micro-RNAs to self-renewal impairment. Differential expression analysis of micro-RNA in the physiological stem cell-to-multi-potent progenitor transition and in Pbx1-deficient stem cells compared to control stem cells revealed miR-127-3p as the most differentially expressed. Furthermore, miR-127-3p was strongly stem cell-specific, being quickly down-regulated upon differentiation and not re-expressed further downstream in the bone marrow hematopoietic hierarchy. Inhibition of miR-127-3p function in Lineage-negative cells, achieved through a lentiviral-sponge vector, led to severe stem cell depletion, as assessed with serial transplantation assays. miR-127-3p-sponged stem cells displayed accelerated differentiation, which was uncoupled from proliferation, accounting for the observed stem cell reduction. miR-127-3p overexpression in Lineage-negative cells did not alter stem cell pool size, but gave rise to lymphopenia, likely due to lack of miR-127-3p physiological downregulation beyond the stem cell stage. Thus, tight regulation of miR-127-3p is crucial to preserve the self-renewing stem cell pool and homeostasis of the hematopoietic system.

Autonomous role of Wiskott-Aldrich syndrome platelet deficiency in inducing autoimmunity and inflammation.

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency characterized by eczema, infections, and susceptibility to autoimmunity and malignancies. Thrombocytopenia is a constant finding, but its pathogenesis remains elusive. OBJECTIVE: To dissect the basis of the WAS platelet defect, we used a novel conditional mouse model (CoWas) lacking Wiskott-Aldrich syndrome protein (WASp) only in the megakaryocytic lineage in the presence of a normal immunologic environment, and in parallel we analyzed samples obtained from patients with WAS. METHODS: Phenotypic and functional characterization of megakaryocytes and platelets in mutant CoWas mice and patients with WAS with and without autoantibodies was performed. Platelet antigen expression was examined through a protein expression profile and cluster proteomic interaction network. Platelet immunogenicity was tested by using ELISAs and B-cell and platelet cocultures. RESULTS: CoWas mice showed increased megakaryocyte numbers and normal thrombopoiesis in vitro, but WASp-deficient platelets had short lifespan and high expression of activation markers. Proteomic analysis identified signatures compatible with defects in cytoskeletal reorganization and metabolism yet surprisingly increased antigen-processing capabilities. In addition, WASp-deficient platelets expressed high levels of surface and soluble CD40 ligand and were capable of inducing B-cell activation in vitro. WASp-deficient platelets were highly immunostimulatory in mice and triggered the generation of antibodies specific for WASp-deficient platelets, even in the context of a normal immune system. Patients with WAS also showed platelet hyperactivation and increased plasma soluble CD40 ligand levels correlating with the presence of autoantibodies. CONCLUSION: Overall, these findings suggest that intrinsic defects in WASp-deficient platelets decrease their lifespan and dysregulate immune responses, corroborating the role of platelets as modulators of inflammation and immunity.

The atypical chemokine receptor ACKR2 drives pulmonary fibrosis by tuning influx of CCR2+ and CCR5+ IFNγ-producing γδT cells in mice.

Chemokines coordinate lung inflammation and fibrosis by acting on chemokine receptors expressed on leukocytes and other cell types. Atypical chemokine receptors (ACKRs) bind, internalize, and degrade chemokines, tuning homeostasis and immune responses. ACKR2 recognizes and decreases the levels of inflammatory CC chemokines. The role of ACKR2 in fibrogenesis is unknown. The purpose of the study was to investigate the role of ACKR2 in the context of pulmonary fibrosis. The effects of ACKR2 expression and deficiency during inflammation and fibrosis were analyzed using a bleomycin-model of fibrosis, ACKR2-deficient mice, bone marrow chimeras, and antibody-mediated leukocyte depletion. ACKR2 was upregulated acutely in response to bleomycin and normalized over time. ACKR2-/- mice showed reduced lethality and lung fibrosis. Bone marrow chimeras showed that lethality and fibrosis depended on ACKR2 expression in pulmonary resident (nonhematopoietic) cells but not on leukocytes. ACKR2-/- mice exhibited decreased expression of tissue-remodeling genes, reduced leukocyte influx, pulmonary injury, and dysfunction. ACKR2-/- mice had early increased levels of CCL5, CCL12, CCL17, and IFNγ and an increased number of CCR2+ and CCR5+ IFNγ-producing γδT cells in the airways counterbalanced by low Th17-lymphocyte influx. There was reduced accumulation of IFNγ-producing γδT cells in CCR2-/- and CCR5-/- mice. Moreover, depletion of γδT cells worsened the clinical symptoms induced by bleomycin and reversed the phenotype of ACKR2-/- mice exposed to bleomycin. ACKR2 controls the CC chemokine expression that drives the influx of CCR2+ and CCR5+ IFNγ-producing γδT cells, tuning the Th17 response that mediated pulmonary fibrosis triggered by bleomycin instillation.

Thrombopoietin/TGF-ß1 Loop Regulates Megakaryocyte Extracellular Matrix Component Synthesis.

Extracellular matrix (ECM) components initiate crucial biochemical and biomechanical cues that are required for bone marrow homeostasis. In our research, we prove that a peri-cellular matrix composed primarily of type III and type IV collagens, and fibronectin surrounds human megakaryocytes in the bone marrow. The data we collected support the hypothesis that bone marrow megakaryocytes possess a complete mechanism to synthesize the ECM components, and that thrombopoietin is a pivotal regulator of this new function inducing transforming growth factor-ß1 (TGF-ß1) release and consequent activation of the downstream pathways, both in vitro and in vivo. This activation results in a dose dependent increase of ECM component synthesis by megakaryocytes, which is reverted upon incubation with JAK and TGF-ß1 receptor specific inhibitors. These data are pivotal for understanding the central role of megakaryocytes in creating their own regulatory niche within the bone marrow environment.

Mesenchymal Stem Cells Reduce Colitis in Mice via Release of TSG6, Independently of Their Localization to the Intestine.

BACKGROUND & AIMS: Mesenchymal stem cells (MSCs) are pluripotent cells that can promote expansion of immune regulatory cells and might be developed for the treatment of immune disorders, including inflammatory bowel diseases. MSCs were reported to reduce colitis in mice; we investigated whether MSC localization to the intestine and production of paracrine factors, including tumor necrosis factor-induced protein 6 (TSG6), were required for these effects. METHODS: MSCs were isolated from bone marrow (BM-MSCs) of 4- to 6-week-old C57BL/6, C57BL/6-green fluorescent protein, or Balb/c Tsg6-/- male mice. Colitis was induced by ad libitum administration of dextran sulfate sodium for 10 days; after 5 days the mice were given intraperitoneal injections of BM-MSCs or saline (controls). Blood samples and intestinal tissues were collected 24, 48, 96, and 120 hours later; histologic and flow cytometry analyses were performed. RESULTS: Injection of BM-MSCs reduced colitis in mice, increasing body weight and reducing markers of intestinal inflammation, compared with control mice. However, fewer than 1% of MSCs reached the inflamed colon. Most of the BM-MSCs formed aggregates in the peritoneal cavity. The aggregates contained macrophages and B and T cells, and produced immune-regulatory molecules including FOXP3, interleukin (IL)10, transforming growth factor-ß, arginase type II, chemokine (C-C motif) ligand 22 (CCL22), heme oxygenase-1, and TSG6. Serum from mice given BM-MSCs, compared with mice given saline, had increased levels of TSG6. Injection of TSG6 reduced the severity of colitis in mice, along with the numbers of CD45+ cells, neutrophils and metalloproteinase activity in the mucosa, while increasing the percentage of Foxp3CD45+ cells. TSG6 injection also promoted the expansion of regulatory macrophages that expressed IL10 and inducible nitric oxide synthase, and reduced serum levels of interferon-γ, IL6, and tumor necrosis factor. Tsg6-/- MSCs did not suppress the mucosal inflammatory response in mice with colitis. CONCLUSIONS: BM-MSCs injected into mice with colitis do not localize to the intestine but instead form aggregates in the peritoneum where they produce immunoregulatory molecules, including TSG6, that reduce intestinal inflammation. TSG6 is sufficient to reduce intestinal inflammation in mice with colitis.

Loss of function of Ribonuclease T2, an ancient and phylogenetically conserved RNase, plays a crucial role in ovarian tumorigenesis.

In recent years, the role played by the stromal microenvironment has been given growing attention in order to achieve a full understanding of cancer initiation and progression. Because cancer is a tissue-based disease, the integrity of tissue architecture is a major constraint toward cancer growth. Indeed, a large contribution of the natural resistance to cancer stems from stromal microenvironment components, the dysregulation of which can facilitate cancer occurrence. For instance, recent experimental evidence has highlighted the involvement of stromal cells in ovarian carcinogenesis, as epitomized by ovarian xenografts obtained by a double KO of the murine Dicer and Pten genes. Likewise, we reported the role of an ancient extracellular RNase, called Ribonuclease T2 (RNASET2), within the ovarian stromal microenvironment. Indeed, hyperexpression of RNASET2 is able to control tumorigenesis by recruiting macrophages (mostly of the anticancer M1 subtype) at the tumor sites. We present biological data obtained by RNASET2 silencing in the poorly tumorigenetic and highly RNASET2-expressing human OVCAR3 cell line. RNASET2 knockdown was shown to stimulate in vivo tumor growth early after microinjection of OVCAR3 cells in nude mice. Moreover, we have investigated by molecular profiling the in vivo expression signature of human and mouse cell xenografts and disclosed the activation of pathways related to activation of the innate immune response and modulation of ECM components. Finally, we provide evidence for a role of RNASET2 in triggering an in vitro chemotactic response in macrophages. These results further highlight the critical role played by the microenvironment in RNASET2-mediated ovarian tumor suppression, which could eventually contribute to better clarify the pathogenesis of this disease.

Flow cytometry applications for the analysis of chemokine receptor expression and function.

Chemokine receptors play an important role in leukocyte migration, both in physiological and pathological conditions, and the interest in new methodologies for their detection is increasing. In this review, we focused on chemokine receptors detection through flow cytometric approaches, including the use of specific antibodies and fluorescent chemokines, and on approaches aimed at the analysis of their functions, from intracellular trafficking to signaling activities.

Agrin is required for survival and function of monocytic cells.

Agrin, an extracellular matrix protein belonging to the heterogeneous family of heparan sulfate proteoglycans (HSPGs), is expressed by cells of the hematopoietic system but its role in leukocyte biology is not yet clear. Here we demonstrate that agrin has a crucial, nonredundant role in myeloid cell development and functions. We have identified lineage-specific alterations that affect maturation, survival and properties of agrin-deficient monocytic cells, and occur at stages later than stem cell precursors. Our data indicate that the cell-autonomous signals delivered by agrin are sensed by macrophages through the α-DC (DG) receptor and lead to the activation of signaling pathways resulting in rearrangements of the actin cytoskeleton during the phagocytic synapse formation and phosphorylation of extracellular signal-regulated kinases (Erk 1/2). Altogether, these data identify agrin as a novel player of innate immunity.

Control of murine Ly6C(high) monocyte traffic and immunosuppressive activities by atypical chemokine receptor D6.

The atypical chemokine receptor D6 is a decoy and scavenger receptor for most inflammatory CC chemokines and prevents the development of exacerbated inflammatory reactions. Here we report that mice lacking D6 expression in the nonhematopoietic compartment have a selective increase in the number of Ly6C(high) monocytes in the circulation and in secondary lymphoid tissues. Under inflammatory conditions, Ly6C(high) monocytes accumulate in increased number in secondary lymphoid organs of D6(-/-) mice in a CCR2-dependent manner. Ly6C(high) monocytes derived from D6(-/-) mice have enhanced immunosuppressive activity, inhibit the development of adaptive immune responses, and partially protect mice from the development of GVHD. Thus, control of CCR2 ligands by D6 regulates the traffic of Ly6C(high) monocytes and controls their immunosuppressive potential.

The critical role of agrin in the hematopoietic stem cell niche.

Hematopoiesis is the process leading to the sustained production of blood cells by hematopoietic stem cells (HSCs). Growth, survival, and differentiation of HSCs occur in specialized microenvironments called "hematopoietic niches," through molecular cues that are only partially understood. Here we show that agrin, a proteoglycan involved in the neuromuscular junction, is a critical niche-derived signal that controls survival and proliferation of HSCs. Agrin is expressed by multipotent nonhematopoietic mesenchymal stem cells (MSCs) and by differentiated osteoblasts lining the endosteal bone surface, whereas Lin(-)Sca1(+)c-Kit(+) (LSK) cells express the α-dystroglycan receptor for agrin. In vitro, agrin-deficient MSCs were less efficient in supporting proliferation of mouse Lin(-)c-Kit(+) cells, suggesting that agrin plays a role in the hematopoietic cell development. These results were indeed confirmed in vivo through the analysis of agrin knockout mice (Musk-L;Agrn(-/-)). Agrin-deficient mice displayed in vivo apoptosis of CD34(+)CD135(-) LSK cells and impaired hematopoiesis, both of which were reverted by an agrin-sufficient stroma. These data unveil a crucial role of agrin in the hematopoietic niches and in the cross-talk between stromal and hematopoietic stem cells.

Flow Cytometry Analysis of IL-1 Receptors and Toll-Like Receptors on Platelets and Platelet-Derived Extracellular Vesicles.

Flow cytometry is largely used for the immunophenotyping and quantification of several cell types or related components including platelets and extracellular vesicles. Platelets and platelet-derived extracellular vesicles (PEVs) are receiving increased interest in inflammatory diseases including sepsis. Thus, in this chapter, we will describe protocols for the flow cytometry analysis of platelets, platelet/neutrophils hetero aggregates, and PEVs mainly focusing on the evaluation of the surface expression of some IL-1 receptor (ILR) and Toll-like receptor (TLR) family members.

Size-advantage of monovalent nanobodies against the macrophage mannose receptor for deep tumor penetration and tumor-associated macrophage targeting.

Rationale: Nanobodies (Nbs) have emerged as an elegant alternative to the use of conventional monoclonal antibodies in cancer therapy, but a detailed microscopic insight into the in vivo pharmacokinetics of different Nb formats in tumor-bearers is lacking. This is especially relevant for the recognition and targeting of pro-tumoral tumor-associated macrophages (TAMs), which may be located in less penetrable tumor regions. Methods: We employed anti-Macrophage Mannose Receptor (MMR) Nbs, in a monovalent (m) or bivalent (biv) format, to assess in vivo TAM targeting. Intravital and confocal microscopy were used to analyse the blood clearance rate and targeting kinetics of anti-MMR Nbs in tumor tissue, healthy muscle tissue and liver. Fluorescence Molecular Tomography was applied to confirm anti-MMR Nb accumulation in the primary tumor and in metastatic lesions. Results: Intravital microscopy demonstrated significant differences in the blood clearance rate and macrophage targeting kinetics of (m) and (biv)anti-MMR Nbs, both in tumoral and extra-tumoral tissue. Importantly, (m)anti-MMR Nbs are superior in reaching tissue macrophages, an advantage that is especially prominent in tumor tissue. The administration of a molar excess of unlabelled (biv)anti-MMR Nbs increased the (m)anti-MMR Nb bioavailability and impacted on its macrophage targeting kinetics, preventing their accumulation in extra-tumoral tissue (especially in the liver) but only partially influencing their interaction with TAMs. Finally, anti-MMR Nb administration not only allowed the visualization of TAMs in primary tumors, but also at a distant metastatic site. Conclusions: These data describe, for the first time, a microscopic analysis of (m) and (biv)anti-MMR Nb pharmacokinetics in tumor and healthy tissues. The concepts proposed in this study provide important knowledge for the future use of Nbs as diagnostic and therapeutic agents, especially for the targeting of tumor-infiltrating immune cells.

Role of Toll interleukin-1 receptor (IL-1R) 8, a negative regulator of IL-1R/Toll-like receptor signaling, in resistance to acute Pseudomonas aeruginosa lung infection.

Toll interleukin-1 receptor (IL-1R) 8 (TIR8), also known as single Ig IL-1 receptor (IL-R)-related molecule, or SIGIRR, is a member of the IL-1R-like family, primarily expressed by epithelial cells. Current evidence suggests that TIR8 plays a nonredundant role as a negative regulator in vivo under different inflammatory conditions that are dependent on IL-R and Toll-like receptor (TLR) activation. In the present study, we examined the role of TIR8 in innate resistance to acute lung infections caused by Pseudomonas aeruginosa, a Gram-negative pathogen responsible for life-threatening infections in immunocompromised individuals and cystic fibrosis patients. We show that Tir8 deficiency in mice was associated with increased susceptibility to acute P. aeruginosa infection, in terms of mortality and bacterial load, and to exacerbated local and systemic production of proinflammatory cytokines (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], IL-1ß, and IL-6) and chemokines (CXCL1, CXCL2, and CCL2). It has been reported that host defense against P. aeruginosa acute lung infection can be improved by blocking IL-1 since exaggerated IL-1ß production may be harmful for the host in this infection. In agreement with these data, IL-1RI deficiency rescues the phenotype observed in Tir8-deficient mice: in Tir8-/- IL-1RI-/- double knockout mice we observed higher survival rates, enhanced bacterial clearance, and reduced levels of local and systemic cytokine and chemokine levels than in Tir8-deficient mice. These results suggest that TIR8 has a nonredundant effect in modulating the inflammation caused by P. aeruginosa, in particular, by negatively regulating IL-1RI signaling, which plays a major role in the pathogenesis of this infectious disease.