HyperCas12a enables highly-multiplexed epigenome editing screens.

Interactions between multiple genes or cis-regulatory elements (CREs) underlie a wide range of biological processes in both health and disease. High-throughput screens using dCas9 fused to epigenome editing domains have allowed researchers to assess the impact of activation or repression of both coding and non-coding genomic regions on a phenotype of interest, but assessment of genetic interactions between those elements has been limited to pairs. Here, we combine a hyper-efficient version of Lachnospiraceae bacterium dCas12a (dHyperLbCas12a) with RNA Polymerase II expression of long CRISPR RNA (crRNA) arrays to enable efficient highly-multiplexed epigenome editing. We demonstrate that this system is compatible with several activation and repression domains, including the P300 histone acetyltransferase domain and SIN3A interacting domain (SID). We also show that the dCas12a platform can perform simultaneous activation and repression using a single crRNA array via co-expression of multiple dCas12a orthologues. Lastly, demonstrate that the dCas12a system is highly effective for high-throughput screens. We use dHyperLbCas12a-KRAB and a ~19,000-member barcoded library of crRNA arrays containing six crRNAs each to dissect the independent and combinatorial contributions of CREs to the dose-dependent control of gene expression at a glucocorticoid-responsive locus. The tools and methods introduced here create new possibilities for highly multiplexed control of gene expression in a wide variety of biological systems.

Lean mass predicts asthma better than fat mass among females.

The obesity phenotype associated with asthma is not known. Our objective was to define the relative contribution of various distributions of fat and lean mass to asthma prevalence. Data were obtained from 2,525 participants (including 1,422 females) who underwent dual-energy X-ray absorptiometry (DEXA) at the year 20 examination in the Coronary Artery Risk Development in Young Adults (CARDIA) cohort. Total, truncal, arm and leg distributions of fat and lean mass were adjusted to the person's height. Self-reported asthma was the outcome. Asthma among females was associated with greater total fat mass, arm fat mass, total lean mass, truncal lean mass and arm lean mass. Among males, none of these mass measures were significantly associated with asthma. Among females, the association with asthma was stronger for total lean mass than for total fat mass. Further, among various regional distributions of lean and fat mass in females, truncal lean mass was the strongest predictor. Total lean mass is more strongly associated with asthma than total fat mass among females. These findings are contrary to the popular perception that excess physiological fat drives the obesity-asthma association. Rather, we hypothesise that ectopic fat within the "lean" tissues drives this association among females.

The role of MIP-1alpha in experimental hypersensitivity pneumonitis.

S. rectivirgula (SR) causes Farmer's Lung Disease, a classic example of hypersensitivity pneumonitis (HP). We utilized a model of experimental hypersensitivity pneumonitis (EHP), antibody to MIP-1alpha and MIP-1alpha -/- mice, to test the hypothesis that MIP-1alpha is essential in the development of EHP. Treatment of C57BI/6 mice with anti-MIP-1alpha antibody did not change the extent of pulmonary histology abnormalities, BALF cell number or characteristics, or BALF concentration of IL12p40, TNF, IL1alpha and IL6, after an i.t. challenge with SR. MIP-1alpha -/- animals responded similarly to wild-type (wt) animals in the extent and nature of pulmonary histologic changes and BALF cell number and type after a single i.t. injection of SR There was a dose-response relationship between the amount of SR and BALF IL12p40, MCP-1 and IL6 in both strains, and MIP-1alpha in wild-type animals. We next transferred SR cultured spleen cells from SR sensitized mice (both wt and MIP-1alpha -/-) to naive recipients. Lung histology and BALF characteristics after SR i.t. challenge of the recipients were used to determine if adoptive transfer had occurred. Cultured cells from MIP-1alpha -/- animals were fully capable of transferring EHP to recipients. There was no difference of BALF TNF, IL6 and IL1alpha between the strains, but there was more MCP-1 and IL12p40 in the MIP-1alpha -/- mice than in the control mice. MIP-1alpha is not necessary for the recruitment of cells into the lung and BALF after i.t. administration of SR, or the development of cells able to adoptively transfer EHP.

Eosinophil traffic in the circulation following allergen challenge.

BACKGROUND: Eosinophils contribute to the pathogenesis of asthma and localize to the lung after allergen exposure by uncertain mechanisms. METHODS: We used intrabronchial instillation of allergen to model the interaction between inhaled allergen and the lung. We measured the number of peripheral blood leukocytes and the expression of VLA-4 (CD49d), Mac-1 (CD11b) and PSGL-1 (CD162) up to 4 h after instillation of allergen into a bronchus of eight atopic asthmatics. For controls, we instilled normal saline into a subset of the asthmatic subjects, and allergen into nonatopic, nonasthmatic subjects. RESULTS: There were changes of total leukocyte number, number of polymorphonuclear leukocytes, lymphocytes, monocytes and eosinophils in all three groups (atopic asthmatics instilled with allergen, atopic asthmatics instilled with saline, nonatopic nonasthmatic subjects instilled with allergen), which were likely related to bronchoscopy. However, the decrease of eosinophils was significant only in the atopic asthmatics instilled with allergen. The remaining eosinophils in the allergen challenged asthmatics were not activated as defined by cell density or change of expression of VLA-4, Mac-1 and PSGL-1. CONCLUSIONS: While eosinophils rapidly and specifically leave the circulation after allergen challenge of atopic asthmatics, the remaining circulating eosinophils are not activated.

Mediators of hypersensitivity pneumonitis.

Inhalation of Saccharopolyspora rectivirgula (S. rectivirgula) causes farmer's lung disease, a classic example of hypersensitivity pneumonitis (HP). HP is characterized by bronchoalveolar lavage fluid (BALF) neutrophilia (within the first 48 hours after inhalation), followed by BALF lymphocytosis. We utilized a well-described murine model of HP to determine the timing of the appearance of the C-C chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha); the inflammatory cytokines tumor necrosis factor (TNF), interleukin-1alpha (IL-1alpha), and interleukin-6 (IL-6); and the Th1 -differentiating cytokine interleukin-12 (IL-12) in BALF. After a single intratracheal administration of S. rectivirgula, there was remarkable BALF neutrophilia (peak 24 to 48 hours), followed by a BALF lymphocytosis (peak 48 to 72 hours) in both C57Bl/6 and BALB/c mice that was preceded by the appearance of MIP-1alpha in BALF (peak 4 to 6 hours) and MCP-1 (peak at 48 hours). In both strains of mice there was a striking increase of BALF IL-12 (peak 48 to 72 hours). There was also an increase in BALF IL-6, IL-1alpha, and TNF that was greater in the BALB/c mice than in the C57Bl/6 mice. S. rectivirgula induced the secretion of MIP-1alpha, MCP-1, IL-6, IL-1alpha, and IL-12 from the murine macrophage cell line J774A.1; MIP-1alpha, IL-6, IL-1alpha, IL-12, and TNF from C57Bl/6 alveolar macrophages; and IL-1alpha, IL-6, and TNF-but not IL-12-from BALB/c alveolar macrophages. We conclude that chemokines and cytokines induced by intratracheal administration of S. rectivirgula precede BALF neutrophilia and lymphocytosis and may cause differentiation of Th1 cells; we also conclude that pulmonary macrophages represent a potential source of these substances.

Th1 cells that adoptively transfer experimental hypersensitivity pneumonitis are activated memory cells.

Cultured murine CD4+ T cell lines from Saccharopolyspora rectivirgula-sensitized donors with cytokine secretion characteristics of Th1 cells can adoptively transfer murine experimental hypersensitivity pneumonitis (EHP), whereas Th2 CD4+ cell lines cannot (Cell Immunol 177:169-175, 1997). To assess the differences between these cell lines that may be related to the ability to transfer EHP, we determined cell surface markers that distinguish naive from activated/memory cells that indicate activation and that mediate endothelial adhesion. Both Th1 and Th2 T cell lines are CD4+, CD11a+, ICAM-1+, and L-selectin negative. Th1 cells are CD49d (alpha 4) and LPAM (alpha 4 beta 7) positive, with 32% and 42% of the apparent membrane site density quantitated as the mean molecules of equivalent soluble fluorochromes (MESF) values of unstimulated spleen cells, respectively. Th2 cells are weakly alpha 4 and alpha 4 beta 7 positive, with 15% and 11% of the MESF of unstimulated spleen cells. Th1 cell lines are CD45Rb negative and CD44+, whereas Th2 cell lines are CD45Rb intermediate and CD44-/low. Th1 cells are CD25 (IL-2 receptor) low and Th2 cells CD25 high. We conclude that Th1 cells capable of transfer are activated/memory T cells, and Th2 cells incapable of transfer lack some characteristics of memory/activated T cells (i.e., increase of CD44 and decrease of CD45Rb). Both Th1 and Th2 cell lines express alpha 4 beta 7 and alpha 4 (Th1 > Th2), suggesting that alpha(4) integrin may be important in conferring ability to cells to adoptively transfer EHP.

Experimental hypersensitivity pneumonitis: influence of Th2 bias.

Cultured murine CD4+ cells from Saccharopolyspora rectivirgula sensitized C3H/HeJ (Th1 bias) donors can adoptively transfer murine experimental hypersensitivity pneumonitis (EHP). We sensitized BALB/c mice (Th2 bias) with S. rectivirgula, obtained spleen and lung associated lymph node (LALN) cells, cultured the cells with specific antigen, and attempted adoptive transfer of EHP. We also treated both C3H/HeJ and BALB/c donor mice with IL4 and anti-IFNgamma before exposure to S. rectivirgula and then cultured cells from both spleen and LALN before attempted transfer of EHP. We found that cultured spleen and lung associated lymph node cells can adoptively transfer EHP in both C3H/HeJ and BALB/c mice as demonstrated by infiltration of the recipient lungs with CD4+ lymphocytes. Treatment of both mouse strains with IL4 and anti-IFNgamma did not change the ability of cultured cells to adoptively transfer EHP. We conclude that EHP induced by S. rectivirgula can occur in animals with either a Th1 or a Th2 bias and is not altered by treatment with IL4 and anti-IFNgamma. This suggests that attributes of the antigen and not genetic background or cytokine environment at the site of initial sensitization determines the results of exposure to S. rectivirgula.

Experimental hypersensitivity pneumonitis: location of transferring cells.

Cultured cells from Micropolyspora faeni-sensitized donors can adoptively transfer murine experimental hypersensitivity pneumonitis (EHP). We sought to determine the location of transferred cells in recipient animals, the influence of the origin of the cultured cells, and the effect of specific intratracheal challenge. We labeled cultured sensitized spleen or lung-associated lymph node (LALN) cells with CFDA-SE, a cytoplasmic stain, before transfer to naive recipients, which were sacrificed 1 h, 1 day, or 4 days thereafter. We also transferred labeled cultured spleen cells to recipients that were challenged with intratracheal M. faeni and sacrificed 4 days later (MF). Controls were recipients of M. faeni-sensitized and cultured cells challenged with intratracheal normal saline (NS) and recipients of ovalbumin (OVA)-sensitized cells cultured with M. faeni and challenged with intratracheal M. faeni (OVA). The number and proportion of cells that were stained were determined in dispersed spleen, peripheral and lung-associated lymph nodes, and lung parenchyma. The extent of the pulmonary inflammatory response was measured by determining the proportion of microscopic fields that were abnormal and the total number of dispersed pulmonary cells. CFDA-SE stained cells uniformly, and stained cells could be detected in recipients for up to 7 days after transfer. CFDA-SE treatment (0.5 microM) did not affect the ability of cells to transfer EHP adoptively. Transferred cells could be detected easily in lung, lung-associated and peripheral lymph nodes, blood, and spleen. Transferred cells localized to the lung at 1 h but then rapidly decreased with no difference between labeled cells from spleen and LALN. After intratracheal M. faeni challenge, there was no difference in the proportion of labeled cells in the lung among any of the groups (MF, NS, or OVA). There was an increase in the number of lung cells in the MF group compared with the control (NS and OVA) groups. We conclude that cells capable of transfer are transiently (1 h) trapped in the lung but are much decreased in the lung by four days after transfer. After intratracheal antigen challenge of recipients, there is a substantial increase in the number of pulmonary cells in animals exhibiting adoptive EHP but not in the control groups. Transferred cells responsible for EHP are increased in the lungs of animals with adoptive EHP.

Effect of glucan on murine lungs.

Glucan, a folded high-molecular-weight polysaccharide, has multiple effects in animals when administered intravenously or intraperitoneally, but not when administered by inhalation. The hypotheses tested were whether intratracheal administration of glucan can cause lung damage and whether some of the resulting lung injury is immunologically mediated. There was a dose-response relationship between the amount of intratracheally injected glucan and the extent of pulmonary histologic abnormalities, which consisted of peribronchiolar and intraalveolar infiltration with chronic inflammatory cells. An attempt to adoptively transfer increased susceptibility to glucan induced lung injury was made. Cells cultured with glucan were transferred into naive recipients before intratracheal glucan exposure. The extent of pulmonary inflammation that occurred as a result of intratracheal injection of glucan was not affected by transfer of cultured cells from glucan-treated animals. However, high concentrations of glucan in culture did produce cells with the appearance of lymphoblasts. These data indicate that glucan induces lung injury, but that there is no evidence of cell mediation of pulmonary injury induced by intratracheal exposure to glucan.

Modulation of T-helper cell populations: potential mechanisms of respiratory hypersensitivity and immune suppression.

Information presented at this symposium indicates that modulation of Th cell responses is one means by which xenobiotics may cause immunotoxicity. A shift from Th1 to Th2 responses can enhance both infectious and allergic disease. Hence, in some cases, a common mechanism may be responsible for effects that are generally considered to be very different. Because cytokines produced in the inflammatory process play a role in modulation of Th cell responses, there is a mechanism by which agents that appear to have only local effects at the portal of entry may, in fact, affect immune responses systemically. An understanding of conditions which trigger certain cytokine responses may be useful not only in understanding inflammation but also in predicting certain kinds of immunosuppressive and allergic responses. Future studies in this area are likely to provide insights into many areas of immunotoxicology.

Immunoglobulin response to intrapulmonary immunization of asthmatics.

Atopic asthmatics, compared to non-atopic individuals, exhibit an increased amount of serum antigen-specific IgE and IgG4 antibody directed toward many aeroallergens. We tested the hypothesis that this difference between atopics and non-atopics extends to the response to intrapulmonary deposition of a neoantigen, keyhole limpets haemocyanin (KLH). We immunized nine atopic asthmatics and nine non-atopic controls with 500 micrograms KLH instilled into a subsegment of the lingula and examined serum anti-KLH, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and IgM and specific antibody production by peripheral blood mononuclear cells for 25 days. We also determined specific antibody in bronchoalveolar lavage fluid (BALF) in both the immunized and a non-immunized lobe 11 days after immunization. We found specific serum antibody in all immunized subjects with no difference between atopics and normals in the amount or kinetics of anti-KLH IgG1, IgG2, IgG3, IgA1, IgA2 and IgM. However, the atopics exhibited more anti-KLH IgG4 than the normal controls. Specific anti-KLH antibody-producing cells were detected in peripheral blood in most subjects at day 8 to 12 after immunization with no difference between atopics and normals. Specific IgA1, IgA2, IgG1 and IgM antibodies were detected in BALF from the immunized lobes but not from the non-immunized lobes of both groups of subjects with no difference between atopics and normals. We conclude that atopic asthmatics respond to intrapulmonary KLH with more serum anti-KLH IgG4 than normal controls, consistent with a bias toward a Th2 response to intrapulmonary exposure to antigen.

Th1 CD4+ cells adoptively transfer experimental hypersensitivity pneumonitis.

Cultured cells from Micropolyspora faeni-sensitized donors can adoptively transfer murine experimental hypersensitivity pneumonitis (EHP). To determine whether the CD4+ cells responsible for transfer have characteristics of Th1 or Th2 cells, we established cell lines from lung-associated lymph nodes of M. faeni-sensitized C3H/HeJ mice by culturing with antigen and either IFN-gamma, IL2, and anti-IL4, or IL4. Cell lines were stimulated regularly with antigen, fresh antigen-presenting cells, and the cytokine/anti-cytokine antibody cocktail. At various times after initiation of culture, cells were injected intravenously into recipients, which were then challenged intratracheally with M. faeni and sacrificed and the extent of pulmonary inflammatory response was determined. IFN-gamma, IL4, and IL10 levels were determined in supernatants of cell cultures stimulated with M. faeni to characterize the cell lines as Th1 (IFN-gamma, but low IL4 and IL10 secretion) or Th2 (IL4 and IL10, but low IFN-gamma secretion). Cell lines were differentiated into either Th1 (IFN-gamma = 310 +/- 45 U/ml, IL4 = 0.10 +/- 0.1 U/ml, IL10 = 1750 +/- 75 pg/ ml, >99% CD4+) cell lines by Day 16 of culture or Th2 cell lines (IFN-gamma = 1.8 +/- 1.0 U/ml, IL4 = 830 +/- 388 U/ml, IL10 = 51,700 +/- 10,900 pg/ml, >96% CD4+) by Day 30. Th1 cell lines were able to adoptively transfer EHP whereas Th2 cell lines were unable to adoptively transfer EHP. The ability to transfer EHP was directly related to the amount of IFN-gamma and inversely to the amount of IL4 secreted by antigen-stimulated cells. We conclude that it is possible to produce CD4+ cell lines with either Th1 or Th2 characteristics from lung-associated lymph nodes of mice exposed to M. faeni and that only Th1 CD4+ cell lines can adoptively transfer EHP.

Experimental hypersensitivity pneumonitis: cellular requirements.

We previously demonstrated that Thy1.2+, CD4+, Ia-T cells are responsible for transfer of murine adoptive experimental hypersensitivity pneumonitis (adoptive EHP). To characterize the culture conditions necessary for development of these cells, we depleted cell cultures of Thy1.2+, CD4+, CD8+, or Ia+ cells using MoAbs and complement or magnetic beads, prior to culture of sensitized C3H/HeJ murine spleen cells (SC) with Micropolyspora faeni. After culture, cells were transferred to recipients which were later challenged intratracheally with M. faeni. The extent of pulmonary inflammatory changes in these animals was determined 4 days after intratracheal (i.t.) challenge with M. faeni. Cultured M. faeni-sensitized SC which had been treated before culture with media, complement only, anti-CD8 plus complement or magnetic beads alone could transfer EHP to naive animals. SC treated with anti-Thy1.2 or anti-CD4 plus complement could not transfer EHP. Treatment of SC with anti-Iak plus magnetic beads diminished the ability of cultured cells to transfer EHP. We conclude that the ability to produce cells able to adoptively transfer EPH is dependent on the presence of Thy1.2+, CD4+, and Ia+ cells, but not CD8+ cells, at the onset of culture.

The law of workplace violence.

In addressing law related to workplace violence and employers' obligations, the authors analyze the quagmire of the often conflicting rights of the victim, the perpetrator, and third parties. The close attention that courts are now giving to workplace violence and the scores of legislative efforts to deal with it are assessed.

Experimental hypersensitivity pneumonitis: in vitro effects of interleukin-2 and interferon-gamma.

Cultured CD4+ cells are responsible for transfer of adoptive murine experimental hypersensitivity pneumonitis (EHP) (ARRD 1992; 146:1582-8). To characterize interactions that occur in vitro that result in cells able to transfer EHP, we added either antibody to IFN-gamma, antibody to IL-2, or 30 or 300 micrograms/ml IFN-gamma at the onset of 72-hour culture of C3H/HeJ spleen cells from either M. faeni or ovalbumin (control) sensitized donors with 30 micrograms/ml Micropolyspora faeni. We determined the phenotype of cells after culture and the amount of IL-2 or IFN-gamma in the culture supernatants, transferred cells to naive recipients, challenged the recipients intratracheally with M. faeni, and determined the extent of pulmonary inflammatory changes 4 days thereafter. Substantial amounts of IL-2 and IFN-gamma were detected in supernatants of cultures from M. faeni-sensitized animals, and lesser amounts were detected in culture supernatants from ovalbumin-sensitized donors. Treatment of cultures of M. faeni-sensitized cells with antibody to IL-2 or IFN-gamma blocked or reduced measurable IL-2 or IFN-gamma for the duration of culture. Treatment with IFN-gamma blocked increased levels of IL-2 at 48 and 72 hours of culture. Cultured M. faeni-sensitized cells adoptively transfer EHP. Cells from cultures depleted of either IL-2 or IFN-gamma or supplemented with IFN-gamma could transfer EHP equally well. We conclude that in vitro maturation of cells capable of adoptive EHP is not dependent on soluble IL-2 or IFN-gamma and is not altered by exogenous IFN-gamma.

Experimental hypersensitivity pneumonitis: effect of Thy1.2+ and CD8+ cell depletion.

We previously demonstrated that recipient CD4+ cells are necessary for expression of adoptive murine experimental hypersensitivity pneumonitis (EHP). In contrast, the acute inflammatory response to intratracheal (i.t.) administration of Micropolyspora faeni (direct EHP) is not CD4+ cell dependent (Am. J. Respir. Crit. Care Med. 1994;149:1286-1294). To further characterize the cells responsible for development of pulmonary inflammation in recipient animals, we depleted recipients of either Thy1.2+ or CD8+ cells before transfer of M. faeni-sensitized cultured C3H/HeJ spleen cells and i.t. challenge with M. faeni. We used the same depletion technique to determine the contribution of these cells to the pulmonary response to i.t. M. faeni in animals that did not receive cultured cells (direct EHP). The nature and extent of pulmonary inflammatory changes in these animals were assayed either 4 d after i.t. challenge with M. faeni in adoptive EHP or 2 d after i.t. challenge with M. faeni in direct EHP. We also tested the hypothesis that our previously demonstrated ablation of adoptive EHP caused by administration of anti-CD4 antibody was due to depletion of recipient CD4+ cells by allowing recovery of recipient CD4+ cells of anti-CD4-treated animals before i.t. challenge. In addition, we allowed Thy1.2+ cell recovery of anti-Thy1.2-treated animals before i.t. challenge. Cultured M. faeni-sensitized spleen cells could adoptively transfer EHP to animals treated with an irrelevant antibody or PBS. Depletion of Thy1.2+ but not CD8+ cells ablated the ability of recipient animals to express adoptive EHP. Direct EHP was not affected by depletion of Thy1.2+ or CD8+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The regulation of pulmonary immunity.

No evidence has emerged which suggests that the principles of immunity derived from studies on cells from other body sites are contradicted in the lung and its associated lymphoid tissue. What is clear, however, is that the environment dictates the types of cells, their relationship to one another, and what perturbing events will set in motion either the development of an "active" immune response or tolerance. Investigating mechanisms for the development of lung immunity has increased our understanding of how human diseases develop and is continuing to suggest new ways to manipulate pulmonary immune responses. Demonstration that lung cells regulate both nonspecific inflammation and immunity through the expression of adhesion molecules and the secretion of cytokines offers hope for ways to design more effective vaccines, enhance microbial clearance in immunosuppressed hosts, and to suppress manifestations of immunologically mediated lung disease. Important lung diseases targeted for intensive research efforts in the immediate future are tuberculosis, asthma, and fibrotic lung disease. Perhaps even the common cold might be conquered. Considering the pace of current research on lung immunity, it may not be too ambitious to predict that these diseases may be conquered in the next decade.