Regulatory T cells prevent inducible BALT formation by dampening neutrophilic inflammation.

Inducible BALT (iBALT) can amplify pulmonary or systemic inflammatory responses to the benefit or detriment of the host. We took advantage of the age-dependent formation of iBALT to interrogate the underlying mechanisms that give rise to this ectopic, tertiary lymphoid organ. In this study, we show that the reduced propensity for weanling as compared with neonatal mice to form iBALT in response to acute LPS exposure is associated with greater regulatory T cell expansion in the mediastinal lymph nodes. Ab- or transgene-mediated depletion of regulatory T cells in weanling mice upregulated the expression of IL-17A and CXCL9 in the lungs, induced a tissue neutrophilia, and increased the frequency of iBALT to that observed in neonatal mice. Remarkably, neutrophil depletion in neonatal mice decreased the expression of the B cell active cytokines, a proliferation-inducing ligand and IL-21, and attenuated LPS-induced iBALT formation. Taken together, our data implicate a role for neutrophils in lymphoid neogenesis. Neutrophilic inflammation is a common feature of many autoimmune diseases in which iBALT are present and pathogenic, and hence the targeting of neutrophils or their byproducts may serve to ameliorate detrimental lymphoid neogenesis in a variety of disease contexts.

Plasmacytoid dendritic cells promote host defense against acute pneumovirus infection via the TLR7-MyD88-dependent signaling pathway.

Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in infants. In human infants, plasmacytoid dendritic cells (pDC) are recruited to the nasal compartment during infection and initiate host defense through the secretion of type I IFN, IL-12, and IL-6. However, RSV-infected pDC are refractory to TLR7-mediated activation. In this study, we used the rodent-specific pathogen, pneumonia virus of mice (PVM), to determine the contribution of pDC and TLR7 signaling to the development of the innate inflammatory and early adaptive immune response. In wild-type, but not TLR7- or MyD88-deficient mice, PVM inoculation led to a marked infiltration of pDC and increased expression of type I, II, and III IFNs. The delayed induction of IFNs in the absence of TLR7 or MyD88 was associated with a diminished innate inflammatory response and augmented virus recovery from lung tissue. In the absence of TLR7, PVM-specific CD8(+) T cell cytokine production was abrogated. The adoptive transfer of TLR7-sufficient, but not TLR7-deficient pDC to TLR7 gene-deleted mice recapitulated the antiviral responses observed in wild-type mice and promoted virus clearance. In summary, TLR7-mediated signaling by pDC is required for appropriate innate responses to acute pneumovirus infection. It is conceivable that as-yet-unidentified defects in the TLR7 signaling pathway may be associated with elevated levels of RSV-associated morbidity and mortality among otherwise healthy human infants.

Toll/IL-1 signaling is critical for house dust mite-specific helper T cell type 2 and type 17 [corrected] responses.

RATIONALE: One of the immunopathological features of allergic inflammation is the infiltration of helper T type 2 (Th2) cells to the site of disease. Activation of innate pattern recognition receptors such as Toll-like receptors (TLRs) plays a critical role in helper T type 1 cell differentiation, yet their contribution to the generation of Th2 responses to clinically relevant aeroallergens remains poorly defined. OBJECTIVES: To determine the requirement for TLR2, TLR4, and the Toll/IL-1 receptor domain adaptor protein MyD88 in a murine model of allergic asthma. METHODS: Wild-type and factor-deficient ((-/-)) mice were sensitized intranasally to the common allergen house dust mite (HDM) and challenged 2 weeks later on four consecutive days. Measurements of allergic airway inflammation, T-cell cytokine production, and airway hyperreactivity were performed 24 hours later. MEASUREMENTS AND MAIN RESULTS: Mice deficient in MyD88 were protected from the cardinal features of allergic asthma, including granulocytic inflammation, Th2 cytokine production and airway hyperreactivity. Although HDM activated NF-kappaB in TLR2- or TLR4-expressing HEK cells, only in TLR4(-/-) mice was the magnitude of allergic airway inflammation and hyperreactivity attenuated. The diminished Th2 response present in MyD88(-/-) and TLR4(-/-) mice was associated with fewer OX40 ligand-expressing myeloid dendritic cells in the draining lymph nodes during allergic sensitization. Finally, HDM-specific IL-17 production and airway neutrophilia were attenuated in MyD88(-/-) but not TLR4(-/-) mice. CONCLUSIONS: Together, these data suggest that Th2- and Th17-mediated inflammation generated on inhalational HDM exposure is differentially regulated by the presence of microbial products and the activation of distinct MyD88-dependent pattern recognition receptors.

Correction: Serine 207 phosphorylated lysyl-tRNA synthetase predicts disease-free survival of non-small-cell lung carcinoma.

[This corrects the article DOI: 10.18632/oncotarget.18053.].

Serine 207 phosphorylated lysyl-tRNA synthetase predicts disease-free survival of non-small-cell lung carcinoma.

It has been shown that various tRNA synthetases exhibit non-canonical activities unrelated to their original role in translation. We have previously described a signal transduction pathway in which serine 207 phosphorylated lysyl-tRNA synthetase (P-s207 LysRS) is released from the cytoplasmic multi-tRNA synthetase complex (MSC) into the nucleus, where it activates the transcription factor MITF in stimulated cultured mast cells and cardiomyocytes. Here we describe a similar transformation of LysRS due to EGFR signaling activation in human lung cancer. Our data shows that activation of the EGFR results in phosphorylation of LysRS at position serine 207, its release from the MSC and translocation to the nucleus. We then generated a P-s207 LysRS rabbit polyclonalantibody and tested 242 tissue micro-array samples derived from non-small-cell lung cancer patients. Highly positive nuclear staining for P-s207 LysRS was noted in patients with EGFR mutations as compared to WT EGFR patients and was associated with improved mean disease-free survival (DFS). In addition, patients with mutated EGFR and negative lymph node metastases had better DFS when P-s207 LysRS was present in the nucleus. The data presented strongly suggests functional and prognostic significance of P-s207 LysRS in non-small-cell lung cancer.

Non-canonical roles of lysyl-tRNA synthetase in health and disease.

Lysyl-tRNA synthetase (LysRS) is a highly conserved enzyme that is part of the translational machinery in all living cells. Besides its canonical role in translation, LysRS gained additional domains and functions throughout evolution. These include its essential role in HIV replication and its roles in transcriptional regulation, cytokine-like signaling, and transport of proteins to the cell membrane. These diverse processes are tightly regulated through post-transcriptional modifications, interactions with other proteins, and targeting to the various cell compartments. The emerging variety of tasks performed by LysRS may therefore be utilized by various processes and pathological conditions that are described in this review, and their ongoing investigation is of extreme importance for our understanding of basic cellular regulatory mechanisms.

A structural hypothesis for the transition between bent and extended conformations of the leukocyte beta2 integrins.

Integrins mediate cell adhesion in response to activation signals that trigger conformational changes within their ectodomain. It is thought that a compact bent conformation of the molecule represents its physiological low affinity state and extended conformations its active state. We have determined the structure of two integrin fragments of the beta2 subunit. The first structure, consisting of the plexin-semaphorin-integrin domain, hybrid, integrin-epidermal growth factor 1 (I-EGF1), and I-EGF2 domains (PHE2), showed an L-shaped conformation with the bend located between the I-EGF1 and I-EGF2 domains. The second structure, which includes, in addition, the I-EGF3 domain, showed an extended conformation. The major reorientation of I-EGF2 with respect to the other domains in the two structures is accompanied by a change of torsion angle of the disulfide bond between Cys(461)-Cys(492) by 180 degrees and the conversion of a short alpha-helix (residues Ser(468)-Cys(475)) into a flexible coil. Based on the PHE2 structure, we introduced a disulfide bond between the plexin-semaphorin-integrin domain and I-EGF2 domains in the beta2 subunit. The resultant alphaLbeta2 integrin (leukocyte function-associated antigen-1) variant was locked in a bent state and could not be detected with the monoclonal antibody KIM127 in Mg(2+)/EGTA. However, it retained the binding activity to ICAM-1. These results provide a structural hypothesis for our understanding of the transition between the resting and active states of leukocyte function-associated antigen-1.

Mutation of a conserved asparagine in the I-like domain promotes constitutively active integrins alphaLbeta2 and alphaIIbbeta3.

The leukocyte beta2 integrins are heterodimeric adhesion receptors required for a functional immune system. Many leukocyte adhesion deficiency-1 (LAD-1) mutations disrupt the expression and function of beta2 integrins. Herein, we further characterized the LAD-1 mutation N329S in the beta2 inserted (I)-like domain. This mutation converted alphaLbeta2 from a resting into a high affinity conformer because alphaLbeta2N329S transfectants adhered avidly to ligand intercellular adhesion molecule (ICAM)-3 in the absence of additional activating agent. An extended open conformation is adopted by alphaLbeta2N329S because of its reactivity with the beta2 activation reporter monoclonal antibodies MEM148 and KIM127. A corresponding mutation in beta3 generated constitutively active alphaIIbbeta3 that adhered to fibrinogen. This Asn is conserved in all human beta subunits, and it resides before the last helix of the I-like domain, which is known to be important in activation signal propagation. By mutagenesis studies and review of existing integrin structures, we conjectured that this conserved Asn may have a primary role in shaping the I-like domain by stabilizing the conformation of the alpha7 helix and the beta6-alpha7 loop in the I-like domain.

Recombinant protein-based enzyme-linked immunosorbent assay and immunochromatographic tests for detection of immunoglobulin G antibodies to severe acute respiratory syndrome (SARS) coronavirus in SARS patients.

An enzyme-linked immunosorbent assay (ELISA) and a rapid immunochromatographic test for detection of immunoglobulin G (IgG) antibodies in severe acute respiratory syndrome (SARS) patients were developed by utilizing the well-characterized recombinant proteins Gst-N and Gst-U274. The ELISA detected IgG antibodies to SARS-CoV in all 74 convalescent-phase samples from SARS patients while weakly cross-reacting to only 1 of the 210 control sera from healthy donors. This finding thus led to a kit sensitivity, specificity, and accuracy of 100, 99.5, and 99.6%, respectively. The test thus provided a positive predictive value (PPV) of 98.7% and a negative predictive value (NPV) of 100%. In addition, the ELISA gave a positive delta of 5.4 and a negative delta of 3.6, indicating an excellent differentiation between positives and negatives. The same recombinant proteins were also applied to a newly developed platform for the development of a 15-min rapid test. The resulting rapid test has an excellent agreement of 99.6%, with a kappa value of 1.00, with the ELISA. Again, this rapid test was able to detect 100% of the samples tested (n = 42) while maintaining a specificity of 99.0% (n = 210). The PPV and NPV for the rapid test thus reached 95.3 and 100%, respectively.