Additional file 1: Supplemental Fig. 1. Effect of chronic exposure to HDM in a urethane-induced LC model. A-F) WT C57BL/6 mice were treated i.n with HDM (n = 8), HI-HDM (n = 10), or with the vehicle (VEH, n = 9) and i.p with urethane as shown in Fig. 1A. A) Quantification of inflammatory cell infiltrates on H&E-stained lung sections as shown in Fig. 1D. B) The tumor multiplicity and C) the tumor area data presented in Fig. 1H–I were subdivided into male and female mice for the three experimental groups. D) Representative images of Wright-Giemsa staining of BALF cytospins. Magnification, 200x (overview panels), 400x (insets). E) BALF total and differential cell counts of BALF cytospins as shown in D. F) Correlation analysis of the number of monocytes/macrophages in the BALF with tumor multiplicity. The Pearson coefficient (R squared) and the corresponding P-value are shown. G) Representative pictures of H&E-stained lung sections of Rag1 KO mice treated i.n with VEH (n = 9) or HDM (n = 9), and i.p with urethane (0.6 mg/g of BW) as shown in Fig. 1A for WT mice. One lobe per mouse is shown. Arrowheads indicate tumors. Scale bars, 1 mm. H) Tumor multiplicity calculated on H&E-stained sections as shown in G. Data are presented as mean ± SEM. Statistical significance was assessed by one-way (A) or two-way (B, C, and E) ANOVA with post hoc Bonferroni’s test, linear regression using Pearson correlation (F), or two-tailed Student’s t-test (H). n.s: non-significant, * P < 0.05, ** P <0.01, *** P <0.001, **** P < 0.0001. Supplemental Fig. 2. Effect of chronic exposure to HDM in a KrasG12D-driven LC model. A) Five-week-old KrasG12D mice were treated i.n with VEH (n = 11) or HDM (n = 12) as indicated in this schematic overview of the study design. At 14 weeks of age and 72h after the last HDM challenge the mice were sacrificed and the BALFs and the lungs were harvested. B) Representative pictures of 4 lung lobes (dorsal view) of a VEH- and an HDM-treated mouse. Scale bars, 0.25 cm. C) Lung weight normalized to mouse body weight (BW). D) BALF total cell counts. E) Representative pictures of H&E-stained lung sections. The lower panels are the same images as the above panels after tumor area quantification using QuPath software. The area within the blue borders was considered positive for lung tumors. The right panels show the boxed region (ROI#1) at higher magnification with areas of alveolar/bronchiolar hyperplasia and numerous mononuclear inflammatory infiltrates. Scale bars, 2 mm (whole lungs) and 0.5 mm (ROI#1). F) Tumor multiplicity calculated on H&E-stained sections as shown in E upper panels. G) Tumor area calculated on H&E-stained sections as shown in E lower panels. H) Quantification of inflammatory cell infiltrates on H&E-stained lung sections of 18-week-old KrasG12D mice treated i.n with VEH, HDM, or HI-HDM as shown in Fig. 2E. I) Representative pictures of H&E-stained lung sections showing the different types of lung lesions observed in 18-week-old
KrasG12D mice treated i.n with VEH, HDM, or HI-HDM as shown in Fig. 2E. Top left panels: Grade 1 lesion of AAH with lepidic growth pattern observed in a
KrasG12D mouse treated with VEH. Scale bars, 0.1 mm (overview panel and inset). Middle left panels: Grade 1 lesion of EH of a respiratory bronchiole with hyperproliferative cells in the alveolar compartment observed in a
KrasG12D mouse treated with VEH. Scale bars, 0.1 mm (overview panel and inset). Middle right panels: Grade 2 AD of the papillary type with uniform nuclei observed in a
KrasG12D mouse treated with VEH. Scale bars, 0.2 mm (overview panel) and 0.1 mm (inset). Right panels: Grade 3 AC of the papillary type with enlarged nuclei, prominent nucleoli, and some areas of tumor cell crowding (field of cells indicated by arrows) observed in a
KrasG12D mouse treated with HDM.Scale bars, 0.25 mm (overview panel) and 0.1 mm (inset).Data are representative of one experiment conducted in six independent cohorts of mice pooled together and are presented as mean ± SEM. Statistical significance was assessed by two-tailed Student’s t-test (C, D, F, and G) or one-way ANOVA with post hoc Bonferroni’s test (H). **
P <0.01, ***
P <0.001, ****
P <0.0001.
Supplemental Fig. 3. Effect of chronic HDM exposure on the lung microenvironment.
KrasG12D mice were treated i.n with VEH or HDM (
n = 3 mice/group) as shown in Fig. 2A and were sacrificed at day 61 (24h after the last i.n treatment). The lungs and the BALFs were recovered and single-cell suspensions were prepared for flow cytometry analyses. A) Representative flow cytometry plots of an HDM-treated mouse illustrating the gating strategy for the flow cytometry analyses. First, dead cells and debris were excluded based on forward scatter area (FSC-A) and side scatter area (SSC-A) values, then live single cells were gated based on forward scatter height (FSC-H) and FCS-A values.Macrophages (MΦ) were then identified as CD11b
+CD11c
–F4/80
+ cells. B) Representative dot plots of cells expressing the pan-myeloid marker CD11b in the BALFs of VEH- and HDM-treated mice. C) Frequencies and D) absolute numbers of CD11b
+ cells as shown in B. E) Representative histograms showing intracellular levels of pro-IL-1β in CD11b
+ cells gated as in B in the BALFs of VEH- and HDM-treated mice. F) Frequencies and G) absolute numbers of pro-IL-1β
+CD11b
+ cells as shown in E. H) Representative dot plots of MDSCs (identified as CD11b
+ cells gated as in B expressing F4/80 and intermediate levels of Gr-1) in the BALFs of VEH- and HDM-treated mice. I) Frequencies and J) absolute numbers of MDSCs as shown in H. K) Representative histograms showing PD-1 expression levels on CD11b
– cells gated as in A. L) Frequencies and M) absolute numbers of PD-1
+CD11b
– cells as shown in K. Data are presented as mean ± SEM. Statistical significance was assessed by two-tailed Student’s t-test. n.s: non-significant, *
P < 0.05, **
P < 0.01.
Supplemental Fig. 4. Effect of HDM and other allergens on IL-1β secretion by BMDMs. A) Schematic overview of the BMDM culture system. Bone marrow (BM) cells were harvested from WT,
Nlrp3,
Casp1, or
Il1b KO mice and were differentiated into BMDMs in presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) for 7 days. BMDM (identified as CD11b
+F4/80
+ cells) purity was assessed by flow cytometry and was ~80% as shown in a representative dot plot. BMDMs were then stimulated for 24h with LPS, HDM, or other allergens, and ATP was added to each well for the last hour of culture. The supernatants were collected and the levels of IL-1β or TNF were analyzed by ELISA. B) WT BMDMs were pretreated for 1h at 37°C with an NLRP3 inhibitor (MCC950) or with a caspase-1 inhibitor (VX-765) at concentrations of 100 nM and 10 μM, respectively before being stimulated with HDM (200 μg/mL) and ATP (5 mM) as shown in A, and the levels of IL-1β or C) TNF in the supernatants were analyzed by ELISA. D) WT BMDMs were stimulated with the indicated concentrations of HDM
Dermatophagoides pteronyssinus (DP) as in B and C, HDM
Dermatophagoides farinae (DF), German cockroach (CR), ragweed pollen (RW), the fungi
Candida albicans (CA) or
Alternaria alternata (AA) and ATP (5 mM) as shown in A. The supernatants were collected and IL-1β production was analyzed by ELISA. IL-1β level in the supernatants of DP-treated cells was used as a reference and assigned to 100%. Data are representative of two (B and C) or three (D) independent experiments performed in triplicate and are presented as mean ± SEM. Statistical significance was assessed by one-way ANOVA with post hoc Bonferroni’s test. n.s: non-significant, *
P < 0.05, **
P < 0.01, ****
P < 0.0001.
Supplemental Fig. 5. Effect of chronic exposure to HDM DF or ovalbumin on lung tumor development. A)
KrasG12D mice were treated i.n with VEH (
n = 4), DF (
n = 9), or ovalbumin (OVA,
n = 6) as indicated in this schematic overview of the study design. B) Representative pictures of H&E-stained lung sections of mice in the three different groups. Scale bars, 2 mm. C) Tumor multiplicity calculated on H&E-stained sections as shown in B. Data are presented as mean ± SEM. Statistical significance was assessed by one-way ANOVA with post hoc Bonferroni’s test. n.s: non-significant, *
P < 0.05, **
P < 0.01.
Supplemental Fig. 6. Effect of IL-1β, CCL2, or NLRP3 neutralization in
KrasG12D mice. A)
KrasG12D mice were treated i.n with VEH or HDM and i.p with an anti-IL-1β, an anti-CCL2, or with the isotype control (ctrl) antibody (Ab), or with the NLRP3 inhibitor MCC950 as indicated in this schematic overview of the study design. B) Representative pictures of H&E-stained lung sections of mice treated with VEH + MCC950 (
n = 7) or HDM + MCC950 (
n = 11). Scale bars, 2 mm. C) Tumor multiplicity calculated on H&E-stained sections as shown in B. D) Tumors on H&E-stained sections as shown in B were classified into three grades (Grade 1, AAH and EH; Grade 2, AD; Grade 3, AC) and each grade was expressed as a percentage of the total. Data are representative of one experiment conducted in three independent cohorts pooled together (B–D) and are presented as mean ± SEM. Statistical significance was assessed by two-tailed Student’s t-test (C) or two-way ANOVA (D) with post hoc Bonferroni’s test. n.s: non-significant.
Supplemental Fig. 7. HDM induces CCL2 production in the BALF of
KrasG12D mice and IL-1β neutralization inhibits this effect. A) ELISA analysis of CCL2 production in BALF recovered from
KrasG12D mice treated i.n with VEH, HDM, or HI-HDM as shown in Fig. 2A (n = 4 mice/group) or B) treated i.n with VEH or HDM and i.p with the anti-IL-1β or the isotype ctrl Ab as shown in Supplemental Fig. 6A (
n = 3 mice/group). Data are representative of one experiment conducted twice independently and are presented as mean ± SEM. Statistical significance was assessed by one-way ANOVA with post hoc Bonferroni’s test. n.s: non-significant, *
P< 0.05, **
P < 0.01, ***
P< 0.001.
Supplemental Fig. 8. Antibody validation for mIF and IHC staining. A) mIF staining of a mouse spleen section showing strong F4/80 and Ki-67 immunoreactivity. As expected, no PanCK was observed in the spleen. B) No background or non-specific binding was observed in the spleen negative control in the absence of the primary Abs. DAPI is shown for context. C) Positive control slide images using human tonsils showing strong PanCK and Ki-67 immunoreactivity. The F4/80 antibody does not work for human tissue. D) No background or non-specific binding was observed in the tonsil negative control in the absence of the primary Abs. DAPI is shown for context. E) Negative controls on a mouse lung section using secondary anti-Tag fluorescent Abs only. No staining was observed in absence of primary Abs. However, some autofluorescence was detected in the PanCK/CL550 (Cy3) channel from the heart tissue and red blood cells. Overlay (top) and single colors (bottom). DAPI nuclear staining (blue), F4/80 (magenta), Ki-67 (cyan) and PanCK (yellow). Scale bars, 0.25 mm (spleen and tonsil samples) and 2 mm (lung samples). F) IHC staining of a mouse lung lobe showing nuclear staining for TTF-1 in airway epithelial cells, including alveolar pneumocytes and club cells. No staining was observed in inflammatory cell infiltrates surrounding the bronchi and bronchioles. Scale bars, 1mm (upper panel) and 0.1 mm (inset). Data are representative of one (F) or two (A–G) independent experiments.
Supplemental Fig. 9. Proposed model for the lung tumor-promoting effect of HDM. Chronic i.n instillation of HDM extracts triggers the activation of the NLRP3 inflammasome and caspase-1 in lung macrophages (MΦ, identified as CD11b
+CD11c
–F4/80
+ cells), which leads to increased secretion of IL-1β in the lung microenvironment and persistent lung inflammation. In addition, HDM induces the accumulation of PD-1
+-myeloid cells (identified as PD-1
+CD11b
+ cells) and myeloid-derived suppressor cells (MDSCs, identified as Gr-1
+CD11b
+ cells) in the lungs, which are known to inhibit anti-tumor immunity and to promote tumor cell proliferation. Neutralization of NLRP3, IL-1β, or CCL2, which could contribute to the recruitment of bone marrow (BM)-derived circulating monocytes and to the replenishment and pro-inflammatory phenotype of lung MΦ, as well as treatment with budesonide, which has anti-inflammatory and anti-proliferative properties, all inhibit the tumor-promoting effect of HDM. Finally, NLRP3 activation, IL-1β production, and the pro-tumorigenic effect of HDM were also significantly decreased but not completely abolished by heat treatment of the HDM extracts, suggesting that both heat-sensitive (e.g., proteolytic enzymes derived from the mites’ digestive system) and -insensitive (e.g., heat-insensitive proteases, LPS, and/or carbohydrates such as chitin) factors in HDM extract contributed to these effects. This schematic representation was created with BioRender.com and Servier Medical Art.
Supplemental Table 1. Reagents used in this study.
Supplemental Table 2. Oligonucleotides used for RT-qPCR analysis.