TLR signaling controls multiple pathways for tolerating the commensal microbiota.

<p>Crosstalk between the immune system and the microbiota can limit disease in three non-mutually exclusive ways; by limiting the migration of microbes into sensitive host tissues by promoting healthy barrier function (i.e. spatial segregation), by changing the community of organisms that colonize the host (some may be disease protective while others may be disease inductive), and by educating the immune system to be tolerant of innocuous members of the microbial community. (A) Intestinal epithelial cells are the first cells to come into contact with luminal bacteria. Multiple studies have demonstrated that a mechanism to avoid overt inflammation toward the intestinal bacteria is to sequester TLRs toward the basal compartment of the IEC. Therefore, TLRs, and subsequently inflammation, are only engaged when bacteria have penetrated host tissue. This represents a passive mechanism to avoid chronic inflammation. (B) Spatial segregation of the commensal microbiota is another mechanism to avoid inflammation within the intestine. A thick, organized mucus layer that is composed of anti-bacterial proteins, mucins, and antibodies creates a barrier between the host tissue and the luminal bacteria. (C) Several examples exist that demonstrate how TLR signaling can influence the diversity of the microbial community. Additionally, a single commensal species, <i>B. fragilis</i>, has been shown to utilize TLR2 signaling to colonize the host tissue. Therefore, a mutation in any one of the TLRs could lead to a loss of colonization by a beneficial commensal and a change in the structure of the microbial community. Changes in microbial community dynamics could lead to a loss of tolerance within the intestine. (D) It is now becoming appreciated that cells of both the innate and adaptive immune system have functional TLR receptors. TLR signaling on various cell types can have differential functional consequences. Indeed, while triggering of TLR signaling on macrophages has long been known to induce inflammation, it was just recently demonstrated that B cell-intrinsic TLR signaling is important for maintaining tolerance within the intestine. Therefore, TLRs represent a way in which the commensal microbiota can communicate with the host and loss of this machinery could disrupt intestinal tolerance.</p

Anti-correlation functional profiling identifies relevant miRNA-target interactions, including miR-150 repression of p53, that regulate MV4-11 cell line growth.

<p>(A) Heat map indicating representative oncogenes whose loss leads to decreased cell growth according to Log2 Fold Change values from lentiCRISPRv2 library screen (first column), and functionally anti-correlated miRNAs that are predicted to target each oncogene. Grey boxes indicate that the miRNA is not predicted to bind the 3’UTR of the oncogene (NT = Not targeted). (B) Heat map indicating representative TSGs whose loss lead to increased cell growth according to our Log2 Fold Change values from lentiCRISPRv2 library screen (first column), and miRNAs predicted to target each TSG whose growth anti-correlated in library. Grey boxes indicates that the miRNA is not predicted to bind the 3’UTR of TSG (NT = Not targeted). (C) Schematic showing miR-150 targeting of the p53 3’UTR. (D) Schematic of the miR-150 hairpin sequence as annotated in miRBase and sgRNA design of the miR-150-targeting lentiCRISPRv2 construct (150-CR1). (E) Expression level of miR-150 in MV4-11 cells infected with EV control or 150-CR1 lentiCRISPRv2 constructs determined by qPCR. Expression normalized to 5s. (F) Western blot of p53 in p53-CR1, 150-CR1, and EV control infected MV4-11 cell lines with actin serving as load control. (G) Competitive growth curve of EV (GFP+), p53-CR1 (GFP+), or 150-CR1 (GFP+) infected MV4-11 cells mixed ~1:1 with WT MV4-11 cells at time point 0. Y-axis = (%GFP+ cells at indicated time point)/(%GFP+ cells initial). (A, B) Only expressed protein-coding genes and microRNAs with p-values <0.05 were analyzed. Data represented as mean +/- SEM. P-values as indicated: *≤0.05, **≤0.01, ***≤0.001, and ns p>0.05. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153689#pone.0153689.s004" target="_blank">S3 Table</a>.</p

Genome-Wide CRISPR-Cas9 Screen Identifies MicroRNAs That Regulate Myeloid Leukemia Cell Growth

<div><p>Mammalian microRNA expression is dysregulated in human cancer. However, the functional relevance of many microRNAs in the context of tumor biology remains unclear. Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to simultaneously test the functions of individual microRNAs and protein-coding genes during the growth of a myeloid leukemia cell line. This approach identified evolutionarily conserved human microRNAs that suppress or promote cell growth, revealing that microRNAs are extensively integrated into the molecular networks that control tumor cell physiology. miR-155 was identified as a top microRNA candidate promoting cellular fitness, which we confirmed with two distinct miR-155-targeting CRISPR-Cas9 lentiviral constructs. Further, we performed anti-correlation functional profiling to predict relevant microRNA-tumor suppressor gene or microRNA-oncogene interactions in these cells. This analysis identified miR-150 targeting of p53, a connection that was experimentally validated. Taken together, our study describes a powerful genetic approach by which the function of individual microRNAs can be assessed on a global level, and its use will rapidly advance our understanding of how microRNAs contribute to human disease.</p></div

An acute, but not germline or conditional, loss of <i>dlg1</i> impairs receptor-mediated actin polymerization.

<p>(A) <i>dlg1^flox/flox</i> or <i>dlg1^flox/flox:CD4^cre</i> CD4⁺ T cells were allowed to conjugate with anti-TCR antibody-coated beads for 20 minutes and stained for actin with rhodamine-phalloidin (<i>top panel</i>). At least 50 cells were scored for actin localization to the T cell/bead interface (<i>bottom panel</i>). (B) Expanded T cells from <i>dlg1^wt;BG</i> or <i>dlg1^ko;BG</i> mice were restimulated with plate-bound anti-CD3 and anti-CD28 antibodies for 5 or 15 minutes. Cells were stained with a combination of anti-CD4, anti-CD8, and FITC-phalloidin and assessed by FACS to determine the relative level of induced actin polymerization in CD4⁺ (<i>top panel</i>) and CD8⁺ (<i>bottom panel</i>) T cell populations. These data are representative of 2 independent experiments, n≥4 for WT and KO samples. (C) Whole cell lysates from Jurkat cells expressing empty vector (empty) or Dlg1 shRNA were immunoblotted with antibodies against Dlg1 or GAPDH, as indicated. (D) Jurkat T cells (green) were transfected with either pCMS3.eGFP.H1p empty vector or pCMS3.eGFP.H1p containing a shDlg1 target sequence. Cells were stimulated with SEE-pulsed (or untreated) Raji B cells (blue) and stained with rhodamine-phalloidin (red) (<i>left panel</i>). At least 50 conjugates were scored for F-actin localization to the T cell/APC interface in each of two independent experiments (<i>right panel</i>). Data represent mean +/− StDev. n.s., not statistically significant.</p

The regulation of T cell surface activation markers is unaffected in <i>dlg1</i> knockout mice.

<p>(A) Splenocytes isolated from <i>dlg1^wt;GT</i> (black) or <i>dlg1^ko;GT</i> (red) mice were activated with indicated concentrations of plate bound anti-CD3 in the presence of anti-CD28 for 24 hours and assessed for the expression of activation markers. Histograms were gated on viable donor derived (CD45.2⁺) CD4⁺ or CD8⁺ T cells and show one representative mouse per genotype (n≥3). (B) Flow cytometry profiles of selected surface markers on purified CD4⁺ T cells from <i>dlg1^flox/flox</i> (top <i>panel</i>) or <i>dlg1^flox/flox:CD4^cre</i> (<i>bottom panel</i>) mice stimulated with T-depleted splenocytes with (unshaded) or without (shaded) anti-CD3 antibody for 24 hrs (n≥3).</p

T cells from <i>dlg1</i> knockout mice show normal TCR-induced tyrosine- and alternative p38- phosphorylation.

<p>(A) Purified CD4⁺ T cells from <i>dlg1^flox/flox</i> or <i>dlg1^flox/flox:CD4^cre</i> mice were stimulated with anti-CD3 antibody for the indicated times. Whole cell lysates were immunoblotted with anti-phosphotyrosine antibody. (B) <i>(Top panel)</i> Expanded T cells from <i>dlg1^wt;BG</i> or <i>dlg1^ko;BG</i> mice were restimulated with anti-CD3 and anti-CD28 antibodies for 30 minutes in the absence or presence of an Insolution p38 (InS) or U0126 Erk (E) inhibitor. Whole cell lysates were then immunoblotted with anti-phospho-p38 (T180/Y182) followed by anti-p38 to assess loading. <i>(Bottom panel)</i> Levels of induced p38 phosphorylation relative to corresponding unstimulated samples were determined by densitometry and normalized according to loading controls (n = 3 each for <i>dlg1^wt;BG</i> and <i>dlg1^ko;BG</i>). Data represent mean +/− StDev. n.s. = not significant, (p = 0.7236).</p

T cell polarization and migration are not hindered by the loss of <i>dlg1</i>.

<p>(A) <i>dlg1^wt;GT</i> or <i>dlg1^ko;GT</i> activated T lymphoblasts were stained with CD43 or CD44 and assessed for protein polarization (<i>left panel</i>). The percentage polarization of CD43 and CD44 to the T cell uropod was determined by scoring 45 cells in two independent experiments (<i>right panel, top and bottom</i>). (B) <i>dlg1^flox/flox or dlg1^flox/flox:CD4^cre</i> CD4⁺ T cells were allowed to conjugate with anti-TCR antibody coated beads for 20 minutes, fixed and stained with the indicated antibodies. The indicated proteins were scored for localization to the DPC (<i>left panel</i>) or IS (<i>right panel</i>). 50–100 conjugates were scored for F-actin localization to the T cell/APC interface in each of 2–3 independent experiments (<i>right panel</i>). Data represent mean +/− StDev. Differences were not statistically significant for any sample pair. (C) <i>dlg1^wt;GT</i> or <i>dlg1^ko;GT</i> activated T lymphoblasts were subjected to time-lapse microscopy and assessed for random migration (<i>top panels</i>). Total distance was determined from DIC images acquired at 1 min intervals by tracking a total of 30 cells over a 30 min period (<i>bottom panels</i>). Data are representative of n = 2 experiments. (D) A modified Boyden chamber was used to assess the percent of <i>dlg1^flox/flox</i> or <i>dlg1^flox/flox:CD4^cre</i> T cells which migrated in response to no chemokine, CXCL12, or CCL19 over 2 hours and was calculated as the ratio of the total cells, to cells that migrated * = p≤0.05. (E) The percentage of Jurkat cells transfected with either empty vector or shDlg1 which migrated in response to no chemokine or CXCL12 for 2 hours was calculated as: number of cells migrated/total number of cells. n.s. = no significiant difference.</p

Dlg1 expression is ablated in the <i>dlg1^ko;BG</i> and <i>dlg1^flox/flox:CD4^cre</i> mouse models.

<p>(A) Representative flow cytometry histograms show differential β-gal levels in fetal liver cells from BG <i>dlg1^+/+</i> (<i>left</i>), <i>BG dlg1^+/−</i> (<i>center</i>), or BG <i>dlg1^−/−</i> (<i>right</i>) pups (n = 10 independent experiments). (B) Representative western blot of Dlg1 expression in whole cell lysates from BG <i>dlg1^+/+</i>, BG <i>dlg1^+/−,</i> or BG <i>dlg1^−/−</i> fetal tissue using antibodies against Dlg1 or p38 (n = 6 independent experiments). (C) Western blot demonstrating Dlg1 expression in total splenocytes obtained from <i>dlg1^wt;BG</i> and <i>dlg1^ko;BG</i> mice 8 weeks post adoptive transfer. Data are representative of 3 independent experiments (D) <i>Left</i>, lymphocytes from Dlg1^flox/flox or Dlg1^flox/flox:CD4^cre mice were enriched for T cells and whole cell lysates immunoblotted with antibodies against Dlg1. Non-specific bands (Non-spec.) from the same gel demonstrate equivalent loading in each lane. <i>Right</i>, splenocytes from Dlg1^flox/flox:CD4^cre mice were enriched or depleted for T cells, and lysates were immunoblotted with antibodies against Dlg1 or GAPDH, as indicated.</p

The acute or conditional loss of <i>dlg1</i> results in differential Th1/Th2-type cytokine production.

<p>(A) Expanded splenocytes from <i>dlg1^wt;BG</i> or <i>dlg1^ko;BG</i> mice were restimulated with plate-bound anti-CD3 and anti-CD28 antibodies or PMA/Ionomycin for 4 hours. Cells were stained with anti-CD8, permeabilized, and subsequently stained with anti-IFN-γ to determine intracellular cytokine levels (n≥3 each for <i>dlg1^wt;BG</i> and <i>dlg1^ko;BG</i> mice). Data represent mean +/− StDev. n.s. = no significance where p>0.05. (B) Expanded CD4⁺ T cells from <i>dlg1^flox/flox</i> or <i>dlg1^flox/flox:CD4^cre</i> mice were restimulated with plate-bound anti-CD3 and anti-CD28 antibodies for 24 hours. Cell supernatants were collected and cytokine production analyzed by ELISA. Standard deviations were calculated from triplicate stimulations and statistical significance determined using a paired Student’s t test. *p<0.05. (C) Th1 or Th2 polarized T cells from C57Bl/6 mice were transduced with either control- or Dlg1-miRNA retrovirus (<i>right panel</i>) and subsequently stimulated with plate-bound anti-CD3/CD28 for 6 hours. Cells were then surface stained with anti-CD4 followed by intracellular staining with antibodies against either IFNγ or TNFα (Th1 cells) or IL-4 (Th2 cells). Cells were analyzed by FACS and gated on CD4+ and GFP+ (miRNA vector) cells to determine the relative level of cytokine production (MFI) in control and knockdown populations (<i>left panel</i>) (IFNγ, TNFα n = 4; IL-4 n = 3). Data represent means from ≥3 experiments ± SEM. *p<0.05.</p

T cells from <i>dlg1</i> germline knockout mice proliferate comparably to wildtype T cells.

<p>(A) <i>dlg1^wt;BG</i> or <i>dlg1^ko;BG</i> derived splenocytes were stained with CFSE and stimulated with various concentrations of anti-CD3 alone or anti-CD3 and anti-CD28 antibodies for 72 hours. Cells were subsequently surface stained with anti-CD8 or anti-CD4 antibodies and T cell populations analyzed by flow cytometry. (B) Naive splenic T lymphocytes isolated from <i>dlg1^wt;GT</i> (black) or <i>dlg1^ko;GT</i> (red) were labeled with CFSE and activated with the indicated concentrations of plate bound anti-CD3 in the presence or absence of anti-CD28. CFSE profiles at 62 hours were gated on CD8⁺ or CD4⁺ T cells and are representative of 2 independent experiments.</p