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<article doi="10.1002/eji.201142105" abstract="A role for the nucleotide-binding oligomerization domain 2 (NOD2) receptor in pulmonary innate immune responses has recently been explored. In the present study, we investigated the role that NOD2 plays in human alveolar macrophage innate responses and determined its involvement in the response to infection with virulent Mycobacterium tuberculosis. Our results showed that NOD2 was expressed in human alveolar macrophages, and significant amounts of IL-1β, IL-6, and TNF-α were produced upon ligand recognition with muramyldipeptide (MDP). NOD2 ligation induced the transcription and protein expression of the antimicrobial peptide LL37 and the autophagy enzyme IRGM in alveolar macrophages, demonstrating a novel function for this receptor in these cells. MDP treatment of alveolar macrophages improved the intracellular growth control of virulent M. tuberculosis; this was associated with a significant release of TNF-α and IL-6 and overexpression of bactericidal LL37. In addition, the autophagy proteins IRGM, LC3 and ATG16L1 were recruited to the bacteria-containing autophagosome after treatment with MDP. In conclusion, our results suggest that NOD2 can modulate the innate immune response of alveolar macrophages and play a role in the initial control of respiratory M. tuberculosis infections."><fig id="2401"><title></title><label>Figure 1</label><graphic href="https://api.sourcedata.io/file.php?figure_id=2401"/><sd-panel panel_id="6733"><div>(A) Intracellular detection of <sd-tag id="sdTag1" source="sdapi" category="entity" entity_type="protein" role="assayed" text="NOD2" ext_ids="Q9HC29" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="NOD2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">NOD2</sd-tag> (bold line in the histogram plot) was performed by <sd-tag id="sdTag2" source="sdapi" category="assay" entity_type="" role="" text="flow cytometry" ext_ids="BAO_0000005" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="flow cytometry" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">flow cytometry</sd-tag> using freshly isolated <sd-tag id="sdTag3" source="sdapi" category="entity" entity_type="cell_type" role="assayed" text="alveolar macrophages" ext_ids="CL:0000583" norm_text="" ext_dbs="CL" in_caption="True" ext_names="alveolar macrophage" ext_tax_ids="" ext_tax_names="" ext_urls=" https://identifiers.org/">alveolar macrophages</sd-tag> selected from a gate set on <sd-tag id="sdTag4" source="sdapi" category="entity" entity_type="cell" role="component" text="large granular bronchoalveolar cells" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">large granular bronchoalveolar cells</sd-tag> (R1, dot plot). One representative experiment out of three experiments is presented.</div><graphic href="https://api.sourcedata.io/file.php?panel_id=6733"/></sd-panel><sd-panel panel_id="6734">(B) Cells were stimulated with 10 μg/mL of <sd-tag id="sdTag7" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="MDP" ext_ids="CHEBI:59414" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="muramyl dipeptide" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">MDP</sd-tag> for 24 h, and <sd-tag id="sdTag6" source="sdapi" category="entity" entity_type="protein" role="assayed" text="NOD2" ext_ids="Q9HC29" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="NOD2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">NOD2</sd-tag> and <sd-tag id="sdTag9" source="sdapi" category="entity" entity_type="protein" role="assayed" text="IκBα" ext_ids="P25963" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="NFKBIA" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">IκBα</sd-tag> proteins were measured in the <sd-tag id="sdTag143" source="sdapi" category="entity" entity_type="subcellular" role="component" text="cytosolic" ext_ids="GO:0005829" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="cytosol" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">cytosolic</sd-tag> fractions by <sd-tag id="sdTag10" source="sdapi" category="assay" entity_type="" role="" text="western blot" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">western blot</sd-tag> analysis. The fold increase relative to unstimulated cells and normalized to <sd-tag id="sdTag11" source="sdapi" category="entity" entity_type="protein" role="normalizing" text="tubulin" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">tubulin</sd-tag> of one representative experiment out of three is reported.<graphic href="https://api.sourcedata.io/file.php?panel_id=6734"/></sd-panel><sd-panel panel_id="6735">(C) The upregulation of <sd-tag id="sdTag14" source="sdapi" category="entity" entity_type="gene" role="assayed" text="NOD2" ext_ids="64127" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="NOD2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">NOD2</sd-tag> gene expression was assessed by <sd-tag id="sdTag13" source="sdapi" category="assay" entity_type="" role="" text="quantitative PCR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">quantitative PCR</sd-tag>. The results are depicted as the mean fold change in <sd-tag id="sdTag15" source="sdapi" category="entity" entity_type="gene" role="assayed" text="NOD2" ext_ids="64127" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="NOD2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">NOD2</sd-tag> gene expression relative to unstimulated cells (n = 11).<graphic href="https://api.sourcedata.io/file.php?panel_id=6735"/></sd-panel><sd-panel panel_id="6736">(D) Subcellular localization of <sd-tag id="sdTag18" source="sdapi" category="entity" entity_type="protein" role="assayed" text="NOD2" ext_ids="Q9HC29" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="NOD2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">NOD2</sd-tag> was ascertained in unstimulated (left, ×80,000) and <sd-tag id="sdTag19" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="MDP" ext_ids="CHEBI:59414" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="muramyl dipeptide" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">MDP</sd-tag>‐stimulated cells (right, ×63,000) using an anti‐<sd-tag id="sdTag20" source="sdapi" category="entity" entity_type="protein" role="assayed" text="NOD2" ext_ids="Q9HC29" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="NOD2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">NOD2</sd-tag> antibody and detected with a secondary antibody coupled to 5‐nm gold particles, indicated with arrowheads. Bar represents 150 nm. Micrographs were obtained from one experiment.<graphic href="https://api.sourcedata.io/file.php?panel_id=6736"/></sd-panel></fig><fig id="2402"><title></title><label>Figure 2</label><graphic href="https://api.sourcedata.io/file.php?figure_id=2402"/><sd-panel panel_id="6750"><sd-tag id="sdTag25" source="sdapi" category="entity" entity_type="protein" role="component" text="NOD2" ext_ids="Q9HC29" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="NOD2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">NOD2</sd-tag> ligation in <sd-tag id="sdTag26" source="sdapi" category="entity" entity_type="cell_type" role="component" text="alveolar macrophages" ext_ids="CL:0000583" norm_text="" ext_dbs="CL" in_caption="True" ext_names="alveolar macrophage" ext_tax_ids="" ext_tax_names="" ext_urls=" https://identifiers.org/">alveolar macrophages</sd-tag> induces pro‐inflammatory cytokine release. Cells were incubated for <sd-pretag id="sdPretag14389508068870" parent-tag-id="155">24 h</sd-pretag> in the presence of 10 μg/mL of <sd-tag id="sdTag27" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="MDP" ext_ids="CHEBI:59414" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="muramyl dipeptide" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">MDP</sd-tag> or 100 ng/mL of <sd-tag id="sdTag28" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="LPS" ext_ids="CHEBI:16412" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="lipopolysaccharide" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">LPS</sd-tag>. Production of <sd-tag id="sdTag29" source="sdapi" category="entity" entity_type="protein" role="assayed" text="IL‐1β" ext_ids="P01584" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="IL1B" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">IL‐1β</sd-tag>, <sd-tag id="sdTag30" source="sdapi" category="entity" entity_type="protein" role="assayed" text="IL‐6" ext_ids="P05231" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="IL6" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">IL‐6</sd-tag>, <sd-tag id="sdTag31" source="sdapi" category="entity" entity_type="protein" role="assayed" text="TNF‐α" ext_ids="P01375" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="TNF" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">TNF‐α</sd-tag>, and <sd-tag id="sdTag32" source="sdapi" category="entity" entity_type="protein" role="assayed" text="IL‐10" ext_ids="P22301" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="IL10" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">IL‐10</sd-tag> was measured in culture supernatants using Milliplex technology. Depicted are box plots with median values and quartiles for each cytokine. The data are representative of two independent experiments (n = 11); *p 0.05 and **p 0.01 using the two‐tailed Wilcoxon signed‐rank test.<graphic href="https://api.sourcedata.io/file.php?panel_id=6750"/></sd-panel></fig><fig id="2403"><title></title><label>Figure 3</label><graphic href="https://api.sourcedata.io/file.php?figure_id=2403"/><sd-panel panel_id="6737"><sd-tag id="sdTag39" source="sdapi" category="entity" entity_type="gene" role="assayed" text="LL37" ext_ids="820" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="CAMP" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">LL37</sd-tag> and <sd-tag id="sdTag40" source="sdapi" category="entity" entity_type="gene" role="assayed" text="IRGM" ext_ids="345611" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="IRGM" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">IRGM</sd-tag> gene expression is upregulated following <sd-tag id="sdTag35" source="sdapi" category="entity" entity_type="gene" role="component" text="NOD2" ext_ids="64127" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="NOD2" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">NOD2</sd-tag> activation (A‐D). <sd-tag id="sdTag41" source="sdapi" category="entity" entity_type="cell_type" role="component" text="Alveolar macrophages" ext_ids="CL:0000583" norm_text="" ext_dbs="CL" in_caption="True" ext_names="alveolar macrophage" ext_tax_ids="" ext_tax_names="" ext_urls=" https://identifiers.org/">Alveolar macrophages</sd-tag> were incubated in the presence of 10 μg/mL of <sd-tag id="sdTag36" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="MDP" ext_ids="CHEBI:59414" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="muramyl dipeptide" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">MDP</sd-tag> or 100 ng/mL of <sd-tag id="sdTag37" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="LPS" ext_ids="CHEBI:16412" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="lipopolysaccharide" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">LPS</sd-tag> for 24 h. Upregulation of (A) <sd-tag id="sdTag42" source="sdapi" category="entity" entity_type="gene" role="assayed" text="LL37" ext_ids="820" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="CAMP" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">LL37</sd-tag> and (D) <sd-tag id="sdTag43" source="sdapi" category="entity" entity_type="gene" role="assayed" text="IRGM" ext_ids="345611" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="IRGM" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">IRGM</sd-tag> gene expression was assessed after specific ligand recognition by <sd-tag id="sdTag38" source="sdapi" category="assay" entity_type="" role="" text="quantitative PCR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">quantitative PCR</sd-tag> using the Taqman system and the ΔΔCT method for relative quantification. The fold change in gene expression relative to unstimulated cells is depicted. Data are representative of two independent experiments (n = 7-12). Bold lines indicate median values.<graphic href="https://api.sourcedata.io/file.php?panel_id=6737"/></sd-panel><sd-panel panel_id="6738">(B, E) <sd-tag id="sdTag44" source="sdapi" category="entity" entity_type="protein" role="assayed" text="LL37" ext_ids="P49913" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="CAMP" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">LL37</sd-tag> and <sd-tag id="sdTag45" source="sdapi" category="entity" entity_type="protein" role="assayed" text="IRGM" ext_ids="A1A4Y4" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="IRGM" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">IRGM</sd-tag> protein levels were measured in <sd-tag id="sdTag144" source="sdapi" category="entity" entity_type="subcellular" role="component" text="cytosolic" ext_ids="GO:0005829" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="cytosol" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">cytosolic</sd-tag> fractions by <sd-tag id="sdTag46" source="sdapi" category="assay" entity_type="" role="" text="western blot" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">western blot</sd-tag> analysis.<graphic href="https://api.sourcedata.io/file.php?panel_id=6738"/></sd-panel><sd-panel panel_id="6739">(C, F) Data are expressed as the protein fold increases relative to unstimulated cells and normalized to <sd-tag id="sdTag52" source="sdapi" category="entity" entity_type="protein" role="normalizing" text="tubulin" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">tubulin</sd-tag> calculated by densitometry and are shown as means ± SEM. One representative experiment out of three experiments is presented.<graphic href="https://api.sourcedata.io/file.php?panel_id=6739"/></sd-panel></fig><fig id="2404"><title></title><label>Figure 4</label><graphic href="https://api.sourcedata.io/file.php?figure_id=2404"/><sd-panel panel_id="6740">(A) Intracellular bacterial burden was measured by quantifying <sd-tag id="sdTag66" source="sdapi" category="assay" entity_type="" role="" text="CFU" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">CFU</sd-tag>, and the intracellular growth index was calculated after 1 and 4 days post infection relative to phagocytosed bacteria at day 0. Data are shown as medians and quartiles and are representative of n = 7 experiments.<graphic href="https://api.sourcedata.io/file.php?panel_id=6740"/></sd-panel><sd-panel panel_id="6741">The production of (B) <sd-tag id="sdTag67" source="sdapi" category="entity" entity_type="protein" role="assayed" text="TNF‐α" ext_ids="P01375" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="TNF" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">TNF‐α</sd-tag>, (C) <sd-tag id="sdTag68" source="sdapi" category="entity" entity_type="protein" role="assayed" text="IL‐6" ext_ids="P05231" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="IL6" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">IL‐6</sd-tag> and (D) <sd-tag id="sdTag69" source="sdapi" category="entity" entity_type="protein" role="assayed" text="IL‐1β" ext_ids="P01584" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="IL1B" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">IL‐1β</sd-tag> was measured in culture supernatants using Milliplex technology. Box plots depicting median values and quartiles are representative of n = 9 experiments.<graphic href="https://api.sourcedata.io/file.php?panel_id=6741"/></sd-panel><sd-panel panel_id="6742">(E) <sd-tag id="sdTag75" source="sdapi" category="entity" entity_type="gene" role="assayed" text="LL37" ext_ids="820" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="CAMP" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">LL37</sd-tag> and (F) <sd-tag id="sdTag76" source="sdapi" category="entity" entity_type="gene" role="assayed" text="IRGM" ext_ids="345611" norm_text="" ext_dbs="NCBI gene" in_caption="True" ext_names="IRGM" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="http://www.ncbi.nlm.nih.gov/gene/">IRGM</sd-tag> gene expression was assessed by <sd-tag id="sdTag77" source="sdapi" category="assay" entity_type="" role="" text="quantitative PCR" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">quantitative PCR</sd-tag> using the Taqman system and the ΔΔCT method for relative quantification. Fold changes in gene expression relative to unstimulated cells are reported. Bold lines indicate median values of n = 9. *p 0.05 and **p 0.01 using the two‐tailed Wilcoxon signed‐rank test.<graphic href="https://api.sourcedata.io/file.php?panel_id=6742"/></sd-panel><sd-panel panel_id="6743">(G) <sd-tag id="sdTag81" source="sdapi" category="entity" entity_type="protein" role="assayed" text="LL37" ext_ids="P49913" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="CAMP" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">LL37</sd-tag> and <sd-tag id="sdTag82" source="sdapi" category="entity" entity_type="protein" role="assayed" text="IRGM" ext_ids="A1A4Y4" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="IRGM" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">IRGM</sd-tag> protein was assessed by <sd-tag id="sdTag83" source="sdapi" category="assay" entity_type="" role="" text="western blot" ext_ids="BAO_0002424" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="western blot" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">western blot</sd-tag>. One representative experiment out of three experiments is presented.<graphic href="https://api.sourcedata.io/file.php?panel_id=6743"/></sd-panel><sd-panel panel_id="6744">(H) The amount of protein normalized to <sd-tag id="sdTag89" source="sdapi" category="entity" entity_type="protein" role="normalizing" text="tubulin" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">tubulin</sd-tag> relative to infected‐only cells was calculated by densitometry. Data are depicted as the means ± SEM and are representative of n = 3 experiments.<graphic href="https://api.sourcedata.io/file.php?panel_id=6744"/></sd-panel></fig><fig id="2405"><title></title><label>Figure 5</label><graphic href="https://api.sourcedata.io/file.php?figure_id=2405"/><sd-panel panel_id="6745">(A, B, D, E, G, H) The subcellular localization of autophagy proteins was ascertained in untreated and <sd-tag id="sdTag97" source="sdapi" category="entity" entity_type="molecule" role="intervention" text="MDP" ext_ids="CHEBI:59414" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="muramyl dipeptide" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">MDP</sd-tag>‐treated cells using (A, B) anti‐<sd-tag id="sdTag98" source="sdapi" category="entity" entity_type="protein" role="assayed" text="IRGM" ext_ids="A1A4Y4" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="IRGM" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">IRGM</sd-tag>, (D, E) anti‐<sd-tag id="sdTag104" source="sdapi" category="entity" entity_type="protein" role="assayed" text="LC3" ext_ids="Q9GZQ8///Q9BXW4///Q9H492" norm_text="" ext_dbs="Uniprot///Uniprot///Uniprot" in_caption="True" ext_names="MAP1LC3B///MAP1LC3C///MAP1LC3A" ext_tax_ids="9606///9606///9606" ext_tax_names="Homo sapiens///Homo sapiens///Homo sapiens" ext_urls="https://www.uniprot.org/uniprot////https://www.uniprot.org/uniprot////https://www.uniprot.org/uniprot/">LC3</sd-tag>, and (G, H) anti‐<sd-tag id="sdTag99" source="sdapi" category="entity" entity_type="protein" role="assayed" text="ATG16L1" ext_ids="Q676U5" norm_text="" ext_dbs="Uniprot" in_caption="True" ext_names="ATG16L1" ext_tax_ids="9606" ext_tax_names="Homo sapiens" ext_urls="https://www.uniprot.org/uniprot/">ATG16L1</sd-tag> antibodies and detected with a secondary antibody coupled to 5‐nm gold particles indicated with arrowheads; bar represents 150 nm.<graphic href="https://api.sourcedata.io/file.php?panel_id=6745"/></sd-panel><sd-panel panel_id="6746">(C, F, I) <sd-tag id="sdTag146" source="sdapi" category="entity" entity_type="molecule" role="reporter" text="Gold" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">Gold</sd-tag> particles co‐localizing with bacteria were manually counted in ten macrophages of each condition. Means ± SEM are depicted and differences between treatments are indicated, *p 0.05 using a two‐tailed paired t‐test. (B) <sd-tag id="sdTag113" source="sdapi" category="assay" entity_type="" role="" text="Transmission electron microscopy (TEM)" ext_ids="" norm_text="" ext_dbs="" in_caption="True" ext_names="" ext_tax_ids="" ext_tax_names="" ext_urls="">Transmission electron microscopy (<sd-pretag id="sdPretag14389508715350" parent-tag-id="158">TEM</sd-pretag>)</sd-tag> magnification ×50,000; the rest of the micrographs are <sd-pretag id="sdPretag14389508935300" parent-tag-id="158">TEM</sd-pretag> magnification ×80,000. Data were generated from one experiment.<graphic href="https://api.sourcedata.io/file.php?panel_id=6746"/></sd-panel></fig><fig id="2406"><title></title><label>Figure 6</label><graphic href="https://api.sourcedata.io/file.php?figure_id=2406"/><sd-panel panel_id="6747"><sd-tag id="sdTag149" source="sdapi" category="entity" entity_type="cell_type" role="component" text="Macrophages" ext_ids="CL:0000235" norm_text="" ext_dbs="CL" in_caption="True" ext_names="macrophage" ext_tax_ids="" ext_tax_names="" ext_urls=" https://identifiers.org/">Macrophages</sd-tag> treated with <sd-tag id="sdTag150" source="sdapi" category="entity" entity_type="molecule" role="component" text="MDP" ext_ids="CHEBI:59414" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="muramyl dipeptide" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">MDP</sd-tag> after <sd-tag id="sdTag151" source="sdapi" category="entity" entity_type="organism" role="component" text="M. tuberculosis" ext_ids="1773" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mycobacterium tuberculosis" ext_tax_ids="1773" ext_tax_names="Mycobacterium tuberculosis" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">M. tuberculosis</sd-tag> infection develop <sd-tag id="sdTag152" source="sdapi" category="entity" entity_type="subcellular" role="assayed" text="autophagy vacuoles" ext_ids="GO:0005776" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="autophagic vacuole" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">autophagy vacuoles</sd-tag>. <sd-tag id="sdTag153" source="sdapi" category="entity" entity_type="cell_type" role="component" text="Alveolar macrophages" ext_ids="CL:0000583" norm_text="" ext_dbs="CL" in_caption="True" ext_names="alveolar macrophage" ext_tax_ids="" ext_tax_names="" ext_urls=" https://identifiers.org/">Alveolar macrophages</sd-tag> were infected with <sd-tag id="sdTag156" source="sdapi" category="entity" entity_type="organism" role="component" text="M. tuberculosis" ext_ids="1773" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mycobacterium tuberculosis" ext_tax_ids="1773" ext_tax_names="Mycobacterium tuberculosis" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">M. tuberculosis</sd-tag> H37Rv at an MOI of 5 for 1 h. Nonphagocytosed bacteria were washed away, and the macrophages were incubated for an additional hour. The cells were then treated with 10 μg/mL of <sd-tag id="sdTag157" source="sdapi" category="entity" entity_type="molecule" role="component" text="MDP" ext_ids="CHEBI:59414" norm_text="" ext_dbs="ChEBI" in_caption="True" ext_names="muramyl dipeptide" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ebi.ac.uk/chebi/searchId.do?chebiId=">MDP</sd-tag> for 24 h. Autophagy indicators (arrowheads) such as (A) <sd-tag id="sdTag147" source="sdapi" category="entity" entity_type="subcellular" role="assayed" text="vesicles" ext_ids="GO:0031982" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="vesicle" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">vesicles</sd-tag> containing organelles (<sd-tag id="sdTag158" source="sdapi" category="assay" entity_type="" role="" text="TEM" ext_ids="BAO_0000455" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="transmission electron microscopy" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">TEM</sd-tag> magnification ×20,000) and (B) onion skin‐like lamellar <sd-tag id="sdTag148" source="sdapi" category="entity" entity_type="subcellular" role="assayed" text="multivesicular bodies" ext_ids="GO:0005771" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="multivesicular body" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">multivesicular bodies</sd-tag> (<sd-pretag id="sdPretag14389508107000" parent-tag-id="158">TEM</sd-pretag> magnification ×16,000) were identified by ultrastructural analysis..<graphic href="https://api.sourcedata.io/file.php?panel_id=6747"/></sd-panel><sd-panel panel_id="6748">(C, D) <sd-tag id="sdTag131" source="sdapi" category="entity" entity_type="subcellular" role="assayed" text="Autophagosomes" ext_ids="GO:0005776" norm_text="" ext_dbs="Gene Ontology" in_caption="True" ext_names="autophagic vacuole" ext_tax_ids="" ext_tax_names="" ext_urls="http://amigo.geneontology.org/amigo/term/">Autophagosomes</sd-tag> containing <sd-tag id="sdTag132" source="sdapi" category="entity" entity_type="organism" role="assayed" text="mycobacteria (Mtb)" ext_ids="1763" norm_text="" ext_dbs="NCBI taxon" in_caption="True" ext_names="Mycobacterium" ext_tax_ids="" ext_tax_names="" ext_urls="http://www.ncbi.nlm.nih.gov/taxonomy/">mycobacteria (Mtb)</sd-tag> are indicated with arrows (C, <sd-tag id="sdTag128" source="sdapi" category="assay" entity_type="" role="" text="TEM" ext_ids="BAO_0000455" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="transmission electron microscopy" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">TEM</sd-tag> magnification ×40,000; D, <sd-tag id="sdTag129" source="sdapi" category="assay" entity_type="" role="" text="TEM" ext_ids="BAO_0000455" norm_text="" ext_dbs="BAO" in_caption="True" ext_names="transmission electron microscopy" ext_tax_ids="" ext_tax_names="" ext_urls="https://bioportal.bioontology.org/ontologies/BAO/?p=classes&conceptid=http%3A%2F%2Fwww.bioassayontology.org%2Fbao%23">TEM</sd-tag> magnification ×12,000). Micrographs were obtained from one experiment. Bar represents 300 nm.<graphic href="https://api.sourcedata.io/file.php?panel_id=6748"/></sd-panel></fig></article>