S100A12 Is Part of the Antimicrobial Network against Mycobacterium leprae in Human Macrophages - PubMed (original) (raw)

. 2016 Jun 29;12(6):e1005705.

doi: 10.1371/journal.ppat.1005705. eCollection 2016 Jun.

Kindra M Kelly-Scumpia 2, Angeline Tilly Dang 1, Jing Lu 3, Rosane Teles 2, Philip T Liu 4, Mirjam Schenk 2, Ernest Y Lee 5, Nathan W Schmidt 5, Gerard C L Wong 5, Euzenir N Sarno 6, Thomas H Rea 7, Maria T Ochoa 7, Matteo Pellegrini 3, Robert L Modlin 1 2

Affiliations

S100A12 Is Part of the Antimicrobial Network against Mycobacterium leprae in Human Macrophages

Susan Realegeno et al. PLoS Pathog. 2016.

Abstract

Triggering antimicrobial mechanisms in macrophages infected with intracellular pathogens, such as mycobacteria, is critical to host defense against the infection. To uncover the unique and shared antimicrobial networks induced by the innate and adaptive immune systems, gene expression profiles generated by RNA sequencing (RNAseq) from human monocyte-derived macrophages (MDMs) activated with TLR2/1 ligand (TLR2/1L) or IFN-γ were analyzed. Weighed gene correlation network analysis identified modules of genes strongly correlated with TLR2/1L or IFN-γ that were linked by the "defense response" gene ontology term. The common TLR2/1L and IFN-γ inducible human macrophage host defense network contained 16 antimicrobial response genes, including S100A12, which was one of the most highly induced genes by TLR2/1L. There is limited information on the role of S100A12 in infectious disease, leading us to test the hypothesis that S100A12 contributes to host defense against mycobacterial infection in humans. We show that S100A12 is sufficient to directly kill Mycobacterium tuberculosis and Mycobacterium leprae. We also demonstrate that S100A12 is required for TLR2/1L and IFN-γ induced antimicrobial activity against M. leprae in infected macrophages. At the site of disease in leprosy, we found that S100A12 was more strongly expressed in skin lesions from tuberculoid leprosy (T-lep), the self-limiting form of the disease, compared to lepromatous leprosy (L-lep), the progressive form of the disease. These data suggest that S100A12 is part of an innate and adaptive inducible antimicrobial network that contributes to host defense against mycobacteria in infected macrophages.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1. Network analysis of RNAseq reveals defense response genes in TLR2/1 and IFN-γ specific modules.

(A) Hierarchical clustering of log-transformed normalized counts of 45 samples obtained from RNAseq. For each sample, the replicate number, stimuli, and time point are indicated. (B) Correlation of treatment conditions (x-axis) to WGCNA module eigengenes (y-axis) displayed in a heat map. The p-values (bottom) for each r correlation value (top) are indicated for each modules and each condition. Red indicates positive correlation and green indicated an inverse correlation. (C) Functional annotation network of significant gene ontology terms (red) from ClueGO analysis associated with representative genes (blue) found in WGCNA modules indicated.

Fig 2

Fig 2. Analysis of TLR2/1L and IFN-γ inducible genes identifies common pathways.

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

Fig 3

Fig 3. RNAseq gene expression is validated by qPCR.

MDMs were stimulated with TLR2/1L (black bars) or IFN-γ (gray bars) for 2, 6, and 24 hours and (A) S100A12, (B) GBP1, and (C) 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, n = 5.

Fig 4

Fig 4. TLR2/1L and IFN-γ inducible S100A12 protein expression.

Intracellular S100A12 protein expression following stimulation of MDMs with TLR2/1L or IFN-γ for 24, 48, and 72 hours. (A) 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) TLR2/1L (n = 9) and (C) IFN-γ (n = 5) stimulated conditions compared to media control at each time point. Data are represented as mean ± SEM, *P _≤_0.05, **P _≤_0.01.

Fig 5

Fig 5. Expression of S100A12 in skin lesions from leprosy patients.

(A) 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) The percentage of diaminobenzidine (DAB)-stained area per nuclear area was calculated for each photomicrograph using ImmunoRatio [30]. 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 (P = 0.006) using GraphPad Prism software and post hoc analysis (Turkey test) is indicated by the asterisk (*P<0.05 and ** P<0.01). (C) 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.

Fig 6

Fig 6. Antimicrobial activity of S100A12 against mycobacteria.

(A) A plot of (NK/(NK + NR)) vs. mean hydrophobicity for all known α-helical antimicrobial peptides in the antimicrobial peptide database (

http://aps.unmc.edu/AP/main.php

). The black circles represent peptides that meet amino acid criterion for membrane curvature generation and exhibit membrane permeating antimicrobial activity. NK and NR 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) M. tuberculosis 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). (C) M. tuberculosis 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). (D) Data from B is represented in % viability relative to media control for in addition to rifampicin control (n = 3). (E) M. leprae was maintained in 7H9 media with S100A12. Shown in AU is the ratio of M. leprae 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. (F) 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. *P _≤_0.05, **P _≤_0.01, ***P _≤_0.001; ns, non-significant.

Fig 7

Fig 7. S100A12 plays a role in macrophage antimicrobial activity against M. leprae.

(A) 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 (n = 9) or IFN-γ (n = 5). (B) MDMs were infected with M. leprae and stimulated with TLR2/1L (n = 9) and IFN-γ (n = 8). M. leprae 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, *P _≤_0.05, **P _≤_0.01, ***P _≤_0.001.

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