Journal: bioRxiv

Article Title: Hijacking of inflammasome responses by the complement system during Pseudomonas aeruginosa – Aspergillus fumigatus sur-infection

doi: 10.64898/2026.02.20.707105

Figure Lengend Snippet: (a) Experimental workflow showing primary murine alveolar macrophages (mAMs) or bone marrow-derived macrophages (BMDMs) first infected with live P. aeruginosa ( P.a ) for 4 hours, followed by antibiotic treatment, then challenged with A. fumigatus ( A.f ) conidia/hyphae, and analyzed at 16–22 hours post-superinfection. (b) Immunoblot of pro–caspase-1 (Pro–CASP1) and its cleaved p20 form in cell lysates from mAMs untreated (medium alone (Med)) or after infection with P.a (MOI of 0.005 or 0.01) and/or with A.f (MOI 5). (c-e) Quantification of cytokines IL-1β (c) , TNFα (d) , and KC (e) in supernatants from mAMs under each conditions as measured by ELISA. (f) Immunoblots for Pro–CASP1 and p20 in cell lysates of BMDMs from the indicated conditions. (g-i) BMDMs cytokine quantification of IL-1β (g) , TNFα (h) , and KC (i) release following P.a priming with increasing multiplicity of infection (MOI), and subsequent A.f superinfection. (j) Quantification of cell death, measured as the number of SytoxGreen positive (Sytox + ) cells, in BMDMs following infection with P.a (MOI 0.005 or 0.01) and/or A.f (MOI 5). (k) Representative images of Sytox-stained BMDMs imaging at 8 and 20 hours post-infection (hpi) with A.f alone or after P.a primary infection “ P.a→A.f ”. Scale bars, 100µm. (l) Time-course analysis of fungal growth (hyphal length in mm/mm²) of A.f (MOI 5) after BMDMs infection, either left uninfected (medium control, Med) or co-infected with P.a (MOI 0.005 or 0.01). (m) Representative images of fungal morphology of A.f (Strain: DAL DSRED), highlighted in bleu, at early (5 hpi) and late (15 hpi) timepoints in BMDMs single A.f infection and in superinfection (MOI 5). Scale bars, 100µm. (n) Macroscopic images of BMDMs culture wells at endpoint (22 hpi) showing visual differences in fungal growth and cellular conditions across infection models (MOI 5 for A.f and MOI 0.005 or 0.01 for P.a ). Data are representative of at least three independent experiments. Error bars indicate mean ± SEM. Statistical significance: *p<0.05, #p<0.01, ****p<0.0001, ns = not significant by ANOVA.

Article Snippet: Cytokine secretion was analyzed by ELISA for mouse IL-1β (DY401, R&D Systems), TNF-α (DY410, R&D Systems), and KC (DY453, R&D Systems).

Techniques: Derivative Assay, Infection, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Imaging, Control

Journal: bioRxiv

Article Title: Hijacking of inflammasome responses by the complement system during Pseudomonas aeruginosa – Aspergillus fumigatus sur-infection

doi: 10.64898/2026.02.20.707105

Figure Lengend Snippet: (a) Quantification of P.a colony-forming units (CFUs) in BMDMs at different time points and multiplicities of infection (MOI 0.005 or 0.01). (b) Mean number of A.f (MOI 5) conidia phagocytosed per BMDM without P.a priming (Med) or following priming with different doses of live P.a (MOI 0.005 or 0.01). (c) A.f CFUs from cultures with or without P.a priming at 4 and 6 hours after fungal infection, indicating enhanced fungal clearance when macrophages are pre-infected with P.a . (d) Immunoblot for pro–caspase-1 and its cleaved p20 fragment in BMDMs lysates stimulated with LPS and/or flagellin, IFNγ, or live P.a. (e) ELISA quantification of IL-1β secretion from BMDMs under the indicated priming (LPS alone, flagellin alone, LPS and flagellin together, or live P.a ) and fungal challenge conditions (+ A.f ). (f) Time-course of A.f hyphal growth (length in mm/mm²) monitored in the presence of BMDMs primed with the different stimuli prior to fungal challenge. (g) Immunoblot for caspase-1 p20 cleavage with live P.a (MOI 0.01), heat-killed (HK) or PFA-fixed P.a (MOI 0.02) and fungal challenge ( A.f ). (h) ELISA quantification of IL-1β release upon live, HK or PFA-fixed P.a (with increased MOI 0.01 and 0.02) with or without second A.f infection (MOI 5). (i) Time-course quantification of A.f hyphal growth with BMDMs primed by live, HK (at MOI 0.02), or fixed P.a (MOI 0.01), in the presence or absence of A.f superinfection (MOI 5). Data are representative of at least three independent experiments. Error bars indicate mean ± SEM. Statistical analysis by one-way or two-way ANOVA with multiple comparisons; ****p < 0.0001.

Article Snippet: Cytokine secretion was analyzed by ELISA for mouse IL-1β (DY401, R&D Systems), TNF-α (DY410, R&D Systems), and KC (DY453, R&D Systems).

Techniques: Infection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Hijacking of inflammasome responses by the complement system during Pseudomonas aeruginosa – Aspergillus fumigatus sur-infection

doi: 10.64898/2026.02.20.707105

Figure Lengend Snippet: (a) Immunoblot for pro–caspase-1 and its cleaved p20 fragment and in BMDMs untreated (Med) or primed with wild-type (WT) or mutant P.a strains (Δ fliC , Δ pscF , Δ pilQ ) at MOI of 0.01, and followed or not by superinfection with A.f (MOI 5). (b) ELISA quantification of IL-1β secretion in supernatants from BMDMs exposed to the indicated P.a mutants (MOI 0.005 and 0.01) and followed or not by superinfection with A.f (MOI 5). (c) Immunoblot for caspase-1 processing and (d) corresponding IL-1β ELISA levels in BMDMs left untreated (Med) or challenged with WT or mutant A.f strains (Δ gt4c , Δ ugm1 ) (MOI 5), following or not P.a priming (MOI 0.01). (e) IL-1β quantification in BMDMs left untreated (Med) or challenged with GAG only or first primed by P.a (MOI 0.01). (f) Immunoblots for caspase-1 cleavage and (g) quantification of IL-1β release in BMDMs untreated (Med) or infected with three independent sets of co-isolated clinical P.a (MOI 0.01) and A.f (MOI 5) strains from cystic fibrosis patients. Data are mean ± SEM of at least three independent experiments. Statistical significance assessed by ANOVA or t-test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Cytokine secretion was analyzed by ELISA for mouse IL-1β (DY401, R&D Systems), TNF-α (DY410, R&D Systems), and KC (DY453, R&D Systems).

Techniques: Western Blot, Mutagenesis, Enzyme-linked Immunosorbent Assay, Infection, Isolation

Journal: bioRxiv

Article Title: Hijacking of inflammasome responses by the complement system during Pseudomonas aeruginosa – Aspergillus fumigatus sur-infection

doi: 10.64898/2026.02.20.707105

Figure Lengend Snippet: (a) Immunoblot analysis showing caspase-1 processing (pro–CASP1 and p20) and ELISA quantification of IL-1β (b) and TNFα (c) secretion in BMDMs from WT, Nlrp3 −/− , and Aim2 −/− mice left uninfected (Med) or after infection with P.a (MOI 0.005 or 0.01), and/or A.f (MOI 5). (d) Time-course of fungal hyphal growth (length/mm²) in WT and Nlrp3 −/− BMDMs left uninfected (Med) or after infection with P.a (MOI 0.005 or 0.01), and/or A.f (MOI 5). (e) Immunoblot analysis showing caspase-1 processing (pro–CASP1 and p20) and (f) ELISA quantification of IL-1β secretion in BMDMs from WT, and Asc −/− mice left uninfected (Med) or after infection with P.a (MOI 0.005 or 0.01), and/or A.f (MOI 5). (g) Immunoblot for caspase-1 in BMDMs treated with caspase-1 inhibitor (VX765), caspase-8 inhibitor (Z-IETD-FMK), or both (VX765+Z-IETD-FMK), under single and sequential infections. (h) IL-1β measurement by ELISA in BMDMs treated with caspase-8 inhibitor Z-IETD-FMK, caspase-1 inhibitor VX765, their combination, left uninfected (Med) or after infection with P.a (MOI 0.005 and 0.01), and/or A.f (MOI 5). (i) Immunoblot analysis showing caspase-1 processing (pro–CASP1 and p20) in BMDMs from WT and Gsdmd −/− mice left uninfected (Med) or after infection with P.a (MOI 0.005 or 0.01), and/or A.f (MOI 5) and (j) corresponding IL-1β quantification for Gsdmd −/− . (k) Quantification of cell death, measured as the number of propidium iodide-positive (PI + ) cells, in Gsdmd −/− BMDMs following infection with P.a (MOI 0.01) and/or A.f (MOI 5). Data are from at least three independent experiments and presented as mean ± SEM. Statistical significance determined by ANOVA; *p<0.05, **p<0.01, ****p<0.0001.

Article Snippet: Cytokine secretion was analyzed by ELISA for mouse IL-1β (DY401, R&D Systems), TNF-α (DY410, R&D Systems), and KC (DY453, R&D Systems).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Infection

Journal: bioRxiv

Article Title: Hijacking of inflammasome responses by the complement system during Pseudomonas aeruginosa – Aspergillus fumigatus sur-infection

doi: 10.64898/2026.02.20.707105

Figure Lengend Snippet: (a) Volcano plot illustrating differentially expressed genes (DEGs) in BMDMs comparing P.a → A.f superinfection vs. A.f single infection at 36 h time point. Upregulated (red), downregulated (blue), and non-significant (grey) genes are indicated; key inflammatory genes are labeled. (b) Gene ontology (GO) enrichment analysis of significantly upregulated transcripts following superinfection, highlighting overrepresented biological process categories related to immune and inflammatory responses in BMDMs sequentially infected by P.a → A.f . (c) Heatmap showing the top 30 up- and downregulated genes in WT BMDMs across samples, stratified by condition ( P. aeruginosa pre-exposure ( P.a ) vs no P.a ) and time point (32 h = 4 h post– A. fumigatus infection; 36 h = 8 h post– A. fumigatus infection). (d–f) Heatmaps showing log2 fold change (log2FC) in WT BMDMs with P. aeruginosa pre-exposure ( P.a ) vs . no P.a at baseline before A. fumigatus infection (28 h), and at 4 h (32 h) and 8 h (36 h) post– A. fumigatus infection. Heatmaps highlight genes encoding (d) inflammasome-related PRRs, (e) inflammasome-associated signaling pathways, and (f) core inflammasome components. (g) Immunoblot analysis of phosphorylated and total SYK, IκBα, and ERK, and of pro-IL-1β, in BMDM lysates collected over a time course (0, 2, 4, 8, 10 hours) post- A.f (MOI 5) alone or post A.f infection in BMDMs already primed by P.a (MOI 0.01) P.a → A.f . β-actin is shown as a loading control.

Article Snippet: Cytokine secretion was analyzed by ELISA for mouse IL-1β (DY401, R&D Systems), TNF-α (DY410, R&D Systems), and KC (DY453, R&D Systems).

Techniques: Infection, Labeling, Protein-Protein interactions, Western Blot, Control

Journal: bioRxiv

Article Title: Hijacking of inflammasome responses by the complement system during Pseudomonas aeruginosa – Aspergillus fumigatus sur-infection

doi: 10.64898/2026.02.20.707105

Figure Lengend Snippet: (a) Immunoblot analysis showing caspase-1 processing (pro–CASP1 and p20 cleavage) and ELISA quantification of IL-1β (b) , TNFα (c) secretion in BMDMs from WT and Myd88 −/− mice left uninfected (Med) or after infection with P.a (MOI 0.005 or 0.01), and/or A.f (MOI 5). (a) Data are means ± SEM (****p<0.0001 by ANOVA). (d) Quantification of IL-1β levels in supernatants from WT THP-1 Dual and MyD88 −/− knockout THP-1 macrophages after left uninfected (Med), infected by P.a alone (MOI 0.01), A.f alone (MOI 5) or sequential infection ( P.a→A.f ). Results are presented as means ± SEM (*p<0.05, ***p<0.001 by ANOVA). (e) Immunoblot of phospho-SYK (p-SYK), total SYK, phospho-IκBα (p-IκBα), total IκBα, phospho-ERK (p-ERK), total ERK, pro-IL-1β, NLRP3, and β-actin over indicated time points after A.f single infection (MOI 5) or P.a→A.f superinfection ( P.a MOI 0.01 and A.f MOI 5). (f) Quantification of A.f hyphal growth (length/mm² over time) in WT and Myd88 −/− BMDMs left uninfected (Med) or after infection with P.a (MOI 0.01), and/or A.f (MOI 5). (g) Volcano plot of RNA-seq analysis comparing WT and Myd88 −/− BMDMs following P.a→A.f superinfection, highlighting upregulated (orange) and downregulated (blue) genes. (h) Gene ontology (GO) enrichment for differentially expressed genes, showing biological processes with greatest fold enrichment in Myd88 −/− BMDMs sequentially infected by P.a→A.f . (i) Venn diagram showing overlap of differentially expressed genes among WT and Myd88 −/− BMDMs across the given timepoints and conditions. (j) Heatmap of top upregulated genes based on fold change (by log2 fold change, ΔLog2FC) involved in immune response. (k) Network node degree heatmap of top genes, underscoring interaction strength and centrality in the regulatory network.

Article Snippet: Cytokine secretion was analyzed by ELISA for mouse IL-1β (DY401, R&D Systems), TNF-α (DY410, R&D Systems), and KC (DY453, R&D Systems).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Infection, Knock-Out, RNA Sequencing

Journal: bioRxiv

Article Title: Hijacking of inflammasome responses by the complement system during Pseudomonas aeruginosa – Aspergillus fumigatus sur-infection

doi: 10.64898/2026.02.20.707105

Figure Lengend Snippet: (a) Quantification of CD11b surface expression (general mean fluorescence intensity, gMFI) in BMDMs following incubation with medium (Med) or P.a, assessed by flow cytometry. (b) Immunoblot showing caspase-1 activation (pro-caspase-1 and p20 cleavage fragment) and (c) corresponding IL-1β levels in WT BMDMs untreated (Med), or infected with A.f alone, P.a alone or sequential infection ( P.a → A.f ), with or without anti-ITGAM antibody blockade. (d) Dot plot of hallmark pathway enrichment for differentially expressed genes, showing fold enrichment, number of genes, and statistical significance by -log10(FDR). (e) Chord diagram of network connectivity among genes (top part of the diagram) and the pathways (bottom part) in immune and inflammatory responses. (f) Volcano plot showing differentially expressed genes in P. aeruginosa –infected WT BMDMs upon A. fumigatus infection, with significantly upregulated (red), downregulated (blue) and non-significant (grey) genes. (g) Immunoblot analysis showing caspase-1 processing (pro–CASP1 and p20 cleavage) and ELISA quantification of IL-1β (h) and TNFα (i) in BMDMs from WT and C3 −/− mice left uninfected (Med) or after infection with P.a (MOI 0.005 or 0.01), and/or A.f ( MOI 5). (j) Immunoblot time course investigating protein phosphorylation of SYK, ERK, IκBα, and pro-IL1β processing in WT and C3 −/− BMDMs after A.f or sequential P.a → A.f infection for the indicated hours post-infection (hpi). β-actin is shown as a loading control.

Article Snippet: Cytokine secretion was analyzed by ELISA for mouse IL-1β (DY401, R&D Systems), TNF-α (DY410, R&D Systems), and KC (DY453, R&D Systems).

Techniques: Expressing, Fluorescence, Incubation, Flow Cytometry, Western Blot, Activation Assay, Infection, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Control

Journal: Nature Communications

Article Title: CLEC5A is a critical receptor in innate immunity against Listeria infection

doi: 10.1038/s41467-017-00356-3

Figure Lengend Snippet: CLEC5A is involved in L. monocytogenes -induced IL-1β production. Mouse macrophages were incubated with live L. monocytogenes (MOI 10) and lysates were collected at 60 min post infection to detect a Phospho-Syk, while b phospho-NFκB p65 were detected at indicated time points. c Mouse macrophages were incubated with live L. monocytogenes , supernatants were collectedand IL-1β secretion was measured by ELISA. d Mouse macrophages incubated with live (left ) or UV-inactivated L. monocytogenes were collected6 and 12 h post infection to detect Il1b transcripts by RT-qPCR. e Caspase-1 activity in mouse macrophages (6 h post Listeria infection) was presented as absorbance at 405 nm ( A 405 ). f The amounts of caspase-1 p10 and p20 released from macrophages were determined by ELISA at 12 h post L. monocytogenes infection. g Mouse macrophages incubated with live L. monocytogenes were collected to detect Nrlp3, Aim2 , and Nlrc4 transcripts by RT-qPCR. All the data were collected and expressed as mean ± s.e.m. from at least three independent experiments. One-way ANOVA was performed. * P < 0.05, ** P < 0.01; for treated versus control or knockout versus wild-type mice

Article Snippet: The monoclonal antibodies used in this study was listed as following: mouse IL-1β ELISA kit (DY 401), human TNF ELISA kit (DY210) and blocking antibody to human TLR2 (MAB2616) were purchased from R&D Systems, antibodies to histone H3 (citrulline R2 + R8 + R17; ab5103, 1:100), myeloperoxidase (MPO, sc-52707 or sc-34159, 1:100), phospho-syk (#2701, 1:1000), phosphor-p65 (#3033, 1:1000), histone 1–4 (Mab 3422, 1:200).

Techniques: Incubation, Infection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Activity Assay, Control, Knock-Out

Journal: Nature Communications

Article Title: CLEC5A is a critical receptor in innate immunity against Listeria infection

doi: 10.1038/s41467-017-00356-3

Figure Lengend Snippet: Co-activation of CLEC5A and TLR2 by L. monocytogenes enhances host immunity. Listeria activates CLEC5A and TLR2 simultaneously to activate both MyD88-p38 and Syk-AKT signaling pathways. Co-activation of CLEC5A and TLR2 in neutrophils enhances CXCL8 and ROS production, and NET formation, whereas co-activation of CLEC5A and TLR2 in macrophages induces inflammasome activation and production of IL-1β to stimulate the development of IL-17A proudcing TCR γδ T-cells

Article Snippet: The monoclonal antibodies used in this study was listed as following: mouse IL-1β ELISA kit (DY 401), human TNF ELISA kit (DY210) and blocking antibody to human TLR2 (MAB2616) were purchased from R&D Systems, antibodies to histone H3 (citrulline R2 + R8 + R17; ab5103, 1:100), myeloperoxidase (MPO, sc-52707 or sc-34159, 1:100), phospho-syk (#2701, 1:1000), phosphor-p65 (#3033, 1:1000), histone 1–4 (Mab 3422, 1:200).

Techniques: Activation Assay, Protein-Protein interactions