TLR2/1L and IFN-γ inducible S100A12 protein expression.

<p>Intracellular S100A12 protein expression following stimulation of MDMs with TLR2/1L or IFN-γ for 24, 48, and 72 hours. <b>(A)</b> Representative histogram at 48 hours from flow cytometry showing percent of S100A12 positive cell population (white) with respect to isotype control (gray) in media, TLR2/1L and IFN-γ treated MDMs. Change in percent S100A12 positive cells in <b>(B)</b> TLR2/1L (<i>n</i> = 9) and <b>(C)</b> IFN-γ (<i>n</i> = 5) stimulated conditions compared to media control at each time point. Data are represented as mean ± SEM, *<i>P</i><i>≤</i>0.05, **<i>P</i><i>≤</i>0.01.</p

Antimicrobial activity of S100A12 against mycobacteria.

<p><b>(A)</b> A plot of (N_K/(N_K + N_R)) vs. mean hydrophobicity for all known α-helical antimicrobial peptides in the antimicrobial peptide database (<a href="http://aps.unmc.edu/AP/main.php" target="_blank">http://aps.unmc.edu/AP/main.php</a>). The black circles represent peptides that meet amino acid criterion for membrane curvature generation and exhibit membrane permeating antimicrobial activity. N_K and N_R are numbers of lysines (K) and arginines (R) on the peptide or protein. Cathelicidin (LL-37) is represented by the blue diamond and S100A12 by the red diamond. <b>(B)</b> <i>M</i>. <i>tuberculosis</i> H37Ra was incubated in the presence of S100A12 protein at the indicated concentrations for 3 days (n = 4). Rifampicin was used as a positive control (20 mg/ml) and IFN-γ protein was used as a negative control. Bacteria were then quantified by CFU assay (n = 4). Each dot represents a replicate sample for each condition indicated (x-axis). <b>(C)</b> <i>M</i>. <i>tuberculosis</i> H37Rv was cultured in the presence of S100A12 protein (0.1μM) or media control for 3 days. Arbitrary units (AU) as determined by the ratio of RNA (16S) to DNA (IS6110) as a measure of viability are shown for H37Rv in media versus S100A12 (n = 3). <b>(D)</b> Data from B is represented in % viability relative to media control for in addition to rifampicin control (n = 3). <b>(E)</b> <i>M</i>. <i>leprae</i> was maintained in 7H9 media with S100A12. Shown in AU is the ratio of <i>M</i>. <i>leprae</i> RNA (16S) to DNA (RLEP) in media control versus S100A12 (0.1μM). Each dot represents a replicate sample for each condition, with a line connecting the AU of each replicate (n = 6). Since there is a large variation in absolute values and ratios, the P-value was determined based on log-transformed values, not log transformed ratios are shown here. <b>(F)</b> Data from D is represented as mean % viability, which was calculated with AU relative to media control. In addition, two other concentrations of S100A12 (1μM, n = 7 and 10μM, n = 3), positive control rifampicin (n = 9), and negative control protein (n = 7) are included. Data are represented as mean % viability ± SEM. *<i>P</i><i>≤</i>0.05, **<i>P</i><i>≤</i>0.01, ***<i>P</i><i>≤</i>0.001; ns, non-significant.</p

Expression of S100A12 in skin lesions from leprosy patients.

<p><b>(A)</b> S100A12 labeling of leprosy skin biopsy specimens by immunoperoxidase. Two representative images are shown for each condition: T-lep, RR, and L-lep, as well as an isotype control for each representative staining. Original magnification: x20. <b>(B)</b> The percentage of diaminobenzidine (DAB)-stained area per nuclear area was calculated for each photomicrograph using ImmunoRatio [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005705#ppat.1005705.ref030" target="_blank">30</a>]. Each dot represents the %DAB-stained area/nuclear area for each individual photomicrograph. Horizontal dotted line represents mean %DAB per nuclear area for n = 5. One-Way ANOVA analysis was performed (<i>P</i> = 0.006) using GraphPad Prism software and post hoc analysis (Turkey test) is indicated by the asterisk (*<i>P</i><0.05 and ** <i>P</i><0.01). <b>(C)</b> Co-expression of S100A12 (red) and CD209 (green) in skin lesion from RR patient. Nucleus was stained with DAPI (blue). Arrows indicate areas of co-expression of S100A12 and CD209; scale bar = 10μm. Original magnification: x40.</p

RNAseq gene expression is validated by qPCR.

<p>MDMs were stimulated with TLR2/1L (black bars) or IFN-γ (gray bars) for 2, 6, and 24 hours and <b>(A)</b> S100A12, <b>(B)</b> GBP1, and <b>(C)</b> IL15 expression is assessed by RNAseq (left) and quantitative PCR (qPCR) (right). RNAseq results are represented as FC determined by normalized counts in stimulated MDMs versus counts in media control for each time point. Quantitative PCR (qPCR) results were determined by calculating relative arbitrary units using ΔΔCT analysis and normalizing to housekeeping gene. Data are represented as mean FC ± SEM, <i>n</i> = 5.</p

S100A12 plays a role in macrophage antimicrobial activity against <i>M</i>. <i>leprae</i>.

<p><b>(A)</b> MDMs were transfected with siRNA targeting S100A12 (siS100A12) or non-targeting siRNA (siCtrl). qPCR results displaying arbitrary unit (AU) of S100A12 mRNA 24 hours after stimulation with TLR2/1L (<i>n</i> = 9) or IFN-γ (<i>n</i> = 5). <b>(B)</b> MDMs were infected with <i>M</i>. <i>leprae</i> and stimulated with TLR2/1L (<i>n</i> = 9) and IFN-γ (<i>n</i> = 8). <i>M</i>. <i>leprae</i> viability was determined by measuring RNA (16S) and DNA element (RLEP) and determining the % viability relative to respective media control. Data are represented as mean % viability ± SEM, *<i>P</i><i>≤</i>0.05, **<i>P</i><i>≤</i>0.01, ***<i>P</i><i>≤</i>0.001.</p

Analysis of TLR2/1L and IFN-γ inducible genes identifies common pathways.

<p><b>(A)</b> TLR2/1L and IFN-γ inducible genes defined as fold-change (FC) greater than 2 and <i>P</i>-value less than 0.01. Overlap indicates number of genes shared by both stimuli. <b>(B)</b> TLR2/1L versus IFN-γ log₂(FC) of max value for antimicrobial response genes. Genes highlighted in light gray represent TLR2/1L only inducible genes, genes in dark gray represent IFN-γ only inducible genes, and genes in blue represent shared TLR2/1L and IFN-γ inducible genes. <b>(C)</b> Functional annotation network of robust TLR2/1L and IFN-γ inducible genes (FC>25 and <i>P</i>-value<0.01). Simplified GO terms are in red, genes are in blue. Size of gene node represents <i>P</i>-value associated with max FC; largest node has smallest <i>P</i>-value in dataset. <b>(D)</b> Distribution of max or min log₂(FC) values versus associated–log₁₀(<i>P</i>-value) of all genes in dataset. Genes of interest are indicated in blue.</p

DataSheet_2_Regulator of G-protein signaling 1 critically supports CD8+ TRM cell-mediated intestinal immunity.docx

Members of the Regulator of G-protein signaling (Rgs) family regulate the extent and timing of G protein signaling by increasing the GTPase activity of Gα protein subunits. The Rgs family member Rgs1 is one of the most up-regulated genes in tissue-resident memory (TRM) T cells when compared to their circulating T cell counterparts. Functionally, Rgs1 preferentially deactivates Gαq, and Gαi protein subunits and can therefore also attenuate chemokine receptor-mediated immune cell trafficking. The impact of Rgs1 expression on tissue-resident T cell generation, their maintenance, and the immunosurveillance of barrier tissues, however, is only incompletely understood. Here we report that Rgs1 expression is readily induced in naïve OT-I T cells in vivo following intestinal infection with Listeria monocytogenes-OVA. In bone marrow chimeras, Rgs1-/- and Rgs1+/+ T cells were generally present in comparable frequencies in distinct T cell subsets of the intestinal mucosa, mesenteric lymph nodes, and spleen. After intestinal infection with Listeria monocytogenes-OVA, however, OT-I Rgs1+/+ T cells outnumbered the co-transferred OT-I Rgs1-/- T cells in the small intestinal mucosa already early after infection. The underrepresentation of the OT-I Rgs1-/- T cells persisted to become even more pronounced during the memory phase (d30 post-infection). Remarkably, upon intestinal reinfection, mice with intestinal OT-I Rgs1+/+ TRM cells were able to prevent the systemic dissemination of the pathogen more efficiently than those with OT-I Rgs1-/- TRM cells. While the underlying mechanisms are not fully elucidated yet, these data thus identify Rgs1 as a critical regulator for the generation and maintenance of tissue-resident CD8+ T cells as a prerequisite for efficient local immunosurveillance in barrier tissues in case of reinfections with potential pathogens.</p

DataSheet_1_Regulator of G-protein signaling 1 critically supports CD8+ TRM cell-mediated intestinal immunity.pdf

Members of the Regulator of G-protein signaling (Rgs) family regulate the extent and timing of G protein signaling by increasing the GTPase activity of Gα protein subunits. The Rgs family member Rgs1 is one of the most up-regulated genes in tissue-resident memory (TRM) T cells when compared to their circulating T cell counterparts. Functionally, Rgs1 preferentially deactivates Gαq, and Gαi protein subunits and can therefore also attenuate chemokine receptor-mediated immune cell trafficking. The impact of Rgs1 expression on tissue-resident T cell generation, their maintenance, and the immunosurveillance of barrier tissues, however, is only incompletely understood. Here we report that Rgs1 expression is readily induced in naïve OT-I T cells in vivo following intestinal infection with Listeria monocytogenes-OVA. In bone marrow chimeras, Rgs1-/- and Rgs1+/+ T cells were generally present in comparable frequencies in distinct T cell subsets of the intestinal mucosa, mesenteric lymph nodes, and spleen. After intestinal infection with Listeria monocytogenes-OVA, however, OT-I Rgs1+/+ T cells outnumbered the co-transferred OT-I Rgs1-/- T cells in the small intestinal mucosa already early after infection. The underrepresentation of the OT-I Rgs1-/- T cells persisted to become even more pronounced during the memory phase (d30 post-infection). Remarkably, upon intestinal reinfection, mice with intestinal OT-I Rgs1+/+ TRM cells were able to prevent the systemic dissemination of the pathogen more efficiently than those with OT-I Rgs1-/- TRM cells. While the underlying mechanisms are not fully elucidated yet, these data thus identify Rgs1 as a critical regulator for the generation and maintenance of tissue-resident CD8+ T cells as a prerequisite for efficient local immunosurveillance in barrier tissues in case of reinfections with potential pathogens.</p

Video_2_Regulator of G-protein signaling 1 critically supports CD8+ TRM cell-mediated intestinal immunity.mp4

Members of the Regulator of G-protein signaling (Rgs) family regulate the extent and timing of G protein signaling by increasing the GTPase activity of Gα protein subunits. The Rgs family member Rgs1 is one of the most up-regulated genes in tissue-resident memory (TRM) T cells when compared to their circulating T cell counterparts. Functionally, Rgs1 preferentially deactivates Gαq, and Gαi protein subunits and can therefore also attenuate chemokine receptor-mediated immune cell trafficking. The impact of Rgs1 expression on tissue-resident T cell generation, their maintenance, and the immunosurveillance of barrier tissues, however, is only incompletely understood. Here we report that Rgs1 expression is readily induced in naïve OT-I T cells in vivo following intestinal infection with Listeria monocytogenes-OVA. In bone marrow chimeras, Rgs1-/- and Rgs1+/+ T cells were generally present in comparable frequencies in distinct T cell subsets of the intestinal mucosa, mesenteric lymph nodes, and spleen. After intestinal infection with Listeria monocytogenes-OVA, however, OT-I Rgs1+/+ T cells outnumbered the co-transferred OT-I Rgs1-/- T cells in the small intestinal mucosa already early after infection. The underrepresentation of the OT-I Rgs1-/- T cells persisted to become even more pronounced during the memory phase (d30 post-infection). Remarkably, upon intestinal reinfection, mice with intestinal OT-I Rgs1+/+ TRM cells were able to prevent the systemic dissemination of the pathogen more efficiently than those with OT-I Rgs1-/- TRM cells. While the underlying mechanisms are not fully elucidated yet, these data thus identify Rgs1 as a critical regulator for the generation and maintenance of tissue-resident CD8+ T cells as a prerequisite for efficient local immunosurveillance in barrier tissues in case of reinfections with potential pathogens.</p

DataSheet_4_Regulator of G-protein signaling 1 critically supports CD8+ TRM cell-mediated intestinal immunity.zip

Members of the Regulator of G-protein signaling (Rgs) family regulate the extent and timing of G protein signaling by increasing the GTPase activity of Gα protein subunits. The Rgs family member Rgs1 is one of the most up-regulated genes in tissue-resident memory (TRM) T cells when compared to their circulating T cell counterparts. Functionally, Rgs1 preferentially deactivates Gαq, and Gαi protein subunits and can therefore also attenuate chemokine receptor-mediated immune cell trafficking. The impact of Rgs1 expression on tissue-resident T cell generation, their maintenance, and the immunosurveillance of barrier tissues, however, is only incompletely understood. Here we report that Rgs1 expression is readily induced in naïve OT-I T cells in vivo following intestinal infection with Listeria monocytogenes-OVA. In bone marrow chimeras, Rgs1-/- and Rgs1+/+ T cells were generally present in comparable frequencies in distinct T cell subsets of the intestinal mucosa, mesenteric lymph nodes, and spleen. After intestinal infection with Listeria monocytogenes-OVA, however, OT-I Rgs1+/+ T cells outnumbered the co-transferred OT-I Rgs1-/- T cells in the small intestinal mucosa already early after infection. The underrepresentation of the OT-I Rgs1-/- T cells persisted to become even more pronounced during the memory phase (d30 post-infection). Remarkably, upon intestinal reinfection, mice with intestinal OT-I Rgs1+/+ TRM cells were able to prevent the systemic dissemination of the pathogen more efficiently than those with OT-I Rgs1-/- TRM cells. While the underlying mechanisms are not fully elucidated yet, these data thus identify Rgs1 as a critical regulator for the generation and maintenance of tissue-resident CD8+ T cells as a prerequisite for efficient local immunosurveillance in barrier tissues in case of reinfections with potential pathogens.</p