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Richard M Locksley

Publications and source records attributed to Richard M Locksley.

At least 19 recordsLinked to original sources
PubMed2026

Humanizing acidic mammalian chitinase variants establish lung immune conditioning and control environmentally driven inflammation and fibrosis.

Chitin, a widespread environmental particle constituent, triggers lung inflammation but is degraded by chitinases. In humans, single-nucleotide polymorphisms (SNPs) in CHIA (acidic mammalian chitinase; AMCase) are associated with lung disease, suggesting that chitinase variants influence responses to airborne particles. Here, we edit the mouse Chia1 locus to generate humanized (hChia) mice harboring common human SNPs. Compared with controls expressing disease-protective SNPs, hChia mice lack robust chitinase activity and fail to degrade natural chitin substrates. Lung-resident lymphocytes and macrophages are spontaneously primed and sensitive to inflammatory triggering by environmental chitin. Immune cell infiltration correlates with airway chitin following challenge, and hChia mice exhibit exacerbated inflammatory and fibrotic lung disease. In humans with acute respiratory failure, alveolar hemorrhage coincides with environmentally derived chitin particles that are susceptible to chitinase degradation, attenuating inflammatory cell responses. Thus, environmental chitin and chitinase activity are crucial determinants of lung immune conditioning with potential therapeutic applications.

AMCase↗
PubMed2006

Neutrophil histamine contributes to inflammation in mycoplasma pneumonia.

Mycoplasmas cause chronic inflammation and are implicated in asthma. Mast cells defend against mycoplasma infection and worsen allergic inflammation, which is mediated partly by histamine. To address the hypothesis that mycoplasma provokes histamine release, we exposed mice to Mycoplasma pulmonis, comparing responses in wild-type and mast cell-deficient KitW-sh/KitW-sh (W-sh) mice. Low histamine levels in uninfected W-sh mice confirmed the conventional wisdom that mast cells are principal sources of airway and serum histamine. Although mycoplasma did not release histamine acutely in wild-type airways, levels rose up to 50-fold above baseline 1 week after infection in mice heavily burdened with neutrophils. Surprisingly, histamine levels also rose profoundly in infected W-sh lungs, increasing in parallel with neutrophils and declining with neutrophil depletion. Furthermore, neutrophils from infected airway were highly enriched in histamine compared with naive neutrophils. In vitro, mycoplasma directly stimulated histamine production by naive neutrophils and strongly upregulated mRNA encoding histidine decarboxylase, the rate-limiting enzyme in histamine synthesis. In vivo, treatment with antihistamines pyrilamine or cimetidine decreased lung weight and severity of pneumonia and tracheobronchitis in infected W-sh mice. These findings suggest that neutrophils, provoked by mycoplasma, greatly expand their capacity to synthesize histamine, thereby contributing to lung and airway inflammation.

Animals↗
PubMed2006

Visualization of IL-12/23p40 in vivo reveals immunostimulatory dendritic cell migrants that promote Th1 differentiation.

IL-12p40 is induced in macrophages and dendritic cells (DC) after activation by microbial TLR ligands and cytokines and constitutes a component of IL-12 and IL-23. In an effort to understand the location and kinetics of these cytokines during the course of an immune response, we generated knockin (gene-targeted) mice that express the p40 gene linked via a viral internal ribosome entry site element with fluorescent reporters, eYFP or eGFP. Macrophages and DC from these mice faithfully reported biallelic p40 induction using the fluorescent marker. s.c. inoculation with Listeria monocytogenes or LPS led to a rapid, but transient, accumulation of p40-expressing DC in draining lymph nodes, which could be blocked by the addition of pertussis toxin. In situ analysis also revealed the accumulation of IL-12p40 protein around high endothelial venules located in close proximity to p40-expressing DC. Consistent with the in vivo findings, in vitro-activated DC that expressed p40 migrated to draining lymph nodes and promoted Th1 differentiation more efficiently than DC that did not express p40. Accordingly, these mice provide a valuable tool for tracking critical functions of DC in vivo and should bestow a useful reagent for exploring the effector biology of these cells in models of infectious disease, cancer immunity, and vaccine development.

Alleles↗
PubMed2006

Type 2 immunity is controlled by IL-4/IL-13 expression in hematopoietic non-eosinophil cells of the innate immune system.

Nippostrongylus brasiliensis infection and ovalbumin-induced allergic lung pathology are highly interleukin (IL)-4/IL-13 dependent, but the contributions of IL-4/IL-13 from adaptive (T helper [Th]2 cells) and innate (eosinophil, basophils, and mast cells) immune cells remain unknown. Although required for immunoglobulin (Ig)E induction, IL-4/IL-13 from Th2 cells was not required for worm expulsion, tissue inflammation, or airway hyperreactivity. In contrast, innate hematopoietic cell-derived IL-4/IL-13 was dispensable for Th2 cell differentiation in lymph nodes but required for effector cell recruitment and tissue responses. Eosinophils were not required for primary immune responses. Thus, components of type 2 immunity mediated by IL-4/IL-13 are partitioned between T cell-dependent IgE and an innate non-eosinophil tissue component, suggesting new strategies for interventions in allergic immunity.

Animals↗
PubMed2006

Activation of the integrated stress response during T helper cell differentiation.

Adaptive immune responses require clonal expansion and differentiation of naive T cells into cytokine-secreting effector cells. After priming via signals through the T cell receptor, naive T helper cells express cytokine mRNA but do not secrete cytokine protein without additional T cell receptor stimulation. Here we show that primed T cells demonstrated phosphorylation of eukaryotic initiation factor 2-alpha (eIF2alpha), a 'collapsed' polysome profile, increased expression of stress-response genes and accumulation of cytoplasmic granules associated with RNA-binding proteins, all features of the integrated stress response. Restimulation of the cells resulted in rapid eIF2alpha dephosphorylation, ribosomal mRNA loading and cytokine secretion. Interference with the function of granule-associated proteins or accumulation of phosphorylated eIF2alpha enhanced release of interleukin 4 during T helper type 2 priming. Therefore, T lymphocytes require components of the integrated stress response to uncouple differentiation from the execution of effector functions.

Animals↗
PubMed2006

Replication initiation from a novel origin identified in the Th2 cytokine cluster locus requires a distant conserved noncoding sequence.

Lineage commitment of Th cells is associated with the establishment of specific transcriptional programs of cytokines. However, how Th cell differentiation affects the program of DNA replication has not been addressed. To gain insight into interplays between differentiation-induced transcription regulation and initiation of DNA replication, we took advantage of an in vitro differentiation system of naive T cells, in which one can manipulate their differentiation into Th1 or Th2 cells. We searched for replication origins in the murine IL-4/IL-13 locus and compared their profiles in the two Th cell lineages which were derived in vitro from the same precursor T cells. We identified a replication origin (ori(IL-13)) downstream from exon 4 of IL-13 and showed that this origin functions in both Th2 and Th1 cells. A distant regulatory element called CNS-1 (conserved noncoding sequence 1) in the IL-4/IL-13 intergenic region coincides with a Th2-specific DNase I-hypersensitive site and is required for efficient, coordinated expression of Th2 cytokines. Replication initiation from ori(IL-13) is significantly reduced in Th1 and Th2 cells derived from CNS-1-deficient mice. However, the replication timing of this locus is consistently early during S phase in both Th1 and Th2 cells under either the wild-type or CNS-1 deletion background. Thus, the conserved noncoding element in the intergenic region regulates replication initiation from a distant replication origin in a manner independent from its effect on lineage-specific transcription but not the replication timing of the segment surrounding this origin.

Animals↗
PubMed2006

T helper cell effector fates--who, how and where?

CD4 helper T cells functionally organize the host immune response by elaborating cytokines, often in patterns that have overlapping effects on other cells. Much interest centers on understanding how these stereotyped cytokine patterns become elaborated and what mechanisms underlie the generation of distinct helper T cell subsets. The past two years have seen advances in understanding of additional subsets, including T helper follicular cells and IL-17-producing T helper cells. Progress has also been achieved in resolving some of the crosstalk that regulates effector fate at the level of distinct transcription factors and chromatin reorganization of the cytokine genes, and a crucial role for gene silencing has been exposed. Finally, the role of innate cells in influencing these processes has become increasingly realized.

Animals↗
PubMed2006

LACK-reactive CD4+ T cells require autocrine IL-2 to mediate susceptibility to Leishmania major.

Mice from most inbred strains are resistant to infection with Leishmania major whereas mice from BALB strains are highly susceptible. Resistance and susceptibility result from the development of Th1 or Th2 cells, respectively. In this report, we document an IL-2 mRNA burst, preceding the reported early IL-4 response, in draining lymph nodes of susceptible mice infected with L. major. Neutralization of IL-2 during the first days of infection redirected Th1 cell maturation and resistance to L. major, through interference with the rapid IL-4 transcription in Leishmania homolog of mammalian RACK1 (LACK)-reactive CD4(+) cells. A burst of IL-2 transcripts also occurred in infected C57BL/6 mice that do not mount an early IL-4 response. However, although the LACK protein induced IL-2 transcripts in susceptible mice, it failed to trigger this response in resistant C57BL/6 mice. Reconstitution experiments using C.B.-17 SCID mice and LACK-reactive CD4(+) T cells from IL-2(-/-) BALB/c mice showed that triggering of the early IL-4 response required autocrine IL-2. Thus, in C57BL/6 mice, the inability of LACK-reactive CD4(+) T cells to express early IL-4 mRNA transcription, important for disease progression, appears due to an incapacity of these cells to produce IL-2.

Adoptive Transfer↗
PubMed2006

TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells.

We describe de novo generation of IL-17-producing T cells from naive CD4 T cells, induced in cocultures of naive CD4 T cells and naturally occurring CD4+ CD25+ T cells (Treg) in the presence of TLR3, TLR4, or TLR9 stimuli. Treg can be substituted by TGFbeta1, which, together with the proinflammatory cytokine IL-6, supports the differentiation of IL-17-producing T cells, a process that is amplified by IL-1beta and TNFalpha. We could not detect a role for IL-23 in the differentiation of IL-17-producing T cells but confirmed its importance for their survival and expansion. Transcription factors GATA-3 and T-bet, as well as its target Hlx, are absent in IL-17-producing T cells, and they do not express the negative regulator for TGFbeta signaling, Smad7. Our data indicate that, in the presence of IL-6, TGFbeta1 subverts Th1 and Th2 differentiation for the generation of IL-17-producing T cells.

Animals↗
PubMed2006

A failure to launch: fuelling cytokine secretion in iNKT cells.

In this issue of Immunity, Bezbradica et al., (2006) uncover an unsuspected role for the cytokine GM-CSF in the thymic development of invariant NKT cells, a role that licenses these cells to secrete effector cytokines upon activation in the periphery.

Animals↗
PubMed2006

Inhibition of T cell activation and autoimmune diabetes using a B cell surface-linked CTLA-4 agonist.

CTL-associated antigen 4 (CTLA-4) engagement negatively regulates T cell activation and function and promotes immune tolerance. However, it has been difficult to explore the biology of selective engagement of CTLA-4 in vivo because CTLA-4 shares its ligands, B7-1 and B7-2, with CD28. To address this issue, we developed a Tg mouse expressing a single-chain, membrane-bound anti-CTLA-4 Ab (scFv) on B cells. B and T cells developed normally and exhibited normal phenotype in the steady state and after activation in these mice. However, B cells from scFv Tg+ mice (scalphaCTLA4+) prevented T cell proliferation and cytokine production in mixed lymphocyte reactions. Additionally, mice treated with scalphaCTLA4+ B cells had decreased T cell-dependent B cell Ab production and class switching in vivo after antigen challenge. Furthermore, expression of this CTLA-4 agonist protected NOD mice from spontaneous autoimmune diabetes. Finally, this disease prevention occurred in Treg-deficient NOD.B7-1/B7-2 double-knockout mice, suggesting that the effect of the CTLA-4 agonist directly attenuates autoreactive T cell activation, not Treg activation. Together, results from this study demonstrate that selective ligation of CTLA-4 attenuates in vivo T cell responses, prevents development of autoimmunity, and represents a novel immunotherapeutic approach for the induction and maintenance of peripheral tolerance.

Animals↗
PubMed2005

Visualizing regulatory T cell control of autoimmune responses in nonobese diabetic mice.

The in vivo mechanism of regulatory T cell (T(reg) cell) function in controlling autoimmunity remains controversial. Here we have used two-photon laser-scanning microscopy to analyze lymph node priming of diabetogenic T cells and to delineate the mechanisms of T(reg) cell control of autoimmunity in vivo. Islet antigen-specific CD4(+)CD25(-) T helper cells (T(H) cells) and T(reg) cells swarmed and arrested in the presence of autoantigens. These T(H) cell activities were progressively inhibited in the presence of increasing numbers of T(reg) cells. There were no detectable stable associations between T(reg) and T(H) cells during active suppression. In contrast, T(reg) cells directly interacted with dendritic cells bearing islet antigen. Such persistent T(reg) cell-dendritic cell contacts preceded the inhibition of T(H) cell activation by dendritic cells, supporting the idea that dendritic cells are central to T(reg) cell function in vivo.

Adoptive Transfer↗
PubMed2005

Cytokine reporter mouse system for screening novel IL12/23 p40-inducing compounds.

Cytokines interleukin (IL) 12 and 23 play critical roles in linking innate and adaptive immune responses. They are members of heterodimeric cytokines, sharing a subunit p40. Although IL12/23 p40 is mainly induced in macrophages and dendritic cells (DCs) after stimulation with microbial Toll-like receptor ligands, methods to monitor the cells that produce IL12/23 p40 in vivo are limited. Recently, the mouse model to track p40-expressing cells with fluorescent reporter, yellow fluorescent protein, has been developed. Macrophages and DCs from these mice faithfully reported p40 induction using the fluorescent marker. Here we took advantage of these reporter mice to screen bio-compounds for p40-inducing activity. After screening hundreds of compounds, we found several extracts inducing IL12/23 p40 gene expression. Treatment of DCs with these extracts induced the expression of MHC class II and co-stimulatory molecules, which implies that these might be useful as adjuvants. Next, the in vivo target immune cells of candidate compounds were examined. The reporter system can be useful to identify cells producing IL12 or IL23 in vivo as well as in vitro. Thus, our cytokine reporter system proved to be a valuable reagent for screening for immunostimulatory molecules and identification of target cells in vivo.

Adjuvants, Immunologic↗
PubMed2005

Inducible costimulator is required for type 2 antibody isotype switching but not T helper cell type 2 responses in chronic nematode infection.

Inducible costimulator (ICOS) has been suggested to perform an important role in T helper cell type 2 (Th2) responses, germinal center formation, and isotype switching. The role of ICOS in chronic Th2 responses was studied in a nematode model with the filarial parasite, Brugia malayi. Contrary to expectations, we did not observe a significant defect in IL-4-producing Th2 cells in ICOS-/- mice or in eosinophil recruitment. We also found that ICOS was not required for the differentiation of alternatively activated macrophages (AAMPhi) that express Ym1 and Fizz1. Although the production of IgE was slightly reduced in ICOS-/- mice, this was not as significant as in CD28-/- mice. In contrast to live infection, the primary response of ICOS-/- mice immunized with soluble B. malayi antigen and complete Freund's adjuvant resulted in significantly fewer IL-4-producing cells in the lymph nodes. As previously reported, we observed a defect in antibody isotype switching toward the IgG1 isotype in ICOS-/- mice during live infection. Interestingly, there was a significant enhancement of parasite-specific IgG3 isotype antibodies. CD28-/- and MHC class II-/- mice also had enhanced parasite-specific IgG3 isotype antibodies. Our results suggest that ICOS is not required to maintain a chronic cellular Th2 response. The primary role of ICOS in a chronic helminth infection could be to drive antibodies toward type 2 isotypes. T-independent antibody response to the parasite could be enhanced in the absence of costimulation and T cell help.

Animals↗
PubMed2005

Interleukin-4 can be a key positive regulator of inflammatory arthritis.

OBJECTIVE: Development of arthritis in the K/BxN mouse model depends on the induction of high titers of antibodies against the enzyme glucose-6-phosphate isomerase (GPI), promoted by CD4(+) T cells expressing a GPI-specific transgenic T cell receptor (TCR). This study was undertaken to determine whether this strong autoantibody response depends on T cell differentiation to the Th1 or Th2 phenotype. METHODS: The roles of Th cell-biasing cytokines were investigated by introducing the interleukin-4 (IL-4) and IL-12-specific subunit p35 (IL-12p35)-knockout mutations into the K/BxN model and evaluating the impact of these deficiencies on disease. The IL-4-expressing cell types in K/BxN mice were revealed by crossing in a knockin alteration, which resulted in green fluorescent protein expression controlled by endogenous IL-4 gene-regulatory elements. Transfer experiments permitted the identification of the IL-4-producing cell type required for arthritis, and quantitative reverse transcriptase-polymerase chain reaction allowed for determination of the cytokine profile of K/BxN T cells. RESULTS: While IL-12p35 appeared dispensable for the development of arthritis, IL-4 was crucial for full development of disease. The GPI-reactive TCR of standard K/BxN mice induced the transcriptional activation of the IL-4 locus in CD4(+) T cells and eosinophils, and CD4(+) T cells were the obligatory source of IL-4 for disease. However, the cytokine profile of K/BxN T cells revealed that K/BxN arthritis is not a "pure" Th2 disease. CONCLUSION: The K/BxN model, although not a classic Th2 disease, depends critically on IL-4. The potential of IL-4 to promote inflammatory arthritis should be considered when proposing therapies for rheumatoid arthritis aimed at biasing T cells toward IL-4 production.

Animals↗
PubMed2005

A two-step process for cytokine production revealed by IL-4 dual-reporter mice.

To monitor IL-4 expression at the single-cell level, we generated mice with insertions of different reporter genes into both copies of the Il4 gene that permitted the simultaneous analysis of IL-4 transcripts via GFP and IL-4 protein secretion by use of huCD2. Innate and adaptive cells competent for IL-4 production were marked by GFP, while cells that presently or recently secreted IL-4 additionally displayed huCD2. After challenge with the strictly enteric helminth, Heligmosomoides polygyrus, GFP-positive innate and adaptive cells disseminated widely, but IL-4 secretion was predominantly mediated by CD4+ T cells in the intestines and draining lymphoid organs. IL-4-competent cells persisted in cured animals, and memory responses reflected rapid cytokine production at the site of rechallenge. These data reveal a two-step process for cytokine production: the first generating poised cells that disseminate systemically and the second inducing the rapid production of the cytokine in response to local stimulation.

Animals↗
PubMed2004

CD200 receptor family members represent novel DAP12-associated activating receptors on basophils and mast cells.

Modulation by balancing activating and inhibitory receptors constitutes an important mechanism for regulating lymphocyte and myeloid cell effector responses. Using a microarray screen during parasitic helminth infection, we identified CD200 receptor-like 3 as a transcript highly expressed in basophils. Novel splice variants were present that generated proteins that differed in surface expression. The second immunoglobulin-like domain, encoded by exon 4, was required for cell surface expression and recruitment of DAP12 to the cell surface. Splice variants also generated unique cytoplasmic domains, which contributed to efficient pairing with DAP12. Despite expression on basophils and mast cells, which are integral components of allergic immunity, the absence of DAP12 did not alter effector cell recruitment or the host response elicited by helminth infection with Nippostrongylus brasiliensis.

Adaptor Proteins, Signal Transducing↗